BRPI0620639A2 - bispecific single chain antibodies and pharmaceutical composition comprising the same - Google Patents

Abstract

SIMPLE BIESPECIFICIAL CHAIN ANTIBODIES AND PHARMACEUTICAL COMPOSITION UNDERSTANDING THE SAME. The present invention relates to bispecific single chain antibodies comprising a first binding domain that binds immunospecifically with the T cell antigen CD3 and a second binding domain that binds immunospecifically with the EphA2 receptor. Such bispecific single chain antibodies are covered by the term "EphA2-BiTEs". The present invention also relates to methods and compositions for the treatment, prevention and / or management of disorders associated with the abnormal expression and / or activity of EphA2. Such disorders include, without limitation, cancer, non-cancerous hyperproliferative cell disorders and infections. The invention discloses vectors that comprise polynucleotides encoding the EphA2-BiTEs of the invention, host cells transformed by them and their use in the production of said EphA2-BiTEs. The present invention also provides compositions, including pharmaceutical compositions, comprising any of said EphA2-BiTEs, polynucleotides or vectors, alone or in combination with one or more therapeutic or prophylactic agents. Methods for scanning said EphA2-BiTEs and kits comprising any of said compositions and diagnostic reagents are also described.

Description

SIMPLE BISPECIFIC CHAIN ANTIBODIES AND PHARMACEUTICAL COMPOSITION UNDERSTANDING THE SAME

The present application claims the benefit of U.S. Provisional Application No. 60 / 753,368, filed December 21, 2005, which is incorporated by reference herein in its entirety.

FIELD OF INVENTION

The present invention relates to single-chain bispecific antibodies comprising a first "binding domain that immunospecifically binds to the T cell CD3 antigen and a second binding domain that immunospecifically binds to the EphA2 receptor. Such bispecific single chain antibodies are encompassed. by the term "EphA2-BiTEs." The present invention further relates to methods and compositions designed for the treatment, prevention and / or management of disorders associated with aberrant EphA2 expression and / or activity. Such disorders include, but are not limited to. a, cancer, non-cancerous hyperproliferative cell disorders, and infections The invention is further related to vectors comprising polynucleotides encoding the EphA2-BiTEs of the invention, host cells transformed with them, and their use in the production of said EphA2-BiTEs. provides compositions, including compositions pharmaceutical agents, comprising any of the following EphA2-BiTEs, polynucleotides or vectors either alone or in combination with one or more prophylactic or therapeutic agents. Also described are screening methods for said EphA2-BiTEs and kits comprising any of the compositions and diagnostic reagents mentioned below.

BACKGROUND OF THE INVENTION

2.1 EphA2

EphA2 is a 130 kDa receptor tyrosine kinase that is expressed in adult epithelium where it is found at low levels and is enriched at cell-cell adhesion sites (Zantek et al., 1999, Cell Growth & Differentiation 10 (9): 629 Lindberg et al., Mol. & Cell. Biol. 10: 6316, 1990). This subcellular localization is thought to play a role in inhibiting contact through the interaction of EphA2 with its ligands (known as EphrinsAl to A5) that are anchored to the cell membrane in adjacent cells (Eph Nomenclature Committee, 1997, Cell 90: 403-04). Cheng et al., 2002, Cytokine & Growth Factor Rev. 13: 75-85). The engagement of EphA2 with its ligand results in EphA2 autophosphorylation and its subsequent degradation (Walker-Daniels et al., 2002, Mol. Cancer. Res. 1 (1): 79-87; Carles-Kinch et al., 2002, Cancer Res. 62 (10): 2840-47). This signaling cascade also initiates below events that negatively regulate attachment to extracellular matrix adhesion molecules and thereby regulate cell growth and migration (Zantek et al., 1999, Cell Growth & Differentiation 10 (9): 629- 38; Miao et al., 2000, Nat. Cell Biol. 2 (2): 62-69; Zelinski et al., 2001, Cancer Res. 61 (5): 2301-06).

EphA2 has been shown to be overexpressed in a number of different tumor types including melanoma, renal cell carcinoma, breast, prostate, colon, esophageal, cervical, lung, ovarian, and bladder cancers (Carles-Kinch et al. , 2002, Cancer Res. 62 (10): 2840-47). Higher levels of EphA2 expression are observed in the more aggressive cells, suggesting a role for EphA2 in disease progression. High EphA2 levels have also been correlated with poor survival for non-small cell lung, esophageal, cervical, and ovarian cancers (Kinch et al., 2003, Clin. Cancer Res. 9 (2): 613-18; Miyazaki et al., 2003, Int. J. Cancer 103 (5) 657-63; Wu et al., 2004, Gynecol. Oncol. 94 (2): 312-19; Thaker et al., 2004, Clin. 10 (15): 5145-50). Additionally, in preclinical models, exogenous EphA2 expression has been shown to be sufficient to yield a non-tumorigenic, tumorigenic cell line in vitro and in vivo (Zelinski et al., 2001, Cancer Res. 61 (5): 2301- 06).

2.2 BiTE® Molecules

Bispecific T cell engagements, or BiTEs®, are a form of bispecific antibodies that are based on single-stranded antibodies arranged one after another (reviewed in Wolf et al., 2005, Drug Discovery Today: in press). They form a single polypeptide chain of approximately 55 kDa and are secreted by Chinese hamster ovary (CHO) cells as a mixture of monomers and dimers. With a branch, BiTEs® binds to the human CD3 epsilon (ε) subunit, a protein component of T-cell receptor signal transduction complex. With a branch, BiTEs® recognizes an antigen on target cells. T cell activation is seen only when BiTEs® are presented to T cells on the surface of target cells.

BiTEs® transiently limit T cells and target cells. T cell activation by BiTEs® involves up-regulation of CD69, CD25 and various cell adhesion molecules, de novo expression and cytokine release (eg, IFN-γ, TNF-α, IL-β, IL-2, IL -4 and IL-10), up-regulation of granzyme and perforin expression, and cell proliferation. BiTEs®-redirected target cell lysis is independent of T cell receptor specificity, presence of MHC class I and β2 microglobulin, and any costimulatory stimuli. This independence of regular T-cell signals and recognition molecules can be explained by BiTEs® induction of regular cytolytic synapses and maximum membrane proximity. Displacement of negative regulatory proteins such as CD45 from BiTE®-induced synapses may alleviate the need for costimulation. BiTEs® present in vitro redirected lysis with previously unstimulated CD8- and CD4-positive peripheral T cells. No activity is seen with CD8- or CD4-positive T cells. CD4 T cells can positively regulate granzyme B and perforin expression when stimulated with BiTEs® and thus contribute to CD8-mediated target cell lysis. In vitro, redirected lysis is seen at low picomolar concentrations, suggesting that very low numbers of BiTE molecules need to be bound to target cells to activate T cells. In mouse SCID models, sub-pg doses of BiTEs® have been proven to completely prevent tumor growth (Dreier et al., 2003, J Immunol. 170: 4397-4402) and for eradicating solid tumors by up to 200 mm3 (Schlereth et al., 2005, Cancer Res. 2005 65 (7): 2882-89 ).

BiTEs® therefore provide a unique opportunity to develop effective and selective antibody-based therapies against EphA2 for the treatment, prevention and / or management of disorders associated with aberrant EphA2 expression and / or activity (eg, cancer, non-hyperproliferative cell disorders). cancerous, and infections).

SUMMARY OF THE INVENTION

The present invention provides bispecific T-cell engaging (ie, EphA2-BiTEs (in particular, EphA2-BiTEs, which are single-chain bispecific antibodies)) that bind immunospecific EphA2 and T-cell CD3 antigen, and methods of using it. to treat, prevent and / or manage disorders associated with aberrant EphA2 expression and / or activity. Such disorders include, for example, cancer, noncancerous hyperproliferative cell disorders, and infections. In one aspect, EphA2-BiTEs are more efficient at eliminating aberrantly expressing EphA2 cells than specific EphA2 antibodies known in the art. In a specific aspect, EphA2-BiTEs are more efficient at eliminating EphA2-expressing cancer cells (in particular, EphA2-expressing malignant cancer cells) than EphA2 antibodies known in the art. In another aspect, EphA2-BiTEs are more efficient at eliminating non-cancerous hyperproliferative cells expressing EphA2 than EphA2 antibodies known in the art. In yet another aspect, the EphA2-BiTEs of the invention are more efficient at eliminating infected cells expressing EphA2 (in particular, respiratory syncytial virus infected cells; "RSV") than EphA2 antibodies known in the art. In another aspect, lower dosages of EphA2-BiTEs than specific EphA2 antibodies known in the art are required to treat, prevent and / or manage disorders associated with aberrant EphA2 expression and / or activity.

The specific EphA2 bispecific T cell engaging agents of the present invention comprise a first binding domain that immunospecifically binds to the T cell CD3 antigen and a second binding domain that immunospecifically binds to EphA2 (hereinafter "EphA2-BiTEs,"). EphA2-BiTE molecules "or" EphA2 bispecific cell engagements). In one advance, the first binding domain immunospecifically binds to CD3. In one specific advance, the first binding domain immunospecifically binds to one or more of any subunit. (eg, the gamma, delta, zeta, or eta subunit.) In a preferred advance, the first binding domain immunospecifically binds to the CD3 epsilon (ε) subunit. immunospecifically binds to the epsilon (ε) subunit of CD3 when said subunit is complexed with the delta subunit of CD3. CD3 binding is de-immunized In another specific advance, the second binding domain immunospecifically binds to the extracellular domain of EphA2. In a preferred embodiment, the second binding domain of EphA2-BiTEs, which are used in cancer treatment, prevention and / or management, immunospecifically binds to EphA2 epitopes that are selectively exposed and / or increased in cancer cells, but not. in noncancerous cells. In another preferred embodiment, the second binding domain of the EphA2-BiTEs of the invention immunospecifically binds to epitopes on EphA2 that are selectively exposed and / or increased in non-cancerous hyperproliferative cells, but not non-hyperproliferative cells. In another preferred embodiment, the second binding domain of the EphA2-BiTEs of the invention immunospecifically binds to epitopes on EphA2 that are selectively exposed and / or increased in infected cells, but not in uninfected cells.

In a specific advance, an EphA2-BiTE of the invention comprises: (1) a first binding domain comprises a heavy variable domain (VH) and a light variable domain (VL) of an antibody that immunospecifically binds to the CD3 cell antigen T; and (2) a second binding domain comprising a VH domain and a VL domain of an antibody that immunospecifically binds to EphA2. In a specific advance, the VH and VL domains of the first binding domain are joined together by a ligand of sufficient strength to allow the domains to bend to allow binding to the T cell CD3 antigen. may comprise, for example, the sequence GEGTSTGS (G2S) 2GGAD (SEQ ID NO.:57). In another specific advance, the VH and VL domains of the second binding domain are joined together by a ligand of sufficient strength to allow the domains to fold to allow binding to EphA2. Still for this advance, such a binder may comprise, for example, the sequence (G4S) 3 (SEQ ID NO: 59). In another specific advance, the first and second binding domains are joined together by a ligand of sufficient strength to allow the domains to fold to allow binding to the Tea EphA2 cell CD3 antigen. Still for this advance, such a binder may comprise, for example, the sequence G4S (SEQ ID NO: 58). In one specific advance, an EphA2-BiTE of the invention is deimmunized anti-CD3xEA2 (VH / VL) (SEQ ID NO: 65). According to the indent in the immediately preceding paragraph, the bond is covalent. In a specific advance, the binders of the invention comprise serine and glycine residues. The EphA2-BiTEs ligands, eg, the ligand between the VH and VL domains of the first CD3 binding domain, the ligand between the VH and VL domains of the second EphA2 binding domain, and the ligand between the first CD3 binding domain and the second EphA2 binding domain may be of any length sufficient to allow the domains to fold to allow binding to the CD3 and EphA2 antigens, respectively. In certain advances, the binders of the invention comprise a length of at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 25 residues, at least 30 residues or more. In further advances, the binders of the invention comprise a length between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, 14 wastes, 2-16 wastes, 2-18 wastes, 2-20 wastes, 2-22 wastes, 2-24 wastes, 2-26 wastes, 2-28 wastes, or 2-30 waste. In certain advances, the first binding domain is 5 'to the second binding domain. In other advances, the second binding domain is 5 'to the first binding domain. In certain advances, the first and second binding domains are single chain antibodies. In a specific advance, the first and second binding domains comprise single stranded Fvs (scFvs).

In a specific advance, the invention provides a single chain bispecific antibody comprising (a) a first heavy chain variable domain and a first light chain variable domain each of an antibody that immunospecifically binds to the CD3 ε chain, said first variable domain covalently linked to said first light chain variable domain by a first binder of sufficient length (eg, GEGTSTGS (G2S) 2GGAD (SEQ ID NO.:57)) such that said first heavy chain variable domain and said first light chain variable domain fold to form a first binding domain that binds to the CD3 β subunit; and (b) a second heavy chain variable domain and a second light chain variable domain of an antibody that immunospecifically binds an exposed EphA2 epitope on the cell surface, said second heavy chain variable domain covalently linked to said second variable domain. light chain by a second binder of sufficient length (eg, (G4S) 3 (SEQ ID NO: 59)) such that said second heavy chain variable domain and said second light chain variable domain bend to form a second domain. binding that binds said EphA2 epitope, characterized in that said first binding domain and said second binding domain are covalently linked by a third binder of a length (eg, G4S (SEQ ID NO: 58)), such that said first binding domain and said second binding domain fold independently of each other.

In specific embodiments, the EphA2-BiTEs of the invention comprise any of the following 5 'to 3' direction arrangements: (1) VHcD3-VLcD3-VHEphA2-VL-EphA2; (2) VLCD3-VHcD3-VHEphA2-VL EphA2 r (3) VLCD3-VHcD3-VLEphA2-VH-EphA2 / (4) VHCD3-VLcD3-VLEphA2-VHEphA2; (5) VHEphA2-VLEphA2-VHcd3-VLcd3; (6) VLEphA2-VHEphA2-VHCD3-VLcd3 / '(7) VLEphA2-VHEphA2-VLcD3-VHcD3; or (8) VHEphA2-VLEphA2-VLcD3-VH-CD3-See, e.g., FIG. 14A for a generic demonstration of the EphA2-BiTE constructs of the invention.

In a specific advance, the first binding domain of an EphA2-BiTE of the invention binds to the subunit and CD3 with a lower affinity than the second binding domain that binds EphA2. In one advance, the dissociation constant (Kd) of the first subunit binding domain and CD3 is between 0.1 x 10-12 M and 0.5 x 10-12 M, 0.1 x 10- 12 Melx 10-12 M, 0.1 x 10-11 Me 0.5 x 10-11 M, 0.1 x 10-11 Melx 10 "11 M, 0.1 x 10-10 M and 0.5 x 10-10 M, 0.1 x 10-10 Melx 10-10 M, 0.1 x 10-9 M and 0.5 x 10-9 M, 0.1 x 10-9 Melx 10-9 M, 0 0.1 x 10-8 Me 0.5 x 10-8 M, 0.1 x 10-8 Mel x 10-8 M, 0.1 x 10-7 M and 0.5 x 10-7 M, 0, 1 x 10-7 Melx 10-7 M, 1 x 10-7 M and 2 x 10-7 M, 1 x 10-7 M and 3 x 10-7 M, 1 x 10-7 M and 4 x 10- 7 M, 1 x 10-7 M and 5 x 10-7 M, 1 x 10-7 M and 6 x 10-7 M, 1 x 10-7 M and 7 x 10-7 M, 1 x 10-7 M and 8 x 10-7 M, 1 x 10-7 M and 9 x 10-7 M, 1 x 10-7 M and 10 x 10-7 M, 0.1 x 10-6 Me 0.5 x 10 -6 M, 0.1 x 10-6 Melx 10-6 M, 1 x 10-6 M and 2 x 10-6 M, 1 x 10-6 M and 3 x 10-6 M, 1 x 10-6 M and 4 x 10-6 M, 1 x 10-6 M and 5 χ 10-6 Μ, 1 χ 10-6 M and 6 χ 10-6 Μ, 1 χ 10-6 M and 7 χ 10-6 Μ , 1 χ 10-6 M and 8 χ 10-6 Μ, 1 χ 10-6 M and 9 χ 10-6 Μ, 1 χ 10-6 M and

10χ 10-6 Μ, 0.1 χ 10-5 Me 0.5 χ 10-5 Μ, 0.1 χ 10-5 Melx 10-5 Μ, 1 χ 10-5 M and 2 χ 10-5 Μ, 1 χ 10-5 M and 3 χ 10-5 Μ, 1 χ 10-5 M and 4 χ 10-5 Μ, 1 χ 10-5 M and 5 χ 10-5 Μ, 1 χ 10-5 M and 6 χ 10-5 Μ, 1 χ 10-5 Μ and 7 χ 10-5 Μ, 1 χ 10-5 M and 8 χ 10-5 Μ, 1 χ 10-5 M and 9 χ 10-5 Μ, 1 χ 10-5 M and 10 χ 10-5 Μ. In a specific advance, the dissociation constant of the first domain that binds to the CD3 subunit ε is 4 χ 10-7 Μ. In another specific advance, the dissociation constant of the second domain that binds to EphA2 is between 0.1 χ 10-12 Me 0.5 χ 10-12 Μ, 0.1 χ 10-12 Melx 10-12 Μ, 0 , 1 χ 10-11 M and 0.5 χ 10-11 Μ, 0.1 χ 10-11 Melx 10-11 Μ, 0.1 χ 10-10 Me 0.5 χ 10-10 Μ, 0.1 χ 10-10 Melx 10-10 Μ, 0.1 χ 10-9 M and 0.5 χ 10-9 Μ, 0.1 χ 10-9 Melx 10-9 Μ, 0.1 χ 10-8 M and 0 0.5 χ 10-8 Μ, 0.1 χ 10-8 Melx 10-8 Μ, 0.1 χ 10-7 Me 0.5 χ 10-7 Μ, 0, 1 χ 10-7 M and 1 χ 10 -7 Μ, 1 χ 10-7 M and 2 χ 10-7 Μ, 1 χ 10-7 M and 3 χ 10-7 Μ, 1 χ 10-7 M and 4 χ 10-7 Μ, 1 χ 10- 7 M and 5 χ 10-7 Μ, 1 χ 10-7 M and 6 χ 10-7 Μ, 1 χ 10-7 M and 7 χ 10-7 Μ, 1 χ 10-7 M and 8 χ 10-7 Μ, 1 χ 10-7 M and 9 χ 10-7 Μ, 1 χ 10-7 M and 10 χ 10-7 Μ, 0.1 χ 10-6 M and 0.5 χ 10-6 Μ, 0, 1 χ 10-6 Melx 10-6 Μ, 1 χ 10-6 M and 2 χ 10-6 Μ, 1 χ 10-6 M and 3 χ 10-6 Μ, 1 χ 10-6 M and 4 χ 10- 6 Μ, 1 χ 10-6 M and 5 χ 10-6 Μ, 1 χ 10-6 M and 6 χ 10-6 Μ, 1 χ 10-6 M and 7 χ 10-6 Μ, 1 χ 10-6 M e 8 χ 10-6 Μ, 1 χ 10-6 M and 9 χ 1 0-6 Μ, Ix 10-6 M and 10 χ 10-6 Μ, 0.1 χ 10-5 Me 0.5 χ 10-5 Μ, 0.1 χ 10-5 Melx 10-5 Μ, 1 χ 10-5 M and 2 χ 10-5 Μ, 1 χ 10-5 M and 3 χ 10-5 Μ, 1 χ 10-5 M and 4 χ 10-5 Μ, 1 χ 10-5 M and 5 χ 10 -5 Μ, 1 χ 10-5 M and 6 χ 10-5 Μ, 1 χ 10-5 M and 7 χ 10-5 Μ, 1 χ 10-5 M and 8 χ 10-5 Μ, 1 χ 10- 5 M and 9 χ 10-5 Μ, 1 χ 10-5 M and 10 χ 10-5 Μ. At a specific advance, the dissociation constant of the second domain that binds to EphA2 is 1.13 χ IO-7 Μ. In another specific advance, an EphA2-BiTE of the invention comprises a first binding domain that binds to the CD3 ε subunit with a K0 of 4 χ 10 7 M and a second binding domain that binds to EphA2 with a K0 of 1.13 χ 10 "7 Μ.

In specific advances, the EphA2 binding domain of an EphA2-BiTE of the invention has a high affinity constant and a low dissociation constant. In an alternative advance, the EphA2 binding domain of an EphA2-BiTE of the invention has a low affinity constant and a high dissociation constant. In another advance, the EphA2 binding domain of an EphA2-BiTE of the invention has a high affinity constant and a high dissociation constant. In another specific advance, the CD3 binding domain of an EphA2-BiTE of the invention binds to the CD3 ε subunit with lower affinity than the second binding domain that binds EphA2.

In a specific advance, the EphA2-BiTEs of the invention first bind to EphA2 and then bind to CD3. In another specific advance, the EphA2-BiTEs of the invention cause lysis of EphA2-expressing target cells as measured by standard cytotoxicity tests known in the art or as described below in Sections 6.2.6 and 6.3. In a specific advance, the EphA2-BiTEs of the invention mediate lysis of target cells (eg, non-cancerous or infected cancer cells expressing EphA2) by at least 10%, at least 15%, at least 20% 25% at least 30% at least 35% at least 40% at least 45% at least 50% at least 55% at least 60% at least 65% at least 70% at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 7 times or at least 10 times relative to the expression level of EphA2 in normal cells, cells of a subject normal, healthy and / or a normal healthy cell population expressing EphA2 as measured by a flow cytometry-based assay as described in Section 6.0 below. In yet another advance, the EphA2-BiTEs of the invention do not activate (e.g., phosphorylate) EphA2 when bound to EphA2 as measured by an immunoprecipitation / Western blot assay as described in Section 6.0 below. At a specific rate, less than 20%, less than 15%, or less than 5% of the EphA2 receptor population is activated (e.g., phosphorylated) when bound to an EphA2-BiTE of the invention.

The present invention further provides compositions comprising the EphA2-BiTEs of the invention. In particular, the present invention provides pharmaceutical compositions comprising the EphA2-BiTEs of the invention and one or more pharmaceutical carriers or excipients. The present invention provides aqueous formulations, lyophilized formulations, gels, and surgical implants containing any of the EphA2-BiTEs of the invention. The present invention also provides kits comprising one or more EphA2-BiTEs of the invention in one or more containers and instructions for use of such EphA2-BiTEs.

The present invention provides compositions and methods for treating, preventing and / or managing cancer associated with aberrant EphA2 expression (eg, overexpression) and / or activity, the methods comprising administering to a subject in need an EphA2-BiTE of invention. In a specific advance, the present invention provides methods for treating, preventing and / or managing cancer associated with aberrant EphA2 expression, methods comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of an EphA2-BiTE of the invention. .

In a breakthrough, cancer to be treated, prevented and / or managed is a cell of epithelial origin. In yet another advancement, a subject's cancer cells being treated, prevented and / or managed overexpress EphA2 relative to said subject's normal cells, cells from a normal, healthy subject, and / or a normal cell population. , healthy. In a preferred embodiment, some EphA2 expressed by cancer cells is bound to a ligand, either as a result of fewer cell-cell contacts, altered subcellular localization, or increase in the amount of ligand-related EphA2. In a preferred advance, the cancer to be treated, prevented and / or managed is malignant.

The EphA2-BiTEs of the invention may be administered in combination with one or more cancer therapies. In particular, the present invention provides methods of treating, preventing and / or managing cancer, methods comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of one or more EphA2-BiTEs of the invention in combination with administration. of a therapeutically or prophylactically effective amount of one or more other therapies. Non-limiting examples of other therapies include chemotherapies, hormone therapies, biological therapies / immunotherapies, radiation, and surgery.

Increased EphA2 expression has been seen to be associated with infections by certain intracellular pathogens, in particular RSV (see, eg, US Appn. Ser. No. 11 / 259,266, filed October 27, 2005, entitled "Use of Modulators of EphA2 and EphrinAl for the Treatment and Prevention of Infections ", which is incorporated herein by reference in its entirety). Accordingly, the invention also provides compositions and methods designed for the treatment, prevention and / or management of a pathogen infection, including, but not limited to, a viral infection, a bacterial infection, a fungal infection and a protozoan infection ( Examples of such pathogens are described in, eg, US Appn. Ser. No. 11 / 259,266, filed October 27, 2005, entitled "Use of Modulators of EphA2 and EphrinAl for Treatment and Prevention of Infections", (and in particularly in paragraphs [006], [0046], [0047], and [0057]) which is incorporated herein by reference in its entirety). In particular, the present invention provides methods for treating, preventing and / or managing an infection where EphA2 expression is up-regulated in infected cells (eg, infected cells expressing EphA2), said methods comprising administering to a subject in need thereof effective amount of one or more EphA2-BiTEs of the invention, and optionally an effective amount of a therapy other than EphA2-BiTEs. In a specific advance, the pathogenic infections to be treated, prevented and / or managed according to the methods of the invention are intracellular pathogenic infections. In another specific advance, the pathogenic infection to be treated, prevented and / or managed according to the methods of the invention is an RSV infection.

In another aspect of the invention, increased expression of EphA2 has been seen to be associated with certain non-cancerous hyperproliferative cell disorders such as, for example, those described in U.S. Pat. Pub. No. US 2005-0059592 Al, entitled "EphA2 and Hyperproliferative Cell Disorders", which is incorporated herein by reference in its entirety. Accordingly, the invention also provides compositions and methods designed for the treatment, prevention and / or management of hyperproliferative cell disorders (nonlimiting examples of such disorders are described in, eg, US Pat. Pub. No. 2005-0059592, entitled " EphA2 and Hyperproliferative Cell Disorders ", (and in particular in paragraph [0035]) which is incorporated herein by reference in its entirety. In particular, the present invention provides methods for treating, preventing and / or managing a hyperproliferative cell disorder where expression of EphA2 is up-regulated in cells affected by such disorder, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention, and optionally an effective amount of therapy without EphA2-BiTE. In a specific advance, the hyperproliferative cell disorder to be treated, prevented and / or managed according to the methods of the invention. Treatment is asthma, COPD, pulmonary fibrosis, asbestosis, IPF, DIP, PIÜ, kidney fibrosis, liver fibrosis, other fibrosis, hyperresponsive bronchial psoriasis, psoriasis, seborrheic dermatitis, cystic fibrosis, or a hyperproliferative endothelial cell disorder. such as restenosis, hyperproliferative vascular disease, Behcet's syndrome, atherosclerosis, macular degeneration, or a hyperproliferative fibroblast disorder.

The methods and compositions of the invention are useful not only in untreated cancer patients, but are also useful in treating cancer patients partially or completely refractory to current standard and experimental cancer therapies, including, but not limited to chemotherapies, hormone therapies. , biological therapies, immunotherapies, radiation therapies and / or surgery as well as to improve the effectiveness of such treatments. Thus, in a preferred embodiment, the invention provides therapeutic and prophylactic methods for the treatment, prevention and / or management of cancer that has been proven to be or may be refractory or unresponsive to therapies other than those comprising the administration of EphA2-BiTEs. of the invention. In a specific advance, one or more EphA2-BiTEs of the invention are administered to a patient refractory or unresponsive to non-EphA2-BiTE-based therapy (ie, a therapy other than EphA2-BiTE) to render the patient non-refractory or responsive. In certain advances, therapy to which the patient was previously refractory or unresponsive may then be administered with therapeutic effect.

The methods and compositions of the invention are useful not only in patients not treated with a noncancerous hyperproliferative cell disorder, but are also useful in treating such patients partially or completely refractory to current standard and experimental therapies for noncancerous hyperproliferative cell disorders, including, but not limited to chemotherapies, hormone therapies, biological therapies, immunotherapies, radiation therapies and / or surgery as well as to improve the effectiveness of other treatments. Thus, in a preferred embodiment, the invention provides therapeutic and prophylactic methods for the treatment, prevention and / or management of non-cancerous hyperproliferative cell disorders that have been found to be or may be refractory or unresponsive to therapies other than those comprising administration. EphA2-BiTEs of the invention. In a specific advance, one or more EphA2-BiTEs of the invention are administered to a patient refractory or unresponsive to non-EphA2-BiTE-based therapy (ie, a therapy other than EphA2-BiTE) to render the patient non-refractory or responsive. In certain advances, therapy to which the patient was previously refractory or unresponsive may then be administered with therapeutic effect.

In another advance, methods and compositions of the invention are useful not only in untreated patients infected with a pathogen (eg, a pathogen virus, bacteria, fungus or protozoan), but are also useful in treating patients partially or completely refractory to current therapies. standard and experimental for infections, including but not limited to antiviral, antibacterial, antifungal and / or other antimicrobial agents. Thus, in a preferred embodiment, the invention provides therapeutic and prophylactic methods for the treatment, prevention and / or management of infections which have been found to be or may be refractory or unresponsive to therapies other than those comprising the administration of EphA2-BiTEs. of the invention. In a specific advance, one or more EphA2-BiTEs of the invention are administered to a patient refractory or unresponsive to non-EphA2-BiTE-based therapy (ie, a therapy other than EphA2-BiTE) to render the patient non-refractory or responsive. In certain advances, therapy in which the patient was previously refractory or unresponsive may then be administered with therapeutic effect.

In addition, the present invention provides methods of tracking EphA2-BiTEs of the invention. In particular, EphA2-BiTEs may be screened for binding to EphA2, particularly the EphA2 extracellular domain, and the T cell CD3 antigen using routine immunological techniques such as, for example, radioimmunoassays (RIAs), immunosorbent assays linked to enzymes (ELISA), flow cytometry tests and those described in Section 6 below. In advance, to identify EphA2-BiTEs, candidate EphA2-BiTEs can be screened for the ability to initiate redirected lysis of positive EphA2 target cells (e.g., cancerous cells, noncancerous hyperproliferative cells, or infected cells expressing EphA2). In another advance, candidate EphA2-BiTEs can be screened for the ability to have antitumor activity in vivo (e.g., in a mouse NOD / SCID xenograft model).

The invention also provides methods of screening single stranded bispecific antibody constructs that bind to other Eph A type Eph receptors, i.e., other Eph receptor BiTEs using the methods described herein to identify EphA2 specific BiTEs. See Eph Nomenclature Committee, 1997, Cell 90 (3): 403-4; and Cheng et al., 2002, Cytokine & Growth Factor Rev. 13: 75-85, each of which is incorporated by reference herein in its entirety, for a list of members of the Eph receptor family that can be used as targets to identify other Eph receptor-specific BiTE molecules.

In another advance, to identify EphA2-BiTEs that preferentially bind an exposed EphA2 epitope on cancer cells, but not on noncancerous cells, noncancerous hyperproliferative cells, or on infected but not uninfected cells, EphA2-BiTEs can also be screened. for the ability to preferentially bind EphA2 which is not bound to a ligand, eg, Ephrin Al, and which is not localized for cell-cell contacts. In a specific advance, the invention provides methods for identifying tissue affected by a disorder associated with EphA2 expression and / or aberrant activity, comprising using an EphA2-BiTE of the invention in an epitope exclusion test (see, eg, Sections 6.2. 4, 6.3.8 and 6.7 .2, infra). According to this advancement, EphA2-BiTEs of the invention bind to accessible EphA2 epitopes or expose themselves only to tissue cells affected by a disorder associated with EphA2 expression and / or aberrant activity (eg, cancerous, noncancerous, hyperproliferative cells). or infected cells expressing EphA2), not normal tissue cells of the same tissue type. Any method known in the art to determine antibody binding / localization in a cell can be used to screen candidate BiTEs for desirable binding properties. In a specific advance, standard tests known in the art, such as nuclear magnetic resonance (NMR) microscopy, immunofluorescence microscopy, flow cytometry, or surface plasmon resonance tests are used to determine the binding characteristics of an EphA2-BiTE. in particular. In this advance, EphA2-BiTEs that weakly bind to EphA2 when it is bound to its ligand and located in cell-cell contacts, but bind well to free EphA2 in a cell are encompassed by the invention. In another specific advance, EphA2-BiTEs are selected for their ability to compete with ligands (e.g., cell-anchored or purified ligands) for binding to EphA2 using cell-based or ELISA assays.

DEFINITIONS

As used herein, the term "aberrant" in the context of EphA2 expression, in certain advances, refers to the expression of EphA2 which is increased in a cell, eg, a cancer cell, a hyperproliferative noncancer cell, or an infected cell. of a subject, relative to the level of EphA2 expression in the normal cells of said subject, cells of a normal healthy subject and / or a population of normal healthy cells. Increased EphA2 expression refers to an increase in EphA2 expression in cells of a subject with a disorder associated with aberrant EphA2 expression, relative to the level of EphA2 expression in normal subject cells, cells of a normal healthy subject. and / or a normal healthy cell population. In a specific advance, the level of EphA2 expression in cells of a subject with a disorder associated with aberrant EphA2 expression is increased by at least 10%, at least 15%, at least 25%, at least 25%. 30% at least 35% at least 40% at least 45% at least 50% at least 55% at least 60% at least 65% at least 70% at least 75% at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 7 times or at least 10 times relative to the level of EphA2 expression in the normal cells of said subject, cells of a normal subject , healthy and / or a normal healthy cell population, as measured by a standard known in the art or as described in Section 6.0 below (eg, a flow cytometry test). In a specific advance, the expression of EphA2 is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45 %, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 % or at least 99% or at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 7 times or at least 10 times in a cancer cell, a hyperproliferative non-cancer cell, or an infected cell, relative to the level of EphA2 expression in the normal cells of said subject, cells of a normal subject, healthy and / or a normal healthy cell population as measured by a standard known in the art or as described in Section 6.0 below (eg, a flow cytometry test). In another advance, the term "aberrant" in the context of EphA2 expression refers to EphA2 expression, characterized by the fact that certain EphA2 epitopes are selectively exposed in a cancer cell, a hyperproliferative non-cancer cell, or an infected cell. of a subject, and not in normal cells of said subject, cells of a normal, healthy subject and / or a normal, healthy cell population. In another advance, the term "aberrant" in the context of EphA2 expression refers to EphA2 expression, characterized in that the subcellular location of EphA2 is altered in a cell (at, eg, sites other than contact sites cell-cell) as measured by a standard known in the art or by methods described in Section 6 below, such as, for example, immunofluorescence labeling.

As used herein, the term "agent" refers to a molecule that has a desired biological effect. Agents include, but are not limited to, protein molecules (eg, peptides, polypeptides, proteins and antibodies (eg, single-chain bispecific antibodies)), vaccines, small molecules (less than 1000 daltons), inorganic or organic compounds, and nucleic acid molecules (including, but not limited to, double stranded or single stranded DNA, or double stranded or single stranded RNA (eg, antisense, RNAi, etc.), aptamers, as well as helix nucleic acid molecules triple). Agents may be derived from or obtained from any known organism (including, but not limited to, animals (eg, mammals (humans and non-human mammals)), plants, bacteria, fungi, and protists, or viruses) or a library of molecules. synthetic.

As used herein, the term "analog" in the context of a protein agent (eg, peptide, polypeptide, protein or antibody) refers to a protein agent that has a similar or identical function as a second protein, but does not necessarily comprise a similar or identical amino acid sequence or structure of the second protein agent. A protein agent having a similar amino acid sequence refers to a protein agent that satisfies at least one of the following: (a) a protein agent having an amino acid sequence that is at least 30%, at least 35%, at least 40% at least 45% at least 50% at least 55% at least 60% at least 65% at least 70% at least 75% at least 80% at least 85% at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a second protein agent; (b) a protein agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a second protein agent of at least 20 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, by at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least minus 150 amino acid residues; and (c) a protein agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence encoding a second protein agent. A protein agent having a structure similar to a second protein agent refers to a protein agent having a similar secondary, tertiary or quaternary structure as the second protein agent. The structure of a protein agent may be determined by methods known to those skilled in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. Preferably, a protein agent of the invention has EphA2-BiTE activity.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (eg, spaces may be introduced in the sequence of a first amino acid sequence or nucleic acid sequence for optimal alignment with a second sequence of amino acids or nucleic acids). The amino acid or nucleotide residues at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity = number of identical overlapping positions / total number of positions x 100%). In one advance, both sequences are the same length.

Determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm used for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Know. U.S.A. 87: 2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Know. U.S.A. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set at, e.g., for score = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set at, e.g., for score = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules of the present invention. To obtain spaced alignments for comparison purposes, Gapped BLAST may be used as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (Jd.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., from XBLAST and NBLAST) may be used (see, e.g., the NCBI website). Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN (version 2.0) program that is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 residue weight table, a space extension penalty of 12, and a space penalty of 4 may be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with no spaces allowed. When calculating percent identity, typically only exact matches are counted. As used herein, the term "analog" in the context of a non-protein analog refers to a second organic or inorganic molecule that has a similar or identical function to a first organic or inorganic molecule and is structurally similar to the first organic or inorganic molecule. .

As used herein, the terms "antibody" or "antibodies" refer to molecules containing an antigen binding site, e.g., immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules containing an antigen binding site. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or a subclass of immunoglobulin molecule. Antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFvs (eg, including monospecific and bispecific, etc.), Fab fragments, F (ab ') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and fragments binding to epitopes of any of above. In a specific advance, an EphA2-BiTE of the invention is a single chain bispecific antibody comprising: (1) a first binding domain comprising a heavy variable domain (VH) and a light variable domain (VL) of an antibody that immunospecifically binds to CD3 T cell antigen; and (2) a second binding domain that immunospecifically binds to EphA2. In another specific advance, the first binding domain and the second binding domain of an EphA2-BiTE of the invention each comprise a single Fv (scFv) chain. The heavy variable domain and / or light variable domain of the first binding domain of an EphA2-BiTE may be obtained or derived from any type of antibody that immunospecifically binds to CD3. The heavy variable domain and / or light variable domain of the second binding domain of an EphA2-BiTE may be obtained or derived from any type of antibody that immunospecifically binds to EphA2. Non-limiting examples of antibody types include synthetic antibodies, monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFvs (eg, including monospecific and bispecific, etc.), Fab fragments, F (ab '), disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. As used herein, the term "binding domain" refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one advance, said domain may comprise the VH and / or VL domain of an antibody chain, preferably at least the VH domain. In another advance, the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain recognizing the EphA2 and CD3 antigens, respectively. In this regard, it is noted that the domains of the binding domains present in the EphA2-BiTE of the invention may not only be derived from antibodies, but also from other EphA2 or CD3 binding proteins, such naturally occurring surface receptors or ligands.

As used herein, the terms "deimmunized", "deimmunization" or grammatically related variants thereof denote a modification of the first and / or second binding domain vis-à-vis an original wild-type construct making said non-immunogenic or wild-type construct. less immunogenic in humans. Disimmunization approaches are well known in the art and are described in, e.g., International Pub. Nos. WO 00/34317 (in particular, pp. 1-14); WO 98/52976 (and in particular, Examples 1-6 on pp. 18-38); WO 02/079415 (and in particular pp. 2-8 and Examples 1-10 on pp. 15-43); and WO 92/10755 (and in particular, pp. 6-9), each of which is incorporated by reference herein in its entirety. The term "unimmunized" is also related to constructs, which are prone to generate T cell epitopes. According to this invention, the term "reduced propensity to generate T cell epitopes" is related to the removal of T cell epitopes leading to Furthermore, "reduced propensity to generate T cell epitopes" refers to amino acid substitution contributing to the formation of T cell epitopes, ie, amino acid substitution that are essential for the formation of a T cell. T cell epitope In other words, "reduced propensity to generate T cell epitopes" is related to reduced immunogenicity or reduced ability to induce antigen dependent T cell proliferation. The term T cell epitope is related to short peptide sequences that can be released during the degradation of peptides, polypeptides or proteins in cells and subsequently presented by MHC molecules to initiate T cell activation (see , eg, International Pub. No. WO 02/066514, which is incorporated by reference herein in its entirety). For MHC class II presented peptides, such T cell activation can then initiate an antibody response by direct stimulation of B cells to produce said antibodies. "reduced propensity to generate T cell epitopes" and / or "deimmunization" can be measured by techniques known in the art. Preferably protein deimmunization may be tested in vitro by a T cell proliferation assay. In this assay, donor peripheral blood mononuclear cells (PBMCs) from donors representing> 80% of HLA-DR alleles worldwide are screened for proliferation in response. either wild-type or unimmunized peptides. Ideally, cell proliferation is only detected by loading antigen-bearing cells with wild-type peptides. Alternatively, deimmunization can be tested by expressing HLA-DR tetramers representing all haplotypes. Such tetramers may be tested for peptide binding or loaded with cell-substituted peptides presented antigen in proliferation assays. To determine whether deimmunized peptides are presented in HLA-DR haplotypes, the binding of, e.g., fluorescence-labeled peptides in PBMCs can be measured. Additionally, deimmunization can be confirmed by determining whether antibodies to the deimmunized molecules have been formed following administration to patients. Preferably, antibody-derived molecules are de-immunized in the tool regions and most CDR regions are not modified to generate reduced propensity for inducible T cell epitopes, so that the binding affinity of the CDR regions is not affected. Even elimination of a T cell epitope results in reduced immunogenicity. In specific advances, the immunogenicity of the binding domain that binds to an antigen of interest (eg, CD3) is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%. , at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 99% or at least 100% compared to an unimmunized control polypeptide or protein or fragment thereof, as measured by the standard test described above (eg, a test T cell proliferation) or known in the art. In summary, the approaches discussed above help to reduce the immunogenicity of bispecific single chain therapeutic antibodies (eg, EphA2-BiTEs) described herein when they are administered to patients having a disorder associated with aberrant EphA2 expression and / or activity (eg, cancer, non-cancerous hyperproliferative cell disorders and infections). For example, the first binding domain that binds to the epsilon CD3 subunit is deimmunized. Preferably, the arrangement of the variable regions in this CD3 binding domain is VH-VL.

As used herein, the term "derivative" in the context of a protein agent (eg, proteins, polypeptides, peptides, and antibodies) refers to a protein agent comprising the amino acid sequence that has been altered by the introduction of substitutions, deletions, and / or amino acid residue additions. The term "derivative" as used herein also refers to a protein agent that has been modified, i.e., by covalently attaching any type of molecule to the protein agent. For example, but not by way of limitation, a derivative of a protein agent may be produced, eg, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to a cell ligand. or another protein, etc. A protein agent derivative may also be produced by chemical modifications using techniques known to those skilled in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a protein agent derivative may contain one or more non-classical amino acids. A protein agent derivative has a function (s) identical to the protein agent from which it is derived.

As used herein, the term "derivative" in the context of a non-protein derivative refers to a second organic or inorganic molecule that is formed based on the structure of a first organic or inorganic molecule. A derivative of an organic molecule includes, but is not limited to, a modified molecule, e.g., by the addition or deletion of a hydroxyl, methyl, ethyl, carboxyl, nitril, or amine group. An organic molecule may also, for example, be esterified, alkylated and / or phosphorylated.

As used herein, the term "effective amount" refers to the amount of a therapy (eg, a therapeutic or prophylactic agent) that is sufficient to reduce and / or improve the severity and / or duration of a disorder associated with expression and / or aberrant EphA2 activity, or a symptom thereof, prevent the advance of said disorder, cause regression of said disorder, prevent recurrence, development, or onset of one or more symptoms associated with said disorder associated with aberrant expression and / or activity. EphA2, or enhance or ameliorate the prophylactic or therapeutic effect (s) of another therapy (eg, another therapeutic or prophylactic agent).

As used herein, the terms "elderly", "elderly," or variations thereof refer to a human being 65 years of age or older, preferably 70 years of age or older.

As used herein, the term "endogenous ligand" or "natural ligand" refers to a molecule that normally binds to a particular receptor in vivo. For example, EphrinA1 is an endogenous ligand of EphA2.

As used herein, the term "EphA2 polypeptide" refers to EphA2, an analog, derivative or fragment thereof, or a fusion protein comprising EphA2, an analog, derivative or fragment thereof. The EphA2 polypeptide may be of any species. In one specific advance, the EphA2 polypeptide is human. In certain advances, the term "EphA2 polypeptide" refers to the mature, processed form of EphA2. In other advances, the term "EphA2 polypeptide" refers to an immature form of EphA2. Accordingly, the antibodies of the invention immunospecifically bind to the portion of the immature form of EphA2 which corresponds to the mature, processed form of EphA2. In a specific advance, the term "EphA2 polypeptide" refers to the extracellular domain of EphA2 or a fragment thereof. In certain advances, in the context of a cell, an EphA2-BiTE of the invention binds to the extracellular domain (i.e., an EphA2 epitope that is exposed on the cell surface) of an EphA2.

EphA2 polypeptide nucleotide and / or amino acid sequences can be found in the literature or public databases, or nucleotide and / or amino acid sequences can be determined using cloning and sequencing techniques known to those skilled in the art. For example, the human EphA2 nucleotide sequence can be found in the GenBank database (see, e.g., Accession Accession Nos. BC037166, M59371 and M36395). The human EphA2 amino acid sequence can be found in the GenBank database (see, e.g., Accession Accession Nos. AAH37166 and AAA53375). Additional non-limiting examples of EphA2 amino acid sequences are listed in the following table.

<table> table see original document page 39 </column> </row> <table>

As used herein, the term "epitope" refers to sites or fragments of a polypeptide or protein having antigenic or immunogenic activity in an animal, preferably in a mammal, and more preferably in a human. In specific advances, the term "epitope" refers to a fragment of an EphA2 polypeptide or a fragment of a CD3 polypeptide having antigenic or immunogenic activity in an animal, preferably in a mammal, and more preferably in a human. An epitope having immunogenic activity is a site or fragment of a polypeptide or protein that evokes an antibody response in an animal. In specific advances, an epitope having immunogenic activity is a fragment of an EphA2 polypeptide or a fragment of a CD3 polypeptide that evokes an antibody response in an animal. An epitope having antigenic activity is a site or fragment of a polypeptide or protein to which an antibody binds immunospecifically as determined by any method well known to one skilled in the art, for example by immunoassays such as RIAs or ELISAs. In specific embodiments, an epitope having antigenic activity is a fragment of an EphA2 polypeptide or a fragment of a CD3 polypeptide to which an antibody binds immunospecifically as determined by any method well known in the art, for example by immunoassay. Antigenic epitopes need not necessarily be immunogenic.

As used herein, the term "fragment" in the context of a protein agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 residues.

contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 amino acid residues contiguous at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or skin> at least 250 residues of

adjoining amino acids from another polypeptide or protein. In a specific advance, the term "fragment" in the context of a protein agent refers to a peptide or polypeptide comprising an amino acid sequence ranging from 10 to 15, 10 to 20, 10 to 30, 10 to 40, 10 to 50 , 10 to 60, 10 to 70, 10 to 80, 10 to 100, 10 to 125, 10 to 150, 10 to 175, 10 to 200, 10 to 250, or 10 to 300 contiguous amino acid residues of another polypeptide or protein .

In a specific advance, a fragment is an EphA2 fragment, or an antibody that immunospecifically binds to an EphA2. In another specific advance, a fragment is a CD3 fragment or an antibody that immunospecifically binds to CD3. In one advance, a fragment of a protein or polypeptide retains at least one function of the protein or polypeptide. In another advance, a fragment of a polypeptide or protein retains at least two, three, four or more functions of the polypeptide or protein. In a preferred embodiment, an antibody fragment that immunospecifically binds to an EphA2 polypeptide or fragment thereof retains the ability to immunospecifically bind to an EphA2 polypeptide or fragment thereof. In another preferred embodiment, an antibody fragment that immunospecifically binds to a CD3 polypeptide or fragment thereof retains the ability to immunospecifically bind to a CD3 polypeptide or fragment thereof. Preferably, antibody fragments are fragments that bind to epitopes.

As used herein, the term "fusion protein" refers to a polypeptide or protein comprising the amino acid sequence of a first polypeptide or protein or fragment, analog or derivative thereof, and the amino acid sequence of a heterologous polypeptide or protein. (ie, a second polypeptide or protein or fragment, analog or derivative thereof different from the first polypeptide or protein or fragment, analog or derivative thereof, or not normally part of the first polypeptide or protein or fragment, analog or derivative thereof). In one advance, a fusion protein comprises a therapeutic or prophylactic agent fused to a heterologous protein, polypeptide or peptide. According to this advance, the heterologous protein, polypeptide or peptide may or may not be a different type of therapeutic or prophylactic agent. For example, two different proteins, polypeptides, or peptides with immunomodulatory activity may be fused together to form the fusion protein. In a preferred embodiment, fusion proteins retain or possess enhanced activity relative to the activity of the parent polypeptide or protein before being fused to a heterologous protein, polypeptide, or peptide. In a specific advance, a fusion protein comprises a first binding domain that binds to the T cell CD3 antigen and a second binding domain that binds to an antigen of interest (e.g., EphA2). According to this advance, a fusion protein is an EphA2-BiTE.

As used herein, the term "humanized antibody" refers to non-human (e.g., murine) antibody forms, preferably chimeric antibodies, which contain minimal sequence from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which the container hypervariable or complementarity determining (CDR) residues are replaced by hypervariable region residues or CDR residues of an antibody of a non-human species (antibody). donor) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and ability. In some instances, one or more human immunoglobulin tool region (FR) residues are replaced by corresponding non-human residues or other residues based on structural modeling, e.g., to improve the affinity of the humanized antibody. Additionally, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least one fragment of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details regarding tool shuffling humanization methods, see Dall'Acqua et al., 2005, Methods 36: 43-60, Jones et al. , 1986, Nature 321: 522-525; Reichmann et al., 1988, Nature 332: 323-329; Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596; and Queen et al. , U.S. Patent No. 5,585,089, and US Pat. Pub. No. 2005-0048617 Al, each of which is incorporated by reference herein in its entirety.

As used herein, the term "hybridize under stringent conditions" describes conditions for hybridization and washing under which nucleotide sequences are at least 30% (preferably 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95% or 98%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

Generally, stringent conditions are selected to be about 5 to 10 ° C lower than the melting thermal point (Tm) for the specific sequence at a given ionic strength and pH. Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the complementary probe targets hybridize to the equilibrium target sequence (since the target sequences are present in excess of 50% of the target probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ions, typically about 0.01 to 1.0 M sodium ion (or other salts) concentration at pH 7.0 to 8.3 and the temperature is at least about 30 ° C for short probes (eg 10 to 50 nucleotides) and at least about 60 ° C for long probes (eg greater than 50 nucleotides) ). Stringent conditions may also be achieved with the addition of destabilizing agents, for example formamide. For selective or specific hybridization, a positive signal is at least twice the hybridization base, preferably 10 times the hybridization base. In a non-limiting example, stringent hybridization conditions are 6X sodium chloride / sodium citrate (SSC) hybridization at about 45 ° C, followed by one or more washes in 0.1X SSC, about 0.2% SDS. at 68 ° C. In a preferred, non-limiting example, stringent hybridization conditions are 6X SSC hybridization at about 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65 ° C (ie. one or more washes at 50 ° C, 55 ° C, 60 ° C or 65 ° C). It is understood that the nucleic acids of the invention do not include hybridizing nucleic acid molecules. under these conditions only to a nucleotide sequence consisting of only nucleotides A or T.

As used herein, the term "hypervariable region" refers to amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues of a "Complementarity Determination Region" or "CDR" (ie residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health , Bethesda, MD. (1991)) and / or those hypervariable loop residues (ie residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917). "Tool Region" or "FR" residues are those variable domain residues other than hypervariable region residues as defined herein.

As used herein, the term "immunomodulatory agent" refers to an agent that modulates an immune system of a subject. In particular, an immunomodulatory agent is an agent that alters the ability of a subject's immune system to respond to one or more foreign antigens. In a specific advance, an immunomodulatory agent is an agent that changes an aspect of a subject's immune system response. In a preferred embodiment of the invention, an immunomodulatory agent is an agent that inhibits or reduces a subject's immune system response (i.e., an immunosuppressive agent).

As used herein, the term "immunospecifically binds to EphA2" and analogous terms in the context of anti-EphA2 antibodies, EphA2-BiTEs, and EphA2-BiTEs binding domains, refer to protein agents, including antibodies (eg, EphA2 -BiTEs which are single-chain bispecific antibodies) and EphA2-BiTEs binding domains (eg, EphA2-BiTEs scFvs which are single-chain bispecific antibodies) that specifically bind to an EphA2 polypeptide, and do not bind specifically to non-EphA2 polypeptides. Preferably, antibodies and EphA2-BiTEs binding domains that specifically bind to an EphA2 polypeptide do not react with other unrelated antigens. In specific advances, anti-EphA2 antibodies of the invention bind to an EphA2 polypeptide with little or no cross-reactivity with other unrelated antigens, as measured by a standard known in the art, such as an ELISA. In certain advances, antibodies and EphA2-BiTEs binding domains that immunospecifically bind to an EphA2 polypeptide may cross react with related antigens (e.g., other types of Eph receptor A and / or B family of Eph receptors). A peptide, polypeptide, protein, antibody, or binding domain that immunospecifically binds to an EphA2 polypeptide may be binding to other lower affinity peptides, polypeptides, or proteins as determined by, eg, immunoassays or other assays known in the art for detect binding affinity. Antibodies or binding domains that immunospecifically bind to an EphA2 polypeptide may cross-react with related antigens. Preferably, antibodies or fragments thereof that immunospecifically bind to an EphA2 polypeptide may be identified, for example, by immunoassays or other techniques known to those skilled in the art. An antibody, or fragment thereof, specifically binds to an EphA2 polypeptide when it binds to a higher affinity EphA2 polypeptide than to any reactive antigen as determined using experimental techniques such as radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays. (ELISAs). See, e.g., Paul, ed., 1989, Fundamental Immunology, 2nd ed., Raven Press, New York on pages 332-336 for a discussion regarding antibody specificity. In another advance, an antibody that binds to an EphA2 polypeptide, which is a fusion protein, immunospecifically binds to the fusion protein fragment that is an EphA2 polypeptide. Preferably, antibodies and EphA2-BiTEs binding domains that immunospecifically bind to an EphA2 polypeptide modulate only one EphA2 activity (s) and do not significantly affect other activities. In a specific advance, antibodies and EphA2-BiTEs binding domains that immunospecifically bind to an EphA2 polypeptide are preferably single chain antibodies (eg, scFvs comprising a VH domain and a VL domain), which may have a low rate of Koff (eg, K0ff less than 3 χ 10-2S-1).

