HK1193989B

HK1193989B - Bispecific antibodies specific for t-cell activating antigens and a tumor antigen and methods of use

Info

Publication number: HK1193989B
Application number: HK14107390.5A
Authority: HK
Inventors: Peter Bruenker; Tanja FAUTI; Christiane Jaeger; Christian Klein; Pablo Umana
Original assignee: 罗切格利卡特公司
Priority date: 2011-08-23
Filing date: 2012-08-21
Publication date: 2018-09-21

Description

Bispecific antibodies specific for T cell activating antigens and tumor antigens and methods of use

Technical Field

The present invention relates to a bispecific antibody specifically binding to a T cell activating antigen and a Tumor Antigen (TA), comprising a first Fab fragment and a second Fab fragment, wherein either the variable or constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody does not comprise an Fc domain; methods for their production, pharmaceutical compositions comprising said antibodies, and their uses.

Background

Selective destruction of individual cells or specific cell types is often desired in a variety of clinical settings. For example, the specific destruction of tumor cells without damage to healthy cells and tissues is a primary goal of cancer therapy. One approach is to selectively induce an immune response against the tumor, which triggers the attack and subsequent destruction of tumor cells by immune effector cells, such as Natural Killer (NK) cells or Cytotoxic T Lymphocytes (CTLs). CTLs constitute the most potent effector cells of the immune system, however they cannot be activated by effector mechanisms mediated by the Fc domain of conventional therapeutic antibodies. In this regard, bispecific antibodies capable of binding to surface antigens on cancer cells and activating invariant components in the T Cell Receptor (TCR) complex have become of interest in recent years. Simultaneous binding of the bispecific antibody to both of its targets forces a transient interaction between the cancer cell and the T cell, resulting in cytotoxic T cell activation and subsequent tumor cell lysis.

Several bispecific antibody formats have been developed and investigated for their suitability for T cell-mediated cancer immunotherapy. Among these, the so-called BiTE (bispecific T-cell engager) molecules have been very well characterized and have shown some promising results already in the clinic (for review see Nagorsen and nagarer)Exp CellRes317,1255-1260 (2011)). BiTE is a tandem scFv molecule in which two scFv molecules are fused by a flexible linker. Other bispecific versions evaluated for T cell engagement include diabodies (Holliger et al, Prot eng9,299-305 (1996)) and derivatives thereof, such as tandem diabodies (Kipriyanov et al, J Mol Biol293,41-66 (1999)). One recent development is the so-called DART (dual affinity retargeting) molecule, which is based on a diabody format but is characterized by a C-terminal disulfide bridge to achieve additional stabilization (Moore et al, blood117,4542-51 (2011)). The so-called triomab, which is an intact heterozygous mouse/rat IgG molecule and is also currently evaluated in clinical trials, represents a larger size version (reviewed in Seimetz et al, Cancer Treat rev36,458-467 (2010)).

However, the bispecific antibodies developed for T cell mediated cancer immunotherapy known to date have significant drawbacks related to their efficacy, toxicity and applicability. Small constructs (such as, for example, BiTE molecules), while effective in cross-linking effector and target cells, have a very short serum half-life, requiring their administration to a patient by continuous infusion. IgG-like formats, on the other hand, while having the great benefit of long half-life, suffer from toxicity associated with the natural effector functions inherent to IgG molecules. This immunogenic potential constitutes another negative feature of IgG-like bispecific antibodies for successful therapeutic development. Finally, one of the major challenges in the general development of bispecific antibodies remains to produce bispecific antibody constructs in clinically sufficient quantities and purities. Mismatches in the heavy and light chains of antibodies with different specificities upon co-expression reduce the yield of correctly assembled constructs and result in many non-functional by-products.

In view of the difficulties and disadvantages associated with currently available bispecific antibodies for T cell mediated cancer immunotherapy, there remains a need for novel improved versions of such molecules. These disadvantages have now been overcome with the novel bispecific antibodies of the present invention. Due to the reduced amount of mismatch by-products, which shows less aggregation than bispecific antibody fragments known in the art, new bispecific antibodies can be readily generated in increased yields. Using the crossover approach, correct LC association can be forced without the need to generate a common (common) light chain. In addition, the new bispecific antibodies have a higher molecular weight compared to many conventional bispecific antibody fragments, thus preventing excessive renal clearance and resulting in improved in vivo half-life. The novel bispecific antibodies are fully functional and have comparable or improved binding and activity to corresponding conventional bispecific antibodies.

The present invention provides bispecific antigen binding molecules designed for T cell activation and redirection that combine good efficacy and productivity with lower toxicity and favorable pharmacokinetic profiles.

Summary of The Invention

The present invention relates to a bispecific antibody specifically binding to a T cell activating antigen and a Tumor Antigen (TA), comprising a first Fab fragment and a second Fab fragment, wherein either the variable or constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody does not comprise an Fc domain.

The antibodies of the invention specifically bind to a tumor antigen on the surface of a tumor cell and simultaneously bind to a T cell activating antigen. Thus, the bispecific antibody is capable of specifically eliciting an immune response at the tumor site, which subsequently leads to apoptosis of the target cell.

In one aspect, bispecific antibodies are provided that specifically bind a T cell activating antigen and a Tumor Antigen (TA), comprising at least two Fab fragments, wherein the first Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen (TA); and the second Fab fragment comprises at least one antigen binding site specific for a T cell activating antigen, wherein either the variable or constant regions of the second Fab heavy and light chains are exchanged; and wherein the bispecific antibody does not comprise an Fc domain.

In particular, the invention relates to bispecific antibodies wherein the T cell activating antigen is a CD3T cell co-receptor (CD3) target antigen.

In one aspect, bispecific antibodies are provided that specifically bind to a CD3T cell co-receptor (CD3) antigen and a Tumor Antigen (TA), comprising at least two Fab fragments, wherein a first Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen (TA); and the second Fab fragment comprises at least one antigen binding site specific for the CD3T cell co-receptor (CD3), wherein either the variable or constant regions of the second Fab heavy and light chains are exchanged; and wherein the bispecific antibody does not comprise an Fc domain. In one embodiment, the first and second Fab fragments are linked via a peptide linker. Preferably, the peptide linker is a (G4S)2 linker.

In one embodiment, the antibody further comprises a third Fab fragment. In another embodiment, the third Fab fragment comprises at least one antigen binding site specific for a tumor antigen. In one embodiment, the third Fab fragment is linked to the N-or C-terminus of the light or heavy chain of the first Fab fragment. In another embodiment, the third Fab fragment is linked to the N or C terminus of the light or heavy chain of the second Fab fragment. In one embodiment, the third Fab fragment is linked to the first or second Fab fragment via a peptide linker. Preferably, the peptide linker is a (G4S)2 linker.

Bispecific antibodies according to the invention are at least bivalent and may be trivalent or multivalent, e.g. tetravalent or hexavalent. In one embodiment, the bispecific antibody is bivalent (1 +1 version), having one binding site each targeting a Tumor Antigen (TA) and a T cell activating antigen. In another embodiment, the bispecific antibody is trivalent (2 +1 version), has two binding sites each targeting a Tumor Antigen (TA) and one binding site targeting a T cell activating antigen, as detailed in the following section. In a preferred embodiment, the T cell activating antigen is CD 3.

In a second object, the present invention relates to a pharmaceutical composition comprising a bispecific antibody of the present invention.

In a third object, the invention relates to a bispecific antibody of the invention for use in the treatment of cancer. In another embodiment, the bispecific antibody is provided for use as a medicament. Preferably, the use is for the treatment of cancer.

In further objects, the present invention relates to a nucleic acid sequence comprising a sequence encoding the heavy chain of a bispecific antibody of the present invention, a nucleic acid sequence comprising a sequence encoding the light chain of a bispecific antibody of the present invention, an expression vector comprising a nucleic acid sequence of the present invention and a prokaryotic or eukaryotic host cell comprising a vector of the present invention. Additionally, a method of producing an antibody is provided, comprising culturing the host cell such that the antibody is produced.

Brief Description of Drawings

FIG. 1: schematic representation of exemplary bispecific antibody versions of the invention. a) The C-terminus of the Fab-Crosfab molecule, b) the N-terminus of the Fab-Crosfab molecule, C) (Fab) the C-terminus of the 2-Crosfab molecule, d) (Fab) the N-terminus of the 2-Crosfab molecule, e) the Fab-Crosfab-Fab molecule.

FIG. 2: analysis of hu Fab (MCSP) -Crossfab (CD3) production and purification: SDS-Page: 4-12% Bis/Tris (NuPage [ invitrogen ]; Coomassie staining): a) 1-marker 12 (invitrogen), 2-non-reduced hu Fab (MCSP) -Crossfab (CD 3); b) 1-marker 12 (invitrogen), 2-reduced hu Fab (MCSP) -Crossfab (CD 3).

FIG. 3: analysis of Fab (MCSP) -Crossfab (CD3) production and purification. Analytical size exclusion chromatography, chromatogram A280 (Superdex 20010/300GL [ GE Healthcare ]; 2mM MOPS pH7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample was injected).

FIG. 4: analysis of hu Fab (MCSP) -Crossfab (CD3) production and purification: SDS-Page: 4-12% Bis/Tris (NuPage [ invitrogen ]; Coomassie staining): a) 1-marker 12 (invitrogen), 2-non-reduced hu Fab (MCSP) -Crossfab (CD 3); b) 1-marker 12 (invitrogen), 2-reduced huFab (MCSP) -Fab (MCSP) -Crossfab (CD 3).

FIG. 5: analysis of hu Fab (MCSP) -Crossfab (CD3) production and purification. Analytical size exclusion chromatography, chromatogram A280 (Superdex 20010/300GL [ GE Healthcare ]; 2mM MOPS pH7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample was injected).

FIG. 6: analysis of hu Fab (MCSP) -Crossfab (CD3) -Fab (MCSP) production and purification. SDS-Page: 4-12% Bis/Tris (NuPage [ invitrogen ]; Coomassie staining): a) 1-marker 12 (invitrogen), 2-non-reduced hu Fab (MCSP) -Crossfab (CD3) -Fab (MCSP); b) 1-marker 12 (invitrogen), 2-reduced huFab (MCSP) -Crossfab (CD3) -Fab (MCSP).

FIG. 7: analysis of hu Fab (MCSP) -Crossfab (CD3) -Fab (MCSP) production and purification. Analytical size exclusion chromatography, chromatogram A280 (Superdex 20010/300GL [ GE Healthcare ]; 2mM MOPS pH7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample was injected).

FIG. 8: analysis of murine Crossfab (CD3) -Fab (MCSP) production and purification. SDS-Page: 4-12% Bis/Tris (NuPage [ invitrogen ]; Coomassie staining): a) 1-marker 12 (invitrogen), 2-non-reducing murine Crossfab (CD3) -fab (mcsp); b) 1-marker 12 (invitrogen), 2-reduced murine Crossfab (CD3) -Fab (MCSP) — Fab (MCSP).

FIG. 9: analysis of murine Crossfab (CD3) -Fab (MCSP) production and purification. Analytical size exclusion chromatography, chromatogram A280 (Superdex 20010/300GL [ GE Healthcare ]; 2mM MOPS pH7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample was injected).

FIG. 10: MDA-MB-killing of tumor cells (as measured by LDH release) at 20 hours of co-culture with human pan T cells (E: T ratio =5: 1) and activation by different concentrations of hu Fab (MCSP) -Crossfab (CD3) (= "Fab-Crossfab"), hu Fab (MCSP) -Crossfab (CD3) -Fab (MCSP) (= "Fab-Crossfab"), hu Fab (MCSP) -Crossfab (CD3) (= "(Fab) 2-Crossfab"), and (scFv)2 (anti-MCSP/anti-huCD 3E) (= "(scFv) 2") bispecific molecules. Constructs with bivalent MCSP targeting showed comparable cytotoxic activity compared to the "(scFv) 2" construct, whereas the "Fab-Crossfab" construct with monovalent MCSP binding was clearly less potent.

FIG. 11: comparison of hu Fab (MCSP) -Crossfab (CD3) (= "(Fab) 2-Crossfab") and (scFv)2 (anti-MCSP/anti-huCD 3e) (= "(scFv) 2") constructs. Depicted is LDH release from MDA-MB-435 tumor cells when co-cultured with human pan T cells (E: T ratio =5: 1) and activated for 21 hours by different concentrations of bispecific construct and corresponding IgG. "(Fab) 2-Crossfab" induces apoptosis in target cells at least as well as the (scFv)2 molecule.

FIG. 12: comparison of hu Fab (MCSP) -Crossfab (CD3) (= "(Fab) 2-Crossfab") and (scFv)2 (anti-MCSP/anti-huCD 3e) (= "(scFv) 2") constructs. Depicted is LDH release from MV-3 human melanoma tumor cells when co-cultured with human PBMC (E: T ratio =10: 1) and activated by bispecific constructs at different concentrations and corresponding IgG for 26 hours. "(Fab) 2-Crossfab" induces apoptosis in target cells at least as well as the (scFv)2 molecule.

FIG. 13: LDH release from B16/F10-huMCSP Fluc2 clone 48 tumor cells induced by primary murine T cells activated with murine Crossfab (CD3) -Fab (MCSP) construct (= (Fab) 2-Crossfab) targeting human MCSP and murine CD 3. The ratio of effector to target cells was 5: 1. At 37 ℃ and 5%CO₂The assay was analyzed after 23.5 hours of incubation. The constructs induce concentration-dependent, T cell-mediated apoptosis of target cells expressing human MCSP.

FIG. 14: LDH release from B16/F10-huMCSP Fluc2 clone 48 tumor cells induced by primary murine T cells activated with murine Crossfab (CD3) -Fab (MCSP) construct (= (Fab) 2-Crossfab) targeting human MCSP and murine CD3 at 50 nM. The ratio of effector to target cells was 5: 1. At 37 ℃ 5% CO₂The assay was analyzed after 23.5 hours of incubation. The constructs induce T cell-mediated apoptosis of target cells expressing human MCSP. At this construct concentration, there was only weak T cell hyperactivation.

FIG. 15: different cytokine levels measured in whole blood supernatants 24 hours after treatment with 1nM different CD3-MCSP bispecific constructs (hu Fab (MCSP) -Crossfab (CD3) (= "(Fab) 2-Crossfab") and (scFv)2 (anti-MCSP/anti-huCD 3e) (= "(scFv) 2") in the presence of (a, B) or absence of (C, D) Colo-38 tumor cells. Mu.l of whole blood was added to each well of the 96-well plate and 30,000 Colo-38 cells were added, as indicated. The major cytokine secreted upon T cell activation in the presence of Colo-38 tumor cells is IL-6, followed by IFN γ. In addition, there was also a large increase in granzyme B levels upon T cell activation in the presence of target cells. Overall, the "(scFv) 2" construct increased TNF and IFN γ and granzyme B levels slightly more than the other bispecific constructs in the presence of target cells (a and B).

There was no significant secretion of Th2 cytokines (IL-10 and IL-4) when the bispecific construct activated T cells in the presence (or absence) of the target cells. There was also weak secretion of IFN γ induced by the "(Fab) 2-Crossfab" construct in the absence of target cells in this assay.

FIG. 16: surface expression level of the late activation marker CD25 on murine pan T cells isolated from splenocytes. As indicated (E: T ratio 10: 1), murine pan T cells were incubated with 50nM murine Crossfab (CD3) -Fab (MCSP) construct (= (Fab) 2-Crossfab) bispecific construct (targeting murine CD3, and human MCSP) in the presence or absence of B16/F10-huMCSP Fluc2 clone 48 tumor target cells. Depicted is the expression level of late activation marker CD25 on CD8+ T cells after 70 hours. Upregulation of CD25 on CD8+ T cells via the (Fab)2-CrossFab construct occurs only in the presence of the target cell. The reference IgG used adjusted to the same molar concentration did not up-regulate CD 25.

FIG. 17: analysis of Fab (CD33) -CrossFab (CD3) production and purification. SDS-Page: a)3-8% Tris/acetate (NuPage [ invitrogen ]; coomassie staining): a) 1-himark (invitrogen), 2-non-reduced Fab (CD33) -CrossFab (CD 3); b)4-12% Bis/Tris (NuPage [ invitrogen ]): 1-marker 12 (invitrogen), 2-reduced Fab (CD33) -CrossFab (CD 3).

FIG. 18: analysis of Fab (CD33) -CrossFab (CD3) production and purification. Analytical size exclusion chromatography, chromatogram A280 (Superdex 20010/300GL [ GE Healthcare ]; 2mM MOPS pH7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample was injected).

FIG. 19: killing of MV-3 tumor cells (as measured by LDH release) at 24 hours of co-culture with human PBMC (E: T ratio =10: 1) and activation by different concentrations of the CD3-MCSP bispecific construct (hu Fab (MCSP) -Crossfab (CD3) designated "1 +1 Fc-free"; and (scFv)2 (anti-MCSP/anti-huCD 3E) (= "(scFv) 2") reference molecule). The "1 +1 Fc-free" construct induced apoptosis in MV-3 target cells at a calculated EC50 of 25.4pM, whereas the "(scFv) 2" reference molecule had a calculated EC50 of 57pM, showing a slightly better potency of the "1 +1 Fc-free" molecule in terms of EC 50.

