EA044092B1 - ANTI-NTB-A ANTIBODIES AND THERAPEUTIC COMPOSITIONS CONTAINING THEM - Google Patents

Description

List of sequences

The sequence listing designated NTBA-00303PR_ST25.txt, which is 29 kilobytes in size, is included by reference.

State of the art

NTB-A is a type I single-pass membrane glycoprotein, also called SLAMF6, a member of the immunoglobulin superfamily (Ig-SF) belonging to the CD2/SLAM subfamily. See, for example, Bottino et al., J. Exp. Med. 194:235–246, 2001. In its extracellular portion, NTB-A is characterized by an N-terminal V-type domain followed by a C2-type domain, while the intracytoplasmic portion contains three tyrosine-based motifs: two immunoreceptor-based switch motifs tyrosine (ITSM; TxYxxV/I) and the classical immunoreceptor tyrosine-based inhibition motif (ITIM; I/V/L/SxYxxL). See id. Through its ITSM motifs, NTB-A binds to the SH2 domain of the SLAM-associated protein SH2D1A and its associated Ewing's sarcoma activated transcript (EAT) 2. See Bottino et al., supra; Falco et al., Eur. J. Immunol. 34: 1663-1672, 2004; Flaig et al., Immunol. 172:6524-6527, 2004.

NTB-A is expressed on natural killer (NK) cells, NK-like T cells, T cells, monocytes, dendritic cells, B cells and eosinophils. See Salort JD. et al. , Immunology Letters 129-136, 2011; Matesanz-Isabel et al. , Immunology Letters 104-112, 2011; Muntz et al. , Journal of Immunology 174: 110-118, 2005; Bottino et al., Journal of Experimental Medicine 194(3):235-246; 2001. NTBA can function through homotypic interactions (i.e., as a self-ligand) and has been shown to act as a positive regulator of NK cell functions through signaling to induce NK cell cytotoxicity. See, for example, Bottino et al., supra; Falco et al., supra; Flaig et al., supra. NTB-A has also been shown to be expressed on B cells from patients with chronic lymphocytic leukemia (CLL) and B-cell lymphoma. See Korver et al., British Journal of Haematology 137:307-318, 2007.

Brief description of graphic materials

In fig. 1 shows an alignment of the mouse 20F3 heavy chain variable region sequences, the humanized sequence with the mouse CDR (boxed) in the human acceptor without back mutations and the humanized variants - HA-HE. CDRs are designated by Kabat.

In fig. 2 shows a sequence alignment of the mouse 20F3 light chain variable region, the humanized sequence with the mouse CDR (boxed) in the human acceptor without back mutations and the humanized variants - LA-LD. CDRs are designated by Kabat.

In fig. Figure 3 presents the results of a study of disseminated multiple myeloma xenograft in the MM.1R cell line in NSG mice (NOD (non-obese diabetes) SCID (severe combined immunodeficiency) gamma). The dose is indicated in the figure. ADC (antibody drug conjugate) is a humanized 20F3PBD (pyrrolebenzdiazepine) antibody drug conjugate.

In fig. Figure 4 presents the results of a study of disseminated multiple myeloma xenograft in the U-266 cell line in NSG mice. The dose is indicated in the figure. ADC (antibody drug conjugate) is a humanized 20F3-PBD (pyrrolebenzdiazepine) antibody drug conjugate.

In fig. Figure 5 presents the results of a study of disseminated multiple myeloma xenograft in the EJM cell line in NSG mice. The dose is indicated in the figure. ADC (antibody drug conjugate) is a humanized 20F3-PBD (pyrrolebenzdiazepine) antibody drug conjugate.

In fig. Figure 6 presents the results of a study of a subcutaneous xenograft of AML (acute myeloid leukemia) on the HNT-34 cell line in SCID mice. The dose is indicated in the figure. ADC (antibody drug conjugate) is a humanized 20F3-PBD (pyrrolebenzdiazepine) antibody drug conjugate.