As used herein, the term "immunospecifically binds to CD3" and analogous terms refer to protein agents that specifically bind to CD3 or a subunit thereof, and do not specifically bind to other antigens. Preferably, antibodies and EphA2-BiTEs binding domains that bind immunospecifically to CD3 do not cross react with unrelated antigens.

As used herein, the term "in combination" in the context of administering a therapy refers to the use of more than one therapy. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject with a disorder associated with aberrant EphA2 expression and / or activity. A first therapy can be given before (eg, 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 minutes). hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior), concurrently or concurrently with, or subsequent to (eg, 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later) administration of a second therapy to a subject. who had, has, or is susceptible to a disorder associated with aberrant EphA2 expression and / or activity. Any additional therapy may be administered in any order with the other additional therapies. In certain advances, EphA2-BiTEs of the invention may be administered in combination with one or more therapies (eg, non-EphA2-BiTEs peptides currently administered to treat, prevent and / or manage the disorder associated with aberrant EphA2 expression and / or activity. , such as, for example, analgesic agents, anesthetic agents, antibiotics, antiviral agents, antifungal agents, anti-protozoal agents, immunomodulatory agents).

As used herein, the terms "increased" or "overexpressed" or "overexpressed" with respect to EphA2 expression refers to an increase in EphA2 expression in cells of a subject with a disorder associated with expression and / or aberrant EphA2 activity, relative to the level of EphA2 expression in normal subject cells, healthy normal subject cells, and / or a healthy normal cell population as measured by any method known in the art, including but not limited to RIAs, ELISA, Western Blot analysis, flow cytometry, or immunohistochemistry using antibodies that immunospecifically bind to EphA2. In a specific advance, the level of EphA2 expression in cells of a subject with a disorder associated with aberrant EphA2 expression and / or activity is increased by at least 10%, at least 15%, at least 20%, at least 25%. % at least 30% at least 35% at least 40% at least 45% at least 50% at least 55% at least 60% at least 65% at least 70% at least 75 % at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times , at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 7 times or at least 10 times relative to the level of EphA2 expression in the normal cells of said subject, a normal healthy subject and / or the normal healthy cell population.

As used herein, the terms "child" or "child" or variations thereof refer to a human being less than 24 months old, preferably less than 12 months, less than 6 months, less than 3 months, less than 2 months old or less than 1 month old. As used herein, the terms "prematurely born child" or "premature child", or variations thereof, refer to a human being born less than 40 weeks of gestation time, preferably less than 35 weeks of gestation time, who is less than 6 months old, preferably less than 3 months old, more preferably less than 2 months old, and more preferably less than 1 month old.

As used herein, the term "isolated" in the context of. An organic or inorganic molecule (whether a small or large molecule), other than a protein agent or nucleic acid, refers to an organic or inorganic molecule substantially free of a different organic or inorganic molecule. Preferably, an organic or inorganic molecule is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% free of a different second organic or inorganic molecule. In a preferred embodiment, an organic and / or inorganic molecule is isolated.

As used herein, the term "isolated" in the context of a protein agent (eg, a peptide, polypeptide, fusion protein, or antibody) refers to a protein agent that is substantially free of cellular material or source source contaminating proteins. cells or tissues from which it is derived, or substantially free of chemical or other chemical precursors when chemically synthesized. The phrase "substantially free of cellular material" includes preparations of a protein agent in which the protein agent is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a protein agent that is substantially free of cellular material includes preparations of a protein agent having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein, polypeptide, peptide, or protein. antibody (also referred to as a "contaminating protein"). When the protein agent is

recombinantly produced, it is also preferably substantially free of culture medium, i.e., the culture medium represents less than about 20%, 10%, or 5% of the protein preparation volume. When the protein agent is produced by chemical synthesis, it is preferably substantially free of chemical or other chemical precursors, i.e., it is separated from chemical or other chemical precursors that are involved in protein synthesis. Accordingly, such preparations of a protein agent have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the protein agent of interest. In a specific advance, protein agents described herein are isolated. In a preferred embodiment, an EphA2-BiTE molecule of the invention is isolated.

As used herein, the term "isolated" in the context of nucleic acid molecules refers to a nucleic acid molecule that is separate from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, is preferably substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical or other chemical precursors when synthesized. chemically. In a specific advance, nucleic acid molecules are isolated. In a preferred embodiment, a nucleic acid molecule encoding an EphA2-BiTE molecule of the invention is isolated.

As used herein, the term "low tolerance" refers to a condition in which the patient suffers from side effects of therapy (s) so that the patient does not benefit from therapy and / or does not continue therapy for cause of adverse effects and / or damage from side effects outweigh the benefit of treatment.

As used herein, the terms "administering", "administering" and "administering" refer to the beneficial effects a subject receives from therapy, which does not result in a cure for a disorder. In certain advances, a subject is administered one or more therapies to "administer" a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, noncancerous hyperproliferative cell disorder or an infection). to prevent the progression or worsening of the disorder (ie, insure the progress of the disease). As used herein, the term "pathology-causing cellular phenotype" refers to a function that a cell affected by a disorder associated with aberrant EphA2 expression and / or activity that causes or contributes to the pathological state of said disorder. Cell Phenotypes

Pathology causes include, but are not limited to, decreased cell / cell interaction, increased extracellular matrix deposition, increased migration, cell survival, and / or proliferation of an enlarged cancer cell, a hyperproliferative cell, or an infected cell (eg, an infected cell). epithelial cell) by a pathogen / infectious agent (eg, bacteria, virus, fungus or protozoan). One or more of these pathological causative cell phenotypes cause or contribute to symptoms in a patient suffering from a disorder associated with aberrant EphA2 expression and / or activity (e.g., cancer, a noncancerous hyperproliferative cell disorder, or an infection).

As used herein, the phrase "pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the US Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

As used herein, the term "potentiating" in the context of administering a therapy to a subject refers to an improvement in the efficacy of a therapy at its common or approved dose.

As used herein, the terms "preventing", "preventing", and "prevention" in the context of therapies administered to a subject refer to the reduction or inhibition of the development, onset, spread or recurrence of a disorder associated with expression and / or aberrant EphA2 activity or a symptom thereof in a subject resulting from the administration of a therapy (eg, a therapeutic or prophylactic agent), or the administration of a combination of therapies (eg, a combination of prophylactic or therapeutic agents). In a specific advance, the terms "preventing", "preventing", and "prevention" in the context of therapies administered to a subject refer to an increase in recurrence time or a decrease in the spread or progression of a disorder associated with expression and / or aberrant EphA2 activity.

As used herein, the term "prophylactic agent" refers to any agent that can prevent the development, recurrence, spread or onset of a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, a non-cancerous hyperproliferative cell disorder, or an infection), or a symptom thereof. In certain advances, the term "prophylactic agent" refers to an EphA2-BiTE. In certain other advances, the term "prophylactic agent" refers to an agent other than an EphA2-BiTE. Preferably, a prophylactic agent is an agent that is known to be useful for or has been or is currently used to prevent or prevent the onset, development, recurrence, progression and / or dispersion of a disorder associated with aberrant expression (eg, overexpression). ) and / or EphA2 activity (eg, cancer, non-cancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof.

As used herein, a "prophylactically effective amount" refers to the amount of a therapy (eg, a prophylactic agent) that is sufficient to prevent the development, recurrence, spread, or onset of a disorder associated with expression and / or activity. aberrant EphA2 (eg, cancer, non-cancerous hyperproliferative cell disorder or an infection), or such a symptom. A prophylactically effective amount may refer to the amount of a therapy (eg, a prophylactic agent) sufficient to prevent the development, recurrence, spread or onset of a disorder associated with aberrant EphA2 expression and / or activity (eg, cancer, a disorder non-cancerous hyperproliferative cell disease or an infection), or a symptom thereof, for example, in those who have previously suffered from such a disorder, or those who are immunocompromised or immunosuppressed, or are genetically predisposed to such a disorder. A prophylactically effective amount may also refer to the amount of a therapy (eg, a prophylactic agent) that provides a prophylactic benefit in preventing a disorder associated with aberrant EphA2 expression and / or activity (eg, cancer, hyperproliferative cell disorder). cancerous or an infection), or such a symptom. Further, a prophylactically effective amount with respect to a therapy (eg, a prophylactic agent of the invention) means amount of therapy (eg, prophylactic agent) alone, or in combination with one or more other therapies (eg, therapies without currently EphA2-BiTE). administered to prevent the disorder associated with aberrant EphA2 expression and / or activity (eg, cancer, non-cancerous hyperproliferative cell disorder or an infection), analgesic agents, anesthetic agents, antibiotics, or immunomodulatory agents) that provide a prophylactic benefit in preventing of disorder associated with aberrant EphA2 expression and / or activity. Used in connection with an amount of an EphA2-BiTE of the invention, the term may encompass an amount that improves general prophylaxis or enhances prophylactic efficacy of or synergy with another therapy (e.g., a prophylactic agent).

As used herein, a "protocol" includes dosage schedules and dosage regimens.

As used herein, the term "refractory" refers to a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, a noncancerous hyperproliferative cell disorder or an infection) which is unresponsive to one or more therapies (eg, therapies currently available). In certain advances, a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, a noncancerous hyperproliferative cell disorder or an infection) is refractory to therapy meaning that at least some significant portion The symptoms associated with this disorder are not eliminated or alleviated by that therapy. Determination of whether such a disorder is refractory can be done either in vivo and / or in vitro by any method known in the art to test the effectiveness of therapy for a disorder associated with aberrant EphA2 expression and / or activity (eg, cancer, a non-cancerous hyperproliferative cell disorder or an infection).

As used herein, the term "side effects" encompasses unwanted and adverse effects of a therapy (e.g., a therapeutic or prophylactic agent). Adverse effects are always unwanted, but unwanted effects are not necessarily adverse. An adverse effect of a therapy (e.g., a therapeutic or prophylactic agent) may be harmful or uncomfortable or dangerous. Examples of side effects include, but are not limited to, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, mouth. dryness, and loss of appetite, rash or swelling at the site of administration, flu-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions. Additional unwanted effects experienced by patients are numerous and known in the art. Several are described in the Physicians' Desk Reference (61st ed., 2007).

As used herein, the term "single chain Fv" or "scFv" refers to antibody fragments comprising the VH and VL domains of an antibody, characterized in that these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a linker polypeptide between the VH and VL domains, which allows scFv to form the desired antigen-binding structure. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). In a specific advance, the EphA2-BiTEs of the invention are comprised of scFvs.

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is an animal, preferably a mammal, such as a non-primate mammal (eg, cows, pigs, horses, cats, dogs, rats, etc.) and a primate (eg, monkey (eg, rhesus monkey , a cinomolgus or chimpanzee monkey) and human), and more preferably a human. In one advance, the subject is a mammal, preferably a human, with a disorder associated with aberrant EphA2 expression and / or activity. Such disorders include, but are not limited to, cancer, noncancerous hyperproliferative cell disorders, and infections. In another advance, the subject is a mammal, preferably a human, at risk of developing a disorder associated with aberrant EphA2 expression and / or activity (e.g., an immunocompromised or immunosuppressed mammal, or a genetically predisposed mammal). In another advance, the subject is not an immunocompromised or immunosuppressed mammal, preferably a human. In another advance, the subject is a mammal, preferably a human, with a lymphocyte count that is below approximately 500 cells / mm3.

As used herein, the term "synergistic" refers to a combination of therapies (eg, prophylactic or therapeutic agents) that is more effective than the additive effects of any two or more simple therapies (eg, one or more prophylactic or therapeutic agents). therapeutics). In one aspect, a synergistic effect of a combination of therapies (eg, a combination of prophylactic or therapeutic agents) allows the use of lower dosages of one or more therapies (eg, one or more prophylactic or therapeutic agents) and / or less management. frequent use of such therapies to a subject with a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, a noncancerous hyperproliferative cell disorder or an infection). In some advances, the ability to use lower dosages of therapies (eg, prophylactic or therapeutic agents) and / or to administer said therapies less frequently reduces the toxicity associated with administering said therapies to a subject without reducing the effectiveness of said therapies in the treatment. preventing or treating a disorder associated with aberrant EphA2 expression and / or activity (eg, overexpression) (eg, cancer, a noncancerous hyperproliferative cell disorder or an infection). In another aspect, a synergistic effect may result in improved efficacy of therapies (eg, prophylactic or therapeutic agents) in the treatment, prevention and / or management of a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity. (eg, cancer, a noncancerous hyperproliferative cell disorder or an infection). In another aspect, the synergistic effect of a combination of therapies (e.g., prophylactic or therapeutic agents) may prevent or reduce adverse or unwanted side effects associated with the use of any single therapy.

As used herein, the term "therapeutic agent" refers to any agent that may be used in the treatment and / or management of a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer). , a non-cancerous hyperproliferative cell disorder or an infection) or one or more symptoms thereof. In certain advances, the term "therapeutic agent" refers to an EphA2-BiTE molecule of the invention. In certain other advances, the term "therapeutic agent" refers to an agent other than an EphA2-BiTE molecule of the invention. Preferably, a therapeutic agent is an agent that is known to be useful for, or has been used or is currently being used for the treatment, prevention and / or management of a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, a non-cancerous hyperproliferative cell disorder or an infection) or one or more symptoms thereof.

As used herein, a "therapeutically effective amount" in the context of cancer refers to the amount of a therapy alone, or in combination with other therapies, that provides a therapeutic benefit in cancer treatment and / or management. In one aspect, a therapeutically effective amount refers to the amount of therapy sufficient to destroy, modify, control or remove primary, regional or metastatic cancerous tissue. In another aspect, a therapeutically effective amount refers to the amount of therapy sufficient to reduce the symptoms of a cancer. In another aspect, a therapeutically effective amount refers to the amount of therapy sufficient to delay or minimize the spread of cancer. In a specific advance, a therapeutically effective amount of a therapy is an amount of a therapy sufficient to inhibit cancer cell growth or proliferation, kill existing cancer cells (eg, cause cancer regression), and / or prevent cell dispersion. cancerous to other tissues or areas (eg, preventing metastasis). In another specific advance, a therapeutic amount of a therapy is an amount of a therapy sufficient to inhibit tumor growth by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%. % at least 30% at least 35% at least 40% at least 45% at least 50% at least 55% at least 60% at least 65% at least 70% at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% as measured by standard method known in the art, or as described in Section 6.4.1 below. Used in connection with an amount of an EphA2-BiTE of the invention, the term may encompass an amount that enhances general therapy, reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapy. In a breakthrough, a therapeutically effective amount of one therapy reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapy by at least 5%, preferably at least 10%, at least 15%, at least 20%. at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control (eg, a negative control such as a buffered saline phosphate) in a test known in the art or described herein.

As used herein, a "therapeutically effective amount" in the context of a non-cancerous hyperproliferative cell disorder refers to the amount of a therapy alone, or in combination with other therapies, that provides a therapeutic benefit in treating and / or managing said disorder. . In one aspect, a therapeutically effective amount refers to the amount of therapy sufficient to destroy, modify, control or remove cells affected by a non-cancerous hyperproliferative cell disorder. In another

In this regard, a therapeutically effective amount refers to that amount of therapy sufficient to reduce the symptoms of a noncancerous hyperproliferative cell disorder. In another aspect, a therapeutically effective amount refers to the amount of therapy sufficient to delay or minimize the spread of a non-cancerous hyperproliferative cell disorder. In a specific advance, the therapeutically effective amount of a therapy is an amount of therapy sufficient to inhibit the growth or proliferation of a noncancerous hyperproliferative cell disorder, kill existing noncancerous hyperproliferative cells (e.g., cause regression of the disorder). In another specific advance, a therapeutically effective amount of a therapy is an amount of a therapy sufficient to inhibit non-cancerous hyperproliferative cell growth by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% as measured by a standard method known in the art. Used in connection with an amount of an EphA2-BiTE of the invention, the term may encompass an amount that enhances general therapy, reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapy. In a breakthrough, a therapeutically effective amount of one therapy reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapy by at least 5%, preferably at least 10%, at least 15%, at least 20%. at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control (eg, a negative control such as a buffered saline phosphate) in a test known in the art or described herein.

As used herein, a "therapeutically effective amount" in the context of an infection refers to that amount of a therapy (eg, a therapeutic agent) sufficient to reduce the severity of an infection, reduce the duration of an infection, improve one or more symptoms of an infection, prevent further infection, cause regression of the infection, or intensify or ameliorate the therapeutic effect (s) of another therapy. In certain advances, a therapeutically effective amount refers to the amount of a therapeutic agent sufficient to reduce or inhibit replication of a pathogen, inhibit or reduce infection of a cell by a pathogen, inhibit or reduce the production of pathogenic proteins, inhibit or reduce the release of a pathogen, inhibit or reduce the spread of a pathogen to other tissues or subjects, or ameliorate one or more symptoms associated with the infection. In one advance, a therapeutically effective amount of a therapeutic agent reduces replication or dispersion of a pathogen by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%. %, at least 35%, at least 40%, at least .45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control (eg, a negative control, such as buffered saline) in a test known in the art or described herein. Tests that can be used to measure replication of a pathogen, e.g., a virus, include, but are not limited to, infectivity and transformation tests as described in, e.g., H.M. Temin and H. Rubin. 1958. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 17: 669-688; and J.W. Hartley and W.P. Rowe 1966. Production of altered cell foci in tissue culture by defective Moloney sarcoma virus particles. Proc. Natl. Acad. Know. 55: 780-786, each of which is incorporated herein by reference in its entirety.

As used herein, the term "therapy" refers to any protocol, method and / or agent that may be used in the treatment, prevention and / or management of a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, a non-cancerous hyperproliferative cell disorder or an infection). In certain advances, the terms "therapies" and "therapy" refer to biological therapy, supportive therapy, and / or other therapies useful in treating, preventing, and / or managing a disorder associated with aberrant expression (eg, about aberrant expression). -expression) and / or EphA2 activity (eg, cancer, a non-cancerous hyperproliferative cell disorder or infection), or one or more symptoms known to one of skill in the art, such as a medical team.

As used herein, the terms "treating", "treating" and "treating" in the context of administering therapy (s) to a subject refer to the reduction or amelioration of the progression, severity, and / or duration of a disorder associated with aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer, a non-cancerous hyperproliferative cell disorder or infection), and / or amelioration of one or more of these symptoms resulting from the administration of one or more therapies. (including, but not limited to, administration of one or more prophylactic or therapeutic agents). In specific advances, the terms "treating", "treating" and "treating" in the context of administering therapy (s) to a subject refer to the reduction or amelioration of the progression, severity, and / or duration of a disorder associated with Aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer) refers to a reduction in cancer cells by at least 5%, preferably at least 10%, at least 15%, at least 20%. , at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control (eg, a negative control such as phosphate buffered saline). In other advances, the terms "treating", "treating" and "treating" in the context of administering therapy (s) to a subject refer to the reduction or amelioration of the progression, severity, and / or duration of a disorder associated with Aberrant expression (eg, overexpression) and / or EphA2 activity (eg, cancer) refers to no change in cancer cell numbers, a reduction in hospitalization time, a reduction in mortality, or an increase in survival of the subject with cancer.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1B: VL and VH domain sequences of the anti-EphA2 antibody EA2. The nucleotide and amino acid sequences of the VL domain (SEQ ID NOS: 1 and 2, respectively) are shown in (A). The VH domain nucleotide and amino acid sequences (SEQ ID NOS: 9 and 10, respectively) are shown in (B). Nucleotide and amino acid sequences of CDRs are indicated in bold and underlined. EA2 antibody has been previously described in U.S. Pat. Pub. No. US 2005-0152899 A1, and is incorporated herein by reference in its entirety.

FIGS. 2A-2B: Sequences of EA5 anti-EphA2 antibody VL and VH domains. The VL domain nucleotide and amino acid sequences (SEQ ID NOS: 17 and 18, respectively) are shown in (A). The VH domain nucleotide and amino acid sequences (SEQ ID NOS: 25 and 26, respectively) are shown in (B). Nucleotide and amino acid sequences of CDRs are indicated in bold and underlined. EA5 antibody has been previously described in U.S. Pat. Pub. No. US 2005-0152899 A1, and is incorporated herein by reference in its entirety.

FIGS. 3A-3B: Structure of EphA2-BiTE cDNA. The cloned insertions in the pEF-DHFR expression vector each comprised a leader with a terminal 5 'Kozak site for increased translation efficiency and a secretory signal sequence (heavy chain murine Ig leader). The leader is followed by four variable Ig domains, as listed above. The linker peptide at the VL-VH or VH-VL junction of anti-EphA2 is 15 amino acids long (three repeats of the G4S motif). The linker peptide connecting EphA2 with CD3 binding specificity comprises a repeat of the G4S motif. Directly adjacent to the fourth domain is a C-terminal hexahistidine (H6) sequence for detection and purification purposes. The indicated restriction enzyme sites were used to clone the EphA2-BiTE constructs in the expression vector.

FIGS. 4A-4B: Structure of pEF-DHFR vector used to express EphA2-BiTEs in CHO cells. The pEF-DHFR vector is a 5.8 kb vector with murine dihydrofolate reductase as the selection marker forming a bicistronic transcription unit together with the gene to be expressed under the control of the human EFla promoter. The eukaryotic expression vector is a derivative of the pMT2PC expression vector.

FIGS. 5A-5B: Elution profile of deimmunized anti-CD3xEA2 (VH / VL) EphA2-BiTE containing protein fractions from a gel filtration column. Peak 1: aggregates, peak 2: dimer, peak 3: monomer.

FIGS. 6A-6B: SDS PAGE and anti-His Western blot label of purified anti-CD3xEA2 (VH / VL) deimmunized EphA2-BiTE fractions. The monomer fraction (lane 7) contained essentially pure EphA2 BiTE. No EphA2-BiTE was detectable in the aggregate.

FIGS. 7A-7B: Flow cytometer binding analysis of cinomolgus-reactive parental anti-CD3 antibodies. PBMC from cinomolgus monkeys were used to detect CD3 binding of parental antibodies cCD3-1 and cCD3-2, both conjugated to FITC. Cells were analyzed by flow cytometry on a FACS-Calibur (Becton Dickinson, Heidelberg). As shown herein, both antibodies exhibit distinct binding to the cinomolgus PBMC T cell fraction.

FIG. 8. Evidence for cross reactivity of EA2 and EA5 with Rhesus EphA2. Activated phosphorylation of EphA2 EA2 and EA5 antibodies in CMMT110 / CL cells, as shown in Western blot.

FIG. 9. Flow cytometric binding analysis of various anti-EphA2 constructs replace BiTE by reacting with CD3 macague. Only those substitutes for the CD3 macaque cCD3-1 antibody-based BiTE exhibit strong binding to CD3 and EphA2. CCD3-2-based constructs show only strong binding to CD3; the EphA2 binding ended up being almost completely suppressed. Thus, cCD3-1-based Birog surrogate constructs will be continued for production, purification and analysis of cytotoxic activity with cinomolgus T cells.

FIG. 10. Flow cytometric binding analysis of parental anti-EphA2 antibodies. A549 cells expressing EphA2 (human lung carcinoma cell line) (A) and MDA-MB-231 (human breast cancer) cells (B) were used. Cells (200,000) were incubated with 10 pg / ml of their antibody for 30 min on ice. The cells were subsequently washed twice in PBS. Primary antibody binding was detected via a phycoerythrin-conjugated specific murine Fc-gamma antibody (Dianova, order no. 115-116-071) diluted 1: 100 in 50 μΐ PBS with 2% FCS. As a negative control, an irrelevant antibody with the same isotype was used (thin line). Cells were analyzed by flow cytometry on a FACS-Calibur (Becton Dickinson, Heidelberg). As shown in the figure, mAb B322 showed the strongest binding signal, followed by EA2 and EA5. The binding capacities of the three different antibodies are presented in a histogram overlap (4th left peak = B322 antibody; 3rd left peak = EA2 antibody; 2nd left peak = EA5 antibody; leftmost control peak).

FIG. 11. Immunohistochemical analysis of binding to EA2 and EA5 for normal tissues. (A) No primary antibody. ΞΑ5 demonstrated discoloration of intercalated discs in heart tissue (B), vascular and stromal multi-organ muscle elements (cytoplasm), colonic epithelium (cytoplasm), and uterine myometrium. Human tissue sections (C) were not stained with EA2 compared to a control monoclonal antibody isotype 1A7.

FIG. 12. Bispecific binding of EphA2-BiTEs in different cell lines as determined by flow cytometry. A549 EphA2 positive cells (human lung carcinoma cell line) and MDA-MB-231 cells (human breast cancer cell line) as well as HP-BALL CD3 positive (human T cell line) were used. FIGS. 13Α-13Β. Iise power redirected by five EphA2-BiTEs in (A) MDA MB 231 (breast), and (B) A549 (lung) cells. The unimmunized anti-CD3xEA2 (VH / VL) BiTE construct consistently showed the highest potency in redirected lysis of breast and lung cancer cell lines.

FIGS. 14A-14C. Sequences of the unimmunized anti-CD3 EphA2-BiTE (VH-VL) χ EA2 (VH-VL). Shown in (A) is a representation of an EphA2-BiTE construct comprising a first binding domain that immunospecifically binds to CD3 and a second binding domain that immunospecifically binds to EphA2. Also represented are link sequences between the domains VKcd3-VLcd3, VLcd3-VHepha2, and VHEphA2-VLEphA2, respectively, in the 5 'to 3' direction (from left to right). The amino acid and nucleotide sequences of each ligand are represented by SEQ ID NOS: 27 and 28, 29 and 30, and 31 and 32, respectively. Presented in (B) is the full length sequence of the nucleotide sequence (SEQ ID NO: 60) and amino acid sequence (SEQ ID NO: 65) of the deimmunized anti-CD3 EphA2-BiTE (VH-VL) χ EA2 (VH) -VL). Rewrited letters represent the restriction sites of EcoRI and SalIf enzymes respectively. Uppercase italic letters indicate the leading sequence, and double underlined letters represent the stop codon. Histidine ligand and tag sequences are underlined. FIG. 15. The cytotoxic activity of two different groups of anti-CD3xEA2 (VH / VL) deimmunized EphA2 BiTE was compared with PBMCs (after depletion of CD16 positive cells) and CD8 + T cells stimulated as effector cells. EphA2 positive cell lines A549 and MDA-MB231 served as target cells. The ratio E: T was 10: 1; and the incubation time was 18 hours. (A) EphA2-BiTE mediated A549 cell lysis with PBMC; (B) EphA2-BiTE mediated A549 cell lysis with stimulated CD8 + T cells; (C) EphA2-BiTE-mediated MDA-MB231 cell lysis with PBMC; and (D) Stimulated CD8 + T-cell mediated MDA-MB231 cell lysis.

FIGS. 16A-16B. Cytotoxic activity variation of EphA2-BiTE with effector cell donor. EphA2-BiTE redirect T cells from different subjects to mediate EphA2 + tumor cell death. The cytotoxicity test used SW480 target cells and CD3 + T cells isolated from 49 donor human subjects. For most human donors, EphA2-BiTE mediated tumor cell death at very low concentrations. The EC50 for EphA2-BiTE was between 1 and 110 ng / ml for all T cell donors tested with a median (horizontal bar) of 24 ng / ml. Thus, for most human T cell donors, EphA2-BiTE is a highly potent molecule with cell killing activity observed at concentrations in the low ng / ml range. FIGS. 17A-17D. Target cell binding specificity - soluble EphA2 fusion protein competes for deimmunized anti-CD3xEA2 (VH / VL) binding. Target binding specificity of anti-CD3xEA2 (VH / VL) deimmunized BiTE was tested in a competition test. As shown in the figure, co-incubation of deimmunized anti-CD3xEA2 (VH / VL) BiTE with a 10-fold excess weight of soluble EphA2 Fc fusion protein completely blocked the binding of BiTE to human lung cancer A549 cells. . This shows that the binding of BiTE to tumor cells is specifically mediated by target EphA2 recognition.

FIG. 18. Target cell specificity of anti-CD3xEA2 (VH / VL) deimmunized EphA2-BiTE: killing of antigen positive cells. The cytotoxic activity of deimmunized anti-CD3xEA2 BiTE depends strictly on the expression of EphA2 in transfected B16F10 target cells; B16F10 EphA2 negative cells are completely resistant to deimmunized anti-CD3xEA2 mediated lysis.

FIGS. 19A-19C. Cytotoxic activity of deimmunized anti-CD3xEA2 (VH./VL) specificity in vitro. (A) Specificity of target cell lysis. CD3 + T-cell enriched PBMC were incubated with positive EphA2 SW480 cells (full symbols) or negative SK-MEL-28 EphA2 cells (open symbols) in the presence of serial dilutions of deimmunized anti-CD3xEA2 (VH./VL) ( squares) or bscCD19xCD3 (triangles) for 42 hours at 37 ° C. (B and C) Parental antibodies to CD3 (DIL2K) and EphA2 (EA2) inhibit bscEphA2xCD3 lysis of EphA2 positive tumor cells. CD3 + T cells were incubated in 200 μl of SW480 cell culture medium in the presence of serial dilutions of unimmunized anti-CD3xEA2 (VH./VL) alone (open squares), or in the presence of 10 μg (triangle) or 100 μg (circle) of DI2K in panel B or EA2 in panel C for 42 hours at 37 ° C; Results are presented as the percentage of lysed target cells. Error bars indicate the standard deviation (SD) of duplicate measurements. Note: Iise magnitude approaches 100% for each unimmunized anti-CD3xEA2 (VH./VL) curve.

FIG. 20. EphA2-BiTE-mediated death of renal carcinoma cells and prostate cancer cells. Cytotoxicity tests have shown EphA2-BiTE-mediated tumor cell death of ACHN (A), Caki 2 (B), PC3 (C), and DUl45 (D) cells with EC50 values of 3, 30, 6, and 0, 8 ng / ml respectively. A CD19-specific BiTE negative control did not redirect T cells to lyse target cells.

FIGS. 21A-21B. Effects of EphA2-BiTE killing on EphA2 levels in target cells. (A) Cytotoxicity tests have shown EphA2-BiTE-mediated tumor cell death of SW480 cells with an EC50 value of 6 ng / ml. A CD19-specific BiTE negative control did not redirect T cells to lyse target cells. (B) After completion of an in vitro cytotoxicity test, the remaining live SW480 target cells were measured for the presence of EphA2 or EphA2-BiTE. The geometric mean fluorescence intensity of living SW480 cells was stained with a control isotype, EphA2 expression (B233 stain), or EphA2-BiTE (BiTE) was plotted versus the inserted BiTE dose. EphA2-BiTE redirected T cells to first kill target cells with high expression of EphA2 levels. Interestingly, not killing all target cells were lysed despite EphA2-BiTE being bound to the cells. Thus, EphA2-BiTE-mediated T-cell killing of most target EphA2 + cells, although a subgroup of EphA2 + cells may be resistant.

FIGS. 22A-22D. Cytotoxicity characterization of deimmunized artti-CD3xEA2 (VH./VL) in vitro. (A) Reduced Iise kinetics. CD3 + T cells were incubated with SW480 cells in the presence of serial dilutions of deimmunized anti-CD3xEA2 (VH./VL) for 4 hours (square), 18 hours (triangle), 24 hours (inverted triangle), or 42 hours (diamond ) at 37 ° C. Error bars indicate standard deviation. EC50 values were estimated from the curves and are listed. (B) Effector influence for target ratio in redirected Iise. CD3 + T cells were incubated with SW480 cells at a ratio of 20: 1 (dark square), 10: 1 (dark triangle), 5: 1 (inverted open triangle), 1: 1 (open diamond), 1: 2 (circle gray), or 1: 5 (gray square) in the presence of serial dilutions of deimmunized anti-CD3xEA2 (VH./VL) for 42 hours at 37 ° C. Error bars indicate standard deviation. EC50 values were estimated from the curves and are listed. (C) Effects of available EphA2 receptor binding sites on redirected lysis potential. The estimated number of EphA2 molecules per cell for tumor lineage (HeyA8, SW480, A549 human carcinoma lineages and M14 and A375 melanoma lineages) was plotted against the respective EC50 values of the percentage of lysed target cells from four separate experiments. . The values of P and r2 of the linear regression curve are listed in the graph. (D) EphA2 surface density effects on redirected Iise. CD3 + T cells were incubated with HeyA8 cell lines (dark squares; 107,000 EphA2 receptors per cell), M14 (gray circle; 2,400 EphA2 receptors per cell), and nd SKMEL-28 (open square; EphA2 receptors below detection limit). ) in the presence of serial dilutions of deimmunized anti-CD3xEA2 (VH./VL) for 18 hours at 37 ° C. Error bars indicate standard deviation. EC50 values were estimated from the curves and are listed.

FIG. 23. Stability of deimmunized anti-CD3xEA2 (VH / VL) EphA2-BiTE in human plasma. Plasma stability of specific BiTE EphA2 was tested under different incubation conditions followed by ED50 determination of cytotoxic activity in a standard chrome release test 51.

FIGS. 24A-E. Target epitope exclusion in cells not transformed by deimmunized anti-CD3xEA2 (VH / VL). Video microscopy was employed to visualize CD8 + T cell attack against untransformed MCFlOA cells in the presence of unimmunized anti-CD3 χ EA2 (VH / VL) BiTE and a control BiTE excluding non-epitopes. (A) Untransformed MCFlOA co-incubated with CD8 + T cells (1: 1 E: T ratio) and 100 ng / ml deimmunized anti-CD3xEA2 (VH / VL). (B) Untransformed MCFlOA co-incubated with CD8 + T cells (1: 1 E: T ratio) without BiTE. (C) Untransformed MCF10A co-incubated with CD8 + T cells (E: T ratio of 1: 1) and 100 ng / ml of a excluding pan-carcinoma control BiTE. not epitope. (D) A549 lung carcinoma cell line co-incubated with CD8 + T cells (1: 1 E: T ratio) and 100 ng / ml deimmunized anti-CD3xEA2 (VH / VL). (E) Same configuration as in (D). The overlay of the transmission microscopy and fluorescence microscopy in the presence of propidium iodide and to visualize lysed cells (light gray).

FIG. 25. Determination of deimmunized anti-CD3xEA2 (VH / VL) target affinity by surface plasmon resonance. The formation and dissociation of BiTE / EphA2 complexes was monitored by surface plasmon resonance using a Biacore 3000 system. The KD is estimated at 45 nM.

FIG. 26. In vivo negative control test groups for deimmunized anti-CD3xEA2 (VH / VL) antitumor in model SW480. 5x106 SW480 cells alone ("SW480 only" and "unimmunized anti-CD3xEA2") or mixed with 2.5 x 106 human CD3 + T cells ("PBS", "non-relevant BiTE", and "add. Iv T cells, PBS ") were injected subcutaneously into female NOD / SCID mice. One group of mice received 2.5 x 10 6 intravenously injected CD3 + T cells ("add. T cells, i.v., PBS"). Mice were treated daily for five consecutive days with BiTE ("deimmunized anti-CD3xEA2 only"), PBS ("PBS"; "add. T cells i.v., PBS"), or a non relevant BiTE. The average tumor volume of six mice / group is presented.

FIG. 27. Daily treatment with deimmunized anti-CD3xEA2 (VH / VL) for five consecutive days induced dose-dependent inhibition of SW480 tumor growth in the presence of effector CD3 + T cells. ** Highly significant (p≤0.01); * significant (p≤0.05) according to Student's t test.

FIG. 28. No effect of peripheral T cells on antitumor effect of deimmunized anti-CD3xEA2 (VH / VL). The average tumor volume of six mice / group is shown. ** Highly significant (p≤0.01).

FIG. 29. VL and VH domain amino acid sequences of anti-EphA2 antibody 233. Presented in (A) is the amino acid sequence of the VL domain (SEQ ID NO: 33). Presented in (B) is the VH domain amino acid sequence (SEQ ID NO: 37). The CDR sequences are on the board. Antibody 233 has been previously described in U.S. Pat. Pub. No. US 2004-0028685 Al, and is incorporated herein by reference in its entirety.

FIG. 30. Amino acid sequences of the VL and VH domains of the anti-EphA2 antibody 3F2. Presented in (A) is the VL domain amino acid sequence (SEQ ID NO: 41). Presented in (B) is the amino acid sequence of the VH domain (SEQ ID NO: 45). The CDR sequences are on the board. Antibody 3F2 has been previously described in U.S. Pat. Appn. Ser. No. 11 / 203,251, filed August 15, 2005, and is incorporated herein by reference in its entirety.

FIG. 31. Linear map of 4H5 scFv insertion site in expression vector MD102.

FIGS. 32A-32B. Sequences of the humanized clone 4H5 VH-VL scFV. Presented in (A) is the nucleotide sequence (SEQ ID NO: 67). Presented in (B) is the amino acid sequence (SEQ ID NO: 68). The CDRs are on the board. The 4H5 antibody has been previously described in U.S. Pat. Appn. Ser. No. 2005-0048617 Al, and is incorporated herein by reference in its entirety.

FIG. 33A-33B. VH VL 2A4 scFv nucleotide and amino acid sequence. Presented in (A) is the nucleotide sequence (SEQ ID NO: 75). Presented in (B) is the amino acid sequence (SEQ ID NO: 76). The CDRs are on the board. FIGS. 34Α-34Β. VH VL 2E7 scFv nucleotide and amino acid sequence. (A) represents the nucleotide sequence (SEQ ID NO: 83); and (B) represents the amino acid sequence (SEQ ID NO: 84).

FIGS. 35A-35B. VH VL 12E2 scFv nucleotide and amino acid sequence. (A) represents the nucleotide sequence (SEQ ID NO: 91); and (B) represents the amino acid sequence (SEQ ID NO: 92).

FIG. 36. ELISA titration of scFv supernatants of affinity optimized combining variants in immobilized human EphA2.

FIG. 37. Amino acid sequence alignment of affinity optimized variants 2A2, 2E7 and 12E2 with humanized 4H5 scFv.

FIG. 38. This figure represents the sequence of the combinatorial primers (L1, L2, L3, L4, L6, H6, H7, H8, H9, H10, H11, H12, and H13) used for affinity optimization of Fab 2G6 (SEQ ID NOS : 99-111, respectively).

FIG. 39. Amino acid sequence alignment of the murine B233, humanized 2G6, and affinity optimized 3F2 mAbs variable regions provided. FIG. 40. Anti-human EphA2 antibodies bind to cinomolgus EphA2. Human purified murine (EA2) and humanized (3F2 and 4H5) anti-human antibodies bound to human EphA2 in colon carcinoma SW480 cells and cinomolgus EphA2 expressed in transfected CHO cells. Bound antibodies were detected by flow cytometry using an anti-mouse Alexafluor 488 or human IgG (H + L) antibody. Antibodies were compared with a non-binding negative control antibody (R347). EA2, 3F2, and 4H5 linked to human and cinomolgus EphA2.

FIG. 41. Selective recognition of tumor cell by an EphA2 antibody subgroup. Untransformed (MCF-10A) and malignant (MDA-MB-231) epithelial mammary cell monolayers were fixed and labeled with the indicated antibodies (G5, EA5, EA2, 3F2, or 4H5), and then visualized using light microscopy. immunofluorescence. Antibodies deleted (yes) or not excluded (no) from MCF-IOA cell cell-cell contact sites are indicated. Thus, the EA5 and EA2 epitopes are selectively excluded by the normal architecture that typifies untransformed epithelial cells, but becomes accessible after malignant transformation.

FIG. 42. G5 VL and VH domain amino acid sequences. Presented in (A) is the VL domain amino acid sequence (SEQ ID NO: 49). Presented in (B) is the amino acid sequence of the VH domain (SEQ ID NO: 53). G5 antibody has been previously described in U.S. Appn. Ser. No. 11 / 165,023, filed June 24, 2005, and is incorporated herein by reference in its entirety.

FIG. 43. Cytotoxic potency comparison of four specific EphA2 BiTEs made from two different production runs of the humanized 3F2 monoclonal antibody. In vitro cytotoxic tests compared the ability of four different 3F2-based BiTE anti-EphA2 constructs to mediate redirected T cell lysis of MDA-MB-231 EphA2 + cells. The table lists power values (i.e., EC50) values for each construct purified from two separate production runs. The deimmunized anti-CD3x3F2 (VH / VL) construct consistently showed the highest potency.

FIG. 44. Independent cytotoxic power comparison of four 3F2-based anti-EphA2 BiTEs. In vitro cytotoxic testing compared the ability of four different 3F2-based BiTE anti-EphA2 constructs to mediate redirected T-cell lysis of SW480 EphA2 + cells. The unimmunized anti-CD3x3F2 (VL / VH) BiTE construct had the highest potency.

FIG. 45. Target cell binding specificity of 4H5-based anti-EphA2 BiTES to cells expressing EphA2 + and CD3 +. The target binding specificity of the various 4H5-based BiTE constructs was examined using the flow cytometry-based assay described above. MDA MB 231 EphA2 + breast cancer cells and HP Ball CD3 + human T cells were used as target cells. Deimmunized anti-CD3xEA2 BiTE (VH / VL) was used as a positive control. As shown in the figure, each of the 4H5-based EphA2-BiTE constructs bound to cells expressing both EphA2 and CD3.

FIG. 46. Target cell binding specificity of mature affinity 4H5 based humanized BiTE 12E2, 2E7 and 2A4 constructs. The target binding specificity of the various 4H5-based BiTE constructs. was examined using the flow cytometry-based test described above. MDA MB 231 EphA2 + breast cancer cells and HP Ball CD3 + human T cells were used as target cells. As shown in the figure, each of the 4H5-based EphA2-BiTE constructs bound to cells expressing both EphA2 and CD3.

FIG. 47. Target cell binding specificity of purified monomers. As shown in the figure, purified monomers of mature affinity-based 4H5-based EphA2-BiTE constructs bound to MDA MB 231 EphA2 + breast cancer cells and target HP Ball CD3 + human T cells at a concentration of 5 μg / ml using the assay. based on flow cytometry as described above.

FIG. 48. Comparison of cytotoxic potency of four EphA2 BiTEs specific to the humanized 4H5 monoclonal antibody. Chromium 51 release-based cytotoxicity tests were performed to determine target cell lysis redirected by the various anti-EphA2 BiTE constructs in the presence of stimulated human CD8 + T cells. The MDA-MB-231 EphA2 positive tumor cell line was loaded with Chromium 51 and served as target cells. The various anti-EphA2 BiTE constructs were titrated over a wide range of concentrations. The duration of the test was 18 hours and the target effector ratio 10: 1. The required EphA2-BiTE concentrations for half of the maximum lysis (i.e., EC50) with different production groups were estimated using a four-parameter nonlinear nesting model.

FIG. 49. This figure provides a direct comparison of the target cell lysis potency of MDA-MB-231 cells of the various 4H5-based EphA2 3F2 (A) and (B) constructs as measured by a Chromium 51-based cytotoxicity test.

FIG. 50. EphA2-BiTEs induced cytotoxicity activity based on mature affinity 4H5 12E4, 2E7 and 2A4. This figure demonstrates the target cell lysis potency of the various 4H5 based EphA2-BiTE constructs (12E4, 2E7 and 2A4). The A549 cells of EphA2 positive tumor cell line were loaded with Chromium 51 and served as target cells. Stimulated human CD8 + T cells served as effector cells. The various anti-EphA2 BiTE constructs were titrated over a wide range of concentrations. The duration of the test was 18 hours and the target effector ratio 10: 1. The required EphA2-BiTE concentrations for half the maximum lysis (i.e., EC50) with different production groups were estimated using a four-parameter nonlinear nesting model.

FIG. 51. EphA2-BiTEs induced cytotoxicity activity based on mature affinity 4H5 12E4, 2E7 and 2A4. This figure demonstrates the target cell lysis potency of the various 4H5 based EphA2-BiTE constructs (12E4, 2E7 and 2A4). The A549 cell lines from EphA2 positive and SW480 tumor cells were loaded with Chromium 51 and served as target cells. Stimulated human CD8 + T cells served as effector cells. The various anti-EphA2 BiTE constructs were titrated over a wide range of concentrations. The duration of the test was 18 hours and the target effector ratio was 10: 1. The required EphA2-BiTE concentrations for half of the maximum lysis (i.e., EC50) with different production groups were estimated using a four-parameter nonlinear nesting model.

FIG. 52. No activation of EphA2 by 4H5 based EphA2-BiTEs as measured by EphA2 phosphorylation. This figure demonstrates that neither the BiTE 2A4 construct nor the BiTE 2E7 construct induced EphA2 phosphorylation in cells expressing EphA2.

FIG. 53. In vivo efficacy of mature affinity 4H5-based EphA2-BiTE constructs. The in vivo potency of the 2A4- and 2E7-BITE constructs was evaluated in immunodeficient NOD / SCID mice with a xenotransplanted model of human colon carcinoma. The human colon carcinoma cell line SW480 has been selected for establishment of a human xenotransplant model, as SW480 cells express EphA2. The results of the study are presented here.

DETAILED DESCRIPTION OF THE INVENTION

As detailed below, the present invention relates to bispecific single chain antibodies comprising a first binding domain that immunospecifically binds to the T cell CD3 antigen and a second binding domain that immunospecifically binds to the EphA2 receptor. Such single chain bispecific antibodies are encompassed by the term "EphA2-BiTEs." The present invention further relates to methods and compositions designed for the treatment, prevention and / or management of disorders associated with aberrant EphA2 expression and / or activity. Such disorders include, but are not limited to, cancer, noncancerous hyperproliferative cell disorders, and infections. The invention further relates to vectors comprising polynucleotides encoding the EphA2-BiTEs of the invention, host cells transformed in this manner, and their use in the production of said EphA2-BiTEs. The invention also provides compositions, including pharmaceutical compositions, comprising any of the EphA2-BiTEs, polynucleotides or vectors then mentioned either alone or in combination with one or more prophylactic or therapeutic agents. Also described are screening methods for said EphA2-BiTEs and kits comprising any of the compositions and diagnostic reagents then mentioned.

EphA2-BiTEs

The present invention provides bispecific T-cell engagements (i.eEphA2-BiTEs (in particular, EphA2-BiTEs which are single-chain bispecific antibodies)) that immunospecifically bind EphA2 and the T-cell CD3 antigen, and methods of using them. to treat, prevent and / or manage disorders associated with aberrant EphA2 expression and / or activity. Such disorders include, for example, cancer, noncancerous hyperproliferative cell disorders, and infections. In one aspect, EphA2-BiTEs are more efficient at eliminating aberrantly expressing EphA2 cells than specific EphA2 antibodies known in the art. In a specific aspect, EphA2-BiTEs are more efficient at eliminating EphA2-expressing cancer cells (in particular, EphA2-expressing malignant cancer cells) than EphA2 antibodies known in the art. In another aspect, EphA2-BiTEs are more efficient at eliminating non-cancerous hyperproliferative cells expressing EphA2 than EphA2 antibodies known in the art. In yet another aspect, the EphA2-BiTEs of the invention are more efficient at eliminating infected cells expressing EphA2 (in particular cells infected with the syncytial respiratory virus; "RSV") than EphA2 antibodies known in the art. In another aspect, lower dosages of EphA2-BiTEs than specific EphA2 antibodies known in the art are required to treat, prevent and / or manage disorders associated with aberrant EphA2 expression and / or activity.

Specific EphA2 BiTEs of the present invention comprise a first binding domain that immunospecifically binds to the T cell CD3 antigen and a second binding domain that immunospecifically binds to EphA2 (hereinafter "EphA2-BiTEs" or "EphA2-BiTE Molecules") . In one advance, the first binding domain immunospecifically binds to CD3. In a specific advance, the first binding domain immunospecifically binds to one or more of any CD3 subunit (e.g., the gamma, delta, zeta, or eta subunit). In a preferred embodiment, the first binding domain immunospecifically binds to the epsilon (ε) subunit of CD3. In a specific advance, the first binding domain immunospecifically binds to the CD3 epsilon (ε) subunit when said subunit is complexed with the delta subunit of CD3. In another advance, the binding domain that binds to CD3 is de-immunized. In another specific advance, the second binding domain immunospecifically binds to the extracellular domain of EphA2. In a preferred embodiment, the second binding domain of EphA2-BiTEs, which are used in cancer treatment, prevention and / or management, immunospecifically binds to epitopes on EphA2 that are selectively exposed and / or increased in cancer cells. not in non cancerous cells. In another preferred embodiment, the second binding domain of the EphA2-BiTEs of the invention immunospecifically binds to epitopes on EphA2 that are selectively exposed and / or increased in non-cancerous hyperproliferative cells, but not in normal cells. In another preferred embodiment, the second EphA2-BiTEs binding domain immunospecifically binds to EphA2 epitopes that are selectively exposed and / or increased in infected cells, but not in uninfected cells.

The present invention provides EphA2-BiTEs in which the first binding domain comprises a heavy variable domain (VH) in a light variable domain (VL) of an antibody that immunospecifically binds to the T cell CD3 antigen and a second binding domain. which comprises a VH domain and a VL domain of an antibody that immunospecifically binds to EphA2. In a specific advance, the VH and VL domains of the first binding domain are joined together by a ligand of sufficient strength that allows the domains to fold to allow binding to the T cell CD3 antigen. The linker may comprise, for example, the sequence GEGTSTGS (G2S) 2GGAD (SEQ ID NO.:57). In another specific advance, the VH and VL domains of the second binding domain are joined together by a ligand of sufficient strength that allows the domains to bend to allow binding to EphA2. Still for this advance, such a binder may comprise, for example, the sequence (G4S) 3 (SEQ ID NO: 59). In another specific advance, the first and second binding domains are joined together by a ligand of sufficient strength that allows the domains to fold to allow binding to the Tea EphA2 cell CD3 antigen. Still for this advance, such a binder may comprise, for example, the sequence G4S (SEQ ID NO: 58). In a specific advance, an EphA2-BiTE of the invention has the amino acid sequence of (SEQ ID NO: 65). In a specific advance, an EphA2-BiTE of the invention comprises a single domain antibody.

According to the preceding paragraph, the bond is covalent. In a specific advance, the binders of the invention comprise serine and glycine residues. The EphA2-BiTEs ligands, eg, the ligand between the VH and VL domains of the first CD3 binding domain, the ligand between the VH and VL domains of the second EphA2 binding domain, and the ligand between the first CD3 binding domain and the second EphA2 binding domain may be of any length sufficient to allow the domains to fold to allow binding to the CD3 and EphA2 antigens, respectively. In certain advances, the binders of the invention comprise a length of at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 25 residues, at least 30 residues or more. In further advances, the binders of the invention comprise a length between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, 14 wastes, 2-16 wastes, 2-18 wastes, 2-20 wastes, 2-22 wastes, 2-24 wastes, 2-26 wastes, 2-28 wastes, or 2-30 waste. In certain advances, the first binding domain is 5 'to the second binding domain. In other advances, the second binding domain is 5 'to the first binding domain. In certain advances, the first and second binding domains are single chain antibodies. In a specific advance, the first and second binding domains comprise single stranded Fvs (scFvs).

In another specific advance, the invention provides a single-stranded bispecific antibody comprising (a) a first heavy chain variable domain and a first first light chain variable domain, each of an antibody that immunospecifically binds the CD3 ε chain said. first heavy chain variable domain covalently linked to said first light chain variable domain by a first binder of sufficient length (eg, GEGTSTGS (G2S) 2GGAD (SEQ ID NO.:57)) such that said first heavy chain variable domain and said first light chain variable domain fold to form a first binding domain that binds the CD3 β subunit; and (b) a second heavy chain variable domain and a second light chain variable domain of an antibody that immunospecifically binds an EphA2 epitope exposed on the cell surface, said second heavy chain variable domain covalently linked to said second variable domain. light chain by a second binder of sufficient length (eg, (G4S) 3 (SEQ ID NO: 59)) such that said second heavy chain variable domain and said second light chain variable domain bend to form a second domain. binding that binds said EphA2 epitope, characterized in that said first binding domain and said second binding domain are covalently linked by a third binder of a length (eg, G4S (SEQ ID NO: 58)) such that said first binding domain and said second binding domain fold independently of each other.

In specific embodiments, the EphA2-BiTEs of the invention comprise any of the following 5 'to 3' direction arrangements: (1) VHCD3-VLCD3-VHEphA2-VL-EphA2; (2) VLcd3-VHcd3-VHEphA2-VL EphA2; (3) VLCD3-VHcD3-VLEphA2-VH-EphA2; (4) VHCD3-VLCD3-VLEphA2-VHEphA2; (5) VHEphA2-VLEphA2-VHcd3-VLcd3; (6) VLEphA2-VHEphA2-VHCD3-VLcd3; (7) VLEphA2-VHEphA2-VLCD3-VHCD3; or (8) VHEphA2-VLEphA2-VLcd3-VH-cd3. See, e.g., FIG. 14A for a generic representation of the EphA2-BiTE constructs of the invention.