FIG. 20: activation of CD4+ or CD8+ T cells in the presence of huMCSP positive MV-3 tumor cells, as determined by the up-regulation of CD69 (a), the respective increase of CD69 positive cells (B), when co-cultured with human PBMC (E: T ratio =10: 1), treated with CD3-MCSP bispecific construct (hufab (MCSP) -Crossfab (CD3), designated "1 +1 Fc-free", and (scFv)2 (anti-MCSP/anti-huCD 3E) (= "(scFv) 2") reference molecule, respectively) for about 24 hours. Overall, the median CD69 was higher on CD8+ T cells compared to CD4+ T cells. There was a clear concentration-dependent increase in the median CD69 and the percentage of CD69 positive cells for both constructs.

FIG. 21: illustration of (scFv)2 reference molecule.

FIG. 22: analysis of (scFv)2 (anti-MCSP/anti-huCD 3e) production and purification. SDS-Page: 4-12% Bis/Tris (NuPage [ invitrogen ]; Coomassie staining): 1-marker 12 (invitrogen), 2-reduced (scFv)2 (anti-MCSP/anti-huCD 3e), 3-non-reduced (scFv)2 (anti-MCSP/anti-huCD 3 e).

FIG. 23: analysis of (scFv)2 (anti-MCSP/anti-huCD 3e) production and purification. Analytical size exclusion chromatography, chromatogram A280 (Superdex 7510/300GL [ GE Healthcare ]; 2mM MOPS pH7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample ((scFv) 2 (anti-MCSP/anti-huCD 3 e))).

Detailed Description

I. Definition of

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. In general, the FRs of a variable domain consist of 4 FR domains: FR1, FR2, FR3, and FR 4. Thus, HVR and FR sequences typically occur in the following order in VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

For purposes herein, an "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework may comprise its identical amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"human consensus framework" refers to a framework representing the amino acid residues most commonly found in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, NIHPubtilization 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is as in Kabat et al, supra for subgroup kappa I. In one embodiment, for the VH, the subgroup is as in Kabat et al, supra, subgroup III.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and/or which forms structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises 6 HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops are present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) are present at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35B (H1), 50-65(H2), and 95-102(H3) (Kabat et al, Sequences of Proteins of immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md (1991)). The terms hypervariable region (HVR) and Complementarity Determining Region (CDR) are used interchangeably herein when referring to the variable region portions that form the antigen-binding region. This particular region has been described by Kabat et al, U.S. Dept. of health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al, J Mol Biol196:901-917(1987), wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. However, the use of either definition to refer to the CDRs of an antibody or variant thereof is intended to be within the scope of the terms as defined and used herein. Suitable amino acid residues encompassing the CDRs as defined by each of the references cited above are listed in table 1 below for comparison. The exact residue number covering a particular CDR will vary with the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues make up a particular CDR.

Table 1: CDR definition¹

CDR	Kabat	Chothia	AbM²
				V_H CDR1	31-35	26-32	26-35
V_H CDR2	50-65	52-58	50-58
				V_H CDR3	95-102	95-102	95-102
V_L CDR1	24-34	26-32	24-34
				V_L CDR2	50-56	50-52	50-56
V_L CDR3	89-97	91-96	89-97

¹The numbering scheme defined for all CDRs in table 1 follows the numbering convention set forth by Kabat et al (see below).

²"AbM" with the lower case letter "b" as used in table 1 refers to the CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

Kabat et al also define a numbering system for the variable region sequences that can be applied to any antibody. One of ordinary skill in the art can explicitly apply this "Kabat numbering" system to any variable region sequence, independent of any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise indicated, reference to the numbering of specific amino acid residue positions in the variable region of an antibody is according to the Kabat numbering system.

In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues", or "SDRs", which are residues that contact the antigen. SDR is contained within a CDR region called a shortened-CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 50-58 of 31-35B, H2 of H1, and 95-102 of H3 (see Almagro and Fransson, Front. biosci.13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. In particular, the term "antibody" includes bispecific antibodies of the invention comprising at least two Fab fragments but no Fc domain.

The term "bispecific" means that the antigen binding molecule is capable of specifically binding at least two different antigenic determinants. In certain embodiments, the bispecific antigen binding molecule is capable of binding two antigenic determinants simultaneously, in particular two antigenic determinants expressed on two different cells.

The term "monovalent binding antigen" means that no more than one antigen is comprised in an antibody that specifically binds to an antigen.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from host cells such as NS0 or CHO cells or from animals (e.g., mice) that are transgenic for human immunoglobulin genes or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have rearranged forms of variable and constant regions. Recombinant human antibodies according to the invention have been subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have undergone humanization. Other forms of "humanized antibodies" encompassed by the invention are those in which the constant regions have been additionally modified or altered from the constant regions of the original antibody to generate properties in accordance with the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of "chimeric antibodies" encompassed by the invention are those in which the constant region has been modified or altered from that of the original antibody to generate the properties according to the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also known as "class switch antibodies". Chimeric antibodies are the expression product of an immunoglobulin gene comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for generating chimeric antibodies involve conventional recombinant DNA and gene transfection techniques, which are well known in the art. See, e.g., Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA81(1984)6851-6855, US5,202,238 and US5,204,244.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For example, scFv antibodies are described in Huston, J.S., Methods in enzymol.203(1991) 46-88. In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain or a VL domain (i.e., capable of assembling together with a VL domain or a VH domain into a functional antigen binding site, and thereby providing the antigen binding properties of a full-length antibody).

As used herein, a "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a light chain constant domain (CL) and a VH domain and a heavy chain first constant domain (CH 1). Bispecific of the inventionThe antibody comprises at least two Fab fragments wherein either the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged. Due to the exchange of either the variable or constant regions, the second Fab fragment is also referred to as the "cross-Fab" fragment or the "xFab" fragment or the "exchange Fab" fragment. Exchanging two different chain compositions of Fab molecules is possible and comprised in the bispecific antibody of the invention: in one aspect, the variable regions of the Fab heavy and light chains are exchanged, i.e., the exchanged Fab molecule comprises one peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH1) and one peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL). Such exchanged Fab molecules are also known as crossFab_(VLVH). On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the exchanged Fab molecule comprises one peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL) and one peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH 1). Such exchanged Fab molecules are also known as crossFab_(CLCH1)。

In one embodiment, the Fab fragment is linked via a peptide linker. By "linked" is meant that the Fab fragments are linked by peptide bonds, either directly or via one or more peptide linkers.

As used within the present invention, the term "peptide linker" refers to a peptide having an amino acid sequence, which is preferably of synthetic origin. These peptide linkers according to the invention are used to link one of the Fab fragments with the C-or N-terminus of another Fab fragment to form a multispecific antibody according to the invention. Preferably, the peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5-100, more preferably 10-50 amino acids in length. In one embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein G = glycine, S = serine, and (x =3, n =3, 4,5 or 6 and m =0, 1, 2 or 3) or (x =4 and n =2, 3,4 or 5 and m =0, 1, 2 or 3), preferably x =4 and n =2 or 3, more preferably x =4 and n = 2. Additionally, the linker may comprise (part of) an immunoglobulin hinge region. In one embodiment, the peptide linker is (G)₄S)₂(SEQ ID: NO 28). Other peptide linkers suitable for the connection of Fab fragments are for example (G)₄S)₆-GG(SEQ ID NO:147)、(SG₃)₂-(SEG₃)₄-(SG₃) -SG (SEQ ID NO:148), or EPKSC (D) - (G)₄S)₂(SEQ ID NOs 145 and 146).

The term "antigen binding domain" refers to the portion of an antigen binding molecule that comprises a region that specifically binds to and is complementary to part or all of an antigen. In the case of larger antigens, the antigen binding molecule may bind only a particular portion of the antigen, which portion is referred to as an epitope. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising 4 conserved Framework Regions (FRs) and 3 hypervariable regions (HVRs) (see, e.g., kingdt et al Kuby Immunology, 6 th edition, w.h.freeman and dc., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using VH or VL domains, respectively, from antibodies that bind the antigen. See, for example, Portolano et al, J.Immunol.150:880-887(1993); Clarkson et al, Nature352:624-628 (1991).

The term "antigen binding site of an antibody" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody comprises amino acid residues from a "complementarity determining region" or "CDR". The "framework" or "FR" regions are those regions of the variable domain which differ from the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of the antibody comprise, from N to C terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. In particular, the CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. CDR and FR regions are determined according to the standard definition of Kabat et al, Sequences of Proteins of immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from "hypervariable loops".

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and may in certain embodiments have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is the region of an antigen to which an antibody binds.

The term "Fc domain" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. For example, in a natural antibody, the Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody (in the IgG, IgA and IgD isotypes); the IgM and IgE Fc domains contain three heavy chain constant domains (C) in each polypeptide chain_HDomains 2-4). The bispecific antibodies of the invention do not contain an Fc domain. As used herein, "Fc domain free" means that the bispecific antibody of the invention does not comprise a CH2, CH3, or CH4 domain; i.e., the constant heavy chain consists only of one or more CH1 domains.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (KD). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and is capable of interacting with an unwanted or non-specific interaction regionAnd (4) separating. The ability of an antigen binding moiety to bind a particular epitope can be measured via enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., Surface Plasmon Resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljebllad et al, Glyco J17,323-329(2000)) and traditional binding assays (Heeley, Endocr Res28,217-229 (2002)). In one embodiment, the extent of binding of the antibody to the unrelated protein is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by SPR. In certain embodiments, an antigen-binding moiety that binds an antigen or an antigen-binding molecule comprising the antigen-binding moiety has a molecular weight of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (K)_D)。

In one embodiment, a bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA) binds to an unrelated protein to less than about 10% of the binding of the antibody to the T cell activating antigen or Tumor Antigen (TA), as measured, for example, by immunoassay (RIA) or flow cytometry (FACS). In certain embodiments, a bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA) has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (K)_D). In certain embodiments, a bispecific antibody that specifically binds to a T cell activating antigen and a Tumor Antigen (TA) binds to a T cell activating antigen or Tumor Antigen (TA) epitope that is conserved among T cell activating antigens or Tumor Antigens (TAs) from different species.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) which result in an improved affinity of the antibody for an antigen compared to a parent antibody that does not possess such alterations.

The term "bispecific antibody that specifically binds to a T cell activating antigen and a Tumor Antigen (TA)" refers to a bispecific antibody that is capable of binding to a T cell activating antigen and a tumor antigen with sufficient affinity such that the antibody is useful for mediating a T cell-mediated immune response in or near a cell expressing the tumor antigen. In a particular embodiment, the T cell activating antigen is a CD3T cell co-receptor (CD3) antigen, particularly human or cynomolgus CD3, most particularly human CD 3. In some embodiments, the T cell activating antigen is the epsilonclon () subunit of CD 3. In other embodiments, the T cell activating antigen is the alpha (α) or beta (β) subunit of CD 3.

In one embodiment, a bispecific antibody that specifically binds to a T cell activating antigen and a Tumor Antigen (TA) competes with monoclonal antibody H2C (described in PCT publication No. wo 2008/119567) for binding to an epitope of CD 3. In another embodiment, a bispecific antibody that specifically binds to a T cell activating antigen and a Tumor Antigen (TA) competes for binding to an epitope of CD3 with monoclonal antibody V9 (described in Rodrigues et al, Int J Cancer Suppl7,45-50(1992) and U.S. Pat. No.6,054,297). In yet another embodiment, a bispecific antibody that specifically binds to a T cell activating antigen and a Tumor Antigen (TA) competes with monoclonal antibody FN18 (described in Nooij et al, Eur jimmunol19,981-984 (1986)) for binding to an epitope of CD 3.

As used herein, an "activating T cell antigen" refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which upon interaction with an antigen binding molecule is capable of inducing T cell activation. Specifically, the interaction of the antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In a specific embodiment, the activating T cell antigen is CD 3.

As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity and expression of activation markers. The T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art as described herein.

As used herein, the term "CD 3T cell co-receptor (CD 3)" refers to a protein complex and is made up of four distinct chains. In mammals, the complex comprises one CD3 γ chain, one CD3 chain, and two CD3 chains. These chains associate with a molecule called the T Cell Receptor (TCR) and zeta chain in T lymphocytes to generate an activation signal. The term "CD 3T cell co-receptor (CD 3)" includes any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), preferably from human sources, unless otherwise indicated. The term encompasses "full-length" unprocessed CD3 as well as any form of CD3 that results from processing in a cell. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In a preferred embodiment, the term CD3T cell co-receptor refers to human or cynomolgus CD3, in particular human CD 3. In some embodiments, the T cell activating antigen is the epsilonclon () subunit of CD 3. In other embodiments, the T cell activating antigen is the alpha (α) or beta (β) subunit of CD 3. An exemplary sequence of human CD3 is given in SEQ ID No. 103.

As used herein, the term "Tumor Antigen (TA)" refers to tumor-associated antigens as well as tumor-specific antigens, i.e., any immunogenic epitope (e.g., protein) expressed by tumor cells. The protein may be expressed by non-tumor cells, but is immunogenic only when expressed by tumor cells. Alternatively, the protein may be expressed by tumor cells, but not normal cells. Preferably, the anti-TA antibodies of the invention bind to the extracellular domain of TA. In a preferred embodiment, the tumor antigen is a human tumor antigen. Exemplary tumor antigens include, but are not limited to, melanoma-associated chondroitin sulfate proteoglycan (MCSP, UniProt Q6UVK1, NCBI accession No. NP _001888), fibroblast activation protein (FAP, UniProt Q12884, Q86Z29, Q99998; NCBI accession No. NP _004451), epidermal growth factor receptor (EGFR, also known as ErbB1 and Her1, UniProt P00533; NCBI accession No. NP _958439, NP _958440), carcinoembryonic antigen (CEA, also known as carcinoembryonic antigen-associated cell adhesion molecule 5 or CD66e; UniProt P06731, NCBI accession No. NP _004354) and CD33 (also known as gp76 or sialic acid-binding Ig-like lectin 3(Siglec-3), Prot P20138, NCBI accession No. NP _001076087, NP _ 001171079).

In one embodiment, a bispecific antibody of the invention comprises at least one antigen-binding site specific for melanoma-associated chondroitin sulfate proteoglycan (MCSP).

In one embodiment, the bispecific antibody of the invention comprises at least one antigen binding site specific for CD 3.

Antibody specificity refers to the selective recognition of a particular epitope of an antigen by an antibody. For example, natural antibodies are monospecific. A "bispecific antibody" according to the invention is an antibody having two different antigen binding specificities. The antibodies of the invention are specific for two different antigens, a T cell activating antigen as the first antigen and a tumor antigen as the second antigen.

As used herein, the term "monospecific" antibody means an antibody having one or more binding sites, each binding to the same epitope of the same antigen.

As used herein, the term "bispecific" antibody means an antibody having at least two binding sites, each binding to a different antigen or a different epitope of the same antigen.

The antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies refer to monoclonal antibodies having binding specificities for at least two different sites. Provided herein are bispecific antibodies having binding specificity for a Tumor Antigen (TA) and a T cell activating antigen. In certain embodiments, a bispecific antibody can bind two different epitopes of TA. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing TA.

As used within this application, the term "valency" or "valency" means the presence of the specified number of binding sites in an antibody molecule. Thus, the terms "bivalent", "tetravalent", and "hexavalent" indicate the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody molecule. Bispecific antibodies according to the present invention are at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent").

The antibodies of the invention have two or more binding sites and are bispecific. That is, the antibody can be bispecific, even in the presence of more than two binding sites (i.e., the antibody is trivalent or multivalent).

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

By "not substantially cross-reactive" is meant that the molecule (e.g., an antibody) does not recognize or specifically bind an antigen that is different from the actual target antigen of the molecule (e.g., an antigen that is closely related to the target antigen), particularly when compared to the target antigen. For example, the antibody may bind less than about 10% to less than about 5% of an antigen that is different from the actual target antigen, or may bind the antigen that is different from the actual target antigen in an amount selected from the group consisting of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5%, and most preferably less than about 0.2% or 0.1% of an antigen that is different from the actual target antigen.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document where it is registered with US Copyright registration number TXU 510087. ALIGN-2 programs are publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, J.Chromatogr.B848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

An "isolated nucleic acid encoding a bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA)" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

As used within this application, the term "amino acid" denotes the naturally occurring group of carboxy α -amino acids, including alanine (three letter code: ala, one letter code: a), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. As such, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is also understood that all progeny may not be exactly identical in DNA content due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. The naked antibody may be present in a pharmaceutical formulation.

An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

As used herein, the term "cytotoxic agent" refers to a compound that inhibits or prevents cellular function and/or causes cell deathOr a destructive substance. Cytotoxic agents include, but are not limited to: radioisotope (e.g. At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed hereinafter.

The term "N-terminal" denotes the last amino acid from the N-terminus and the term "C-terminal" denotes the last amino acid from the C-terminus.

The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective and free of other components having unacceptable toxicity to a subject who will receive administration of the formulation.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and regression or improved prognosis. In some embodiments, antibodies of the invention are used to delay the development of or slow the progression of disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "effective amount" of a pharmaceutical agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result over the necessary dosage and period of time.

