HK1184165B - Cd33 binding agents - Google Patents

Description

CD33 binding agents

Technical Field

The present invention relates to immunotherapy based on the elimination of myeloid cells. In particular, the invention relates to CD33 binding agents for use in such therapies, e.g., in the treatment of myeloid cell malignancies and myelodysplastic syndrome (MDS).

Prior Art

CD33 was identified as a marker for myeloid leukemia in the early 1980 s (Andrews et al, Blood62,24-132,1983). CD33 is a cell surface antigen specifically expressed on myeloid lineage cells, including myeloid leukemia cells. It is the smallest member of the siglec (sialic acid-binding Ig-related lectin) family. CD33 is expressed on early multilineage hematopoietic progenitor cells and bone marrow monocyte precursors. It has no multipotent hematopoietic stem cells (Andrews et al, Journal of Experimental medicinal section 169,1721-1731,1989). It is down-regulated on mature granulocytes but retained on macrophages, monocytes and dendritic cells (Andrews et al, Blood62,24-132,1983). In addition to myelomonocytic cells, CD33 has been shown to be expressed on human mast cells and Blood basophils (Valent et al, Blood15;73(7):1778-85, 1989). Monoclonal antibodies against CD33 are useful for the diagnosis of leukemia and for therapeutic targeting and ex vivo sweeping of bone Marrow for autografting in Acute Myelogenous Leukemia (AML) (Duzkale et al, Biol Blood Marrow Transplant.9(6):364-72, 2003). Initial efforts at therapeutic targeting focused on the development of immunotoxins using anti-CD 33 antibodies conjugated to the toxin ricin. Immunotoxin approaches are apparent because CD33 internalizes rapidly upon binding to an antibody (Audran et al, J Immunol methods.188(1):147-54, 1995).

CD33 is a 67KD transmembrane glycoprotein. The sialic acid binding extracellular domain of CD33 is involved in cell-cell adhesion. Intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) confer cytostatic signals, affecting proliferation and cell survival. The actual signaling pathway of CD33 is poorly understood, but is thought to be involved in the recruitment of ITIM and ITIM-like motifs and tyrosine phosphatases (von Gunten et al, Ann.N.Y.Acad.Sci.1143:61-82,2008). Murine CD33 homologues have been defined, but their functional comparability to human CD33 has been questioned (Brinkman-Van der Linden et al, Mol cell biol.,23(12):4199-206, 2003). The functional role of human CD33 on normal and malignant leukocytes is still unknown.

Several publications have described that CD33 is a stable cell surface marker expressed on primary AML and CML cells in 70% to 100% of test patients (Plesa et al, Cancer112(3),572 + 80,2007; Hauswitt et al, Eur J Clin invest. Jan73-82,2007; Scheinberg et al, Leukemia, Vol.3, 440 + 445, 1989). CD33 is characterized by its ability to self-renew and maintain blood in malignant myeloid progenitor cells (which represent the majority of malignant cells in the peripheral blood and bone marrow of leukemia patients) and leukemic stem cells (i.e., a relatively small number of poorly differentiated cells in bone marrow)At the level of leukemia clones). The clearance of leukemic stem cells is considered to be a key mechanism for sustained tumor-free survival. Immunotoxins targeting CD33(humanized IgG conjugated with toxin chalcone (chalicemicin)₄Antibody) for treating AML patients by delivering their toxic payload to CD 33-positive AML cells (Amadori et al, Cancer Treat rev.34(1):49-60,2008). In phase II clinical trials for the treatment of AML and MDS, initial clinical signs of efficacy from phase I dose escalation studies and reported tolerogenic events were used to evaluate Lintuzumab (Lintuzumab) (SGN-33, HuM195, a "naked" CD 33-specific humanized monoclonal antibody) (Raza et al abstract No. 983, 14)^thEHA Congress, 6 months 4 to 7 days 2009).

Targeting of an AML cell line in vitro with CD 33-specific HuM195 reduces TNF- α -induced secretion of inflammatory cytokines such as IL-8, MCP-1 and RANTES (Sutherland et al, Mabs1:5,481-490, 2009). The relevance of this effect for AML therapy is not known, but modulating the cytokine milieu of the tumor microenvironment may be beneficial for the therapeutic efficacy of the antibody. In addition, antibodies induce antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) of AML cell lines in vitro (Sutherland et al, Mabs1:5,481-490, 2009). ADCC is considered to be a crucial mechanism for the anti-tumor activity of antibodies in hematological malignancies. Data from clinical trials using the CD 20-specific monoclonal antibody Rituximab (Rituximab) show the importance of effector cell-mediated mechanisms for the treatment of B-cell malignancies in response to antibody therapy (Weng and Levy, J ClinOncol.21(21): 3940-E7, 2003).

In summary, it has been shown that the CD33 antigen is expressed on normal cells of the myeloid monocytic lineage and frequently on tumor cells in myeloid leukemia. In phase I trials with antibodies against CD33 (lintuzumab), first signs of clinical efficacy were observed without serious adverse events. However, clinical development of lintuzumab was discontinued after the results of phase II trials in combination with chemotherapy did not produce the desired improvement in efficacy. Thus, there is a clear need to develop improved therapeutic modalities targeting CD 33.

In view of the prior art, there is a need to provide further improved therapies for myeloid cell malignancies and MDS, especially acute myeloid leukemia.

In particular, there is a need to provide further improved antagonistic binding agents against CD33 for the treatment of cancer, in particular AML.

Summary of The Invention

The present invention provides novel CD33 binding agents that bind human CD33, and which are defined by

a) Has a heavy chain variable region comprising CDR1, CDR2 and CDR3 and a light chain variable region comprising CDR4, CDR5 and CDR6, wherein CDR1 has an amino acid sequence selected from SeqID Nos. 1-14, CDR2 has an amino acid sequence selected from SeqID Nos. 14-28, CDR3 has an amino acid sequence selected from SeqID Nos. 29-42, CDR4 has an amino acid sequence selected from SeqID Nos. 43-56, CDR5 has an amino acid sequence selected from SeqID Nos. 57-70, CDR6 has an amino acid sequence selected from SeqID Nos. 71-84, or

b) Recognizes an epitope within the amino acid sequence FFHPIPYYDKNSPVHGYW (SeqIDno:141) of human CD 33.

The invention further provides a CD33 binding agent, wherein the kinetics of internalization of the CD33 binding agent is such that at least 30% (preferably 40%) of the initial amount of antibody remains on the cell surface of HL60 cells 4 hours after incubation.

The invention further provides a CD33 binding agent, wherein the heavy chain variable region comprises an amino acid sequence selected from SeqID Nos. 85-98 and the light chain variable region comprises an amino acid sequence selected from SeqID Nos. 99-112.

The invention further provides CD33 binding agents wherein the heavy chain has an amino acid sequence selected from the group consisting of SeqID No 113-126 and the light chain has an amino acid sequence selected from the group consisting of SeqID No 127-140.

The invention further provides at F_cA CD33 binding agent having a mutation in the domain that enhances ADCC.

More preferred embodiments are outlined in the following description and claims.

It has been found that the CD33 binding agents of the invention have a high affinity for human CD33 and further have favorable internalization kinetics characterized in that the CD33 binding agent can be present long-term upon binding to CD33 on the surface of target cells, which can translate into favorable ADCC activity.

The inventors have also found that the CD33 binding agents of the invention bind to a different epitope of the extracellular domain of CD33 compared to lintuzumab. Without wishing to be bound by any particular theory, it is believed that this is the reason for the different internalization kinetics of the CD 33-binding agents of the present invention and lintuzumab.

Drawings

Figures 1-3 show internalization of exemplary CD33 binding agents of the invention on HL60 cells compared to lintuzumab.

Figure 4 shows the internalization rate of two exemplary antibodies of the invention on HL60 cells compared to lintuzumab.

Figure 5 shows ADCC performance on HL60 cells for two exemplary antibodies of the invention compared to lintuzumab.

Detailed Description

CD33 binding agents

The term "binding agent" as used herein means a protein or peptide that specifically binds to a target antigen. The binding agent can be, for example, an antibody, a derivative of the antibody, or another agent that specifically binds to a target antigen. The binding agent can also be a V comprising an Fv region or a portion thereof (e.g., a V of an antibody that specifically binds a target antigen_HOr V_LOr a CDR). In a preferred embodiment herein, the binding agent is an antibody.

The term "CD 33 binding agent" as used herein refers to a binding agent that specifically binds to CD33, typically a portion of the extracellular domain of human CD 33.

The term "antibody" as used herein refers to (a) immunoglobulin polypeptides and immunologically active portions of immunoglobulin polypeptides (i.e., polypeptides of the immunoglobulin family that contain an antigen binding site that immunospecifically binds to a specific target antigen, or fragments thereof), or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments that immunospecifically bind to a target antigen. Antibodies are generally described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988). The term "antibody" refers to intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit a desired biological activity (e.g., antigen binding). The antibody may be of any class or class (e.g. IgG, IgE, IgM, IgD and IgA) or subclass (IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2), preferably of the IgG class, more preferably of the IgG 1.

An "intact" antibody is one that comprises an antigen-binding variable region, as well as a light chain constant domain and a heavy chain constant domain, depending on the antibody class. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof.

An "antibody fragment" comprises a portion of an antibody, including an antigen binding or variable region or a portion thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And F_vFragment, V_HAnd V_LAntigen binding fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single chain antibodies, scFv-Fc, SMTP, and multispecific antibodies formed from antibody fragments.

"heavy chain variable region" or "V_H"is meant to include CDR1, CDR2 and CDR3 and the surrounding backboneA portion of a heavy chain of a region.

"light chain variable region" or "V_LBy "is meant the portion of the light chain that includes CDR4, CDR5 and CDR6 and the surrounding framework regions.

By "CDR" is meant the hypervariable regions of the heavy and light chains which determine the complementarity/binding specificity of an antibody or antibody fragment. The order of the CDRs in this application is only numerical.

An "epitope" herein means a portion of an antigen that is recognized by an antibody or antibody fragment. In particular, this term refers to the portion of CD33 that is recognized by an antibody.

As used herein, "mAb" refers to a monoclonal antibody.

An antibody may have one or more "effector functions," which refer to those biological activities attributed to the Fc region of the antibody (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include CIq binding; complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LA domain, wherein said domain is present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a peptide at V_HAnd V_LA polypeptide linker between the domains that enables the scFv to form the desired antigen binding structure. For reviews of scFv see Pl ü ckthun, The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, Springer-Verlag, New York, pp.269-315 (1994).

A binding agent, such as an antibody that "is directed against," "binds," or "specifically binds" an antigen of interest (i.e., a target antigen), is a binding agent that is capable of binding the antigen with sufficient affinity such that the binding agent is useful for targeting cells expressing the antigen. Typically, the binding agent is present in an amount of at least about 1X 10⁷M^-1Is bound with affinity, andbinds to the predetermined antigen with an affinity that is at least two times greater than the affinity for binding to non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein).

As used herein, "antibody derivative" refers to an antibody as defined above, which is modified by covalent attachment of a heterologous molecule (e.g., by attachment of a heterologous polypeptide), or by glycosylation, deglycosylation, acetylation, or phosphorylation, or other modifications not normally associated with antibodies. In some embodiments, the heterologous molecule is not a therapeutic agent. In some embodiments, the heterologous molecule does not itself exhibit cytostatic or cytotoxic effects.

Further comprehensive reference to all terms and procedures used herein is Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press; 3 rd edition (1 month 15 days 2001).

CD33 binding agents specifically bind to the receptor CD33 associated with a given target cell population. CD33 is a member of the sialoadhesin family, which is expressed on cells of the hematopoietic lineage (including myeloid precursors, monocytes, macrophages, dendritic cells and mast cells). CD33 is also expressed on tumor cells associated with myeloproliferative or mast cell proliferative diseases, including acute myelogenous leukemia and myelodysplastic syndromes, and leukemia stem cells. Antibodies targeting CD33 and their uses have been described in general (see, e.g., Pierelli et al, 1993, Br. J. Haematol.84: 24-30; Matutes et al, 1985, Hemaiol. Oncol.3: 179-186; Taussig et al, 2005, Blood106: 4086-4092; Florian et al, 2006, Leuk. Lyamph.47: 207-222).

In some embodiments, the CD33 binding agent is an antibody (e.g., a monoclonal antibody). Useful monoclonal antibodies can be a group of antibodies homologous to CD33 (e.g., the extracellular domain of human CD 33). Monoclonal antibodies (mabs) may be made by using any technique known in the art. Including but not limited to initially consisting ofAnd Milstein (1975, Nature256:495-497), the human B-cell hybridoma technology (Kozbor et al, 1983, Immunology Today4:72), and the EBV-hybridoma technology (Cole et al, 1985, Monoclonal Antibodies and Cancer Therapy, AnanR.Liss, pp.77-96). The antibodies may belong to any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo.

Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and functionally active antibody fragments of any one thereof.

Useful CD33 antibodies include antibodies that can achieve therapeutic effects by various mechanisms known in the art, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). For example, antibodies can mediate ADCC by interacting with immune effector cells (e.g., NK cells, monocytes, and macrophages).