As is well known, an antibody fragment that contains a complete antigen recognition and binding domain may under certain circumstances consist of a dimer of one heavy chain variable domain and one light chain variable domain (VH and VL) in combination. not covalent. In that configuration corresponding to that found in native antibodies, the three complementarity determining regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity for the antibody. Tool regions (FRs) flanking the CDRs have a tertiary structure that is essentially conserved in native immunoglobulins in species as diverse as humans and mice. These RFs serve to hold the CDRs and their proper orientation. Constant domains are not required for the binding function, but may assist in stabilizing the VH-VL interaction. Even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind antigen with high conformational stability (see, eg, Dumoulin et al., 2002, Protein Science 11: 500- 515). Accordingly, said domain of the binding domain of an EphA2-BiTE of the invention may comprise a pair of VH-VL domains of either the same or different immunoglobulins. The order of VH and VL domains in the polypeptide chain is not decisive for the present invention; The invention encompasses all possible arrangements of the variable domains. It is important, however, that the VH and VL domains are arranged so that the antigen binding domain can fold correctly to recognize and bind the antigen. In a specific advance, the EphA2 binding domain domains may be of the same or a different immunoglobulin.

Accordingly, the EphA2-BiTEs of the invention may comprise any arrangement of the heavy variable domain and light variable domain of each antibody (eg, the VH and VL domains of an antibody that immunospecifically binds to CD3 and the VH and VL domains of an antibody). which immunospecifically binds to another antigen of interest (eg, EphA2)), and each domain may be located at the N-terminal or C-terminal portion of the BiTE molecule. For example, and not by way of limitation, an EphA2-BiTE molecule of the invention may comprise the following arrangements as outlined in the following table:

<table> table see original document page 97 </column> </row> <table>

Said binding domains discussed above are preferably connected by a flexible binder, preferably by a linker polypeptide disposed between said domains, characterized in that the linker polypeptide comprises peptide-linked plural amino acids of sufficient length to overcome the distance between the binder. The C-terminal end of one of said domains comprising said binding domains and the N-terminal end of other domains comprising said binding domains when the polypeptide of the invention assumes a suitable conformation to bind when disposed in aqueous solution. Preferably, said linker polypeptide comprises a plurality of glycine, alanine and / or serine residues. It is further preferred that said linker polypeptide comprises a plurality of consecutive copies of an amino acid sequence. Usually, the linker polypeptide comprises from 1 to 15 amino acids, although linker polypeptides of more than 15 amino acids may also function. In certain advances, the binders of the invention comprise a length of at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 25 residues, at least 30 residues or more. In further advances, the binders of the invention comprise a length between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, 14 wastes, 2-16 wastes, 2-18 wastes, 2-20 wastes, 2-22 wastes, 2-24 wastes, 2-26 wastes, 2-28 wastes, or 2-30 waste. Examples of binders that may be used according to the methods of the invention are found in FIG. 3 (SEQ ID NOS .-. 57-59). In an advance of the invention, an antibody or linker that immunospecifically binds to EphA2 may comprise a portion of the BiTE molecule. For example, the VH and / or VL (preferably an scFV) of an antibody that immunospecifically binds to EphA2 can be fused to an anti-CD3 binding moiety, such as that of the molecule described above, thus creating a single chain bispecific antibody that targets EphA2. In addition to the VH and / or VL domains of an EphA2 antibody, other EphA2-binding molecules may comprise EphA2-BiTE, for example receptors or ligands (e.g., an Ephrin).

In a specific advance, an EphA2-BiTE of the invention is a single chain bispecific antibody comprising a first binding domain that immunospecifically binds CD3, and a second binding domain that immunospecifically binds EphA2. In a specific advance, the first binding domain comprises a de-immunized version of the VH and VL domains of an antibody that immunospecifically binds to CD3. Also in accordance with this advance, the second binding domain comprises the EA2 VH and VL domains. Preferably, the variable region arrangement of an EphA2-BiTE of the invention is as follows: VH-VL in the CD3 binding domain, and VH-VL in the EphA2 binding domain. Also in accordance with this advance, in a specific advance, the second binding domain comprises the V2 and / or VL domain of EA2, EA3, EA4, EA5, 3F2, 4H5, 2A4, 2E7, 12E2, Eph099B-102.147, Eph099B- 208,261, Eph099B-210,248, Eph099B-233,152, Eph101,530,241, 233 or G5.

Methods of making the antibodies from which the EphA2-BiTES (in particular the EphA2-BiTEs which are single chain bispecific antibodies) of the invention are derived are well known in the art and can be found, for example, in US 2004- 0091486 A1 (May 13, 2004), US 2004-0028685 A1 (February 12, 2004) and WO 99/5440 (and references disclosed therein), each of which is incorporated herein by reference in its entirety. For information related to affinity optimized EphA2 agonistic antibody variants 2A4, 2E7 and 12E2, see also U.S. Provisional Appn. No.60 / 751,964, filed December 21, 2005, entitled "Afinity Optimized EphA2 Agonistic Antibodies and Methods of Use Thereof," which is incorporated herein by reference in its entirety.

EphA2 Antibodies

As discussed above, the invention encompasses bispecific T cell engagements (ie, EphA2-BiTEs (in particular, EphA2-BiTEs which are single-chain bispecific antibodies) comprising at least two EphA2 and CD3 antigen specific binding domains, respectively. The EphA2-BiTEs of the invention comprise a first binding domain that binds to the T cell CD3 antigen and a second binding domain that binds to an EphA2 polypeptide or fragment thereof. immunospecifically binding to EphA2 is a single chain Fv (scFv) As used herein, the term "single chain Fv" or "scFv" refers to antibody fragments comprising the VH and VL domains of an antibody, characterized by these domains are present in a single polypeptide chain.Fv polypeptide generally further comprises a linker polypeptide between the VH and VL domains that allow so that scFv forms the desired structure for antigen binding. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). In one advance, the binding domain of an EphA2-BiTE that immunospecifically binds to EphA2 is derived from an scFV produced from any antibody, including the EphA2 antibodies described herein.

In specific advances, EphA2 antibodies used to generate EphA2-BiTEs include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding domain that immunospecifically binds to an EphA2 antigen, eg, a or more complementarity determining regions (CDRs) of an anti-EphA2 antibody. The EphA2 antibodies from which the EphA2-BiTEs binding domain are derived may be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgGi, IgG2, IgG3, IgG4, IgAi and IgA2) or immunoglobulin molecule subclass.

In other specific advances, the EphA2 antibodies used to generate the EphA2-BiTEs encompassed by the invention are EA2 (see FIG. 1), EA3, EA4, EA5 (see FIG. 2), 3F2 (see FIG. 30), 4H5 (see 32), 2A4 (FIG. 33), 2E7 (FIG. 34), 12E2 (FIG. 35), Eph099B-102.147, Eph099B-208.261, Eph099B-210.248, Eph099B-233.152, Eph10100.530.241, 233 (FIG. 29) and G5 (FIG. 42). See also, e.g., U.S. Patent Pub. Nos. US 2004-0091486 Al (May 13, 2004), US 2004-0028685 Al (Feb. 12, 2004), US 2005-0059592 Al (Mar. 17, 2005), US 2005-0048617 Al, U.S. Appn. Ser. 11 / 165,023, filed June 24, 2005 and 11 / 203,251, filed August 15, 2005, each of which is incorporated herein by reference in its entirety. Hybridoma-producing EA2 (EA2.31) and EA5 (EA5.12 strain) antibodies of the invention were deposited with the American Type Culture Collection (ATCC, PO Box 1549, Manassas, VA 20108) on May 22, 2002, and designated PTA-4380 and PTA-4381 access numbers, respectively and incorporated by reference. Eph099B-102.147, Eph099B-208.261, and Eph099B-210.248 were deposited with the ATCC on August 7, 2002 and assigned access numbers (PTA-4572, PTA-4573, and PTA-4574, respectively). Eph099B-233,152 was deposited with the ATCC on May 12, 2003 and designated accession number PTA-5194, and Eph101. 530,241 was filed with the ATCC on September 26, 2002 and designated accession number PTA-4724. All previously mentioned deposits were made under the terms of the Budapest Treaty on International Recognition of the Deposit of Microorganisms for Patent Procedure ( Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures), and the accession numbers and dates corresponding to the respective antibodies are incorporated herein by reference in their entirety.

In a specific advance, the antibodies used to create the EphA2-BiTEs using the methods of the invention are human or humanized versions of the aforementioned EphA2 antibodies. Such humanized EphA2 antibodies and production methods are described in, e.g., U.S. Patent Appln. Publ. No. US 2005-0048617 Al, and Dall'Acqua et al., 2005, Methods 36: 43-60, each of which is incorporated herein by reference in its entirety. In another specific advance, the EphA2 antibody used to generate an EphA2-BiTE of the invention is EA2. In yet another specific advance, the antibodies used to create an EphA2-BiTE using the methods of the invention are humanized versions with optimized affinity of the aforementioned antibodies. Accordingly, the binding domain that immunospecifically binds to EphA2 of an EphA2-BiTE of the invention is derived from 2A4, 2E7 and 12E2. For information related to affinity optimized EphA2 agonistic antibody variants 2A4, 2E7 and 12E2, see also U.S. Provisional Appn. No. 60 / 751,964, filed December 21, 2005, entitled "Afinity Optimized EphA2 Agonistic Antibodies and Methods of Use Thereof", which is incorporated herein by reference in its entirety.

The sequences of some EphA2 antibodies are described in FIGS. 1, 2, 29, 30, 32, 33, 34, 35, 37 or 39, or in publications describing the sequences of the EphA2 antibodies cited above. Methods for making the EphA2 antibodies from which the EphA2-BiTEs of the invention are generated are described, for example, in U.S. Patent Pub. Nos. US 2004-091486 A1 (May 13, 2004), US 2004-0028685 A1 (February 12, 2004), US 2005-0059592 A1 (March 17, 2005), US 2005-0048617 Al, U.S. Appn. Ser. 11 / 165,023, filed June 24, 2005 and 11 / 203,251, filed August 15, 2005, each of which is incorporated herein by reference in its entirety.

The present invention provides antibody-derived EphA2-BiTEs which immunospecifically bind to an EphA2 polypeptide, characterized in that said antibodies may comprise a VH CDR having an amino acid sequence of any of the VH CDRs underlined, for example, in FIGS. 1, 2, 29, 30, 32, 33, 34, 35, 37 or 39, or in publications describing the EphA2 antibodies cited above. In particular, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to EphA2, which binding domain comprises (or alternatively consists of) one, two, three, four, five or more VH CDRs having a sequence of. amino acids of any of the VH CDRs listed in, for example, FIGS. 1, 2, 29, 30, 32, 33, 34, 35, 37 or 39, or in publications describing the sequences of the EphA2 antibodies cited above.

The present invention provides antibody-derived EphA2-BiTEs which immunospecifically bind to an EphA2 polypeptide, characterized in that said antibodies may comprise a VL CDR having an amino acid sequence of any of the VL CDRs underlined, for example, in FIGS. 1, 2, 29, 30, 32, 33, 34, 35, 37 or 39, or in publications describing the EphA2 antibodies cited above. In particular, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to EphA2, which binding domain comprises (or alternatively consists of) one, two, three, four, five or more VL CDRs having a sequence of. amino acids of any of the VL CDRs listed, for example, in FIGS. 1, 2, 29, 30, 32, 33, 34, 35, 37 or 39, or in publications describing the sequences of the EphA2 antibodies cited above.

The present invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to EphA2, which binding domain (or alternatively consists of) a CDR1 VH and a CDR1 VL; one CDR1 VH one CDR2 VL; a CDR1 VH and a CDR3 VL; a CDR2 VH and a CDR1 VL; CDR2 VH and CDR2 VL; a CDR2 VH and a CDR3 VL; a CDR3 VH and a CDR1 VH; one CDR3 VH and one CDR2 VL; one CDR3 VH and one CDR3 VL; one VH1 CDR1, one CDR2 VH and one CDR1 VL; one CDR1 VH, one CDR2 VH and one CDR2 VL; one CDR1 VH, one CDR2 VH and one CDR3 VL; one CDR2 VH, one CDR3 VH and one CDR1 VL, one CDR2 VH, one CDR3 VH and one CDR2 VL; one CDR2 VH, one CDR2 VH and one CDR3 VL; one CDR1 VH, one CDR1 VL and one CDR2 VL; one CDR1 VH, one CDR1 VL and one CDR3 VL; one CDR2 VH, one CDR1 VL and one CDR2 VL; one CDR2 VH, one CDR1 VL and one CDR3 VL; one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR3 VH, one CDR1 VL and one CDR3 VL; one CDR1 VH, one CDR2 VH, one CDR3 VH and one CDR1 VL; a CDR1 VH, a CDR2 VH, a CDR3 VH and a CDR2 VL; a CDR1 VH, a CDR2 VH, a CDR3 VH and a CDR3 VL; one CDR1 VH, one CDR2 VH, one CDR1 VL and one CDR2 VL; one CDR1 VH, one CDR2 VH, one CDR1 VL and one CDR3 VL; one CDR1 VH, one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR1 VH, one CDR3 VH, one CDR1 VL and one CDR3 VL; one CDR2 VH, one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR2 VH, one CDR3 VH, one CDR1 VL and one CDR3 VL; one CDR2 VH, one CDR3 VH, one CDR2 VL and one CDR3 VL; one CDR1 VH, one CDR2 VH, one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR1 VH, one CDR2 VH, one CDR3 VH, one CDR1 VL and one CDR3 VL; one CDR1 VH, one CDR2 VH, one CDR1 VL, one CDR2 VL7 and one CDR3 VL; a CDR1 VH, a CDR3 VH, a CDR1 VL, a CDR2 VL, and a CDR3 VL; one CDR2 VH, one CDR3 VH, one CDR1 VL, one CDR2 VL, and one CDR3 VL; or any combination thereof of the VH CDRs and VL CDRs described, for example, in FIGS. 1, 2, 29, 30, 32, 33, 34, 35, 37 or 39, or in publications describing the sequences of the EphA2 antibodies cited above. In further advances, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to EphA2, which binding domain (or alternatively consists of) a VH domain and a VL domain of any of the above-mentioned EphA2 antibodies, and for example as described in FIGS. 1, 2, 29, 30, 32, 33, 34, 35 or 37, or in publications describing the sequences of the EphA2 antibodies cited above. In a specific advance, the VH and VL domains are from antibodies 2A4, 2E7 and 12E2. See, e.g., FIGS. 33, 34, 35 and 37.

In a specific advance, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to EphA2, which binding domain is derived from antibodies that immunospecifically bind to an EphA2 polypeptide, characterized in that said antibodies have a rate constant association or kon rate (antibody (Ab) + antigen (Ag) -Ab-Ag) of at least 104 M-1S-1, at least 105M-1S-1, at least 1.5 X 105 M-1s- 1, at least 2 X 105M-1S-1, at least 2.5 X 105M-1S-1, at least 5 X 105 M-1s-1, at least 106 M-1s-1, at least 5 X 106 M -1s-1, at least 107 M-1S-1, at least 5 X 107 M-1s-1, or at least 108 M-1s-1, or in a range of about 105 to 108 M-1s-1 , and a range of about 1.5 X 105 M-1s-1 to 1 X 107 M-1s-1, in a range of about 2 X 105 to 1 X 106 M-1s-1, or in a range from about 4.5 X 10 5 to 10 7 M -1 s -1. In certain advances, an antibody that immunospecifically binds to an EphA2 polypeptide has a kon of at least 104 M-1S-1, at least 2 X 10 -5 M-1s-1, at least 2.5 X 10 -5 M -1s-1, at least 5 X 10 "5 M-1S-1, at least 10" 6 M-1S-1, at least 5 X 10 "6 M-1S-1, at least 10" 7 M-1S -1, at least 5 X 10 7 M-1S-1, or at least 10 8 M-1S-1 as determined by a surface plasmon resonance test.

In another specific advance, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to EphA2, which binding domain is derived from antibodies that immunospecifically bind to an EphA2 polypeptide, characterized in that said antibodies have a rate kQff (antibody (Ab) + antigen (Ag) -> Ab-Ag) of less than 10 "3 s" 1, less than 5 X 10 "3 s" 1, less than 10 "4 s" 1, less than 2 χ 10 "4 s" 1, less than 5 X 10 ~ 4 s "1, less than 10" 5 s "1, less than 5 X 10" 5 s "1, less than 10" 6 s "1, less than X 10 "6 s" 1, less than 10 "7 s" 1, less than 5 X 10 "7 s" 1, less than 10 "8 s" 1, less than 5 X 10 "8 s" 1, less than 10 "9 s" 1, less than 5 X 10 "9 s" 1, or less than 10 "10 s" 1, or in a range of about 10 "3 to 10" 10 s "1, in a range of about 10 "4 to 10" 8 s "1, or in a range of about 10" 5 to 10 "8 s" 1. In certain advances, an antibody that immunospecifically binds to an EphA2 polypeptide has a koff of 10 "2 s" 1, or less than 5 X 10 "3 s" 1, or less than 10 "4 s" 1, as determined by a surface plasmon resonance test.

In another specific advance, the invention provides EphA2-BiTEs comprising a binding domain that binds immunospecifically to EphA2, a binding domain is derived from antibodies that immunospecifically bind to an EphA2 polypeptide, characterized in that said antibodies have a constant of affinity or Ka (kon / koff) of at least 102 M-1, at least 5 X 102 M-1, at least 103 M-1, at least 5 X 103 M-1, at least 104 M-1 5 X 104 M-1, at least 105 M-1, at least 5 X 105 M-1, at least 106 M-1, at least 5 X 106 M-1, at least 107 M-1, at least 5 X 107 M-1, at least 108 M-1, at least 5 X 108 M-1, at least 109 M-1, at least 5 X 109 M-1, at least 1010 M-1, at least 5 X 1010 M -1, at least 1011 M-1, at least 5 X 1011 M-1, at least 1012 M-1, at least 5 X 1012 M-1, at least 1013 M-1, at least 5 X 1013 M-1 , at least 1014 M-1, at least 5 X 1014 M-1, at least 1015 M-1, or at least 5 X 1015 M-1, or in a range of about 102 to 5 X 105 M-1, in a range of about 104 to 1 X 1010 M-1, or in a range of about 105 to 1 X 108 M-1 as determined by a surface plasmon resonance.

In another specific advance, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to EphA2, which binding domain is derived from antibodies that immunospecifically bind to an EphA2 polypeptide, characterized in that said antibodies have a constant dissociation or Kd (koff / kon) of less than 10-5 M, less than 5 X 10-5 M, less than 10-6 M, less than 5 X 10-6 M, less than 10-7 M, less 5 X 10-7 M, less than 10-8 M, less than 5 X 10-8 M, less than 10-9 M, less than 5 X 10-9 M, less than 10-10 M, less than 5 X 10-10 M, less than 10-11 M, less than 5 X 10-11 M, less than 10-12 M, less than 5 X 10 -12 Μ, less than 10-13 M, less than 5 X 10 -13 M, less than 10-14 M, less than 5 X 10-14 M, less than 10-15 M, or less than 5 X 10-15 M or in a range of about 10-2 M to 5 X 10-5 M, in a range of about 10-6 to 10-15 M, or in a range of about 10-8 to 10-14 M, as determined by a plasma plasmon resonance test. surface.

In a specific advance, the invention provides EphA2-BiTEs that immunospecifically bind to an EphA2 polypeptide, 10 characterized in that said EphA2-BiTEs have an association constant rate or kon (antibody (Ab) + antigen (Ag) rate. ) -> Ab-Ag) of at least 10 4 M "1S" 1, at least 105 M-1S-1, at least 1,5 X 10 5 M-1S "!, At least 2 X 10 5 M-1S" 1, at least 2,5 X 105 M-1S "1, at least 5 X 105 M-1S-1, at least 106 M-1S-1, at least 5 X 106 M-1S-1, at least 107 M-1S -1, at least 5 X 107 M-1S-1, or at least 108 M-1S-1, or in a range of about 105 to 108 M-1S-1, in a range of about 1.5 X 105 M-1S-1 to 1 X 107 M-1S-1, in a range of about 2 X 105 to 1 X 106 M-1S-1, or in a range of about 4.5 X 105 to 107 M In certain advances, an EphA2-BiTE that immunospecifically binds to an EphA2 polypeptide has a kon of at least 104 M-1s_1, at least 2 X 105 M-1S-1, at least 2.5 X 105 M-1S-1 at least 5 X 105 M-1S-1 at least 106 M-1S-1, at least 5 X 106 M-1s-1, at least 107 M-1S-1, at least 5 X 107 M-1S-1, or at least 108 M-1S-1 as determined by a Surface plasmon resonance testing In another specific advance, the invention provides EphA2-BiTEs that immunospecifically bind to an EphA2 polypeptide, characterized in that said EphA2-BiTEs have a k0ff (antibody (Ab) + antigen (Ag) rate). -> Ab-Ag) less than 10-3 s-1, less than 5 X 10-3 s-1, less than 10-4 s-1, less than 2 χ 10-4 s-1, less than 5 X 10-4 s-1, less than 10-5 s-1, less than 5 X 10-5 s-1, less than 10-6 s-1, less than 5 X 10-s-1, less than 10 -7 s-1, less than 5 X 10-7 s-1, less than 10-8 s-1, less than 5 X 10-8 s-1, less than 10-9 s-1, less than 5 X 10-9 s-1, or less than 10-10 s-1, or in a range of about 10-3 to 10 s-1, in a range of about 10-4 to 10-8 s-1, or in a range of about 10-5 to 10-8 s-1. In certain advances, an EphA2-BiTE that immunospecifically binds to an EphA2 polypeptide has a koff of 10-2 s-1, less than 5 X 10-3 s-1, or less than 10-4 s-1, as determined by a surface plasmon resonance test.

In another specific advance, the invention provides EphA2-BiTEs which immunospecifically bind to an EphA2 polypeptide, characterized in that said EphA2-BiTEs have an affinity constant or Ka (kon / koff) of at least 102 M-1. at least 5 X 102 M-1, at least 103 M-1, at least 5 X 103 M-1, at least 104 M-1, at least 5 X 104 M-1, at least 105 M-1, at least at least 5 X 105 M-1, at least 106 M-1, at least 5 X 106 M-1, at least 107 M-1, at least 5 X 107 M-1, at least 108 M-1, at least 5 X 108 M-1, at least 109 M-1, at least 5 X 109 M-1, at least 1010 M-1, at least 5 X 1010 M-1, at least 1011 M-1, at least 5 X 1011 M-1, at least 1012 M-1, at least 5 X 1012 M-1, at least 10 ^ 13 M "1, at least 5 X 10 ^ 13 M" 1, at least 10 ^ 14 M "1, at least at least 5 X 10 ^ 14 M "1, at least 10 ^ 15 M" 1, or at least 5 X 10 ^ 15 M "1, or in a range of about 10 ^ 2 to 5 X 10 ^ 5 M" 1 , in a range of about 10 ^ 4 to 1 X 10 ^ 10 M "1, or in a range of about 10 ^ 5 to 1 X 10 ^ 8 M-1 as determined by a surface plasmon resonance test.

In another specific advancement, the invention provides EphA2-BiTEs which immunospecifically bind to an EphA2 polypeptide, characterized in that said EphA2-BiTEs have a dissociation constant or Kd (k0ff / k0n) of less than 10.5 M minus 5 X 10 "5 M, less than 10" 6 M, less than 5 X 10 "6 M, less than 10" 7 M, less than 5 X 10 "7 M, less than 10" 8 M, less than 5 X 10 "8 M, less than 10" 9 M, less than 5 X 10 "9 M, less than 10" 10 M, less than 5 X 10 "10 M, less than 10" 11 M, less than 5 X 10 "11 M, less than 10" 12 M, less than 5 X 10 "12 M, less than 10" 13 M, less than 5 X 10 "13 M, less than 10" 14 M, less than 5 X 10 "14 M, less than 10 "15 M, or less than 5 X 10" 15 M or in a range of about 10 "2 M to 5 X 10" 5 M, in a range of about 10 "6 to 10" 15 M, or in a range of about 10 "8 to 10" 14 M, as determined by a surface plasmon resonance test.

CD3 Antibodies

In specific advances of the invention, the EphA2-BiTEs

comprise a binding domain that binds

immunospecifically to the CD3 T cell antigen. Said

CD3 T cell antigen may be of any species (e.g., human). In a specific advance, the EphA2-BiTEs of the invention comprise a binding domain that immunospecifically binds to one or more CD3 subunits (e.g., the gamma, delta, zeta, or eta subunit). In a preferred embodiment, the first binding domain immunospecifically binds to the epsilon (ε) subunit of CD3. In a specific advance, the first binding domain immunospecifically binds to the CD3 epsilon (ε) subunit when said subunit is complexed with the delta subunit of CD3.

In one specific advance, the CD3 binding domain of an EphA2-BiTE of the invention is de-immunized. Disimmunization approaches are well known in the art and are described in, e.g., International Pub. Nos. WO 00/34317 (and in particular, pp. 1-14); WO 98/52976 (and in particular,

Examples 1-6 on pp. 18-38); WO 02/079415 (in particular, pp. 2-8 and Examples 1-10 on pp. 15-43); and WO 92/10755 (and in particular, pp. 6-9), each of which is incorporated herein by reference in its entirety.

In a specific advance, the binding domain that immunospecifically binds to CD3 is a single chain Fv (scFv). As used herein, the term "single chain Fv" or "scFv" refers to antibody fragments comprising the VH and VL domains of an antibody, characterized in that these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a VH and VL domain linker polypeptide that allows scFv to form the desired antigen-binding structure. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). In one advance, the EphA2-BiTEs of the invention are derived from scFvs produced from any of the EphA2 antibodies described below.

In specific advances, the CD3 antibodies used to generate EphA2-BiTEs include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding domain that immunospecifically binds to a CD3 antigen, eg, one or more complementarity determining regions (CDRs) of an anti-CD3 antibody. The CD3 antibodies from which the EphA2-BiTEs CD3 binding domain are derived may be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. Such CD3 antibodies may be of any species (e.g., rat, mouse, man, etc.).

In a specific advance, the CD3 binding domain of an EphA2-BiTE of the invention may be produced as described in International Publication No. WO 99/54440 (p. 3 and Figure 8), which is incorporated herein by reference in its entirety. Anti-CD3 antibodies from which said binding domain is derived are also described, for example, in Kipriyanov, 1998, Int. J. Cancer 77: 763-772 (p. 763-765); Dreier et al., 2002, Int. J. Cancer 100: 690-697 (ρ. 691), each of which is incorporated herein by reference in its entirety.

The present invention provides antibody-derived EphA2-BiTEs which immunospecifically bind to a CD3 polypeptide, characterized in that said antibodies may comprise a VH CDR having an amino acid sequence of any of the VH CDRs described, for example, in publications describing the CD3 antibodies or binding fragments cited above. In particular, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to CD3, which binding domain (or alternatively consists of) one, two, three, four, five or more VH CDRs having a binding domain. amino acid sequence of any of the VH CDRs described, for example, in publications describing the CD3 antibodies or binding fragments cited above.

The present invention provides antibody-derived EphA2-BiTEs which immunospecifically bind to a CD3 polypeptide, characterized in that said antibodies may comprise a VL CDR having an amino acid sequence from any of the described VL CDRs, for example, the publications describing the CD3 antibodies or binding fragments cited above. In particular, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to CD3, which binding domain (or alternatively consists of) one, two, three, four, five or more VL CDRs having a binding domain. amino acid sequence of any of the VL CDRs described, for example, in publications describing the CD3 antibodies or binding fragments cited above.

The present invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to a CD3 polypeptide, which binding domain comprises (or alternatively consists of) a CDR1 VH and a CDR1 VL; a CDR1 VH and a CDR2 VL; a CDR1 VH and a VL CDR3; a CDR2 VH and a CDR1 VL; CDR2 VH and CDR2 VL; a CDR2 VH and a VL CDR3; a CDR3 VH and a CDR1 VH; one CDR3 VH and one CDR2 VL; a CDR3 VH and a VL CDR3; one VH1 CDR1, one CDR2 VH and one CDR1 VL; one CDR1 VH, one CDR2 VH and one CDR2 VL; a CDR1 VH, a CDR2 VH and a VL CDR3; one CDR2 VH, one CDR3 VH and one CDR1 VL, one CDR2 VH, one CDR3 VH and one CDR2 VL; one CDR2 VH, one CDR2 VH and one VL CDR3; one CDR1 VH, one CDR1 VL and one CDR2 VL; a CDR1 VH, a CDR1 VL and a VL CDR3; one CDR2 VH, one CDR1 VL and one CDR2 VL; one CDR2 VH, one CDR1 VL and one VL CDR3; one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR3 VH, one CDR1 VL and one VL CDR3; one CDR1 VH, one CDR2 VH, one CDR3 VH and one CDR1 VL; a CDR1 VH, a CDR2 VH, a CDR3 VH and a CDR2 VL; a CDR1 VH, a CDR2 VH, a CDR3 VH and a VL CDR3; one CDR1 VH, one CDR2 VH, one CDR1 VL and one CDR2 VL; a CDR1 VH, a CDR2 VH, a CDR1 VL and a VL CDR3; one CDR1 VH, one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR1 VH, one CDR3 VH, one CDR1 VL and one VL CDR3; one CDR2 VH, one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR2 VH, one CDR3 VH, one CDR1 VL and one VL CDR3; one CDR2 VH, one CDR3 VH, one CDR2 VL and one VL CDR3; one CDR1 VH, one CDR2 VH, one CDR3 VH, one CDR1 VL and one CDR2 VL; one CDR1 VH, one CDR2 VH, one CDR3 VH, one CDR1 VL and one VL CDR3; one CDR1 VH, one CDR2 VH, one CDR1 VL, one CDR2 VL, and one VL CDR3; one CDR1 VH, one CDR3 VH, one CDR1 VL, one CDR2 VL, and one VL CDR3; one CDR2 VH, one CDR3 VH, one CDR1 VL, one CDR2 VL, and one CDR3 VL; or any combination thereof of the VH CDRs and VL CDRs of the CD3 antibodies described above.

In further advances, the invention provides EphA2-BiTEs derived from antibodies that immunospecifically bind CD3, characterized in that said antibodies may comprise a VH domain and a VL domain of a CD3 antibody described above.

In a specific advance, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to a CD3 polypeptide, which binding domain is derived from antibodies that immunospecifically bind to a CD3, characterized in that said antibodies have an association rate or kon (antibody (Ab) + antigen (Ag) -> Ab-Ag) rate constant of at least 104 M-1S-1, at least 105 M-1s-1, at least 1,5 X 105 M-1S-1, at least 2 X 105 M-1S-1, at least 2.5 X 105 M-1S-1, at least 5 X 105 M-1S-1, at least 106 M-1S- 1, at least 5 X 106 M-1s-1, at least 107 M-1s-1, at least 5 X 107 M-1s-1, or at least 108 M-1s-1, or in a range of about 105 to 108 M-1s-1, in a range of about 1.5 X 105 M-1s-1 to 1 X 107 M-1s-1, in a range of about 2 X 105 to 1 X 10 "6M -1S-1, or in a range of about 4.5 X 10 "5 to 10" 7M-1S-1. In certain advances, an antibody that immunospecifically binds to an EphA2 polypeptide may sui a kon of at least 10 "4 M-1s-1, at least 2 X 10" 5 M-1S-1, at least 2.5 X 10 "5M-1S-1, at least 5 X 10" 5M- 1S-1, at least 10 "6M-1S-1, at least 5 X 10" 6 M-1s-1, at least 10 "7 M-1S-1, at least 5 X 10" 7 M-1s-1 , or at least 10 8 M-1s-1 as determined by a surface plasmon resonance test.

In another specific advance, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to CD3, which binding domain is derived from antibodies that immunospecifically bind to a CD3 polypeptide, characterized in that said antibodies have a k0ff rate (antibody (Ab) + antigen (Ag) → Ab-Ag) of less than 10 "-3 s-1, less than 5 X 10" -3 s-1, less than 10 "-4 s" -1 less than 2 χ 10 "-4 s" -1, less than 5 X 10 "-4 s-1, less than 10" -5 s "-1, less than 5 X 10" -5 s "-1, less than 10 "-6 s" -1, less than 5 X 10 "-6 s" -1, less than 10 "-7 s" -1, less than 5 X 10 "-7 s" -1, less than 10 "-8 s" -1, less than 5 X 10 "-8 s" -1, less than 10 "-9 s" -1, less than 5 X 10 "-9 s" -1, or less than 10 "-10 s" -1, or in a range of about 10 "-3 to 10" -10 s "-1, in a range of about 10" -4 to 10 "-8 s" -1, or in a range of about 10 "-5 to 10" -8 s "-1. In certain advances, an antibody that immunospecifically binds to an EphA2 polypeptide has a koff of 10 "-2 s" -1, less than 5 X 10 "-3 s" -1, or less than 10 "-4 s "-1 as determined by a surface plasmon resonance test. In another specific advance, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to CD3, which binding domain is derived from antibodies that immunospecifically bind to a CD3 polypeptide, characterized in that said antibodies possess a affinity constant or Ka (kon / koff) of at least 102 M-1, at least 5 X 102 M-1, at least 103 M-1, at least 5 X 103 M-1, at least 104 M-1, at least 5 X 104 M-1, at least 105 M-1, at least 5 X 105 M-1, at least 106 M-1, at least 5 X 106 M-1, at least 107 M-1, at least 5 X 107 M-1, at least 108 M-1, at least 5 X 108 M-1, at least 109 M-1, at least 5 X 109 M-1, at least 1010 M-1, at least 5 X 1010 M-1, at least 1011 M-1, at least 5 X 1011 M-1, at least 1012 M-1, at least 5 X 1012 M-1, at least 1013 M-1, at least 5 X 1013 M -1, at least 1014 M-1, at least 5 X 1014 M-1, at least 1015 M-1, or at least 5 X 1015 M-1, or in a range of about 102 to 5 X 105 M-1, in a range of about 104 to 1 X 1010 M-1, or in a range of about 105 to 1 X 108 M-1 as determined by a surface plasmon resonance.

In another specific advance, the invention provides EphA2-BiTEs comprising a binding domain that immunospecifically binds to CD3, which binding domain is derived from antibodies that immunospecifically bind to a CD3 polypeptide, characterized in that said antibodies possess a dissociation constant or Kd (koff / kon) of less than 10 M, less than 5 X 10-5 M, less than 10ˉ6 Μ, less than 5 X 10ˉ6 Μ, less than 10ˉ7 Μ, less than 5 X 10ˉ7 M, less 10ˉ8 M, less than 5 X 10ˉ8 M, less than 10ˉ9 M, less than 5 X 10ˉ9 M, less than 10 X 10ˉ10 M, less than 5 X 10ˉ10 M, less than 5 X 10ˉ11 M, less than 10ˉ12 M, less than 5 X 10ˉ12 M, less than 10ˉ13 M, less than 5 X 10ˉ13 M, less than 10ˉ14 M, less than 5 X 10ˉ14 M, less than 10ˉ15 M, or less than 5 X 10ˉ15 M about 10ˉ2 M to 5 X 10ˉ5 M, in a range of about 10 "to 10ˉ15 M, or in a range of about 10ˉ8 to 10ˉ14 M, as determined by a resonance test surface plasmon INSTANCE.

In a specific advance, the invention provides EphA2-BiTEs that immunospecifically bind to a CD3 polypeptide, characterized in that said EphA2-BiTEs have an association rate or kon rate constant (antibody (Ab) + antigen (Ag) - Ab-Ag) of at least 104 Mˉ1sˉ1, at least 105 Mˉ1Sˉ1, at least 1.5 X 105 Mˉ1sˉl, at least 2 X 105Mˉ1S "ˉ1, at least 2.5 X 105Mˉ1Sˉ1, at least 5 X 105Mˉ1Sˉ 1, at least 106 Mˉ1Sˉ1, at least 5 X 106 Mˉ1Sˉ1, at least 107 Mˉ1sˉ1, at least 5 X 107 Mˉ1sˉ1, or at least 108 Mˉ1Sˉ1, or in a range of about 105 to 108 Mˉ1sˉ1, in a range of about 1.5 X 105 MˉV1 at 1 X 107 Mˉ1Sˉ1, in a range of about 2 X 105 to 1 X 106 Mˉ1sˉ1, or in a range of about 4.5 X 105 to 107Mˉ1Sˉ1. In certain advances, an EphA2-BiTE 25 that binds immunoespecif EphA2 polypeptide has a kon of at least 104 Mˉ1sˉ1, at least 2 X 105 Mˉ 1Sˉ1, at least 2.5 X 105 M-1s-1, at least 5 X 105 Mˉ1Sˉ1, at least 106 Mˉ1sˉ1, at least 5 X 106 Mˉ1sˉ1, at least 107 Mˉ1sˉ1, at least 5 X 107 Mˉ1Sˉ1, or at least 108 Mˉ1Sˉ1 as determined by a surface plasmon resonance test.

In another specific advance, the invention provides EphA2-BiTEs that immunospecifically bind to a CD3 polypeptide, characterized in that said EphA2-BiTEs have a kQff (antibody) (Ab) + antigen (Ag) - ^ Ab-Ag) rate. less than 10 "3 s-1, less than 5 X 10" s-, less and 10 "-4S-; less than 2x10" 1, less than 5 x 10 "-3s" -1, less than 10 "-4s "1 less than 5 x10" -4 less than 10 "-6s" -1, less than 5 x 10 "-6s-1, less than 10" -7 s-1, 5x 10 "-7 s-1, less 10 "-8s" -1 less than 5 x 10 "-8 s" -1, less than 10 "-9s" -1, less than 5 x 10 "-9 s" -1 or less and 10 "-10 or in a range of about 10 "13 to 10" -10 s'1, in a range of about 10 "-4a to 10" -8s "-1, or in a range of about 10 -5 to 10" 8 s "1. In certain advances, an EphA2-BiTE that immunospecifically binds to an EphA2 polypeptide has a kGff of 10 s "1, less than 5 X 10" 3s "-1 or less than 10" -4, as determined by a resonance test. of surface plasmon.

In another specific advance, the invention provides EphA2-BiTEs that immunospecifically bind to a CD3 polypeptide, characterized in that said EphA2-BiTEs have an affinity constant or Ka (k0n / k0ff) of at least 10 "2M-1 at least. 5 X 10 "2 M" 1, at least 10 "3M" 1, at least 5 X 10 3 M "1, at least 10 4 M" 1, at least 5 X 10 "4 M" 1, at least 10 "5 M" 1, at least 5 X10 "5M" 1, at least 10 "6M" 1, at least 5 X 10 "6M'1, at least 10" 7M "1, at least 5 X10" 7 M "1, at least 10" 8M "1, at least 5 X 10" 8 M "1, at least 10 9 M" 1, at least 5 X 10 9 M "1, at least 10 10 M" 1, at least 5 X 10 10 M "1, at least 10 11 M "1, at least 5 X 10 11 M" 1, at least 10 12 M "1, at least 5 X 10 12 M" 1, at least 10 13 M -1, at least 5 X 10 13 M "1, at least 10 14 M" 1 , at least 5 X 10 14 M "1, at least 10 15 M" 1, or at least 5 X 10 15 M "1, or in a range of about 10 2 to 5 X 10 5 M -1, in a range of about 10 4 at 1 X 10 10 M "1, or in a range of about 10 5 to 1 X 10 8 M" 1, with determined by a surface plasmon resonance test.

In another specific advancement, the invention provides EphA2-BiTEs that immunospecifically bind to a CD3 polypeptide, characterized in that said EphA2-BiTEs have a dissociation constant or Kd (k0ff / k0n) of less than 10 5 M minus 5 X 10-5 M, less than 10 "6 M, less than 5 X 10" 6 M, less than 10 "7 M, less than 5 X 10 ~ 7 M, less than 10" 8 M, less than 5 X 10 "8 M7 less than 10" 9 M, less than 5 X 10 10 "9 M, less than 10" 10 M, less than 5 X 10 "10 M, less than 10" 11 M, less than 5 X 10 " 11 M, less than 10 "12 M, less than 5 X 10" 12 M, less than 10 "13 M, less than 5 X 10" 13 M, less than 10 "14 M, less than 5 X 10" 14 M less than 10 15 M or less than 5 X 10 15 M or in a range of about 10 2 M to 5 X 10 5 M in a range of about 10 6 to 10 15 M , or in a range of about 10 "to 10" 14 M, as determined by a surface plasmon resonance test.

EphA2-BiTE Conjugates The present invention further relates to bispecific T-cell engagements (ie, EphA2-BiTEs (in particular, EphA2-BiTEs which are single-chain bispecific antibodies) comprising at least one additional domain, said domain being linked by linkers. covalent or non-covalent The additional domain may be of a predefined specificity or function Thus, the present invention relates to the use of recombinantly fused or chemically conjugated EphA2-BiTEs of the invention (including both covalent and non-covalent conjugations ) to a heterologous polypeptide (or fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. Fusion does not necessarily have to be direct, but can occur through s Linker sequences For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating antibodies to specific antibodies to particular cell surface receptors. See e.g., International Publication WO 93/21232; EP 439,095; Naramura et al., 1994, Immunol. Lett. 39: 91-99; U.S. Patent 5,474,981; Gillies et al., 1992, PNAS 89: 1428-1432; and Fell et al., 1991, J. Immunol. 146: 2446-2452, which are incorporated herein by reference in their entirety. The present invention further includes compositions comprising heterologous polypeptides fused or conjugated to EphA2-BiTEs fragments. For example, heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F (ab) 2 fragment, or fragment thereof. Methods for fusing or conjugating polypeptides to antibody fragments are known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP 307,434; EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, PNAS 88: 10535-10539; Zheng et al. , 1995, J. Immunol. 154: 5590-5600; and Vil et al. , 1992, PNAS 89: 11337-11341 (said references incorporated by reference in their entirety).

Additional fusion proteins, eg from any of the aforementioned EphA2-BiTEs, may be generated through gene shuffling, motif shuffling, exon scrambling, and / or codon shuffling techniques (collectively referred to as "DNA scrambling"). ). DNA shuffling may be employed to alter the antibody activities of the invention (e.g., antibodies with higher affinities or lower dissociation rates). See generally U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol. 8: 724-33; Harayama, 1998, Trends Biotechnol. 16:76; Hansson, et al., 1999, J. Mol. Biol. 287: 265; and Lorenzo and Blasco, 1998, BioTechniques 24: 308 (each of these patents and publications are incorporated herein by reference in their entirety). EphA2-BiTEs, or encoded EphA2-BiTEs, may be altered by random error-prone PCR mutagenesis, random nucleotide insertion, or other methods prior to recombination. One or more fragments of a polynucleotide encoding an antibody or antibody fragment, fragments which immunospecifically bind to EphA2 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

In addition, EphA2-BiTEs or fragments thereof may be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, several of which are commercially available. As described in Gentz et al., 1989, PNAS 86: 821, for example, hexahistidine provides for convenient purification of the fusion protein. Other useful peptide tags for purification include, but are not limited to, the haemagglutinin tag "HA", which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37: 767) and the "flag" tag. " In other advances, EphA2-BiTEs of the present invention or fragments or variants thereof are conjugated to a diagnostic or detectable agent. Such EphA2-BiTEs may be useful for monitoring or predicting the development or progression of a cancer as part of a clinical testing procedure, such as determining the effectiveness of a particular therapy. Additionally, such antibodies may be useful for monitoring or predicting the development or progression of a precancerous condition associated with EphA2 overexpressing cells (eg, high grade intraepithelial prostate neoplasia (NIP), fibrocystic breast disease fibroadenoma , or nevi compound). In one advance, an exposed EphA2 antibody epitope is conjugated to a diagnostic or detectable agent. In a more specific advance, the antibody is an EphA2-BiTE.

Such diagnosis and detection may be accomplished by combining the antibody with detectable substances including, but not limited to various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as, but not limited to, umbeliferone, fluorescein, fluorescein isothiocyte, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to luciferase, luciferin, and aequorin; radioactive materials such as, but not limited to, bismuth (213Bi), carbon (14C), chromium (51Cr), cobalt (57Co), fluorine (18F), gadolinium (153Gd, 159Gd), gallium (68Ga, 67Ga), germanium (68Ge), holmium (166Ho), indium (115In, 113In, 112In, 111In), iodine (131If 125I, 123I, 121I), lanthanum (140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo) , Palladium (103Pd), Phosphorus (32P), Praseodymium (142Pr), Promethium (149Pm), Rhenium (186Re, 188Re), Rhodium (105Rh), Ruthenium (97Ru), Samarium (153Sm), Scandium (47Sc), Selenium ( 75Se), Strontium (85Sr), Sulfur (35S), Technetium (99Tc), Titanium (201Ti), Tin (113Sn, 117Sn), Tritium (3H), Xenon (133Xe), Ytterbium (169Yb, 175Yb), Yttrium (90Y ), zinc (65 Zn); positron emitting metals using various positron emission tomographs, and paramagnetic non-radioactive metal ions.

In one advance, the location of an EphA2-BiTE to diseased tissue (s) (e.g., a tumor) can be determined by detecting a labeled form of EphA2-BiTE in the tissue. In a specific advance, a labeled EphA2-BiTE is detected in vivo in a subject according to a method comprising the steps of: (a) administering to the subject an effective amount of a labeled EphA2-BiTE, and (b) detecting the EphA2 - BiTE labeled in the subject after sufficient time to allow EphA2-BiTE to concentrate on sites in the subject where EphA2 is expressed. Accordingly, labeled EphA2-BiTE may be administered to the subject according to any method suitable in the art, for example parenterally or intraperitoneally. Also, according to this advance, the effective amount of labeled EphA2-BiTE is the amount that allows detection of EphA2-BiTE in the subject. This amount will vary depending on the subject in question, the label used, and the detection method employed. For example, it is understood in the art that the size of the subject and the imaging system used will determine the amount of labeled EphA2-BiTE required to detect EphA2-BiTE in a subject using imaging means. In the case of a radiolabelled EphA2-BiTE for a human, the amount of labeled EphA2-BiTE administered is measured in terms of radioactivity, for example from about 5 to 20 milicuries of 99tc. The time interval after administration of labeled EphA2-BiTE which is sufficient to allow labeled EphA2-BiTE to concentrate on sites in the subject where EphA2 is expressed will vary depending on a number of factors, for example the type of label used, mode of administration, and the body part of the subject from which the image is obtained. In a particular advance, the time interval that is sufficient is 6 to 48 hours, 6 to 24 hours, or 6 to 12 hours. In another advance, the time range is 5 to 20 days or 5 to 10 days.

The presence of a labeled EphA2-BiTE can be detected in the subject using imaging means known in the art. In general, the imaging media employed depends on the type of label used. Experts will be able to determine the appropriate means to detect a particular label. Methods and devices that may be used include, but are not limited to, computed tomography (CT), whole body scanner, such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In a specific advance, EphA2-BiTE is labeled with a radioisotope and is detected in the patient using a radiation response surgical instrument (Thurston et al., U.S. Patent No. 5,441,050). In another advance, EphA2-BiTE is labeled with a fluorescent compound and is detected in the patient using a fluorescence response scanner. In another advance, EphA2-BiTE is labeled with a positron emitting metal and is detected in the patient using positron emission tomography. In yet another advance, EphA2-BiTE is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

The present invention further encompasses uses of EphA2-BiTEs or fragments thereof conjugated to a therapeutic agent.

An EphA2-BiTE of the invention may be conjugated to a non-macromolecular therapeutic component, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromet, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycinine dehydroneprocaine, actinomycinine, , tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and the like or homologues thereof. Therapeutic Agents

include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thioepa chlorambucil, melfalan, carmustine (BCNU) and lomustine (CCNU) ), cyclotosfamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly

daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and antramycin (AMC)), and anti-mitotic agents (e.g. vincristine and vinblastine).

In addition, an EphA2-BiTE may be conjugated to a therapeutic agent or drug arrangement that modifies a given biological response. Therapeutic agents or drug arrangements should not be construed as limited to classical chemical therapeutic agents. For example, the drug arrangement may be a protein or polypeptide having a desired biological activity. Such proteins may

include, for example, a toxin such as abrina, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein, such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, plasminogen tissue activator, an apoptotic agent, eg, TNF-a, TNF-β, AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., 1994, J. Iminunol., 6: 1567), and VEGI (see, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, eg angiostatin or endostatin; or a biological response modifier such as, for example, a lymphokine (eg, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin -6 ("IL-6" ), macrophage colony granulocyte stimulating factor ("GM-CSF"), and colony granulocyte stimulating factor ("G-CSF")), or a growth factor (eg, growth hormone ("GH") ).

In addition, an EphA2-BiTE may be conjugated to therapeutic components, such as radioactive materials or macrocyclic chelators useful for conjugating radiomethion ions (for examples of radioactive materials, see above). In certain advances, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N, N ', N' ', N' '- tetraacetic acid (DOTA) which may be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4: 2483-90; Peterson et al., 1999, Bioconjug. Chem. 10: 553; and Zimmerman et al. , 1999, Nucl. Med. Biol. 26: 943-50 each is incorporated by reference in its entirety. In a specific advance, the conjugated EphA2-BiTE comprises a domain that preferentially binds an exposed EphA2 epitope on cancer cells, but not on non-cancer cells, or on infected cells, but not on uninfected cells (ie, epitope antibody). EphA2 exposed), and a second domain which preferentially binds CD3.

Techniques for conjugating therapeutic components to antibodies are well known. Moieties may be conjugated to antibodies by any method known in the art, including, but not limited to, aldehyde / Schiff binding, sulfhydryl binding, acid-labile binding, cis-aconityl binding, hydrazone binding, enzymatically degradable binding (see generally, Garnett, 2002, Adv. Drug Deliv. Rev. 53: 171-216). Additional techniques for conjugating therapeutic components to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Mareei Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies λ84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); Analysis, Results, And Future Prospective Of The 132/346

Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, "In Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev 62: 119-58 Methods for fusing or conjugating antibodies to polypeptide components are known in the art See, eg, US Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,944; EP 307,434; EP 3 67,166; International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, PNAS 88: 10535-10539; Zheng et al., 1995, J 154: 5590-5600, and Vil et al., 1992, PNAS 89: 11337-11341. The fusion of an antibody to a component need not necessarily be direct, but may occur through linker sequences. are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4: 2483-90; Peterson et al., 1999, Bioconjug. Chem. 10: 553; Zimmerman et al., 1999, Nucl. Med Biol 26:94 3-50; Garnett, 2002, Adv. Drug Deliv. Rev. 53: 171-216, each of which is incorporated herein by reference in its entirety.

EphA2-BiTEs may also be attached to solid supports, which are particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

EphA2-BiTE Polynucleotides of the Invention The present invention provides the polynucleotide sequences encoding the EphA2-BiTES described herein. In a specific advance, the EphA2-BiTEs-encoding polynucleotides of the invention comprise a first nucleotide sequence encoding a first binding domain and a second nucleotide sequence encoding a second binding domain, and nucleotide sequences encoding a ligand sequence that binds the first and second connection domains. See, e.g., FIG. 3 for a general representation of an EphA2-BiTE polynucleotide construct. The present invention also provides EphA2-BiTEs encoding polynucleotide sequences in which nucleotide sequences encoding the first and / or second binding domain hybridize to the nucleotide sequences of one or more variable domains of an anti-EphA2 antibody known in the art or herein. described (eg, EA2, 4H5, 2A4, 2E7 or 12E2) and / or an anti-CD3 antibody known in the art or described herein.

In a breakthrough, EphA2-BiTEs produced from polynucleotides that hybridize to EphA2-BiTEs encoding polynucleotides that modulate EphA2 expression and / or induce redirected lysis of T-overexpressing cells in a test well known in the art or described herein. In another advance, EphA2-BiTEs used in the methods of the invention include polypeptides made from polynucleotides that hybridize to polynucleotides encoding EphA2-BiTEs fragments. Hybridization conditions include, but are not limited to, stringent hybridization conditions such as filter-linked DNA hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washings in 0.2X SSC / 0.1% SDS at about 50-65 ° C, highly stringent conditions such as 6X SSC filter-bound DNA hybridization at about 45 ° C, followed by one or more washes at 0 ° C, 1X SSC / 0.2% SDS at about 60 ° C, or any other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, FM et al., Eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY on pages 6.3.1 to 6.3.6 and 2.10.3).

Since the EphA2-BiTE nucleotide sequence used in the methods of the invention is determined, the nucleotide sequence can be engineered using methods well known in the art for nucleotide sequence manipulation, eg, recombinant DNA techniques, site directed mutagenesis. , PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated herein by reference in their entirety), to generate polypeptides having a different amino acid sequence, for example to create amino acid substitutions, deletions, and / or insertions. In specific advances, such polypeptides have at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 amino acid substitutions, deletions, and / or insertions. Possible substitutions include conservative substitutions, in which the amino acid sequence is modified by substituting one or more amino acids with different amino acids that have similar chemical characteristics or structures and / or do not significantly alter the biological function of the peptide.