As used herein, the term "cancer" refers to a proliferative disease, such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer of the stomach, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, cancer of the vagina, carcinoma of the vulva, hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell cancer, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, cancer of the gallbladder, Central Nervous System (CNS) neoplasms, spinal tumors, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and ewing's sarcoma, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers.

The term "package insert" is used to refer to instructions for use typically contained in commercial packaging for a therapeutic product that contains information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings relating to the use of such therapeutic products.

Compositions and methods

In one aspect, the invention is based, in part, on a bispecific antibody comprising a first antigen-binding site specific for a T cell activating antigen and a second antigen-binding site specific for a Tumor Antigen (TA). The antibodies of the invention are useful, for example, in the treatment of cancer.

A. Exemplary bispecific antibodies that bind to T cell activating antigen and Tumor Antigen (TA)

The present invention relates to bispecific antibodies that combine a T cell activating antigen binding site with a second antigen binding site that targets a Tumor Antigen (TA). The antibodies of the invention specifically bind to a tumor antigen on the surface of a tumor cell and simultaneously bind to an antigen on the surface of a cytotoxic T lymphocyte. Preferably, the antigen is a CD3T cell co-receptor (CD3) antigen. The bispecific antibody is capable of specifically eliciting an immune response at the tumor site, which subsequently leads to apoptosis of the target cell.

In a particular embodiment according to the invention, the T cell activating bispecific antibody is capable of simultaneously binding to a tumor cell antigen and an activating T cell antigen. In one embodiment, the T cell activating bispecific antibody is capable of cross-linking a T cell and a tumor cell by simultaneously binding to a tumor cell antigen and an activating T cell antigen. In an even more particular embodiment, such simultaneous binding results in tumor cell lysis. In one embodiment, such simultaneous binding results in T cell activation. In other embodiments, such simultaneous binding results in a cellular response of T lymphocytes (particularly cytotoxic T lymphocytes) selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and activation marker expression. In one embodiment, binding of the T cell activating bispecific antibody to the activating T cell antigen does not result in T cell activation in the absence of simultaneous binding to the target cell antigen.

In one embodiment, the T cell activating bispecific antibody is capable of redirecting the cytotoxic activity of the T cell to the target cell. In a particular embodiment, the redirecting is independent of MHC-mediated peptide antigen presentation by the target cell and/or specificity of the T cell.

In particular, the T cell according to any embodiment of the invention is a cytotoxic T cell. In some embodiments, the T cell is CD4⁺Or CD8⁺T cells, in particular CD8⁺T cells.

In one embodiment, a bispecific antibody is provided that specifically binds a T cell activating antigen and a Tumor Antigen (TA), comprising a first Fab fragment and a second Fab fragment, wherein either the variable or constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody does not comprise an Fc domain.

In a particular embodiment, the T cell activating antigen is a CD3T cell co-receptor (CD3) antigen, particularly human or cynomolgus CD3, most particularly human CD 3. In some embodiments, the T cell activating antigen is a subunit of CD 3. In other embodiments, the T cell activating antigen is the alpha or beta subunit of CD 3.

In one aspect, bispecific antibodies are provided that specifically bind to a CD3T cell co-receptor (CD3) antigen and a Tumor Antigen (TA), comprising at least two Fab fragments, wherein a first Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen (TA); and the second Fab fragment comprises at least one antigen binding site specific for the CD3T cell co-receptor (CD3), wherein either the variable or constant regions of the second Fab heavy and light chains are exchanged; and wherein the bispecific antibody does not comprise an Fc domain.

In one embodiment, the first and second Fab fragments are linked via a peptide linker. Preferably, the peptide linker is a peptide having an amino acid sequence of at least 5, preferably 5 to 100, more preferably 10 to 50 amino acids in length. In one embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein G = glycine, S = serine, and (x =3, n =3, 4,5 or 6, and m =0, 1, 2 or 3) or (x =4, n =2, 3,4 or 5 and m =0, 1, 2 or 3), preferably x =4 and n =2 or 3, more preferably x =4, n = 2. In one embodiment, the peptide linker is (G4S)₂. A peptide linker is used to link the first and second Fab fragments.

In one embodiment, the first Fab fragment is linked to the C or N terminus of the second Fab fragment.

In one embodiment, the first Fab fragment is linked to the N-terminus of the second Fab fragment. When the first Fab fragment is linked to the N-terminus of the second Fab fragment, it may be a different bispecific antibody molecule depending on whether the variable or constant domains of the heavy and light chains of the second Fab fragment are exchanged.

In one embodiment, the variable domain of the second Fab fragment is exchanged (i.e., the second Fab fragment is a CrossFab_(VHVL)) And the C-terminus of the heavy or light chain of the first Fab fragment is linked to the N-terminus of the VLCH1 chain of the second Fab fragment. Preferably, the C-terminus of the heavy chain of the first Fab fragment is linked to the N-terminus of the VLCH1 chain of the second Fab fragment. Thus, in one embodiment, a bispecific antibody comprises three chains: the light chain of the first Fab fragment (VLCL), the heavy chain of the first Fab fragment (which is linked to the VLCH1 chain of the second Fab fragment via a peptide linker (VHCH 1-linker-VLCH 1)) and the VHCL chain of the second Fab fragment.

In another embodiment, the constant domains of the second Fab fragment are exchanged (i.e., the second Fab fragment is a CrossFab_(CLCH1)) And the C-terminus of the heavy or light chain of the first Fab fragment is linked to the N-terminus of the VHCL chain of the second Fab fragment. Preferably, the first Fab fragmentThe C-terminus of the heavy chain of the fragment is linked to the N-terminus of the VHCL chain of the second Fab fragment. Thus, in one embodiment, a bispecific antibody comprises three chains: the light chain of the first Fab fragment (VLCL), the heavy chain of the first Fab fragment (which is linked to the VHCL chain of the second Fab fragment via a peptide linker) (VHCH 1-linker-VHCL) and the VLCH1 chain of the second Fab fragment.

In one embodiment, the first Fab fragment is linked to the C-terminus of the second Fab fragment. When the first Fab fragment is linked to the C-terminus of the second Fab fragment, it may be a different bispecific antibody molecule depending on whether the variable or constant domains of the heavy and light chains of the second Fab fragment are exchanged.

In one embodiment, the variable domain of the second Fab fragment is exchanged (i.e., the second Fab fragment is a CrossFab_(VHVL)) And the CH1 domain of the second Fab fragment is linked to the N-terminus of the heavy or light chain of the first Fab fragment. Preferably, the CH1 domain of the second Fab fragment is linked to the N-terminus of the heavy chain of the first Fab fragment. Thus, in one embodiment, a bispecific antibody comprises three chains: the light chain of the first Fab fragment (VLCL), the VLCH1 chain of the second Fab fragment (which is linked to the heavy chain of the first Fab fragment via a peptide linker) (VLCH 1-linker-VHCH 1) and the VHCL chain of the second Fab fragment.

In another embodiment, the constant domains of the second Fab fragment are exchanged (i.e., the second Fab fragment is a CrossFab_(CLCH1)) And the CL domain of the second Fab fragment is linked to the N-terminus of the heavy or light chain of the first Fab fragment. Preferably, the CL domain of the second Fab fragment is linked to the N-terminus of the heavy chain of the first Fab fragment. Thus, in one embodiment, a bispecific antibody comprises three chains: the light chain of the first Fab fragment (VLCL), the VHCL chain of the second Fab fragment (which is linked to the heavy chain of the first Fab fragment via a peptide linker) (VLCH 1-linker-VHCH 1) and the VLCH1 chain of the second Fab fragment.

Bispecific antibodies according to the invention are at least bivalent and may be trivalent or multivalent, e.g. tetravalent or hexavalent. In one embodiment, the bispecific antibody is bivalent (1 +1 version), having one binding site each targeting a Tumor Antigen (TA) and a T cell activating antigen. In another embodiment, the bispecific antibody is trivalent (2 +1 version), has two binding sites each targeting a Tumor Antigen (TA) and one binding site targeting a T cell activating antigen, as detailed in the following section.

In one embodiment, the antibody further comprises a third Fab fragment. In one embodiment, the third Fab fragment comprises at least one antigen binding site specific for a tumor antigen. In one embodiment, the antigen binding site of the third Fab fragment is specific for the same tumor antigen as the antigen binding site of the first Fab fragment.

In one embodiment, the third Fab fragment is linked to the N-or C-terminus of the first Fab fragment. In one embodiment, the third Fab fragment is linked to the first Fab fragment via a peptide linker. Preferably, the peptide linker is (G4S)₂And (4) a joint.

In one embodiment, the third Fab fragment is linked to the N or C terminus of the light or heavy chain of the first Fab fragment. Depending on which end of the first Fab fragment is linked to the second Fab fragment (as detailed above), the third Fab fragment is linked on the opposite (free) end of the first fragment.

In one embodiment, the bispecific antibody of the invention comprises three Fab fragments, wherein the Fab fragment and the linker are linked in the following order from N-terminus to C-terminus: fab fragment 3-linker-Fab fragment 1-linker-Fab fragment 2, wherein either the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged. In this embodiment, the C-terminus of the third Fab fragment is linked to the N-terminus of the first Fab fragment. As detailed above, the Fab fragments may be linked to each other via heavy or light chains. In one embodiment, the C-terminus of the heavy chain of the third Fab fragment is linked to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker; and the C-terminus of the first Fab fragment is linked to the N-terminus of the second Fab fragment, wherein either the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged. Different bispecific antibody molecules are possible depending on whether the variable or constant domains of the heavy and light chains of the second Fab fragment are exchanged.

In one embodiment, the variable domain of the second Fab fragment is exchanged (i.e., the second Fab fragment is a CrossFab_(VHVL)And the chains of the three Fab fragments were connected in the following order from N-terminus to C-terminus: VHCH 1-linker-VHCH 1-linker-VLCH 1. In one embodiment, the bispecific antibody comprises four chains: a light chain of a third Fab fragment (VLCL), a light chain of the first Fab fragment (VLCL), a heavy chain of the third fragment (which is linked to the heavy chain of the first Fab fragment, which is itself linked to the VLCH1 chain of the second Fab fragment via a peptide linker) (VHCH 1-linker-VHCH 1-linker-VLCH 1) and the VHCL chain of the second Fab fragment.

In one embodiment, the constant domains of the second Fab fragment are exchanged (i.e., the second Fab fragment is a CrossFab_(CLCH1)) And the chains of the three Fab fragments were connected in the following order from N-terminus to C-terminus: VHCH 1-linker-VHCH 1-linker-VHCL. In one embodiment, the bispecific antibody comprises four chains: a light chain of a third Fab fragment (VLCL), a light chain of the first Fab fragment (VLCL), a heavy chain of the third fragment (which is linked to the heavy chain of the first Fab fragment, which is itself linked to the VHCL chain of the second Fab fragment via a peptide linker) (VHCH 1-linker-VHCH 1-linker-VHCL) and the VLCH1 chain of the second Fab fragment.

In one embodiment, the bispecific antibody of the invention comprises three Fab fragments, wherein the Fab fragment and the linker are linked in the following order from N-terminus to C-terminus: fab fragment 2-linker-Fab fragment 1-linker-Fab fragment 3, wherein either the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged. In this embodiment, the N-terminus of the third Fab fragment is linked to the C-terminus of the first Fab fragment. As detailed above, Fab fragments may be linked to each other via heavy or light chains. In one embodiment, the N-terminus of the heavy chain of the third Fab fragment is linked to the C-terminus of the heavy chain of the first Fab fragment via a peptide linker; and the N-terminus of the first Fab fragment is linked to the C-terminus of the second Fab fragment, wherein either the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged. Different bispecific antibody molecules are possible depending on whether the variable or constant domains of the heavy and light chains of the second Fab fragment are exchanged.

In one embodiment, the variable domain of the second Fab fragment is exchanged (i.e., the second Fab fragment is a CrossFab_(VHVL)) And the chains of the three Fab fragments were connected in the following order from N-terminus to C-terminus: VLCH 1-linker-VHCH 1-linker-VHCH 1. In one embodiment, the bispecific antibody comprises four chains: a light chain of the third Fab fragment (VLCL), a light chain of the first Fab fragment (VLCL), a VLCH1 chain of the second Fab fragment (which is linked to the heavy chain of the first fragment, which is itself linked to the heavy chain of the first Fab fragment via a peptide linker) (VLCH 1-linker-VHCH 1-linker-VHCH 1) and a VHCL chain of the second Fab fragment.

In one embodiment, the constant domains of the second Fab fragment are exchanged (i.e., the second Fab fragment is a CrossFab_(CLCH1)) And the chains of the three Fab fragments were connected in the following order from N-terminus to C-terminus: VHCL-linker-VHCH 1-linker-VHCH 1. In one embodiment, the bispecific antibody comprises four chains: a light chain of the third Fab fragment (VLCL), a light chain of the first Fab fragment (VLCL), a VHCL chain of the second Fab fragment (which is linked to the heavy chain of the first fragment, which is itself linked to the heavy chain of the first Fab fragment via a peptide linker) (VHCL-linker-VHCH 1-linker-VHCH 1) and a VLCH1 chain of the second Fab fragment.

In another embodiment, the third Fab fragment is linked to the N or C terminus of the light or heavy chain of the second Fab fragment. In one embodiment, the third Fab fragment is linked to the second Fab fragment via a peptide linker. Preferably, the peptide linker is (G4S)₂And (4) a joint. As detailed above, the Fab fragments may be linked to each other via heavy or light chains.

In one embodiment, the bispecific antibody of the invention comprises three Fab fragments, wherein the Fab fragment and the linker are linked in the following order from N-terminus to C-terminus: fab fragment 1-linker-Fab fragment 2-linker-Fab fragment 3, wherein either the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged. In one embodiment, the N-terminus of the third Fab fragment is linked to the C-terminus of the second Fab fragment.

In another embodiment, the C-terminus of the heavy chain of the third Fab fragment is linked to the N-terminus of the second Fab fragment via a peptide linker; and the N-terminus of the first Fab fragment is linked to the C-terminus of the second Fab fragment, wherein either the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged.

Different bispecific antibody molecules are possible depending on whether the variable or constant domains of the heavy and light chains of the second Fab fragment are exchanged.

In one embodiment, the variable domain of the second Fab fragment is exchanged (i.e., the second Fab fragment is a CrossFab_(VHVL)) And the chains of the three Fab fragments were connected in the following order from N-terminus to C-terminus: VHCH 1-linker-VLCH 1-linker-VHCH 1. In one embodiment, the bispecific antibody comprises four chains: a light chain of a third Fab fragment (VLCL), a light chain of the first Fab fragment (VLCL), a heavy chain of the third fragment (which is linked to the N-terminus of the VLCH1 chain of the second Fab fragment, and the C-terminus of the VLCH1 chain is linked to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker) (VHCH 1-linker-VLCH 1-linker-VHCH 1) and a VHCL chain of the second Fab fragment.

In one embodiment, the constant domains of the second Fab fragment are exchanged (i.e., the second Fab fragment is a CrossFab_(CLCH1)) And the chains of the three Fab fragments were connected in the following order from N-terminus to C-terminus: VHCH 1-linker-VHCL-linker-VHCH 1. In one embodiment, the bispecific antibody comprises four chains: a light chain of the third Fab fragment (VLCL), a light chain of the first Fab fragment (VLCL), a heavy chain of the third fragment (which is linked to the N-terminus of the VHCL chain of the second Fab fragment, and the C-terminus of said VHCL chain is linked to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker) (VHCH 1-linker-VHCL-linker-VHCH 1) and the VLCH1 chain of the second Fab fragment.

In one embodiment, the antigen binding site of the third Fab fragment is specific for the same tumor antigen as the antigen binding site of the first Fab fragment, and the bispecific antibody of the invention comprises a peptide linker in the following order (or from N-terminus to C-terminus)End-to-end or C-to-N) linked three Fab fragments: fab_(TA)linker-Fab_(TA)linker-xFab_{(T cell activating antigen)}Wherein Fab_(TA)Represents a Fab fragment having an antigen binding site specific for a tumor antigen, while xFab_{(T cell activating antigen)}Represents a Fab fragment having an antigen binding site specific for a T cell activating antigen, wherein either the variable or constant regions of the heavy and light chains are exchanged.

In one embodiment, the antigen binding site of the third Fab fragment is specific for the same tumor antigen as the antigen binding site of the first Fab fragment, and the bispecific antibody of the invention comprises three Fab fragments linked via a peptide linker in the following order (either in the N-terminal to C-terminal direction or in the C-terminal to N-terminal direction): fab_(TA)linker-xFab_{(T cell activating antigen)}linker-Fab_(TA)Wherein Fab_(TA)Represents a Fab fragment having an antigen binding site specific for a tumor antigen, while xFab_{(T cell activating antigen)}Represents a Fab fragment having an antigen binding site specific for a T cell activating antigen, wherein either the variable or constant regions of the heavy and light chains are exchanged.

In one embodiment, the bispecific antibody comprises an antigen binding moiety that competes with monoclonal antibody V9 for binding to an epitope of CD 3. See, e.g., Rodigues et al, Int J Cancer Suppl 7(1992), 45-50; US6,054,297, herein incorporated by reference in its entirety.

In one embodiment, the bispecific antibody comprises an antigen binding moiety that competes with monoclonal antibody FN18 for binding to an epitope of CD 3. See Nooij et al, Eur J Immunol19 (1986), 981-.