Recombinant antibodies (e.g., chimeric and humanized monoclonal antibodies) comprise human and non-human portions, which can be made using standard recombinant DNA techniques. (see, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; and Boss et al, U.S. Pat. No. 4,816,397; both cases are incorporated herein by reference in their entirety). A "humanized antibody" is a non-human species antibody molecule having one or more Complementarity Determining Regions (CDRs) of a non-human species and framework regions of a human immunoglobulin molecule. (see, e.g., Queen, U.S. Pat. No. 5,585,089, the entire disclosure of which is incorporated herein by reference). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using the methods set forth in: international publication No. WO 87/02671; european patent publication No. 0184187; european patent publication No. 0171496; european patent publication No. 0173494; international publication No. WO 86/01533; U.S. Pat. nos. 4,816,567; european patent publication No. 012023; berter et al, 1988, Science240: 1041-; liu et al, 1987, Proc. Natl. Acadset. USA84: 3439-3443; liu et al, 1987, J.Immunol.139: 3521-3526; sun et al, 1987, Proc. Natl. Acad. Sci. USA84: 214-218; nishimura et al, 1987, cancer. Res.47: 999-1005; wood et al, 1985, Nature314: 446-449; shaw et al, 1988, J.Natl.cancer Inst.80: 1553-1559; morrison,1985, Science229: 1202-1207; oi et al, 1986, BioTechniques4: 214; U.S. Pat. nos. 5,225,539; jones et al, 1986, Nature321: 552-525; verhoeyan et al, 1988, Science239: 1534; and Beidler et al, 1988, J.Immunol.141: 4053-4060: each case is incorporated by reference herein in its entirety.

Human monoclonal antibodies can be made by any of a variety of techniques known in the art (see, e.g., Teng et al, 1983, Proc. Nail. Acad. Sci. USA.80: 7308-.

Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but can express human heavy and light chain genes. The transgenic mice are immunized in the normal manner with the selected antigen (e.g., all or a portion of the CD33 polypeptide). Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma techniques. The transgenic mice contain human immunoglobulin transgenes that rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced using this technique. For a review of this technology for the production of human antibodies, see, e.g., Lonberg and Huszar (1995, int. Rev. Immunol.13: 65-93). For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, e.g., U.S. patent No. 5,625,126; 5,633,425 No; U.S. Pat. No. 5,569,825; 5,661,016 No; and U.S. Pat. No. 5,545,806. Other human antibodies are available, for example, from Medarex (Princeton, NJ) which are obtained by immunizing mice.

Fully human antibodies recognizing selected epitopes can also be generated using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody (e.g., a mouse antibody) is used to guide the selection of fully human antibodies that recognize the same epitope. (see, e.g., Jespers et al, 1994, Biotechnology12: 899-. Human antibodies can also be generated using a variety of techniques known In The art, including phage display libraries (see, e.g., Hoogenboom and Winter,1991, J.MoI.biol227:381; Marks et al, 1991, JMoI Biol222:581; quant and Carter,2002, The rise of monoclonal antibodies assay protocols, In Anti-IgE and allogenic diseases, Jardieu and Fick J. editor, Marcel Dekker, New York, NY, Chapter 20, p. 427-469).

Useful antibody fragments include, but are not limited to, F (ab ')2 fragments, Fab' fragments, Fab fragments, Fv, Single Chain Antibodies (SCA) (e.g., as described in U.S. Pat. No. 4,946,778: Bird,1988, Science242: 423-42; Huston et al, 1988, Proc. Natl. Acad. Sci. USA85: 5879-5883; and Ward et al 1989, Nature334: 544-54), scFv-Fc, FvdsFvdsFvs, minibodies (minibody), diabodies, triabodies, tetradiabodies, SMIPs (see, e.g., published U.S. patent application 2005-0238646), and any other molecule that comprises one or more CDRs and has the same specificity as an antibody.

In other embodiments, the antibody is a fusion protein of the antibody linked to another protein, or a functionally active fragment thereof. For example, an antibody or antibody fragment can be fused via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus to an amino acid sequence of another protein (or portion thereof, typically at least 10,20, or 50 amino acid portions of a protein) that is not the antibody or antibody fragment. In some embodiments, the antibody or fragment thereof may be covalently linked to another protein at the C-terminus of the variable or constant domain.

Antibodies can be modified by, for example, covalently linking any type of molecule, so long as the covalent linkage allows the antibody to retain its immunospecificity for binding to an antigen. For example, the derivative of the antibody may have been further modified by: such as glycosylation, deglycosylation, acetylation, pegylation, phosphorylation, amidation, de-effectuation by known protecting/blocking groups (deraelastation), proteolytic cleavage, linkage to another protein, and the like. Any of a variety of chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. In addition, the derivative may contain one or more unnatural amino acid.

In particular embodiments, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. (see, for example, U.S. patent publication Nos. 2006 & 0003412 & 2006 & 0008882). Amino acid sequence variants of the antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and/or substitutions can be made to achieve the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of a site of favorable mutagenesis in an antibody is referred to as "alanine scanning mutagenesis" as described in Cunningham and Wells (1989, Science244: 1081-1085). Here, a residue or group of the target residue (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (typically alanine or polyalanine) to affect the interaction of the amino acid with the antigen. The amino acid position exhibiting functional sensitivity to substitution is then modified by introducing additional or other variants at or against the substitution site. Thus, the nature of the mutation itself need not be predetermined at the time of the site of introduction of the amino acid sequence variant. For example, to analyze the performance of a mutation at a given site, alanine scanning mutagenesis or random mutagenesis is performed at the target codon or region and the expressed antibody variants are screened for the desired activity.

Amino acid sequence insertions can include amino and/or carboxy-terminal fusions (ranging in length from 1 residue to polypeptides containing hundreds 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 or antibodies fused to a cytotoxic polypeptide.

Another type of antibody is an amino acid substitution variant of an antibody. These variants are variants in which at least one amino acid residue in the antibody molecule is replaced by a different residue. The sites of most interest for substitution mutagenesis include hypervariable regions, but framework region alterations are also encompassed.

Substantial modification of the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect in retaining the following characteristics: (a) the structure of the polypeptide backbone in the substituted region, e.g., in a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Natural residues are classified into the following classes according to common side chain properties:

(1) hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilicity: cys, ser, thr;

(3) acidity: asp, glu;

(4) alkalinity: asn, gin, his, lys, arg;

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

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail exchanging a member of one of the classes for another class.

It is desirable to modify the effector function of an antibody, for example, to enhance antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC) of the antibody. In particular, ADCC activity can be enhanced by introducing amino acid mutations in the constant region of the antibody (Lazar et al, PNAS103,11, 4005-. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody, for example, see published U.S. patent application No. 2006-. Alternatively or additionally, cysteine residues may be introduced into the Fc region, thereby forming interchain disulfide bonds in this region. The homodimeric antibody thus produced may have improved internalization capability and/or enhanced CDC and ADCC. (see, for example, Caron et al, 1992, J Exp. Med176: 1191-. Homodimeric antibodies with enhanced anti-tumor activity can also be made using heterobifunctional cross-linkers as described in Wolff et al, 1993, Cancer Research53:2560-2565, or antibodies with dual Fc regions can be engineered, whereby the antibodies have enhanced complement lysis and ADCC capabilities. (see, e.g., Stevenson et al, 1989, Anti-Cancer Drug Design3: 219-230).

Numerous modifications of the Fc region have been proposed in the art both in the scientific literature and in the patent literature, such as EP0307434, WO9304173, WO9734631, WO9744362, WO9805787, WO9943713, WO9951642, WO9958572, WO02060919, WO03074679, WO2004016750, WO2004029207, WO 20040351, WO2004074455, WO2004035752, WO2004099249, WO2005077981, WO2005092925, WO2006019447, WO2006031994, WO2006047350, WO2006053301, WO2006088494 and WO 2007041635.

In a preferred embodiment, the antibody of the invention is an Fc variant having an amino acid substitution at positions 332 and/or 239 and/or 236. In a preferred embodiment, the antibody of the invention has a mutation in the Fc domain selected from the group consisting of

i) A single substitution at position 332, preferably I332E;

ii) a combination of substitutions at positions 239 and 332, preferably S239D/I332E;

iii) a combination of substitutions at positions 236 and 332, preferably G236A/I332E;

iv) combinations of substitutions at positions 236, 239 and 332, preferably G236A/S239D/I332E.

In this context, it is especially preferred to introduce mutations at one or more positions in the Fc domain selected from the amino acids at positions 332 and/or 239 and/or 236, according to the Kabat EU numbering index. Substitutions at positions 239 and 332, especially S239D/I332E, are especially preferred.

The Fc variants in the antibodies of the invention are defined in terms of amino acid modifications that make up them. Thus, for example, I332E is an Fc variant having substitution I332E relative to the parent Fc polypeptide. Likewise, S239D/I332E defines an Fc variant having substitutions S239D and I332E relative to the parent Fc polypeptide, and S239D/I332E/G236A defines an Fc variant having substitutions S239D, I332E and G236A relative to the parent Fc polypeptide.

To extend the serum half-life of the antibody, for example, salvage receptor binding epitopes can be incorporated into the antibody (particularly antibody fragments), as described in U.S. patent No. 5,739,277. The term "salvage receptor binding epitope" as used herein refers to an IgG molecule (e.g., IgG) that is responsible for extending the in vivo serum half-life of the IgG molecule₁、IgG₂、IgG₃Or IgG₄) An epitope of the Fc region.

Antibodies may be glycosylated at conserved positions in their constant regions (see, e.g., Jefferis and Lund,1997, chem. Immunol.65:111-128: Wright and Morrison,1997, TibTECH15: 26-32). The oligosaccharide side chains of immunoglobulins can affect protein function (see, e.g., Boyd et al, 1996, MoI. Immunol.32: 1311-1318; Wittwe and Howard,1990, biochem.29:4175-4180), and intramolecular interactions between parts of the glycoprotein that can affect conformation and provide a three-dimensional surface of the glycoprotein (see, e.g., Jefferis and Lund, supra; Wys and Wagner,1996, Current opin. Biotech.7: 409-416). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on specific recognition structures. For example, it has been reported that in galactosylated IgG the oligosaccharide moiety "flips" out of the internal-C_H2 sterically and terminally located N-acetylglucosamine residues are useful for binding to mannose binding proteins: (See, e.g., Malhotra et al, 1995, Nature Med.1: 237-. Removal of oligosaccharides from CAMPATH-1H (recombinant humanized murine monoclonal IgG1 antibody, which recognizes the CDw52 antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO) cells by a glycosylpeptidase completely reduced complement-mediated lysis (CMCL or CDC) (Boyd et al, 1996, MoI. Immunol.32:131,1-1318), whereas selective removal of sialic acid using neuraminidase did not reduce CMCL. It has also been reported that glycosylation of antibodies can affect ADCC. In particular, CHO cells with tetracycline-regulated expression of β (1.4) -N-acetylglucosaminyltransferase III (GnTIII) (a glycosyltransferase that catalyzes the formation of an aliquot of GIcNAc) have been reported to have improved ADCC activity (see, for example, Umana et al, 1999, Nature Biotech.17: 176-180).

Glycosylation of antibodies is usually N-linked or O-linked. N-linked means that the carbohydrate moiety is attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of the tripeptide sequences in a polypeptide may result in a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid (most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used).

A glycosylation variant of an antibody is a variant that alters the glycosylation pattern of the antibody. Altering means clearing one or more carbohydrate moieties found in the antibody, adding one or more carbohydrate moieties to the antibody, altering the composition of glycosylation (i.e., glycosylation pattern), degree of glycosylation, and the like.

Glycosylation sites can be readily added to an antibody (for N-linked glycosylation sites) by altering the amino acid sequence so that the antibody contains one or more of the above-described tripeptide sequences. Alterations (for O-linked glycosylation sites) may also be achieved by adding (or substituting) one or more serine or threonine residues in the original antibody sequence. Similarly, removal of glycosylation sites can be accomplished by amino acid changes within the native glycosylation sites of the antibody.

Typically, the amino acid sequence is altered by altering the underlying nucleic acid sequence. Such methods include, but are not limited to, isolating a previously prepared variant or non-variant form of the antibody from a natural source (in the case of a natural amino acid sequence variant), or by oligonucleotide-mediated (or site-directed) mutation, PCR mutation, or cassette mutation.

Glycosylation (including glycosylation patterns) of antibodies can also be altered without altering the amino acid sequence or the basic nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell type used to express recombinant glycoproteins (e.g., antibodies) as potential therapeutic agents is rarely a native cell, significant changes in the glycosylation pattern of the antibody can be expected. (see, for example, Hse et al, 1997, biol. chem.272: 9062-9070). In addition to the choice of host cell, factors that influence glycosylation during recombinant production of antibodies also include growth patterns, media formulations, culture density, oxygenation, pH, purification protocols, and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (see, e.g., U.S. Pat. Nos. 5,047,335; 5,510,261; 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from glycoproteins using, for example, endoglycosidase h (endo h). In addition, recombinant host cells can be genetically engineered, for example, to be deficient in the processing of certain types of polysaccharides. Such and similar techniques are well known in the art.

The glycosylation structure of an antibody can be readily analyzed by conventional carbohydrate analysis techniques, including lectin chromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharide composition analysis, sequential enzymatic digestion, and HPAEC-PAD based on charge separation of oligosaccharides using high pH anion exchange chromatography. Methods of releasing oligosaccharides for analytical purposes are also known and include, but are not limited to, enzymatic treatment (typically performed using peptide-N-glycosidase F/endo-beta-galactosidase), elimination of structures that use caustic environments to release primarily O-linkages, and chemical methods of releasing N-linked and O-linked oligosaccharides using anhydrous hydrazine.

Amino acid residues in an antibody may also have modifications (e.g., substitutions, deletions, or additions) that interact with an Fc receptor. In particular, amino acid residues in antibodies may have modifications identified as being involved in the interaction between an anti-Fc domain and the FcRn receptor (see, e.g., international publication No. WO 97/34631).