Standard techniques known to those skilled in the art may be used to introduce mutations into the nucleotide sequence encoding an EphA2-BiTE of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that results in amino acid substitutions. Preferably, the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. In a preferred embodiment, the derivatives having conservative amino acid substitutions are made on one or more predicted nonessential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similarly charged side chain. Families of amino acid residues having similarly charged side chains have been defined in the art. These families include

amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), chains apolar sideways (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched polar side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded EphA2-BiTE can be inserted into an expression vector and expressed (e.g., in a heterologous host cell) and protein activity can be determined as described below.

The present invention also encompasses the use of single chain bispecific antibodies comprising the amino acid sequence of any of the EphA2-BiTEs described herein with mutations (eg, one or more amino acid substitutions) in the first or second domain tool or variable regions binding.

Preferably, such mutations maintain or enhance the avidity and / or affinity of the EphA2-BiTEs for EphA2 and / or CD3 to which they immunospecifically bind. Standard techniques known to those skilled in the art (e.g., immunoassay or ELISA assays) can be used to test the degree of binding between an EphA2-BiTE polypeptide and its binding partner.

Methods of Producing EphA2-BiTEs

Recombinant Expression of an EphA2-BiTE

The EphA2-BiTEs of the invention may be produced by any method known in the art or described herein, in particular by chemical synthesis or preferably by recombinant expression techniques described above (see, eg, WO 99/54440, which is incorporated herein by reference in its entirety). See also Section 6, infra, for detailed examples of methods for producing EphA2-BiTEs of the invention.

The polynucleotides of the invention may be used alone or as part of a vector to express the polypeptides of the invention (eg, EphA2-BiTEs) in cells, for example, in gene therapy or diagnosis of disorders associated with aberrant expression (eg, overexpression). ) and / or EphA2 activity (eg, cancer, a non-cancerous hyperproliferative cell disorder or an infection). Polynucleotides or vectors containing the DNA sequence (s) encoding any of the polypeptides of the invention are introduced into cells which, in contrast, produce the polypeptide of interest (e.g., an EphA2-BiTE). Gene therapy, which is based on introducing therapeutic genes into cells by ex vivo or in vivo techniques, is one of the most important gene transfer applications.

Thus, recombinant expression of an EphA2-BiTE of the invention requires the construction of an expression vector containing a polynucleotide sequence encoding the EphA2-BiTE. See, e.g., FIG. 3. Polynucleotides encoding the EphA2-BiTEs described herein may be obtained and sequenced by any method known in the art. For example, a polynucleotide encoding an EphA2-BiTE using in the methods of the invention may be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier et al., 1994, BioTechniques 17: 242), which briefly involves synthesis. of overlapping oligonucleotides containing sequence fragments encoding the polypeptide, annealing and ligating such oligonucleotides, and then amplifying the PCR-linked oligonucleotides.

Once a polynucleotide encoding an EphA2-BiTE of the invention has been obtained, the vector for producing the EphA2-BiTE molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein expressing a polynucleotide containing a nucleotide sequence encoding EphA2-BiTE are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing EphA2-BiTE coding sequences and appropriate transcriptional control and translation signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention thus provides replicable vectors comprising a nucleotide sequence encoding an EphA2-BiTE of the invention, an antibody heavy or light chain, an antibody light or heavy chain variable domain or fragment thereof, or a heavy chain or mild CDR operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, eg, International Publication Nos. WO 86/05807 and WO 89/01036; and US Patent No. 5,122,464) and the antibody variable domain. they may be cloned into such a vector for expression of the entire heavy chain, whole light chain, or both whole heavy and light chains.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an EphA2-BiTE of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an EphA2-BiTE of the invention, fragments thereof (e.g., first and / or second binding domains of an EphA2-BiTE) operably linked to a heterologous promoter.

A variety of host-vector expression systems may be used to express the EphA2-BiTEs of the invention or fragments thereof (eg, a first and / or second binding domain of an EphA2-BiTE) (see, eg, US Patent No. 5,807,715). Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the appropriate coding nucleotide sequences, express an EphA2-BiTE molecule of the invention. in situ These include, but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA expression vectors, or cosmid DNA containing EphA2-BiTE coding sequences; yeasts (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing EphA2-BiTE coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., bacillovirus) containing EphA2-BiTE coding sequences; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing sequences EphA2-BiTE coders; or mammalian cell systems (eg, COS, CHO, BHK, 293, NSO, and 3T3 cells) anchoring recombinant expression constructs containing promoters derived from the mammalian cell genome (eg, metallothionein promoter) or mammalian virus ( eg, late adenovirus promoter; vaccinia virus promoter 7.5K). Preferably, bacterial cells, such as Escherichia coli, and more preferably eukaryotic cells, are used for expression of a recombinant EphA2-BiTE molecule. For example, cells of

mammals, such as Chinese hamster ovary (CHO) cells, together with a vector, such as the primary intermediate human cytomegalovirus early gene promoter element is an effective antibody expression system (Foecking et al., 1986, Gene 45: 101; and Cockett et al., 1990, BioTechnology 8: 2). In a specific advance, the expression of nucleotide sequences encoding EphA2-BiTEs is regulated by a constitutive promoter, induced promoter or tissue specific promoter. Alternatively, the polynucleotides of the invention may be expressed in a transgenic plant expression system, such as, for example, the LEX System ™ system described in US Patent No. 6,040,498, international application published February 18, 1999 (WO 99/07210), and international application published February 7, 2002 (WO 02/10414). Another transgenic plant expression system that can be used for the polynucleotides of the invention is the Plantibodiesm technology described in U.S. Patent Nos. 5,202,422; 5,639,947; 5,959,177; and 6,417,429.

In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use of the EphA2-BiTE molecule or fragment thereof being expressed. For example, when a large amount of such a protein must be produced, for the generation of pharmaceutical compositions of an EphA2-BiTE molecule, vectors that direct the expression of high levels of readily purified fusion protein products may be desired. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12: 1791), in which the EphA2-BiTE coding sequence can be individually linked to the vector in the frame. with the coding region Iac Z such that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24: 5503-5509); and the like. PGEX vectors can also be used to express foreign polypeptides as glutathione 5-transferase (GST) fusion proteins. In general, such fusion proteins are soluble and can be easily purified from lysed cells by glutathione-agarose matrix adsorption and binding followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST component. Another microbial system that can be used to express the polynucleotides of the invention is Pfex Expression technology which is based on new Pseudomonas fluorescens strains, as described in Squires et al., BioProcess Int'l p. December 54, 2004; and Squires et al., Specialty Chemicals July / August 2004.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The EphA2-BiTE coding sequence can be individually cloned into nonessential regions (e.g. the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g. the polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems may be used. In cases where an adenovirus is used as an expression vector, the EphA2-BiTE coding sequence of interest may be linked to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartitioned leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg, E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the EphA2-BiTE molecule in infected hosts (eg, see Logan & Shenk, 1984, PNAS 8). 1: 355-359). Specific initiation signals may also be required for efficient translation of inserted EphA2-BiTE coding sequences. These signals include the ATG initiation codon and adjacent sequences. Additionally, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of all insertion. These exogenous translation control signals and initiation codons can be from a variety of sources, both natural and synthetic. Expression efficiency can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153: 516-544).

In addition, a host cell line that modulates expression of inserted sequences, or modifies and processes the gene product in the desired specific manner may be chosen. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems may be chosen to ensure correct modification and processing of the expressed foreign protein. To this extent, eukaryotic host cells that have the cellular machinery for correct processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20, NS1, and T47D, NSO (one cell line) cells. murine myeloma that does not endogenously produce any immunoglobulin chain), CRL7030 and HsS78BsT cells.

For high yield and long term production of recombinant proteins, stable expression is preferred. For example, cell lines stably expressing the EphA2-BiTE molecule may be constructed. Instead of using expression vectors that contain sources. In viral replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, constructed cells can be grown for 1-2 days in an enriched medium and then exchanged for a selective medium. The selectable marker on recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form Ioci which, in contrast, can be cloned and expanded into cell lines. This method can be advantageously used to construct cell lines expressing the EphA2-BiTE molecule. Such constructed cell lines may be particularly useful in screening and evaluating compositions that interact directly or indirectly with the EphA2-BiTE molecule.

A number of selection systems may be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), glutamine synthase, hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992). , Proc. Natl. Acad. Sci. USA 48: 202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 8-17) genes can be employed in tk-, gs-, hgprt- or aprt- respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers methotrexate resistance (Wigler et al., 1980, PNAS 77: 357; O'Hare et al., 1981, PNAS 78: 1527 ); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, PNAS 78: 2072); neo, which confers resistance to aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573; Mulligan, 1993, Science 260: 926; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191; May, 1993, TIB TECH 11: 155-); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in chapters 12 and 13, Dracopoli et al. (eds), Current

Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1, which are hereby incorporated by reference in their entirety.

Expression levels of an EphA2-BiTE molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in DNA cloning, Vol.3 (Academic Press, New York, 1987)). When a marker in the EphA2-BiTE expressing vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the EphA2-BiTE gene, EphA2-BiTE production will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).

The host cell can be co-transfected with two expression vectors of the invention, the first vector encoding a first binding domain of an EphA2-BiTE or an antibody variable heavy domain (eg, an anti-EphA2 antibody or an anti-EphA2 antibody). -CD3) and the second vector encoding a second binding domain of an EphA2-BiTE or a light variable domain of an antibody (eg, an anti-EphA2 antibody or an anti-CD3 antibody). The first and second EphA2-BiTE binding domains may be purified using techniques known in the art and the two domains may be chemically linked by methods known in the art.

The host cell may be transfected with an expression vector of the invention encoding an EphA2-BiTE of the invention. The vector may contain a single vector that encodes, and is capable of expressing either the light variable domains of an antibody (eg, anti-EphA2 antibody or anti-CD3 antibody), or both first and second binding domains of an EphA2-BiTE. (Proudfoot, 1986, Nature 322: 52; and Kohler, 1980, PNAS 77: 2197). The coding sequences for the variable domains or binding domains may comprise cDNA or genomic DNA.

Once an EphA2-BiTE of the invention or a binding domain thereof has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example by chromatography (eg, ion exchange). , ion affinity, particularly by affinity for specific antigen after Protein A, and measuring column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Further, the EphA2-BiTEs of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or, conversely, known in the art to facilitate purification. In a specific advance, recombinantly produced EphA2-BiTEs of the invention comprise a first binding domain and a second binding domain, characterized in that the first binding domain comprises a VH domain and a VL domain joined together by a ligand. sufficient strength to allow the domains to fold to allow binding to the T cell CD3 antigen. In another specific advance, the second binding domain comprises the VH domain and a VL domain, and the VH and VL domains are linked together. by a binder of sufficient strength to allow the domains to bend to allow binding to EphA2. In another specific advance, the first and second binding domains are joined together by a ligand of sufficient strength to allow the domains to bend to allow binding to CD3 and EphA2. In certain advances, the first and second binding domains are scFvs. In other advances, the binding domain that binds to CD3 is unimmunized.

Prophylactic / Therapeutic Methods

The present invention relates to pharmaceutical compositions and prophylactic and therapeutic regimens designed to treat, prevent and / or manage disorders associated with aberrant EphA2 expression and / or activity (eg, cancer, non-cancerous hyperproliferative cell disorders and infections) and a subject. comprising administering one or more EphA2-BiTEs. In a specific advance, the EphA2-BiTEs of the invention are administered to a subject to treat, prevent and / or manage disorders associated with aberrant EphA2 expression and / or activity (e.g., cancer, non-cancerous hyperproliferative cell disorders and infections). In certain advances, the EphA2-BiTEs of the invention are administered in combination with one or more therapies. In certain advances, one or more EphA2-BiTEs of the invention are administered to a mammal, preferably a human, concurrently with one or more other therapies. Preferably, such therapies are useful for treating such disorders. The term "concurrently" is not limited to administering therapies at exactly the same time, but rather should mean that the EphA2-BiTEs of the invention and other therapy are administered to a subject in a sequence and at a time interval such that antibodies of the invention may act together with the other therapy to provide an increased benefit over being administered otherwise. For example, each therapy may be administered at the same time or sequentially in any order at different times; however, if not administered at the same time, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect. Each therapy may be administered separately, in any appropriate manner and by any suitable route. In other advances, the EphA2-BiTEs of the invention are administered before, concomitantly or after surgery. Preferably, surgery completely removes localized tumors or reduces the size of large tumors. Surgery can also be done as a preventative measure or to relieve pain.

In several advances, therapies are given in less than 1 hour of! about 1 hour apart, from about 1 hour to about 2 hours apart, from about 2 hours to about 3 hours apart, from about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, from about 5 hours to about 6 hours apart, from about 6 hours to about 7 hours apart, from about 7 hours to about 8 hours apart , from about 8 hours to about 9 hours apart, from about 9 hours to about 10 hours apart, from about 10 hours to about 11 hours apart, from about 11 hours to about 12 hours no more than 24 hours apart or no more than 48 hours apart. In preferred advances, two or more components are administered at the same patient visit.

The dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. Dosage and frequency will also typically vary according to patient-specific factors depending on the specific therapies administered, the severity and type of cancer, the route of administration, as well as the patient's age, body weight, response, and medical history. . Suitable regimens may be selected by one of skill in the art considering such factors and following, for example, dosages reported in the literature and recommended in the Physicians Desk Desk Reference (61st ed., 2007).

Patient Population

Cancer patients

The invention provides methods for treating, preventing and / or managing cancer by administering to a subject a therapeutically or prophylactically effective amount of one or more EphA2-BiTEs of the invention. In another advance, the EphA2-BiTEs of the invention may be administered in combination with one or more other therapies. The subject is an animal, preferably a mammal, such as a non-primate (eg, cows, pigs, horses, cats, dogs, mice, etc.) and a primate (eg, monkey, such as a cinomolgus monkey and a human). . In a preferred advance, the subject is a human.

Specific examples of cancers that may be treated by the methods encompassed by the invention include, but are not limited to, cancers that overexpress EphA2. In a further advance, the cancer is of epithelial origin. Examples of such cancers are lung, colon, prostate, breast, and skin cancer. Additional cancers are set forth as an example and not by limitation in Section 5.4.1.2 below. In particular advances, methods of the invention may be used to treat, prevent and / or manage metastasis of primary tumors.

The methods and compositions of the invention comprise administering one or more EphA2-BiTEs of the invention to subjects / patients suffering from or expected to have cancer, eg, have a genetic predisposition to a particular type of cancer, have been exposed to carcerigen, or are in remission of a particular cancer. As used herein, "cancer" refers to primary or metastatic cancers. Such patients may or may not have been previously treated for cancer. The methods and compositions of the invention may be used as any line of therapy, e.g., a first, second, third, etc. therapy line. Also included in the invention is the treatment of patients receiving other cancer therapies and the methods and compositions of the invention may be used before adverse effects or intolerance of such other cancer therapies occurs. The invention also encompasses methods for administering one or more EphA2-BiTEs of the invention to treat, prevent and / or manage symptoms in refractory patients. In a certain advance, cancer being refractory to therapy means that at least some significant portion of the cancer cells are not killed or imprisoned. Determination of whether cancer cells are refractory can be done either in vivo or in vitro by any method known in the art to test the effectiveness of cancer cell therapy (s) using means accepted in the art of "refractory" in such a context. In several advances, a cancer is refractory where the number of cancer cells has not been significantly reduced or increased. The invention also encompasses methods for administering one or more EphA2-BiTEs to prevent cancer onset or recurrence in cancer predisposed patients.

In particular advances, the EphA2-BiTEs of the invention, or other therapeutics that reduce EphA2 expression, are administered to reverse the resistance or reduced sensitivity of cancer cells to certain hormone therapies, radiation and chemical therapeutics, thereby again sensitizing. cancer cells to one or more of these agents, which may then be administered (or continue to be administered) to treat or administer cancer, including preventing metastasis.

In alternating advances, the invention provides methods for treating cancer of patients by administering one or more EphA2-BiTEs of the invention in combination with any other therapy or to patients who have been shown to be refractory to other treatments but not to these treatments. In certain advances, patients being treated by the methods of the invention are patients already being treated with chemotherapy, radiotherapy, hormone therapy, or biological therapy / immunotherapy. These patients include refractory patients and those with cancer despite treatment with existing cancer therapies. In other advances, patients have been treated and have no disease activity and one or more EphA2-BiTEs of the invention are administered to prevent cancer recurrence.

In preferred advances, the existing treatment is chemotherapy. In particular advances, existing treatment includes administration of chemotherapies including, but not limited to, methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposide , bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, etc. These patients include patients treated with radiotherapy, hormone therapy and / or biological therapy / immunotherapy. Also among these patients are those who underwent surgery to treat cancer.

Alternatively, the invention also encompasses methods for treating patients receiving or receiving radiotherapy. These include patients being treated or previously treated with chemotherapy, hormone therapy and / or biological therapy / immunotherapy. Also among these patients are those who underwent surgery to treat cancer. In further advances, the invention encompasses methods for treating patients on or who have had hormonal therapy and / or biological therapy / immunotherapy. These include patients being treated or treated with chemotherapy and / or radiotherapy. Also among these patients are those who underwent surgery to treat cancer.

Additionally, the invention also provides methods of treating cancer as an alternative to chemotherapy, radiotherapy, hormone therapy, and / or therapy.

Biological / immunotherapy where therapy has been shown or may be very toxic, i.e., results in unacceptable or unbearable side effects for the subject being treated. The subject being treated with the methods of the invention may optionally be treated with other cancer treatments, such as surgery, chemotherapy, radiotherapy, hormone therapy or biological therapy, depending on which treatment has been found to be unacceptable or unbearable.

In further advances, the invention provides for the administration of one or more EphA2-BiTEs of the invention without any other cancer therapies for treating cancer, but has been shown to be refractory to such treatments. In specific advances, patients refractory to other cancer therapies are administered with one or more EphA2-BiTEs of the invention in the absence of cancer therapies. In other advances, patients with a precancerous condition associated with EphA2 overexpressing cells may be administered the EphA2-BiTEs of the invention to treat the disorder and decrease the likelihood that the disorder will progress to malignant cancer. In specific advances, the precancerous condition is high-grade prostate intraepithelial neoplasia (NIP), breast fibroadenoma, fibrocystic disease, or nevi compound.

Cancer

Reported cancers and disorders that can be treated, prevented and / or managed by the methods and compositions of the present invention include, but are not limited to: leukemias, such as, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia and myelodysplastic syndrome; chronic leukemia such as, but not limited to, chronic (granulocytic) myelocytic leukemia, chronic lymphocytic leukemia, hair cell leukemia; polycythemia vera; lymphomas, such as, but not limited to Hodgkin's syndrome, non-Hodgkin's lymphoma; multiple myelomas, such as but not limited to latent multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacitoma, and extramedullary plasmacitoma; Waldenström macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, giant cell malignant tumor, bone fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemoma, rhabdomyosarcoma, synuvial sarcoma; brain tumors, such as, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary cerebral lymphoma; breast cancer including, but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, spinal cord cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer ; adrenal cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, such as, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as, but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulva cancer, such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers, such as, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers, such as, but not limited to, squamous cancer, adenocarcinoma, cystic adenoid carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacitoma, verrucous carcinoma, and oat (small cell) carcinoma; stomach cancers, such as, but not limited to, adenocarcinoma, fungus (polypoid), ulcerant, superficial dispersion, diffuse spread, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as, but not limited to, hepatocellular carcinoma and hepatoblastoma; gallbladder cancers, such as adenocarcinoma; cholangiocarcinomas, such as, but not limited to, papillary, nodular, and diffuse; lung cancers, such as non-small cell lung cancer, squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; testicular cancers such as, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonic carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor), prostate cancers such as, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; criminal cancers; oral cancers, such as, but not limited to, squamous cell carcinoma; basal cancers; salivary gland cancers such as, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenocystic carcinoma; pharyngeal cancers, such as, but not limited to, squamous cell, and warty cancer; skin cancers, such as, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial dispersion melanoma, nodular melanoma, lentigo malignant melanoma, lentiginous acral melanoma; kidney cancers, such as, but not limited to, renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterer); Wilms tumor; bladder cancers such as, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchiogenic carcinoma, sweat carcinoma of the papillary gland, papillary adenocarcinoma, carcinoma of the papillary gland , see Fishraan et al., 1985, Medicine, 2nd Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovering Viking Penguin, Penguin Books USA, Inc., United States of America).

Accordingly, the methods and compositions of the invention are also useful in the treatment, prevention and / or management of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, ovarian lung, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoactantoma, seminoma, thyroid follicular cancer and teratocarcinoma. It is further contemplated that cancers caused by apoptotic aberrations could also be treated by the methods and compositions of the invention. Such cancers may include, but are not limited to, follicular lymphomas, p53 mutated carcinomas, tumors dependent on breast, prostate, and ovarian hormones, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In specific advances, changes in malignancy or deproliferation (such as metaplasias and dysplasias), or hyperproliferative cell disorders, are treated or prevented in the skin, lung, colon, breast, prostate, bladder, kidney, pancreas, ovary, or uterus. In other specific advances, sarcoma, melanoma, or leukemia are treated or prevented.

In specific advances, cancers to be treated, prevented and / or managed by the methods and compositions of the invention are of epithelial origin. In other advances, cancer is malignant and over-expressed EphA2. In a specific advance, cancer comprises aberrantly expressing EphA2 cells. In other advances, the disorder being treated is a precancerous condition associated with cells that overexpress EphA2. In one specific advance, the precancerous condition is high-grade prostate intraepithelial neoplasia (NIP), breast fibroadenoma, fibrocystic disease, or nevi compound.

In specific advances, the methods and compositions of the invention are used for the treatment, prevention and / or management of breast, colon, ovarian, lung, and prostate cancers and skin cancer, such as melanoma. Breast Cancer Treatment

In specific advances, breast cancer patients are administered an effective amount of one or more EphA2-BiTEs of the invention. In a further advance, the EphA2-BiTEs of the invention may be administered in combination with an effective amount of one or more other agents useful for breast cancer therapy including, but not limited to: doxorubicin, epirubicin, the combination of doxorubicin and cyclophosphamide ( AC), the combination of cyclophosphamide, doxorubicin and 5-fluorouracil (CAF), the combination of cyclophosphamide, epirubicin and 5-fluorouracil (CEF), herceptin, tamoxifen, the combination of tamoxifen and cytotoxic chemotherapy, taxanes (such as docetaxel and paclitaxel) ). In a further advance, EphA2-BiTEs of the invention may be administered with taxanes plus standard doxorubicin and cyclophosphamide for treatment with node-positive localized breast cancer adjuvant.

In a specific advance, patients with precancerous breast fibroadenoma or fibrocystic disease are administered an EphA2-BiTE of the invention to treat the disorder and decrease the likelihood that it will progress to malignant breast cancer.

Treatment of Colon Cancer In specific advances, patients with colon cancer are administered an effective amount of one or more EphA2-BiTEs of the invention. In another advance, the antibodies of the invention may be administered in combination with an effective amount of one or more other agents useful for colon cancer therapy, including, but not limited to: the combination of 5-FU and leucovorin, the combination of 5-FU and levamisole, irinotecan (CPT-11) or the combination of irinotecan, 5-FU and leucovorin (IFL).

Prostate Cancer Treatment

In specific advances, prostate cancer patients are administered an effective amount of one or more EphA2-BiTEs of the invention. In a further advance, the EphA2-BiTEs of the invention may be administered in combination with an effective amount of one or more other agents useful for prostate cancer therapy including, but not limited to: external beam radiotherapy, interstitial implantation of radioisotopes (ie. , I125, palladium, iridium), leuprolide or other LHRH agonists, non-steroidal antiandrogens (flutamide, nilutamide, bicalutamide), steroidal antiandrogens (cyproterone acetate), the combination of leuprolide and flutamide, estrogens such as DES, chlorothrin estrogen, , USP conjugated estrogens, DES-diphosphate, radioisotopes such as strontium-89, the combination of external beam radiotherapy and strontium-89, second-line hormone therapies such as aminoglutethimide, hydrocortisone, flutamide withdrawal, progesterone, and ketoconazole , low dose prednisone, or other chemotherapy regimens reported to produce subjective improvement symptoms and reduction in PSA level, including docetaxel, paclitaxel, estramustine / docetaxel, estramustine / etoposide, estramustine / vinblastine, and estramustine / paclitaxel.

In a specific advance, patients with high grade precancerous intraepithelial prostatic neoplasia (NIP) are administered an EphA2-BiTE of the invention to treat the disorder and decrease the likelihood that this will progress to malignant prostate cancer.

Melanoma Treatment

In specific advances, melanoma patients are administered an effective amount of one or more EphA2-BiTEs of the invention. In another advancement, the EphA2-BiTEs of the invention may be administered in combination with an effective amount of one or more other agents useful for melanoma cancer therapy including, but not limited to: dacarbazine (DTIC), nitrosoureas such as carmustine ( BCNU) and lomustine (CCNU), agents with modest single-agent activity, including vinca alkaloids, platinum compounds, and taxanes, the Dartmouth regimen (cisplatin, BCNU, and DTIC), alpha-interferon (IFN-A), and interleukin -2 (IL-2). In a specific advance, an effective amount of one or more EphA2-BiTEs of the invention may be administered in combination with melphalan alone (L-PAM) isolated hyperthermic limb perfusion (ILP), with or without tumor necrosis factor alpha (TNF-α). alpha) to patients with multiple brain metastases, bone metastases, and spinal cord compression to achieve symptom relief and some shrinkage of the tumor with radiotherapy.

In a specific advance, patients with precancerous nevi compound are administered an EphA2-BiTE of the invention to treat the disorder and decrease the likelihood that it will progress to malignant melanoma.

Ovarian Cancer Treatment

In specific advances, ovarian cancer patients are administered an effective amount of one or more EphA2-BiTEs of the invention. In another advancement, the EphA2-BiTEs of the invention may be administered in combination with an effective amount of one or more other agents useful for ovarian cancer therapy including, but not limited to: intraperitoneal radiotherapy, such as P32 therapy, abdominal radiotherapy. and total pelvic, cisplatin, the combination of paclitaxel (Taxol) or docetaxel (Taxotere) and cisplatin or carboplatin, the combination of cyclophosphamide and cisplatin, the combination of cyclophosphamide and carboplatin, the combination of 5-FU and leucovorin, etoposide, doxorubicin liposomal , gemcitabine or topotecan. It is contemplated that an effective amount of one or more EphA2-BiTEs of the invention is administered in combination with administration of Taxol to patients with platinum refractory disease. Included are the treatment of refractory ovarian cancer patients including administration of: ifosfamide in patients with platinum-refractory disease, hexamethylmelamine (HMM) as recovery chemotherapy following failure of platinum-based combination regimens, and tamoxifen in patients. with detectable levels of cytoplasmic estrogen receptor in their tumors.

Lung Cancer Treatment

In specific advances, patients with small cell lung cancer are administered an effective amount of one or more EphA2-BiTEs of the invention. In another advance, the EphA2-BiTEs of the invention may be administered in combination with an effective amount of one or more other agents useful for lung cancer therapy including, but not limited to: thoracic radiotherapy, cisplatin, vincristine, doxorubicin, and etoposide. , alone or in combination, the combination of cyclophosphamide, doxorubicin, vincristine / etoposide, and cisplatin (CAV / EP), local mitigation with endobronchial laser therapy, endobronchial stents, and / or brachytherapy.

In other specific advances, non-small cell lung cancer patients are administered an effective amount of one or more EphA2-BiTEs of the invention in combination with an effective amount of one or more other useful agents for lung cancer therapy including but not limited to: palliative radiotherapy, the combination of cisplatin, vinblastine and mitomycin, the combination of cisplatin and vinorelbine, paclitaxel, docetaxel or gemcitabine, the combination of carboplatin and paclitaxel, interstitial radiotherapy for endobronchial lesions or stereotactic radiosurgery.

Other Prophylactic / Therapeutic Agents

In some advances, therapy by administering one or more EphA2-BiTEs of the invention is combined with administration of one or more therapies, such as, but not limited to, chemotherapies, radiotherapies, hormonal therapies, and / or biological therapies / immunotherapies. . Prophylactic or therapeutic agents include, but are not limited to, protein molecules, including, but not limited to, peptides, polypeptides, proteins, including post-translationally modified proteins, antibodies, etc .; or small molecules (less than 1000 Daltons), inorganic or organic compounds; or nucleic acid molecules including, but not limited to, double stranded or single stranded DNA, or double stranded or single stranded RNA, as well as triple helix nucleic acid molecules. Prophylactic or therapeutic agents may be derived from any known organism (including, but not limited to, animals, plants, bacteria, fungi, and protists, or viruses) or a library of synthetic molecules. Such therapies may be administered before, concomitantly, or after administration of one or more EphA2-BiTEs of the invention.

In a specific advance, the methods of the invention encompass administration of an EphA2-BiTE of the invention in combination with administration of one or more prophylactic / therapeutic agents that are kinase inhibitors such as, but not limited to, ABL, ACK, AFK, AKT (eg, AKT-I, AKT-2, and AKT-3), ALK, AMP-PK, ATM, Aurorai, Aurora2, bARK1, bArk2, BLK, BMX, BTK, CAK, CaM Kinase, CDC2, CDK , CK, COT, C TD, DNA-PK, EGF-R, ErbB-1, ErbB-2, ErbB-3, ErbB-4, ERK (eg, ERK1, ERK2, ERK3, ERK4, ERK5, ERK6, ERK7) , ERT-PK, FAK, FGR (eg, FGF1R, FGF2R), FLT (eg, FLT-I, FLT-2, FLT-3, FLT-4), FRK, FYN, GSK (eg, GSK1, GSK2, GSK3 -alpha, GSK3-beta, GSK4, GSK5), combined protein G receptor kinases (GRKs), HCK, HER2, HKII, JAK (eg, JAK1, JAK2, JAK3, JAK4), JNK (eg, JNK1, JNK2, JNK3 ), KDR, KIT, IGF-1 receptor, IKK-1, IKK-2, INSR (insulin receptor), IRAK1, IRAK2, IRK, ITK, LCK, LOK, LYN, MAPK, MAPKAPK-1, MAPKAPK-2 , MEK, MET, MFPK, MHCK, MLCK, MLK3, NEU, NIK, alpha re PDGF receptor, PDGF beta receptor, PHK, PI-3 kinase, PKA, PKB, PKC, PKG, PRK1, PYK2, p38 kinases, pl35tyk2, p34cdc2, p42cdc2, p42mapk, p44mpk, RAF, RET, RIP, RIP-2 , RK, RON, RS kinase, SRC, SYK, S6K, TAK1, TEC, TIE1, TIE2, TRKA, TXK, TYK2, UL13, VEGFR1, VEGFR2, YES, YRK, ZAP-70, and all subtypes of these kinases (see eg , Hardie and Hanks (1995) The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.). In preferred embodiments, an EphA2-BiTE of the invention is administered in combination with the administration of one or more prophylactic / therapeutic agents which are inhibitors of Eph receptor kinases (e.g., EphA2). In a more preferred embodiment, an EphA2-BiTE of the invention is administered in combination with the administration of one or more prophylactic / therapeutic agents which are EphA2 inhibitors.

In another specific advance, the methods of the invention encompass administration of an EphA2-BiTE of the invention in combination with administration of one or more prophylactic / therapeutic agents that are angiogenesis inhibitors, such as, but not limited to: Angiostatin (plasminogen fragment). ); antithrombin III

antiangiogenic; Angiozyme; ABT-627; Bay 12-9566; Benefits; Bevacizumab; BMS-275291; cartilage derived inhibitor (CDI); FALLS; CD59 complement fragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen fragment XVIII); fibronectin fragment; Gro-beta; Halofuginone; Heparinases; hexasaccharide Heparin fragment; HMV833; human chorionic gonadotropin (hCG); IM-862; alpha / beta / gamma Interferon; Interferon-induced protein (IP-IO); Interleukin-12; Kringle 5 (plasminogenic fragment); Marimastat; Metalloproteinase Inhibitors (TIMPs); 2-Methoxeestradiol; MMI 270 (CGS 27023A); MoAb IMC-1C11; Neovastat; NM-3; Panzera; PI-88; placenta ribonuclease inhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4); Prinomastat; 16kD Prolactin fragment; proliferin-related protein (PRP); PTK 787 / ZK 222594; Retinoids; Solimastat; squalamine; SS 3304; SU 5416; SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1 (TSP-1); TNP-470; transforming growth factor beta (TGF-β); Vasculostatin; Vasostatin calreticulin fragment); ZD6126; ZD6474; farnesyl transferase (FTI) inhibitors; and bisphosphonates.

In another specific advance, the methods of the invention encompass administration of an EphA2-BiTE of the invention in combination with administration of one or more prophylactic / therapeutic agents which are anticancer agents such as, but not limited to: acivicin, aclarubicin. , acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethantrone acetate, aminoglutethimide, amsacrine, anastrozole, antramycin, asparaginase, asperline, azacytidine, azetepa, azotomycin, bisimaltidate bisnaphtidate, benzodalamide , bizelesine, bleomycin sulphate, brachyrine sodium, bropyrimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine, cybatrate, cybatrazine , dactinomycin, daunorubicin hydrochloride, decarbazine, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, eprolpropyl hydrochloride esorubicin, estramustine, estramustine sodium phosphate, ethanidazole, etoposide, etoposide phosphate, etoprine, fadrozola hydrochloride, fazarabine, phenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocytabine, phosquidone gemhydrochloridine, hydrochloride of idarubicin, ifosfamide, ilmophosine, interleukin 2 (including recorabinant interleuquin 2, or rIL2), alpha-2a interferon, alpha-2b interferon, alpha-nl interferon, alpha-n3 interferon, gamma-I b interferon , iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leu acetate prolide, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, mercaptopurine, methotrexate, sodium methotrexate, metoprine, mitoprine, metoprine mitochromine, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nitrosoureas, nocodazola, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine sulphide, pipamustine sulphide pyroxantrone, plicamycin, plomestane, porfimer sodium, porphyromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, riboprine, rogletimide, safingol, safingol hydrochloride, semustine, sphatromenohydrate, sphacrosulfonate, spiroplatin strept onigrine, streptozocin, sulofenur, talisomycin, sodium tecogalan, tegafur, teloxantrone hydrochloride, temoporphine, teniposide, teroxirone, testolactone, tiamiprine, thioguanine, thiotepa, thiazofurine, tirapazamine, toremifene trimatoate trimethoxytrate acetate, , triptorelin, tubulozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinglicinate sulfate, vinleurosine sulfate, vinorelin tartrate vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3, 5-ethinyluracil, abiraterone, aclarubicin, acylfulveno, adecipenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrografolide, angiogenesis inhibitors, D antagonist, G antagonist, antarelix, morphogenetic anti-dorsal protein-1, antiandrogens, antiestrogens, antineoplastin gene, apidicolin glyphide apoptosis regulators, apurinic acid, ara CDP-DL-PTBA, arginine deaminase, asulacrin, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetrone, azatoxin, azatirosine, baccatin III derivatives, balanol, batimastat, BCR / GLA antagonists, benzochlorines, benzoilstaurosporine, beta lactam derivatives, beta-alethine, betacyclamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisanthrene, b isaziridinilspermine, bisnafide, bistratene A, byzelesine, breflate, bropyrimine, budotitan, butionine sulfoximin, calcipotriol, calfostin C, camptothecin derivatives, canaripox IL-2, capecitabine, carboxamide-amino-triazole, carboxiamidotria3, carboxiamidotria3 of cartilage, carzelesin, casein kinase inhibitors (ICOS), castanospermine, cecropin B, cetrorelix, chloroquinoxaline sulphonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analogs, colisicin A, colismicin B, combretastatin A4, conagenin, crambescidine 816, chrysnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentantraquinones, cycloplatam, cypemicine, cytarabine ocphosphate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemine B, dexorxone diaxide, dexosphonine B , didemnin B, didox, diethylorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, phenyl spiromustine, docetaxel, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, estrogen antagonist, estrogen estrogen etoposide phosphate, exemestane, fadrozola, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicine hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrinin, ganaphilatin nitraphelinase, ganzolinate texaphyrinase, ganzolinate , gemcitabine, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, oxyphene, idramantpna, ilmophosine, ilomastat, imidazoacridones, imiquimod, insulin-receptor-1-receptor agonist-enhancing peptide-1 interferon, interferons, interleukines, io benguana, iododoxorubicin, ipomeanol, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplaquinolide, cahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leucolide factor 7 + estrogen + progesterone, leuprorelin, Levamisole, liarozole, linear polyamine analogue, lipophilic disaccharide peptide, platinum lipophilic compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, laphrothecin, lurothecin, laparothinine lithic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspine, matrilysin inhibitors, metalloproteinase matrix inhibitors, menogaril, merbarone, meterelin, methioninase, MIF inhibitor, mifepristone, miltefosine, double-strand RNA, mismatch, mitoguazone, mitolactol, mitomycin analogs , mitonafide, fibroblast mitotoxin growth factor saporin, mitoxantrone, mopharotene, molgramostim, monoclonal antibody, human chorionic gonadotropin, monophosphoryl lipid A + miobacterium cell wall sk, mopidamol, multiple drug resistance gene inhibitor, drug-based therapy multiple tumor suppressor 1, mustard anticancer agent, micaperoxide B, mycobacterial cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, naloxone + pentazocine, napavine, nafterpine, nartograstim, nedaplatin acid, nedaplatin, nemedahydrin, nedaplatin , neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, antioxidant nitroxide, nitruline, 06-benzylguanine, octreotide, oquicenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin oxapatlin, paconplatinum, , paclitaxel analogs, paclitaxel derivatives, palauamine, pal mitoylrizoxin, pamidronic acid, panaxitriol, panomiphene, parabactin, pazeliptin, pegaspargase, peldesin, pentosan sodium polysulfate, pentostatin, pentrozole, perflubron, perfosfamide, perylyl alcohol, phenazinomycin, phenylacetate, phosphatanitrin pyrazide hydrochloride, piciboline placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, porfimer sodium, porphyromycin, prednisone, propyl bis-acridone, prostaglandin J2, proteaome inhibitors, protein A-based immune modulator , protein kinase C inhibitor, protein kinase C inhibitors, microalgal, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, glitter, pyrazoloacridine, pyridoxylated conjugate hemoglobin polyoxyethylene, raf antagonists, raltitrexate, ramosetron, ras farnesyl protein transfer inhibitors ras inhibitors, r inhibitor as- GAP, demethylated reteliptin, rhenium Re 186 etidronate, rhizoxin, ribozymes, RII retinamide, rogletimide, rohituquine, romurtide, roquinimex, rubiginone BI, ruboxyl, safingol, saintopine, sarCNU, sarcophytol A, sargramostim, Sdi 1 inhibitor, sdi 1, inhibitor 1 senescence derivative, sense oligonucleotides, transduction signal inhibitors, transduction signal modulators, single chain antigen binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedine binding protein, sonermin , sparphic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipiamide, stromelysin inhibitors, sulfinosin, vasoactive overactive intestinal peptide antagonist, suradist, suramine, swainsonine , synthetic glycosaminoglycans, talimustine, tamoxifen metiodide, tauromustine, taxol, tazarotene, sodium tecogalan, tegafur, telurapyrilium, telomerase inhibitors, temoporphine, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, taliblastine, thalidomide, thiocoraline, thioguanine, thrombopoietin, mimetic thrombopoietin, thymorphine thymorphine hormone, thymorphine thymorphine, thymorphine thymorphine etiopurpurin, tirapazamine, titanocene bichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, typhine ubase, typhine ubase inhibitors urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, vector system, erythrocyte gene therapy, velaresol, veramine, verdins, verteporfin, vinorelbine, vinxaltin, VITAXIN®, vorozola, zanoterone, zeniplatin, zilascorb, and zinostatin estimalamer. Additional preferred anticancer drugs are 5-fluorouracil and leucovorin.

In more particular advances, the present invention also comprises the administration of one or more EphA2-BiTEs of the invention in combination with the administration of one or more therapies, such as, but not limited to, anti-cancer agents, such as those described in Table 1, preferably for the treatment of breast, ovarian, melanoma, prostate, colon and lung cancers, as described above. TABLE 1

<table> table see original document page 180 </column> </row> <table> <table> table see original document page 181 </column> </row> <table> <table> table see original document page 182 < / column> </row> <table> <table> table see original document page 183 </column> </row> <table> <table> table see original document page 184 </column> </row> <table> <table> table see original document page 185 </column> </row> <table> <table> table see original document page 186 </column> </row> <table>

The invention also encompasses the administration of the EphA2-BiTEs of the invention in combination with radiotherapy comprising the use of x-rays, gamma rays and other radiation sources to destroy cancer cells. In preferred embodiments, radiation treatment is administered as an external radiation beam or teletherapy characterized by the fact that radiation is directed from a remote source. In other preferred advances, radiation treatment is administered as internal therapy or brachytherapy characterized by the fact that a radioactive source is placed within the body, near the cancer cells or tumor mass.

Cancer therapies and their dosages, routes of administration and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (61st ed., 2007).

Patients with Hyperproliferative Cell Disorders The present invention provides methods for treating, preventing and / or managing hyperproliferative cell disorder or a symptom thereof, methods comprising administering to a subject one or more EphA2-BiTE of the invention alone or in combination with therapies that do not. an EphA2-BiTE. The subject is an animal, preferably a mammal, such as non-primate (eg, cows, pigs, horses, cats, dogs, rats, etc.) and a primate (eg, monkey, such as a cinomolgus monkey). or human). In a preferred advance, the subject is a human.

In a specific advance, the hyperproliferative cell disorder is not cancer. Non-limiting examples of hyperproliferative cell disorders to be treated, prevented and / or managed by the methods of the invention are described, e.g., in U.S. Pat. Pub. No. 2005-20059592, entitled "EphA2 and Hyperproliferative Cell Disorders", which is incorporated by reference herein in its entirety. Accordingly, the invention also provides compositions and methods designed for the treatment, prevention and / or management of hyperproliferative cell disorders (nonlimiting examples of such disorders are described in, eg, US Pat. Pub. No. 2005-0059592, entitled " EphA2 and Hyperproliferative Cell Disorders ", which is incorporated by reference herein in its entirety).

In particular, the present invention provides methods for treating, preventing and / or managing a hyperproliferative cell disorder where EphA2 expression is up-regulated in cells affected by such a disorder, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention, and optionally an effective amount of a therapy other than an EphA2-BiTE. In a preferred embodiment, the hyperproliferative cell disorder to be treated, prevented and / or managed according to the methods of the invention are asthma, COPD, pulmonary fibrosis, asbestosis, IPF, DIP, UIP, renal fibrosis, liver fibrosis, other fibrosis, hyperresponsive bronchial, psoriasis, seborrheic dermatitis, cystic fibrosis, or an endothelial hyperproliferative cell disorder, such as restenosis, hyperproliferative vascular disease, Behcet's syndrome, atherosclerosis, macular degeneration, or a hyperproliferative fibroblast disorder.

Patients with Inflammatory and / or Autoimmune Disorders

The present invention provides methods for treating, administering and / or preventing an inflammatory or autoimmune disorder or a symptom thereof, methods comprising administering to a subject one or more EphA2-BiTE of the invention alone or in combination with therapies other than an EphA2. - BiTE. The subject is preferably a mammal such as a primate (e.g., monkey (e.g., a rhesus monkey, cinomolgus monkey or chimpanzee), or human). In a preferred advance, the subject is a human. In particular, the present invention provides methods for treating, preventing, and / or managing an inflammatory or autoimmune disorder where EphA2 expression is up-regulated in cells affected by such disorder, said methods comprising administering to a subject in need an amount of one or more EphA2-BiTEs of the invention, and optionally an effective amount of a therapy other than an EphA2-BiTEs. In specific advances, the inflammatory or autoimmune disorder to be treated is a disorder described in, eg, International Publication No. WO 00/78815, International Publication No. WO 02/070007 Al, dated September 12, 2002, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV Beta3 Antagonists", International Publication No. WO 03/075957 Al, dated September 18, 2003, entitled "The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3 Antagonists in combination" with Other Agents, "US Patent Pub. No. US 2002/0168360 Al, dated November 14, 2002, entitled" Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin ανβ3 Antagonists in Combination with Other Prophylactic or Therapeutic Agents " , and International Publication No. WO 03/075741 A2, dated September 18, 2003, entitled, "Methods of Preventing or Treating Disorders by Administe. ring an Integrin ανβ3 Antagonist in combination with HMG-CoA Reductase Inhibitor or Bisphosphonate ", each of which is incorporated herein by reference in its entirety. Accordingly, the EphA2-BiTEs of the invention are administered in combination with an effective amount of VITAXIN® (Medlmmune, Inc., International Publication No. WO 00/78815, International Publication No. WO 02/070007 Al, dated September 12, 2002, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV Beta3 Antagonists", International Publication No. WO 03/075957 Al, dated September 18, 2003, entitled "The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3 Antagonists in combination with Other Agents, US Patent Pub. No. US 2002/0168360 Al, dated November 14, 2002, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin ανβ3 Antagonists in Combination" with Other Prophylactic or Therapeutic Agents, "and International Publication No. WO 03/075741 A2, dated September 18, 2003, entitled," Methods of Prevention ". ng or Treating Disorders by Administering an Integrin ανβ3 Antagonist in combination with an HMG-CoA Reductase Inhibitor or a Bisphosphonate ", each of which is incorporated herein by reference in its entirety). In another specific advance, the invention provides methods for treating, preventing and / or managing an inflammatory or autoimmune disorder or one or more symptoms thereof, said methods comprising administering to a subject in need an effective amount of one or more EphA2-BiTEs. of the invention in combination with an effective amount of siplizumab (Medlmmune, Inc., International Pub. No. WO 02/069904, which is incorporated herein by reference in its entirety). In another specific advance, the invention provides methods for treating, preventing and / or managing an inflammatory or autoimmune disorder or one or more symptoms thereof, said methods comprising administering to a subject in need an effective amount of one or more EphA2-BiTEs. of the invention in combination with an effective amount of an anti-inflammatory agent described in Section 5.4.2.4, infra.

Infected Patients

The present invention provides methods for treating, preventing and / or managing an infection (in particular an intracellular infection), or a symptom thereof, methods comprising administering one or more EphA2-BiTE of the invention alone or in combination with therapies other than one. EphA2-BiTE. The subject is preferably a mammal, such as non-primate (eg, cows, pigs, horses, cats, dogs, rats, etc.) and a primate (eg, monkey (eg, rhesus monkey, cinomolgus or chimpanzee monkey) and human ). In a preferred advance, the subject is a human.

The methods of the invention comprise administering one or more EphA2-BiTEs of the invention to patients suffering from or expected to suffer from (e.g., patients with a genetic predisposition to or patients previously suffering from) an infection. Such patients may have been previously treated or are currently being treated for infection, e.g., with therapy without EphA2-BiTE. In a further advance, the methods of the invention comprise administering one or more EphA2-BiTEs of the invention to patients who are immunocompromised or immunosuppressed.

At some stage, an EphA2-BiTE is not administered to patients who are immunocompromised or immunosuppressed. According to the invention, an EphA2-BiTE may be used as any therapy line, including, but not limited to, a first, second, third and fourth line of therapy.

Further, according to the invention, an EphA2-BiTE may be used before any adverse effect or intolerance of therapies without EphA2-BiTE occurs. The invention encompasses methods for administering one or more EphA2-BiTEs of the invention to prevent the onset or recurrence of an infection.

In a breakthrough, the invention also provides methods of treating, preventing and / or managing an infection as alternatives to current therapies. In a specific advance, current therapy has been shown or may be very toxic (i.e., results in unacceptable or unbearable side effects) to the patient. In another breakthrough, an EphA2-BiTE decreases side effects compared to current therapy. In another advance, the patient was shown refractory to a current therapy. In such advances, the invention provides for the administration of one or more EphA2-BiTEs of the invention without any other anti-infection therapies. In certain advances, one or more EphA2-BiTEs of the invention may be administered to a patient in need rather than another therapy to treat an infection. In a breakthrough, the invention provides methods of treating, preventing and / or managing an active infection. In another advance, the invention provides methods of treating, preventing and / or managing a latent infection. In another advance, the invention provides methods of preventing the recurrence of an acute infection. In yet another advancement, the invention provides methods of treating, preventing and / or managing a chronic infection.

The present invention also encompasses methods for administering one or more EphA2-BiTEs of the invention to treat, prevent and / or manage symptoms of infections in patients who are or have become refractory to therapies without EphA2-BiTEs. Determination of whether the infection is refractory may be made in vivo or in vitro by any method known in the art to test the effectiveness of a therapy on cells affected by the infection, particularly epithelial cells, or in patients who are or have become refractory to therapies. without EphA2-BiTE.

Viral Infections

Increased EphA2 expression has been seen to be associated with infections by certain intracellular pathogens, in particular RSV (see, eg, US Appn. Ser. No. 11 / 259,266, filed October 27, 2005, entitled "Use of Modulators of EphA2"). and EphrinAl for the Treatment and Prevention of Infections ", which is incorporated herein by reference in its entirety). Accordingly, the invention also provides compositions and methods designed for the treatment, prevention and / or management of a pathogen infection, including, but not limited to, a viral infection, such as, for example, an RSV infection. One or more EphA2-BiTEs of the invention and compositions comprising said EphA2-BiTEs may be administered to a subject to treat, prevent and / or manage a viral infection or one or more symptoms thereof. In a preferred embodiment, the viral infection to be treated, prevented and / or managed according to the methods of the present invention is intracellular viral infections. One or more EphA2-BiTEs of the invention and compositions comprising said antibodies may be administered in combination with one or more other therapies (eg, one or more prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention to a subject prone to or with a viral infection useful for treating, preventing, and / or managing a viral infection. Non-limiting examples of such therapies include the agents described in Section 5.4.2.5, infra.

In a specific advance, the invention provides methods of treating, preventing and / or managing a viral infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention. . In another advance, the invention provides a method of treating, preventing and / or managing a viral infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention. and an effective amount of one or more therapies (eg, one or more prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention.

In certain advances, an effective amount of one or more EphA2-BiTEs of the invention is administered in combination with an effective amount of one or more therapies (eg, one or more prophylactic or therapeutic agents) currently being used, having been used, or being known to be useful in treating, preventing and / or managing a viral infection or one or more symptoms thereof to a subject in need. Therapies for a viral infection include, but are not limited to, antiviral agents such as acyclovir, amantadine, oseltamivir, ribaviran, palivizumab, and anamivir. In certain advances, an effective amount of one or more EphA2-BiTEs of the invention is administered in combination with one or more supportive measures to a subject in need to treat, prevent and / or manage a viral infection or one or more symptoms thereof. Non-limiting examples of supportive measures include air humidification by an ultrasonic nebulizer, aerosolized racemic epinephrine, oral dexamethasone, intravenous fluids, intubation, fever reducers (eg, ibuprofen, acetomethaphin), and antibiotic and / or antifungal therapy (ie to prevent or treat secondary bacterial infections).

Any type of viral infection or condition resulting from or associated with a viral infection may be treated, prevented and / or managed according to the methods of the invention, said methods comprising administering an effective amount of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of another therapy (eg, a non-EphA2-BiTEs therapeutic or prophylactic agent of the invention). Examples of viruses that cause viral infections include, but are not limited to, retroviruses (eg, human T-cell lymphotrophic virus (HTLV) types I and II and human acquired immunodeficiency virus (HIV, eg, HIV-I and HIV-2). )), herpes virus (eg, herpes simplex virus (HSV) types I and II, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), arenavirus (eg, lassa fever virus), paramyxovirus (eg, virus morbillivirus, human respiratory syncytial virus, mumps, hMPV, and pneumovirus), adenovirus, buniavirus (eg, hantavirus), cornavirus, filovirus (eg, Ebola virus), flavivirus (eg, hepatitis C virus (HCV), fever virus yellow, and Japanese encephalitis virus), hepadnavirus (eg, hepatitis B virus (HBV)), orthomiovirus (eg, influenza A virus, BeCe PIV), papovavirus (eg, papillomavirus), picornavirus (eg, rhinovirus, enterovirus and virus hepatitis A), poxvirus, reovirus (eg, rotavirus), togavirus (eg, rubella virus), and rabdovirus (eg ., rabies virus). Biological responses to a viral infection include, but are not limited to, elevated IgE antibody levels, increased T cell proliferation and / or infiltration, increased B cell proliferation and / or infiltration, epithelial hyperplasia, and mucin production. In a specific advance, the invention also provides methods of treating, preventing and / or managing viral infections that are associated with or cause the common cold, viral pharyngitis, viral laryngitis, viral croup, viral bronchitis, influenza, parainfluenza viral diseases ("PIV"). ") diseases (eg, croup, bronchiolitis, bronchitis, pneumonia), respiratory syncytial virus (" RSV ") diseases, metapneumavirus diseases, and adenovirus diseases (eg, feverish respiratory disease, croup, bronchitis, pneumonia), said method comprising administering an effective amount of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of another therapy.

In a specific advance, influenza virus infections, IVP infections, hMPV infections, adenovirus infections, and / or RSV infections, or one or more symptoms thereof are treated, prevented and / or managed according to the methods of the invention. . In a specific advance, the invention provides methods for treating, preventing and / or managing an RSV infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of alone or in combination with one or more antiviral agents such as, but not limited to, amantadine, rimantadine, oseltamivir, znamivir, ribaviran, RSV-IVIG (ie, intravenous infusion of immune globulin) (RESPIGAM ™), and palivizumab and those antibodies described in US Pat. Appn. Ser. 09 / 996,288 and 09 / 996,265, both entitled "Methods of Administering / Dosing Anti-RSV Antibodies for Prophylaxis and Treatment", filed November 28, 2001. In certain advances, viral infection treated, prevented and / or managed accordingly. with the methods of the invention is not an RSV infection.