In one embodiment, the bispecific antibody comprises an antigen binding moiety capable of competing with monoclonal antibody CH2527 (sequence IDs 157 and 158) or an affinity matured variant thereof for binding to an epitope of CD 3.

In one embodiment, the bispecific antibody comprises a second Fab fragment that specifically binds CD3, wherein the heavy chain variable region comprises CDR1SEQ id No.10 or SEQ id No.32, CDR2SEQ id No.11 or SEQ id No.33, and CDR3SEQ id No.12 or SEQ id No. 34; and wherein the light chain variable region comprises CDR1SEQ ID No.7 or SEQ ID No.29, CDR2SEQ ID No.8 or SEQ ID No.30, and CDR3SEQ ID No.9 or SEQ ID No. 31.

In one embodiment, the bispecific antibody comprises a second Fab fragment that specifically binds CD3, wherein the heavy chain variable region comprises CDR1SEQ id No.10, CDR2SEQ id No.11, and CDR3SEQ id No. 12; and wherein the light chain variable region comprises CDR1SEQ id No.7, CDR2SEQ id No.8 and CDR3SEQ id No. 9.

In one embodiment, the bispecific antibody comprises a second Fab fragment that specifically binds CD3, wherein the heavy chain variable region comprises CDR1SEQ id No.32, CDR2SEQ id No.33, and CDR3SEQ id No. 34; and wherein the light chain variable region comprises CDR1SEQ id No.29, CDR2SEQ id No.30, and CDR3SEQ id No. 31.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No.20 or SEQ id No. 36; wherein the light chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No.19 or SEQ id No.35, or a variant thereof which retains functionality. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ id No. 20; and the light chain variable region comprises the amino acid sequence SEQ id No.19 or a variant thereof that retains functionality.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ id No. 36; and the light chain variable region comprises the amino acid sequence seq id No.35 or a variant thereof which retains functionality.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the heavy chain variable region comprises the amino acid sequence SEQ id No. 158; and the light chain variable region comprises the amino acid sequence seq id No.157 or a variant thereof that retains functionality. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to a second Fab fragment of CD3, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 158; wherein the light chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No.157, or a variant thereof which retains functionality. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the heavy chain variable region sequence is the affinity matured variant of SEQ id No.158 and wherein the light chain variable region sequence is the affinity matured variant of SEQ id No. 157. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.158 and/or SEQ id No.157 are independently exchanged.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein said heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No.22 or SEQ ID No.38 or a functionally preserved variant thereof. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No.22 or SEQ ID no38, and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA), comprising one or more of the amino acid sequences defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No. 22. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of the second Fab fragment specifically binding to CD3, wherein said heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No.22, and a light chain and a heavy chain of the first Fab fragment specific for a Tumor Antigen (TA), comprising one or more of the amino acid sequences as defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No.21 or SEQ ID No. 37. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No.21 or SEQ ID No.37, and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA), comprising one or more of the amino acid sequences defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No. 21. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of the second Fab fragment specifically binding to CD3, wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No.21, and a light chain and a heavy chain of the first Fab fragment specific for a Tumor Antigen (TA), comprising one or more of the amino acid sequences as defined in any of the embodiments described herein.

In yet another specific embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically binds to the second Fab fragment of CD3, said heavy chain comprising a heavy chain constant region comprising the amino acid sequence SEQ ID No.22 or SEQ ID No. 38; and the light chain comprises a light chain constant region comprising the amino acid sequence SEQ ID No.21 or SEQ ID No. 37.

In yet another specific embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically binds to the second Fab fragment of CD3, said heavy chain comprising a heavy chain constant region comprising the amino acid sequence SEQ ID No. 22; and the light chain comprises a light chain constant region comprising the amino acid sequence SEQ ID No. 21.

In yet another specific embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of the second Fab fragment specifically binding to CD3 (said heavy chain comprising a heavy chain constant region comprising the amino acid sequence SEQ ID No. 22; and said light chain comprising a light chain constant region comprising the amino acid sequence SEQ ID No. 21), and a light chain and a heavy chain of the first Fab fragment specific for a Tumor Antigen (TA), comprising one or more of the amino acid sequences as defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain which specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.19 and variable heavy chain SEQ ID No.20, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.22, and a light chain constant region comprising the amino acid sequence SEQ ID No. 21.

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of the second Fab fragment specifically binding to CD3 (which comprises variable light chain SEQ ID No.19 and variable heavy chain SEQ ID No.20, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.22, and a light chain constant region comprising the amino acid sequence SEQ ID No. 21), and a light chain and a heavy chain of the first Fab fragment specific for a Tumor Antigen (TA) (which comprises one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain which specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.35 and variable heavy chain SEQ ID No.36, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.38, and a light chain constant region comprising the amino acid sequence SEQ ID No. 37.

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of the second Fab fragment specifically binding to CD3 (which comprises variable light chain SEQ ID No.35 and variable heavy chain SEQ ID No.36, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.38, and a light chain constant region comprising the amino acid sequence SEQ ID No. 37), and a light chain and a heavy chain of the first Fab fragment specific for a Tumor Antigen (TA) (which comprises one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain which specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.157 and variable heavy chain SEQ ID No.158, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.22, and a light chain constant region comprising the amino acid sequence SEQ ID No. 21.

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain which specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.157 or an affinity matured variant thereof and variable heavy chain SEQ ID No.158 or an affinity matured variant thereof, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.22, and a light chain constant region comprising the amino acid sequence SEQ ID No. 21. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.158 and/or SEQ id No.157 are independently exchanged.

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of the second Fab fragment specifically binding to CD3 (which comprises variable light chain SEQ ID No.157 and variable heavy chain SEQ ID No.158, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.22, and a light chain constant region comprising the amino acid sequence SEQ ID No. 21), and a light chain and a heavy chain of the first Fab fragment specific for a Tumor Antigen (TA) (which comprises one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of the second Fab fragment specifically binding to CD3 (which comprises variable light chain SEQ ID No.157 or an affinity matured variant thereof and variable heavy chain SEQ ID No.158 or an affinity matured variant thereof, and a heavy chain constant region comprising the amino acid sequence SEQ ID No.22, and a light chain constant region comprising the amino acid sequence SEQ ID No. 21), and a heavy chain of the first Fab fragment (which comprises one or more of the amino acid sequences defined in any of the embodiments described herein) specific for a Tumor Antigen (TA). In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.158 and/or SEQ id No.157 are independently exchanged.

In one embodiment, the tumor antigen is selected from the group consisting of: melanoma associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), Fibroblast Activation Protein (FAP) and CD 33. In a preferred embodiment, the tumor antigen is MCSP.

In one embodiment, the T cell activating bispecific antibody comprises at least one antigen binding site specific for melanoma associated chondroitin sulfate proteoglycan (MCSP). In another embodiment, the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties capable of competing with monoclonal antibody M4-3ML2 (sequences ID161 and 162) or affinity matured variants thereof for binding to an epitope of MCSP.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to a first Fab fragment of MCSP, wherein the variable heavy chain comprises CDR1SEQ id No.4, CDR2SEQ id No.5, CDR3SEQ id No. 6; and the variable light chain comprises CDR1SEQ id No.1, CDR2SEQ id No.2, and CDR3SEQ id No. 3.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to a first Fab fragment of MCSP, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 14; and the light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 13.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to a first Fab fragment of MCSP, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 161; and the light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 162.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the first Fab fragment of MCSP, wherein the heavy chain variable region sequence is an affinity matured variant of SEQ id No.161 and wherein the light chain variable region sequence is an affinity matured variant of SEQ id No. 162. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the first Fab fragment of MCSP, wherein the heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No. 16. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment which specifically binds MCSP (wherein said heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No. 16), and a light chain and a heavy chain of a second Fab fragment specific for CD3 (which comprises one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the first Fab fragment of MCSP, wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No. 15. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second antibody that specifically binds MCSP (wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No. 15), and a light chain and a heavy chain of a second Fab fragment specific for CD3 (comprising one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to the first Fab fragment of MCSP, wherein the heavy chain constant region comprises the amino acid sequence SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence of SEQ ID No. 15.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to a first Fab fragment of MCSP, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID No. 14; and the light chain variable region comprises the amino acid sequence SEQ ID No.13, and wherein the heavy chain constant region comprises the amino acid sequence SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence SEQ ID NO 15.

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising a variable light chain SEQ ID No.19 and a variable heavy chain SEQ ID No. 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising variable light chain SEQ ID No.13 and variable heavy chain SEQ ID No. 14.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to a first Fab fragment of MCSP, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID No. 161; and the light chain variable region comprises the amino acid sequence SEQ ID No.162, and wherein the heavy chain constant region comprises the amino acid sequence SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence SEQ ID NO 15.

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising a variable light chain SEQ ID No.19 and a variable heavy chain SEQ ID No. 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising variable light chain SEQ ID No.161 and variable heavy chain SEQ ID No. 162.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain that specifically bind to a first Fab fragment of MCSP, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID No.161 or an affinity matured variant thereof; and the light chain variable region comprises the amino acid sequence SEQ ID No.162 or an affinity matured variant thereof, and wherein the heavy chain constant region comprises the amino acid sequence SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence SEQ ID NO 15. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising a variable light chain SEQ ID No.19 and a variable heavy chain SEQ ID No. 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising variable light chain SEQ ID No.161 or an affinity matured variant thereof and variable heavy chain SEQ ID No.162 or an affinity matured variant thereof. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.158 and variable heavy chain SEQ ID No. 157; and a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising variable light chain SEQ ID No.161 and variable heavy chain SEQ ID No. 162.

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.158 or an affinity matured variant thereof and variable heavy chain SEQ ID No.157 or an affinity matured variant thereof; and a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising variable light chain SEQ ID No.161 or an affinity matured variant thereof and variable heavy chain SEQ ID No.162 or an affinity matured variant thereof. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of one or more of SEQ id No.157, SEQ id No.158, SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the variable heavy chain comprises CDR1SEQ id No.4, CDR2SEQ id No.5, CDR3SEQ id No. 6; and the variable light chain comprises CDR1SEQ id No.1, CDR2SEQ id No.2, and CDR3SEQ id No. 3.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 14; and the light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 13.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 161; and the light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ id No. 162.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain variable region sequence is an affinity matured variant of SEQ id No.161 and wherein the light chain variable region sequence is an affinity matured variant of SEQ id No. 162. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No. 16. In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds to MCSP (wherein the heavy chain comprises a constant region comprising the amino acid sequence SEQ ID No. 16), and a light chain and a heavy chain of a second Fab fragment specific for CD3 (comprising one or more of the amino acid sequences defined in any of the embodiments described herein), and a light chain and a heavy chain of a first Fab fragment specific for MCSP (comprising one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No. 15. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second antibody that specifically binds MCSP (wherein the light chain comprises a constant region comprising the amino acid sequence SEQ ID No. 15), and a light chain and a heavy chain of a second Fab fragment specific for CD3, and a light chain and a heavy chain of a first Fab fragment specific for MCSP (which comprises one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain constant region comprises the amino acid sequence SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence SEQ ID No. 15.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID No. 14; and the light chain variable region comprises the amino acid sequence SEQ ID No.13, and wherein the heavy chain constant region comprises the amino acid sequence SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence SEQ ID NO 15.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID No. 161; and the light chain variable region comprises the amino acid sequence SEQ ID No.162, and wherein the heavy chain constant region comprises the amino acid sequence SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence SEQ ID NO 15.

In one embodiment, the bispecific antibody comprises a third Fab fragment comprising a light chain and a heavy chain that specifically binds MCSP, wherein the heavy chain variable region sequence is an affinity matured variant of SEQ ID No.161 and wherein the light chain variable region sequence is an affinity matured variant of SEQ ID No.162, and wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID No. 16; and the light chain constant region comprises the amino acid sequence SEQ ID NO 15. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising a variable light chain SEQ ID No.19 and a variable heavy chain SEQ ID No. 20; and the light and heavy chains of a first Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.13 and variable heavy chain SEQ ID No. 14), and the light and heavy chains of a third Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.13 and variable heavy chain SEQ ID No. 14).

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising a variable light chain SEQ ID No.19 and a variable heavy chain SEQ ID No. 20; and the light and heavy chains of a first Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.162 and variable heavy chain SEQ ID No. 161), and the light and heavy chains of a third Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.162 and variable heavy chain SEQ ID No. 161).

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising a variable light chain SEQ ID No.19 and a variable heavy chain SEQ ID No. 20; and the light and heavy chains of a first Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.162 or an affinity matured variant thereof and variable heavy chain SEQ ID No.161 or an affinity matured variant thereof), and the light and heavy chains of a third Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.162 or an affinity matured variant thereof and variable heavy chain SEQ ID No.161 or an affinity matured variant thereof). In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.157 and variable heavy chain SEQ ID No. 158; and the light and heavy chains of a first Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.162 and variable heavy chain SEQ ID No. 161), and the light and heavy chains of a third Fab fragment specific for MCSP (which comprises variable light chain SEQ ID No.162 and variable heavy chain SEQ ID No. 161).

In yet another embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain that specifically bind to the second Fab fragment of CD3, comprising variable light chain SEQ ID No.157 and variable heavy chain SEQ ID No. 158; and a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising variable light chain SEQ ID No.162 or an affinity matured variant thereof and variable heavy chain SEQ ID No.161 or an affinity matured variant thereof; and a light chain and a heavy chain of a third Fab fragment specific for MCSP comprising variable light chain SEQ ID No.162 or an affinity matured variant thereof and variable heavy chain SEQ ID No.161 or an affinity matured variant thereof. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.161 and/or SEQ id No.162 are independently exchanged.

In yet another embodiment, the bispecific antibody comprises one or more amino acid sequences selected from the group consisting of seq id no: SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.41 and SEQ ID NO. 43.

In one embodiment, the bispecific antibody comprises SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No. 27.

In one embodiment, the T cell activating bispecific antibody comprises at least one antigen binding site specific for Epidermal Growth Factor Receptor (EGFR). In another embodiment, the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties capable of competing with monoclonal antibody GA201 for binding to an EGFR epitope. See PCT publication WO2006/082515, incorporated herein by reference in its entirety. In one embodiment, the antigen binding site specific for EGFR comprises heavy chain CDR1SEQ ID No.68, heavy chain CDR2SEQ ID No.69, heavy chain CDR3SEQ ID No.70, light chain CDR1SEQ ID No.71, light chain CDR2SEQ ID No.72, and light chain CDR3SEQ ID No. 73. In yet another embodiment, the antigen binding site specific for EGFR comprises a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.74 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.75, or a variant thereof that retains functionality.

In yet another embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen binding site specific for EGFR comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.74 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.75, or a variant thereof that retains functionality, and a light and heavy chain of a second Fab fragment specific for CD3 comprising one or more of the amino acid sequences defined in any of the embodiments described herein.

In yet another embodiment, the bispecific antibody comprises a first and a third Fab fragment (which comprise an antigen binding site specific for EGFR comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.74 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.75, or a variant thereof that retains functionality), and a light chain and a heavy chain of a second Fab fragment specific for CD3 (which comprise one or more of the amino acid sequences defined in any of the embodiments described herein).

In one embodiment, the T cell activating bispecific antibody comprises at least one antigen binding site specific for Fibroblast Activation Protein (FAP). In another embodiment, the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties capable of competing with monoclonal antibody 3F2 for binding to an epitope of FAP. See european patent application No. ep10172842.6, incorporated herein by reference in its entirety. In one embodiment, the antigen binding site specific for FAP comprises heavy chain CDR1SEQ ID No.76, heavy chain CDR2SEQ ID No.77, heavy chain CDR3SEQ ID No.78, light chain CDR1SEQ ID No.79, light chain CDR2SEQ ID No.80, and light chain CDR3SEQ ID No. 81. In yet another embodiment, the antigen-binding site specific for FAP comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.82 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.83, or a variant thereof that retains functionality.

In yet another embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen-binding site specific for FAP comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.82 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.83, or a variant thereof that retains functionality, and a light and heavy chain of a second Fab fragment specific for CD3 comprising one or more of the amino acid sequences defined in any of the embodiments described herein.

In yet another embodiment, the bispecific antibody comprises a first and a third Fab fragment (which comprise an antigen-binding site specific for FAP comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.82 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.83, or a variant thereof that retains functionality), and a light chain and a heavy chain of a second Fab fragment (which comprise one or more of the amino acid sequences defined in any of the embodiments described herein) specific for CD 3.

In one embodiment, the T cell activating bispecific antibody comprises at least one antigen binding site specific for carcinoembryonic antigen (CEA). In another embodiment, the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties capable of competing with monoclonal antibody CH1A1A for binding to an epitope of CEA. In one embodiment, the T cell activating bispecific antibody comprises at least one, typically two or more, monoclonal antibodyCH1A1A clone 98/99(CH1A 1)_(98/99)) An antigen binding moiety that competes for binding to the CEA epitope. See PCT patent application No. PCT/EP2010/062527, incorporated herein by reference in its entirety. In one embodiment, the antigen binding site specific for CEA comprises heavy chain CDR1SEQ ID No.84, heavy chain CDR2SEQ ID No.85, heavy chain CDR3SEQ ID No.86, light chain CDR1SEQ ID No.87, light chain CDR2SEQ ID No.88, and light chain CDR3SEQ ID No. 89. In yet another embodiment, the antigen binding site specific for CEA comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.90 or SEQ ID No.159 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.91 or SEQ ID No.160, or a variant thereof that retains functionality.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of the first Fab fragment that specifically binds CEA, wherein the heavy chain variable region comprises the affinity maturation variant of SEQ ID No. 159; and the light chain variable region comprises an affinity matured variant of SEQ ID NO. 160. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.159 and/or SEQ id No.160 are independently exchanged.