In its broadest aspect, the present invention relates to CD33 binding agents that bind human CD33, and which are defined by the following definitions

a) Has a heavy chain variable region comprising CDR1, CDR2 and CDR3 and a light chain variable region comprising CDR4, CDR5 and CDR6, wherein CDR1 has an amino acid sequence selected from SeqID Nos. 1-14, CDR2 has an amino acid sequence selected from SeqID Nos. 15-28, CDR3 has an amino acid sequence selected from SeqID Nos. 29-42, CDR4 has an amino acid sequence selected from SeqID Nos. 43-56, CDR5 has an amino acid sequence selected from SeqID Nos. 57-70, CDR6 has an amino acid sequence selected from SeqID Nos. 71-84, or

b) Recognizes an epitope within the amino acid sequence FFHPIPYYDKNSPVHGYW (SeqIDno:141) of human CD 33.

The invention further provides a CD33 binding agent, wherein the kinetics of internalization of the CD33 binding agent is such that at least 30% of the initial amount of antibody remains on the cell surface of HL60 cells 4 hours after incubation.

The CD33 binding agents of the invention have been found to bind different epitopes to lintuzumab, see example 4 herein. Both epitopes (SeqID No:141 and SeqID No:142) are non-overlapping epitopes. It is believed that the different epitopes on the extracellular domain of CD33, which are recognized by the CD33 binding agents of the invention and lintuzumab, are responsible for the differences in the internalization behavior and ADCC performance of the CD33 binding agents of the invention and lintuzumab (see examples 2 and 3 herein).

In a preferred embodiment, at least 40% of the initial amount of CD 33-binding agent remains on the cell surface at 4 hours after incubation.

In a preferred embodiment, the heavy chain variable region comprises an amino acid sequence selected from SeqID Nos. 85-98 and the light chain variable region comprises an amino acid sequence selected from SeqID Nos. 99-112.

In a preferred embodiment, the CD33 binding agent comprises a heavy chain having an amino acid sequence selected from the group consisting of SeqID No 113-126 and a light chain having an amino acid sequence selected from the group consisting of SeqID No 127-140.

In a preferred embodiment, the affinity of the CD33 binding agent for both human CD33 and cynomolgus (cynomolgus) CD33 is K_DLess than or equal to 10 nM.

In a preferred embodiment, the CD33 binding agent is a humanized binding agent.

In a preferred embodiment, the CD33 binding agent is a fully human binding agent.

In a preferred embodiment, the CD 33-binding agent further comprises an effector function.

In a preferred embodiment, the effector function is that of F_cDomain mediation.

In a preferred embodiment, F of a CD33 binding agent_cThe structural domain comprises one or more regulatory F_cMutations in the function of the domains.

In a preferred embodiment, F_cThe modulation of the function of the domain is an ADCC enhancement of at least 10%, preferably 50% or 100%.

Particularly preferred CD33 binding agents of the invention are listed in table 1:

TABLE 1

Cancer treatment

Several publications have described CD33 as a cell surface marker on primary AML and CML cells expressed on malignant cells in 70% to 100% of patients (Scheinberg et al, 1989, hausewirt et al, 2007, Plesa et al, 2007, Webber et al, 2008). CD33 is expressed on malignant myeloid progenitor cells (which represent the majority of malignant cells in the peripheral blood and bone marrow of leukemia patients), and leukemia stem cells (i.e., a relatively small number of poorly differentiated cells in bone marrow characterized by the ability to self-renew and maintain the leukemic lineage level). The clinical feasibility of using antibodies to target CD33 has been derived from(Gemtuzumab ozogamizin, an antibody-calicheamicin conjugate approved for the treatment of relapsed AML patients who are not suitable for other treatment options). An alternative approach to CD33 was the development of lintuzumab (SGN-33, HuM195), a humanized IgG1 monoclonal antibody that showed early signs of efficacy in phase I clinical trials (Raza et al, 2009). In summary, there is a large body of preclinical and clinical data that underscores the relevance and feasibility of targeted treatment of AML and other CD33 positive malignancies with CD 33.

Acute Myeloid Leukemia (AML) is a malignancy of the myeloid lineage of white blood cells. This hematologic tumor is a disease of the blood and bone marrow that is usually fatal within weeks to months if untreated. There were estimated prevalence of 30000 AMLs in the united states and 47000 AMLs in the european union (10-year prevalence data confirmed by Mattson-Jack, 2010). AML is the most prevalent form of adult acute leukemia (about 90%), which comprises about 33% of new leukemia cases. The median age of patients diagnosed with AML was 67 years. AML accounts for about 1.2% of cancer deaths in the united states.

AML causes non-specific symptoms such as weight loss, fatigue, fever and night sweats. AML is diagnosed by hematological testing, bone marrow examination, and laboratory tests to determine the AML subtype and to determine treatment decisions.

The therapy for AML is highly dependent on the age and physical condition of the patient. Patients who can tolerate intensive induction (and subsequent reinforcement and maintenance) of chemotherapy are strongly treated with a combination of cytotoxic drugs. The likelihood of a complete response in the patient is about 75%. In this patient population, the therapeutic goal is cure. In addition, about half of the patients develop AML relapse within the year after a complete response is achieved. The long-term cure rate is within 30 percent.

However, higher age does not allow for intensive induction therapy at the time of co-morbid diagnosis or presence, which leads to palliative therapeutic goals. Thus, the remission rate in elderly patients with AML is significantly reduced. Median survival of elderly patients with AML is less than 6 months.

In one aspect, CD33 binding agents may be used to treat cancer, for example, by delaying cancer progression and/or reducing cancer-associated cachexia, or preventing or delaying the recurrence of a hematologic malignancy (e.g., leukemia) in a mammal, preferably a human patient. The CD33 binding agent can be administered alone or in combination with another therapeutic agent. In some embodiments, the CD33 binding agent is co-administered with a standard of care chemotherapy. The CD33 binding agent may be administered in unconjugated form (i.e., not conjugated to a cytotoxin) or in conjugated form.

In this section, a "patient" is a human or other mammal undergoing treatment for cancer or who has been diagnosed with cancer.

In some embodiments, a CD33 binding agent may be used to delay cancer progression and/or reduce cancer-associated cachexia in a patient by administering an effective dose of a CD33 binding agent to a patient in need thereof. Without being limited to a particular mechanism, the CD 33-binding agent binds to effector or helper cells (e.g., monocytes, macrophages, dendritic cells, and neutrophils) of the myeloid or monocytic lineage, thereby inhibiting or reducing the production of various cytokines, chemokines, and growth factors from the effector or helper cells and/or tumor cells. The cytokines, chemokines and growth factors that promote tumor cell growth and proliferation and/or cause cancer cachexia include, but are not limited to, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-gamma (IFN-gamma), Vascular Endothelial Growth Factor (VEGF), Leukemia Inhibitory Factor (LIF), monocyte chemoattractant protein-1 (MCP-1), RANTES, interleukin-10 (IL-10), interleukin-12 (IL-12), matrix metalloproteinase 2(MMP2), IP-10, and/or macrophage inflammatory protein 1 alpha (MIP1 alpha). CD33 binding agents may also reduce the sites of macrophage migration to tumor cells.

In some embodiments, administration of an effective dose of a CD 33-binding agent to a patient can reduce the level of at least one cytokine, chemokine, or growth factor that promotes the growth and proliferation of tumor cells, promotes migration of non-malignant effector cells (e.g., tumor-associated macrophages (TAMS)) near the tumor site, and/or causes cancer cachexia. In particular embodiments, the cytokine, chemokine, or growth factor is, for example, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-gamma (IFN-gamma), Vascular Endothelial Growth Factor (VEGF), Leukemia Inhibitory Factor (LIF), monocyte chemoattractant protein-1 (MCP-1), RANTES, interleukin-10 (IL-10), interleukin-12 (IL-12), matrix metalloproteinase 2(MMP2), IP-10, and/or macrophage inflammatory protein 1 alpha (MIP1 alpha).

In another embodiment, methods are provided for delaying cancer progression by administering to a patient an effective regime of a CD33 binding agent that specifically binds to CD 33. Due to the administration of the CD33 binding agent, cancer progression is delayed by, for example, reducing the proliferative growth of tumor cells, reducing metastasis, reducing the content of at least one cytokine, chemokine, or growth factor, reducing non-malignant effector cells in the vicinity of tumor cells, and the like.

In another embodiment, a method is provided for reducing tumor burden in a patient by administering to the patient an effective regime of a CD33 binding agent that specifically binds to CD 33. The tumor burden is prevented or reduced as a result of the administration of the CD33 binding agent, for example, by reducing the size or mass of the tumor, decreasing the level of at least one cytokine, chemokine, or growth factor, reducing non-malignant effector cells in the vicinity of the tumor cells, inhibiting the migration of macrophages in the vicinity of the tumor cells, reducing the number of non-malignant effector cells (e.g., TAMS or macrophages) in the tumor, and the like.

In another embodiment, there is provided a method of reducing tumor burden or delaying cancer progression in a patient by administering to the patient an effective regime of a CD33 binding agent that specifically binds to CD 33. Due to the administration of CD33 binding agents, tumor burden in a patient is prevented or reduced by, for example, recruiting immune effector cells (such as NK cells or macrophages or monocytes) that can destroy tumor cells by an immune-mediated mechanism.

Antibody-dependent cellular cytotoxicity (ADCC) is an immune effector cell-mediated mechanism that confers anti-tumor activity to monoclonal antibodies (Weiner GJ. monoclonal antibodies of action in immune Res.2007;39(1-3): 271-8). The relevance of ADCC to anti-tumor efficacy has been demonstrated in preclinical models (e.g., mouse tumor models) (e.g., Clynes RA, Tower TL, PrestalG, Ravetch JV. inhibition Fc receptors modulators of cellular toxicity against human tumor targets of tissues Nat. Med. 2000, 4 months; 6(4): 443-6). Clinical trial data support the relevance of ADCC to the clinical efficacy of therapeutic antibodies (e.g., Weng WK, Levy R Two immunologlobulinG fragment C receptor polymorphism indexes receptor expression torituximab in tissues with follicullar lymphoma. J Clin Oncol. 11/1/2003; 21 (3940-7. Epub 9/15/2003). The interaction of monoclonal antibodies with Fc receptors on immune cells contributes to ADCC. The Fc of an antibody can be modified to exhibit greater affinity for Fc receptors (e.g., Presta LG Engineering of therapeutic antibodies to minizeim specificity and optize function. adv Drug Deliv. 2006, 8/7/2006; 58(5-6):640-56.Epub, 23/5/2006). This stronger affinity for Fc receptors results in enhanced ADCC activity, which may lead to enhanced anti-tumor efficacy in patients.

In various embodiments described in this section, CD 33-binding agents can be used to treat CD 33-positive cancers (i.e., cancers comprising cancer cells that overexpress CD33 on their cell surface or express CD33 to an extent deemed acceptable for therapy with CD33 antibodies). CD33 binding agents can also be used to treat cancers that do not overexpress CD33 on non-malignant effector cells relative to normal tissue of the same type. The cancer can be, for example, a non-hematologic malignancy or a hematologic malignancy. In particular embodiments, the hematologic malignancy can be a CD33 positive tumor and can be, for example, acute lymphoid leukemia, acute myeloid leukemia, chronic myelomonocytic leukemia, erythroleukemia, acute megakaryoblastic leukemia, histiocytic lymphoma, myeloid sarcoma, mast cell proliferative disorder, or myelodysplastic syndrome (MDS). In some embodiments, the hematological malignancy is a CD 33-positive malignancy, such as acute myelogenous leukemia or myelodysplastic syndrome (MDS).

In various embodiments described in this paragraph, the CD33 binding agent can be an unconjugated anti-CD 33 antibody. For example, the antibody may be a fully human antibody, a humanized antibody, or a chimeric antibody, such as a chimeric or humanized M195 antibody. The antibody may also be another antibody, such as an antibody that competes with the M195 antibody for specific binding to CD 33. The antibody may also bind to the same or a different epitope as the M195 antibody.

In other embodiments, the CD 33-binding agent can bind to (i.e., be conjugated to) a cytotoxin. The cytotoxin can be, for example, a peptide toxin such as saporin, ricin, chlorotoxin, pseudomonas exotoxin, pseudomonas endotoxin, or diphtheria toxin. The cytotoxin may also be a chemical (i.e., non-peptidyl) toxin, such as calicheamicin (calicheamicin), doxorubicin (doxorubicin), camptothecin (camptothecin), daunorubicin (daunorubicin), or other DNA binding agent. The cytotoxin may also be an auristatin (auristatin), maytansinoid (maytansinoid), dolastatin (dolastatin), or other microtubule-blocking agent.

A CD33 binding agent that is an anti-CD 33 antibody may be administered to a patient intravenously or subcutaneously at a dose of 0.1mg/kg or less to about 25mg/kg, preferably 1.0mg/kg to about 10 mg/kg. CD33 binding agents that are fragments of anti-CD 33 antibodies or other CD33 binding proteins may be administered at doses equivalent to 0.1mg/kg to about 25mg/kg, 1.0mg/kg to about 10mg/kg of intact antibody. The CD33 binding agent can be administered to the patient intravenously or subcutaneously on a schedule, i.e., in the patient, e.g., daily, weekly, biweekly, every three weeks (i.e., every three weeks), or monthly, or combinations thereof. The CD33 binding agent can be administered for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, or longer, as desired. In some embodiments, the treatment period of the CD33 binding agent described above is followed by a maintenance period, wherein doses of the CD33 binding agent are administered at a lower frequency than during the treatment period. For example, maintenance doses may be administered weekly, biweekly, every three weeks, or monthly for a period of 1 to 6 months. The dose in the maintenance phase may be the same as the dose in the treatment phase.