In a specific advance, the invention provides methods for treating, preventing and / or managing a PIV infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the PIV. invention alone or in combination with an effective amount of one or more antiviral agents such as, but not limited to, amantadine, rimantadine, oseltamivir, znamivir, ribaviran, and palivizumab. In another specific advance, the invention provides methods for treating, preventing and / or managing an hMPV infection or one or more symptoms thereof, said methods comprising administering an effective amount of one or more antibodies of the invention alone or in combination with an effective amount. of one or more antiviral agents such as, but not limited to, amantadine, rimantadine, oseltamivir, znamivir, ribaviran, and palivizumab to a subject in need thereof. In another specific advance, the invention provides methods for treating, preventing and / or managing influenza, said methods comprising administering an effective amount of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of an antiviral agent, such as but not limited to zanamivir (RELENZA®), oseltamivir (TAMIFLU®), rimantadine, and amantadine (SYMADINE®; SYMMETREL®) to a subject in need thereof.

The invention provides methods for preventing the development of asthma in a subject suffering from or suffering from a respiratory viral infection, said methods comprising administering an effective amount of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of another therapy. . In a specific advance, the subject is an elderly person (i.e., a human who is 65 years of age or older), a prematurely born infant, an infant, or a child. In another specific advance, the subject has suffered or suffers from RSV infection. In a specific advance, the infection is not a respiratory viral infection. In a further advance, the infection is not an RSV infection.

In a specific advance, the invention provides methods for treating, preventing and / or managing one or more secondary responses to a primary viral infection, said methods comprising administering an effective amount of one or more EphA2-BiTEs of the invention alone or in combination with a effective amount of other therapies (eg, other prophylactic or therapeutic agents). Examples of secondary responses to a primary viral infection, particularly a primary respiratory viral infection, include, but are not limited to, asthma-like response to mucosal stimuli, elevated total respiratory resistance, increased susceptibility to secondary bacterial, fungal, and viral infections. by protozoa, and development of such conditions as, but not limited to, pneumonia, croup, and febrile bronchitis. In a specific advance, the invention provides methods for treating, preventing and / or managing an acute viral infection. In a further advance, the invention provides methods for treating, preventing and / or managing a latent viral infection. In further advancements, the invention provides methods for treating, preventing and / or managing an HIV infection or an HBV infection.

In a specific advance, the invention provides methods of treating, preventing and / or managing a viral infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention. in combination with an effective amount of VITAXIN® (Medlmmune, Inc., International Publication No. WO 00/78815, International Publication No. WO 02/070007 Al, dated September 12, 2002, entitled "Methods of Preventing or Treating Inflammatory" or Autoimmune Disorders by Administering Integrin AlphaV Beta3 Antagonists, "International Publication No. WO 03/075957 Al, dated September 18, 2003, titled" The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3 Antagonists in Combination with Other Agents, "US Patent Pub. No. US 2002/0168360 Al, dated November 14, 2002, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Int." egrin ανβ3 Antagonists in combination with Other Prophylactic or Therapeutic Agents ", and International Publication No. WO 03/075741 A2, dated 18 September 2003, entitled," Methods of Preventing or Treating Disorders by Administering an Integrin ανβ3 Antagonist in combination with HMG-CoA Reductase Inhibitor or Bisphosphonate ", each of which is incorporated herein by reference in its entirety). In another specific advance, the invention provides methods for treating, preventing and / or managing a viral infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention. in combination with an effective amount of siplizumab (Medlmmune, Inc., International Pub. No. WO 02/069904, which is incorporated herein by reference in its entirety). In another advance, the invention provides methods of treating, preventing and / or managing a viral infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs in combination with an effective amount of one or more anti-IL-9 antibodies, such as those described in US Pat. Pub. No. 20050002934 (Jan. 6, 2005), which is incorporated herein by reference in its entirety. In yet another advancement, the invention provides methods for treating, preventing and / or managing a viral infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention. in combination with an effective amount of two or more of the following: VITAXIN®, an anti-IL-9 antibody and / or siplizumab.

In an advance, an effective amount of one or more EphA2-BiTEs of the invention is administered in combination with an effective amount of one or more anti-IgE antibodies to a subject to treat, prevent and / or manage a viral infection or one or more. symptoms of this. In a specific advance, an effective amount of one or more antibodies of the invention is administered in combination with an effective amount of anti-IgE TNX901 antibody to a subject to treat, prevent and / or manage a viral infection or one or more symptoms thereof. In a specific advance, an effective amount of one or more antibodies of the invention is administered in combination with an effective amount of rhuMAb-E25 omalizumab anti-IgE antibody to a subject to treat, prevent and / or manage a viral infection or one or more. symptoms of this. In another advance, an effective amount of one or more EphA2-BiTEs of the invention is administered in combination with an effective amount of HMK-12 anti-IgE antibody to a subject to treat, prevent and / or manage a viral infection or one or more. symptoms of this. In a specific advance, an effective amount of one or more EphA2-BiTEs of the invention is administered in combination with an effective amount of anti-IgE 6HD5 antibody to a subject to treat, prevent and / or manage a viral infection or one or more symptoms. of that. In another advance, an effective amount of one or more antibodies of the invention is administered in combination with an effective amount of MAb Hu-901 anti-IgE antibody to a subject to treat, prevent and / or manage a viral infection or one or more symptoms. of that.

The invention encompasses methods for preventing the development of viral infections in a patient expected to suffer from a viral infection or at increasing risk of such infection, eg, patients with suppressed immune systems (eg, organ transplant recipient, AIDS patients, patients undergoing chemotherapy, the elderly, prematurely born infants, infants, children, patients with obstructive esophageal carcinoma, patients with tracheobronchial fistula, patients with neurological diseases (eg, stroke, amyotrophic lateral sclerosis, multiple sclerosis, and myopathies), and patients already suffering from viral infection). Patients may or may not have been previously treated for a viral infection.

The EphA2-BiTEs of the invention, compositions, or combination therapies of the invention may be used as any line of therapy, including, but not limited to, the first, second, third, fourth, or fifth line of therapy for treating, preventing and / or manage a viral infection or one or more symptoms of it. The invention also includes methods of treating, preventing and / or managing a viral infection, or one or more symptoms thereof in a patient undergoing therapies for other diseases or disorders associated with increased EphA2 expression. The invention encompasses methods of treating, preventing and / or managing a viral infection, or one or more symptoms thereof in a patient before any adverse effects or intolerance to therapies without EphA2-BiTEs of the invention develop. The invention also encompasses methods of treating, preventing and / or managing a viral infection or such symptom in refractory patients. In certain advances, a patient with a viral infection is refractory to therapy when the infection has not been significantly eradicated and / or the symptoms have not been significantly alleviated. Determination of whether a patient is refractory may be made either in vivo or in vitro by any method known in the art to test the effectiveness of an infection treatment using means accepted in the art of "refractory" in such a context. In several advances, a patient with a viral infection is refractory when viral replication has not decreased or increased. The invention also encompasses methods of preventing the onset or recurrence of viral infections in patients at risk of developing such infections. The invention also encompasses methods of treating, preventing and / or managing a viral infection or such symptom in patients who are susceptible to adverse reactions to conventional therapies. The invention further encompasses methods for treating, preventing and / or managing a viral infection for which no antiviral therapy is available.

The invention encompasses methods for treating, preventing and / or managing a viral infection or such symptom in a patient seen as refractory to therapies without EphA2-BiTEs of the invention, but is no longer in such therapies. In certain advances, patients being administered or treated according to the methods of this invention are patients already being treated with antibiotics, antivirals, antifungals, or other biological therapy / immunotherapy. Among these

patients are refractory patients, patients who are too young for conventional therapies, and patients with recurrent viral infections despite administration or treatment with existing therapies.

The present invention encompasses methods for treating, preventing and / or managing a viral infection, or one or more symptoms thereof as an alternative to other conventional therapies. In specific advances, the patient being administered or treated according to the methods of the invention is refractory to other therapies or susceptible to adverse reactions of such therapies. The patient may be a person with a suppressed immune system (eg, postoperative patients, chemotherapy patients, and patients with immunodeficiency disease), a person with impaired renal or hepatic function, the elderly, children, infants, prematurely born infants, people with neuropsychiatric disorders or those taking psychotropic drugs, people with a history of seizures, or people on medication who would negatively interact with conventional agents used to treat, prevent and / or manage a viral infection or one or more symptoms thereof. Therapies for viral infection and their dosages, routes of administration and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (61st ed., 2007).

Bacterial infections

The invention provides a method of treating, preventing and / or managing a bacterial infection, in particular an intracellular bacterial infection, or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2. -BiTEs of the invention. Preferably, cells infected with intracellular bacteria have increased EphA2 expression. In another advance, the invention provides a method of treating, preventing and / or managing a bacterial infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of invention and an effective amount of one or more therapies (eg, one or more prophylactic or therapeutic agents) without EphA2-BiTEs of the invention. In a preferred embodiment, the bacterial infections to be treated, prevented and / or managed according to the methods of the present invention are intracellular bacterial infections.

Any type of intracellular bacterial infection or condition resulting from or associated with a bacterial infection (e.g., a respiratory infection) may be treated, prevented and / or managed according to the methods of the invention. Examples of intracellular bacteria that cause infections include, but are not limited to, Mycobacterium tuberculosis, Mycobacterium leprae, Salmonella enterica serovar Typhi, Brucella sp, Legionella sp, Listeria monoeytogenes, Franeisella tularensis, Riekettsia riekettsii; Riekettsia prowazekii; Riekettsia typhi; Riekettsia tsutsugamushi; Chlamydia traehomatis; Chlamydia psittaei; and Chlamydia pneumoniae. In certain advances, an intracellular bacterial infection treated, prevented and / or managed according to the methods of the invention is not a respiratory bacterial infection. In other advances, an intracellular bacterial infection treated, prevented and / or managed according to the methods of the invention is not an infection with Salmonella species. In yet other advances, an intracellular bacterial infection treated, prevented and / or managed according to the methods of the invention is not a Salmonella dublin infection.

In a specific advance, the invention provides methods for treating, preventing and / or managing an intracellular bacterial infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of invention. In another advance, the invention provides a method of treating, preventing and / or managing an intracellular bacterial infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs. of the invention and an effective amount of one or more therapies (eg, prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention.

In certain advances, the invention provides methods for treating, preventing and / or managing a bacterial infection or one or more of the symptoms, said methods comprising administering to a subject in need thereof one or more EphA2-BiTEs of the invention in combination and effective amount. of one or more therapies (eg, one or more prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention used to treat, prevent and / or manage bacterial infections. Infection Therapies

Bacterial infections, particularly, bacterial infections include, but are not limited to, antibacterial agents (eg, aminoglycosides (eg, gentamicin, tobramycin, amikacin, netylimycin) aztreonam, celfalosporins (eg, cefaclor, cefadroxil, cephalexin, cefazolin), clindamycin, erythromycin, penicillin (eg, penicillin V, crystalline penicillin G, penicillin G procaine), spectinomycin, and tetracycline (eg, chlortetracycline, doxycycline, oxytetracycin)) and supportive therapy such as supplemental and mechanical ventilation. In certain advances, one or more EphA2-BiTEs of the invention are administered in combination with one or more supportive measures to a subject in need thereof to treat, prevent and / or manage a bacterial infection or one or more symptoms thereof. Non-limiting examples of supportive measures include ultrasonic nebulizer air humidification, aerosolized racemic epinephrine, oral dexamethasone, intravenous fluids, intubation, fever reducers (eg, ibuprofen, acetomethaphine), and more preferably antibiotic or antiviral therapy (ie to prevent or treat secondary infections).

The invention provides methods for treating, preventing and / or managing a biological response to a bacterial infection, such as, but not limited to, elevated IgE antibody levels, proliferation, degranulation, and / or mastitis infiltration, proliferation and / or increasing B cell infiltration, and increasing T cell proliferation and / or infiltration, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of a or more therapies (eg a therapeutic or prophylactic agent) without EphA2-BiTEs of the invention. The invention also provides methods of treating, preventing and / or managing respiratory conditions caused by or associated with bacterial infections, such as, but not limited to, pneumonia, recurrent aspiration pneumonia, legionellosis, pertussis, meningitis, or tuberculosis, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of another therapy. In a specific advance, the methods of the invention are used to treat, prevent and / or manage a bacterial infection caused by Mycobacteria or one or more symptoms thereof, said method comprising administering to a subject in need of an effective amount of one or more. EphA2-BiTEs of the invention alone or in combination with an effective amount of one or more other therapies (e.g., one or more prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention.

In a specific advance, the invention provides methods for treating, preventing and / or managing one or more secondary conditions or responses to a primary bacterial infection, preferably a primary bacterial infection, said method comprising administering to a subject in need thereof in a effective amount of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of other therapies (e.g., other prophylactic or therapeutic agents). Examples of secondary conditions or responses to a primary bacterial infection, particularly a bacterial infection, include, but are not limited to, asthma-like responses to mucosal stimulation, high total resistance, susceptibility to increased viral, bacterial, fungal, and protozoal secondary infections. , and development of such conditions as, for example, but not limited to, pneumonia, croup, and febrile bronchitis. In a specific advance, the methods of the invention are used to treat, prevent and / or manage a bacterial infection, or one or more symptoms thereof, said methods comprising administering to a subject in need of an effective amount of one or more EphA2-. BiTEs of the invention in combination with an effective amount of VITAXIN® (Medlmmune, Inc., International Publication No. WO 00/78815, International Publication No. WO 02/070007 Al, dated September 12, 2002 entitled ssMethods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV Beta3 Antagonists, "International Publication No. WO 03/075957 Al, dated September 18, 2003, entitled ssThe Prevention or Treatment of Cancer Using Integrin AlphaVBeta3 Antagonists in Combination with Other Agent s", US Patent Pub. No. US 2002/0168360 Al, dated November 14, 2002, entitled ssMethods of Preventing or Treating Inflammatory or Autoimmune Diso rders by Administering Integrin ανβ3 Antagonists in Combination with Other Prophylactic or Therapeutic Agents, "and International Publieation No. WO 03/075741 A2, dated September 18, 2003, titled, ssMethods of Preventing or Treating Disorders by Administering an Integrin ανβ3 Antagonist in Combination with an HMG-CoA Reductase Inhibitor or a Bisphosphona te ", each of which is incorporated herein by reference in its entirety).

In another specific advance, the methods of the invention are used to treat, prevent and / or manage a bacterial infection, or one or more symptoms thereof, said methods comprising administering to a subject in need thereof in an effective amount of one or more EphA2. -BiTEs of the invention in combination with an effective amount of siplizumab (Medlmmune, Inc., International Pub. No. WO 02/069904). In another advance, the methods of the invention are used to treat, prevent and / or manage a bacterial infection, or one or more symptoms thereof, said methods comprising administering to a subject in need thereof in an effective amount of one or more EphA2-. BiTEs of the invention in combination with an effective amount of one or more anti-IL-9 antibodies (for example, one of the anti-IL-9 antibodies described in US Pat. Pub. No. 20050002934 (January 6, 2005), the which is incorporated herein by reference in its entirety). In still another advancement, the invention provides methods for treating, preventing and / or managing a bacterial infection, or one or more symptoms thereof, said methods comprising administering to a subject in need thereof in an effective amount of one or more EphA2-. BiTEs of the invention in combination with an effective amount of two or more of the following: VITAXIN®, siplizumab, and / or anti-Il-9 antibodies.

The invention encompasses methods for preventing the development of bacterial infection in a patient who is expected to suffer from a bacterial infection or at increased risk of such infection, for example, patients as suppressed immune systems (e.g., organ transplant recipients, AIDS patients, patients undergoing chemotherapy, the elderly, premature infants, infants, children, patients with obstructed esophageal carcinoma, patients with tracheobronchial fistula, patients with neurological diseases (eg, stroke, lateral sclerosis amyotrophic disease, multiple sclerosis, and myopathies), and patients already suffering from an infection). Patients may or may not have been previously treated for an infection.

The EphA2-BiTEs of the invention or combination of therapies of the invention may be used as any line of therapy, including but not limited to the first, second, third, fourth or fifth line of therapies, to treat, prevent and / or manage an infection. bacterial disease, or one or more symptoms thereof. The invention also includes methods for treating, preventing and / or managing a bacterial infection, or one or more symptoms thereof in a patient undergoing therapies for other diseases or disorders. The invention encompasses methods for treating, preventing and / or managing a bacterial infection, or one or more symptoms thereof, in a patient before any adverse effects or intolerance to therapies other than EphA2-BiTEs of the invention develops. The invention also encompasses methods for treating, preventing and / or managing a bacterial infection or symptom thereof in refractory patients. In certain advances, a patient with a bacterial infection is refractory to therapy when the infection has not been significantly eradicated and / or the symptoms have not been significantly alleviated. Determination of whether a patient is refractory may be made either in vivo or in vitro by any method known in the art to test the effectiveness of a treatment of infections using refractory meanings and such context accepted in the art. In several advances, a patient with a bacterial infection is refractory when bacterial replication is not diminished or increased. The invention also encompasses methods of preventing the onset or recurrence of bacterial infection in patients at risk of developing such an infection. The invention also encompasses methods for treating, managing and / or preventing a bacterial infection or a symptom thereof in patients who are susceptible to adverse reactions to conventional therapies. The invention further encompasses methods for treating, preventing, and / or managing bacterial infections for which no antibacterial therapy is available.

The invention encompasses methods for treating, preventing, and / or managing bacterial infections or a symptom thereof in a patient who has been refractory to therapies other than EphA2-BiTEs of the invention, but are no longer in these therapies. In certain advances, patients being managed or treated according to the methods of this invention are patients already being treated with anti-inflammatory agents, antibiotics, antivirals, antifungals, antiprotozoal agents, or other biological therapies / immunotherapies. These patients include refractory patients, patients who are too young for conventional therapies, and patients with recurrent bacterial infections despite management or treatment with existing therapies.

The present invention encompasses methods of treating, preventing and / or managing a bacterial infection, or one or more symptoms thereof as an alternative to other conventional therapies. In specific advances, a patient being managed or treated according to the methods of the invention is refractory to other therapies or susceptible to adverse reactions from these therapies. The patient may be a person with a suppressed immune system (eg, postoperative patient, chemotherapy patients, and patients with immunodeficiency disease), a person with impaired renal or liver function, the elderly, children, infants, premature infants, people with neuropsychiatric disorders or those taking psychotropic drugs, people with a history of seizures, or people on medication who would negatively interact with conventional agents used to treat, prevent and / or manage a bacterial infection, or one or more symptoms. this one.

Therapies for bacterial infections and their dosages, routes of administration and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (6Ist edition, 2007).

Fungal Infections One or more EphA2-BiTEs of the invention may be administered according to methods of the invention to a subject for treating, preventing and / or managing a fungal infection or one or more symptoms thereof. In a preferred advance, fungal infected cells have increased EphA2 expression. One or more EphA2-BiTEs of the invention may also be administered to a subject to treat, manage, and / or ameliorate a fungal infection and / or one or more symptoms thereof in combination with one or more other therapies (for example, one or more prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention which are useful for treating, preventing and / or managing a fungal infection or one or more symptoms thereof. In a preferred embodiment, fungal infections may be treated, prevented and / or managed according to the methods of the present invention in intracellular fungal infections.

Any type of fungal infection or condition resulting from or associated with the fungal infection may be treated, prevented and / or managed according to the methods of the invention. Examples of fungi that cause fungal infections include, but are not limited to, Absidia species (eg Absidia corymbifera and Absidia ramosa), Aspergillus species (eg Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus), Basidiobolus ranarum, Blastomyces dermatitidis, Candida species (eg Candida albicans, Candida glabrata, Candida kerr, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Candida stellid) immitis, Conidiobolus species, Cryptococcus neoforms, Cunninghamella species, dermatophytes, Histoplasma eapsulatum, Microsporum gypseum, Mueor pusillus, Paraeoceidioides brasiliensis, Pseudallescheria boydii, Rhinosporidium seeberi, Pneumoeystus rhiz eari, Rhumo poreopiz rhizopus Rhizopus mierosporus), Saccharomyee species s, Sporothrix sehenekii, zygomycetes, and classes such as,. for example, Zygomycetes, Aseomieetos, Basidiomieetos, Deuteromieetos, and Oomieetos. In a specific advance, the fungal infection is not a respiratory fungal infection.

In a specific advance, the invention provides a method for treating, preventing and / or managing a fungal infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof in an effective amount of one or more EphA2-BiTEs. of the invention. In another advance, the invention provides a method of treating, preventing and / or managing a fungal infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the and an effective amount of one or more other therapies (e.g., one or more prophylactic or therapeutic agents) other than the EphA2-BiTEs of the invention. In certain advances, an effective amount of one or more antibodies is administered in combination with an effective amount of one or more therapies (for example, one or more prophylactic or therapeutic agents) other than the EphA2-BiTEs of the invention which are currently being used, have been used, or are known to be useful in treating, preventing and / or managing a fungal infection, preferably fungal infection, for a subject in need thereof. Therapies for yeast infections include, but do not. antifungal agents such as azole drugs eg miconazole, ketoconazole (NIZORAL®), caspofungin acetate (CANCIDAS®), imidazole, triazols (eg fluconazole (DIFLUCAN®)), and itraconazole (SPORANOX) ®)), polyenes (eg nystatin, amphotericin B colloidal dispersion ("ABCD") (AMPHOTEC ©), liposomal amphotericin B (AMBISONE®)), potassium iodide (KI), pyrimidine (eg flucytosine (ANCOBON ®)), and voriconazole (VFEND®). In certain advances, an effective amount of one or more EphA2-BiTEs of the invention are administered in combination with one or more sustaining measures to a subject in need thereof to treat, prevent and / or manage a fungal infection or one or more symptoms thereof. Non-limiting examples of supportive measures include air humidification by an ultrasonic nebulizer, aerosolized racemic epinephrine, oral dexamethasone, intravenous fluids, intubation, fever reducers (eg, ibuprofen and acetomethaphine), and antiviral or antibacterial therapy (ie. to prevent or treat secondary viral or bacterial infections).

The invention also provides methods for treating, preventing and / or managing a biological response to a fungal infection such as, but not limited to, elevated IgE antibody levels, elevated nerve growth factor (NGF) levels, mast cell proliferation. , degranulation, and / or infiltration, increased proliferation and / or infiltration of B cells, and increased proliferation and / or infiltration of T cells, said methods comprising administering an effective amount of one or more EphA2-BiTEs alone or in combination. with one or more other therapies.

In a specific advance, the invention provides methods for treating, preventing and / or managing one or more secondary conditions or responses to primary fungal infection, preferably a primary fungal infection, said method comprising administering to a subject in need thereof in an effective amount. of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of other therapies (e.g., other prophylactic or therapeutic agents). Other than the EphA2-BiTEs of the invention. Examples of secondary conditions or responses to primary fungal infections, particularly but not limited to primary fungal infections, asthma-type responsiveness to mucosal stimulation, elevated total resistance, increased susceptibility to secondary viral, fungal, and fungal infections, and development of such conditions as, for example, but not limited to pneumonia, croup, and febrile bronchitis.

In a specific advance, the invention provides methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention. in combination with an effective amount of VITAXIN® (Medlmmune, Inc., International Publication No. WO 00/78815, International Publication No. WO 02/070007 Al, dated September 12, 2002, entitled "Methods of Preventing or Treating Inflammatory" or Autoimmune Disorders by Administering Integrin AlphaV Beta3 Antagonists, "International Publication No. WO 03/075957 Al, dated September 18, 2003, titled" The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3 Antagonists in Combination with Other Agents, "US Patent Pub. No. US 2002/0168360 Al, dated November 14, 2002, titled ysMethods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administ ering Integrin ανβ 3 Antagonists in Combination with Other Prophylactic or Therapeutic Agent ", and International Publieation No. WO 03/075741 A2, dated September 18, 2003, titled, ssMethods of Preventing or Treating Disorders by Administering an Integrin ανβ3 Antagonist in Combination with an HMG-CoA Reductase Inhibitor or a Bisphosphonate ", each being incorporated herein by reference in its entirety) to a subject in need thereof.

In another advancement, the invention provides methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the present invention. combination with an effective amount of siplizumab (Medlmmune, Inc.,

International Pub. No. WO 02/069904) to a subject in need thereof. In another advance, the invention provides methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs in combination with an effective amount of one or more anti-IL-9 antibodies (for example, one or more of the anti-IL-9 antibodies described in US Pat. Pub. No. 20050002934 (January 6, 2005)), which is incorporated here by reference in its entirety). In another advance, the invention provides methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs in combination with an effective amount of two or more of the following: VITAXIN®, Siplizumab and / or anti-IL-9 antibodies. The invention encompasses methods for preventing the development of fungal infections in a patient who is expected to suffer from a fungal infection, or at increased risk of such infection. Such subjects include, but are not limited to, patients with suppressed immune systems (e.g., organ transplant recipients, AIDS patients, patients undergoing chemotherapy, patients with obstructive esophageal carcinoma, patients with fistula). tracheobronchial disease, patients with neurological diseases (eg, caused by stroke, amyotrophic lateral sclerosis, multiple sclerosis, and myopathies), and patients already suffering from a condition, particularly an infection). Patients may or may not have been previously treated for an infection. In a specific advance, the patient suffers from bronchopulmonary dysplasia, congenital heart disease, cystic fibrosis, and / or acquired or congenital immunodeficiency. In another specific advance, the patient is a prematurely born infant, an infant, a child, an older human, or a human in a group home, nursing home, or some other type of institution. The invention also encompasses methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof in patients who are susceptible to adverse reactions to conventional antifungal therapies for conditions for which no therapies are available.

The EphA2-BiTEs of the invention or combination of therapies of the invention may be used as any line of therapy, including but not limited to the first, second, third, fourth, or fifth line of therapy, to treat, prevent and / or manage an infection. fungal disease or one or more symptoms of this. The invention also includes methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof in a patient undergoing therapies for other diseases or disorders. The invention encompasses methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof in a patient before any adverse effects or intolerance to therapies other than EphA2-BiTEs of the invention develops. The invention also encompasses methods for treating, preventing and / or managing a fungal infection or a symptom thereof in refractory patients. In certain advances, a patient with a fungal infection is refractory to therapy when the infection has not been significantly eradicated and / or the symptoms have not been significantly alleviated. Determination of whether a patient is refractory may be made either in vivo or in vitro by any method known in the art to test the effectiveness of a treatment of infections, using "refractory" meanings accepted in such art. In several advances, a patient with a fungal infection is refractory when fungal replication has not been decreased or has been increased. The invention also encompasses methods of preventing the onset or recurrence of fungal infections in patients at risk of developing such infections. The invention also encompasses methods for treating, preventing and / or managing a fungal infection or a symptom thereof in patients who are susceptible to adverse reactions to conventional therapies. The invention further encompasses methods for treating, preventing and / or managing fungal infections for which antifungal therapy is not available.

The invention encompasses methods for treating, preventing and / or managing a fungal infection, or a symptom thereof, in a patient who has proven to be refractory to therapies other than EphA2-BiTEs of the invention but no longer in these therapies. In certain advances, patients being managed or treated according to the methods of this invention are patients already being treated with antibiotics, antivirals, antifungals, or other biological therapy / immunotherapy. These patients include refractory patients, patients who are too young for conventional therapies, and patients with recurrent fungal infections despite management and treatment with existing therapies.

The present invention provides methods for treating, preventing and / or managing a fungal infection or one or more symptoms thereof as an alternative to other conventional therapies. In specific advances, the patient being managed or treated according to the methods of the invention is refractory to other therapies or susceptible to adverse reactions of such therapies. The patient may be a person with a suppressed immune system (eg, postoperative patients, chemotherapy patients, and patients with immunodeficiency disease), a person with impaired renal or liver function, the elderly, children, infants, premature infants, people with neuropsychiatric disorders or those taking psychotropic drugs, people with a history of seizures, or people on medication who would negatively interact with conventional agents used to treat, prevent and / or manage a bacterial infection, or one or more symptoms. this one.

Therapies for fungal infections and their dosages, routes of administration and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (61st edition, 2007).

Protozoan Infections

One or more EphA2-BiTEs of the invention may be administered according to methods of the invention to a subject for treating, preventing and / or managing a protozoal infection or one or more symptoms thereof. In a preferred advance, protozoan infected cells have increased EphA2 expression. One or more EphA2-BiTEs of the invention may also be administered to a subject to treat, manage, and / or ameliorate a protozoal infection or one or more symptoms thereof in combination with one or more therapies (e.g., one or more prophylactic agents. or therapeutic) other than EphA2-BiTEs of the invention which are useful for treating, preventing and / or managing a fungal infection or one or more symptoms thereof. In a preferred embodiment, protozoan infections to be treated, prevented and / or managed according to the methods of the present invention are intracellular protozoan infections.

Any type of protozoal infection or condition resulting from or associated with a protozoan infection may be treated, prevented and / or managed according to the methods of the invention. Examples of protozoa that cause infections include, but are not limited to, Leishmania; Trypanosoma; Giardia; Trichomonas; Entamoeba; Dientamoeba; Naegleria and Acanthamoeba; Babesia; Plasmodium; Isospora; Sarcocystis; Toxoplasma; Enterocytozoon; Balantidium; and Pneumocystis.

In a specific advance, the invention provides a method for treating, preventing and / or managing a protozoal infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs. of the invention. In another advance, the invention provides a method for treating, preventing and / or managing a protozoan infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention and an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention. In certain advances, an effective amount of one or more EphA2-BiTEs is administered in combination with an effective amount of one or more therapies (for example, one or more prophylactic or therapeutic agents) other than the EphA2-BiTEs of the invention, which are currently being used, have been used, or are known to be useful in treating, preventing and / or managing a protozoan infection, to a subject in need thereof. In certain advances, an effective amount of one or more EphA2-BiTEs of the invention are administered in combination with one or more sustaining measures to a subject in need thereof to treat, prevent and / or manage a protozoal infection or one or more symptoms thereof. . Non-limiting examples of supportive measures include air humidification by an ultrasonic nebulizer, aerosolized racemic epinephrine, oral dexamethasone, intravenous fluids, intubation, fever reducers (eg, ibuprofen and acetomethaphine), and antiviral or antibacterial therapy (ie. to prevent or treat secondary viral or bacterial infections).

The invention also provides methods for treating, preventing and / or managing a biological response to a protozoan infection such as, but not limited to, elevated IgE antibody levels, elevated nerve growth factor (NGF) levels. mast cell proliferation, degranulation, and / or infiltration, increased B cell proliferation and / or infiltration, and increased T cell proliferation and / or infiltration, said methods comprising administering an effective amount of one or more EphA2- BiTEs alone or in combination with one or more therapies.

In a specific advance, the invention provides methods for treating, preventing and / or managing one or more secondary conditions or responses to a primary infection, preferably a primary protozoal infection, said method comprising administering to a subject in need thereof an effective amount. of one or more EphA2-BiTEs of the invention alone or in combination with an effective amount of other therapies (e.g., other prophylactic or therapeutic agents) other than EphA2-BiTEs of the invention.

In a specific advance, the invention provides methods for treating, preventing and / or managing a protozoan infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention in combination with an effective amount of VITAXIN® (Medlmmune, Inc., International Publication No. WO 00/78815, International Publication No. WO 02/070007 A1, dated September 12, 2002, entitled "Methods of Preventing or Treating"). Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV Beta3 Antagonists ", International Publication No. WO 03/075957 Al, dated September 18, 2003, entitled" The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3 Antagonists in Combination with Other Agents, "US Patent Pub. No. US 2002/0168360 Al, dated November 14, 2002, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Disorders" by Administering Integrin ανβ3 Antagonists in Combination with Other Prophylactic or Therapeutic Agent ", and International Publication No. WO 03/075741 A2, dated September 18, 2003, titled, ysMethods of Preventing or Treating Disorders by Administering an Integrin ανβ3 Antagonist in Combination with an HMG-CoA Reductase Inhibitor or a Bisphosphona te ", each being incorporated herein by reference in its entirety) to a subject in need thereof.

In another advance, the invention provides methods for treating, preventing and / or managing a protozoan infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs of the invention. in combination with an effective amount of siplizumab (Medlmmune, Inc., International Pub. No. WO 02/069904) to a subject in need thereof. In another advance, the invention provides methods for treating, preventing and / or managing a protozoal infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs in combination. with an effective amount of one or more anti-IL-9 antibodies (for example, the anti-IL-9 antibodies described in US Pat. Pub. No. 20050002934 (Jan. 6, 2005)), which is incorporated herein by reference in its entirety). In another advance, the invention provides methods for treating, preventing and / or managing a protozoal infection or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more EphA2-BiTEs in combination. with an effective amount of two or more of the following: VITAXIN®, siplizumab and / or anti-IL-9 antibodies.

The invention encompasses methods for preventing the development of protozoal infections in a patient who is expected to suffer from a protozoan infection, or at increased risk of such protozoan infection. Such subjects include, but are not limited to, patients with suppressed immune systems (eg, organ transplant recipients, AIDS patients, chemotherapy patients, cancer patients, tracheobronchial fistula patients, patients with neurological disorders). (eg, caused by stroke, amyotrophic lateral sclerosis, multiple sclerosis, and myopathies), and patients already suffering from a condition, particularly an infection. At a specific advance, the patient suffers from bronchopulmonary dysplasia, congenital heart disease, cystic fibrosis , and / or acquired or congenital immunodeficiency In another specific advance, the patient is a prematurely born infant, an infant, a child, an older human, or a human in a residential group, nursing home, or some other type of The invention also encompasses methods for treating, preventing and / or managing a protozoan infection or a m or more such symptoms in patients who are susceptible to adverse reactions to conventional antiprotozoal therapies for conditions for which no therapies are available.

The EphA2-BiTEs of the invention or combination of therapies of the invention may be used in any line of therapy, including but not limited to the first, second, third, fourth or fifth line of therapy, to treat, prevent and / or manage an infection. by protozoa or one or more symptoms of this. The invention also includes methods for treating, preventing and / or managing a protozoal infection or one or more symptoms thereof in a patient undergoing therapies for other diseases or disorders. The invention encompasses methods for treating, preventing and / or managing a protozoal infection, or one or more symptoms thereof in a patient before any adverse effects or intolerance to therapies other than EphA2-BiTEs of the invention develops. The invention also encompasses methods for treating, preventing and / or managing a protozoan infection or a symptom thereof in refractory patients. In certain advances, a patient with a protozoan infection is refractory to therapy when the infection has not been significantly eradicated and / or the symptoms have not been significantly alleviated. Determination of whether a patient is refractory can be made either in vivo or in vitro by any method known in the art to test the effectiveness of an infection treatment using "refractory" meanings in such art-accepted context. In several advances, a patient with a protozoan infection is refractory when protozoan replication has not been decreased or increased. The invention also encompasses methods of preventing the onset or recurrence of protozoal infections in patients at risk of developing such infections. The invention also encompasses methods for treating, preventing and / or managing a protozoan infection or a symptom thereof in patients who are susceptible to adverse reactions to conventional therapies. The invention further encompasses methods for treating, preventing and / or managing protozoal infections for which no antiprotozoal therapies are available.

The invention encompasses methods for treating, preventing and / or managing a protozoan infection or a symptom thereof in a patient who has been shown to be refractory to non-EphA2-BiTEs therapies of the invention but no longer in these therapies. In certain advances, patients being managed or treated according to the methods of this invention are patients already being treated with antibiotics, antivirals, antiprotozoans, or other biological therapy / immunotherapy. These patients include refractory patients, patients who are too young for conventional therapies, and patients with recurrent protozoan infections despite management or treatment with existing therapies.

The present invention provides methods for treating, preventing and / or managing a protozoan infection or one or more symptoms thereof as an alternative to other conventional therapies. In specific advances, the patient being managed or treated according to the methods of the invention is refractory to other therapies or susceptible to adverse reactions of such therapies. The patient may be a person with a suppressed immune system (eg, postoperative patients, chemotherapy patients, and patients with immunodeficiency disease), a person with impaired renal or liver function, the elderly, children, infants, premature infants, people with neuropsychiatric disorders or those taking psychotropic drugs, people with a history of seizures, or people on medication who would negatively interact with conventional agents used to treat, prevent and / or manage a fungal infection or one or more symptoms of this disorder. .

Protozoan infection therapies and their dosages, routes of administration and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (61st edition, 2007).

Anti-Inflammatory Therapies

Any anti-inflammatory agent, including agents useful in therapies for inflammatory disorders, well known to one skilled in the art may be used in the compositions and methods of the invention. Non-limiting examples of anti-inflammatory agents include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, anticholinergics (eg, atropine sulfate, atropine methylnitrate, and ipratropium bromide (ATROVENT ™)), agonists. beta2 (e.g., abuterol (VENTOLIN ™ and PROVENTIL ™), bitolterol (TORNALATE ™), levalbuterol (XOPONEX ™), metaproterenol (ALUPENT ™), pyrbuterol (MAXAIR ™), terbutlaine (BRETHAIRE ™ and BRETHINE ™), albuterol (PROVENTIL) ™, REPETABS ™, and VOLMAX ™), formoterol (FORADIL AEROLIZER ™), and salmeterol (SEREVENT ™ and SEREVENT DISKUS ™)), and methylxanthines (e.g. theophylline (UNIPHYL ™, THEO-DUR ™, SLO-BID ™, and TEHO-42 ™)). Examples of NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREX ™), diclofenac (VOLTAREN ™), ethodolac (LODINE ™), phenoprofen (NALFON ™), indomethacin (INDOCIN ™), ketoralac (TORADOL ™) , oxaprozine (DIAPROet), nabumetone (RELAFEN ™), sulindac (CLINORIL ™), tolmentin (TOLECTIN ™), rofecoxib (VIOXX ™), naproxen (ALEVE ™, NAPROSYN ™), ketoprofen (ACTRON ™) and RELFENET ™ (RELAFEN ™) . Such NSAIDs function by inhibiting a cyclooxygenase enzyme (e.g., COX-I and / or COX-2). Examples of steroidal anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRON ™), corticosteroids (e.g. methylprednisolone (MEDROL ™)), cortisone, hydrocortisone, prednisone (PREDNISONE ™ and DELTASONE ™), prednisolone ( PRELONE ™ and PEDIAPRED ™), triamcinolone, azulfidine, and eicosanoid inhibitors (e.g., prostaglandins, thromboxanes, and leukotrienes (e.g. montelukast (SINGULAIR ™), zafirlukast (ACCOLATE ™), pranlukast (ONON ™), or zile Anti-inflammatory therapies and their dosages, routes of administration, and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (61st edition, 2007).

Antiviral Therapies

Any antiviral agent well known to one skilled in the art may be used in the compositions and methods of the invention. Non-limiting examples of antiviral agents include proteins, polypeptides, peptides, fusion protein antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and / or reduce the association of a virus with its receptor, the internalization of a virus in a cell, replicating a virus, or releasing viruses from a cell. In particular, antiviral agents include, but are not limited to, nucleoside analogs (e.g., zidovudine, acyclovir, ganciclovir, vidarabine, oxidoidine, trifluoridine, and ribavirin), foscarnide, amantadine, rimantadine, saquinavir, indinavir, ritonavir, interferons and other interferons, and AZT.

In specific advances, the antiviral agent is an immunomodulatory agent that is immunospecific for a viral antigen. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide and protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, RSV G glycoprotein, influenza virus neuraminidase, influenza virus, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) which is capable of eliciting an immune response. Antibodies useful in this invention for the treatment of a viral infection include, but are not limited to, antibodies against pathogenic virus antigens, including by way of example and not limitation: adenoviridae (e.g., mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (eg levivirus, enterobacterial phage MS2, allolevirus), poxviridae (eg chordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxvirinae), papovaviridae (eg, polyomavirus and papillomavirus), paramyxoviridae (eg, paramyxovirus, influenza 1 paravirus, mobilivirus (eg, measles virus), rubulavirus (eg) , pneumonovirinae (eg pneumovirus, human respiratory syncytial virus), and metapneumovirus (eg tire avian movirus and human metapneumovirus)), picornaviridae (eg enterovirus, rhinovirus, hepatovirus (eg human hepatitis A virus, cardiovirus, and apthovirus)), reoviridae (eg ortoreovirus, orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus , and orizavirus), retroviridae (eg mammalian type B retrovirus, mammalian type C retrovirus, bird type C retrovirus, type B retrovirus group, BLV-HTLV retrovirus, lentivirus (e.g. human immunodeficiency and human immunodeficiency virus 2), spumavirus), flaviviridae (eg hepatitis C virus), hepadnaviridae (eg hepatitis vírus virus), togaviridae (eg alfavirus (eg sindbis virus) and rubivirus (eg rubella virus)), rhabdoviridae (eg vesiculovirus, lissavirus, ephemerovirus, cytorabdovirus, and necleorabdovirus), arenaviridae (eg arenavirus, color virus lymphocytic iomeningitis, Ippy virus, and lassa virus), and coronaviridae (eg, coronavirus and torovirus).

Specific examples of available antibodies useful for the treatment of a viral infection include, but are not limited to, PR0542 (Progenics) which is a CD4 fusion antibody useful for the treatment of HIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a useful human antibody for the treatment of hepatitis B virus; and Protovir (Protein Design Labs, Inc., CA) which is a humanized IgG1 antibody useful for the treatment of cytomegalovirus (CMV); and palivizumab (SYNAGIS®; Medlmmune, Inc .; International Publication No. WO 02/43660) which is a humanized antibody useful for the treatment of RSV.

In a specific advance, the antiviral agents used in the compositions and methods of the invention inhibit or reduce a virus infection, inhibit or reduce replication of a virus that causes an infection, or inhibit or reduce the spread of a virus that causes a virus infection. other cells or subjects. In another specific advance, the antiviral agents used in the compositions and methods of the invention inhibit or reduce RSV, hMPV, or PIV infection, inhibit or reduce RSV, hMPV, or PIV replication, or inhibit or reduce the spread of RSV, hMPV. , or PIV to other cells or subjects. Examples of such agents and methods of treating RSV, hMPV, and / or PIV infections include, but are not limited to, nucleoside analogs such as zidovudine, acyclovir, gangciclovir, vidarabine, oxidoidine, trifluridine, and ribavirin. , as well as foscarneto, amantadine, rimantadine, saquinavir, indinavir, ritonavir, and alpha interferons. See U.S. Prov. Patent App. No. 60 / 398,475 filed July 25, 2002, entitled "Methods of Treating and Preventing RSV, HMPV, and PIV Using Anti-RSVf Anti-HMPVr and Anti-PIV Antibodies", and US Patent App. No. 10 / 371,122 filed February 21, 2003, which are incorporated herein by reference in their entirety.

In specific advances, the viral infection is RSV and the viral antigen is an antibody that immunospecifically binds to an RSV antigen. In certain advances, the anti-RSV antigen antibody immunospecifically binds to an RSV Group A RSV antigen. In other advances, the anti-RSV antigen antibody immunospecifically binds to an RSV Group B RSV antigen. In other advances, an antibody binds to an RSV antigen from one Group and cross-reacts as analogous antigen from the other Group. In particular advances, the anti-RSV antigen antibody immunospecifically binds to an RSV nucleoprotein, RSV phosphoprotein, RSV matrix protein, small hydrophobic RSV protein, RSV RNA dependent polymerase, RSV F protein, and / or RSV G protein. In specific further advances, the anti-RSV antibody binds to allelic variants of an RSV nucleoprotein, an RSV nucleocapsid protein, an RSV phosphoprotein, an RSV matrix protein, a glycoprotein of RSV adhesion, an RSV fusion glycoprotein, an RSV nucleocapsid protein an RSV matrix protein, a small hydrophobic RSV protein, an RSV RNA-dependent polymerase, an RSV F protein, an RSV L protein, an RSV P protein, and / or an RSV G protein.

It should be recognized that antibodies that immunospecifically bind to an RSV antigen are known in the art. For example, palivizumab (SYNAGIS®) is a humanized monoclonal antibody currently used for the prevention of RSV infection in pediatric patients. In a specific advance, an antibody to be used with the methods of the present invention is palivizumab or an antibody binding fragment thereof (e.g., a fragment containing one or more complementarity determining regions (CDRs) and preferably the palivizumab variable domain ). The palivizumab amino acid sequence is described, for example, in Johnson et al., 1997, J. Infection 176: 1215-1224, and US Patent No. 5,824,307 and International Application Publication No. WO 02/43660, entitled nMethods of Administering / Dosing Anti-RSV Antibodies for Prophylaxis and Treatment ", by Young et al., Which are incorporated herein by reference in their entirety.

One or more antibodies or antigen-binding fragments thereof that immunospecifically bind to an RSV antigen comprise an Fc domain with a greater affinity for the FcRn receptor than the palivizpmab Fc domain may also be used according to the invention. Such antibodies are described in U.S. Pat. Appn. No. 10 / 020,354, filed December 12, 2001, which is incorporated herein by reference in its entirety. Additionally, one or more of the RSV A4B4 anti-antigen antibodies; P12f2 P12f4; Plld4; Ale9; A12a6; Al3c4; Al7d4; A4B4; 1X-493L1; FR H3-3F4; M3H9; Y10H6; DG; AFFF; AFFF (I); 6H8; L1-7E5; L2-15B10; A13all; Alh5; A4B4 (1); A4B4-F52S; or A4B4L1FR-S28R may be used according to the invention. These antibodies are described in International Application Publication No. WO 02/43660 entitled "Methods of Administering / Dosing Anti-RSV Antibodies for Prophylaxis and Treatment" by Young et al., And US Provisional Patent Application 60 / 398,475 filed 25. July 2002, entitled "Methods of Treating and Preventing RSV, HMPV, and PIV Using Anti-RSV, Anti-HMPV, and Anti-PIV Antibodies" which are incorporated herein by reference in their entirety. In certain advances, RSV antigen antibodies are RSV antigen antibodies from or are prepared by the methods of U.S. Application No: 09 / 724,531, filed November 28, 2000; 09 / 996,288, filed November 28, 2001; and U.S. Pat. Publication No. US2003 / 0091584 Al, published May 15, 2003, all entitled "Methods of Administering / Dosing Anti-RSV Antibodies for Prophylaxis and Treatment", by Young et al., Which are incorporated herein by reference in their entirety. . Methods and compositions for stabilized antibody formulations that may be used in the methods of the present invention are described in U.S. Provisional Application Nos. 60 / 388,921, filed June 14, 2002, and 60 / 388,920, filed June 14, 2002, which are incorporated herein by reference in their entirety.

Antiviral therapies and their dosages, routes of administration and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (61st edition, 2007). Additional information on respiratory viral infections is available in the Cecil Textbook of Medicine (18th edition, 1988).

Antibacterial Therapies

Antibacterial agents and therapies well known to one skilled in the art for the treatment, prevention and / or management of bacterial infections may be used in the compositions and methods of the invention. Nonlimiting examples of antibacterial agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit or reduce a bacterial infection, inhibit or reduce the replication of bacteria, or inhibit or reduce the spread of bacteria to other subjects. In particular, examples of antibacterial agents include, but are not limited to, penicillin, cephalosporin, imipenem, axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol, erythromycin, clindamycin, tetracycline, streptomycin, tobramycin, gentamycin, amikacin, spectamycin, spectamycin trimethoprim, norfloxacin, rifampin, polymyxin, amphotericin B, nystatin, ketoconazole, isoniazid, metronidazole, and pentamidine.

In a preferred advance, the antibacterial agent is an agent that inhibits or reduces bacterial infection, inhibits or reduces replication of a bacterium that causes an infection, or inhibits or reduces the spread of a bacterium that causes an infection to other subjects. In cases where the bacterial infection is a mycoplasma infection (eg, pharyngitis, tracheobronchitis, and pneumonia), the antibacterial agent is preferably a tetracycline, erythromycin, or spectinomycin. In cases in which the bacterial infection is tuberculosis, the antibacterial agent is preferably rifampcin, isonaizide, pyrazinamide, ethambutol, and streptomycin.

Antibacterial therapies and their dosages, routes of administration and recommended use are known in the art and have been described in such literature as the Physicians' Desk Reference (61st edition, 2007). Additional information on respiratory infections and antibacterial therapies is available in Cecil Textbook of Medicine (18th edition, 1988).

5.4.2.7 Antifungal Therapies

Antifungal agents and therapies well known to one skilled in the art for treating, preventing and / or managing a fungal infection or one or more symptoms thereof (e.g., a fungal respiratory infection) may be used in the compositions and methods of the invention. Nonlimiting examples of antifungal agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and / or reduce fungal infection, inhibit and / or reduce the replication of fungi, or inhibits and / or reduces the spread of fungi to other subjects. Specific examples of antifungal agents include, but are not limited to, azole drugs (eg, miconazole, ketoconazole (NIZORAL®), caspofungin acetate (CANCIDAS®), imidazole, triazoles (eg fluconazole (DIFLUCAN®)), and itraconazole (SPORANOX®)), polyene (eg nystatin, amphotericin B (FUNGOSZONE®), amphotericin B lipid complex ("ABLC") (ABELCET®), amphotericin B colloidal dispersion ("ABCD") (AMPHOTEC®) , liposomal amphotericin B (AMBISONE®)), potassium iodide (Kl), pyrimidines (eg, flucytosine (ANCOBON®)), and voriconazole (VFEND®). See, for example, Table 3, below for a list of specific antifungal agents and their recommended dosages.

Table 3. Antifungal Agents

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In certain advances, the antifungal agent is an agent that inhibits or reduces a fungal infection, inhibits or reduces replication of a fungus that causes an infection, or inhibits or reduces the spread of a fungus that causes an infection to other subjects. In cases where the fungal infection is Blastomyces dermatitidis, the antifungal agent is preferably itraconazole, amphotericin B, fluconazole, or ketoconazole. In cases where the fungal infection is pulmonary aspergilloma, the antifungal agent is preferably amphotericin B, liposomal amphotericin B, itraconazole, or fluconazole. In cases where the fungal infection is histoplasmosis, the antifungal agent is preferably amphotericin B, itraconazole, fluconazole, or ketoconazole. In cases where the fungal infection is coccidioidomycosis, the antifungal agent is preferably fluconazole or amphotericin B. In cases where the fungal infection is cryptococcosis, the antifungal agent is preferably amphotericin B, fluconazole, or a combination of both agents. In cases where the infection is chromomycosis, the antifungal agent is preferably itraconazole, fluconazole, or flucytosine. In cases where the fungal infection is mucormycosis, the antifungal agent is preferably amphotericin B or liposomal amphotericin B. In cases in which the pulmonary or respiratory fungal infection is pseudoalesqueriasis, the antifungal agent is preferably itraconazole or miconazole.

Antifungal therapies and their dosages, routes of administration, and recommended use are known in the art and have been described in such literature as, for example, Dodds et al., 2000 Pharmacotherapy 20 (11) 1335-1355, the Physicians' Desk Reference (61st edition, 2007) and the Merck Manual of Diagnosis and Therapy (17th edition, 1999).

Antiprotozoal Therapies

Antiprotozoal agents and therapies well known to one skilled in the art for treating, preventing and / or managing a protozoal infection or one or more symptoms thereof (for example, a respiratory infection associated with a protozoan infection) may be used in the compositions and methods of the invention. Non-limiting examples of antiprotozoal agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and / or reduce protozoan infection, inhibit and / or reduce protozoan replication, or inhibits and / or reduces the spread of protozoa to other subjects. Specific examples of antiprotozoal agents include, but are not limited to, chloroquine phosphate (Aralen ™); quinine sulfate plus one of the following: doxycycline, tetracycline, or clindamycin; atovaquone-proguanyl (Malarone ™); Mefloquina (Lariam ™); metronidazole (Flagyl); tinidazole (Tindamax); 5-nitroimidazole (ornidazole), and agents described in U.S. Patent No. 6,440,936.

In certain advances, the antiprotozoan agent is an agent that inhibits or reduces a protozoan infection, inhibits or reduces replication of a protozoan that causes an infection, or inhibits or reduces the spread of a protozoan that causes an infection in other subjects. In cases in which protozoan infection is trichomoniasis, the antiprotozoal agent is preferably metronidazole (Flagyl), tinidazole (Tindamax), or 5-nitroimidazole (ornidazole). In cases where protozoal infection is malaria, the antiprotozoal agent is preferably chloroquine phosphate (Aralen ™); quinine sulfate plus one of the following: doxycycline, tetracycline, or clindamycin; quinidine gluconate plus one of the following: docicycline, tetracycline, or clindamycin; Fansidar ™; Malarone ™ (atovaquone 250 mg plus proguanyl 100 mg); or Mefloquina (Larium ™).

Antiprotozoal therapies and their dosages, routes of administration, and recommended use are known in the art and have been described in such literature as, for example, Dodds et al., 2000 Pharmacotherapy 20 (11) 1335-1355, The Physicians Desk Reference (61st edition) , 2007); the Merk Manual of Diagnosis and Therapy (17th edition, 1999); and publications provided by the Center for Disease Control and Prevention (CDC; http://www.cdc.gov) (Atlanta, GA).