In yet another embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen-binding site specific for CEA comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.90 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.91, or a variant thereof that retains functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more of the amino acid sequences defined in any of the embodiments described herein.

In yet another embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen-binding site specific for CEA comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.159 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.160, or a variant thereof that retains functionality, and a light and heavy chain of a second Fab fragment specific for CD3 comprising one or more of the amino acid sequences defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of the first Fab fragment that specifically binds CEA, wherein the heavy chain variable region comprises the affinity matured variant of SEQ ID No.159 and the light chain variable region comprises the affinity matured variant of SEQ ID No. 160; and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more of the amino acid sequences defined in any of the embodiments described herein. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.159 and/or SEQ id No.160 are independently exchanged.

In yet another embodiment, the bispecific antibody comprises first and third Fab fragments comprising an antigen binding site specific for CEA comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.90 or SEQ ID No.159 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.91 or SEQ ID No.160, or a variant thereof that retains functionality; and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more of the amino acid sequences defined in any of the embodiments described herein. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.159 and/or SEQ id No.160 are independently exchanged.

In yet another embodiment, the bispecific antibody comprises a first and a third Fab fragment comprising an antigen binding site specific for CEA wherein the heavy chain variable region comprises the affinity matured variant of SEQ ID No.159 and the light chain variable region comprises the affinity matured variant of SEQ ID No. 160. In this embodiment, an affinity matured variant means that 1, 2,3 or 4 amino acids of SEQ id No.159 and/or SEQ id No.160 are independently exchanged.

In one embodiment, the T cell activating bispecific antibody comprises at least one antigen binding site specific for CD 33. In one embodiment, the antigen binding site specific for CD33 comprises heavy chain CDR1SEQ ID No.92, heavy chain CDR2SEQ ID No.93, heavy chain CDR3SEQ ID No.94, light chain CDR1SEQ ID No.95, light chain CDR2SEQ ID No.96, and light chain CDR3SEQ ID No. 97. In yet another embodiment, the antigen binding site specific for CD33 comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.98 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.99, or a variant thereof that retains functionality.

In yet another embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen binding site specific for CD33 comprising a heavy chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.98 and a light chain variable region sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No.99, or a variant thereof that retains functionality; and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more of the amino acid sequences defined in any of the embodiments described herein.

In a specific embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.100, SEQ ID NO.101 and SEQ ID NO. 102.

In one embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.151, SEQ ID NO.152 and SEQ ID NO. 153.

In yet another embodiment, the bispecific antibody comprises one or more amino acid sequences selected from the group consisting of seq id no: SEQ ID NO.100, SEQ ID NO.101, SEQ ID NO.151, SEQ ID NO.152 and SEQ ID NO. 153.

In one embodiment of the invention, the bispecific antibody is a humanized antibody, as described in detail below.

In another embodiment of the invention, the bispecific antibody is a human antibody, as described in detail below.

In a third object, the invention relates to a bispecific antibody of the invention for use in the treatment of cancer. In another embodiment, a bispecific antibody is provided for use as a medicament. Preferably, the use is for the treatment of cancer.

In further objects, the present invention relates to a nucleic acid sequence comprising a sequence encoding a heavy chain of a bispecific antibody according to the invention, a nucleic acid sequence comprising a sequence encoding a light chain of a bispecific antibody according to the invention, an expression vector comprising a nucleic acid sequence according to the invention and a prokaryotic or eukaryotic host cell comprising a vector according to the invention. Additionally, methods of producing an antibody are provided, comprising culturing the host cell such that the antibody is produced.

In a specific embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52, SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.66, and SEQ ID NO. 67.

In a specific embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.107, SEQ ID NO.108, SEQ ID NO.109, SEQ ID NO.110, SEQ ID NO.111, and SEQ ID NO. 112.

In a specific embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.113, SEQ ID NO.114, SEQ ID NO.115, SEQ ID NO.116, SEQ ID NO.117, SEQ ID NO.118, SEQ ID NO.119, and SEQ ID NO. 120.

In a specific embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.121, SEQ ID NO.122, SEQ ID NO.123, SEQ ID NO.124, SEQ ID NO.125, SEQ ID NO.126, SEQ ID NO.127, and SEQ ID NO. 128.

In a specific embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.129, SEQ ID NO.130, SEQ ID NO.131, SEQ ID NO.132, SEQ ID NO.133, SEQ ID NO.134, SEQ ID NO.135, and SEQ ID NO. 136.

In a specific embodiment, the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of seq id no: SEQ ID NO.105, SEQ ID NO.106, SEQ ID NO.137, SEQ ID NO.138, SEQ ID NO.139, SEQ ID NO.140, SEQ ID NO.141, SEQ ID NO.142, SEQ ID NO.143, SEQ ID NO.144, SEQ ID NO.154, SEQ ID NO.155 and SEQ ID NO. 156.

In yet another aspect, a bispecific antibody according to any of the above embodiments may incorporate any of the features described in sections 1-5 below, singly or in combination:

1. affinity of antibody

The affinity of the T cell activating bispecific antibody for the target antigen can be determined by Surface Plasmon Resonance (SPR) using standard instruments such as the BIAcore instrument (GE Healthcare) according to the methods set forth in the examples, and the receptor or target protein can be obtained, such as by recombinant expression. Alternatively, the binding of a T cell activating bispecific antibody to a different receptor or target antigen can be assessed, for example, by flow cytometry (FACS), using a cell line expressing the particular receptor or target antigen.

In certain embodiments, a bispecific antibody provided herein has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (KD).

According to one embodiment, the KD is determined using a surface plasmon resonance assay-2000 or-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For the kinetic measurements, inInjected at 25 ℃ at a flow rate of about 25. mu.l/min into a solution containing 0.05% polysorbate 20 (TWEEN-20)^TM) Two-fold serial dilutions of Fab (0.78 nM to 500 nM) in surfactant PBS (PBST). Using a simple one-to-one langmuir binding model (Evaluation Software version3.2) calculation of Association Rate (ka or k) by Simultaneous fitting of Association and dissociation sensorgrams_on) And dissociation rate (kd or k)_off). At a ratio of k_off/k_onThe equilibrium dissociation constant (KD) was calculated. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay⁶M^-1s^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as an Aviv Instruments spectrophotometer or 8000 series SLM-AMINCO^TMMeasurement in a stirred cuvette in a spectrophotometer (ThermoSpectronic) measured the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBS ph7.2 at 25 ℃ in the presence of increasing concentrations of antigen.

2. Chimeric and humanized antibodies

In certain embodiments, the bispecific antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No.4,816,567, and Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and Methods for their production are reviewed, for example, in Almagro and Fransson, Front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature332:323-329(1988), Queen et al, Proc.Nat' l Acad.Sci.USA86:10029-10033(1989), U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409, Kashmiri et al, Methods36:25-34(2005) (SDR (a-CDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (describes "resurfacing"); dall' Acqua et al, Methods36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods36:61-68(2005) and Klimka et al, Br.J. cancer83:252-260(2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of a specific subset of human antibodies from the light or heavy chain variable regions (see, e.g., Carter et al Proc. Natl. Acad. Sci. USA,89:4285(1992); and Presta et al J.Immunol.,151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

3. Human antibodies

In certain embodiments, the bispecific antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. In general, human antibodies are described in van Dijk and van de Winkel, Curr, Opin, Pharmacol.5:368-74(2001), and Lonberg, Curr, Opin, Immunol.20: 450-.

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describes K-MTechnology, and U.S. patent application publication No. US2007/0061900, which describesA technique). The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for generating human Monoclonal antibodies have been described (see, e.g., Kozbor J.Immunol.,133:3001(1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol.,147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiaondai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, Histologyand Histopathlogy, 20(3): 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

4. Library-derived antibodies

Bispecific antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such Methods are reviewed, for example, in Hoogenboom et al, Methods in molecular Biology178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature348:552-554, Clackson et al, Nature352:624-628(1991), Marks et al, J.mol.biol.222:581-597(1992), Marks and Bradbury in Methods in molecular Biology248:161-175(Lo eds., Human Press, Totowa, NJ,2003), Sidhu et al, J.mol.338 (2): 299-2004 (2004); Lee et al, J.mol.340 (5):1073 (Act.) and Fec. 124101.55-55-34 (USA), and Methods in U.72 (USA) 2004).

In some phage display methods, the repertoire of VH and VL genes, respectively, is cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the non-immune repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self antigens in the absence of any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373, and U.S. patent publication Nos. 2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

5. Antibody variants

In certain embodiments, amino acid sequence variants of the bispecific antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of bispecific antibodies. Amino acid sequence variants of bispecific antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the bispecific antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.

a)Substitution, insertion, and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". More substantial variations are provided in table 1 under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, e.g., retained/improved antigen binding or reduced immunogenicity.

TABLE 1

Initial residue	Exemplary substitutions	Preferred alternatives
			Ala(A)	Val;Leu;Ile	Val
Arg(R)	Lys;Gln;Asn	Lys
			Asn(N)	Gln;His;Asp,Lys;Arg	Gln
Asp(D)	Glu;Asn	Glu
			Cys(C)	Ser;Ala	Ser
Gln(Q)	Asn;Glu	Asn
			Glu(E)	Asp;Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn;Gln;Lys;Arg	Arg
Ile(I)	Leu, Val, Met, Ala, Phe, norleucine	Leu
			Leu(L)	Norleucine, Ile, Val, Met, Ala, Phe	Ile
Lys(K)	Arg;Gln;Asn	Arg
			Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Trp;Leu;Val;Ile;Ala;Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val;Ser	Ser
Trp(W)	Tyr;Phe	Tyr
			Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile, Leu, Met, Phe, Ala, norleucine	Leu

According to common side chain properties, amino acids can be grouped as follows:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207: 179. 196 (2008)), and/or SDRs (a-CDRs), where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom, et al, Methods in Molecular Biology178:1-37 (O' Brien et al, eds., HumanPress, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. Such changes may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1, 2, or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex identifies the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

Of an Fc domain variant.

b)Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create a cysteine engineered bispecific antibody, e.g., a "thioMAb," in which one or more residues of the bispecific antibody are replaced with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the bispecific antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, cysteine may be substituted for any one or more of the following residues: v205 for the light chain (Kabat numbering) and a118 for the heavy chain (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.

c)Antibody derivatives

In certain embodiments, the bispecific antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of bispecific antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for therapy under specified conditions, and the like.

In another embodiment, conjugates of a bispecific antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

B. Recombinant methods and compositions

The T cell activating bispecific antibody of the invention may be obtained, for example, by solid phase peptide synthesis (e.g., Merrifield solid phase peptide synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the T cell activating bispecific antibody (fragment), e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector (preferably an expression vector) comprising one or more polynucleotides of the invention is provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for the T cell activating bispecific antibody (fragment) and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, e.g., techniques described in Maniatis et al, MOLECULAR CLONING, A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989), and Ausubel et al, CURRENT PROTOCOLS MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector may be a plasmid, part of a virus or may be a nucleic acid fragment. The expression vector comprises an expression cassetteWherein a polynucleotide (i.e. a coding region) encoding a T cell activating bispecific antibody (fragment) is cloned (in operable association with a promoter and/or other transcriptional or translational control elements). As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of the coding region (if present), but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides which are separated post-translationally or co-translationally into the final protein via proteolytic cleavage. In addition, the vector, polynucleotide or nucleic acid of the invention may encode a heterologous coding region, fused or unfused with a polynucleotide encoding a T cell activating bispecific antibody (fragment) of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains. Operable association occurs when the coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in a manner such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, if a promoter is capable of effecting transcription of a nucleic acid encoding a polypeptide, the promoter region will be in operable association with the nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. OpenerIn addition to promoters, other transcriptional control elements such as enhancers, operators, repressors, and transcriptional termination signals can be operably associated with a polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., the immediate early promoter, along with intron-a), simian virus 40 (e.g., the early promoter), and retroviruses (such as, for example, Rous (Rous) sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit-globulin-derived, and other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (specifically, internal ribosome entry sites or IRES, also known as CITE sequences). The expression cassette may also comprise other features, such as an origin of replication and/or chromosomal integration elements, such as retroviral Long Terminal Repeats (LTRs) or adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

The polynucleotide and nucleic acid coding regions of the invention may be associated with additional coding regions that encode secretion or signal peptides that direct the secretion of the polypeptide encoded by the polynucleotide of the invention. For example, if secretion of the T cell activating bispecific antigen binding molecule is desired, a DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the T cell activating bispecific antibody or fragment thereof of the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein upon initiation of export of the growing protein chain across the rough endoplasmic reticulum. One of ordinary skill in the art knows that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of such a sequence that retains the ability to direct secretion of the polypeptide with which it is operably associated, is used. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.

DNA encoding short protein sequences that can be used to facilitate later purification (e.g., histidine tag) or to aid in labeling of T cell activating bispecific antibodies can be incorporated into or at the terminus of the T cell activating bispecific antibody (fragment) encoding polynucleotide.

In a particular embodiment, host cells are provided that comprise one or more polynucleotides of the invention. In certain embodiments, host cells comprising one or more vectors of the invention are provided. The polynucleotide and vector may incorporate any of the features described herein with respect to the polynucleotide and vector, respectively, alone or in combination. In one such embodiment, the host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide encoding (part of) a T cell activating bispecific antibody of the invention. As used herein, the term "host cell" refers to any kind of cell system that can be engineered to produce the T cell activating bispecific antibody or fragment thereof of the present invention. Host cells suitable for replicating T cell activating bispecific antibodies and supporting their expression are well known in the art. Where appropriate, such cells may be transfected or transduced with a particular expression vector, and a large number of vector-containing cells may be cultured for seeding a large-scale fermentor to obtain a sufficient amount of T cell-activating bisSpecific antibodies are used in clinical applications. Suitable host cells include prokaryotic microorganisms such as E.coli, or various eukaryotic cells such as Chinese hamster ovary Cells (CHO), insect cells, and the like. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be separated from the bacterial cell paste in the soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, NatBiotech22,1409-1414(2004), and Li et al, Nat Biotech24,210-215 (2006). Host cells suitable for the expression (glycosylation) of polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified for use with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978 and 6,417,429 (PLANTIBODIIES described for the production of antibodies in transgenic plants^TMA technique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al, J Gen Virol36,59(1977)), baby hamster kidney cells (BHK), mouse Sertoli (Sertoli) cells (TM4 cells, as described, for example, in Mather, Biol Reprod23,243-251 (1980)), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), bovine murine (buffalo rat) liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (as described, for example, in Mather et al, Annals N.Y. Acad Sci383,44-68 (1982)), MRC5 cells, and MR 4 cells. Other useful mammalian host cell lines include the Chinese hamster ovary(CHO) cells, including dhfr-CHO cells (Urlaub et al, ProcNatl Acad Sci USA77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in molecular Biology, volume 248 (b.k.c.lo, Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells such as mammalian culture cells, yeast cells, insect cells, bacterial cells, plant cells, and the like, but also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide comprising an antigen binding domain such as the heavy or light chain of an antibody may be engineered such that the other antibody chain is also expressed, such that the product of expression is an antibody having both a heavy chain and a light chain.

In one embodiment, a method of producing a T cell activating bispecific antibody according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding a T cell activating bispecific antibody (as provided herein) under conditions suitable for expression of the T cell activating bispecific antigen binding molecule, and recovering the T cell activating bispecific antibody from the host cell (or host cell culture broth).

The components of the T cell activating bispecific antibody are genetically fused to each other. The T cell activating bispecific antibody can be designed such that its components are fused to each other directly or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and the efficacy can be tested. Examples of linker sequences between the different components of the T cell activating bispecific antibody are found in the sequences provided herein. Additional sequences may also be included to incorporate cleavage sites to separate the individual components of the fusion (if desired), such as endopeptidase recognition sequences.