Treatment in remission of hematologic malignancies

In another aspect, methods are provided for preventing or delaying the recurrence of a hematologic malignancy (e.g., leukemia) in a patient by administering to the patient in remission from the hematologic malignancy an effective dose of a CD33 binding agent, which can prevent or delay substantial hematologic malignancy recurrence. CD33 binding agents specifically bind to CD33 on the surface of hematological malignancies (i.e., leukemia cells) and/or to non-malignant effector cells.

In the context of the present disclosure, a "patient" is typically a human that has undergone treatment for a hematological malignancy or has been diagnosed as having a hematological malignancy. In some embodiments, the hematologic malignancy is a CD 33-positive hematologic malignancy. Hematological malignancies include, but are not limited to, leukemias (e.g., Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), chronic myelomonocytic leukemia, hairy cell leukemia). Related hematological disorders include, but are not limited to, myelodysplastic syndrome (MDS), myelofibrosis, myeloproliferative diseases (e.g., polycythemia vera (PV, PCV, or PRV), Essential Thrombocythemia (ET)), and light chain amyloid diseases.

The term "CD 33-positive hematologic malignancy" refers to a hematologic malignancy characterized by expression of CD33 on the surface of malignant cells. CD33 positive hematologic malignancies include, but are not limited to, Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), chronic myelomonocytic leukemia, platelet leukemia, myelodysplastic syndrome, myeloproliferative disorders, refractory anemia, pre-leukemic syndrome, lymphoid leukemia, or undifferentiated leukemia.

In some embodiments, the methods comprise administering to a patient in remission from a CD 33-positive hematological malignancy an effective regime of a CD33 binding agent, thereby preventing or delaying recurrence of the hematological malignancy. In some embodiments, the patient lacks detectable cells of a hematological malignancy. As used herein, "lack of detectable cells" is determined by standard diagnostic or prognostic methods. Patients in remission from AML typically exhibit regression of the abnormal clinical features, restoration of normal blood counts and normal hematopoiesis in the bone marrow (<5% blast, neutrophil count >1,000-. See, for example, The Merck Manual, sec.11, ch.138 (17 th edition, 1997): estey,2001, Cancer92(5): 1059-.

The CD 33-binding agent can be, for example, an antibody that specifically binds CD33 and the hematologic malignancy can be Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), chronic myelomonocytic leukemia, thymic (thymic) leukemia, myelodysplastic syndrome, myeloproliferative disorders, refractory anemia, pre-leukemic syndrome, lymphoid leukemia, or undifferentiated leukemia.

In some embodiments, the patient in remission from a hematological malignancy does not undergo a bone marrow transplant. In other embodiments, the patient in remission from a hematological malignancy has undergone a bone marrow transplant. The bone marrow transplant may be an autologous bone marrow transplant or an allogeneic bone marrow transplant.

The antibodies of the invention are particularly suitable for treating the following cancer types:

bone marrow cancers, including but not limited to: acute lymphoblastic leukemia "ALL", acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia "AML", acute promyelocytic leukemia "APL", acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute undifferentiated leukemia, chronic myelogenous leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple myeloma.

Acute and chronic leukemias that are amenable to treatment with CD33 binding agents include: lymphoblasts, myeloid lines, lymphocytes, myeloid leukemia, and platelet leukemia. Other myelodysplastic syndromes, myeloproliferative disorders, refractory anemia, pre-leukemic syndromes, lymphoid leukemias, or undifferentiated leukemias can be treated with CD33 binding agents.

In combination with other active substances

Depending on the condition to be treated, the CD 33-binding agents of the invention may be used alone or in combination with one or more other therapeutic agents, especially selected from DNA damaging agents, DNA demethylating agents or tubulin-binding agents, or therapeutically active compounds that inhibit angiogenesis, signal transduction pathways or cancer cell mitotic checkpoints or have immunomodulatory functions (IMIDs).

The additional therapeutic agent may be administered concurrently with the CD33 binding agent (optionally as a component of the same pharmaceutical formulation), or before or after the CD33 binding agent.

In certain embodiments, the additional therapeutic agent may be, but is not limited to, one or more inhibitors selected from the EGFR family, VEGFR family, VEGF, IGF-1R, insulin receptor, AuroraA, AuroraB, PLK, and PI3 kinases, FGFR, PDGFR, Raf, KSP, or PDK 1.

Other examples of other therapeutic agents are inhibitors of CDK, Akt, Src, Bcr-Abl, cKit, cMet/HGF, Her2, Her3, c-Myc, Flt3, HSP 90; (ii) a hedgehog antagonist; inhibitors of JAK/STAT, Mek, mTor, NF κ B, proteasome, Rho; an inhibitor of Wnt signaling or an inhibitor of Notch signaling or ubiquitination pathway.

Other examples of other therapeutic agents are inhibitors of DNA polymerase, topoisomerase II; multiple tyrosine kinase inhibitors, CXCR4 antagonists, IL3RA inhibitors, RAR antagonists, KIR inhibitors, immunotherapeutic vaccines, TUB inhibitors, Hsp70 inducers, IAP family inhibitors, DNA methyltransferase inhibitors, TNF inhibitors, ErbB1 receptor tyrosine kinase inhibitors, multi-kinase inhibitors, JAK2 inhibitors, RR inhibitors, apoptosis inducers, HGPRTase inhibitors, histamine H2 receptor antagonists, and CD25 receptor agonists.

Examples of Aurora inhibitors are, but are not limited to, PHA-739358, AZD-1152, AT-9283, CYC-116, R-763, VX-667, MLN-8045, PF-3814735, SNS-314, VX-689, GSK-1070916, TTP-607, PHA-680626, MLN-8237, BI847325, and ENMD-2076.

Examples of PLK inhibitors are GSK-461364, BI2536 and BI 6727.

Examples of RAF inhibitors are BAY-73-4506 (also a VEGFR inhibitor), PLX-4032, RAF-265 (also a VEGFR inhibitor), sorafenib (also a VEGFR inhibitor), XL-281, Nevavar (also a VEGFR inhibitor), and PLX 4032.

Examples of KSP inhibitors are iparib (ispinesib), ARRY-520, AZD-4877, CK-1122697, GSK-246053A, GSK-923295, MK-0731, SB-743921, LY-2523355, and EMD-534085.

Examples of src and/or bcr-abl inhibitors are dasatinib (dasatinib), AZD-0530, bosutinib (bosutinib), XL-228 (also IGF-1R inhibitors), nilotinib (nilotinib) (also PDGFR and cKit inhibitors), imatinib (imatinib) (also cKit inhibitors), NS-187, KX2-391, AP-2457 (also EGFR, FGFR, Tie2, Flt3 inhibitors), KM-80 and LS-104 (also Flt3, Jak2 inhibitors).

An example of a PDK1 inhibitor is AR-12.

An example of a Rho inhibitor is BA-210.

Examples of PI3 kinase inhibitors are PX-866, PX-867, BEZ-235 (also an mTor inhibitor), XL-147, and XL-765 (also an mTor inhibitor), BGT-226, CDC-0941.

Examples of cMet or HGF inhibitors are XL-184 (also a VEGFR, cKit, Flt3 inhibitor), PF-2341066, MK-2461, XL-880 (also a VEGFR inhibitor), MGCD-265 (also a VEGFR, Ron, Tie2 inhibitor), SU-11274, PHA-665752, AMG-102, AV-299, ARQ-197, MetMAb, CGEN-241, BMS-777607, JNJ-38877605, PF-4217903, SGX-126, CEP-17940, AMG-458, INCB-028060, and E-7050.

An example of a c-Myc inhibitor is CX-3543.

Examples of Flt3 inhibitors are AC-220 (also cKit and PDGFR inhibitors), KW-2449, LS-104 (also bcr-abl and Jak2 inhibitors), MC-2002, SB-1317, lestaurtinib (also VEGFR, PDGFR, PKC inhibitors), TG-101348 (also JAK2 inhibitors), XL-999 (also cKit, FGFR, PDGFR and VEGFR inhibitors), sunitinib (also PDGFR, VEGFR and cKit inhibitors), and tandutinib (also PDGFR and cKit inhibitors).

Examples of HSP90 inhibitors are tanespimycin (tanespimamycin), adriamycin (alvespimycin), IPI-504, STA-9090, MEDI-561, AUY-922, CNF-2024 and SNX-5422.

Examples of JAK/STAT inhibitors are CYT-997 (also interacting with tubulin), TG-101348 (also Flt3 inhibitors), and XL-019.

Examples of Mek inhibitors are ARRY-142886, AS-703026, PD-325901, AZD-8330, ARRY-704, RDEA-119, and XL-518.

Examples of mTor inhibitors are sirolimus (temsirolimus), deforolimus (which also acts as a VEGF inhibitor), everolimus (which is also a VEGF inhibitor), XL-765 (which is also a PI3 kinase inhibitor), and BEZ-235 (which is also a PI3 kinase inhibitor).

Examples of Akt inhibitors are perifosine (perifosine), GSK-690693, RX-0201, and triciribine (triciribine).

Examples of cKit inhibitors are masitinib, OSI-930 (also acting as a VEGFR inhibitor), AC-220 (also as Flt3 and PDGFR inhibitors), tandutinib (also as Flt3 and PDGFR inhibitors), axitinib (also as VEGFR and PDGFR inhibitors), sunitinib (also as Flt3, PDGFR, VEGFR inhibitors) and XL-820 (also acting as VEGFR and PDGFR inhibitors), imatinib (also as a bcr-abl inhibitor), nilotinib (also as a bcr-abl and PDGFR inhibitor).

Examples of hedgehog antagonists are IPI-609, CUR-61414, GDC-0449, IPI-926 and XL-139.

Examples of CDK inhibitors are celecoxib (seliciclib), AT-7519, P-276, ZK-CDK (which also inhibits VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509, PHA-848125, and SCH-727965.

Examples of proteasome inhibitors are bortezomib (bortezomib), carfilzomib (carfilzomib) and NPI-0052 (also NF κ B inhibitors).

Examples of proteasome inhibitors/inhibitors of the NF-. kappa.B pathway are bortezomib (bortezomib), carfilzomib (carfilzomib), NPI-0052, CEP-18770, MLN-2238, PR-047, PR-957, AVE-8680 and SPC-839.

An example of an inhibitor of the ubiquitination pathway is HBX-41108.

Examples of demethylating agents are 5-azacitidine (5-azacitidine) and decitabine (decitabine).

Examples of anti-angiogenic agents are inhibitors of FGFR, PDGFR and VEGF (R) and thalidomide (thalidomide) selected from, but not limited to, bevacizumab (bevacizumab), motesanib (motesanib), CDP-791, SU-14813, tiratinib (telatinib), KRN-951, ZK-CDK (also a CDK inhibitor), ABT-869, BMS-690514, RAF-265, IMC-KDR, IMC-18F1, IMiD, thalidomide, CC-4047, lenalidomide (lenalidomide), ENMD-0995, IMC-56363, Ki-23057, brivarnib (brivanib), cediranib (cediranib), 1B3, CP-868596, IMC-3G3, R-3 (also kic 2 inhibitor), sunitinib (also kic 3), and Flt (also Flt inhibitor), and also kifitinib (thlidinib inhibitor), also Flt 1530, and also bevacizumab inhibitor (also being inhibitors of Bt), and PKI-3, and Zd-493-3, Vatalanib (vatalanib), tandutinib (also Flt3 and cKit inhibitors), pazopanib (pazopanib), PF-337210, aflibercept (aflibercept), E-7080, CHIR-258, sorafenib tosylate (also Raf inhibitor), vandetanib (vandetanib), CP-547632, OSI-930, AEE-788 (also EGFR and Her2 inhibitors), BAY-57-9352 (also Raf inhibitor), BAY-73-4506 (also Raf inhibitors), XL-880 (also cMet inhibitors), XL-647 (also EGFR and EphB4 inhibitors), XL-820 (also cKit inhibitors), nilotinib (also cKit and brc-abl inhibitors), CYT-116, PTC-299, BMS-584622, CEP-11981, dovitinib (dovitinib), CY-2401401, ENMD-2976 and BIBF 1120.

The additional therapeutic agent may also be selected from an EGFR inhibitor, which may be a small molecule EGFR inhibitor or an anti-EGFR antibody. Examples of anti-EGFR antibodies are, but are not limited to, cetuximab (cetuximab), panitumumab (panitumumab), nimotuzumab (nimotuzumab), zatuzumab (zalutumumab); examples of small molecule EGFR inhibitors are gefitinib (gefitinib), erlotinib (erlotinib), vandetanib (also VEGFR inhibitors) and afatinib (also Her2 inhibitors). Another embodiment of an EGFR modulator is an EGF fusion toxin.

Other EGFR and/or Her2 inhibitors that may be used in combination with the CD33 binding agents of the invention are lapatinib (lapatinib), trastuzumab (trastuzumab), pertuzumab (pertuzumab), XL-647, neratinib (neratinib), BMS-599626ARRY-334543, AV-412, mAB-806, BMS-690514, JNJ-26483327, AEE-788 (also a VEGFR inhibitor), AZD-8931, ARRY-380ARRY-333786, IMC-11F8, Zemab, TAK-285, AZD-4769, and afatinib (a dual inhibitor of Her2 and EGFR).