EphA2-BiTEs Biological Activity

Various in vitro and in vivo tests known in the art and described in detail below in Section 6 may be used to determine the biological activity of EphA2-BiTEs produced by the methods of the invention. Such in vitro and in vivo assays allow for the filtration and identification of EphA2-BiTEs with desired biological activities such as, for example, target cell specificity and efficient lysis of said cells. In particular, the invention provides methods for determining target cell specificity (e.g., the ability of EphA2-BiTEs to immunospecifically bind to cells expressing EphA2) using various immunoassays. The binding characteristics of the EphA2-BiTEs of the invention may also be determined using, for example, surface plasmon resonance to identify the affinity constants (KD, Kon and Koff) of the EphA2-BiTEs produced by the methods of the invention. The present invention further provides assays that can be used to filter out EphA2-BiTEs with specific biological activities, such as the ability to bind to target cells (e.g., tumor cells showing increased EphA2 expression) to mediate. in vitro lysis using various cytotoxicity assays. Such tests may be used to determine toxicity and efficacy. of the EphA2-BiTEs of the invention (e.g., to determine the lethal dose for 50% of the EphA2-BiTEs (LD50) treated cell population). Also provided are tests that can be used to determine the ability of the EphA2-BiTEs of the invention to mediate tumor cell death in vivo using animal models.

Tests to determine target cell specificity

Several assays known to one skilled in the art can be used to determine the ability of the EphA2-BiTEs of the invention to immunospecifically bind to cells expressing EphA2 and CD3 (e.g., cells showing enhanced EphA2 expression or cells in which certain epitopes of EphA2 are selectively exposed). Such assays include, for example, RIAs, ELISA, Western Blot, immunohistochemistry, immunofluorescence and flow cytometry tests. For example, as described in Section 6.2.3 below, flow cytometry based assays can be used to determine bispecific cell binding of the various EphA2-BiTE constructs. EphA2 positive cell lines, such as A54 9 cells (human lung carcinoma cells) and MDA-MB-231 cells (human breast cancer cells) can be used as target cells, and HP-Ball positive cells for CD3 (human T cell line) can be used as effector cells. The cells may be mixed together and incubated with an EphA2-BiTE of interest. Flow cytometry can be used to determine the binding intensities for each EphA2 positive target cell.

Epitope exclusion analyzes can be conducted using immunofluorescence labeling to determine if the EphA2-BiTEs produced by the methods of the invention retain epitope exclusion as the parental antibodies from which they are derived. See Section 6.2.4 below.

Surface plasmon resonance tests

Surface plasmon resonance tests can be performed to determine the binding characteristics of the EphA2-BiTEs of the invention. The affinity and dissociation constants as well as the association and dissociation rates (ka, kD, kon and koff) of the EphA2-BiTEs produced by the methods of the invention can be identified using this test. See, for example, 6.8.3 below. For example, surface plasmon resonance tests may be performed using the CM5 Sensor Chip (Biacore AB, üppsala, Sweden) which contains a carboxymethyl dextran (CM) matrix and a Biacore® 3000 surface plasmon resonance biosensor. (SPR) (Biacore AB, Uppsala, Sweden). CD3εγ is covalently associated with the CM dextran matrix using amine chemical coupling. A reference surface is created by default of the CD3εγ coupling step. An EphA2-Fc is captured via a high affinity interaction between the Fc portion of EphA2Fc and the goat anti-human IgG (Fc) (KPL, Inc., Gaithersburg, MD). (Fc) of anti-human IgG goat is covalently associated with CM dextran matrix using chemical amine coupling. Two specific surfaces for (Fc) anti-human IgG are then created. One of these surfaces is used as a reference surface while the other surface is used to create an EphA2 Fc-specific surface. Different concentrations of bscEphA2 x CD3 are then prepared by serial dilution in HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20 surfactant). EphA2-BiTEs are injected in a stream-serial manner through the CD3εγ-specific or EphA2-specific surface and its corresponding reference surface. Dissociation of bound EphA2-BiTEs is monitored in the presence of HBS-EP. Remaining bound material was removed with 10 mM disodium tetraborate pH 8.5, 1 M NaCl (for CD3εγ specific surface) or 10 mM glycine pH 1.7 (for EphA2 Fc specific surface).

Cytotoxicity Tests As illustrated in Section 6.3.1 below, flow cytometry-based redirected cell cytotoxicity tests can be performed to determine the cytotoxic activity of EphA2-Bite with effector cell donors. For example> CD3 + T-cell enriched human peripheral blood mononuclear cells ("PBMCs") may be isolated from healthy donors. Target cells, for example, cells expressing EphA2, are labeled with a fluorescent membrane dye such as (Di0C18 (3) or "DiO"). The cells are incubated together with an EphA2-BiTE of interest and are analyzed by flow cytometry after the addition of propidium iodide (PI). Cell lysis of targets can then be calculated as the percentage of PI-positive stained DiO positive cells.

In a specific advance, an EphA2-BiTE of the invention mediates the lysis of cells associated with aberrant EphA2 expression and / or activity (e.g., cancer cells, noncancerous hyperproliferative cells, or infected cells expressing EphA2). According to this advance, such cells are reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% , at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least at least 5 times, at least 7 times or at least 10 times relative to the level of normal cells of the same cell line or subject, cells of a normal healthy subject and / or a normal healthy cell population. In another specific advance, EphA2-BiTEs do not mediate lysis of normal tissue cells or normal tissue cells of a healthy or control subject.

The biological activity (eg, anticancer, antihyperproliferation, or anti-infective activities) of the therapies used in accordance with the present invention may also be determined by the use of various experimental animal models for the study of cancer, for example. , the SCID mouse model or transgenic mouse where a mouse EphA2 is replaced by the human EphA2, human xenograft nude mice, animal models described in Section 6 below, or any animal model (including hamsters, rabbits, etc.) known in the art. art and described in Relevance of Tumor Models for Anticancer Drug Development (1999, eds. Fiebig and Burger); Contributions to Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991, eds. Boven and Winograd); and Anticancer Drug Development Guide (1997 ed. Teicher), incorporated herein by reference in their entirety.

In specific advances, the cytotoxic effects of the EphA2-BiTEs of the invention may also be tested in vivo with a mouse model such as the non-diabetic combined severe immunodeficiency (NOD / SCID) mouse model using a tumor xenograft such as for example, human colon carcinoma cell line SW480. The SW480 cell line can be selected for establishment of a human xenoimplant model because SW480 cells express EphA2. Briefly, animals may be injected with a mixture of target cells (eg SW480 cells) and effector cells (human donor CD3 + T cells from healthy donors) or a target cell alone without effector cells. Tumor growth kinetics can be measured between the two treatment groups. See, for example, Section 6.4.1 below.

Accordingly, the toxicity and efficacy of the prophylactic and / or therapeutic protocols of the present invention may be determined by standard pharmaceutical procedures in experimental cell or animal cultures, for example to determine LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). One dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the LD50 / ED50 ratio. Prophylactic and / or therapeutic agents exhibiting high therapeutic indices are preferred. While prophylactic and / or therapeutic agents exhibiting toxic side effects may be used, care should be taken to design a delivery system that directs such agents to the site of affected tissue to minimize potential damage to uninfected cells and thus reduce side effects. Specific examples of methods The biological activity of the EphA2-BiTEs of the invention are also provided in Section 6 of Examples, infra.

Data obtained from cell culture tests and

Animal studies may be used in formulating a dosage scale for prophylactic and / or therapeutic agents for use in humans. The dosage of such agents preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration employed. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture tests. The dose may be formulated in animal models to achieve a plasma circulating concentration scale that includes the IC50 (i.e., the concentration of the tested component that reaches half of the maximum inhibition of symptoms) as determined in cell culture. Such information may be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

Compounds for use in therapies may be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, hamsters, etc., for example, the animal models described. above. The compounds may then be used in appropriate clinical trials.

Additionally, any tests known to those skilled in the art may be used to assess the prophylactic and / or therapeutic utility of the corobinatory therapies described herein for treating or preventing a disorder associated with aberrant EphA2 expression and / or activity (e.g., cancer, a non-hyperproliferative cell disorder or an infection).

Compositions

The present invention provides compositions comprising one or more of the EphA2-BiTEs of the invention. The compositions of the invention also include large scale drug compositions useful in the manufacture of pharmaceutical compositions (e.g. impure or non-sterile compositions) and pharmaceutical compositions (i.e. compositions that are suitable for administration to a subject or patient) which may be used in the preparation of unit dosage forms. Such compositions comprise the prophylactically or therapeutically effective amount of a prophylactic and / or therapeutic agent described herein or a combination of these agents and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise the prophylactically or therapeutically effective amount of one or more EphA2-BiTEs of the invention and a pharmaceutically acceptable carrier. In a further advance, the composition of the invention further comprises an additional therapy which is not an EphA2-BiTE.

In a specific advance, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which therapy is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, synthetic or vegetable origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. similar. Water is a preferable carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone gel, sodium stearate, glycerol monostearate, talc, sodium chloride, milk powder, glycerol, propylene, glycol. , water, ethanol and the like. The composition, if desired, may also contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

Generally, the ingredients of the compositions of the invention are supplied either separately or mixed together in unit dosage form, for example as a freeze-dried or water-free concentrated dry powder in a hermetically sealed container such as an ampoule or sachet indicating the amount of the active agent. Where the composition is to be infused, it may be administered with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, a sterile or saline injection vial may be employed so that the ingredients may be mixed prior to administration.

The compositions of the invention may be formulated as neutral or saline forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonia, calcium. , iron hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

Various delivery systems are known and may be used to administer an EphA2-BiTE of the invention or a combination of an EphA2-BiTE of the invention and a prophylactic or therapeutic agent useful for treating, preventing and / or managing a disorder associated with aberrant expression. (e.g., increased expression) and / or EphA2 activity (e.g., cancer, a non-cancerous hyperproliferative cell disorder, or an infection), for example microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment ( see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods for administering a prophylactic or therapeutic agent of the invention include, but are not limited to, parenteral (e.g. intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g. intranasal, inhaled, and oral routes) administration. ). In a specific advance, prophylactic or therapeutic agents of the invention are administered intramuscularly, intravenously, or subcutaneously. Prophylactic or therapeutic agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through the mucocutaneous or epithelial lines (e.g., oral, rectal and intestinal mucosa, etc.) and may be administered together. with other biologically active agents. Administration may be systemic or local.

In a specific advance, it may be desirable to administer the prophylactic or therapeutic agents of the invention locally in the area in need of treatment; this may be achieved, for example, and not by limitation, by local infusion, by injection, or by an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes such as, for example. , sialastic membranes, or fibers.

In yet another advance, the prophylactic or therapeutic agent may be delivered in a controlled release or extended release system. In one advance, a pump may be used to achieve controlled or prolonged release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another advancement, polymeric materials may be used to achieve controlled or prolonged release of the EphA2-BiTEs of the invention or fragments thereof (see for example. Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974), Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23 : 61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; International Publication Nos. WO 99/15154 and WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxy ethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate). , poly (methacrylic acid), polyglycolides (PLG), polyanhydrides, poly (N-vinyl pyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactides (PLA), poly (lactide co-glycolides) ) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in the sustained release formulation is inert, free from leached impurities, stock stable, sterile, and biodegradable. In yet another advancement, the sustained or controlled release system may be placed in close proximity to the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Releaser supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533). Any technique known to one skilled in the art may be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, for example, ü.S. Patent No. 4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698; Ning et al., 1996, Radiotherapy & Oncology 39: 179-189; Song et al. , 1995, PDA Journal of Pharmaceutical Science & Technology 50: 372-397; Cleek et al., 1997, Pro. Int'l. Symp. Control Bioact. Mater. 24: 853-854; and Lam et al., 1997, Proc. IntfI. Symp. King Control. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in its entirety. Formulations

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

Thus, the EphA2-BiTEs of the invention and their physiologically acceptable salts and solvents may be formulated for administration by inhalation or insufflation (either by mouth or nose) or oral, parenteral or mucosal administration (such as mouth, vaginal, rectal). , sublingual). In a preferred advance, local or systemic parenteral administration is used.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (eg pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose). ; fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry build up product in water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (eg almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffering salts, flavoring agents, coloring agents and sweeteners as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For oral administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, prophylactic or therapeutic agents for use in accordance with the present invention are conveniently delivered in the form of a pressurized packet aerosol spray presentation or a nebulizer, using a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane. , dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing that the valve deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a mixture of powders of the compound and a suitable base powder such as lactose or starch.

Prophylactic or therapeutic agents may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers, with an added condom. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example sterile pyrogen-free water, prior to use.

Prophylactic or therapeutic agents may also be formulated in rectal compositions such as suppositories or retention yams, for example containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations previously described, prophylactic or therapeutic agents may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or

intramuscularly) or by intramuscular injection. Thus, for example, prophylactic or therapeutic agents may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example as a sparingly salt. soluble.

The invention also provides that a prophylactic or therapeutic agent is packaged in a hermetically sealed container such as an ampoule or sachet indicating the amount. In one advance, the prophylactic or therapeutic agent is provided with a sterile freeze-dried dry concentrate or water-free concentrate in a hermetically sealed container and may be reconstituted, for example, with water or saline to the appropriate concentration for administration to a subject.

In a preferred embodiment of the invention, the formulation and administration of various chemotherapeutic, biological / immunotherapeutic and hormonal agents are known in the art and often described in the Physicians' Desk Reference (61st edition, 2007). For example, in certain specific advances of the invention, the therapeutic agent of the invention may be formulated and provided as shown in Table 2.

In other advances of the invention, radioactive therapeutic agents such as radioactive isotopes may be given orally as liquid in capsules or as a beverage. Radioactive isotopes may also be formulated for intravenous injections. The skilled oncologist may determine the preferred formulation and route of administration.

In certain advances the EphA2-BiTEs of the invention are formulated at 1 mg / ml, 5 mg / ml, 10 mg / ml, and 25 mg / ml for intravenous injections and at 5 mg / ml, 10 mg / ml, and 80 mg. mg / ml for repeated subcutaneous administration and intramuscular injection.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The package may, for example, comprise metal or plastic foil, such as a bubble wrap. The package or dispensing device may be accompanied by instructions for administration.

Dosage and Frequency of Administration

Frequency and dosage will also vary according to patient-specific factors depending on the specific therapies (eg, specific therapeutic or prophylactic agent or agents) administered, the severity of the disorder, disease or condition, route of administration, as well as age. , body, weight, response, and the patient's past medical history. For example, the dosage of a prophylactic or therapeutic agent or composition of the invention that will be effective in treating, preventing, and / or managing a disorder associated with aberrant expression (e.g., increased expression) and / or EphA2 activity (e.g. cancer, a non-cancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, may be determined by administering a composition to an animal model such as, for example, the animal models described herein or known herein. from those art experts. In addition, in vitro tests may optionally be employed to help identify optimal dosage ranges. Appropriate regimens may be selected by an art expert in considering such factors and following, for example, dosages reported in the literature and recommended in the Physiciansr Desk Reference (61st edition, 2007).

Exemplary doses of a small molecule include amounts in milligrams or micrograms of the small molecule per kilogram of the subject or sample weight (for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram up to about 5 milligrams per kilogram, or about 1 microgram per kilogram to up to 50 micrograms per kilogram). For antibodies, proteins, polypeptides, peptides and fusion proteins comprised by an invention, the dosage administered to a patient is typically from 0.0001 mg / kg to 100 mg / kg body weight of a patient. Preferably, the dosage administered to a patient is between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg at 0 , 25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg or 0, 01 to 0.10 mg / kg body weight of the patient. Generally, human antibodies have a longer half-life within the human body than antibodies of other species due to the immune response to foreign polypeptides. Thus, small dosages of human antibodies and less frequent administrations are often possible. In addition, the dosage and frequency of administration of the antibodies of the invention or fragments thereof may be reduced by increasing the absorption and tissue penetration of the antibodies by modification such as, for example, lipidification.

Exemplary dosages of the EphA2-BiTEs of the invention administered to a patient are typically 0.01 µg to 100 mg. A particularly preferred dosage is 0.1 µg to 10 mg, more preferably 1 µg to 100 µg, and more preferably 3 µg to 10 µg. In a specific advance, the dosage of the EphA2-BiTEs (e.g. antibodies, compositions, or combination therapies of the invention) administered to treat, prevent and / or manage a disorder associated with aberrant expression (e.g. increased expression) and / or EphA2 activity (eg, cancer, a non-cancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, in a patient is 100 mg / kg or less, 50 mg / kg or less, 25 mg / kg kg or less, 10 mg / kg or less, 1 mg / kg or less, 500 pg / kg or less, 400 pg / kg or less, 300 pg / kg or less, 200 pg / kg or less, 150 pg / kg or less, preferably 125 pg / kg or less, 100 pg / kg or less, 95 pg / kg or less, 90 pg / kg or less, 85 pg / kg or less, 80 pg / kg or less, 75 pg / kg or less, 70 pg / kg or less, 65 pg / kg or less, 60 pg / kg or less, 55 pg / kg or less, 50 pg / kg or less, 45 pg / kg or less, 40 pg / kg or 35 pg / kg or less, 30 pg / kg or less, 25 pg / kg or less, 20 pg / kg or less, 1 5 pg / kg or less, 10 pg / kg or less, 5 pg / kg or less, 2.5 pg / kg or less, 2 pg / kg or less, 1.5 pg / kg or less, 1 pg / kg or less, 0.5 pg / kg or less, or 0.5 pg / kg or less of a patient's body weight. In another advance, the dosage of the EphA2-BiTEs or combination of therapies of the invention administered to treat, prevent and / or manage a disorder associated with aberrant expression (eg, increased expression) and / or EphA2 activity (e.g., cancer, a non-cancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, in a patient is a unit dose of 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2, 5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2 0.5 mg.

In other advances, a subject is administered with one or more doses of an effective amount of one or more EphA2-BiTEs of the invention, where a dose in an effective amount achieves a serum titration of at least 0.1 µg / ml. 0.5 µg / ml at least 1 µg / ml at least µg / ml at least 5 µg / ml at least 6 µg / ml at least 15 µg / ml at least 20 µg / ml µg / ml, at least 25 µg / ml, at least 50 µg / ml, at least 100 µg / ml, at least 125 µg / ml, at least 150 µg / ml, at least 175 µg / ml, at least 200 µg at least 225 µg / ml, at least 250 µg / ml, at least 275 µg / ml, at least 300 µg / ml, at least 325 µg / ml, at least 350 µg / ml, at least 375 µg / ml ml, or at least 400 µg / ml of the EphA2-BiTEs of the invention. In still further advances, a subject is administered a dose of an effective amount of one or more EphA2-BiTEs of the invention to achieve a serum titration of at least 0.1 µg / ml, at least 0.5 µg / ml. 1 µg / ml at least 2 µg / ml at least 5 µg / ml at least 6 µg / ml at least 10 µg / ml at least 15 µg / ml at least 20 µg / ml at least 25 µg / ml, at least 50 µg / ml, at least 100 µg / ml, at least 125 µg / ml, at least 150 µg / ml, at least 175 µg / ml, at least 200 µg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml, or at least 400 µg / ml of antibodies and a subsequent dose of an effective amount of one or more EphA2-BiTEs of the invention is administered to maintain a serum titration of at least 0.1 µg / ml, 0.5 µg / ml, 1 µg / ml / at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 g / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml at least 350 μg / ml, at least 375 μg / ml, or at least 400 μg / ml. According to these advances, a subject may be administered with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more subsequent doses.

In a specific advance, the invention provides methods for treating, preventing and / or managing a disorder associated with aberrant expression (e.g., increased expression) and / or EphA2 activity (e.g., cancer, a non-cancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, said method comprising administering to a subject in need thereof a dose of at least 10 µg, preferably at least 15 µg, at least 20 µg, at least 25 µg, at least 30 µg , at least 35 µg, at least 40 µg, at least 45 µg, at least 50 µg, at least 55 µg, at least 60 µg, at least 65 µg / at least 70 µg, at least 75 µg, at least 80 µg / at least 85 µg, at least 90 µg, at least 95 µg, at least 100 µg, at least 105 µg, at least 110 µg, at least 115 µg, at least 120 µg, at least 150 µg.? at least 300 µg, at least 400 µg, at least 500 µg, at least 1 mg at least 5 mg, at least 10 mg, at least 25 mg, at least 50 mg, or at least 100 mg of one or more EphA2-BiTEs, combination therapies, or compositions of the invention. In another advancement, the invention provides a method for treating, preventing and / or managing a disorder associated with aberrant expression (e.g., increased expression) and / or EphA2 activity (e.g. cancer, a non-cancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, said methods comprising administering to a subject in need thereof a dose of at least 10 µg, preferably at least 15 µg, at least 20 µg, at least 25 µg, at least 30 µg , at least 35 µg, at least 40 µg, at least 45 µg, at least 50 µg, at least 55 µg, at least 60 µg, at least 65 pg, at least 70 pg, at least 75 µg, at least 80 µ9 , at least 85 µg, at least 90 µg, at least 95 µg, at least 100 µg, at least 105 µg, at least 110 µg, at least 115 µg, or at least 120 µg of one or more EphA2-BiTEs, combination of therapies, or compositions of the invention as a continuous infusion once the preferably 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 10 days, once every two weeks, once every three weeks, or once a month.

The present invention provides methods for treating, preventing, and / or managing a disorder associated with aberrant expression (e.g., increased expression) and / or EphA2 activity (e.g., cancer, a noncancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, said method comprising: (a) administering to a subject in need thereof one or more doses of a prophylactically or therapeutically effective amount of one or more EphA2-BiTEs, combination therapies, or compositions of the invention; and (b) monitoring the level / plasma concentration of said EphA2-BiTEs administered to said subject following administration of a number of doses of said EphA2-BiTEs. Additionally, said certain dose numbers are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of a prophylactically or therapeutically effective amount of one or more EphA2-BiTEs, compositions, or combination of therapies of the invention. In another advance, the dose is administered as a continuous intravascular infusion.

In a specific advance, the invention provides a method for treating, preventing and / or managing a disorder associated with aberrant expression (e.g., increased expression) and / or EphA2 activity (e.g. cancer, a non-cancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, said method comprising: (a) administering to a subject in need thereof a dose of at least 10 pg (preferably at least 15 pg, at least 20 µg, at least 25 pg, at least 30 pg, at least 35 pg, at least 40 pg, at least 45 pg, at least 50 pg, at least 55 pg, at least 60 pg, at least 65 pg, at least 70 pg, at least 75 pg, at least 80 pg, at least 85 pg, at least 90 pg, at least 95 pg, or at least 100 pg) of one or more EphA2-BiTEs of the invention; and (b) administering one or more subsequent doses to said subject when the plasma level of EphA2-BiTE administered to said subject is less than 0.1 pg / ml, preferably less than 0.25 pg / ml, less than than 0.5 pg / ml, less than 0.75 pg / ml, or less than 1 pg / ml. In another advancement, the invention provides a method for treating, preventing and / or managing a disorder associated with aberrant expression (e.g., increased expression) and / or EphA2 activity (e.g., cancer, a noncancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, said method comprising: (a) administering to a subject in need thereof one or more doses of at least 10 pg (preferably at least 15 pg, at least 20 pg, at least 25 pg , at least 30 pg, at least 35 pg, at least 40 pg, at least 45 pg, at least 50 pg, at least 55 pg, at least 60 pg, at least 65 pg, at least 70 pg, at least 75 pg at least 80 pg, at least 85 pg, at least 90 pg, at least 95 pg, or at least 100 pg) of one or more antibodies of the invention; (b) monitoring the plasma level of the EphA2-BiTEs of the invention administered to said subject after administration of a number of doses; and (c) administering a subsequent dose of the EphA2-BiTEs of the invention when the plasma level of EphA2-BiTE administered to said subject is less than 0.1 pg / ml, preferably less than 0.25 μg / ml, lower than than 0,5 pg / ml, less than 0,75 pg / ml, or less than 1 μg / ml. In one advance, said number of doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of an effective amount of one or more EphA2-BiTEs of the invention or may administered as a continuous intravascular infusion.

Therapies (e.g., prophylactic or therapeutic agents) other than the EphA2-BiTEs of the invention which have been or are currently being used to treat, prevent and / or manage a disorder associated with aberrant expression (eg, increased expression). ) and / or EphA2 activity (e.g. cancer, a non-cancerous hyperproliferative cell disorder, or an infection) or one or more symptoms thereof may be administered in combination with one or more EphA2-BiTEs according to the methods of the invention for treat, prevent, and / or manage a disorder associated with aberrant expression (eg, increased expression) and / or EphA2 activity (eg, cancer, a noncancerous hyperproliferative cell disorder, or an infection) or one or more symptoms thereof. Preferably, the dosages of the prophylactic or therapeutic agents used in combination with therapies of the invention are lower than those which have been or are currently being used to treat, prevent and / or manage a disorder associated with aberrant expression (e.g., increased expression). and / or EphA2 activity (e.g., cancer, a noncancerous hyperproliferative cell disorder, or an infection) or one or more symptoms thereof. The recommended dosages of agents commonly used for the treatment, prevention and / or management of a disorder associated with aberrant expression (eg, increased expression) and / or EphA2 activity (eg, cancer, a noncancerous hyperproliferative cell disorder, or an infection), or one or more symptoms thereof, may be obtained from any reference in the art including, but not limited to, Hardman et al., eds., 2001, Goodman & Gilman's The Pharmacological Basis Of Basis Of Therapeutics, IOth ed., Mc-Graw-Hill, New York; Physicians' Desk Reference (61st edition, 2007), Medical Eeonomics Co., Inc., Montvale, NJ, which are incorporated herein by reference in their entirety.

In various advances, therapies (eg, prophylactic or therapeutic agents) are administered as a continuous infusion, less than 5 minutes between them, less than 30 minutes between them, 1 hour between them, about 1 hour between them. , from about 1 to about 2 hours between them, around 2 hours to around 3 hours between them, around 3 hours to around 4 hours between them, around 4 hours to around 5 hours between them, around 5 hours to around 6 hours between them, around 6 hours to around 7 hours between them, around 7 hours to around 8 hours between them, around from 8 hours to around 9 hours between them, around 9 hours to around 10 hours between them, around 10 hours to around 11 hours between them, around 11 hours to around 12 hours hours between them, around 12 hours to 18 hours between them, 18 hours to 24 hours between them, 24 hours to 36 hours between them, 36 hours to 48 hours between them, 48 hours 52 hours between them, 52 hours to 60 hours between them, 60 hours to 72 hours between them, 72 hours to 84 hours between them, 84 hours to 96 hours between them, or 96 hours to 120 hours between them. In preferred advances, two or more therapies are administered within the same patient visit.

In certain advances, one or more antibodies of the invention and one or more other therapies (e.g., prophylactic or therapeutic agents) are cyclically administered. Cyclic therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally followed by administering a third therapy (e.g., prophylactic or therapeutic agent) for a period of time and so on, and repeating this sequential administration, i.e. the cycle in order to reduce the development of resistance to one of the therapies, to prevent or reduce the side effects of one or more of the therapies, and / or to increase the effectiveness of the therapies. In certain advances, administration of the same EphA2-BiTEs of the invention may be repeated and administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In other advances, administration of a therapy (e.g., a prophylactic or therapeutic agent) other than an EphA2-BiTE of the invention may be repeated and administration may be separated by at least 1 day, 2 days, 3 days, 5 days. days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

Kits

The invention provides a pharmaceutical package or kit comprising one or more containers filled with one or more EphA2-BiTEs of the invention, polynucleotides encoding said EphA2-BiTEs, and vectors expressing said polynucleotides. Additionally, one or more other prophylactic or therapeutic agents useful for treating a disorder associated with aberrant expression (i.e., increased expression) and / or EphA2 activity (e.g., cancer, a non-cancerous hyperproliferative cell disorder, or an infection ) may also be included in the pharmaceutical package or kit. The invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container (s) may be a note in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, such note reflecting the agency's approval of the manufacture, use, or sale for human administration. .

The present invention provides kits that can be used in the above methods. In one advance, a kit comprises, in one or more containers, one or more EphA2-BiTEs of the invention and instructions for use. In a further advance, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of a disorder associated with aberrant expression (i.e., increased expression) and / or EphA2 activity (e.g., cancer, a hyperproliferative cell disorder). cancer, or an infection) in one or more containers. In a specific advance, EphA2-BiTE is deimmunized anti-CD3xEA2 (VH / VL). In certain advances, the other prophylactic or therapeutic agent is a chemotherapeutic. In other advances, the prophylactic or therapeutic agent is a biological or hormonal therapy. In still other advances, the prophylactic or therapeutic agent is an antiviral, antifungal, antiprotozoal, antibacterial, or anti-autoimmune agent.

The present invention further provides kits comprising any of the diagnostic compositions discussed above.

EXAMPLES The following non-limiting examples demonstrate the production of EphA2-BiTEs and their characterization.

EphA2-BiTE GENERATION

Construction of Eight EphA2-Specific BiTEs from Two Monoclonal Antibodies

Domain-coding Plasmids

variables of two anti-EphA2 murine antibodies called EA2 and EA5 as described in U.S. Pub. No. US 2004-0091486 A1 and Appln. Serial No. 11 / 544,322, filed October 6, 2006, were used to construct EphA2-BiTE.

See also FIGS. 1 and 2, respectively. An anti-CD3 single chain antibody, a de-immunized derivative of the human CD2-specific murine monoclonal antibody L2K called diL2K was used for fusion with EphA2 single chain antibodies (see Dreier et al., 2002, Int. J.

Cancer 100: 690-697, which is incorporated herein by reference in its entirety). DNAs c for eight different BiTE constructs were generated by PCR based fusion. BiTE constructs had the following variable domain (VH and VL) arrays and antibody placement

EphA2 and anti-CD3 Single Chain: BiTE Constructs Made

<table> table see original document page 281 </column> </row> <table>

The structure of BiTE cDNA is shown in FIG. 3. The cloned insertions in the pEF-DHFR expression vector each comprising a leader with a 5'-terminal Kozak site for increased translation efficiency and a secretory signal sequence (murine Ig heavy chain leader). The leader is followed by four variable Ig domains listed above. The linker peptide at the anti-EphA2 V1-Vh or VH-VL junction is 15 amino acids long (three repeats of G4S motif; SEQ ID NO: 59). The linker peptide connecting the EphA2 with CD3 binding specificity comprises a repeat of the G4S motif (SEQ ID NO: 58). Directly adjacent to the fourth domain is a hexahistidine (H6) C-terminal sequence (SEQ ID NO: 66) for detection and purification purposes. The indicated restriction enzyme sites were used to clone the BiTE constructs in the expression vector.

Vectors for BiTE Expression in CHO Cells

The pEF-DHFR vector is a 5.8 kb vector with murine dihydrofolate reductase as the selection marker forming a bi-cistronic transcriptional unit together with the gene to be expressed under the control of the human promoter EFlα. The eukaryotic expression vector is a derivative of the pMT2PC expression vector.

The EFla promoter is followed by a multiple cloning site, an internal ribosomal entry site (IRES), the DHFR gene, and a polyadenylation signal. Additional control sequences present in the vector are the origin of bacterial replication (ORI) and the gene for ampicillin resistance (bla). In the following table, the various elements of the cDNA expression vector are expression are summarized.

<table> table see original document page 282 </column> </row> <table> <table> table see original document page 283 </column> </row> <table> <table> table see original document page 284 < / column> </row> <table>

A description of the vector is shown in FIG. 4

CHO Container Cell Line

The Chinese hamster ovary (CHO) CH0 / dhfrˉ cell line, CRL 9096, was purchased from the American Type Culture Collection ("ATCC"; Manassas, Virginia), and was characterized by Q-One, UK. Hypoxanthine and thymidine t / 6in 10 should be added as supplements to the culture medium because the CHO / dhFr cell line is deficient in dihydrofolate reductase.

BiTE Expression in CHO Cells

Following transfection of CHO / dhfr "cells with the eight expression vectors encoding EphA2-based BiTE constructs, each cell pool was grown in May of HyQ PF CHO nucleoside free liquid soybean (with 4.0 mM L-Glutamine with Pluronic F - 68 to 0.1%; Cat. # SH30359.02; HyClone) for selection of DHFR-positive transfectants Subsequently, the transfected cell pool was subjected to a single gene amplification step using DHFR inhibitor methotrexate (MTX) at a concentration of 20 nM as a nucleoside free medium supplement Purification of Culture Supernatants EphA2-BiTEs

Ãkta® (Amersham) FPLC system and Unicorn® computer program were used for chromatography. Immobilized metal affinity chromatography ("IMAC") was performed using a Fractogel © (Merck) column which was loaded with ZnCl2 according to the protocol provided by the manufacturer. The column was equilibrated with buffer A (20 mM sodium phosphate buffer pH 7.5, 0.4 M NaCl) and the cell culture supernatant (500 mL) was applied to the column (10 mL) at a rate flow rate 3 ml / min. The column was washed with buffer A to remove unbound sample. Bound proteins were eluted using a two step gradient of buffer B (2mM sodium phosphate buffer pH 7.5, 0.4 M NaCl, 0.5 M Imidazole) as follows:

Step 1: 10% Buffer B in 6 column volumes;

Step 2: 100% Buffer B in 6 column volumes.

Protein fractions eluted by step 2 were pooled for further purification.

Gel filtration chromatography was performed on a Sephadex 200 HiPrep (Amersham) column equilibrated with PBS (Gibco) (FIG. 5). Eluted protein samples (at a flow rate of 1 ml / min) were subjected to detection by standard SDS-PAGE and Western Blot (FIG. 6). Prior to purification, the column was calibrated for molecular weight determination (molecular weight marker kit, Sigma MW GF-200. Protein concentrations were determined using protein dye for testing (MicroBCA, Pierce) and IgG (Biorad) as protein. standard.

BiTE EphA2s Monkey CD3 Reactive Substitutes for Toxicity Studies

Cytometric Binding Analysis of Cinomolgus-Reactive Parental Antibodies

As a starting point for the EphA2-BiTE constructs to be tested for toxicology in cinomolgus monkeys, peripheral blood mononuclear cells ("PBMCs") of cinomolgus monkeys were used to detect CD3 binding of two cinomolgus reactive parent CD3 antibodies, cCD3. -1 and cCD3-2, both conjugated to FITC. For each sample 200,000 cells were incubated with their antibodies for 30 minutes on ice. Subsequently the cells were washed twice with PBS. As negative control an irrelevant antibody was used (black line). CD3 binding is represented by gray lines in histogram overlays. Cells were analyzed by flow cytometry on a FACS-Calibur (Becton Dickinson, Heidelberg). Both antibodies showed distinct binding to the cinomolgus BMC T-cell fraction. See FIG. 7. Substitute Molecules with a Substituted Target Specificity

The variable domains of the parental anti-CD3 antibodies cCD3-1 and cCD3-2 were used to construct substituted target specificity BiTE molecules. Single chain antibodies in different domain arrays were created by PCR based fusion. The following BiTE constructs were generated by combining these specificities with a surrogate target specificity:

<table> table see original document page 287 </column> </row> <table>

Cloning and transfection of surrogate constructs were performed as described above.

Evidence for Cross-Reactivity of Monoclonal Antibodies EA2 and EA5 with Rhesus EphA2

The Rhesus EphA2 sequencing of the

Rhesus CMMT110 / CL strain (97.7% human EphA2 homology) and partial sequencing of cinomolgus spleen cells (> 99% human EphA2 homology) suggested that human EphA2-specific monoclonal antibodies EA2 and EA5 may be capable of cross-reacting with non-human primate EphA2 molecules. Indeed, EA2 and EA5 antibodies activated EphA2 phosphorylation in CMMT110 / CL cells (FIG. 8), and weakly bound to EphA2 from CMMT110 / CL cell surfaces as measured by flow cytometry (change in MFI versus isotype control 1.2-1.8 times).

Construction of EA2 and EA5 BiTEs from CD3 Reactive Monkeys

Plasmids with coding sequences for EA2 and EA5 anti-EphA2 antibody variable chains and monkey single CD3 specific antibodies cCD3-1 and cCD3-2 were constructed by PCR-based fusion. The following 12 BiTE molecules were made:

<table> table see original document page 288 </column> </row> <table> <table> table see original document page 289 </column> </row> <table>

Cloning and transfection of surrogate constructs were performed as described above.

Flow Cytometric Binding Analysis of Various BiTE Constructs Anti-EphA2 Substitutes Reacting with Monkey CD3

For each sample 200,000 cells (T cells or EphA2 + tumor cells) were incubated with 50 µL of CHO cell culture supernatant transfected with the substitute BiTE constructs for 30 minutes on ice. Cells were subsequently washed twice with PBS and bound constructs were detected via their c-terminal histidine tags with Penta His murine antibody (diluted at 5pg / ml in 50 μΐ PBS with 2% fetal calf serum; Qiagen; Order No. 34660) followed by a wash step and a phycoerythrin-conjugated murine gamma FC-specific antibody (Dianova, order number 115-116-071), diluted 1: 100 in 50 μΐ PBS with 2% fetal calf serum (gray line). As a negative control, untransfected cell culture supernatants were used (black line). Cells were analyzed by FACS-Calibur flow cytometry (Becton Dickinson, Heidelberg). As shown in FIG. 9, only those monkey CD3 antibody-based substitute BiTE constructs cCD3-1 showed strong binding to CD3 and EphA2. CCD3-2-based constructs showed only strong binding to CD3; binding to EphA2 was almost completely suppressed.

EphA2-BiTE CHARACTERIZATION

Flow Cytometry Binding Analysis of Anti-EphA2 Parental Antibodies

Flow cytometric analysis was performed to estimate the binding strength of parental anti-EphA2 B233, EA2, and EA5 parent antibodies (FIG. 10). See FIG. 3 for VL and VH domain sequences of the B233 monoclonal antibody. A549 cells expressing EphA2 (human lung carcinoma cell line) and MDA-MB-231 (human breast cancer) cells were used. 200,000 cells were incubated with 10 μg / ml of their antibody for 30 minutes on ice. The cells were subsequently washed twice with PBS. Primary antibody binding was detected via a phycoerythrin-conjugated murine Fc-gamma-specific antibody (Dianova, order number 115-116-071) diluted in 50 µL PBS with 2% fetal calf serum. As a negative control, an irrelevant antibody with the same isotype was used (thin line). Cells were analyzed by flow cytometry on a FACS-Calibur (Becton Dickinson, Heidelberg).

Monoclonal antibody B233 showed the strongest binding signal followed by EA2 and EA5. The binding capacities of the three different antibodies are shown in a histogram overlay.

Tissue Cross Reactivity (TCR) Parental Antibodies EA2 and EA5 Anti-EphA2

Frozen human tissue sections were stained with human EphA2 reactive monoclonal antibodies EA5 and EA2 using immunohistochemical methods to determine if the antibodies specifically bind to normal tissue. Briefly, a primary and secondary antibody pre-complex was produced with the disconnected secondary binding sites blocked with appropriate gamma globulin species. Frozen sections were adhered to 16 amines in 10% formalin and washed with Tris Ix buffered saline (TBS) with 0.01% Tween 20. Endogenous peroxidases were blocked with a solution containing glucose oxidase (Sigma, β-D (+) - glucose (Sigma), and sodium azide (Sigma). An avidin / biotin vector kit (Vector Labs) blocked avidin / biotin reactive sites). The slides were then incubated with a blocking protein solution consisting of bovine serum albumin, casein, and normal goat serum.Tissue sections were incubated with pre-complexed antibodies and then washed with Vectastain ABC Elite Kit (Vector Labs). DAB-treated TBS 1x (Sigma) and counterstained with hematoxylin Tissue sections were dehydrated, coverslipped, and imaged As shown in FIG. 11, EA5 demonstrated labeling of interleaved discs in cardiac tissue (FIG. 11B ) vascular and stromal multiple organ smooth elements (cytoplasm), colon epithelium (cytoplasm), and uterine myometrium). Human tissue sections (FIG. 11 C) were not labeled with EA2 as compared to a 1A7 monoclonal antibody isotype control, and as summarized by the Table below.

<table> table see original document page 292 </column> </row> <table> <table> table see original document page 293 </column> </row> <table>

Neg = Negative; 1+ - weak; 2+ - moderate; 3+ = strong; 4+ = intense

MDA 231 cells were used with a positive control for this test.

Bispecific Cell Binding of EphA2-BiTE Constructs

EphA2 positive A549 cells (human lung carcinoma cell line) and MDA-MB-231 cells (human breast cancer) as well as CD3 positive HP-BALL cells (human T cell line) were used. 200,000 cells were incubated with 10 µg / ml of purified BiTE monomer from four different BiTE EphA2 constructs for 30 minutes on ice. Cells were subsequently washed twice in PBS and bound constructs were detected via their C-terminal hexahistidine tags with a penta-His murine antibody (diluted 1:20 in 50 µL PBS with 2% fetal calf serum; Qiagen; order number 34660) followed by a wash step and a phycoerythrin-conjugated murine Fc-gamma specific antibody (Dianova, order number 115-116-071) diluted 1: 100 in 50 μL PBS with 2% fetal calf serum (line Grey). As a negative control, fresh cell culture medium from untransfected CHO cells instead of transfectant culture supernatant was used (black line). Cells were analyzed by flow cytometry on a FACS-Calibur (Becton Dickinson, Heidelberg). See FIG. 12

The following Table summarizes the relative binding intensities of all eight EphA2-BiTE constructs generated as determined by flow cytometry.

<table> table see original document page 294 </column> </row> <table> <table> table see original document page 295 </column> </row> <table>

All eight BiTE EA2 constructs showed more or less strong bonds with their anti-CD3 SCA to CD3 moieties expressed in HP Ball cells, indicating proper expression and folding of the anti-CD3 moiety. All four EA2 monoclonal antibody-derived BiTEs showed binding to EphA2-expressing breast and lung cancer cell lines, while only one EA5 monoclonal antibody-derived BiTE retained EphA2-specific binding activity. EA2 monoclonal antibody thus appeared to be more suitable for construction of BiTE than EA5 monoclonal antibody.

Epitope Exclusion by BiTE EphA2s EphA2 positive MCFOA positive non-transformed breast epithelial lineage and MDA-MB-231 breast carcinoma were used in an immunofluorescence labeling experiment to determine whether BiTE EphA2 constructs (used at 10 μg / ml) retained epitope exclusion as their parent monoclonal antibodies EA2 and EA5. The results of the analysis are summarized in the Table below. Deimmunized anti-CD3 EA2 (VH / VL) BiTE 'showed stronger epitope exclusion.

<table> table see original document page 296 </column> </row> <table> Productivity of Monomeric EphA2-BiTEs

The productivity of five active BiTE EphA2s was calculated from the monomer production of a small-scale rolling bottle production. Productivity up to 1.1 mg / l 'purified monomers was obtained (see Table below).

The yield of a medium scale production (2 χ 10 L reactor) of a set of amplified CHO cells (20 nM MTX) transfected with deimmunized BiTE anti-CD3xEA2 (VH / VL) construct was 1.1 mg of purified BiTE monomer / L cell culture supernatant.

<table> table see original document page 297 </column> </row> <table> <table> table see original document page 297 </column> </row> <table>

EphA2-BiTE Five Construct Redirected Lysis Power

Chromium-51 Release-based cytotoxicity tests were performed to determine objective cell lysis redirected by the various EphA2-BiTE constructs in the presence of human T cells. Briefly, PBMCs as a source of effector T cells were obtained by Ficoll density gradient centrifugation. NK cells were depleted from PBMC by CD16-directed magnetic beads to prevent T cell-dependent cell lysis. EphA2-positive tumor cell lines MDA-MB231 and A549 were loaded with Chromium-51 and served as target cells. The various EphA2-BiTE constructs were titrated over a wide range of concentrations. The duration of the test was 18 hours, and the effector-to-target ratio (E: A) 10: 1. See FIG. 13

The following Table summarizes the BiTE concentrations required for half maximum redirected cell lysis (i.e., EC50) of cell lysis (i.e., EC50) of EphA2 positive MDA-MB231 and A549 target cell lines, respectively, as determined in two independent experiments. The following Table summarizes the BiTE concentrations required for half maximum redirected cell lysis (i.e., EC50) of cell lysis (i.e., EC50) from EphA2 positive MDA-MB231 and A54 9 target cell lines, respectively, as determined. in two independent experiments.

<table> table see original document page 299 </column> </row> <table>

The deimmunized BiTE anti-CD3xEA2 (VH / VL) construct consistently showed the highest potency in redirected lysis of breast and lung cancer cell lines.

BiTE Anti-CD3xEA2 (VH / VL) Selection Unimmunized For

Additional Characterization

Since deimmunized anti-CD3xEA2 (VH / VL) was found to be the most potent EphA2-BiTE construct in redirected lysis (ED50 values between 6 and 25 ng / mL with both target cell lines), and had the second best monomer productivity (-, 8-1.1 mg / mL) of all five BiTE constructs that showed bispecific binding activity, it was selected for further characterization. See FIG. 14 for the nucleotide and amino acid sequences of the deimmunized anti-CD3xEA2 BiTE construct.

ADDITIONAL CHARACTERIZATION OF EphA2-BiTE "DEIMMUNIZED ANTI-CD3 χ EA2 (VH / VL)"

Cytotoxic Activity Variation with Production Lot and Effector Cell Donor

The cytotoxic activity of two different production lots of deimmunized EphA2-BiTE anti-CD3xEA2 (VH / VL) was compared using PBMCs (after depletion of CD16 positive cells) and stimulated CD8 + T cells as effector cells. EphA2 2 A549 and MDA-MB-231 positive cell lines served as target cells. The E: A ratio was 10: 1, and the incubation time was 18 hours. See FIG. 15

The following table summarizes the maximum half-lysis (i.e. EC50) values for redirected objective cell lysis obtained from the above dose-response analyzes with BiTE EphA2 from different production batches using either PBMC or stimulated CD8 + T cells as effector cells. .

<table> table see original document page 301 </column> </row> <table>

The range of ED50 values for objective redirected cell lysis was tested for the disimmunized EphA2-BiTE anti-CD3xEA2 (VH / VL) with seven human PBMC donors (NK cell depleted).

<table> table see original document page 301 </column> </row> <table> <table> table see original document page 301 </column> </row> <table>

Deimmunized EphA2 anti-CD3xEA2 BiTE (VH / VL) showed considerably higher cytotoxicity with stimulated CD8 + T cells than with depleted NK cell PBMC. Variation in biological activity between two different lots ranged from 1.4 to 3.2 times. Potency variation was, however, most pronounced with human PBMC donors, where ED50 values between 1.9 and 23.9 ng / mL were observed. Specific efficacy by T cell donor can therefore be a major source of BiTE activity variation.

Flow cytometry-based redirected cell cytotoxicity tests were also performed to determine the variation of EphA2-BiTE cytotoxic activity with effector cell donors. CD3 + T cell enriched PBMCs were used as effector cells and SW480 EphA2 + colon carcinoma cells used as target cells. Coming soon, human PBMC

CD3 + T-cell enriched cells (RosetteSep; StemCell Technologies) were isolated from healthy donors by Ficoll density gradient centrifugation. Target cells were labeled with 3,3'-dioctadecyloxacarbocyanine fluorescent green dye (DiOC18 (3) or "DiO" (Molecular Probes)) for 5 minutes at 37 ° C. Effector and target cell mixtures were combined at a 5: 1 ratio and transferred to a round bottomed 96-well plate. Only half or serial dilutions of each BiTE construct was added to appropriate wells, incubated for 18 or 42 hours at 37 ° C, and analyzed by flow cytometry after addition of propidium iodide (PI) to a final concentration of 1 pg / ml. . Objective cell lysis was calculated as the percentage of PI-positive stained DiO positive cells. All incubations were conducted in duplicate. EC50 value calculations were conducted using a four-parameter nonlinear fit model.

As shown in FIG. 16, EphA2-BiTE redirected T cells from different subjects to mediate the death of EphA2 + tumor cells. The flow cytometry-based cytotoxicity test used SW480 target cells and CD3 + T cells isolated from 49 individual human donors. For most human donors, EphA2-BiTE mediated tumor cell death at very low concentrations. The EC50 for EphA2-BiTE was between 1 and 110 ng / mL for all T cell donors tested with a median (horizontal bar) of 24 ng / mL. Thus, for most human T cell donors, EphA2-BiTE is a very potent molecule with killing activity observed at concentrations in the low ng / mL range. Target Cell Binding Specificity: Soluble EphA2 Fusion Protein Competes Binding with Deimmunized anti-CD3xEA2 (VH / VL)

The target binding specificity of deimmunized anti-CD3xEA2 (VH / VL) BiTE was tested in a competition test. In this regard, 200,000 EphA2-positive A549 cells were incubated with 5 μg / ml of the EA2-based BiTE construct both in the presence and absence of 50 pg / ml of ice-soluble EphA2 Fc fusion protein for 30 minutes. The cells were subsequently washed twice in PBS. Binding of the construct was then detected via its C-terminal hexahistidine tags using a murine penta-His antibody (diluted 1:20 in 50 μ com PBS with 2% fetal calf serum; Qiagen; order number 34660) followed by a step of wash and a phycoerythrin-conjugated murine Fc-gamma-specific antibody (Dianova, order number 115-116-071) diluted 1: 100 in 50 μΐ PBS with 2% fetal calf serum (gray line). As a negative control, only secondary antibodies were used (black line). Cells were analyzed by flow cytometry on a FACS Calibur instrument (Becton Dickinson, Heidelberg).

As shown in FIG. 17, co-incubation of the deimmunized EphA2-BiTE anti-CD3xEA2 (VH / VL) with a 10-fold overweight of soluble EphA2 Fc fusion protein completely blocked the binding of EphA2-BiTE to A549 human lung cancer cells. This shows that targeting of EphA2-BiTE to tumor cells is specifically mediated by EphA2 target recognition.

EphA2-BiTE Anti-CD3xEA2 (VH / VL) Targeted Cell Specificity Deimmunized: Antigen-Positive Cell Death

To ensure retention of target specificity after conversion of the original IgG into BiTE format, antigen positive and negative cell cytotoxicity testing was performed. For this purpose, EphA2-transfected B16F10 cells, a mouse melanoma cell line, were compared with transfected cells. A standard chromium release test using effected human CD8 T cells as effectors at an E: A ratio of 10: 1 and a test duration of 18 hours was performed at various concentrations of the EphA2-BiTE construct. See FIG. 18

<table> table see original document page 305 </column> </row> <table>

Specificity of objective cell lysis was further substantiated by deimmunized anti-CD3xEA2 BiTE mediating the lysis of SW480 EphA2 + human colon carcinoma cells and non-EphA2 negative SK-MEL28 melanoma cells. See FIG. 19. Parental antibodies specific for both EphA2 and CD3 blocked deimmunized anti-CD3xEA2 BiTE-mediated lysis. Thus, the cytotoxic activity of deimmunized anti-CD3xEA2 BiTE strictly depends on the expression of EphA2 in the target cells; EphA2 negative cells are completely resistant to deimmunized anti-CD3xEA2 BiTE mediated lysis.

Death of Renal Cancer and Carcinoma Cells from

Prostate Mediated by Epha2-Bite

Flow cytometry-based redirected cell cytotoxicity tests were conducted using CD3 + T-cell enriched human peripheral blood mononuclear cells (PBMC) as effector cells and renal carcinoma cell lines EphA2 + ACHN and Caki 2 and PC3 and DU145 prostate cell lines as target cells. Briefly, CD3 + T-cell enriched human PBMC (RosetteSep; StemCell Technologies) were isolated from healthy donors by Ficoll density gradient centrifugation. Target cells were labeled with 3,3'-dioctadecyloxacarbocyanine fluorescent green dye (DiOC18 (3) or "DiO" (Molecular Probes)) for 5 minutes at 37 ° C. Effector and target cell mixtures were combined at one ratio 5: 1 and transferred to a 96-well round bottom plate. Only half or serial dilutions of each BiTE construct was added to appropriate wells, incubated for 42 hours at 37 ° C, and analyzed by flow cytometry after addition of propidium iodide (PI) to a final concentration of 1 pg / mL. Objective cell lysis was calculated as the percentage of PI-positive stained DiO positive cells. All incubations were conducted in duplicate.

EC50 value calculations were conducted using a four-parameter nonlinear fit model.

As shown in FIG. 20, EphA2-BiTE mediated T-cell mediated cell lysis in both renal carcinoma cells and prostate cancer cells.

EphA2-BiTE Killing Effects on EphA2 Levels in Target Cells

The cytotoxicity test was performed as described above. Once the cytotoxicity test was completed, cultures were placed on ice and left untreated, or treated with 10 pg / ml non-binding isotype 1A7 mouse monoclonal antibody or a B233 human anti-EphA2 mouse monoclonal antibody (see Dall ' Acqua et al., 2005, Methods 36: 43-60, which is incorporated herein by reference in its entirety). An APC-conjugated anti-pentahistidine antibody bound to EphA2-BiTE which remained in untreated cells. An APC-conjugated mouse anti-IgG (H + L) antibody resolved the amount of isotype or B233 bound to target cells. The geometric mean fluorescence intensity of the isotype control EphA2 and EphA2BiTE versus the given dose of EphA2-BiTE was plotted. See FIG. 21

Cytotoxicity Dependence on Time, Effector to Target Ratio, and Receptor Binding Sites

To explore the kinetics and potency of deimmunized anti-CD3xEA2 (VH / VL), several parameters of tumor-redirected cell lysis were evaluated. An analysis of the time course of target cell death revealed that in the presence of anti-CD3xEA2 (VH / VL) lysis was limited by unstimulated CD3 + T cells after 18 hours, and that maximal mortality (> 80% lysis) occurred at 42 hours (FIG. 22A). In most experiments, the magnitude of T-cell redirected lysis exceeded 80% of the target cells. As an additional measure of potency, the effector cell to target cell ratio was investigated. Reasons E: A of 1:! Up to 20: 1 gave similar high lithic activities, while E: A ratios of 1: 2 and 1: 5 still led to redirected lysis of SW480 tumor cells, albeit in a reduced percentage (FIG. 22B). Notably, the estimated EC50 values remained largely constant despite variations in incubation times (FIG. 22A; 1 to 9 ng / mL) or E: A ratio (FIG. 22B; 2 to 7 ng / mL).