In certain embodiments, the one or more antigen binding moieties of the T cell activating bispecific antibody comprise at least an antibody variable region capable of binding an antigenic determinant. The variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods for generating polyclonal and monoclonal Antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a laboratory manual," Cold Spring harbor laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. patent No.4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy and variable light chains (see, e.g., U.S. patent No.5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used for the T cell activating bispecific antibody of the present invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the invention may be of murine, primate or human origin. If the T cell activating antibody is intended for human use, a chimeric form of the antibody may be used in which the constant regions of the antibody are from a human. Humanized or fully human forms of antibodies can also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No.5,565,332 to Winter). Humanization can be achieved by a variety of methods, including but not limited to (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions, with or without retention of critical framework residues (e.g., those important for retaining better antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) onto human frameworks and constant regions, or (c) grafting intact non-human variable domains, but "cloak" them with human-like moieties by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Frontbiosci13,1619-1633(2008), and also described, for example, in Riechmann et al, Nature332,323-329(1988); queen et al, Proc Natl Acad Sci USA86, 10029-; U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; jones et al, Nature321,522-525(1986); morrison et al, ProcNatl Acad Sci81,6851-6855(1984); morrison and Oi, Adv Immunol44,65-92(1988); verhoeyen et al, Science239,1534-1536(1988); padlan, Molec Immun31(3),169-217(1994); kashmiri et al, Methods36,25-34(2005) (describing SDR (a-CDR) grafting); padlan, Mol Immunol28,489-498(1991) (description "resurfacing"); dall' Acqua et al, Methods36,43-60(2005) (description "FR shuffling"); and Osbourn et al, Methods36,61-68(2005) and Klimka et al, Br J cancer83,252-260(2000) (describing the "guided selection" approach to FR shuffling). Human antibodies and human variable regions can be generated using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin pharmacol5,368-74(2001), and Lonberg, Curr Opin immunol20,450-459 (2008). The human variable region can form part of and be derived from human Monoclonal antibodies produced by the hybridoma method (see, e.g., Monoclonal antibody production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody with a human variable region in response to antigen challenge (see, e.g., Lonberg, Nat Biotech23,1117-1125 (2005)). Human antibodies and human variable regions can also be generated by cloning variable region sequences via Fv isolated from human derived phage display library selections (see, e.g., Hoogenboom et al, Methods in molecular Biology178,1-37 (O' Brien et al, eds., human Press, Toewa, NJ,2001); and McCafferty et al, Nature348,552-554; Clackson et al, Nature352,624-628 (1991)). Phage typically display antibody fragments, either as single chain fv (scfv) fragments or as Fab fragments.

In certain embodiments, bispecific antibodies of the invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. patent application publication No.2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of the T cell activating bispecific antibodies of the invention to bind to a particular antigenic determinant may be measured via enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as the surface plasmon resonance technique (analyzed on the BIACORE T100 system) (Liljeblad et al, Glyco J17,323-329(2000)) and traditional binding assays (Heeley, Endocr Res28,217-229 (2002)). Competition assays can be used to identify antibodies, antibody fragments, antigen binding domains or variable domains that compete with a reference antibody for binding to a particular antigen, e.g., antibodies that compete with the V9 antibody for binding to CD 3. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by the reference antibody. A detailed exemplary method for locating epitopes to which antibodies bind is provided in Morris (1996) "Epitope Mapping Protocols," Methods in molecular biology vol.66(human Press, Totowa, NJ). In one exemplary competition assay, an immobilized antigen (e.g., CD3) is incubated in a solution comprising a first labeled antibody (e.g., V9 antibody) that binds the antigen and a second unlabeled antibody that is tested for its competition with the first labeled antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen was incubated in a solution comprising the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The T cell activating bispecific antibody prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens to which the T cell activating bispecific antibody binds may be used. For example, for affinity chromatography purification of the T cell activating bispecific antibody of the invention, a matrix with protein a or protein G may be used. The T cell activating bispecific antibody can be isolated using protein a or G affinity chromatography and size exclusion chromatography in series, essentially as described in the examples. The purity of the T cell activating bispecific antibody can be determined by any of a variety of well known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

C. Assay method

Bispecific antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.

1. Binding assays and other assays

In one aspect, bispecific antibodies of the invention are tested for antigen binding activity, e.g., by known methods such as ELISA, Western blot, and the like.

On the other hand, competition assays can be used to identify antibodies that compete for binding to a Tumor Antigen (TA) or a T cell activating antigen with a particular anti-TA antibody or antibody specific for the T cell activating antigen, respectively. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the epitope bound by the particular anti-TA antibody or antibody specific for a T cell activating antigen. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol.66(HumanaPress, Totowa, N.J.).

2. Activity assay

In one aspect, assays are provided for identifying bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) that have biological activity. The biological activity may include, for example, lysis or induction of apoptosis of the target cell. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, bispecific antibodies of the invention are tested for such biological activity. Assays for detecting cell lysis (e.g., by measuring LDH release) or apoptosis (e.g., using TUNEL assay) are well known in the art.

D. Immunoconjugates

The invention also provides immunoconjugates comprising the bispecific antibodies herein that bind to a T cell activating antigen and a Tumor Antigen (TA) conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman et al, Cancer Res.53:3336-3342(1993); and Lode et al, Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel (paclitaxel), larotaxel, tesetaxel, and ortataxel; trichothecenes (trichothecenes); and CC 1065.

In another embodiment, the immunoconjugate comprises a bispecific antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin (ricin) a chain, abrin (abrin) a chain, modeccin (modecin) a chain, α -sarcin (sarcin), aleurites fordii (aleurites fordii) toxic protein, dianthus caryophyllus (dianthin) toxic protein, phytolacca americana (phytolacocaa) protein (PAPI, PAPII and PAP-S), Momordica charantia (momordia) inhibitor, curculin (curcin), crotin (crotin), saponaria officinalis (pachrina) inhibitor, trichomonas campestris (trichothecin) inhibitor, trichothecin (trichothecin) inhibitor, trichothecin (trichothecin), or fragment thereof, Phenomycin, enomycin and trichothecenes.

In another embodiment, the immunoconjugate comprises a bispecific antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

A variety of bifunctional protein coupling agents may be used to generate conjugates of bispecific antibodies and cytotoxic agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Res52: 127-.

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any bispecific antibody that binds to a T cell activating antigen and a Tumor Antigen (TA) provided herein can be used to detect the presence of a T cell activating antigen and/or a Tumor Antigen (TA) in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue.

In one embodiment, bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) are provided for use in a diagnostic or detection method. In yet another aspect, a method of detecting the presence of T cell activating antigen 3 and/or a Tumor Antigen (TA) in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with a bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA) described herein under conditions permissive for binding of the bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA) to the T cell activating antigen and/or Tumor Antigen (TA), and detecting whether a complex is formed between the bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA) and the T cell activating antigen and/or Tumor Antigen (TA). Such methods may be in vitro or in vivo. In one embodiment, a bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA) is used to select a subject suitable for treatment with the bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA), e.g., where Tumor Antigen (TA) is a biomarker for selecting patients.

Exemplary disorders that can be diagnosed using the antibodies of the invention include cancer.

In certain embodiments, labeled bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes³²P、¹⁴C、¹²⁵I、³H. And¹³¹I. fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine (rhodamine) and its derivatives, dansyl, umbelliferone, fluorescein derivatives, rhodamine derivatives, and rhodamine derivatives,Luciferases, for example, firefly luciferase and bacterial luciferase (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to an enzyme employing a hydrogen peroxide oxidation dye precursor such as HRP), lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical formulations

Pharmaceutical formulations of bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) as described herein are prepared by mixing such bispecific antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds. (1980)) in a lyophilized formulation or in an aqueous solution. Generally, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal composites (e.g. metal-coated metal composites)Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise an interstitial drug dispersant such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) ())Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

An exemplary lyophilized antibody formulation is described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those compounds whose activities are complementary and do not adversely affect each other. Such active components are suitably present in combination in amounts effective for the desired purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

G. Therapeutic methods and compositions

Any bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA) provided herein can be used in a therapeutic method.

In one aspect, bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) for use as a medicament are provided. In still further aspects, bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) are provided for use in treating cancer. In certain embodiments, bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) are provided for use in a method of treatment. In certain embodiments, the invention provides a bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA) for use in a method of treating an individual with cancer, the treatment comprising administering to the individual an effective amount of the bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides the use of a bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA) in the manufacture or manufacture of a medicament. In one embodiment, the medicament is for treating cancer. In yet another embodiment, the medicament is for use in a method of treating cancer, comprising administering to an individual having cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides a method of treating cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of a bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides a pharmaceutical formulation comprising any of the bispecific antibodies provided herein that bind to a T cell activating antigen and a Tumor Antigen (TA), e.g., for use in any of the above therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the bispecific antibodies that bind to a T cell activating antigen and a Tumor Antigen (TA) provided herein and at least one additional therapeutic agent, e.g., as described below.

The bispecific antibodies of the invention can be used alone or in combination with other agents in therapy. For example, a bispecific antibody of the invention can be co-administered with at least one additional therapeutic agent.

Such combination therapies noted above encompass both combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agents and/or adjuvants. Bispecific antibodies of the invention may also be used in combination with radiotherapy.

The bispecific antibody (and any other therapeutic agent) of the present invention may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

The bispecific antibodies of the present invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular condition being treated, the particular mammal being treated, the clinical status of the individual patient, the cause of the condition, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to practitioners. The bispecific antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other drugs will depend on the amount of antibody present in the formulation, the type of condition being treated, and other factors discussed above. These agents are generally used at the same dosages and routes of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route, as empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of the bispecific antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the prophylactic or therapeutic purpose for which the bispecific antibody is administered, previous therapy, the patient's clinical history and response to the bispecific antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient in one or a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg-15 mg/kg (e.g., 0.1mg/kg-10 mg/kg) of the bispecific antibody may be administered to the patient as a first candidate amount, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of the condition occurs. Bispecific antibodies will be in a range of about 0.05mg/kg to about 10mg/kg at one exemplary dose. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient. The above dose can be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives from about 2 to about 20 doses, or, e.g., about 6 doses, of the bispecific antibody). An initial higher loading dose may be administered followed by a lower dose or doses. However, other dosage regimens may be used. The progress of the treatment is readily monitored by conventional techniques and assays.

It is to be understood that any of the above formulations or therapeutic methods can be performed using the immunoconjugates of the invention in vitro, instead of or in addition to a bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA).

H. Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and/or diagnosis of the conditions described above. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition effective, alone or in combination with another composition, in the treatment, prevention, and/or diagnosis of a condition, and may have a sterile access port (e.g., the container may be a vial or intravenous solution bag having a stopper penetrable by a hypodermic needle). At least one active agent in the composition is a bispecific antibody of the invention. The label or package insert indicates the use of the composition to treat the selected condition. In addition, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises a bispecific antibody of the present invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be appreciated that any of the above preparations may include an immunoconjugate of the invention in place of, or in addition to, a bispecific antibody that binds a T cell activating antigen and a Tumor Antigen (TA).

Example III

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in view of the general description provided above.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Example 1: preparation of Fab (MCSP) -CrossFab (CD3)

The resulting heavy and light chain variable region DNA sequences were subcloned into the respective recipient mammalian expression vectors in-frame with the pre-inserted constant heavy chain or constant light chain. Antibody expression is driven by the MPSV promoter and carries a synthetic polyA signal sequence at the 3' end of the CDS. In addition, each vector contains the EBV OriP sequence.

The molecules were produced by co-transfecting HEK293-EBNA cells with mammalian expression vectors using calcium phosphate transfection. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. Alternatively, HEK293-EBNA cells grown in suspension were transfected with polyethylenimine. Cells were transfected with the corresponding expression vectors at a 1:1:1 ratio ("vector CH 1-VH-CK-VH": vector light chain CH1-VL ").

For transfection with calcium phosphate, cells were cultured in T flasks as adherent monolayer cultures using DMEM medium supplemented with 10% (v/v) FCS and transfected when they reached 50% to 80% confluence. For transfection in T150 flasks, 1.5X10 was added 24 hours prior to transfection⁷The individual cells were seeded in 25ml DMEM medium supplemented with FCS (final concentration 10% v/v) and then the cells were plated in medium with 5% CO₂The atmosphere was maintained in an incubator at 37 ℃ overnight. For each T150 flask to be transfected, the plasmid vector DNA was prepared by mixing 94. mu.g of total plasmid vector DNA, a final volume of 469. mu.l of water and 469. mu.l of 1M CaCl in the corresponding proportions₂Preparation of DNA, CaCl by solution mixing₂And a solution of water. To this solution was added 938. mu.l 50mM HEPES, 280mM NaCl, 1.5mM Na₂HPO₄Immediately mixed for 10 seconds and left at room temperature for 20 seconds. The suspension was diluted with 10ml of DMEM supplemented with 2% (v/v) FCS and T150 was added to replace the existing medium. Then an additional 13ml of transfection medium was added. Cells were incubated at 37 ℃ with 5% CO₂Incubate for about 17 to 20 hours, then replace the medium with 25ml DMEM, 10% FCS. Approximately 7 days after medium replacement, conditioned medium was harvested by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% (w/v) and stored at 4 ℃.

For transfection with polyethyleneimine, HEK293EBNA cells were cultured in CD CHO media in serum-free suspension. For 500ml flask production, 4 × 10 was inoculated 24 hours prior to transfection⁸And one HEK293EBNA cell. For transfection, cells were centrifuged at 210Xg for 5 min and the supernatant was replaced with pre-warmed 20ml CD CHO medium. The expression vector was mixed in 20ml of CD CHO medium to a final 200. mu.g DNA amount. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated for 10 minutes at room temperature. The cells were then mixed with DNA/PEI solution, transferred to a 500ml flask and incubated with 5% CO₂The incubation was carried out in an atmosphere incubator at 37 ℃ for 3 hours. After the incubation time 160ml of F17 medium was added and the cells were cultured for 24 hoursThen (c) is performed. 1 day after transfection, 1mM valproic acid (valric acid) and 7% Feed1(Lonza) were added. After 7 days the culture supernatant was collected for purification by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and stored at 4 ℃.

Secreted proteins were purified from cell culture supernatants by affinity chromatography using protein a and protein G affinity chromatography followed by a size exclusion chromatography step. For affinity chromatography, the supernatant was loaded onto a HiTrap protein a HP column (CV =5ml, GE Healthcare) coupled to a HiTrap protein G HP column (CV =5ml, GE Healthcare), each column having been equilibrated with 30ml of 20mM sodium phosphate, 20mM sodium citrate, ph 7.5. Unbound protein was removed by washing both columns with 6 column volumes of 20mM sodium phosphate, 20mM sodium citrate ph 7.5. A further necessary washing step is then to wash only the HiTrap protein G HP column with at least 8 column volumes of 20mM sodium phosphate, 20mM sodium citrate ph 7.5. The target protein was eluted from the HiTrap protein G HP column by a stepwise gradient with 7 column volumes of 8.8mM formic acid ph 3.0. The protein solution was neutralized by the addition of 1/10M sodium phosphate 0.5M, pH 8.0. The target protein was concentrated and filtered and then loaded onto a HiLoad Superdex200 column (GE Healthcare) equilibrated with a solution of 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine, pH 6.7.

The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. By SDS-PAGE in the presence and absence of reducing agent (5mM1, 4-dithiothreitol) with Coomassie (SimpleBlue)^TMSafeStain, Invitrogen) to analyze antibody purity and molecular weight. The use was according to the manufacturer's instructions (4-12% Tris-acetate gel or 4-12% Bis-Tris)Pre-gel systems (Invitrogen, USA). Analytical size exclusion column (GEHealthcare, Sweden) using Superdex20010/300GL in 2mM MOPS, 150mM NaCl, 0.02% (w/v) NaN₃pH7.3 at 25 ℃ in a running buffer ofThe collective content.

Exemplary Fab-Crossfab molecules (consisting of three chains: VHCH1(MCSP) -VLCH1(CD 3)_V9) = SEQ ID NO:25, VLCL (MCSP) = SEQ ID NO:17 and VHCL (CD3)_V9) 23, SEQ ID NO; with the orientation shown in fig. 1 a) is shown in fig. 2 and 3. This molecule is further referred to as Fab (MCSP) -Crossfab (CD3) or huFab (MCSP) -Crossfab (CD 3).

Example 2: preparation of Fab (MCSP) -CrossFab (CD3) and Fab (MCSP) -CrossFab (CD3) -Fab (MCSP)

The molecules were produced by co-transfecting HEK293-EBNA cells with mammalian expression vectors using calcium phosphate transfection. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. Alternatively, HEK293-EBNA cells grown in suspension were transfected with polyethylenimine. Cells were transfected with the corresponding expression vectors at a 1:2:1 ratio ("vector CH 1-VH-CH 1-VH-CK-VH": vector light chain CH1-VL ").

For transfection with calcium phosphate, cells were cultured in T flasks as adherent monolayer cultures using DMEM medium supplemented with 10% (v/v) FCS and transfected when they reached 50% to 80% confluence. For transfection in T150 flasks, 1.5X10 was added 24 hours prior to transfection⁷The individual cells were seeded in 25ml DMEM medium supplemented with FCS (final concentration 10% v/v) and then the cells were plated in medium with 5% CO₂The atmosphere was maintained at 37 ℃ overnight in an incubator. For each T150 flask to be transfected, the plasmid vector DNA was prepared by mixing 94. mu.g of total plasmid vector DNA, 469. mu.l of water and 469. mu.l of 1M CaCl in the corresponding proportions₂Preparation of DNA, CaCl by solution mixing₂And a solution of water. To this solution was added 938. mu.l 50mM HEPES, 280mM NaCl, 1.5mM Na₂HPO₄Immediately mixed for 10 seconds and left at room temperature for 20 seconds. The suspension was diluted with 10ml of DMEM supplemented with 2% (v/v) FCS and T150 was added to replace the existing medium. Then an additional 13ml of transfection medium was added. Cells were incubated at 37 ℃ with 5% CO₂Incubate for about 17 to 20 hours, then replace the medium with 25ml DMEM, 10% FCS. Approximately 7 days after medium replacement, conditioned medium was harvested by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% (w/v) and stored at 4 ℃. For transfection with polyethyleneimine, HEK293EBNA cells were cultured in CD CHO media in serum-free suspension. For 500ml shake flask production, 4X10 was inoculated 24 hours before transfection⁸And one HEK293EBNA cell. For transfection, cells were centrifuged at 210Xg for 5 minutes and the supernatant was replaced with pre-warmed 20ml CD CHO medium. The expression vector was mixed in 20ml of CD CHO medium to a final 200. mu.g DNA amount. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated for 10 minutes at room temperature. The cells were then mixed with DNA/PEI solution, transferred to 500ml shake flasks and incubated with 5% CO₂The incubation was carried out in an atmosphere incubator at 37 ℃ for 3 hours. After the incubation time 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% Feed1(Lonza) were added. After 7 days the culture supernatant was collected for purification by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and stored at 4 ℃.