DNA polymerase inhibitors that may be used in combination with the CD33 binding agents of the invention are Ara-C/cytarabine (cytarabine), Clolar/chlorophenazine (cloffarabine).

DNA methyltransferase inhibitors that may be used in combination with the CD33 binding agents of the present invention are Vidaza/azacitidine.

An apoptosis-inducing agent that may be used in combination with the CD33 binding agents of the present invention is Trisenox/arsenic trioxide.

Topoisomerase II inhibitors that may be used in combination with the CD33 binding agents of the invention are idarubicin (idarubicin), daunorubicin and mitoxantrone (mitoxantrone).

A RAR antagonist that may be used in combination with the CD33 binding agents of the present invention is Vesanoid/vitamin a acid (tretinoin).

The HGPRTase inhibitors that may be used in combination with the CD33 binding agents of the present invention are Mercapto/mercaptopurine (mercaptoprine).

The histamine H2 receptor antagonist that may be used in combination with the CD33 binding agents of the present invention is Ceplene/histamine dihydrochloride.

A CD25 receptor agonist that may be used in combination with the CD33 binding agents of the present invention is IL-2.

Other drugs may also be selected from drugs that target the IGF-1R and insulin receptor pathways. The drugs include antibodies that bind IGF-1R (e.g., CP-751871, AMG-479, IMC-A12, MK-0646, AVE-1642, R-1507, BIIB-022, SCH-717454, rhu Mab IGFR) and novel chemical entities that target the kinase domain of IGF1-R (e.g., OSI-906 or BMS-554417, XL-228, BMS-754807).

Other drugs that may be used in therapy in advantageous combination with the CD33 binding agents of the invention are molecules targeting CD20, including CD 20-specific antibodies (such as rituximab, LY-2469298, ocrelizumab (ocrelizumab), MEDI-552, IMMU-106, GA-101(= R7159), XmAb-0367, ofatumumab), radiolabeled CD20 antibodies (such as tositumumab (tositumumab) and ibritumomab (ibritumomab tixetan)) or other CD 20-directed proteins (such as SMIP Tru015, PRO-131921, FBT-a05, vetuzumab (tuvelzumab), R-7159).

The CD33 binding agent may be combined with inhibitors of other surface antigens expressed on leukocytes, in particular antibodies or antibody-like molecules, such as anti-CD 2 (cetilizumab), anti-CD 4 (zanolimumab), anti-CD 19(MT-103, MDX-1342, SAR-3419, XmAb-5574), anti-CD 22 (epratuzumab), anti-CD 23 (lumiximab), anti-CD 30 (imatumumab), anti-CD 32B (MGA-321), anti-CD 38(HuMax-CD38), anti-CD 40(SGN40), anti-CD 52 (alemtuzumab), anti-CD 80 (galiximab)).

Other drugs to be combined with the CD33 binding agent are immunotoxin-like BL-22 (anti-CD 22 immunotoxin), inotuzumab ozogamicin (anti-CD 23 antibody-calicheamicin conjugate), rft5.dga (chain a anti-CD 25 ricin toxin), SGN-35 (anti-CD 30-auristatin E conjugate), and gemumab (anti-CD 33 calicheamicin conjugate), MDX-1411 (anti-CD 70 conjugate), or radiolabeled antibodies (e.g., 90Y-eperisomab) (anti-CD 22 radioimmunoconjugate).

In addition, the CD33 binding agents may be combined with immunomodulators, drugs that induce apoptosis or modify signal transduction pathways (e.g., antibodies), such as the TRAIL receptor modulators mapatumab (mapatumumab) (TRAIL-1 receptor agonist), lexatuzumab (lexatuzumab) (TRAIL-2 receptor agonist), tegafuzumab (tigatuzumab), Apomab, AMG-951, and AMG-655; anti-HLA-DR antibodies (e.g., 1D09C3), anti-CD 74, osteoclast differentiation factor ligand inhibitors (e.g., denosumab), BAFF antagonists (e.g., AMG-623a), or Toll-like receptor (e.g., TLR-4 or TLR-9) agonists.

Other drugs that may be used in combination with the CD33 binding agents of the invention are selected from, but are not limited to, hormones, hormone analogs and anti-hormones (e.g., tamoxifen (tamoxifen), toremifene (toremifene), raloxifene (raloxifene), fulvestrant (fulvestrant), megestrol acetate (megestrol acetate), flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), cyproterone acetate (cyproterone acetate), finasteride (finasteride), buserelin acetate (buserelin acetate), fludrocortisone (fluucortestinone), fluoxymesterone (fluoroxymesterone), medroxyprogesterone (medroxyprogesterone), oxyprogesterone (oxyphenoxifenesin), oxyprogesterone (oxyphenoxifenethazine)/oxyphenoxifenesin (oxyphenoxipride), oxyphenoxipride (oxyphenoxifenesin), oxyphenoxipride (oxytropamide), oxyphenoxipride), oxyphenoxine (oxytropamide), oxyphenoxipride), oxyphenoxides (oxytropamide), ketoxipride (oxytropamide (ketoxipride), ketolide (oxytropamide), ketoxipride, ketoxipridotropine, ketoxipride, Dexamethasone (dexamethasone), aminoglutethimide (amiglutethimide)), aromatase inhibitors (e.g., anastrozole (anastrozole), letrozole (letrozole), liazole (liarozole), exemestane (exemestane), atamestane (atamestane), formestane (formestane)), LHRH agonists and antagonists (e.g., goserelin acetate (goserelin acetate), leuprolide (letrolide), abarelix (abarelix), cetrorelix (cetrorelix), deslorelin (deslorelin), histrelin (histrelin), triptorelin (triptorelin), antimetabolites (e.g., methotrexate (methotrexate), trimetrexate (trimetrexate), megestrol (meperidine), 5-fluorouracil (5-desouracil), 5-doxidine (5-doxetadine), 5-doxycycline (5-azalide), gemcitabine (5-azalide), and analogs of doxidine (doxamide), doxylamine (5-and doxylamine (doxylamine, doxine, and analogs of these, Purine and adenosine analogues such as mercaptopurine, thioguanine (thioguanine), azathioprine (azathioprine), cladribine (cladribine) and pentostatin (pentostatin), cytarabine, fludarabine (fludarabine), clofazine, etc.; antitumor antibiotics (e.g., anthracyclines such as doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C, bleomycin, actinomycin D (dactinomycin), plicamycin, spicamycin, actinomycin-D (actimycin D), mitoxantrone idarubicin, pixantrone, streptozocin, adriamycin); platinum derivatives (e.g., cisplatin, oxaliplatin (oxaliplatin), carboplatin (carboplatin), lobaplatin (lobaplatin), satraplatin (satraplatin)); alkylating agents (e.g., estramustine (estramustine), semustine (semustine), mechlorethamine (meclorethamine), melphalan (melphalan), chlorambucil (chlorambucil), busulfan (busulphan), dacarbazine (dacarbazine), cyclophosphamide (cyclophophamide), ifosfamide (ifosfamide), hydroxyurea (hydroxyurea), temozolomide (temozolomide), nitrosoureas (nitrosoureas), thiotepa (thiotepa), such as carmustine (carmustine) and lomustine (lomustine)); antimitotic agents (e.g., vinca alkaloids such as vinblastine (vinblastine), vindesine (vindesine), vinorelbine (vinorelbine), vinflunine (vinflunine) and vincristine (vinchristine), and taxanes (taxanes) such as paclitaxel (paclitaxel), docetaxel (docetaxel) and formulations thereof, larotaxel (larotaxel), simetaxel (simotaxel) and epothilones such as ixabepilone (ixabepilone), patupilone (patupilone), ZK-EPO); topoisomerase inhibitors (e.g. epipodophyllotoxins (epipodophyllotoxins), such as etoposide (etoposide) and albopicos (etoposides), teniposide (teniposide), amsacrine (amsacrine), topotecan (topotecan), irinotecan (irinotecan), banohydroquinone (banoxantrone), camptothecin) and various chemical agents, such as retinoic acid derivatives, amifostine (amifostine), anagrelide (anagrelide), interferon alpha, interferon beta, interferon gamma, interleukin-2, procarbazine (procarbazine), N-methylhydrazine, mitotane (mitotane), and poriferine (porfimer), bexarotene (bexatene), celecoxib, ethylenimine/methyl-melamine, triethylenemelamine, triethylenethiomine, hexamethyl-amidase, hexamethyl, and L-amidase (L), methidadone (mebendazole), and various chemical agents such as retinoic acid derivatives, carnosine (irinotecan), carnosine (bane), and (sulfamethoxazole), interferon beta, interferon gamma, interleukin-2, and (mitoxanilide), and (amicarbazide), preferably, Desmethyl etherol azole (desmethyisonidazole), pimonidazole (pimonidazole), etanidazole (etanidazole), nitroazol-pholine (nimorazole), RSU1069, EO9, RB6145, SR4233, nicotinamide (nicotinamide), 5-bromodeoxyuridine (5-bromodeoxyuridine), 5-iododeoxyuridine (5-iododeoxyuridine), bromodeoxycytidine (bromodeoxycytidine), erythrohydroxynonyladenine (erythrohydroxyxynonyl-adenine), anthracenedione (anthracenedione), GRN-163L (competitive telomerase template antagonist), SDX-101 (SDX agonist), Tabostat (talbostat) (inhibitor), furazolidine (forodesine) (inhibitor), apraximin (apraximin) (antisense inhibitor), caspase (apremicin) (antisense inhibitor), antisense endostatin (TNF- α receptor (PPAR) 2 (antisense thereto), alkaline receptor (TNF-2) and neutralizing agent (alkaline receptor) (7-52), TNF- α -2 (antisense thereto), and alkaline receptor (alkaline receptor) (alkaline receptor antagonist), and (alkaline receptor antagonist for TNF-2, alkaline receptor (alkaline receptor) and alkaline receptor (alkaline receptor) for TNF-2, alkaline receptor (alkaline receptor, alkaline receptor (alkaline receptor) for binding, alkaline receptor for binding, olbaccarat (obatoclax) (Bcl2 inhibitor), nizastaurin (PKC beta modulator), Vorinostat (vorinostat) (HDAC inhibitor), Romidepsin (HDAC inhibitor), AT-101(Bcl-2/Bcl-xL inhibitor), pridopeptide neo (plipidemic peptide), SL-11047 (polyamine metabolism modulator).

The CD 33-binding agents of the invention can also be used in combination with other therapies, including surgery, stem cell transplantation, radiotherapy, endocrine therapy, biological response modifiers, hyperthermia and cryotherapy, and drugs that reduce any adverse effects (e.g., antiemetics), G-CSF, GM-CSF; such as hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives, Npe6, tin protoporphyrin (tinoteporphyrin), pheeosporide-a bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine, and the like.

Pharmaceutical compositions and methods of administration

The CD33 binding agent can be in any form that allows for administration of the composition to a patient. For example, the composition may be in solid or liquid form. The preferred mode of administration is parenteral by infusion or injection (intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of administration, such as inhalation, transdermal, intranasal, buccal, oral, and intratumoral, may also be employed. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal (intrastemal) injections or infusion techniques. In one aspect, the composition is administered parenterally. In yet another aspect, the compound is administered intravenously.

The pharmaceutical compositions can be formulated such that the compounds are bioavailable when the compositions are administered to a patient. The composition may take the form of one or more dosage units, for example, wherein a container of the compound in aerosol form may contain a plurality of dosage units.

The materials used to prepare the pharmaceutical compositions can be non-toxic in the amounts employed. It will be apparent to those skilled in the art that the optimum dosage of the active ingredient in the pharmaceutical composition will depend on a number of factors. Relevant factors include, but are not limited to, the type of patient (e.g., human), the particular form of the compound, the mode of administration, and the composition used.

The pharmaceutically acceptable carrier or vehicle can be particulate, such that the composition is in powder form, for example. The carrier can be a liquid, where the composition is, for example, an injectable liquid. The composition may be in liquid form, for example, for parenteral injection. In compositions for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, and isotonic agents may also be included.

The liquid composition, whether liquid, suspension, or other similar form, may also include one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils (e.g., synthetic mono-or diglycerides which may be used as a solvent or suspending medium), polyethylene glycols, glycerol, cyclodextrins, propylene glycol or other solvents; stabilizers, such as amino acids; surfactants such as polysorbates; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting isotonicity, such as sodium chloride or dextrose. Parenteral compositions may be enclosed in ampoules (disposable pumps or multi-dose vials made of glass, plastic or other material). Physiological saline is an exemplary adjuvant. The injectable compositions are preferably sterile.

The CD33 binding agent may also be dried (freeze-dried, spray-freeze-dried, dried by means of near-critical or supercritical gases, vacuum-dried, air-dried), precipitated or crystallized or encapsulated in microcapsules prepared in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), macroemulsions by, for example, coacervation techniques or interfacial polymerization using, for example, hydroxymethylcellulose or gelatin and poly- (methylmethacrylate), respectively, or precipitated or immobilized on a carrier or surface, for example, by pcmc techniques (protein-coated microcrystals). The technique is disclosed in Remington, the science and Practice of Pharmacy, 21 st edition (Ed. Hendrickson R.).

The amount of the composition that is effective to treat a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help determine the optimal dosage range. The exact dosage employed in the composition will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

The composition comprises an effective amount of the drug or medicament such that a suitable dosage is obtained. Typically, this amount is at least about 0.01% of the drug or agent by weight of the composition. When intended for oral administration, this amount may range from about 0.1% to about 80% by weight of the composition. In one aspect, the oral composition may comprise from about 4% to about 50% of the compound, by weight of the composition. In yet another aspect, the compositions of the present invention are prepared such that the parenteral dosage unit contains from about 0.01% to about 2% by weight of the compound.