Tumor target cells, which express different levels of EphA2 on their surfaces, were evaluated to determine if there was a surface target density limit required for anti-CD3xEA2 (VH / VL) activity. The efficiency of T cell redirected lysis was observed for all EphA2 expressing cell lines (FIG. 22C), including M14 cells, which expressed as little as 2,400 EphA2 molecules per cell (FIG. 22D). The magnitude of anti-CD3xEA2 (VH / VL) mediated lysis was similar for target cells despite their surface target densities (FIG. 22D). However, the density of EphA2 on the surface of target cells did have an impact on the efficiency of redirected lysis. A trend was observed in which the potency of anti-CD3xEA2 (VH / VL) increased as the number of EphA2 binding sites increased in tumor cells (FIG. 22C). Taken together, these findings suggest that anti-CD3xEA2 (VH / VL) may potently and specifically redirect unstimulated human T cells to lyse EphA2-expressing tumor cells, even when there are low levels of binding sites available in the tumor.

Anti-CD3xEA2 (VH / VL) Biim Stability Deimmunized in Human Plasma

Plasma stability of EphA2-specific BiTE was tested under different incubation conditions followed by the determination of the ED50 cytotoxic activity in a standard chromium-51 release test. A group of human blood-derived plasmas from five healthy donors were collected by EDTA-coated syringes. Cellular components were removed by centrifugation and the upper plasma phase was collected and subsequently pooled. Deimmunized anti-CD3xEA2 (VH / VL) BiTE was either incubated at 37 ° C or 4 ° C in the presence or absence of plasma. As controls, BiTE was diluted immediately prior to the RPMI-1640 plasma or medium cytotoxicity test, respectively. MDA-MB231 served as target cells; NK cell depleted PBMCs were used as effector cells. The effector: target ratio (E: A) 'was 10: 1. The duration of the test was 18 hours. See FIG. 23

<table> table see original document page 310 </column> </row> <table>

Deimmunized anti-CD3xEA2 (VH / VL) proved stable as no mojaritarian loss of cytotoxic activity could be detected after incubation in human plasma for 24 hours at 31 ° C.

Target Epitope Exclusion in Unimmunized anti-CD3xEA2 (VH / VL) Untransformed Cells

Videomicroscopy was employed to visualize CD8 + T cell attack against untransformed MCFlOA cells in the presence of anti-CD3 χ EA2 deimmunized BiTE (VH / VL) and a non-exclusionary epitope control BiTE (see FIG. 24). Target cells were seeded for growth as adherent cells 24 hours before video recording began on 48-well plates. Directly prior to recording, a mixture of CD8 + and BiTE T cells were added to the culture wells. Videomicroscopy was recorded for 20 hours at approximately one photo per minute. Propidium iodide (1 μg / ml) was added to the wells after 18 hours. Individual photos were converted to an AVI video movie, and transmitted light and fluorescent light photos were taken.

Videomicroscopic analysis over the duration of the 18 hour test exemplified the EphA2-specific BiTE epitope exclusion. While pan-positive BiTE used as a positive control attacked untransformed MCFlOA cells along the entire intact cell monolayer (FIG. 24C), EphA2-specific BiTE showed T-cell activity only at the edge of the confluent cell layer where exclusion epitope through neighboring cells is not possible (FIG. 24A). In the area of an intact monolayer, very low T cell activity was noted, as seen in the absence of BiTE (FIG. 24B) where T cells are only equally distributed over the monolayer. A monolayer of A549 carcinoma cells, which do not support exclusion of EphA2 epitopes, was completely destroyed by T cells in the presence of deimmunized anti-CD3xEA2 (VH / VL). Addition of propidium iodide at the end of the test allows visualization of dead carcinamo cells in T cell groups. Such intensely stained cell groups were present at the end of the test in each well except in control wells without BiTE.

Anti-CD3xEA2 (VH / VL) Binding Constants cleared by EphA2 Targets

The formation and dissociation of BiTE / EphA2 complexes was monitored by surface plasmon resonance using a Biacore 3000 system. For this purpose, Fc EphA2 fusion protein was immobilized on a sensor chip with a carboxymethylated CM5 dextran matrix. Optimal immobilization conditions were identified by pH scanning (pH 4.0 sodium acetate at pH 5.5; disodium tetraborate pH 8.5). The optimal pH value was pH 4.0. After immobilization of 1000 RU to flow 2 cells, 5,000 RU to flow 3 cells and 400 RU to flow 4 cells, excess reactive groups were quenched by ethanolamine. The affinity of anti-EphA2 BiTE to immobilized EphA2 was determined by injecting different concentrations of the de-immunized VHVL anti-CD3xEA2 diluted analyte in HBS-EP buffer. To regenerate the surface, REGEN buffer (50 mM NaOH; 20 mM MES; 1 mM NaCl pH 6.4) was employed.

<table> table see original document page 312 </column> </row> <table> IN VIVO EFFICIENCY OF ANTI-CD3XEA2 (VH / VL) BIT SPECIFIC EPHA2

Deimmunized anti-CD3xEA2 (VH / VL) is specific for human CD3 and primate EphA2 and therefore does not bind to mouse CD3 or EphA2. Anti-CD3xEA2 anti-CD3xEA2 (VH / VL) anti-tumor EphA2 can then only be studied in NOD / SCID mice with a human colon carcinoma xenograft model.

SW480 NOD / SCID Xenograft Model

The human colon carcinoma cell line SW480 was selected for the establishment of a xenograft model since SW480 cells expressing EphA2 and deimmunized anti-CD3xEA2 (VH / VL) were shown to redirect SW480 cell lysis in vitro.

5x10 6 SW480 cells were mixed with 2.5 x 106 human CD3 + T cells from healthy donors in a final volume of 0.2 mL PBS resulting in an E: A ratio of 1: 2. Effector T cell / SW480 cell mixtures were injected subcutaneously into the right flank of each NOD / SCID mouse. SW480 Tumors Growing

subcutaneously they were measured three times a week with a caliper in two perpendicular dimensions and tumor volumes calculated according to the formula: tumor volume = [(width2 * length) / 2]. 6.4.2 Study Design

Six animals per group were treated intravenously with PBS control vehicle, non-relevant BiTE control or anti-CD3xEA2 (VHVL) deimmunized for five consecutive days starting one hour after subcutaneous CD3 + T cell inoculation and SW480 tumor cell inoculation according to Table below.

Animals in groups I and J additionally received 2.5 x 10 6 CD3 + effector cells by injection into the caudal vein 5 minutes after inoculation of SW480 tumor cells to stimulate the presence of peripheral T cells.

<table> table see original document page 314 </column> </row> <table> <table> table see original document page 315 </column> </row> <table>

Anti-CD3xEA2 (VH / VL) Anti-Tum In vivo Anti-Tum

Specificity and Reproducibility of Model SW480

Inoculation of SW480 cells only without effector cells followed by treatment with PBS (group A) or deimmunized anti-CD3xEA2 (VH / VL) led to the first palpable tumors on day 4 after inoculation and mice had to be sacrificed on day 30. due to the large tumor masses (> 1 cm3). There was no difference in tumor growth kinetics between the PBS and deimmunized anti-CD3xEA2 (VH / VL) treatment groups indicating that BiTE EphA2 had no antitumor effect in the absence of effector cells. See FIG. 26

Inoculation of mixtures of SW480 tumors and human T-cells followed by treatment with PBS control vehicle (group C) or non-relevant BiTE control (group D) ·, which shares the deimmunized anti-CD3xEA2 (VH / VL) CD3 ligand arm but it has a different target arm also did not affect tumor growth demonstrating that neither the anti-CD3 BiTE arm alone nor T cells alone had any antitumor activity. Thus, treatment with the non relevant BiTE control showed the same effect as treatment with the PBS vehicle. See FIG. 26

Finally, intravenous injection of additional CD3 + T cells 5 minutes prior to tumor cell inoculation mimicking the presence of peripheral T cells did not influence tumor growth (group I). See FIG. 26

No significant differences in tumor growth were observed among all control conditions tested below. This also shows the robustness and reproducibility of the SW480 NOD / SCID tumor growth model with the selected tumor cell doses. See FIG. 26. Dose-Dependent Inhibition of Tumor Growth by Deimmunized Anti-CD3xEA2 (VH / VL)

Treatment with deimmunized anti-CD3xEA2 (VH / VL) induced a dose-dependent inhibition of SW480 tumor growth in the presence of effector CD3 + T cells.

While treatment with 1 pg of unimmunized anti-CD3-EA2 showed almost no effect on tumor growth, all other doses tested (5, 20 and 100 pg daily for five days) caused significant inhibition of tumor growth. At daily doses of 5 pg, tumor volume was significantly smaller on days 3-9, and significantly lower on days 18, 20, and 27 compared with the non-relevant BiTE control group. Tumor volume of mice treated with 20 µg deimmunized anti-CD3xEA2 (VH / VL) was significantly lower when compared to those of non-relevant BiTE control-treated mice at all time points analyzed. Finally, treatment with 5x100 pg of unimmunized anti-CD3xEA2 (VH / VL) led to highly significant differences at all time points analyzed and showed no tumor growth for two weeks after BiTE treatment. Treatment with non relevant control BiTE showed the same effect as treatment with PBS vehicle. See FIG. 27

No Effect of Peripheral T-Cells on Anti-Tumor Effect of Deimmunized Anti-CD3xEA2 (VH / VL) In contrast to the situation in a xenoimplant model where no human T-cell is present in the periphery, the efficacy of deimmunized anti-CD3xEA2 can be influenced. by circulating T cells that can trap the molecule in the periphery and thereby reduce specific targeting for unimmunized anti-CD3xEA2 (VH / VL) tumors. To mimic this scenario, two groups of mice were additionally injected intravenously with human T cells to provide a peripheral human T cell population.

The i.v. administration of additional human T cells had no influence on the efficacy of deimmunized anti-CD3xEA2 (VH / VL). SW480 tumor growth in groups F (treatment with 20 μg deimmunized anti-CD3xEA2 (VH / VL) / injection) and J (treatment with 20 μg deimmunized anti-CD3xEA2 (VH / VL) / injection after iv injection of 2 , 5x106 additional T cells) was almost identical. In both groups, treatment with deimmunized anti-CD3xEA2 (VH / VL) induced highly significant tumor growth inhibition when compared to PBS-treated mice or PBS-treated mice and additional circulating T cells. See FIG. 28

GENERATION AND CHARACTERIZATION OF HUMANIZED ANTI-EPHA2 BITE

The following information details the generation and characterization of humanized anti-EphA2 antibodies used in the construction of anti-EphA2 BiTEs. Candidate biTEs were constructed from antibodies that were humanized, capable of binding to cinomolgus and human EphA2, and not binding to normal human cardiac tissue.

Two murine anti-EphA2 monoclonal antibodies, B233 (see FIG. '29) and EA2 (FIG. 1), were humanized and produced 3F2 (derived from B233; FIG. 30) and 4H5 (derived from EA2). Affinity optimization was performed as described in detail below, and in DallfAcqua et al., 2005, Methods 36: 43-60, which is incorporated herein by reference in its entirety.

EphA2 Antibody Affinity Optimization

4H5 Affinity Optimization

The following information details the affinity optimization of the humanized 4H5 human antiEphA2 monoclonal antibody to produce the three affinity optimized variants 2A4, 2E7, and 12E2. For more details regarding the production of affinity optimized variants, and in particular 2A4, 2E7, and 12E2, please see U.S. Provisional Appn. No. 60 / 751,964, filed December 21, 2005, entitled "Afinity Optimized Eph A2 Agonistic Antibodies and Methods of Use Thereof", which is incorporated herein by reference in its entirety.

Reagents All chemical reagents are of analytical grade. Restriction enzymes and DNA modifying enzymes, T4 ligase and T7 DNA polymerase were purchased from New England Biolabs, Inc. (Beverly, MA). Custom oligonucleotides were synthesized by Invitrogen (Carlsbad, CA). Human Fc-EphA2 fusion protein (consisting of a human EphA2 ectodomain fused to the Fc portion of a human IgG1) was expressed in human embryonic kidney (HEK) 293 cells and purified by protein G affinity chromatography using standard protocols. . Biotinylation of human Fc-EphA2 was performed using an EZ-Link Sulfo-NHS-LC biotinylation set according to the manufacturer's instructions (Pierce, Rockford, IL).

Humanization of Murine EA2 Anti-EphA2 Monoclonal Antibody by Frame Shuffling Technology

Humanization of the parental murine monoclonal antibody EA2 was performed using frame shuffling technology as described in detail in Dall'Acqua et al., 2005, Methods 36: 43-60, and in US Patent Pub. No. US-2005 / 0048617 A1, each of which is incorporated herein by reference in its entirety. Essentially, CDR regions of both EA2 VL and EA2 VH regions are transplanted into libraries of germline human framework sequences in a combinatorial form, creating a mosaic of humanized variants that retain binding to EphA2. Such a humanized clone, 4H5, exhibited an approximately 20-fold increase in affinity compared to chimeric EA2 Fab. This clone was chosen as the affinity maturation template and was subsequently optimized as described below, resulting in variants 2A4, 2E7 and 12E2.

4H5 scFv Affinity Optimization

ScFv Mold Construction

The variable regions of the humanized monoclonal antibody 4H5 were cloned with an scFv fragment into an M13 expression vector (Dall'Acqua et al., 2005, Methods 36: 43-60, which is incorporated herein by reference in its entirety). The light variable region of 4H5 was combined with the 3 'end of the 4H5 variable heavy chain by a [(Gly) 4Ser] 3 linker, followed by a FLAG marker and a His marker at its C-terminal end (FIG. 31 ). Constructs were generated using PCR and the following primers to amplify the variable regions in separate reactions:

Medi-VH8: TTC TAT GCG GCC CAG CCG GCC CAG

CTG TTG SAG TCT G (5 'primer to amplify VH, S = C / G) (SEQ ID NO: 120)

Medi-JH1: GGA GCC GCC GCC GCC AGA ACC ACC ACC ACC TGA GGA GAC GGT GAC CAG GGT GCC (31 Primer for Amplifying VH) (SEQ ID NO: 121) Medi-VKl: GGC GGC GGC TCC GGT GGT GGT TCT GAC ATC CAG WTG ACC CAG TCT CC (5 'primer for VL, W = A / T) (SEQ ID NO: 122)

Medi-JK4: TGG AAT TCG GCC CCC GAG GCC ACG TTT GAT CTC CAC CTT GGT CCC (3 'primer for VL) (SEQ ID NO: 123), where underlined sequences correspond to ligand [Gly) 4Ser] 3, and letters bold italics denote the Sfi I restriction site. Overlapping PCR was used to construct the scFv fragment which was then restricted by Sfi I and cloned into the MD 102 vector. The murine parental EA2 variable regions were cloned in the same manner. to serve as a scFv control. The 4H5 scFv construct was then expressed in CJ236 to produce uridine + ssDNA as described in Wu and An, 2003, Methods Mol. Biol. 207: 213-234, which is incorporated herein by reference in its entirety. This 4H5 U + ssDNA scFv was used as a template for the following affinity optimization mutagenic reactions. The 4H5 scFv nucleotide and amino acid sequences are described in FIG. 32

ScFv affinity optimization by parsimonious randomization of each CDR region

Each amino acid from all 6 Determinant Regions of

Complementarity (CDR) was individually, randomly mutated using two amino acid-separated libraries (Wu and An, 2003, Methods Mol. Biol. 207: 213-234, which is incorporated herein by reference in its entirety). By coding either 8 amino acids (NSS) or 12 amino acids (NWS) at each CDR amino acid position, each individual degenerate primer was used in a single hybridization mutagenesis reaction (Wuf 2003, Methods Mol. Biol. 207: 197- 212, and Dall'Acqua et al., 2005, Methods 36: 43-60, each of which is incorporated herein by reference in its entirety), and then combined to generate a corresponding CDR library. Briefly, each degenerate primer was phosphorylated, then used at a 10: 1 ratio with 4H5 single stranded uridinylated U + DNA model (prepared as described in Wu and An, 2003, Methods Mol. Biol. 207: 213-234 ) in an annealing reaction where the temperature was lowered from 95 ° C to 55 ° C for 1 hour. T4 ligase and T7 DNA polymerase were added to the annealing reaction and the reaction was incubated for 1.5 hours at 37 ° C. The products synthesized for each amino acid of each CDR were pooled, however the NSS and NWS libraries were kept secreted and filtered. regardless. Typically, 1 µl of the synthesized DNA from the pooled CDR libraries was then electroporated into XLl-Blue for field plating of XLl-Blue bacteria or production of scFv fragments as described in Wu, 2003, Methods Mol. Biol. 207: 197-212.

Library Filtering

Primary filter The primary filter consisted of a single-point ELISA (SPE) which was performed using supernatants containing soluble secreted scFv protein prepared from 1 ml of bacterial culture grown in 96-well deep plates and infected with individual recombinant M13 clones. essentially as described in Wu, 2003, Methods Mol. Biol. 207: 197-212, and DallfAcqua et. al., 2005, Methods 36: 43-60. Briefly, this Capture ELISA involves coating individual wells of a 96-well Maxisorp Immunoplate with approximately 30 ng of an anti-FLAG mouse antibody (Sigma), blocking with 3% BSA / PBS for 2 hours at 37 ° C. and incubating with samples (secreted soluble scFv) for 2 hours at room temperature. 150-600 ng / well Fc-EphA2 of biotinylated humum was then added for 2 hours at room temperature. This was followed by incubation with neutravidine-strong root peroxidase (HRP) conjugate (Pierce, IL) for 40 minutes at room temperature. HRP activity was detected with tetra methyl benzidine substrate (TMB) and the reaction was quenched with 0.2 M H2SO4. Plates were read at 450 nm.

Primary Filter Results

Typically, clones exhibiting an OD 450 nm signal approximately twice as large as that of parental 4H5 scFv were grown on a 15 mL scale, and retested by the same ELISA in duplicate wells to confirm the positive result. Repeat clones were then sequenced and assayed using an activity ELISA (see below) to estimate the increase in human EphA2 binding multiplication.

Secondary Filter

In order to further characterize the previously identified shift-optimized affinity variants, a secondary filter using secreted scFv fragments expressed from 15 ml bacterial cultures (see Wu, 2003, Methods Mol. Biol. 207: 197-212) was performed. . More precisely, two ELISAS were used: (i) an activity ELISA in which individual wells of a 96-well Maxisorp Immunoplate were coated with 3% BSA / PBS for 2 hours at 37 ° C. Serially 2-fold diluted samples were collected. then added and incubated for 1 hour at room temperature. Incubation with a goat anti-human kappa conjugate made with Horseradish peroxidase (HRP) then followed. HRP activity was detected with TMB substrate and the reaction quenched with 0.2 M H2SO4. Plates were read at 450 nm; (ii) an ELISA anti-scFv quantitation which was performed essentially as described in Wu, 2003, Methods Mol. Biol. 207: 197-212. Briefly, individual wells from a 96-well Ni NTA plate (Qiagen) incubated with 2-fold or standard (50-0.78 ng / mL) serial diluted samples. Incubation with a mouse anti-FLAG conjugate with Horseradish peroxidase (HRP) then followed. HRP activity was detected with TMB substrate and the reaction quenched with 0.2 M H2SO4. Plates were read at 450 nm.

Secondary Filter Results

The two-part secondary ELISA filter described above allowed the comparison of 4H5 scFv and affinity optimized variants in terms of binding to human EphA2 by normalizing their scFv concentrations 10. All variant clone scFv variants with optimized affinity optimized exhibited better binding to human EphA2 as compared to the parental scFv of 4H5 (data not shown).

Construction and Characterization of Combinatorial Variants from CDR Affinity Optimized Clones

To design combinatorial variants with additional binding improvements, all single amino acid changes that improved binding compared to the 4H5 parental scFv by activity / quantitative ELIAS were combined to create a small focused combinatorial library. Soon, degenerate primers encoding all identified amino acid changes as well as the parent amino acid in the same position were designed. In a ringing reaction where all primers were included and synthesis followed (supra), a combinatorial library was constructed and filtered as previously described supra. Primary EphA2 Filtration Results

Typically, clones displaying a 50 nm OD 450 signal approximately twice that of 4H5 parental scFv were re-grown on a 15 mL scale, and re-assayed by ELISA (described above) in duplicate wells to confirm the positive result. Sixteen combinatorial variants were then selected and sequenced identifying 11 unique combinations of CDR amino acid changes thus making each variant different by one to three amino acids at the level of its primary sequence.

Secondary EphA2 Filtration Results

The 11 unique combinatorial variants described above were analyzed by a secondary filter as described above to estimate the improvement in binding affinities of the combinatorial variants. All variants had significantly improved affinities for human EphA2 when compared to 4H5 scFv. The nucleotide and amino acid sequences of the three affinity optimized combinatorial variants 2A4, 2E7 and 12E2 are shown in FIGS. 33, 34 and 35, respectively. Data for the three affinity optimized combinatorial variants 2A4, 2E7 and 12E2 are shown from FIG. 36. FIG. 37 shows the amino acid sequence alignment of affinity optimized variants 2A4, 2E7 and 12E2 with the humanized 4H5 scFv.

Binding Analysis

2A4, 2E7 and 12E2 as well as parental scFv from EA2 and humanized 4H5 scFv were induced for expression in E. coli in an IL culture volume. Supernatants containing secreted soluble scFv fragments were centrifuged to remove cell debris then passed through an anti-FLAG (Sigma) column to purify and isolate the variant proteins. Purified affinity optimized variants were analyzed by surface plasmon resonance detection using a BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala, Sweden). Humanized variants optimized for EA2 affinity exhibited a 110-150 fold increase in affinity compared to EA2 anti-EphA2 parental scFv. For affinity measurement results, see the Table in Section 6.6, infra.

Murine B233 Anti-EphA2 Antibody Affinity Optimization of Human

Humanization of the parent monoclonal antibody molecule B233 was performed using frame shuffling technology as described in detail by Dall'Acqua et al., 2005, Methods 36: 43-60, which is incorporated herein by reference in its entirety. Essentially, CDR regions from both B233 VL and B233 VH regions were transplanted into human frame sequences of germline sequences in a combinatorial form, creating a mosaic of humanized variants that retain binding to EphA2. Such a humanized clone, 2G6, exhibited an approximately 10-fold loss of affinity as compared to chimeric B233 monoclonal antibody. This clone was chosen as the affinity maturation template and was subsequently optimized as described below, resulting in the 3F2 variant.

Construction and Characterization of Combinatorial Variants

To design a combinatorial variant with enhanced human EphA2 binding, all of the simple amino acid changes that improved binding compared to parental B233 were combined to create a small focused combinatorial library. Briefly, degenerate primers (see FIG. 38) encoding all identified amino acid changes as well as the parent amino acid in the same position were designed. An annealing reaction was performed where all primers were included and the synthesis followed, essentially as described in Dall'Acqua et al., 2005, Methods 36: 43-60 and Wu, 2003. Methods Mol. Biol. 207: 197-212, both of which are incorporated herein by reference in their entirety. A combinatorial library was built and then filtered.

Library Filtering Primary Filter

The primary filter consisted of a single point ELISA (SPE) which was performed using periplasmic Fab extracts prepared with 1mL grown bacterial cultures in 96-well plates and infected with individual recombinant M13 clones essentially as described in Dall'Acqua et al., 2005, Methods 36: 43-60, and Wu, 2003. Methods Mol. Biol. 207: 197-212. Briefly, this Capture ELISA involves coating individual wells of a 96-well Maxisorp Immunoplate with approximately 20 ng of a goat anti-human Fab antibody, blocking with 3% BSA / PBS for 2 hours at 37 ° C and incubating with samples (periplasma expressed Fabs) for 2 hours at room temperature. 300 ng / well Fc-EphA2 of biotinylated humum was then added for 2 hours at room temperature. This was followed by incubation with neutravidine-strong root peroxidase (HRP) conjugate (Pierce, IL) for 40 minutes at room temperature. HRP activity was detected with tetra methyl benzidine substrate (TMB) and the reaction was quenched with 0.2 M H2SO4. Plates were read at 450 nm.

Primary Filter Results

Typically, clones displaying a 50 nm OD 45 signal at least twice greater than that of parental 2G6 were grown on a 15 mL scale, and re-assayed by ELISA in duplicate wells to confirm the positive result. Repeat clones were then sequenced and assayed by use of an activity ELISA (see below) to estimate multiplications in increased binding to human EphA2.

Secondary Filter

In order to further characterize previously identified optimized affinity variants (see above), a secondary filter using Fab fragments expressed in periplasmic extracts of 15 ml bacterial cultures (see Wu, 2003, Methods Mol. Biol. 207: 197-212 ) was performed. More precisely, two ELISAs were used: (i) an activity ELISA in which individual wells of a 96-well Maxisorp Immunoplate were coated with ~ 1 μg of humc Fc-EphA2 and blocked with 3% BSA / PBS for 2 hours at 31 ° C. Serially diluted 2-fold samples were then added and incubated for 1 hour at room temperature. Incubation with a human goat anti-kappa conjugate made with Horseradish peroxidase (HRP) then followed. HRP activity was detected with TMB substrate and the reaction quenched with 0.2 M H2SO4. Plates were read at 450 nm; (ii) a human anti-Fab quantitation ELISA which was performed essentially as described in Wu, 2003, Methods Mol. Biol. 207: 197-212. Briefly, individual wells from a 96-well Biocoat plate (BD Biosciences, CA) were incubated with 2-fold or standard serially diluted samples (human IgG Fab, 100-1.56 ng / mL). Incubation with a goat anti-human kappa conjugate made with Horseradish peroxidase (HRP) then followed. HRP activity was detected with TMB substrate and the reaction quenched with 0.2 M H2SO4. Plates were read at 450 nm.

Secondary Filter Results

The two-part secondary ELISA filter described in the section above allowed the comparison of the 2G6 Fab and the affinity-optimized variants in terms of binding to human EphA2. One of these affinity optimized combinatorial Fab variants was chosen for further analysis (variant named 3F2 hereafter). The amino acid sequence of the variable regions of murine B233 monoclonal antibodies, humanized 2G6, and affinity optimized 3F2 were aligned in FIG. 39

Cloning, Expression, and Purification of Various Versions of Humanized and Affinity-Optimized B233 Monoclonal Antibodies in a Human IgGl Format

The variable regions of scrambled frame 2G6 humanized clone and affinity optimized humanized variant 3F2 were PCR amplified from M13 phage vectors encoding the corresponding V regions using pfu DNA polymerase. They were then individually cloned into mammalian expression vectors encoding an untranslated, promoter and 5 'enhancer region immediately next to major human cytomegalovirus (hCMVie) (Boshart et al., 1985, Cell 41: 521-530, which is incorporated here by reference in its entirety). In this system, a human γ1 chain is secreted along with a human κ chain (Johnson et al., 1997, Infect. Dis. 176: 1215-1224, which is incorporated herein by reference in its entirety). The different constructs are transiently expressed in HEK 293 cells and harvested 72 and 144 hours after transfection. The secreted soluble human IgG1s were purified from the conditioned medium directly with 1mL protein A and protein G HiTrap columns according to the manufacturer's instructions (APBiotech, Inc., Piscataway, NJ). Purified human IgG1s (typically> 95% homogeneity as judged by SDS-PAGE) were dialyzed against phosphate buffered saline (PBS), instantly frozen and stored at -70 ° C.

Biacore Analysis

The interaction of soluble IgGs B233, 2G6, and 3F2 with immobilized EphA2 Fc was monitored by surface plasmon resonance detection using a Biacore® 3000 instrument (Pharmacia Biosensor, Uppsala, Sweden) essentially as written in WF Dall'Acqua et al. ., 2005, Methods 36 (2005) 43-60. See also Section 6.6.2, infra. Affinity measurements of chimeric B233, humanized 2G6, and affinity optimized 3F2 are given in the Table below.

<table> table see original document page 333 </column> </row> <table> <table> table see original document page 334 </column> </row> <table>

EphA2-BiTEs Affinity Measures

The following describe the affinity constants of the EphA2-BiTEs of the invention as measured by surface plasmon resonance.

Human Anti-CD3

Surface plasmon resonance measurement using immobilized soluble CD3sy is provided. Biphasic association of BiTE molecules to CD3sy prevents accurate binding rate calculations and affinity constants.

Anti-CD3 χ EA2 Deimmunized 400-500 nM VH / VL (est.)

Anti-EphA2 From Human

Surface plasmon resonance measurements using surface immobilized EphA2 Fc are provided: Anti-CD3 χ EA2 VH / VL deimmunized (MT) 45 nM

Anti-CD3 χ EA2 VH / VL Deimmunized (Medi) 113 nM

ScFvs Affinity Measurements (K0)

The Table below provides a summary of the binding kinetics of human anti-EphA2 scFvs. Association of anti-EphA2 monomeric scFv to EphA2 as determined by surface plasmon resonance binding. Affinity optimization yielded three scFvs with 20-30 fold increase in affinity (KD) to human EphA2 when compared to 4H5.

<table> table see original document page 335 </column> </row> <table>

CHARACTERIZATION OF HUMAN ANTI-EPHA2 HUMANIZED BITES

Flow Cytometry Binding Analysis of Humanized Parental Antibodies Anti-EphA2 to Human EphA2 and Cinomolgus Subconfluent Colon Carcinoma Monolayers SW480 or Cinomolgus Transfected CHO Cells were rapidly trypsinized, washed, counted, plated in 96 well plates round bottom, and stained with 10 μg / mL of a negative control antibody (R347), human anti-EphA2 murine antibody, EA2 (Coffman 2003), or humanized antibodies, 3F2 and 4H5. Cells were resuspended in AlexaFluor 488 anti-mouse or H + L anti-human IgG (Invitrogen) and then analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). As shown in FIG. 40, EA2, 3F2, and 4H5 each bound to human and Cinomolgus EphA2.

Epitope Exclusion Properties of Humanized Parental Antibodies Anti-EphA2

MCF-10a or MDA-MB-231 cell monolayers were cultured on glass coverslips for at least 24 hours at 37 ° C before staining. Cell monolayers were fixed in 4% paraformaldehyde (2 minutes, 25 ° C) prior to incubation with primary antibody (clone G5 (negative control), EA5, EA2, 3F2, or 4H5) for 30 minutes followed by subsequent anti-staining. Goat-made mouse IgG to AlexaFluor 488 or goat-made human anti-IgG to AlexaFluor 488 (The Jackson Laboratory). The cells were fixed for immunofluorescence microscopy analysis. As shown in FIG. 41, 3F2 and 4H5 bound to EphA2 in both untransformed and transformed breast epithelial cells. Thus, 3F2 and 4H5 did not share EA2's unique ability to bind to an EphA2 epitope accessible on malignant cells but selectively excluded by the normal architecture of untransformed epithelial cells. The amino acid sequences of the VL and VH domains of the G5 antibody are shown in FIG. 42.

Tissue Cross Reactivity Analysis

Evaluation of the tissue cross-reactivity of humanized anti-EphA2 antibodies to human cardiac tissue determined that 3F2 and 4H5 did not bind to normal cardiac tissue at concentrations up to 10 pg / ml.

Tissue cross reactivity was also evaluated for the scFv 2A4, 12E2, and 2E7 combinatorial variants. None of the scFv combinatorial variants (2A4, 12E2, 2E7) stained normal cardiac tissue (2 donors) at concentrations up to 50 10 pg / ml (data not shown).

Characterization of BiTE Anti-Constructs 3F2 from EphA2

The 3F2-based BiTE constructs produced are described in the Table below and were further characterized by cytotoxicity tests.

<table> table see original document page 337 </column> </row> <table> <table> table see original document page 338 </column> </row> <table>

Chromium-51 release-based cytotoxicity tests were performed to determine objective cell lysis redirected by the various EphA2 anti-construct BiTE in the presence of stimulated human CD8 + T cells. EphA2-positive MDA-MB-231 tumor cells were loaded with chromium-51 and served as target cells. The various BiTE anti-EphA2 constructs were titrated over a wide range of concentrations. The test lasted 18 hours, and the effector-to-target ratio was 10: 1. EphA2-BiTe concentrations at half the maximum Iise (ie EC 50) with different production lots were estimated using a four-parameter nonlinear fit model. See FIG. 43

Flow cytometry-based redirected cell cytotoxicity tests were conducted as listed above using CD3 + T-cell enriched human peripheral blood mononuclear cells (PBMC) as effector cells and EphA2 + SW480 colon carcinoma cells. See FIG. 44.

In vitro cytotoxicity measurement (FIGS. 43 and 44) of the four 3F2-based EphA2 anti-construct single chain BiTEs (Table above) suggested that the potency (ie EC50) of BiTE 3F2 was not greater than the murine BiTE EA2 . These results established that humanized anti-EphA2 BiTEs redirected human T cells to lyse EphA2 + tumor cells.

Characterization of BiTE 4H5 EphA2 Anti-Constructs

The EphA2 4H5 anti-construct BiTEs produced are described in the Table below and were further characterized.

<table> table see original document page 339 </column> </row> <table>

Target Cell Binding Specificity of 4H5-based anti-EphA2 BiTEs to EphA2 + and CD3 + Expressing Cells

The target binding specificity of the various 4H5 based anti-EphA2 BiTEs was examined using the flow cytometry based assay described above. EphA2 + MDA MB 231 breast cancer cells and CD3 + HP Ball human T cells were used as target cells. Deimmunized BiTE anti-CD3xEA2 (VH / VL) was used as a positive control. As shown in FIG. 45, each of the 4H5-based EphA2-BiTE constructs bound to cells expressing both EphA2 and CD3.

Target Cell Binding Specificity of 4H5 Affinity-Matured Humanized BiTE Constructs 12E2, 2E7, and 2A4

The target binding specificity of the various 4H5 based anti-EphA2 BiTEs was examined using the flow cytometry based assay described above. EphA2 + MDA MB 231 breast cancer cells and CD3 + HP Ball human T cells were used as target cells. As shown in FIG. 46, each of the 4H5-based EphA2-BiTE constructs bound to cells expressing both EphA2 and CD3.

As shown in FIG. 47, purified monomers of the affinity-matured 4H5-based EphA2-BiTE constructs bound to EphA2 + MDA MB 231 breast cancer target cells and CD3 + HP Ball human T cells at a concentration of 5 μg / ml using the test based in flow cytometry as described above.

To determine the ability of affinity matured 4H5-based scFvs, MCF-10a monolayers or MDA-MB-231 cells were cultured on glass coverslips for at least 24 hours at 37 ° C before staining. Cell monolayers were fixed in 4% para formaldehyde (2 minutes, 25 ° C) prior to incubation with primary scFv antibody (2A4, 2E7 or 12E2) for 30 minutes followed by subsequent labeling with AlexaFluor-conjugated goat mouse anti-IgG 488 or goat-made human anti-IgG conjugated to AlexaFluor 488 (The Jackson Laboratory). Cells were fixed for immunofluorescence microscopy analysis. ScFvs 2A4 and 12E2 bound to EphA2 in both untransformed and transformed breast epithelial cells. ScFv 2E7, however, shared EA2's unique ability to bind to an EphA2 epitope accessible on malignant cells but not selectively excluded by the normal architecture of untransformed epithelial cells (data not shown).

Comparison of Cytotoxic Power of Four EphA2 Specific BiTEs of Humanized Monoclonal Antibody 4H5

Chromium-51 release-based cytotoxicity tests were performed to determine objective cell lysis redirected by the various EphA2 anti-construct BiTE in the presence of stimulated human CD8 + T cells. See FIG. 48. EphA2-positive tumor cell line MDA-MD231 was loaded with chromium-51 and served as target cell. The various anti-EphA2 BiTE constructs were titrated over a wide range of concentrations. The duration of the test was 18 hours, and the effector-to-target ratio 10: 1. BiTE to EphA2 concentrations required at half the maximum Iise (ie EC50) with different production lots were estimated using a four-parameter nonlinear fit model. FIG. 49 provides a direct comparison of the directed cellular lysis potency of the various 3F2 and 4H5 based EphA2 constructs. Chromium-51 based cytotoxicity tests were performed as described above.

4H5-Based EphA2 BiTEs-Induced Cytotoxic Activity by Affinity Matured 12E4, 2E7 and 2A4

FIGS. 50 and 51 demonstrate the directed cellular lysis potency of the various 4H5-based EphA2 BiTEs (12E4, 2E7 and 2A4). EphA2 A549 and SW480 positive tumor cell lines were loaded with chromium-51 and served as target cells. Stimulated human CD8 + T cells (FIG.

40) and unstimulated human CD3 + (FIG. 51) served as effector cells. The various anti-EphA2 BiTE constructs were titrated over a wide range of concentrations. The duration of the test was 18 hours (FIG. 50) or 42 hours (FIG. 51), and the effector-to-allvo ratio 10: 1.

(FIG. 50) or 5: 1 (FIG. 41). BiTE to EphA2 concentrations required at half the maximum Iise (ie EC50) with different production lots were estimated using a four-parameter nonlinear fit model.

No EphA2 Activation by 4H5 Based EphA2 BiTEs

FIG. 52 demonstrates that neither the BiTE 2A4 nor 2E7 construct induced EphA2 phosphorylation in cells expressing EphA2. EphA2 + SW480 and A549 cells were treated with either BiTE 2A4 or BiTE 2E7, EA5 IgG (positive control), or R347 (negative control) for 15 minutes at the indicated concentrations. Cell extracts were then immunoprecipitated using the anti-EphA2 D7 monoclonal antibody (Upstate, Charlottesville, VA) and probed using the 4G10 anti-phosphotyrosine antibody (üpstate). Sample extraction, immunoprecipitation and western blot analysis were performed as previously detailed (Coffman K. et al., 2003, Cancer Res. 63: 7907-12; and is incorporated by reference herein in its entirety). For all experiments, protein levels were measured using Coomassie standard tests (Pierce, Rockford, IL), and equal amounts of protein were resolved by SDS-PAGE (10%) and transferred to PVDF membranes (Invitrogen, Carlsbad, CA). ).

In Vivo Effectiveness of 4H5-Based Affinity-matched EphA2 BiTE Constructs

The in vivo potency of the BiTE 2A4 and 2E7 constructs were evaluated in NOD / SCID immunodeficient mice with a human carcinoma colon xenograft model (described above in Section 6.4 and below in Section 6.8.7). The SW480 human colon carcinoma strain was selected for establishment of the xenograft model since SW480 cells express EphA2. Study Project

5x10 6 SW480 cells were mixed with 2.5x10 6 human CD3 + T cells from the same donor in a final volume of 0.2 ml PBS resulting in an E: A ratio of 1: 2. The effector / SW480 T-cell mixtures were injected subcutaneously into the right flank of each NOD / SCID mouse. Subcutaneously growing SW480 tumors were measured three times a week with a caliper in two perpendicular dimensions and tumor volumes calculated according to the formula: tumor volume = [(width2 * length) / 2]. Animals were treated intravenously with BiTE 2A4 or BiTE 2E7 at a concentration of 1, 5, 20, and 100 μg / dose, PBS control vehicle, or 100 μg / dose deimmunized anti-CD3xEA2 (VHVL) as a positive control by five consecutive days starting one hour after subcutaneous inoculation of CD3 + T cells and SW480 tumor cells. The results of the study are described in FIG. 53

ADDITIONAL DESCRIPTION OF TESTS CARRIED OUT HERE

Cell Lineages and Culture

CHO dhfr-, SW480, MCF-7, PC3, M14, A549, MDA-MB-231, MCFlOA, MDA-MB-468, SK-MEL-28, ACHN cells were obtained from ATCC and grown according to their recommendation. . Hey8 was an affable gift from Dr. Anil Sood, M.D. Anderson Cancer Center. Immunohistochemistry and Immunofluorescence Microscopy

Frozen human tissue sections were stained with the human EphA2 reactive monoclonal antibody, EA2, to determine if the antibody specifically binds to normal tissue. Soon, a primary and secondary antibody complex was produced, unbound secondary binding sites were blocked with human gamma globulins. Frozen sections were adhered to slides in 10% formalin and washed with tris buffered saline (TBS) 1x with 0.01% Tween 20. Endogenous peroxidases were blocked with a solution containing glucose oxidase (Sigma), β-D (+) - glucose (Sigma), and sodium azide (Sigma). An avidin / biotin vector set (Vector Labs) blocked reactive avidin / biotin sites. The slides were then incubated with a protein blocking solution consisting of bovine serum albumin, casein, and normal goat serum. Tissue sections were incubated with pre-complexed antibodies and then treated with Vectastain ABC Elite set (Vector Labs), washed with TBS 1x, DAB treated (Sigma), and counterstained with Mayer's hematoxylin. Tissue sections were dehydrated, coverslipped, and imaged.

Surface Plasmon Resonance Analysis with Biosensor

All studies were performed using CM5 chip sensor (Biacore AB, Uppsala, Sweden) which contains a carboxymethyl dextran (CM) matrix and a Biacore® 3000 surface plasmon resonance biosensor (Biacore AB, Uppsala, Sweden). ). 0ϋ3εγ was covalently associated with the CM dextran matrix using amine chemical coupling. A reference surface was created by default from the CD3sy association step. EphA2 Fc was captured via a high affinity interaction between the EphA2Fc Fc portion and a goat anti-human IgG (Fc) (KPL, Inc., Gaithersburg, MD). (Fc) goat anti-human IgG was covalently associated with the CM dextran matrix using chemical amine coupling. Two specific surfaces for (Fc) anti-human IgG were created. One of these surfaces was used as a reference surface while the other surface was used to create an EphA2 Fc-specific surface. Different concentrations of bscEphA2 χ CD3 were prepared by serial dilution in HBS-EP (0.01 M HEPES pH 7.4, 0.15 Mf NaCl 3 mM EDTA, 0.005% P20 surfactant). EphA2 biTEs were injected in a serial flow manner along the CD3y specific or EphA2 specific surface and its corresponding reference surface. Dissociation of bound EphA2 BiTEs was monitored in the presence of HBS-EP. Remaining bound material was removed with 10 mM disodium tetraborate pH 8.5, 1 M NaCl (for CD3 specific surface) or 10 mM glycine pH 1.7 (for EphA2 Fc specific surface).

Cell Surface Antigen Density

The number of cell surface EphA2 binding sites was estimated using Qifikit (DakoCytomation). Briefly, subconfluent cell monolayers were rapidly trypsinized, washed, counted, plated in round bottom 96-well plates, and stained with 100 µl of a two-fold serial dilution of a human anti-EphA2 antibody, B233 (Coffman 2003). . Mouse IgG-conjugated cells and calibration beads were resuspended in AlexaFluor 647 anti-mouse IgG H + L (Invitrogen) and then analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). The number of surface binding sites was estimated by nonlinear regression analysis from the bead calibration curve.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using nothing but routine experimentation, various equivalents to the specific advances of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in this specification are hereby incorporated by reference in the specification to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference herein. LIST OF SEQUENCES

I) GENERAL INFORMATION:

I.a) Applicant: Medlmmune, Inc. Micromet Inc.

II) Title of the Invention: SIMPLE BIESPECIFIC CHAIN ANTIBODIES AND PHARMACEUTICAL COMPOSITION UNDERSTANDING THE SAME

III) Reference File: 10271-175-228

IV.a) Zionist Priority Data: 60 / 753,368

IV.b) Date of completion of previous form: 2005-12-21

V) Number of Sequences: 123 (one hundred and twenty three)

VI) Program for reading: FastSEQ for Windows Version 4.0

2) GENERAL SEQUENCE INFORMATION:

I.a) Sequence identifier number: SEQ ID No: 1

II.a) Size: 321

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Light Chain Nucleotide Sequence

IV) Sequence: SEQ ID NO. 1

gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat aactatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg 180 aggttcagtg gcagtggatc ggtcccatca tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatatgg gaatttatta ttgtctgaaa tatgatgagt gttcggaggg 300 gggaccaagc tggaaataaa ttccgtacac 321

I.a) Sequence identifier number: SEQ ID No: 2

II.a) Size: 107

II.b) Type: PRT

II.c) organism: Artificial Sequence III)

III.a) Other information: EA2 Variable Light Chain Amino Acid Sequence

IV) Sequence: SEQ ID NO. 2

Asp Ile Lys Met Thr Gln Be Pro Be Met Be Tyr Wing Be Read Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Wing Be Gln Asp Ile Asn Asyr Tyr 20 25 30 Read Le Be Trp 35 Phe Gln Lys Pro 40 Gly Lys Ser Pro Lys 45 Thr Leu Ile Tyr Arg 50 Wing Asn Arg Leu Val 55 Asp Gly Val Pro Ser 60 Arg Phe Ser Gly Ser 65 Gly Ser Gly Gln Asp 70 Tyr Ser Leu Thr Ile 75 Ser Ser Leu Glu Tyr 80 Glu Asp Met Gly Ile 85 Tyr Tyr Cys Leu Lys 90 Tyr Asp Glu Phe Pro 95 Tyr Thr Phe

I.a) Sequence identifier number: SEQ ID No: 3

II.a) Size: 33 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Light Chain CDR1 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 3

aaggcgagtc aggacattaa taactattta age 33

I.a) Sequence identifier number: SEQ ID No: 4 II.a) size: 11 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III. a) Other information: Variable Light Chain CDR1 Amino Acid Sequence EA2

IV) Sequence: SEQ ID NO. 4 Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Ser 15 10

I.a) Sequence identifier number: SEQ ID No: 5 II.a) size: 21

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: EA2 Variable Light Chain CDR2 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 5th

cgtgcaaaca gattggtaga t ??? 21

I.a) Sequence identifier number: SEQ ID No: 6

II.a) Size: 7

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Light Chain CDR2 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 6 Arg Wing Asn Arg Leu Val Asp

1 5

I.a) Sequence identifier number: SEQ ID No: 7

II.a) Size: 27 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Light Chain CDR3 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 7th

ctgaaatatg atgagtttcc gtacacg 27

I.a) Sequence identifier number: SEQ ID No: 8

II.a) Size: 9

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain CDR3 Amino Acid Sequence EA2

IV) Sequence: SEQ ID NO. 8th

Read Lys Tyr Asp Glu Phe Pro Tyr Thr 1 5

I.a) Sequence identifier number: SEQ ID No: 9

II.a) Size: 345 Il.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: EA2 Variable Heavy Chain Nucleotide Sequence

IV) Sequence: SEQ ID NO. 9th

gacgtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt agctatacca tgtcttgggt tcgccagact 120

ccggagaaga ggctggagtg ggtcgcaacc attagtagtg gtggtactta cacctactat 180

ccagacagtg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtac aagagaagct 300

atctttactt actggggcca agggactctg gtcactgtct ctgca 345

I.a) Sequence identifier number: SEQ ID No: 10

II.a) Size: 115

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Heavy Chain Amino Acid Sequence

IV) Sequence: SEQ ID NO. 10

Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Wing Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser 35 Trp Val Arg Gln Thr 40 Pro Glu Lys Arg Leu 45 Glu Trp Val Wing Thr 50 Ile Being Ser Gly Gly 55 Thr Tyr Thr Tyr Tyr 60 Pro Asp Being Val Lys 65 Gly Arg Phe Thr Ile 70 Being Arg Asp Asn Wing 75 Lys Asn Thr Leu Tyr 80 Leu Gln Met Ser Ser 85 Leu Lys Ser Glu Asp 90 Thr Wing Met Tyr Tyr 95 Cys Thr Arg Glu Wing Ile Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Wing 115 100 105 110

I.a) Sequence ID: SEQ ID No: 11 II.a) Size: 30 Il.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Heavy Chain CDR1 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 11 ggattcactt tcagtagcta taccatgtct 30

I.a) Sequence identifier number: SEQ ID No: 12

II.a) Size: 10 II.b) Type: PRT

II.c) organism: Artificial Sequence III)

III.a) Other information: Variable Heavy Chain CDR1 Amino Acid Sequence EA2

IV) Sequence: SEQ ID NO. 12

Gly Phe Thr Phe Ser Be Tyr Thr Met Ser 15 10

I.a) Sequence identifier number: SEQ ID No: 13

II.a) Size: 48 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Heavy Chain CDR2 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 13

accattagta gtggtggtac ttacacctac tatccagaca gtaagggc 48

I.a) Sequence identifier number: SEQ ID No: 14

II.a) Size: 17

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain CDR2 Amino Acid Sequence EA2

IV) Sequence: SEQ ID NO. 14th

Thr Ile Be Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 15 10 15

Gly

I.a) Sequence identifier number: SEQ ID No: 15 40 II.a) size: 18

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA2 Variable Heavy Chain CDR3 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 15

gaagctatct ttacttac 18

I.a) Sequence identifier number: SEQ ID No: 16

II.a) Size: 6 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain CDR3 Amino Acid Sequence EA2

IV) Sequence: SEQ ID NO. 16 Glu Wing Ile Phe Thr Tyr

1 5

I.a) Sequence identifier number: SEQ ID No: 17

II.a) Size: 336

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA5.12 Variable Light Chain Nucleotide Sequence

IV) Sequence: SEQ ID NO. 17

gatgtkgtka tgacbcagac tccactcact ttgtcggtta ccattggaca accagcctct 60 atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggaa cagattttac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tattactgcg tgcaaggttc acattttccg 300 336 tggacgttcg gtggaggcac caagctggaa atcaaa

I.a) Sequence identifier number: SEQ ID No: 18

II.a) Size: 112

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III. a) Other information: EA5.12 Variable Light Chain Amino Acid Sequence

IV) Sequence: SEQ ID NO. 18 Asp Val Val Met Thr Gln Thr Pro Read Leu Thr Be Val Thr Ile Gly 1 5 10 15 Gln Pro Wing Be 20 Ile Ser Cys Lys Ser 25 Ser Gln Ser Leu Read 30 Tyr Ser Asn Gly Lys 35 Thr Tyr Leu Asn Trp 40 Leu Leu Gln Arg Pro 45 Gly Gln Ser Lys Pro 50 Arg Leu Ile Tyr Leu 55 Val Ser Lys Asp 60 Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Wing Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Gly 85 90 95 Be His Phe Pro 100 Trp Thr Phe Gly Gly 105 Gly Thr Lys Leu Glu 110 Ile Lys

I.a) Sequence ID: SEQ ID No: 19 II.a) Size: 27 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA5.12 Variable Light Chain CDR1 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 19 aagtcaagtc agagcctctt atatagt 27

I.a) Sequence identifier number: SEQ ID No: 20

II.a) Size: 16

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain Amino Acid EA5.12 Sequence CDR1

IV) Sequence: SEQ ID NO. 20

Lys Be Ser Gln Be Read Leu Tyr Be Asn Gly Lys Thr Tyr Leu Asn 15 10 15

I.a) Sequence identifier number: SEQ ID No: 21

II.a) Size: 21 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain CDR2 Nucleotide Sequence EA5.12 IV) Sequence: SEQ ID NO. 21

ctggtgtcta aactggactc t ??? 21

I.a) Sequence identifier number: SEQ ID No: 22

II.a) Size: 7 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain CDR2 Amino Acid Sequence EA5.12

IV) Sequence: SEQ ID NO. 22 Leu Val Ser Lys Leu Asp Ser

1 5

I.a) Sequence identifier number: SEQ ID No: 23 II.a) size: 2 7

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA5.12 Variable Light Chain CDR3 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 23 gtgcaaggtt cacattttcc gtggacg 27

I.a) Sequence identifier number: SEQ ID No: 24

II.a) Size: 9

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain CDR3 Amino Acid Sequence EA5.12

IV) Sequence: SEQ ID NO. 24 Val Gln Gly Being His Phe Pro Trp Thr 1 5

I.a) Sequence identifier number: SEQ ID No: 25

II.a) Size: 345 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA5.12 Variable Heavy Chain Nucleotide Sequence

IV) Sequence: SEQ ID NO. 25

gaggtccagc tgcagcagtc tggacctgag ctagtgaaga ctggggcttc agtgaagata 60 tcctgcaagg cttctggtta ctcattcact ggttactaca tgcactgggt caagcagagc 120 catggaaaga gccttgagtg gattggatat attagttgtt acaatggtgt tactagctac 180 aaccagaagt tcaagggcaa ggccacattt actgtagaca catcctccag cacagcctac 240 atgcagttca acagcctgac atctgaagac tctgcggtct attactgtgc aagatctcat 300 gctatggact actggggtca aggaacctca gtcaccgtct cctca 345

I.a) Sequence identifier number: SEQ ID No: 26

II.a) Size: 115

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence - Variable Heavy Chain Region EA5.12