Secreted proteins were purified from cell culture supernatants by affinity chromatography using protein a and protein G affinity chromatography followed by a size exclusion chromatography step.

For affinity chromatography, the supernatant was loaded onto a HiTrap protein a HP column coupled to a HiTrap protein G HP column (CV =5ml, GE Healthcare) CV =5ml, GE Healthcare (above, each column had been equilibrated with 30ml of 20mM sodium phosphate, 20mM sodium citrate, ph 7.5. Unbound protein was removed by washing both columns with 6 column volumes of 20mM sodium phosphate, 20mM sodium citrate ph 7.5. An additional necessary washing step is then to wash only the HiTrap protein G HP column with at least 8 column volumes of 20mM sodium phosphate, 20mM sodium citrate ph 7.5. The target protein was eluted from the HiTrap protein G HP column by a stepwise gradient with 7 column volumes of 8.8mM formic acid ph 3.0. The protein solution was neutralized by the addition of 1/10M sodium phosphate 0.5M, pH 8.0. The target protein was concentrated and filtered and then loaded onto a HiLoad Superdex200 column (GE Healthcare) equilibrated with a solution of 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine, pH 6.7.

The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. By SDS-PAGE in the presence and absence of reducing agent (5mM1, 4-dithiothreitol) with Coomassie (SimpleBlue)^TMSafeStain, Invitrogen) to analyze antibody purity and molecular weight. The use was according to the manufacturer's instructions (4-12% Tris-acetate gel or 4-12% Bis-Tris)Pre-gel systems (Invitrogen, USA). Analytical size exclusion column (GEHealthcare, Sweden) using Superdex20010/300GL in 2mM MOPS, 150mM NaCl, 0.02% (w/v) NaN₃The aggregate content of the antibody samples was analyzed at 25 ℃ in the running buffer of pH7.3 and compared with the antibody fragment (scFv)2 of the prior art (results are shown in the table below).

HMW = high molecular weight; LMW = low molecular weight

Exemplary Fab-Fab-Crossfab molecules (consisting of four chains: VHCH1(MCSP) -VHCH1(MCSP) -VLCH1(CD 3)_V9) = SEQ ID NO 26, two VLCL (MCSP) chains = SEQ ID NO 17 and one VHCL (CD3)_V9) Strand = SEQ id No. 23; with the orientation shown in fig. 1 c) is shown in fig. 4 and 5. This molecule is further referred to as Fab (MCSP) -Fab (MCSP) -Crossfab (CD3) or hu Fab (MCSP) -Crossfab (CD 3).

Exemplary Fab-Crossfab-Fab molecules (consisting of four chains: VHCH1(MCSP) -VLCH1(CD 3)_V9) -VHCH1(MCSP) = SEQ ID NO:27, two vlcl (MCSP) chains = SEQ ID NO:17 and one VHCL (CD3)_V9) Strand = SEQ id No. 23; with the orientation shown in fig. 1 e) is shown in fig. 6 and 7. This molecule is further referred to as Fab (MCSP) -Crossfab (CD3) or hu Fab (MCSP) -Crossfab (CD 3).

Exemplary Crossfab-Fab-Fab molecules (consisting of four chains: VLCH1(CD 3)_2C11) -VHCH1(MCSP) -VHCH1(MCSP) = SEQ ID NO:42, two vlcl (MCSP) chains = SEQ ID NO:17 and one VHCL (CD3)_2C11) Chain = SEQ ID NO 43; with the orientation shown in fig. 1 d) is shown in fig. 8 and 9. This molecule is further referred to as murine Crossfab (CD3) -Fab (MCSP).

Example 3: preparation of Fab (CD33) -CrossFab (CD3)

For transfection with calcium phosphate, cells were cultured in T flasks using DMEM medium supplemented with 10% (v/v) FCS as adherent monolayer culture,and transfection was performed when they reached 50% to 80% confluence. For transfection in T150 flasks, 1.5X10 was added 24 hours prior to transfection⁷The individual cells were seeded in 25ml DMEM medium supplemented with FCS (final concentration 10% v/v) and then the cells were plated in medium with 5% CO₂The atmosphere was maintained at 37 ℃ overnight in an incubator. For each T150 flask to be transfected, the plasmid vector DNA was prepared by mixing 94. mu.g of total plasmid vector DNA, a final volume of 469. mu.l of water and 469. mu.l of 1M CaCl in the corresponding proportions₂Preparation of DNA, CaCl by solution mixing₂And a solution of water. To this solution was added 938. mu.l 50mM HEPES, 280mM NaCl, 1.5mM Na₂HPO₄Immediately mixed for 10 seconds and left at room temperature for 20 seconds. The suspension was diluted with 10ml of DMEM supplemented with 2% (v/v) FCS and T150 was added to replace the existing medium. Then an additional 13ml of transfection medium was added. Cells were incubated at 37 ℃ with 5% CO₂Incubate for about 17 to 20 hours, then replace the medium with 25ml DMEM, 10% FCS. Approximately 7 days after medium replacement, conditioned medium was harvested by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% (w/v) and stored at 4 ℃.

For transfection with polyethyleneimine, HEK293EBNA cells were cultured in CD CHO media in serum-free suspension. For 500ml shake flask production, 4X10 was inoculated 24 hours before transfection⁸And one HEK293EBNA cell. For transfection, cells were centrifuged at 210Xg for 5 minutes and the supernatant was replaced with pre-warmed 20ml CD CHO medium. The expression vector was mixed in 20ml CDCHO medium to a final 200. mu.g DNA amount. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated for 10 minutes at room temperature. The cells were then mixed with DNA/PEI solution, transferred to 500ml shake flasks and incubated with 5% CO₂The incubation was carried out in an atmosphere incubator at 37 ℃ for 3 hours. After the incubation time 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% Feed1(Lonza) were added. After 7 days the culture supernatant was collected for purification by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and stored at 4 ℃.

For affinity chromatography, the supernatant was loaded onto a HiTrap protein a HP column (CV =5ml, GE Healthcare) coupled to a HiTrap protein G HP column (CV =5ml, GE Healthcare), each column having been equilibrated with 30ml of 20mM sodium phosphate, 20mM sodium citrate, ph 7.5. Unbound protein was removed by washing both columns with 6 column volumes of 20mM sodium phosphate, 20mM sodium citrate ph 7.5. An additional necessary washing step is then to wash only the HiTrap protein G HP column with at least 8 column volumes of 20mM sodium phosphate, 20mM sodium citrate ph 7.5. The target protein was eluted from the HiTrap protein G HP column by a stepwise gradient with 7 column volumes of 8.8mM formic acid ph 3.0. The protein solution was neutralized by the addition of 1/10M sodium phosphate 0.5M, pH 8.0. The target protein was concentrated and filtered and then loaded onto a HiLoad Superdex200 column (GE Healthcare) equilibrated with a solution of 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine, pH 6.7.

The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. By SDS-PAGE in the presence and absence of reducing agent (5mM1, 4-dithiothreitol) with Coomassie (SimpleBlue)^TMSafeStain, Invitrogen) to analyze antibody purity and molecular weight. The use was according to the manufacturer's instructions (4-12% Tris-acetate gel or 4-12% Bis-Tris)Pre-gel systems (Invitrogen, USA). Analytical size exclusion column (GEHealthcare, Sweden) using Superdex20010/300GL in 2mM MOPS, 150mM NaCl, 0.02% (w/v) NaN₃The aggregate content of the antibody samples was analyzed at 25 ℃ in the running buffer of pH 7.3.

Exemplary Fab-Crossfab molecules (consisting of three chains: VHCH1(CD33) -VLCH1(CD 3)_V9) 102, VLCL (CD33) = SEQ ID NO 100 and VHCL (CD3)_V9)=SEQ ID NO 23 or SEQ ID NO 101; with the orientation shown in fig. 1 a) is shown in fig. 17 and 18. This molecule is further referred to as Fab (CD33) -CrossFab (CD3) or huFab (CD33) -CrossFab (CD 3).

Example 4: preparation of reference molecule (scFv)2

Cloning and Generation

The resulting heavy and light chain variable region DNA sequences were subcloned into respective recipient mammalian expression vectors. Antibody expression is driven by the MPSV promoter and carries a synthetic polyA signal sequence at the 3' end of the CDS. In addition, each vector contains the EBV OriP sequence.

HEK293-EBNA cells were transfected with mammalian expression vectors using polyethylenimine to produce the molecule. HEK293EBNA cells were cultured in CD CHO media in serum-free suspension. For 500ml shake flask production, 4X10 was inoculated 24 hours before transfection⁸And one HEK293EBNA cell. For transfection, cells were centrifuged at 210Xg for 5 minutes and the supernatant was replaced with pre-warmed 20ml CDCHO medium. The expression vector was mixed in 20ml of CD CHO medium to a final 200. mu.g DNA amount. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated for 10 minutes at room temperature. The cells were then mixed with DNA/PEI solution, transferred to 500ml shake flasks and incubated with 5% CO₂The incubation was carried out in an atmosphere incubator at 37 ℃ for 3 hours. After the incubation time 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% Feed1(Lonza) were added. After 7 days the culture supernatant was collected for purification by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and stored at 4 ℃.

Purification of (scFv)2 (anti-MCSP/anti-huCD 3)

Secreted proteins were purified from cell culture supernatants by affinity chromatography using immobilized metal ion affinity chromatography (IMAC) followed by a size exclusion chromatography step.

Before the first purification step is carried out, the interfering components are removed from the supernatant by diafiltration using a tangential flow filtration system, Sarcojet (Sartorius), equipped with a 5.000MWCO membrane (Sartocon Slice Cassette, Hydrosart; Sartorius). The supernatant was concentrated to 210ml and then diluted in 1L of 20mM sodium phosphate, 500mM sodium chloride pH 6.5. The protein solution was again concentrated to 210 ml. This procedure was repeated twice to ensure complete buffer replacement.

For affinity chromatography, the retentate of the diafiltration process was loaded onto a NiNTA Superflow Cartridge (CV =5mL, Qiagen) equilibrated with 25mL20mM sodium phosphate, 500mM sodium chloride, 15mM imidazole pH 6.5. Unbound protein was removed by washing with at least 2 column volumes of 20mM sodium phosphate, 500mM sodium chloride, 15mM imidazole ph6.5 and then 3 column volumes of 20mM sodium phosphate, 500mM sodium chloride, 62.5mM imidazole ph 6.5. The target protein was eluted in two column volumes of 20mM sodium phosphate, 500mM sodium chloride, 125mM imidazole pH 6.5. The column was then washed with 20mM sodium phosphate, 500mM sodium chloride, 250mM imidazole pH 6.5.

The target protein was concentrated and then loaded with 25mM KH₂PO₄125mM NaCl, 200mM arginine pH6.7 on a HiLoad Superdex75 column (GE Healthcare).

The yields, aggregate content after the first purification step and final monomer content are shown in the table above. Comparison of the aggregate content after the first purification step indicates the superior stability of the Fab-Crossfab construct in comparison to (scFv) 2.

Characterization of (scFv)2

The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. By SDS-PAGE in the presence and absence of reducing agent (5mM1, 4-dithiothreitol) with Coomassie (SimpleBlue)^TMSafeStain, Invitrogen) to analyze antibody purity and molecular weight. The use was according to the manufacturer's instructions (4-12% Tris-acetate gel or 4-12% Bis-Tris)Pre-gel systems (Invitrogen, USA). Analytical size exclusion column (GEHealthcare, Sweden) using Superdex7510/300GL in 2mM MOPS, 150mM NaCl, 0.02% (w/v) NaN₃The aggregate content of the antibody samples was analyzed at 25 ℃ in the running buffer of pH 7.3.

A schematic representation of the (scFv)2 molecule is shown in FIG. 21.

Exemplary (scFv)2 molecules (anti-MCSP/anti-huCD 3; consisting of two single chain Fv: VL-VH (MCSP) and VH-VL (CD3)_V9) 149) and analysis of the production and purification are shown in fig. 22 and 23. This molecule is further referred to as (scFv)2 (anti-MCSP/anti-huCD 3 e).

Example 5: isolation of primary human Pan T cells from PBMC

Peripheral Blood Mononuclear Cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coats) obtained from local blood banks or fresh blood of healthy human donors.

Enrichment of T cells from PBMC was performed using the pan T cell isolation kit II (Miltenyi Biotec # 130-. Briefly, cell pellets were diluted in 40. mu.l cold buffer (PBS-sterile filtered with 0.5% BSA, 2mM EDTA) per 1 million cells and incubated with 10. mu.l biotin-antibody mixture per 1 million cells for 10 minutes at 4 ℃.

30. mu.l of cold buffer and 20. mu.l of avidin beads were added per 1 million cells, and the mixture was incubated at 4 ℃ for another 15 minutes.

Cells were washed by adding 10-20 times the labeled volume followed by centrifugation at 300g for 10 minutes. Up to 1 million cells were resuspended in 500. mu.l buffer.

Implementation with LS column (Miltenyi Biotec #130-Magnetic separation of human pan-T cells was noted. The resulting T cell populations were automatically counted (ViCell) and 5% CO at 37 ℃₂Stored in AIM-V medium in an incubator until the assay begins (no more than 24 hours).

Example 6: isolation of murine pan T cells from splenocytes

Spleens were isolated from C57BL/6 mice, transferred into GentleMACC C tubes (Miltenyi Biotech # 130-.

The cell suspension was passed through a pre-separation filter to remove residual undissociated tissue particles. After centrifugation at 400g for 4 min at 4 ℃, ACK lysis buffer was added to lyse the red blood cells (5 min incubation at room temperature). The remaining cells were washed twice with MACS buffer, counted and used to isolate murine pan T cells. Negative (magnetic) selection was performed with the pan T cell isolation kit from Miltenyi Biotec (# 130-. The resulting T cell populations were automatically counted (ViCell) and immediately used for further assays.

Example 7: redirected T cell cytotoxicity mediated by crosslinked bispecific constructs targeting CD3 on T cells and MCSP on tumor cells (LDH release assay)

Bispecific constructs targeting CD3 and human tumor cells on human or mouse T cells were analyzed for their potential to induce T cell-mediated apoptosis of target cells by LDH release assay.

Briefly, target cells (human Colo-38, human MDA-MB-435, human melanoma MV-3 or murine B16/F10-humCCFLuc 2 clone 48 cells, all expressing human MCSP) were harvested with either cell dissociation buffer (MCSP is trypsin sensitive) or trypsin (and then spread out the day before), washed and resuspended in the appropriate cell culture medium (see details of the different figures). 20,000-30,000 cells per well were plated in round bottom 96-well plates and respective antibody dilutions (in triplicate) were added as indicated. Effector cells were added to obtain the final E: T ratio of 5:1 (for human pan T cells), 10:1 (for human PBMC).

In addition, 1-10. mu.g/ml PHA-M (Sigma # L8902), an isoagglutinin mixture, isolated from Phaseolus vulgaris (Phaseolus vulgaris) was used as a mitogenic stimulator to induce human or cynomolgus T cell activation. For murine T cells, a 5% solution of "rat T cells stimulated with ConA" (BD # 354115) was used as a positive control for T cell activation.

For standardization, maximum lysis (= 100%) of target cells was achieved by incubating the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (= 0%) refers to target cells that were co-incubated with effector cells but without any constructs or antibodies.

At 37 deg.C, 5% CO₂After an overnight incubation of at least 18 hours, LDH released into the supernatant by target cells for apoptosis/necrosis was measured with LDH detection kit (Roche applied science, # 11644793001) according to the manufacturer's instructions.

Bispecific with Fab (MCSP) -Crossfab (CD3) and Fab (MCSP) -Crossfab (CD3) Construct performed LDH release assay

The purified fab (MCSP) -Crossfab (CD3), fab (MCSP) -Crossfab (CD3) and (scFv)2 (anti-MCSP/anti-huCD 3e) reference molecules were analyzed for their potential to induce T-cell mediated apoptosis in tumor target cells when cross-linked to constructs that bind to the respective antigens on the cells via two targeting moieties. Briefly, the humMCSP-expressing MDA-MB-435 human melanoma target cells were harvested in cell dissociation buffer, washed and resuspended in AIM-V medium (Invitrogen # 12055-. 30,000 cells per well were plated in round bottom 96-well plates and the respective antibody dilutions at the indicated concentrations were added. All constructs and controls were adjusted to the same molar concentration.