For intravenous administration, the composition may comprise from about 1mg to about 50mg of the drug or agent per kg of patient body weight. In one aspect, the composition can include from about 1mg, 1.5mg, or 2.5mg to about 50mg of drug or agent per kg of patient body weight. In another aspect, the amount administered can range from about 1mg, 1.5mg, or 2.5mg to about 25mg of drug or agent per kg of body weight.

In some embodiments, the dose administered to the patient is from less than 0.1mg/kg to about 50mg/kg of the patient's body weight. (conversion to mg/mm)²When it is used, 1.8m²BSA and 80kg body weight).

As discussed herein, CD33 binding agents can be administered to patients intravenously or subcutaneously on a schedule such as daily, weekly, biweekly, every three weeks, or monthly. For example, the CD33 binding agent may be administered weekly for a period of 2 to 10 weeks, typically 3 to 6 weeks. In some embodiments, the dosing regimen of the CD33 binding agent maintains a serum concentration of the antibody of at least 5 μ g/ml or at least 10 μ g/ml during the dosing cycle. CD33 binding agents may be administered, for example, for 1 to 8 or more cycles. In some embodiments, the CD33 binding agent is administered chronically to the individual.

For example, the invention includes methods of treating cancer (e.g., myeloid leukemia) by weekly administration of 0.1mg/kg to 50mg/kg, e.g., about 1.5-8 or 2.5-8mg/kg of an anti-CD 33 antibody of the invention. This treatment may generally last for about 1 to 3 months, usually about 2 months. In one embodiment, the dosing schedule is maintained until a sharp decrease is noted. For example, administration may last up to about 6 months. This treatment may be followed by a less frequent dosing schedule, including, for example, dosing every two weeks (or twice a month). This administration schedule may be maintained for 1, 2, 3,4, 5,6 months or longer to maintain acute reduction and/or remission.

In some embodiments, a prophylactic drug may be administered with a CD33 binding agent to minimize infusion reactions. Suitable prophylactic agents include, for example, methylprednisolone (methylprednisone), diphenhydramine (diphenhydramine), acetaminophen (acetaminophen), or other suitable agents. The prophylactic agent may be administered prior to or concurrently with the CD33 binding agent.

The drug or agent may be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or cutaneous mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration may be systemic or local. Various delivery systems are known, e.g., encapsulated in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer the compounds. In certain embodiments, more than one drug or agent or composition is administered to the patient.

Depending on the need for the drug or agent, it may be desirable to administer one or more drugs or agents or compositions locally to the area in need of treatment. This may be achieved by (e.g., and without limitation) local infusion during surgery; topical application after surgery (e.g., in conjunction with wound dressings); by injection: by means of a catheter; by means of suppositories; or by means of an implant of porous, non-porous or gel-like material, including membranes or fibers such as silastic membranes. In one embodiment, administration can be achieved by direct injection at the site (or pre-site) of the cancer, tumor, or neoplastic or pre-neoplastic tissue.

The drug or medicament or composition may be delivered by a controlled release system such as a pump or various polymeric materials. In yet another embodiment, the controlled Release system may be placed in proximity to the target of the drug or pharmaceutical agent or composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of controlled Release, Vol.2, pp.115-138 (1984)). Other controlled release systems may be used as discussed in the review by Langer (1990, Science249: 1527) -1533).

Depending on the drug or medicament desired, the drug or medicament is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to an animal, particularly a human. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic buffered aqueous solution. The composition may also include a solubilizing agent, if desired. Compositions for intravenous administration may optionally include a local anesthetic (e.g., lidocaine (lignocaine)) to relieve pain at the maintenance of the injection.

The compositions of the therapeutic agents may also be administered in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs depending on the dosage form being received. Orally administered compositions may contain one or more optional agents (e.g., sweetening agents, such as fructose, aspartame or saccharin, flavoring agents, such as peppermint, oil of wintergreen, or cherry, coloring agents, and preserving agents) to provide pharmaceutically palatable preparations. Further, if in tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding osmotically active driver compounds (drivingcompounds) are also suitable for use in orally administered drugs or medicaments. In these latter platforms, fluid from the environment surrounding the capsule is drawn up by the drive compound, which expands to transfer the drug or pharmaceutical composition through the aperture. The delivery platform can provide a substantially zero order delivery profile compared to the peaked profile of an immediate release formulation. Time delay materials such as glyceryl monostearate or glyceryl stearate may also be used.

The compositions may include various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms an envelope around the active ingredient. The material forming the coating is typically an inert material and may be selected from, for example, sugar, shellac, or other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

The composition can be administered to a patient in need thereof at a frequency or for a duration of time determined by the attending physician. The composition can be administered over a period of 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 1 month, 2 months, or longer. It is understood that the composition may be administered over any period of time between 1 day and two months or longer.

Production of antibodies

Antibodies can be produced using any method used for the synthesis of antibodies, in particular, for example, by recombinant expression or chemical synthesis.

Recombinant expression of an antibody or fragment or derivative thereof typically involves construction of a nucleic acid encoding the antibody. If the nucleotide sequence of the antibody is known, the nucleic acid encoding the antibody or polypeptide thereof can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al, 1994, BioTechniques17: 242), which include synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and subsequent amplification of the ligated oligonucleotides by, for example, PCR.

Alternatively, a nucleic acid molecule encoding an antibody or polypeptide thereof can be generated from a suitable source. If a pure line containing nucleic acid encoding a particular antibody cannot be obtained, but the sequence of the antibody is known, the nucleic acid encoding the antibody can be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cell expressing an immunoglobulin) by, for example, PCR amplification using synthetic primers that hybridize to the 3 'and 5' ends of the sequence or by cloning using oligonucleotide probes specific for a particular gene sequence.

If Antibodies specifically recognizing a particular antigen are not available (or the source of the cDNA library used to clone the nucleic acid encoding the immunoglobulin is not available), Antibodies specific for the particular antigen can be generated by any method known in the art (e.g., by immunizing a patient), or suitable animal models (e.g., rabbits or mice) to generate polyclonal Antibodies, or more preferably by generating Monoclonal Antibodies, as described, for example, by Kohler and Milstein (1975, Nature256: 495: 497), or as described by Kozbor et al (1983, Immunology Today4:72) or Cole et al (1985, Monoclonal Antibodies and cDNA Therapy, Alan R.Liss corporation, pp 77-96). Alternatively, clones encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries for clones that bind Fab fragments of a particular antigen (e.g., as described in Huse et al, 1989, Science246, 1275-1281) or by screening antibody libraries (e.g., see Clackson et al, 1991, Nature352: 624; Hane et al, 1997, Proc. Natl. Acad. Sci. USA94: 4937).

After obtaining the nucleic acid sequence encoding at least the variable domain of the antibody, it can be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody (see, for example, International publication No. WO 86/05807; WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors are available that contain an intact light or heavy chain, and thus can express an intact antibody molecule. Subsequently, the antibody-encoding nucleic acid can be used to introduce nucleotide substitutions or deletions necessary to substitute (or delete) one or more variable region cysteine residues involved in the intrachain disulfide bond with a thiol-free amino acid residue. Such modifications can be effected by any method known in the art for introducing specific mutations or deletions into a nucleotide sequence, such as, but not limited to, chemical mutagenesis and in vitro directed mutagenesis (see, e.g., Hutchinson et al, 1978, J.biol. chem.253: 6551).

In addition, techniques for generating "chimeric antibodies" have been developed (see, e.g., Morrison et al, 1984, Proc. Natl. Acad. Sci. USA81: 851-855; Neuberger et al, 1984, Nature312: 604-608; Takeda et al, 1985, Nature314: 452-454). Chimeric antibodies are molecules whose different parts are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region (e.g., humanized antibodies).

After obtaining the nucleic acid sequence encoding the antibody, vectors for producing the antibody can be produced by recombinant DNA techniques using techniques known in the art. Expression vectors containing antibody-encoding sequences and appropriate transcriptional and translational control signals can be constructed as known to those skilled in the art. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in: sambrook et al (1990, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Sambrook et al, 2001; Molecular Cloning, A Laboratory Manual, 3 rd edition, Cold Spring Harbor, N.Y.) and Ausubel et al (eds., 1993-2006, Current protocols in Molecular Biology, John Wiley & Sons, NY).

Expression vectors comprising the antibody nucleotide sequences or antibody nucleotide sequences can be transferred to host cells by conventional techniques (e.g., electroporation, liposomal mobilization, calcium phosphate precipitation, or transduction), and the resulting cells are subsequently cultured by conventional techniques to produce the antibodies. In particular embodiments, expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

Especially for the expression of recombinant immunoglobulin molecules, the host cell for the expression of the recombinant antibody may be a bacterial cell, such as e.g.E.coli (Escherichia coli), or preferably a eukaryotic cell. In particular, the combination of mammalian cells (e.g., Chinese hamster ovary Cells (CHO)) with vectors containing the major mid-early Gene promoter component from human cytomegalovirus is an efficient expression system for immunoglobulins (see, e.g., Foecking et al, 1986, Gene45: 101; Cockett et al, 1990, Biotechnology8: 2). The CHO cell line may be, for example, DG44 or CHO-S. In another embodiment, antibodies can be expressed using the CHEF system. (see, for example, U.S. patent No. 5,888,809).

A variety of other host-expression vectors can be employed to express the antibody. The host expression system represents a vehicle that can produce and subsequently purify the coding sequence of an antibody, but also represents cells that can express an antibody immunoglobulin molecule in situ when transformed or transfected with the appropriate nucleotide coding sequence. Such systems include, but are not limited to, microorganisms (e.g., bacteria, such as e.g., e.coli and bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces pichia (Saccharomyces pichia)) transformed with a recombinant yeast expression vector containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing immunoglobulin coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmids); or mammalian cell systems (e.g., COS, CHO-S, BH, 293T or 3T3 cells) with recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinovirus 7.5K promoter).

In bacterial systems, a variety of expression vectors may be advantageously selected depending on the intended use of the expressed antibody. For example, where large quantities of the protein are to be produced, it may be desirable to direct expression of vectors which facilitate purification of the fusion protein product in large quantities. Such vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al, 1983, EMBO J.2:1791-94) in which the antibody coding sequence can be ligated into the vector individually and in frame with the lac Z coding region, thereby allowing the production of a fusion protein; pIN vector (Inouye and Inouye, 1985, Nucleic Acids Res.13: 3101-3109; Van Heeke and Schuster, 1989, J.biol.chem.24: 5503-5509); and so on. pGEX vectors are also useful for expressing foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, the fusion protein is soluble and readily purified from lysed cells by: adsorbed and bound to glutathione-agarose bead matrix, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites so that the selected target gene product can be released from the GST moiety.

In the insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) or similar virus from Drosophila melanogaster (Drosophila melanogaster) can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda (Spodoptera frugiperda) cells. Antibody coding sequences can be individually cloned into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a variety of viral-based expression systems can be employed. In the case of using an adenovirus as an expression vector, the antibody coding sequence of interest can be linked to an adenovirus transcription/translation control complex (e.g., late promoter and tripartite leader sequence). This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertions in non-essential regions of the viral genome (e.g., regions E1 or E3) result in recombinant viruses that are viable and capable of expressing immunoglobulin molecules in infected hosts. (see, e.g., Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA81: 355-359). Specific initiation signals may also be required in order to efficiently translate the inserted antibody coding sequence. The signal includes the ATG initiation codon and adjacent sequences. In addition, the initiation codon is operably linked to the reading frame of the desired coding sequence to ensure complete translation of the insert. The exogenous translational control signals and initiation codons can have a variety of natural and synthetic origins. Expression efficiency can be enhanced by the inclusion of appropriate transcription enhancer components, transcription terminators, and the like (see, e.g., Bittner et al, 1987, Methods in enzymol.153: 51-544).

In addition, the host cell strain may be selected to regulate the expression of the inserted sequence, or to modify and manipulate the gene product in a particular manner as desired. Such modifications (e.g., glycosylation) and treatment (e.g., cleavage) of the protein product may be important for the function of the protein. Different host cells have unique and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with cellular mechanisms for the appropriate processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO (e.g., DG44 or CHO-S), VERY, BH, HeIa, COS, MDCK, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578 Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody can be engineered. Rather than using an expression vector containing the origin of viral replication, a host cell can be transformed with DNA under the control of appropriate expression control components (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, the engineered cells can be grown in enriched media (enriched media) for 1 to 2 days and then switched to selective media. The selectable marker of the recombinant plasmid can confer resistance to this selection and allow the cell to stably integrate the plasmid into its chromosome and grow, forming a cell that can then be colonized and expanded into a foci of a cell line. This method can be advantageously used to engineer antibody-expressing cell lines. The engineered cell lines are particularly useful for the screening and evaluation of tumor antigens that interact directly or indirectly with antibodies.