IV) Sequence: SEQ ID NO. 26

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala 1 5 10 15 Ser Val Lys Ile 20 Ser Cys Lys Wing 25 Serly Gly Tyr Ser Phe Thr 30 Gly Tyr Tyr Be Glu Trp Ile 35 40 45 Gly Tyr 50 Ile Be Cys Tyr Asn 55 Gly Val Thr Be Tyr 60 Asn Gln Lys Phe Asn Ser 85 Leu Thr Be Glu Asp 90 Ser Wing Val Tyr Tyr 95 Cys Wing Arg Be His 100 Wing Met Asp Tyr Trp 105 Gly

I.a) Sequence identifier number: SEQ ID No: 27

II.a) Size: 15

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA5.12 Variable Heavy Chain CDR1 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 27

ggttactaca tgcac 15

I.a) Sequence identifier number: SEQ ID No: 28 II.a) size: 5 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain CDR1 Amino Acid Sequence EA5.12

IV) Sequence: SEQ ID NO. 28 Gly Tyr Tyr Met His 1 5

I.a) Sequence identifier number: SEQ ID No: 29

II.a) Size: 51

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III. a) Other information: EA5.12 Variable Heavy Chain CDR2 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 29

tatattagtt gttacaatgg tgttactagc tacaaccaga agttcaaggg c

I. a) Sequence identifier number: SEQ ID No: 30

II.a) Size: 17

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain CDR2 Amino Acid Sequence EA5.12

IV) Sequence: SEQ ID NO. 30

Tyr Ile Being Cyr Tyr Asn Gly Val Thr Being Tyr Asn Gln Lys Phe Lys

15 10 15

Gly

I.a) Sequence identifier number: SEQ ID No: 31

II.a) Size: 18 Il.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EA5.12 Variable Heavy Chain CDR3 Nucleotide Sequence

IV) Sequence: SEQ ID NO. 31

tctcatgcta tggactac 18

I.a) Sequence identifier number: SEQ ID No: 32 II.a) size: 6 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain CDR3 Amino Acid Sequence EA5.12

IV) Sequence: SEQ ID NO. 32 Be His Wing Met Asp Tyr 1 5

I.a) Sequence identifier number: SEQ ID No: 33 II.a) size: 123

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: Variable Light Chain Amino Acid Sequence 233 IV) Sequence: SEQ ID NO. 33

Asp Ile Val Leu Thr Thr Gln Be Pro Wing Thr Read Le Be Val Thr Pro Gly 1 5 10 15 Asp Ile Val Leu Thr Thr Wing Pro 25 Wing 25 Thr Leu Be Val Thr 30 Pro Gly Asp Be Val 35 Asn Leu Be Cys Arg 40 Wing Ser Gln Ser Ile 45 Ser Asn Asn Leu His 50 Trp Tyr Gln Gln Lys 55 Ser His Glu Ser Pro 60 Arg Leu Read Ile Lys Tyr Val Phe Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 65 70 75 80 Ser Gly Ser Gly Thr 85 Asp Phe Thr Leu Be 90 Ile Asn Be Val Glu 95 Thr Glu Asp Phe Gly 100 Met Tyr Phe Cys Gln 105 Gln Be Asn Ser Trp 110 Pro Read Thr Phe Gly 115 Wing Gly Thr Lys Leu 120 Glu Leu Lys

I.a) Sequence identifier number: SEQ ID No: 34 II.a) size: 11 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain CDR1 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 34 Arg Wing Be Gln Ser Ile Ser Asn Asn Leu His 15 10

I.a) Sequence identifier number: SEQ ID No: 35 II.a) size: 7

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain CDR2 Amino Acid Sequence 233

IV) Sequence: SEQ ID NO. 35

Tyr Val Phe Gln Ser Ile Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 36

II.a) Size: 9 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain CDR3 Amino Acid Sequence 233 IV) Sequence: SEQ ID NO. 36

Gln Gln Ser Asn Ser Trp Pro Read Thr 1 5

I.a) Sequence identifier number: SEQ ID No: 37

II.a) Size: 120

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain Amino Acid Sequence 233

IV) Sequence: SEQ ID NO. 37

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Ser Met Asn 35 Trp Val Arg Gln Pro 40 Pro Gly Lys Ala Leu 45 Glu Trp Leu Gly Phe 50 Ile Arg Asn Lys Ala 55 Asn Asp Tyr Thr Thr 60 Glu Tyr Be Ala Ser 65 Val Lys Gly Arg Phe 70 Thr Ile Be Arg Asp 75 Asn Ser Gln Ser Ile 80 Leu Tyr Leu Gln Met 85 Asn Wing Read Arg Wing 90 Glu Asp Ser Wing Thr 95 Tyr Tyr Val Cys Tyr Pro Arg Tyr His Wing Met Asp Ser Trp Gly Gln Gly Thr Ser 115 100 Val Thr Val Ser Ser 120 105 110

I.a) Sequence identifier number: SEQ ID No: 38

II.a) Size: 5

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain CDR1 Amino Acid Sequence 233

IV) Sequence: SEQ ID NO. 38 Asp Tyr Ser Met Asn 1 5

I.a) Sequence identifier number: SEQ ID No: 39

II.a) Size: 19

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: Variable Heavy Chain CDR2 Amino Acid Sequence 233

IV) Sequence: SEQ ID NO. 39 Phe Ile Arg Asn Lys Wing Asn Asp Tyr Thr Thr Glu Tyr Ser Wing 15 10 15

Val Lys Gly

I.a) Sequence identifier number: SEQ ID No: 40

II.a) Size: 9 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III. a) Other information: Variable Heavy Chain CDR3 Amino Acid Sequence 233

IV) Sequence: SEQ ID NO. 40 Tyr Pro Arg Tyr His Wing Met Asp Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 41 II.a) size: 107 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Light Chain Amino Acid Sequence 3F2

IV) Sequence: SEQ ID NO. 41

Wing 1 Ile Gln Leu Thr 5 Gln Ser Pro Be Ser 10 Leu Ser Wing Ser Val 15 Gly Asp Arg Val Thr 20 Ile Thr Cys Arg Wing 25 Ser Gln Ser Ile Ser 30 Asn Asn Read His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Read Ile 35 40 45 Tyr Tyr 50 Gly Phe Gln Be Ile 55 Be Gly Val Pro Be 60 Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Thr Leu Thr Ile Be Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Wing Thr 85 Tyr Tyr Cys Gln 90 Wing Asn Ser Trp Pro 95 Leu Thr Phe Gly Gly 100 Gly Thr Lys Leu Glu 105 Ile Lys

I.a) Sequence identifier number: SEQ ID No: 42

II.a) Size: 11 Il.b) Type: PRT

II.c) organism: Artificial Sequence III)

III.a) Other information: Variable Light Chain CDR1 Amino Acid Sequence 3F2

IV) Sequence: SEQ ID NO. 42

Arg Wing Be Gln Be Ile Be Asn Asn Leu His 15 10

I.a) Sequence identifier number: SEQ ID No: 43 II.a) size: 7 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: 3F2 Variable Light Chain CDR2 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 43

Tyr Gly Phe Gln Ser Ile Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 44

II.a) Size: 9

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: 3F2 Variable Light Chain CDR3 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 44

Gln Gln Wing Asn Ser Trp Pro Read Thr 1 5

I.a) Sequence identifier number: SEQ ID No: 45

II.a) Size: 120

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Variable Heavy Chain Amino Acid Sequence 3F2

IV) Sequence: SEQ ID NO. 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 15 10 15

Be Read Arg Read Be Cys Wing Wing Be Gly Phe Thr Val Be Asp Tyr 20 25 30

Being Met Asn Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe 50 Ile Arg Asn Lys Wing 55 Asn Wing Tyr Thr Thr 60 Glu Tyr Being Wing Being Val Lys Gly Arg Phe Thr Ile Being Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met 85 Asn Ser Leu Lys Thr 90 Glu Asp Thr Wing Val 95 Tyr Tyr Cys Thr Thr 100 Tyr Pro Arg Tyr His 105 Ala Met Asp Ser Trp 110 Gly Gln Gly Thr Met 115 Val Thr Val Ser Ser 120

I.a) Sequence identifier number: SEQ ID No: 4 6

II.a) Size: 5

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: 3F2 Variable Heavy Chain Amino Acid CDR1 Sequence

IV) Sequence: SEQ ID NO. 46 Asp Tyr Ser Met Asn

1 5

I.a) Sequence identifier number: SEQ ID No: 47

II.a) Size: 19

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: 3F2 Variable Heavy Chain CDR2 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 47

Phe Ile Arg Asn Lys Wing Asn Wing Tyr Thr Thr

15 10 15

Val Lys Gly

I.a) Sequence identifier number: SEQ ID No: 48

II.a) Size: 9

II.b) Type: PRT

II. c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: 3F2 Variable Heavy Chain CDR3 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 48 Tyr Pro Arg Tyr His Ala Met Asp Ser 1 5 I.a) Sequence identifier number: SEQ ID No: 49

II.a) Size: 107

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: G5 Variable Light Chain Amino Acid Sequence

IV) Sequence: SEQ ID NO. 49

Asp Ile Gln Met Thr Gln Be Pro Be Be Read Be Wing Be Val Gly 1 5 10 15 Asp Arg Val Thr 20 Ile Thr Cys Wing 25 Be Gln Be Ile Be 30 Asn Asn Read His Trp 35 Tyr Gln Lys Pro 40 Gly Lys Wing Pro Lys 45 Leu Leu Ile Lys Tyr Val Phe Gln Be Ile Be Gly Val Pro Be Arg Phe Be Gly 50 55 60 Be Gly Be Gly Thr Asp Phe Thr Ile Be Serve Leu Gln Pro 65 70 75 80 Glu Asp Phe Wing Thr 85 Tyr Cyr Gln Gln 90 Ser Asn Ser Trp Pro 95 Leu Thr Phe Gly Gly 100

I.a) Sequence identifier number: SEQ ID No: 50

II.a) Size: 11

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III. a) Other information: G5 Variable Light Chain CDR1 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 50

Arg Wing Be Gln Be Ile Be Asn Asn Leu His 1 5 10

I.a) Sequence identifier number: SEQ ID No: 51

II.a) Size: 7 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: G5 Variable Light Chain CDR2 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 51 Tyr Val Phe Gln Ser Ile Ser 1 I.a) Sequence identifier number: SEQ ID No: 52

II.a) Size: 9 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: G5 Variable Light Chain CDR3 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 52

Gln Gln Ser Asn Ser Trp Pro Read Thr 1 5

I.a) Sequence identifier number: SEQ ID No: 53

II.a) Size: 120

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: G5 Variable Heavy Chain Amino Acid Sequence

IV) Sequence: SEQ ID NO. 53

Gln Met Gln Read Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Val 20 Ser Cys Lys Ala Ser 25 Gly Phe Thr Phe 30 Asp Tyr Ser Met Asn 35 Trp Val Arg Gln Ala 40 Arg Gly Gln Arg Leu 45 Glu Trp Ile Gly Phe 50 Ile Arg Asn Lys Wing 55 Asn Tyr Thr Thr 60 Glu Tyr Be Wing Ser Val Lys Glu Leu 85 Ser Be Leu Arg Ser 90 Glu Asp Thr Wing Val 95 Tyr Tyr Cys Wing Arg 100 Tyr Pro Arg Tyr His 105 Wing Met Asp Ser Trp 110 Gly Gln Gly Thr Ser 115 Val Thr Val Ser Ser 120

I.a) Sequence identifier number: SEQ ID No: 54 II.a) size: 5 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: G5 Variable Heavy Chain Amino Acid CDR1 Sequence

IV) Sequence: SEQ ID NO. 54 Asp Tyr Ser Met Asn 1 5

I.a) Sequence identification number: SEQ ID No: 55 II.a) Size: 19 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic: III.a) Other information: G5 Variable Heavy Chain CDR2 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 55 Phe Ile Arg Asn Lys Wing Asn Asp Tyr Thr Thr Glu Tyr Ser Wing 1 5 10 15

Val Lys Gly

I.a) Sequence identifier number: SEQ ID No: 56

II.a) Size: 9 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic:

III.a) Other information: G5 Variable Heavy Chain Amino Acid Sequence G5

IV) Sequence: SEQ ID NO. 56

Tyr Pro Arg Tyr His Wing Met Asp Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 57 II.a) size: 18 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EphA2-BiTe construct linker amino acid sequence

IV) Sequence: SEQ ID NO. 57

Gly Glu Gly Thr Gly Thr Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 1 10 10 15

Asp wing

I.a) Sequence identification number: SEQ ID No: 58 II.a) Size: 5 II.b) Type: PRT II.c) Organism: Artificial Sequence III) Characteristic:

III.a) Other information: EphA2-BiTe construct linker amino acid sequence

IV) Sequence: SEQ ID NO. 58 Gly Gly Gly Gly Ser

1 5

I.a) Sequence identifier number: SEQ ID No: 59

II.a) Size: 15 II.b) Type: PRT

II.c) organism: Artificial Sequence III)

III. a) Other information: EphA2-BiTe construct linker amino acid sequence

IV) Sequence: SEQ ID NO. 59

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser 15 10 15

I.a) Sequence ID: SEQ ID No: 60 II.a) Size: 1554 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Deimmunized anti-CD3 (VH-Vl) χ EA2 (VH-VL) Nucleotide Sequence

IV) Sequence

gaattcacca

cactccgacg

aaggtgtcct

caggcacctg

aattacgcag

gcctacatgg

tattatgatg

tcaggcgaag

attgtactga

agctgcagag

gcacccaaaa

agtggcagtg

gctgccactt

aaggtggaga

ggcttagtga

agtagctata

accattagta

atctccagag

gacacagcca

ctggtcactg

SEQ

tgggatggag tccaactggt gcaaggcttc gacagggtct acagcgtcaa aactgagcag atcattactg gtactagtac cccagtctcc ccagtcaaag gatggattta ggtctgggac attactgcca tcagggggggtgtgtgtgt

ID NO. 60

cggtgcggggggggggggggg

ctcttcttgg gctgaagtga tttactaggt ggatacatta acaatcacta gaggacactg tggggccaag ggaagtggag tctctgtctc atgaactggt aaagtggctt ctcacaatca agtaacccgc tggggcgtgtgtcgtgtcgtgtcgtg

tagcaacagc aaggggggggggggggggggggggggg

tacggtgta ggcctcagtg ctgggtaagg tggttatact caccagcaca ctgtgcaaga caccgtctcc agcagacgac tgccaccctg gccgggcaag tgctcgcttc ggctgaagat tggcggggggggggggggggggggggggggggggggggg

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 ggttctgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga 1260 gtcactatca cttgcaaggc gagtcaggac attaataact atttaagctg gttccagcag 1320 aaaccaggga aatctcctaa gaccctgatc tatcgtgcaa acagattggt agatggggtc 1380 ccatcaaggt tcagtggcag tggatctggg caagattatt ctctcaccat cagcagcctg 1440 gagtatgaag atatgggaat ttattattgt ctgaaatatg atgagtttcc gtacacgttc 1500 ggagggggga ccaagctgga aataaaacat catcaccatc atcattaggt cgac 1554

I.a) Sequence identifier number: SEQ ID No: 61

II. a) size: 55

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EphA2-BiTe construct linker nucleotide sequence

IV) Sequence: SEQ ID NO. 61

ggcgaaggta ctagtactgg ttctggtgga agtggaggtt caggtggagc agacg 55

I.a) Sequence ID: SEQ ID No: 62 II.a) Size: 19 Il.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EphA2-BiTe construct linker nucleotide sequence

IV) Sequence: SEQ ID NO. 62 tccggaggtg gtggatccg ??? 19

I.a) Sequence identifier number: SEQ ID No: 63

II.a) Size: 4 8

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EphA2-BiTe construct linker nucleotide sequence

IV) Sequence: SEQ ID NO. 63

tccggtggtg gtggttctgg cggcggcggc tccggtggtg gtggttct 48

I.a) Sequence identifier number: SEQ ID No: 64

II.a) Size: 21

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: EphA2-BiTe construct linker nucleotide sequence IV) Sequence: SEQ ID NO. 64

catcatcacc atcatcatta g ??? 21

I.a) Sequence identifier number: SEQ ID No: 65 II.a) size: 492

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Anti-CD3 (VH-VL) χ EA2 (VH-VL) EphA2-Bite deimmunized Amino Acid Sequence

IV) Sequence: SEQ ID NO. 65

Asp Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 1 5 10 15

Be Val Lys Val Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Ile 35 40 45

Gly Tyr Ile Asn Pro To Be Arg Gly Tyr Thr Asn Tyr Asa Wing Be Val 50 55 60

Lys Gly Arg Phe Thr Ile Thr Thr Asp Lys Be Thr Be Thr Wing Tyr 65 70 75 80

Met Glu Read Be Ser Read Arg Be Glu Asp Thr Wing Thr Tyr Tyr Cys 25 85 90 95

Wing Arg Tyr Tyr Asp Asp His Tyr Cys Read Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Be Ser Gly Glu Gly Thr Ser Be Gly Ser Gly 115 120 125

Gly Ser Gly Gly Gly Gly Gly Wing Asp Asp Ile Val Leu Thr Gln Ser 130 135 140

Pro Wing Thr Read Be Read Be Pro Gly Glu Arg Wing Thr Read Read Be Cys 145 150 155 160

Arg Wing Be Gln Be Val Be Tyr Met Asn Trp Tyr Gln Gln Lys Pro 35 165 170 175

Gly Lys Wing Pro Lys Arg Trp Ile Tyr Asp Thr Be Lys Val Wing Ser

180 185 190

Gly Val Pro Wing Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser 195 200 205

Leu Thr Ile Asn Be Leu Glu Wing Glu Asp Wing Wing Thr Tyr Cys 210 215 220

Gln Gln Trp To Be Asn Pro Read Thr Phe Gly Gly Gly Thr Lys Val 225 230 235 240

Glu Ile Lys Ser Gly Gly Gly Gly Ser Asp Val Lys Leu Val Glu Ser 245, 250 255

Gly Gly Gly Gave Val Lys Pro Gly Gly Gave Leu Lys Gave Cys Wing

260 265 270

Wing Be Gly Phe Thr Phe Be Be Tyr Thr Met Be Trp Val Arg Gln 275 280 285

Thr Pro Glu Lys Arg Leu Glu Trp Val Wing Thr Ile Ser Be Gly Gly 290 295 300

Thr Tyr Thr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 305 310 315 320

Arg Asp Asn Ala Lys Asn Thr Read Tyr Read Gln Met Be Ser Read Leys 325 330 335

Be Glu Asp Thr Wing Met Tyr Tyr Cys Thr Arg Glu Wing Ile Phe Thr

340 345 350

Tyr Trp Gly Gln Gly Thr Read Le Val Val Be Ser Gly Gly Gly Gly 355 360 365 Be Gly 370 Gly Gly Gly Be Gly 375 Gly Gly Gly Be Asp 380 Ile Lys Met Thr Gln Be Pro Be Met Tyr Ala Be Read Gly Glu Arg Val Thr Ile 385 390 395 400 Thr Cys Lys Ala Ser 405 Gln Asp Ile Asn Asn 410 Tyr Leu Ser Trp Phe 415 Gln Lys Pro Gly 420 Lys Ser Pro Lys Thr 425 Leu Ile Tyr Arg Ala 430 Asn Arg Leu Val Asp 435 Gly Val Pro Ser Arg 440 Phe Ser Gly Ser Gly 445 Ser Gly Gln Asp Tyr 450 Ser Leu Thr Ile Ser 455 Ser Leu Glu Tyr Glu 460 Asp Met Gly Ile Tyr Cys Leu Lys Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly 465 470 475 480 Thr Lys Leu Glu Ile 485 Lys His His His 490 His His

I. a) Sequence identifier number: SEQ ID No: 66 II.a) size: 6 II.b) type: PRT II.c) organism: Artificial Sequence III) characteristic: III.a) other information: hex sequence C-terminal cDNA-histidine of EphA2-BiTe

IV) Sequence: SEQ ID NO. 66

His His His His His 1 5

Ia) Sequence identifier number: SEQ ID No: 67 II.a) Size: 711 II.b) Type: DNA II.c) Organism: Artificial Sequence III) Characteristic: III.a) Other information: Nucleotide Sequence4h5 VH VL scFV

IV) Sequence: SEQ ID NO. 67 caggtgcagc tgttggagtc

caggtgcagc tgttggagtc tgggggaggc tcctgtgcag cctctggatt cacctttagc cctggacaag cgcttgagtg gatgggaacc ccagacagtg tgaagggccg attcaccatc ctgcaaatga acagcctgag agccgaggac atctttactt actggggccg tggcaccctg ggcggcggcg gctccggtgg tggtggttct ctgtctgcat ctgtaggaga cagagtcacc aactatttaa gctggtacca gcagaaacct gcaaacagat tggtagatgg ggtcccagac tttaccctca caattaataa catagaatct tatgatgtgt ttccgtacac gttcggccaa

ttggtacagc agctatacca attagtagtg tccagagaca acggctgtgt gtcaccgtct gacatccagt atcacttgca ggccaggctc aggttcagtg gaggatgctg gggaccaagg

ctggggggtc tgtcttgggt gtggtactta acgccaagaa attactgtgc cctcaggtgg tgacccagtc aggcgagtca ccaggctcct gcagcgggta catattactt tggagatcaa

cctgagactc gcgacaggcc cacctactat ctcactgtat gagagaagct tggtggttct tccatcctcc ggacattaat catctatcgt tggaacagat ctgtctgaaa

60 120 180 240 300 360 420 480 540 600 660 711 I. (a) Sequence identifier number: SEQ ID No: 68

II. a) Size: 237 II.b) Type: PRT

II.c) organism: Artificial Sequence III)

III.a) Other information: Amino Acid Sequence4H5 VH VL scFv

IV) Sequence: SEQ ID NO. 68 Gln Val Gln Leu Leu Read Glu Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Be Cys Wing Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Wing Leu Glu Trp Met 35 40 45 Gly Thr Ile Be Ser Gly Gly Thr Tyr Thr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Wing Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95 Wing Arg Glu Wing Ile Phe Thr Tyr Trp Gly Arg Gly Thr Gly Gly 115 120 125 Gly Be Asp Ile Gln Read Thr Gln Be Pro Be Be Read Le Be Be 130 135 140 Val Gln Gln Lys Pro Gly Gln Pro Wing Arg Read 165 170 175 Leu Ile Tyr Arg Wing Asn Arg Leu Val Asp Gly Val Pro Asp Arg Phe 180 185 190 Ser Gly Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile 195 200 205 Glu Ser Glu Asp Wing Tyr Tyr Phe Cys 220 Pro Tyr Thr Phe Gly Gln

225 230 235

I.a) Sequence identifier number: SEQ ID No: 69

II.a) Size: 5 II.b) Type: PRT

II.c) organism: Artificial Sequence III)

III.a) Other information: Amino Acid Sequence4H5 VH VL scFv CDRl (VH)

IV) Sequence: SEQ ID NO. 69

Ser Tyr Thr Met Ser 1 5 I.a) Sequence identifier number: SEQ ID No: 70

II.a) Size: 17

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 4H5 VH VL scFv CDR2 (VH)

IV) Sequence: SEQ ID NO. 70

Thr Ile Be Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 15 10 15

Gly

I.a) Sequence identifier number: SEQ ID No: 71

II.a) Size: 6

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 4H5 VH VL scFv CDR3 (VH)

IV) Sequence: SEQ ID NO. 71

Glu Wing Ile Phe Thr Tyr 1 5

I.a) Sequence identifier number: SEQ ID No: 72

II.a) Size: 11

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III. a) Other information: Amino Acid Sequence 4H5 VH VL scFv CDRl (VL)

IV) Sequence: SEQ ID NO. 72

Lys Wing Ser Gln Asp Ile Asn Asn Tyr Leu Ser 1 5 10

I.a) Sequence identifier number: SEQ ID No: 73

II.a) Size: 7

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 4H5 VH VL scFv CDR2 (VL) IV) Sequence: SEQ ID NO. 73

Arg Wing Asn Arg Leu Val Asp 1 5

I.a) Sequence identifier number: SEQ ID No: 74

II.a) Size: 9 II.b) Type: PRT

II.c) organism: Artificial Sequence III)

III.a) Other information: Amino Acid Sequence 4H5 VH VL scFv CDR3 (VL)

IV) Sequence: SEQ ID NO. 74

Read Lys Tyr Asp Val Phe Pro Tyr Thr 1 5

I.a) Sequence identifier number: II.a) size: 711 Il.b) type: DNA

II.c) organism: Artificial Sequence

SEQ ID No: 75

III) Characteristic:

III.a) Other information VL scFv

2A4 VH Nucleotide Sequence

IV) Sequence

caggtgcagc

tcctgtgcag

cctggacaag

ccagacagtg

ctgcaaatga

atctttactc

ggcggcggcg

ctgtctgcat

aactatcaca

gcaaacagat

tttaccctca

SEQ

tgttggagtc

cctctggatt

cgcttgagtg

tgaagggccg

acagcctgag

actggggccg

gctccggtgg

ctgtaggaga

gctggtacca

tggtcgatgg

caattaataa

tataatgtgt ttccgtacac

ID NO. 7 tgggggaggc cacctttagc gatgggaacc attcaccatc agccgaggac tggcaccctg tggtggttct cagagtcacc gcagaaacct ggtcccagac catagaatct gttcggccaa

ttggtacagc agctatacca attagtagtc tccagagaca acggctgtgt gtcaccgtct gacatccagt atcacttgca ggccaggctc aggttcagtg gaggatgctg gggaccaagg

ctggggggtc tgtcttgggt gtggtactta acgccaagaa attactgtgc cctcaggtgg tgacccagtc aggcgagtca ccaggctcct gcagcgggta catattactt tggagatcaa

cctgagactc 60 gcgacaggcc 120 cacctactat 180 ctcactgtat 240 gagagaagct 300 tggtggttct 360 tccatcctcc 420 ggacattaat 480 catctatcgt 540 tggaacagat 600 ctgtctgaaa 660 to 711

I.a) Sequence identifier number: SEQ ID No: 76

II.a) Size: 237 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2A4 VH VL scFv

IV) Sequence: SEQ ID NO. 76

Gln Val Gln Leu Read Glu Be Gly Gly Gly Leu Val Gln Pro Gly Gly 15 10 15 Be Read Le Arg Leu Be Cys Wing Ala Be Gly Phe Thr Phe Be Ser Tyr 20 25 30 Thr Met Be Trp Val Arg Gln Ala Pro Gly Gln Wing Leu Glu Trp Met 35 40 45 Gly Thr Ile Be Ser Arg Arg Gly Thr Tyr Thr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Wing Lys Asn Ser Leu Tyr 65 70 75 80 Be Leu Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95 Wing Arg Glu Wing Ile Phe Thr His Trp Gly Arg Gly Thr Leu Val Thr 100 105 110 Val Be Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 115 120 125 Gly Be Asp Ile Gln Read Thr Gln Be Pro Be Ser Be Read Le Be Ser 130 130 140 Val Gly Asp Arg Val Thr Ile Thr Cys Lys Wing Be Gln Asp Ile Asn 145 150 155 160 Asn Tyr His Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Wing Arg Read 165 170 175 Read Ile Tyr Arg Wing Asn Arg Read Val Asp Gly Val Pro Asp Arg Phe 180 185 190 Ser Gly Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile 195 200 205 Glu Ser Glu Asp Wing Tyr Tyr Phe Cys Leu Lys Tyr Asn Val Phe 210 215 220 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235

I.a) Sequence identifier number: SEQ ID No: 77

II.a) Size: 5 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2A4 VH VL scFv CDRl (VH)

IV) Sequence: SEQ ID NO. 77

Ser Tyr Thr Met Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 78 II.a) size: 17 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2A4 VH VL scFv CDR2 (VH)

IV) Sequence: SEQ ID NO. 78 Thr Ile Ser Be Arg Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 15 10 15 Gly

I.a) Sequence identifier number: SEQ ID No: 79 II.a) size: 6 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2A4 VH VL scFv CDR3 (VH)

IV) Sequence: SEQ ID NO. 79 Glu Wing Ile Phe Thr His 1 5

I.a) Sequence identifier number: SEQ ID No: 80 II.a) size: 11

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2A4 VH VL scFv CDRl (VL)

IV) Sequence: SEQ ID NO. 80

Lys Ala Ser Gln Asp Ile Asn Asn Tyr His Ser 1 5 10

I.a) Sequence identifier number: SEQ ID No: 81

II.a) Size: 7 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2A4 VH VL scFv CDR2 (VL)

IV) Sequence: SEQ ID NO. 81 Arg Wing Asn Arg Leu Val Asp

1 5

I.a) Sequence identifier number: SEQ ID No: 82

II.a) Size: 9 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic: III.a) Other information: Amino Acid Sequence 2A4 VH VL scFv CDR3 (VL)

IV) Sequence: SEQ ID NO. 82

Read Lys Tyr Asn Val Phe Pro Tyr Thr 1 5

I.a) Sequence identifier number: SEQ ID No: 83

II.a) Size: 711

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Nucleotide Sequence 2E7 VH VL scFv

IV) Follow caggtgcagc tcctgtgcag cctggacaag J-ccagacagtg ctgcaaatga atctttactc

SEQ

tgttggagtc

cctctggatt

cgcttgagtg

tgaagggccg

acagcctgag

actggggccg

gctccggtgg

ctgtaggaga

gctggtacca

tggtcgatgg

caattaataa

ttccgtacac

ID NO. 83

tgggggaggc cacctttagc gatgggaacc attcaccatc agccgaggac tggcaccctg tggtggttct cagagtcacc gcagaaacct ggtcccagac catagaatct gttcggccaa

ttggtacagc agctatacca attagtagtc tccagagaca acggctgtgt gtcaccgtct gacatccagt atcacttgca ggccaggctc aggttcagtg gaggatgctg gggaccaagg

ctggggggtc tgtcttgggt gtggtactta acgccaagaa attactgtgc cctcaggtgg tgacccagtc aggcgagtca ccaggctcct gcagcgggta catattactt tggagatcaa

cctgagactc 60 gcgacaggcc 120 cacctactat 180 ctcactgtat 240 gagagaagct 300 tggtggttct 360 tccatcctcc 420 ggacattaat 480 catctatcgt 540 tggaacagat 600 ctgtctgaaa 660 to 711

I.a) Sequence identification number:

II.a) Size: 333 II.b) Type: PRT

II.c) organism: Artificial Sequence

SEQ ID No: 84

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2E7 VH VL scFv

IV) Sequence: SEQ ID NO. : 84 Gln Val Gln Leu Read Le Glu Be Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Be Cys Wing Ala Ser Gly Phe Thr Phe Ser Be Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Wing Leu Glu Trp Met 35 40 45 Gly Thr Ile Be Being Arg Gly Thr Tyr Thr Tyr Pro Asp Being Val 50 55 60 Lys Gly Arg Phe Thr Ile Being Arg Asp Asn Wing Lys Asn Being Leu Tyr 70 75 80 Leu Gln Met Asn Ser Leu Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95 Gln Val Gln Leu Read Glu Be Gly Gly Gly Leu Val Gln Pro Gly Gly 100 105 110 Ser Leu Arg Leu Ser Cys Wing Gly Phe Thr Phe Ser Ser Tyr 115 120 125 Thr Met 130 Ser Trp Val Arg Gln 135 Wing Pro Gly Gln Wing 140 Leu Glu Trp Met Gly Thr Ile Being Ser Gyr Thr Tyr Tyr Tyr Pro Asp Ser Val 145 150 155 160 Lys Gly Arg Phe Thr 165 Ile Be Arg Asp Asn 170 Wing Lys Asn Be Read 175 Tyr Read Gln Met Asn 180 Be Read Arg Wing lu 185 Asp Thr Wing Val Tyr 190 Tyr Cys Wing Arg Glu 195 Wing Ile Phe Thr His 200 Trp Gly Arg Gly Thr 205 Leu Val Thr Val Ser 210 Ser Gly Gly Gly Gly 215 Ser Gly Gly Gly Gly 220 Ser Gly Gly Gly Gly Ser Asp Ile Gln Read Thr Gln Be Pro Be Ser Be Read Ala Be 225 230 235 240 Val Gly Asp Arg Val 245 Thr Ile Thr Cys Lys 250 Wing Be Gln Asp Ile 255 Asn Asn Tyr Gly Be 260 Trp Tyr Gln Lys 265 Pro Gly Gln Wing Pro 270 Arg Leu Read Le Ile Tyr 275 Arg Wing Asn Arg Leu 280 Val Asp Gly Val Pro 285 Asp Arg Phe Be Gly 290 Be Gly Tyr Gly Thr 295 Asp Phe Thr Leu Thr 300 Ile Asn Ile Glu Be Glu Asp Wing Ala Tyr Tyr Phe Cys Read Lys Tyr Asn Arg Phe 305 310 315 320 Pro Tyr Thr Phe Gly 325 Gln Gly Thr Lys Val 330 Glu Ile Lys

I.a) Sequence identifier number: SEQ ID No: 85 II.a) size: 5 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2E7 VH VL scFv CDRl (VH)

IV) Sequence: SEQ ID NO. 85 Ser Tyr Thr Met Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 86 II.a) size: 17

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2E7 VH VL scFv CDR2 (VH)

IV) Sequence: SEQ ID NO. 86

Thr Ile Be Ser Arg Gly Thr Tyr Thr Tyr Pro Asp Be Val Lys 1 5 10 15

Gly I.a) Sequence identifier number: SEQ ID No: 87 II. a) Size: 6 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2E7 VH VL scFv CDR3 (VH)

IV) Sequence: SEQ ID NO. 87 Glu Wing Ile Phe Thr His

1 5

I.a) Sequence identifier number: SEQ ID No: 88

II.a) Size: 11 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2E7 VH VL scFv CDRl (VL)

IV) Sequence: SEQ ID NO. 88

Lys Ala Ser Gln Asp Ile Asn Asn Tyr Gly Ser 15 10

I.a) Sequence identifier number: SEQ ID No: 89

II.a) Size: 7

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2E7 VH VL scFv CDR2 (VL)

IV) Sequence: SEQ ID NO. 89 Arg Wing Asn Arg Leu Val Asp

1 5

I.a) Sequence identifier number: SEQ ID No: 90

II.a) Size: 9 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 2E7 VH VL sCFv CDR3 (VL) IV) Sequence: SEQ ID NO. 90 Read Lys Tyr Asn Arg Phe Pro Tyr Thr 1 5

I.a) Sequence identification number:

II.a) Size: 711Il.b) Type: DNA

II.c) organism: Artificial Sequence

SEQ ID No: 91

III) Characteristic:

III.a) Other information: Nucleotide Sequence 12E2 VH VL scFv

IV) Sequence: SEQ

caggtgcagc tgttggagtc

tcctgtgcag cctctggatt

cctggacaag cgcttgagtg

ccagacagtg tgaagggccg

ctgcaaatga acagcctgag

atctttactt actggggccg

ggcggcggcg gctccggtgg

ctgtctgcat ctgtaggaga

aactatttaa gctggtacca

gcaaacagat tgttcgatgg

tttaccctca caattaataa

tatgatcggt ttccgtacac

ID NO. 91

tgggggaggc cacctttagc gatgggaacc attcaccatc agccgaggac tggcaccctg tggtggttct cagagtcacc gcagaaacct ggtcccagac catagaatct gttcggccaa

ttggtacagc agctatacca attagtagtc tccagagaca acggctgtgt gtcaccgtct gacatccagt atcacttgca ggccaggctc aggttcagtg gaggatgctg gggaccaagg

ctggggggtc tgtcttgggt gtggtactta acgccaagaa attactgtgc cctcaggtgg tgacccagtc aggcgagtca ccaggctcct gcagcgggta catattactt tggagatcaa

cctgagactc gcgacaggcc cacctactat ctcactgtat gagagaagct tggtggttct tccatcctcc ggacattaat catctatcgt tggaacagat ctgtctgaaa a

I.a) Sequence identifier number: SEQ ID No: 92

II. a) Size: 237 II.b) Type: PRT

II.c) organism: Artificial Sequence

60 120 180 240 300 360 420 480 540 600 660 711

III) Characteristic:

III. a) Other information: Amino Acid Sequence 12E2 VH VL scFv

IV) Sequence: SEQ ID NO. 92 Gln Val Gln Leu Read Glu Be Gly Gly Gly Leu Val Gln Be Gly Gly 1 5 10 15 Be Read Leu Arg Read Le Be Cys Wing Be Gly Phe Thr Phe Be Ser Tyr 20 25 30 Thr Met Be Trp Val Arg Gln Pro Gly Gln Wing Leu Glu Trp Met 35 40 45 Gly Thr Ile Be Ser Arg Arg Gly Thr Tyr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Wing Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95 Wing Arg Glu Wing Ile Phe Thr Tyr Trp Gly Arg Gly Thr Leu Val Thr 100 105 110 Val Be Ser Gly Gly Gly Gly Serly Gly Gly Gly Serly Gly Gly Gly 115 120 125 Gly Be Asp Ile Gln Read Thr Gln Be Pro Be Be Read Be Wing 130 130 140

Val Gly Asp Arg Val Ile Thr Thr Cys Lys Ala Ser Gln Asp Ile Asn 145 150 155 160 Asn Tyr Leu Be Trp Tyr Gln Lys Pro Gly Gln Ala Pro Arg Leu 165 170 175 Leu Ile Tyr Arg Wing Asn Arg Leu Phe Asp Gly Val Pro Asp Arg Phe 180 185 190 Ser Gly Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile 195 200 205 Glu Ser Glu Asp Ala Wing Tyr Tyr Phe Cys Leu Lys Tyr Asp Arg Phe 210 215 220 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu

I.a) Sequence identifier number: SEQ ID No: 93

II.a) Size: 5 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 12E2 VH VL scFv CDRl (VH)

IV) Sequence: SEQ ID NO. 93

Ser Tyr Thr Met Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 94 II.a) size: 17 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 12E2 VH VL scFv CDR2 (VH)

IV) Sequence: SEQ ID NO. 94

Thr Ile Be Ser Arg Gly Thr Tyr Thr Tyr Pro Asp Ser Val Lys 15 10 15

Gly

I.a) Sequence identifier number: SEQ ID No: 95

II.a) Size: 6 II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 12E2 VH VL scFv CDR3 (VH) IV) Sequence: SEQ ID NO. 95

Glu Wing Ile Phe Thr Tyr 1 5

I.a) Sequence identifier number: SEQ ID No: 96

II.a) Size: 11 II.b) Type: PRT II.c) Organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 12E2 VH VL scFv CDRl (VL)

IV) Sequence: SEQ ID NO. 96

Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Ser 15 10

I.a) Sequence identifier number: SEQ ID No: 97 II.a) size: 7 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 12E2 VH VL scFv CDR2 (VL)

IV) Sequence: SEQ ID NO. 97

Arg Wing Asn Arg Read Le Phe Asp

1 5

I.a) Sequence identifier number: SEQ ID No: 98

II.a) Size: 9

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Amino Acid Sequence 12E2 VH VL scFv CDR3 (VL)

IV) Sequence: SEQ ID NO. 98

Read Lys Tyr Asp Arg Phe Pro Tyr Thr 45 1 5

I.a) Sequence identifier number: SEQ ID No: 99

II.a) Size: 33

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: Combinatorial Primer Ll used in 2G6 Fab affinity optimization

IV) Sequence: SEQ ID NO. 99

ccagtgtagg ttgttgctaa tactttggct ggc 33

I.a) Sequence identifier number: SEQ ID No: 100

II.a) Size: 33

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: L2 Combinatorial Primer used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 100 ccagtgtagg ttgttgtdaa tactttggct ggc 33

I.a) Sequence identifier number: SEQ ID No: 101

II.a) Size: 42

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: L3 Combinatorial Primer used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 101

gggaccccag agatgdactg gaagvcatac ttgatcagga gc 42

I.a) Sequence ID: SEQ ID No: 102 II.a) Size: 42 II.b) Type: DNA II.c) Organism: Artificial Sequence

III) Characteristic:

III. a) Other information: L4 Combinatorial Primer used for 2G6 Fab affinity optimization

IV) Sequence: SEQ ID NO. 102

gggaccccag agatgdactg gaagstatac ttgatcagga gc 42

I.a) Sequence identification number: SEQ ID No: 103 II. a) size: 35 II. b) type: DNA II. c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: L5 Combinatorial Primer used for 2G6 Fab affinity optimization

IV) Sequence: SEQ ID NO. 103 gagcggccag ctgttggmct gttgacagta atatg 35

I.a) Sequence identifier number: SEQ ID No: 104

II.a) Size: 46 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: H6 Combinatorial Primer used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 104

gcctgtcgca cccasttcat ggagtaatcg gwaaaggtga atccag 46

I.a) Sequence identifier number: SEQ ID No: 105 II.a) size: 4 6 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Combinatorial Primer H7 used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 105 Gly Cys Cys Thr Gly Cys Thr Cly Gys Cys Wing Cys Cys Cys Wing Gly Gly 15 10 15

Trp Cys Wing Thr Gly Gly Wing Gly Thr Wing Wing Thr Cys Gly Wing

20 25 30

Wing Wing Wing Wing Gly Gly Thr Wing Wing Wing Thr Cys Wing Wing Gly Wing 35 40 45

I.a) Sequence identifier number: SEQ ID No: 106

II.a) Size: 46 II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: H8 Combinatorial Primer used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 106

gcctgtcgca cccasttcat ggagtaatcg tcaaaggtga atccag 4 6

I.a) Sequence ID: SEQ ID No: 107 II.a) Size: 4 6 Il.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Combinatorial Primer H9 used for 2G6 Fab affinity optimization IV) Sequence: SEQ ID NO. 107 gcctgtcgca cccaggwcat ggagtaatcg tcaaaggtga atccag 46

I.a) Sequence identifier number: SEQ ID No: 108

II.a) Size: 4 9

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Combinatorial Primer H10 used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 108

Gly Wings Thr Gly Cys Wings Cys Thr Gly Wings Cys Thr Wings Cys Thr Gly 1 5 10 15

Thr Thr Gly Thr Thr Gly Thr Wing Gly Lys Cys Thr Thr Thr Wing Gly Cys

20 25 30

Thr Thr Thr Gly Thr Thr Cys Thr Wing Wing Thr Wing Wing Wing Thr 35 40 45

Cys

I.a) Sequence identifier number: SEQ ID No: 109

II.a) Size: 49

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: H11 Combinatorial Primer used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 109 gatgcactgt actctgttgt gtaggaatta gctttgtttc taataaatc 49

I.a) Sequence identifier number: SEQ ID No: 110

II.a) Size: 4 5

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: H6 Combinatorial Primer used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 110

gttccttggc cccaggagtc catagcatga trcctagggt atctc 45

I.a) Sequence identifier number: SEQ ID No: 111

II.a) Size: 45

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic: III. a) Other information: H13 Combinatorial Primer used for affinity optimization of Fab 2G6

IV) Sequence: SEQ ID NO. 111 gttccttggc cccacaggtc catagcatga trcctagggt atctc 45

I.a) Sequence identifier number: SEQ ID No: 112

II.a) Size: 120

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Humanized 2G6 Variable Heavy Chain Amino Acid Sequence

IV) Sequence: SEQ ID NO. 112 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu 20 Ser Cys Ala Wing 25 Gly Phe Thr Val Ser 30 Asp Tyr Ser Met Asn 35 Trp Val Arg Gln Wing 40 Pro Gly Lys Gly Leu 45 Glu Trp Ile Gly Phe 50 Ile Arg Asn Lys Wing 55 Asn Asp Tyr Thr Thr 60 Glu Tyr Be Wing Be Val Lys Gly Arg Phe Thr Ile Be As Asp Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met 85 Asn Ser Leu Lys Thr 90 Glu Asp Thr Val Wing 95 Tyr Tyr Cys Thr Thr 100 Tyr Pro Tyr His 105

I.a) Sequence identifier number: SEQ ID No: 113

II.a) Size: 5

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Humanized 2G6 Variable Heavy Chain Amino Acid CDR1 Sequence

IV) Sequence: SEQ ID NO. 113

Asp Tyr Ser Met Asn 1 5

I.a) Sequence identifier number: SEQ ID No: 114

II.a) Size: 19

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: Humanized Variable Chain 2G6 Heavy Chain CDR2 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 114 Phe Ile Arg Asn Lys Wing Asn Asp Tyr Thr Thr Glu Tyr Ser Wing Ser 15 10 15 Val Lys Gly

I.a) Sequence identifier number: SEQ ID No: 115 II.a) size: 9 II.b) type: PRT II.c) organism: Artificial Sequence

III) Characteristic: III.a) Other information: Humanized 2G6 Variable Heavy Chain CDR3 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 115

Tyr Pro Arg Tyr His Wing Met Asp Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 116 II.a) size: 106 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Humanized 2G6 Variable Light Chain Amino Acid Sequence

IV) Sequence: SEQ ID NO. 116

Ile 1 Gln Leu Thr Gln 5 Ser Pro Be Ser Leu 10 Ser Wing Ser Val Gly 15 Asp Arg Val Thr Ile 20 Thr Cys Arg Wing Ser 25 Gln Ser Ile Ser Asn 30 Asn Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Read Leu Ile Tyr 35 40 45 Tyr Val 50 Phe Gln Be Ile Ser 55 Gly Val Pro Be Arg 60 Phe Be Gly Ser Gly Be Gly Thr Asp Phe Thr Leu Thr Ile Ser Be Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr 85 Tyr Cys Gln Gln Be 90 Asn Ser Trp Pro Leu 95 Thr Phe Gly Gly Gly 100 Thr Lys

I.a) Sequence identifier number: SEQ ID No: 117

II.a) Size: 11 II.b) Type: PRT

II.c) organism: Artificial Sequence III)

III.a) Other information: Humanized 2G6 Variable Light Chain CDR1 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 117

Arg Wing Be Gln Be Ile Be Asn Asn Leu His 1 5 10

I.a) Sequence identifier number: SEQ ID No: 118

II.a) Size: 7

II.b) Type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Humanized 2G6 Variable Light Chain CDR2 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 118 Tyr Val Phe Gln Ser Ile Ser 1 5

I.a) Sequence identifier number: SEQ ID No: 119 II.a) size: 9 II.b) type: PRT

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Humanized 2G6 Variable Light Chain CDR3 Amino Acid Sequence

IV) Sequence: SEQ ID NO. 119 Gln Gln Ser Asn Ser Trp Pro Read Thr 1 5

I.a) Sequence identifier number: SEQ ID No: 120

II.a) Size: 4 3

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Medi-VH8 5 'primer used to amplify VH

IV) Sequence: SEQ ID NO. 120

ttctatgcgg cccagccggc ccaggtgcag ctgttgsagt ctg 43

50

I.a) Sequence identifier number: SEQ ID No: 121

II.a) Size: 54

II.b) Type: DNA II.c) Organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Medi-JHl 3 'primer used to amplify VH

IV) Sequence: SEQ ID NO. 121 ggagccgccg ccgccagaac caccaccacc tgaggagacg gtgaccaggg tgcc 54

I.a) Sequence identifier number: SEQ ID No: 122

II.a) Size: 53

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Medi-VKl 5 'primer used for VL

IV) Sequence: SEQ ID NO. 122 ggcggcggcg gctccggtgg tggtggttct gacatccagw tgacccagtc tcc 53

I.a) Sequence identifier number: SEQ ID No: 123

II.a) Size: 45

II.b) Type: DNA

II.c) organism: Artificial Sequence

III) Characteristic:

III.a) Other information: Medi-JK4 3 'primer used for VL

IV) Sequence: SEQ ID NO. 123 tggaattcgg cccccgaggc cacgtttgat ctccaccttg gtccc 45

Claims (33)

1. Bispecific single chain antibody, characterized in that it comprises: (a) first heavy chain variable domain (VH domain) and first light chain variable domain (VL domain), each of an immunospecifically binding antibody with CD3, said first VH domain covalently bonds with said first VL domain by means of a first binder of sufficient length so that said first VH domain and said first VL domain fold to form a first binding domain that binds to CD3; and (b) second VH domain and second VL domain, of an antibody that immunospecifically binds to EphA2 exposed on the cell surface, said second VH domain covalently binds to said second VL domain by means of a second binder of sufficient length such that said second VH domain and said second VL domain fold to form a second binding domain that binds to said EphA2 epitope; wherein said first binding domain and said second binding domain are covalently linked by a third linker in length such that said first binding domain and said second binding domain independently of one another bend. Bispecific single chain antibody according to claim 1, characterized in that the first binding domain, which immunospecifically binds to CD3, binds to the epsilon (ε) subunit of CD3. A bispecific single chain antibody according to claim 2, characterized in that the first binding domain, specific for the β subunit of CD3, is located at the N-terminal part related to the second binding domain. A bispecific single chain antibody according to claim 2, characterized in that the first binding domain, specific for the β subunit of CD3, is located at the C-terminal part related to the second binding domain. Bispecific single chain antibody according to claim 2, characterized in that the first binding domain and the second binding domain are arranged in the order VHCD3-VLcD3-VHEphA2-VLEphA2. A bispecific single chain antibody according to claim 2, characterized in that the first binding domain, which immunospecifically binds with the CD3 β subunit, is de-immunized. Bispecific single chain antibody according to claim 2, characterized in that the VH and / or VL domains of the second binding domain are the VH and / or VL domains of EA2, EA3, EA4, EA5, 3F2, 4H5, 2A4, 2E7, 12E2, EphO99B-102.147, Eph099B-208.261, Eph099B-210.248, Eph099B-233.152, Eph10100.530.241, 233 or G5. Bispecific single chain antibody according to claim 2, characterized in that the sequence length of the first, second and third linkers comprises at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues. , at least 25 wastes or at least 30 wastes. A bispecific single chain antibody according to claim 2, characterized in that said first binder between the first heavy chain variable domain and the first light chain variable domain of said first binding domain with the CD3 subunit ε comprises the sequence SEQ ID NO: 57. A bispecific single chain antibody according to claim 2, characterized in that said second linker between the second heavy chain variable domain and the second light chain variable domain of said second binding domain with EphA2 comprises the sequence SEQ ID NO: 59. A bispecific single chain antibody according to claim 2, characterized in that the third linker between said first binding domain and said second binding domain comprises the sequence SEQ ID NO: 58. A bispecific single chain antibody according to claim 2, characterized in that the VH and / or VL domains of the first binding domain are from the humanized anti-CD3 antibody. Bispecific single chain antibody according to claim 2, characterized in that the VH and / or VL domains of the second binding domain are from the humanized EphA2 antibody. A bispecific single chain antibody according to claim 2, characterized in that said first binding domain binds to the β subunit of CD3 with a lower affinity than said second binding domain binds. to EphA2. A bispecific single chain antibody according to claim 14, characterized in that the dissociation constant of the first binding domain, which binds to the β subunit of CD3, is 4 χ 10 ^ -77 Μ. A bispecific single chain antibody according to claim 14, characterized in that the dissociation constant of the second binding domain, which binds with EphA2, is 1.13 χ 10 ^ -7 Μ. A bispecific single chain antibody according to claim 2, characterized in that it comprises the sequence of SEQ ID NO: 65. Pharmaceutical composition, characterized in that it comprises the bispecific single chain antibody as defined in any one of claims 1 to 17 and a pharmaceutically acceptable carrier. A method of treating, preventing or managing cancer, wherein cancer cells express EphA2, in individuals in need thereof, comprising administering to said individual a therapeutically effective amount of the single chain antibody. bispecific as defined in any one of claims 1 to 17 and a pharmaceutically acceptable carrier. A method according to claim 19, characterized in that said bispecific single chain antibody binds to EphA2 when expressed in the cell, without cell-to-cell contact. A method according to claim 19, characterized in that said cancer is metastatic cancer. Method according to claim 19, characterized in that it comprises the administration of an additional anti-cancer therapy other than bispecific single chain antibody. A method according to claim 22, characterized in that the additional anti-cancer therapy is selected from the group consisting of chemotherapy, biological therapy, immunotherapy, radiotherapy, hormone therapy and surgery. A method of treating, preventing or managing infections in individuals in need thereof, comprising administering to said individual a therapeutically effective amount of the bispecific single chain antibody as defined in any one of claims 1. 17 is a pharmaceutically acceptable carrier. A method according to claim 24, characterized in that said infection is intracellular pathogen infection. The method of claim 24, wherein said infection is respiratory syncytial virus (RSV) infection. Method according to claim 24, characterized in that it comprises the administration of additional therapy. A method according to claim 27, characterized in that said additional therapy is antiviral therapy, antifungal therapy, antibacterial therapy or antiprotozoal therapy. A method of treating, preventing or managing non-cancerous hyperproliferative cell disorders in individuals in need thereof, comprising administering to said subject a therapeutically effective amount of the bispecific single chain antibody as defined. in any one of claims 1 to 17 and a pharmaceutically acceptable carrier. A method according to claim 29, characterized in that said non-cancerous hyperproliferative cell disorders are asthma, COPD, pulmonary fibrosis, asbestosis, EPF, DIP, UIP, renal fibrosis, liver fibrosis, other fibrosis, bronchial hyperresponsiveness, psoriasis, seborrheic dermatitis, cystic fibrosis, or hyperproliferative endothelial cell disorders, such as restenosis, hyperproliferative vascular disease, Behcet's Syndrome, atherosclerosis, macular degeneration, or hyperproliferative fibroblast disorders. Method according to claim 29, characterized in that it comprises the administration of additional therapy. A method according to claim 31, characterized in that said additional therapy is antiviral or immunomodulatory agent therapy. A method according to claim 19, 24 or 29, characterized in that said individual is a human being.