Human pan-T effector cells were added to obtain a final 5: 1E: T ratio. As a positive control for human pan-T cell activation, 1. mu.g/ml PHA-M (Sigma # L8902) was used. For standardization, the maximum lysis (= 100%) of target cells was determined by incubating the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (= 0%) refers to target cells that were co-incubated with effector cells but without any constructs or antibodies.

At 37 ℃ 5% CO₂After 20 hours overnight incubation, LDH released into the supernatant from target cells for apoptosis/necrosis was measured with LDH detection kit (Roche applied science, # 11644793001) according to the manufacturer's instructions.

As depicted in figure 10, the construct with bivalent MCSP targeting showed comparable cytotoxic activity compared to the (scFv)2 (anti-MCSP/anti-huCD 3e) construct, whereas the fab (MCSP) -Crossfab (CD3) construct with monovalent MCSP binding was significantly less potent.

Human melanoma with Fab (MCSP) -Crossfab (CD3) bispecific construct and MDA-MB-435 LDH release assay by target cells

Purified fab (MCSP) -Crossfab (CD3) and (scFv)2 (anti-MCSP/anti-huCD 3e) reference molecules were analyzed for their potential to induce T cell-mediated apoptosis in tumor target cells when cross-linked to constructs that bind to the respective antigens on the cells via two targeting moieties.

Briefly, the humMCSP-expressing MDA-MB-435 human melanoma target cells were harvested in cell dissociation buffer, washed and resuspended in AIM-V medium (Invitrogen # 12055-. 30,000 cells per well were plated in round bottom 96-well plates and the respective antibody dilutions at the indicated concentrations were added. All constructs and controls were adjusted to the same molar concentration.

Human pan-T effector cells were added to obtain a final 5: 1E: T ratio. As a positive control for human pan-T cell activation, 5. mu.g/ml PHA-M (Sigma # L8902) was used. For standardization, the maximum lysis (= 100%) of target cells was determined by incubating the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (= 0%) refers to target cells that were co-incubated with effector cells but without any constructs or antibodies.

At 37 ℃ 5% CO₂After overnight incubation for 21 hours, LDH released into the supernatant from target cells for apoptosis/necrosis was measured with LDH detection kit (Roche applied science, # 11644793001) according to the manufacturer's instructions.

As depicted in figure 11, fab (MCSP) -Crossfab (CD3) induced apoptosis in target cells at least as well as the (scFv)2 (anti-MCSP/anti-huCD 3e) molecule.

Bispecific constructs using Fab (MCSP) -Crossfab (CD3) and MV-3 human melanoma target cells LDH release assay performed

Purified fab (MCSP) -Crossfab (CD3) and (scFv)2 (anti-MCSP/anti-huCD 3e) molecules were analyzed for their potential to induce T cell-mediated apoptosis in tumor target cells when cross-linked to constructs that bind to the respective antigens on the cells via two targeting moieties.

Briefly, MV-3 human melanoma target cells expressing huMCSP were harvested with trypsin the day before the initiation of the LDH release assay. Cells were washed and resuspended in the appropriate cell culture medium. 30,000 cells per well were plated in round bottom 96 well plates. The next day, the supernatant was discarded and 100. mu.l/well AIM-V medium (Invitrogen # 12055-091) and the respective antibody dilutions at the indicated concentrations were added. All constructs and controls were adjusted to the same molar concentration.

Human PBMC effector cells were added to obtain a final E: T ratio of 10: 1. As a positive control for human pan-T cell activation, 5. mu.g/ml PHA-M (Sigma # L8902) was used. For standardization, the maximum lysis (= 100%) of target cells was determined by incubating the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (= 0%) refers to target cells that were co-incubated with effector cells but without any constructs or antibodies.

At 37 ℃ 5% CO₂After overnight incubation for 26 hours, LDH released into the supernatant from target cells for apoptosis/necrosis was measured with LDH detection kit (Roche applied science, # 11644793001) according to the manufacturer's instructions.

As depicted in figure 12, fab (MCSP) -Crossfab (CD3) induced apoptosis in target cells at least as well as the (scFv)2 (anti-MCSP/anti-huCD 3e) molecule.

LDH with Fab (MCSP) -Crossfab (CD3) bispecific construct and MV-3 human melanoma target cells Release assay

The LDH release assay was performed as described above. Figure 19 shows the killing of huMCSP positive MV-3 tumor cells when co-cultured with human PBMCs (E: T ratio =10: 1), treated with fab (MCSP) -Crossfab (CD3), (scFv)2 (anti-MCSP/anti-huCD 3E) reference molecules, respectively, for about 24 hours.

LDH release assay with murine Crossfab (CD3) -Fab (MCSP) bispecific construct Method of

Purified murine Crossfab (CD3) -fab (MCSP) targeting murine CD3 and human MCSP were analyzed for its potential to induce T cell mediated apoptosis in tumor target cells upon cross-linking of constructs bound to the respective antigens on the cells via two targeting modules.

Briefly, huMCSP-expressing B16/F10-humcsflur 2 clone 48 tumor target cells were harvested with cell dissociation buffer, washed and resuspended in RPMI1640 medium comprising 1 × NEAA, 10mM Hepes, 50 μm2-B-ME and 1mM sodium pyruvate.

20,000 cells per well were plated in round bottom 96-well plates and the respective antibody dilutions at the indicated concentrations were added. Both the bispecific construct and the different IgG controls were adjusted to the same molar concentration. As an additional control for murine T cell activation, the "ConA-stimulated T cells" (BD # 354115) were used, diluted 1:160 with the assay vehicle.

Murine pan T effector cells isolated from splenocytes (C57 BL/6 mice) were added to obtain a final E: T ratio of 10: 1. For standardization, the maximum lysis (= 100%) of target cells was determined by incubating the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (= 0%) refers to target cells that were co-incubated with effector cells but without any constructs or antibodies.

At 37 ℃ 5% CO₂After 70 hours of incubation, LDH released into the supernatant from target cells for apoptosis/necrosis was measured with LDH detection kit (Roche Applied Science, # 11644793001) according to the manufacturer's instructions.

As depicted in figure 13, the bispecific constructs induced concentration-dependent LDH release from target cells, comparable to the positive control "T cells stimulated with ConA".

Briefly, huMCSP-expressing B16/F10-huMCSP Fluc2 clone 48 tumor target cells were harvested with cell dissociation buffer, washed and resuspended in RPMI1640 medium comprising 1 × NEAA, 10mM Hepes, 50 μm2-B-ME and 1mM sodium pyruvate.

20,000 cells per well were plated in round bottom 96-well plates and respective antibody dilutions were added at a final concentration of 50 nM. Both the bispecific construct and the different IgG controls were adjusted to the same molar concentration.

Murine pan T effector cells isolated from splenocytes (C57 BL/6 mice) were added to obtain a final E: T ratio of 10: 1. To assess the level of hyperactivation of murine T cells in the absence of target cells, control wells were plated with 50nM bispecific construct and T cells.

For standardization, the maximum lysis (= 100%) of target cells was determined by incubating the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (= 0%) refers to target cells that were co-incubated with effector cells but without any constructs or antibodies.

As depicted in figure 14, the bispecific constructs induced strong LDH release from the target cells. In the absence of target cells, there was only a slight increase in LDH compared to untreated murine T cells co-incubated with target cells (reflecting T cell hyperactivation). None of the control IgG induced LDH release from the target cells.

Example 8: cytokine release assay (CBA analysis)

To assess de novo secretion of different cytokines upon activation of T cells with the CD3 bispecific construct in the presence or absence of target cells, human PBMCs were isolated from buffy coat (buffy coat) and 30 ten thousand cells per well were plated in round bottom 96 well plates. Alternatively, 280 μ l of whole blood from a healthy donor was plated into each well of a deep well 96-well plate.

Tumor target cells (e.g., MDA-MB-435 cells for the CD3-MCSP bispecific construct) were added to obtain a final E/T ratio of 10: 1. Bispecific constructs and controls were added as indicated. At 37 ℃ 5% CO₂After incubation for up to 24 hours, the assay plate is centrifuged at 350g for 5 minutes and the supernatant is transferred to a new deep well 96-well plate for subsequent analysis.

CBA analysis was performed using the following combination of CBA Flex sets according to the manufacturer's instructions for FACS cantonii: human granzyme B (BD 560304), human IFN-. gamma.Flex kit (BD 558269), human TNF Flex kit (BD 558273), human IL-10Flex kit (BD 558274), human IL-6Flex kit (BD 558276), human IL-4Flex kit (BD 558272).

Cytokine release assay with MCSP-CD3 bispecific construct

The following purified bispecific constructs targeting human MCSP and human CD3 were analyzed for their ability to induce T cell-mediated de novo cytokine secretion in the presence (a, B) and absence (C, D) of tumor target cells: fab (MCSP) -Crossfab (CD3) and (scFv)2 (anti-MCSP/anti-huCD 3e) reference molecules.

Briefly, 280 μ l of whole blood from a healthy donor was plated into each well of a deep well 96-well plate. 30,000 Colo-38 tumor target cells expressing human MCSP were added, as well as different bispecific constructs and IgG controls at a final concentration of 1 nM. Cells were incubated at 37 ℃ with 5% CO₂Incubate for 24 hours, then 350Xg centrifugal 5 minutes. The supernatant was transferred to a new deep well 96-well plate for subsequent analysis.

CBA analysis was performed according to the manufacturer's instructions for FACS cantonii using the following combination of CBA Flex sets: human granzyme B (BD 560304), human IFN-. gamma.Flex kit (BD 558269), human TNF Flex kit (BD 558273), human IL-10Flex kit (BD 558274), human IL-6Flex kit (BD 558276), human IL-4Flex kit (BD 558272).

Figure 15 depicts the different cytokine levels measured in the supernatant after 24 hours of treatment with 1nM different CD3-MCSP bispecific constructs (fab (MCSP) -Crossfab (CD3) and (scFv)2 (anti-MCSP/anti-huCD 3 e)) in the presence of (a, B) or absence of (C, D) Colo-38 tumor cells. 280 μ l of whole blood and 30,000 Colo-38 cells were added to each well of a 96-well plate as indicated.

The major cytokine secreted upon T cell activation in the presence of Colo-38 tumor cells is IL-6, followed by IFN γ. In addition, there was a large increase in granzyme B levels upon T cell activation in the presence of target cells. Overall, the (scFv)2 (anti-MCSP/anti-huCD 3e) construct increased levels of TNF and IFN γ and granzyme B slightly more in the presence of target cells (a and B) than the other bispecific constructs.

There was no significant secretion of Th2 cytokines (IL-10 and IL-4) upon activation of T cells by bispecific constructs in the presence (or absence) of target cells.

In this assay, there is also weak secretion of IFN γ induced by the fab (mcsp) -Crossfab (CD3) construct in the absence of target cells.

Cytokine release assay with MCSP-murine CD3 bispecific construct

Purified huMCSP-muCD3 targeted bispecific molecules such as murine Crossfab (CD3) -fab (MCSP) were tested by flow cytometry for its potential to upregulate the late activation marker CD25 on CD8+ T cells in the presence of tumor cells expressing human MCSP.

Briefly, MCSP positive B16/F10-huMCSP Fluc2 clone 48 tumor cells were harvested with cell dissociation buffer, counted and examined for viability. Cells were adjusted to 0.3X10 per ml of RPMI1640 medium (including 1X NEAA, 10mM hepes, 50 μm2-b-ME, 1mM sodium pyruvate)⁶One (viable) cell was added to each well of a round-bottom 96-well plate with a pipette 100. mu.l of this cell suspension (as indicated). 50 μ l (diluted) bispecific construct was added to the wells containing cells to achieve a final concentration of 50 nM. Human murine T effector cells were isolated from splenocytes (C57 BL/6 mice) and adjusted to 3X10 per ml AIM-V medium⁶Individual (viable) cells. 50 μ l of this cell suspension was added to each well of the assay plate (see above) to obtain a final E: T ratio of 10: 1. For analysis, wells containing 50nM of the respective bispecific molecule together with the T effector but no target cells were included if the bispecific construct was able to activate T cells only in the presence of target cells expressing huMCSP.

At 37 ℃ 5% CO₂After 70 hours incubation, cells were centrifuged (5 min, 350 Xg) and washed twice with 150. mu.l/well PBS containing 0.1% BSA.

Surface staining was performed for CD8a (rat IgG2 a; clone 53-6.7; BioLegend # 100712) and CD25 (rat IgG2 b; clone 3C 7; BD # 553075) according to the supplier's recommendations. Cells were washed twice with 150. mu.l/well PBS containing 0.1% BSA and fixed with 100. mu.l/well fixing buffer (BD # 554655) for 15 min at 4 ℃.

After centrifugation, the samples were resuspended in 200. mu.l/well PBS containing 0.1% BSA and analyzed with a FACS CantoII instrument (Software FACS Diva).

Figure 16 shows that the murine Crossfab (CD3) -fab (mcsp) construct induces up-regulation of CD25 only in the presence of target cells.

Example 9: expression of surface activation markers on primary human T cells upon engagement of bispecific constructs

To examine the specific activation of T cells only upon binding of the CD3 bispecific construct in the presence of tumor target cells, primary human PBMCs (isolated as described above) were incubated with the indicated concentrations of the bispecific construct for at least 24 hours in the presence or absence of tumor antigen positive target cells.

Briefly, 3x10 was added to each well of a flat-bottomed 96-well plate containing huMCSP positive target cells (MV-3 tumor cells) or culture medium⁵Primary human PBMC. The final effector to target cell (E: T) ratio was 10: 1. The cells were incubated at 37 ℃ with indicated concentrations of the CD3-MCSP bispecific construct (Fab (MCSP) -Crossfab (CD3) designated "1 +1 Fc-free" and (scFv)2 (anti-MCSP/anti-huCD 3e) reference molecule designated "(scFv) 2") at 5% CO₂Incubate for the indicated incubation time. Effector cells were stained for CD8 and either the early activation marker CD69 or the late activation marker CD25 and analyzed with facscan toti.

The results of this experiment are shown in figure 20.

While there has been shown and described what are at present the preferred embodiments of the invention, it is to be clearly understood that the invention is not limited thereto but may be variously embodied and practiced otherwise within the scope of the appended claims.

Sequence of

While there has been shown and described what is at present the preferred embodiment of the invention, it is to be clearly understood that the invention is not limited thereto but may be variously embodied and practiced otherwise within the scope of the appended claims. Legend: GA201= EGFR conjugate, 3F2= FAP conjugate, CH1A1A = CEA conjugate.

Protein sequences

DNA sequence

While there has been shown and described what is at present the preferred embodiment of the invention, it is to be clearly understood that the invention is not limited thereto but may be variously embodied and practiced otherwise within the scope of the appended claims.

Claims

1. A bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA), comprising a first Fab fragment and a second Fab fragment connected via a peptide linker, wherein either the variable regions of the second Fab heavy and light chain are exchanged or the constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody does not comprise an Fc domain such that the heavy chain constant region consists only of one or more CH1 domains, wherein the peptide linker is a (G4S)2 linker.

2. The bispecific antibody of claim 1, wherein the first Fab fragment comprises at least one antigen binding site specific for a tumor antigen; and the second Fab fragment comprises at least one antigen binding site specific for a T cell activating antigen.

3. The bispecific antibody of claim 1 or 2, wherein the T cell activating antigen is a CD3T cell co-receptor (CD3) antigen.

4. The bispecific antibody of any one of claims 1 to 3, wherein the N-terminus of the second Fab fragment is linked to the C-terminus of the first Fab fragment.

5. The bispecific antibody of any one of claims 1 to 4, further comprising a third Fab fragment.

6. The bispecific antibody of claim 5, wherein the third Fab fragment comprises at least one antigen binding site specific for a tumor antigen.

7. The bispecific antibody of claim 5 or 6, wherein the third Fab fragment is linked to the first Fab fragment.

8. The bispecific antibody of claim 7, wherein the C-terminus of the third Fab fragment is linked to the N-terminus of the first Fab fragment.

9. The bispecific antibody of claim 5 or 6, wherein the third Fab fragment is linked to the second Fab fragment.

10. The bispecific antibody of claim 9, wherein the N-terminus of the third Fab fragment is linked to the C-terminus of the first Fab fragment.

11. The bispecific antibody of any one of claims 5 to 10, wherein the third Fab fragment is linked via a peptide linker.

12. The bispecific antibody of any one of claims 1 to 11, wherein the tumor antigen is selected from the group consisting of: melanoma associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), Fibroblast Activation Protein (FAP) and CD 33.

13. The bispecific antibody of claim 12, wherein the tumor antigen is MCSP.

14. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 13.

15. The bispecific antibody of any one of claims 1 to 13 for use in the treatment of cancer.

16. The bispecific antibody of any one of claims 1 to 13 for use as a medicament.

17. Use of a bispecific antibody according to any one of claims 1 to 13 for the manufacture of a medicament.

18. The use of claim 17, wherein the medicament is for the treatment of cancer.

19. A prokaryotic or eukaryotic host cell comprising a vector comprising a nucleic acid molecule encoding the light chain and the heavy chain of the bispecific antibody of any one of claims 1 to 13.

20. A method of producing an antibody comprising culturing the host cell of claim 19 such that the antibody is produced.