A variety of selection systems may be used. For example, herpes simplex virus thymidine kinase (see, e.g., Wigler et al, 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (see, e.g., Szybalska and Szybalski, 1992, Proc. Natl Acad. Sci. USA48:202), and adenine phosphoribosyltransferase (see, e.g., Lowy et al, 1980, Cell22:817) genes may be used in tk-, hgprt-, or aprt-cells, respectively. Similarly, antimetabolite resistance can be used as a basis for selection of the following genes: DHFR which confers resistance to methotrexate (see, e.g., Wigler et al, 1980, Proc. Natl. Acadsi. USA77: 3567-70; O' Hare et al, 1981, Proc. Natl. Acad. Sci. USA78: 1527-31); gpt, which confers mycophenolic acid resistance (see, e.g., Mulligan and Berg, 1981, proc. Nail. Acad. Sci. USA78: 2072-76); neo, which confers resistance to the aminoglycoside G-418 (see, e.g., Clinical Pharmacy12: 488-505; Wu and Wu, 1991, Biotherapy3: 87-95; Tolstoshev,1993, arm. Rev. Pharmacol. Toxicol.32: 573-596; Mullgan, 1993, Science260: 926-932; Morgan and Anderson,1993, Ann. Rev. biochem.62: 191-217; and May,1993, TIB TECH11(5):155-215) and hygro, which confer resistance to hygromycin (see, e.g., Santerre et al, 1984, Gene30: 147-50). Methods well known in the art of recombinant DNA technology that can be used are described in the following references: ausubel et al (eds., 1993 + 2006, Current Protocols Molecular biology. John Wiley & Sons, NY; Kriegler,1990, Gene Transfer and expression. A laboratory Manual, Stockton Press, NY; and chapters 12 and 13, Dracopoli et al (eds., 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; and Colberre-Garapin et al, 1981,7.MoI.biol.150: 1-14).

The degree of antibody expression can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vector based on gene amplification for The expression of cloned genes in mammalian cells in DNA cloning, Vol.3 (Academic Press, New York, 1987)). Where the marker in the vector system expressing the antibody is amplifiable, increasing the amount of inhibitor present in the culture of the host cell increases the copy number of the marker gene. The yield of antibody can also be increased due to the association of the amplified region with the nucleotide sequence of the antibody (see, e.g., Crouse et al, 1983, mol. cell. biol.3: 257-66).

The host cell may be co-transfected with two expression vectors, a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. Both vectors may contain the same or different selectable markers that allow for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used to encode both heavy and light chain polypeptides. In this case, the light chain is usually placed before the heavy chain to avoid an excess of toxic free heavy chain (see, e.g., Proudfoot,1986, Nature322: 562-65; Kohler,1980, Proc. Natl. Acad. Sci. USA77: 2197-9). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

After recombinant expression of the antibody, it may be purified using any method suitable for purifying the antibody, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly protein a affinity chromatography for a specific antigen), and molecular sieve (sizing) column chromatography), centrifugation, differential solubilization, or by any other standard technique for purifying proteins.

A comprehensive reference to all steps used to prepare Monoclonal Antibodies of the invention is Yokoyama et al, "Production of Monoclonal Antibodies", Current Protocols in Immunology, Unit2.5, 2006.

Examples

Example 1, affinity for CD33

Affinity K of CD33 binding agents for human and cynomolgus monkey CD33 in cell lines HL60 and HEK 293-cynomolgus monkey CD33_DRespectively, 10nM or less.

14 CD33 binding agents (fully human monoclonal antibodies as listed in Table 1 as numbers 1 to 14, respectively) against human and cynomolgus CD33 were identified by FACS Scatchard analysis (Scatchard analysis) on CD33 expressing cells (AML-derived HL60 cell line, recombinant HEK 293-cynomolgus CD33 cell line), as described by Brockhoff et al (cytometric. 1994, 9/1; 17(1): 75-83). Briefly, CD33 binder dilutions (80. mu.l) were prepared in 96-well plates starting at 100-400nM in the first well, followed by 11 dilution steps (1:2,40+ 40. mu.l). Add 50. mu. lCD33 dilution of binding agent to FACS tubes and 150. mu.l cells (0.8X 10) per FACS tube⁶/ml=1.2×10⁵Individual cells/tube). The cells were mixed gently and incubated on ice for 1 hour. Thereafter, 50. mu.l of FITC-conjugated secondary antibody (concentration 15. mu.g/ml; mouse mAb anti-human IgG) was added, mixed and incubated on ice for 30 minutes. Thereafter 4ml of PBS containing 0.02% acid (ph7.2) were added, the cells were pelleted and resuspended in 300. mu.l PBS (pH7.2) and analyzed using BD FACS Canto. All experimental steps were performed on wet ice and all CD33 binder dilutions were in PBS/0.5% BSA +0.02% acidAnd (4) preparing. FACS correction was performed using Quantum FITC MESF (Premix) beads (Bangs laboratories). All specimens were measured using the same FACS parameters. The ratio of bound IgG to free IgG was calculated from MFI values at different CD33 binder concentrations and plotted as scatchard plots. The regression line is drawn through the resulting data points, and the slope of this line corresponds to the negative value of the association constant. The results are listed in table 2.

TABLE 2

Example 2: kinetics of internalization

Internalization of an antibody refers to a decrease in the amount of antibody/antigen complex on the cell surface of a target cell after incubation with the antibody. Internalization assays were performed using a HL60 cell line expressing CD 33. Cells were incubated with a fixed amount of CD33 binding agent (10 μ g/ml of fully human monoclonal antibodies numbered 1 to 14 as listed in table 1) at 37 ℃ for a predetermined period of time (0 hours, 1 hour, 4 hours, 24 hours) to internalize the antibody/antigen complex. At the indicated time points, acid was added to the incubation mixture to prevent further internalization. Thereafter, a fixed amount of CD33 binding agent was added to saturate all CD33 antigenic sites on the cell surface. The total amount of CD33 binding agent bound to the cell surface was determined by FACS analysis using FITC conjugated anti-human IgG secondary antibodies. The time point 0 hours was used to determine the initial level of CD33 antibody/antigen complex on the surface and was defined as 100%. The results are listed in table 3 and are shown in fig. 1-3.

TABLE 3

Lintuzumab was included as a reference antibody. According to the published data, the lintuzumab/CD 33 complex internalizes rapidly upon lintuzumab binding. After a4 hour incubation period, only about 20% of the initial amount of CD 33/lintuzumab complex remained on the cell surface. It could demonstrate that, surprisingly, the internalization of all 14 CD 33-binding agents of the invention slowed down compared to lintuzumab.

Example 3: ADCC Activity

The slowing of the rate of internalization translates into an enhancement of ADCC activity in vitro. To assess the effect of slowed internalization on ADCC activity of CD33 binding agents (fully human monoclonal antibodies numbered 1 through 14 as listed in table 1), target cells (HL60) were incubated with CD33 binding agents for 0 hours, 1 hour, 4 hours, and 24 hours. ADCC assays were then performed using IL-2 stimulated PBMC as effector cells and antibody coated HL60 cells as target cells. For all experiments, a mAb concentration of 30. mu.g/ml was used. Effector cells were co-cultured in quadruplicate or triplicate with target cells in 96-well round-bottom microtiter plates in the presence of CD33 binding agent, using a final volume of 200 μ Ι/well of assay medium consisting of 10% human serum and 1% BSA (1: 1 ratio between them) in RPMI. First, effector cells (freshly isolated PBMC cells, placed in 10% human serum (100 μ l/well) in RPMI) were plated, followed by target cells and CD33 binder solution (diluted in 1% BSA (50 μ l) in RPMI). As controls, effector cells were cultured in assay medium alone (effector cell control) and target cells were cultured in assay medium alone (spontaneous lysis) or in assay medium supplemented with 1% triton x-100 (maximal lysis). The CO-cultures were incubated at 37 ℃ for 3 hours in a wet CO2 incubator. At the end of the incubation, the incubated cells were removed from the medium by centrifugation (200 Xg, i.e., 1000 rpm; 10 minutes) at room temperature. Cell-free supernatants (100. mu.l/well) were transferred to corresponding wells of a 96-well flat-bottom plate. To determine LDH activity in the supernatant, 100 μ l of the reaction mixture (250 μ l of catalyst freshly mixed with 11.25ml of dye solution) was added to each well and incubated at room temperature in the dark for 30 minutes. The absorbance was then measured as described below.

ADCC activity was measured using a cytotoxicity detection kit (LDH; Roche 11644793001). The detection of cytotoxicity is based on the measurement of LDH enzyme activity released from plasma membrane-damaged cells. LDH released into culture supernatant reduces the tetrazolium salt of the kit to formazan(formalzan). Measurement of formazan at 490nm against a reference wavelength of 650nm in an ELISA plate readerMaximum absorption of the dye. To determine the percent cell-mediated cytotoxicity, the mean absorbance in quadruplicates or triplicates was calculated and the background was subtracted. Substituting the corrected value into the following equation to calculate ADCC (%):

(effector/target cell mixture-effector cell control-spontaneous release) divided by (maximum release-spontaneous release)

ADCC activity at time point 0 hours (antibody pre-incubation without target cells) was defined as 100% ADCC activity. ADCC activity was calculated for different time points of antibody pre-incubation relative to time point 0 hours and expressed as relative cytotoxicity (%).

The slowed internalization of CD33 binding agents compared to lintuzumab results in enhanced ADCC activity compared to lintuzumab. In summary, the slowed internalization leads to enhanced ADCC activity. Internalization of the CD33 binding agent is inversely correlated with ADCC activity of the CD33 binding agent, which may indicate an advantage with respect to the clinical activity of the CD33 binding agent. The results for internalization kinetics in this experiment are depicted in figure 4 and the results for ADCC activity are depicted in figure 5.

Example 4: mapping of epitopes (mapping)

The binding epitope of the CD33 binding agents described herein relative to the epitope of lintuzumab was determined by hydrogen exchange mass spectrometry (HXMS).

The method is used for determining the amide skeleton hydrogen and D of the CD33 protein₂Susceptibility to O exchange. The experiment was performed using recombinant CD33 protein alone and CD33 protein (hereinafter referred to as "antibody" in this example) supplemented with CD33 binder/lintuzumab. The region of the CD33 protein that showed significant anti-exchange protection due to binding antibody was thereby identified. By determining the resolution of the method using peptides produced by pepsin digestion, for example, the resulting amino acid sequence may be larger than the actual epitope of the antibody. The CD 33-derived peptides were identified by other control experiments using unchanged samples using standard accurate mass and HPLC MS/MS techniques.

For the protein + antibody samples, the CD33 protein and antibody were incubated for 15 minutes at room temperature. The final molar ratio of antibody/CD 33 was 2: 1. Using the LEAP robot system (exchange plate at 25 degrees C, sample/quenching plate at 4 degrees C) to 80L exchange buffer (10mM NaH)₂PO₄In D₂O, pH =7 or 10mM NaH₂PO₄In H₂O, pH =7), 8 μ l of the sample was added, mixed, and exchanged at 25 ℃ for various times (15, 60, 120, 240, and 600 seconds). Then 80. mu.l of this solution was transferred to 80. mu.l of quench buffer (1M urea, 0.1M TCEP-HCl) at 4 ℃ and mixed. Subsequently 90. mu.l of this solution were transferred to 10. mu.l of pepsin (4mg/ml) at 4 ℃ and mixed. After 2 min, 60 μ l of this solution was injected onto a Michrom C18 trap column. Subjecting the column to H₂O +0.1% TFA was washed at 100. mu.l/min for 2 min. The valve was then switched and the column was eluted onto a Phenomenex Jupiter C5 column (1.0X 50mm, 5 μm, 300A). Mobile phase a was water/acetonitrile/TFA (99/0.95/0.05) and mobile phase B was acetonitrile/water/TFA (95/4.95/0.05). The flow rate was 100. mu.l/min. The gradient is: 0 minute (0% B), 6 minutes (40% B), 7 minutes (40% B), 8 minutes (90% B), 10 minutes (90% B), 11 minutes (0% B). The LEAP system pre-cooled the mobile phase to 4 ℃ and also maintained the trap and analytical columns at 4 ℃. For MS experiments (for quantification and D)₂Exchange of O buffer) for 14 minutes at 60,000 resolution using the 300-2000 single scan method. For MS/MS experiments (for identification)And H₂O buffer exchanged peptides) 7 scans were used for 14 minutes. The first scan is a full range scan of 300-2000 at 30,000 resolution. The subsequent scans were CID scans of the 6 strongest ions from scan 1. The isolation width was 1.5amu, the collision energy was 35V, and the activation time was 30 msec. Pepsin peptides were identified using the fragment data and the program protocol discover (Thermo). The identified peptides were analyzed using an internal program that calculated the average mass of the exchanged peptides.

All CD33 binding agents protected the same peptide fragment with amino acid sequence FFHPIPYYDKNSPVHGYW (SeqID No:141) (Table 4). The sequence of CD33 protected by the CD33 binding agents described herein is different from and does not overlap with the peptide sequence of CD33 protected by binding lintuzumab (MDPNFWLQVQE, SeqID No: 142). In computer modeling using the crystal structure of SIGLEC-5, SIGLEC family members homologous to CD33 revealed binding epitopes for all antibodies in the proximal domain of the protein, wherein the binding epitope for lintuzumab is different from the binding epitope for the CD33 binding agent described herein. In summary, the CD33 binding agents described in the present patent application bind to different epitopes than lintuzumab.

Numbering	Pure series ID number	CD33 epitope
			1	280-03-08	FFHPIPYYDKNSPVHGYW
2	280-21-09	FFHPIPYYDKNSPVHGYW
			3	280-29-12	FFHPIPYYDKNSPVHGYW
4	280-31-01	FFHPIPYYDKNSPVHGYW
			5	280-31-01(mut)	FFHPIPYYDKNSPVHGYW
6	280-34-02	FFHPIPYYDKNSPVHGYW
			7	280-50-01	FFHPIPYYDKNSPVHGYW
8	280-50-01(mut)	FFHPIPYYDKNSPVHGYW
			9	280-61-07	FFHPIPYYDKNSPVHGYW
10	283-03-03	FFHPIPYYDKNSPVHGYW
			11	283-05-01	FFHPIPYYDKNSPVHGYW
12	283-07-03	FFHPIPYYDKNSPVHGYW
			13	283-11-03	FFHPIPYYDKNSPVHGYW
14	283-14-01	FFHPIPYYDKNSPVHGYW
			15	Lintuzumab	MDPNFWLQVQE

TABLE 4

Claims (49)

1. A CD33 binding agent that binds human CD33, which CD33 binding agent is an antibody or antibody derivative that specifically binds to an epitope within the amino acid sequence FFHPIPYYDKNSPVHGYW (SeqID No:141) of human CD33, wherein said CD33 binding agent is selected from the group consisting of

An antibody comprising CDR1 of SeqID No:1, CDR2 of SeqID No:15, CDR3 of SeqID No:29, CDR4 of SeqID No:43, CDR5 of SeqID No:57 and CDR6 of SeqID No:71,

an antibody comprising CDR1 of SeqID No:2, CDR2 of SeqID No:16, CDR3 of SeqID No:30, CDR4 of SeqID No:44, CDR5 of SeqID No:58 and CDR6 of SeqID No:72,

an antibody comprising CDR1 of SeqID No:3, CDR2 of SeqID No:17, CDR3 of SeqID No:31, CDR4 of SeqID No:45, CDR5 of SeqID No:59 and CDR6 of SeqID No:73,

an antibody comprising CDR1 of SeqID No:4, CDR2 of SeqID No:18, CDR3 of SeqID No:32, CDR4 of SeqID No:46, CDR5 of SeqID No:60 and CDR6 of SeqID No:74,

an antibody comprising CDR1 of SeqID No. 5, CDR2 of SeqID No. 19, CDR3 of SeqID No. 33, CDR4 of SeqID No. 47, CDR5 of SeqID No. 61 and CDR6 of SeqID No. 75,

an antibody comprising CDR1 of SeqID No. 6, CDR2 of SeqID No. 20, CDR3 of SeqID No. 34, CDR4 of SeqID No. 48, CDR5 of SeqID No. 62 and CDR6 of SeqID No. 76,

an antibody comprising CDR1 of SeqID No. 7, CDR2 of SeqID No. 21, CDR3 of SeqID No. 35, CDR4 of SeqID No. 49, CDR5 of SeqID No. 63 and CDR6 of SeqID No. 77,

an antibody comprising CDR1 of SeqID No. 8, CDR2 of SeqID No. 22, CDR3 of SeqID No. 36, CDR4 of SeqID No. 50, CDR5 of SeqID No. 64 and CDR6 of SeqID No. 78,

an antibody comprising CDR1 of SeqID No. 9, CDR2 of SeqID No. 23, CDR3 of SeqID No. 37, CDR4 of SeqID No. 51, CDR5 of SeqID No. 65 and CDR6 of SeqID No. 79,

an antibody comprising CDR1 of SeqID No. 10, CDR2 of SeqID No. 24, CDR3 of SeqID No. 38, CDR4 of SeqID No. 52, CDR5 of SeqID No. 66 and CDR6 of SeqID No. 80,

an antibody comprising CDR1 of SeqID No. 11, CDR2 of SeqID No. 25, CDR3 of SeqID No. 39, CDR4 of SeqID No. 53, CDR5 of SeqID No. 67 and CDR6 of SeqID No. 81,

an antibody comprising CDR1 of SeqID No. 12, CDR2 of SeqID No. 26, CDR3 of SeqID No. 40, CDR4 of SeqID No. 54, CDR5 of SeqID No. 68 and CDR6 of SeqID No. 82,

an antibody comprising CDR1 of SeqID No. 13, CDR2 of SeqID No. 27, CDR3 of SeqID No. 41, CDR4 of SeqID No. 55, CDR5 of SeqID No. 69 and CDR6 of SeqID No. 83,

antibodies comprising CDR1 of SeqID No. 14, CDR2 of SeqID No. 28, CDR3 of SeqID No. 42, CDR4 of SeqID No. 56, CDR5 of SeqID No. 70 and CDR6 of SeqID No. 84.

2. A CD33 binding agent that binds human CD33, which CD33 binding agent is an antibody or antibody derivative that specifically binds to an epitope within the amino acid sequence FFHPIPYYDKNSPVHGYW (SeqID No:141) of human CD33, wherein said CD33 binding agent is selected from the group consisting of

An antibody comprising the heavy chain variable region of SeqID No:85 and the light chain variable region of SeqID No:99,

an antibody comprising the heavy chain variable region of SeqID No:86 and the light chain variable region of SeqID No:100,

an antibody comprising the heavy chain variable region of SeqID No:87 and the light chain variable region of SeqID No:101,

an antibody comprising the heavy chain variable region of SeqID No:88 and the light chain variable region of SeqID No:102,

an antibody comprising the heavy chain variable region of SeqID No. 89 and the light chain variable region of SeqID No. 103,

an antibody comprising the heavy chain variable region of SeqID No:90 and the light chain variable region of SeqID No:104,

an antibody comprising the heavy chain variable region of SeqID No. 91 and the light chain variable region of SeqID No. 105,

an antibody comprising the heavy chain variable region of SeqID No:92 and the light chain variable region of SeqID No:106,

an antibody comprising the heavy chain variable region of SeqID No:93 and the light chain variable region of SeqID No:107,

an antibody comprising the heavy chain variable region of SeqID No:94 and the light chain variable region of SeqID No:108,

an antibody comprising the heavy chain variable region of SeqID No:95 and the light chain variable region of SeqID No:109,

an antibody comprising the heavy chain variable region of SeqID No:96 and the light chain variable region of SeqID No:110,

an antibody comprising the heavy chain variable region of SeqID No:97 and the light chain variable region of SeqID No:111,

an antibody comprising the heavy chain variable region of SeqID No:98 and the light chain variable region of SeqID No: 112.

3. A CD33 binding agent that binds human CD33, which CD33 binding agent is an antibody or antibody derivative that specifically binds to an epitope within the amino acid sequence FFHPIPYYDKNSPVHGYW (SeqID No:141) of human CD33, wherein said CD33 binding agent is selected from the group consisting of

An antibody comprising the heavy chain of SeqID No:113 and the light chain of SeqID No:127,

an antibody comprising the heavy chain of SeqID No:114 and the light chain of SeqID No:128,

an antibody comprising the heavy chain of SeqID No:115 and the light chain of SeqID No:129,

an antibody comprising the heavy chain of SeqID No:116 and the light chain of SeqID No:130,

an antibody comprising the heavy chain of SeqID No:117 and the light chain of SeqID No:131,

an antibody comprising the heavy chain of SeqID No:118 and the light chain of SeqID No:132,

an antibody comprising the heavy chain of SeqID No:119 and the light chain of SeqID No:133,

an antibody comprising the heavy chain of SeqID No:120 and the light chain of SeqID No:134,

an antibody comprising the heavy chain of SeqID No:121 and the light chain of SeqID No:135,

an antibody comprising the heavy chain of SeqID No:122 and the light chain of SeqID No:136,

an antibody comprising the heavy chain of SeqID No:123 and the light chain of SeqID No:137,

an antibody comprising the heavy chain of SeqID No:124 and the light chain of SeqID No:138,

an antibody comprising the heavy chain of SeqID No:125 and the light chain of SeqID No:139,

an antibody comprising the heavy chain of SeqID No:126 and the light chain of SeqID No: 140.

4. The CD33 binding agent of claim 1, wherein the kinetics of internalization of the CD33 binding agent is such that at least 30% of the initial amount of the CD33 binding agent is retained on the cell surface of HL60 cells 4 hours after incubation.

5. The CD33 binding agent of claim 2, wherein the kinetics of internalization of the CD33 binding agent is such that at least 30% of the initial amount of the CD33 binding agent is retained on the cell surface of HL60 cells 4 hours after incubation.

6. The CD33 binding agent of claim 3, wherein the kinetics of internalization of the CD33 binding agent is such that at least 30% of the initial amount of the CD33 binding agent is retained on the cell surface of HL60 cells 4 hours after incubation.

7. The CD33 binding agent of claim 1, wherein the kinetics of internalization of the CD33 binding agent is such that at least 40% of the initial amount of the CD33 binding agent is retained on the surface of HL60 cells 4 hours after incubation.

8. The CD33 binding agent of claim 2, wherein the kinetics of internalization of the CD33 binding agent is such that at least 40% of the initial amount of the CD33 binding agent is retained on the surface of HL60 cells 4 hours after incubation.

9. The CD33 binding agent of claim 3, wherein the kinetics of internalization of the CD33 binding agent is such that at least 40% of the initial amount of the CD33 binding agent is retained on the surface of HL60 cells 4 hours after incubation.

10. The CD33 binding agent of claim 1, wherein the CD33 binding agent has a K to both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

11. The CD33 binding agent of claim 2, wherein the CD33 binding agent has a K to both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

12. The CD33 binding agent of claim 3, wherein the CD33 binding agent has a K to both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

13. The CD33 binding agent of claim 4, wherein the CD33 binding agent has a K to both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

14. The CD33 binding agent of claim 5, wherein the CD33 binding agent has a K for both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

15. The CD33 binding agent of claim 6, wherein the CD33 binding agent has a K for both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

16. The CD33 binding agent of claim 7, wherein the CD33 binding agent has a K for both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

17. The CD33 binding agent of claim 8, wherein the CD33 binding agent has a K for both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

18. The CD33 binding agent of claim 9, wherein the CD33 binding agent has a K to both human CD33 and cynomolgus monkey CD33_DAn affinity of less than or equal to 10 nM.

19. The CD33 binding agent of any one of claims 1-18, wherein the CD33 binding agent is a humanized binding agent.

20. The CD33 binding agent of any one of claims 1-18, wherein the CD33 binding agent is a fully human binding agent.

21. The CD33 binding agent of any one of claims 1-18, wherein the CD33 binding agent further comprises an effector function.

22. The CD33 binding agent of claim 19, wherein the CD33 binding agent further comprises an effector function.

23. The CD33 binding agent of claim 20, wherein the CD33 binding agent further comprises an effector function.

24. The CD33 binding agent of claim 21, wherein the effector function is conferred by F_cDomain mediation.

25. The CD33 binding agent of claim 22, wherein the effector function is conferred by F_cDomain mediation.

26. The CD33 binding agent of claim 23, wherein the effector function is conferred by F_cDomain mediation.

27. The CD33 binding agent of claim 21, wherein the CD33 binding agent is at the F_cThe inclusion of one or more regulatory domains in the domain modulates F_cMutations in domain function.

28. The CD33 binding agent of claim 22, wherein the CD33 binding agent is at the F_cThe inclusion of one or more regulatory domains in the domain modulates F_cMutations in domain function.

29. The CD33 binding agent of claim 23, wherein the CD33 binding agent is at the F_cThe inclusion of one or more regulatory domains in the domain modulates F_cMutations in domain function.

30. The CD33 binding agent of claim 27, wherein the modulation of Fc domain function is at least 10% enhancement of ADCC.

31. The CD33 binding agent of claim 28, wherein the modulation of Fc domain function is at least 10% enhancement of ADCC.

32. The CD33 binding agent of claim 29, wherein the modulation of Fc domain function is at least 10% enhancement of ADCC.

33. The CD33 binding agent of claim 30, wherein the modulation of Fc domain function is at least 50% enhancement of ADCC.

34. The CD33 binding agent of claim 31, wherein the modulation of Fc domain function is at least 50% enhancement of ADCC.

35. The CD33 binding agent of claim 32, wherein the modulation of Fc domain function is at least 50% enhancement of ADCC.

36. The CD33 binding agent of claim 33, wherein the modulation of Fc domain function is at least 100% enhancement of ADCC.

37. The CD33 binding agent of claim 34, wherein the modulation of Fc domain function is at least 100% enhancement of ADCC.

38. The CD33 binding agent of claim 35, wherein the modulation of Fc domain function is at least 100% enhancement of ADCC.

39. The CD33 binding agent of claim 3, wherein the CD33 binding agent is at the F_cThe inclusion of one or more regulatory domains in the domain modulates F_cMutation of the function of the structural Domain, F_cThe mutation in the domain is at one or more positions selected from the amino acids at positions 332 and/or 239 and/or 236 indexed according to Kabat EU numbering.

40. The CD33 binding agent of claim 39, wherein the F_cMutations in the domains are a combination of substitutions at positions 239 and 332.

41. The CD33 binding agent of claim 40, wherein the F_cThe mutation in the domain is S239D/I332E.

42. A DNA molecule comprising a DNA molecule encoding the CD33 binding agent of any one of the preceding claims.

43. An expression vector comprising the DNA molecule of claim 42.

44. A host cell carrying one or more vectors according to claim 43.

45. A method of producing a CD33 binding agent according to any one of claims 1 to 41, comprising transfecting a host cell with one or more vectors according to claim 43, culturing the host cell and recovering and purifying the antibody molecule.

46. A pharmaceutical composition comprising as active ingredients one or more CD33 binding agent according to any one of claims 1 to 41 and at least one physiologically acceptable carrier.

47. The pharmaceutical composition of claim 46, further comprising one or more additional therapeutic agents.

48. The pharmaceutical composition of any one of claims 46 to 47, for use in depleting cells expressing CD33 on a surface.

49. Use of a CD33 binding agent according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 46 to 47 in the manufacture of a medicament for use in a method of depleting cells of the myeloid lineage that express CD33 in a patient, the method comprising administering to the patient one or more CD33 binding agents according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 46 to 47.