In fig. Figure 7 presents the results of a study of disseminated multiple myeloma xenograft in the MM.1R cell line in NSG mice. The dose is indicated in the figure. The ADC is a drug-antibody conjugate of humanized 20F3-austatin.

In fig. Figure 8 presents the results of a study of disseminated multiple myeloma xenograft in the U-266 cell line in NSG mice. The dose is indicated in the figure. Both ADCs are humanized 20F3-austatin antibody-drug conjugates.

In fig. Figure 9 presents the results of a study of disseminated multiple myeloma xenograft in the EJM cell line in NSG mice. The dose is indicated in the figure. Both ADCs are humanized 20F3-austatin antibody-drug conjugates.

In fig. Figure 10 presents the results of a study of a subcutaneous xenograft of AML (acute myeloid leukemia) on the HNT-34 cell line in SCID mice. The dose is indicated in the figure. Both ADCs are humanized 20F3-austatin antibody-drug conjugates.

In fig. Figure 11 presents the results of a study of a subcutaneous xenograft of non-Hodgkin's lymphoma on the Raji cell line in SCID mice. The dose is indicated in the figure. Both ADCs are

- 1 044092 humanized antibody-drug conjugates 20F3-aypucmamuH.

In fig. Figure 12 presents the results of a study of a subcutaneous xenograft of non-Hodgkin's lymphoma on the WSU-DLCL2 cell line in SCID mice. The dose is indicated in the figure. Both ADCs are humanized 20F3-ayristatin antibody-drug conjugates.

Definitions

An antibody drug conjugate refers to an antibody conjugated to a cytotoxic agent or a cytostatic agent. Typically, drug-antibody conjugates bind to a target antigen (eg, NTB-A) on the cell surface, followed by internalization of the drug-antibody conjugate into the cell and release of the drug.

A polypeptide or polypeptide chain is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than 10 amino acid residues are usually called peptides.

A protein is a macromolecule containing one or more polypeptide chains. The protein may also contain non-peptide components such as carbohydrate groups.

Carbohydrates and other non-peptide substituents may be added to the protein by the cell in which the protein is made and will vary depending on the type of cell. Proteins are defined herein in terms of their basic amino acid structures; substituents such as carbohydrate groups are usually not indicated but may nevertheless be present.

The terms amino-terminal and carboxy-terminal refer to positions within the boundaries of polypeptides. When the context permits, these terms are used with reference to a specific sequence or portion of a polypeptide to indicate proximity or relative position. For example, a particular sequence positioned carboxyl terminus to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily located at the carboxyl terminus of the complete polypeptide.

The term antibody refers to immunoglobulin proteins produced by the body in response to the presence of an antigen and that bind to the antigen, as well as antigen-binding fragments and modified variants thereof. Therefore, the term antibody includes, for example, intact monoclonal antibodies (eg, antibodies produced using hybridoma technology) and antigen binding antibody fragments, such as F(ab')2 and Fab fragments. Also included are genetically engineered intact antibodies and fragments, such as chimeric antibodies, humanized antibodies, single chain Fv fragments, single chain antibodies, diabodies, mini antibodies, linear antibodies, multivalent or multispecific (eg, bispecific) hybrid antibodies, and the like. Thus, the term antibody is used expansively to include any protein that contains the antigen-binding site of an antibody and is capable of specifically binding to its antigen. The term antibody also includes the antibody itself (naked antibodies) or an antibody conjugated to a cytostatic or cytotoxic drug.

The term genetically engineered antibodies means antibodies in which the amino acid sequence differs from the amino acid sequence of the native antibody. Because of the importance of recombinant DNA techniques, antibody creation should not be limited to the amino acid sequences found in natural antibodies; antibodies can be redesigned to achieve desired characteristics. Possible variations are numerous and range from changes in only one or more amino acids to complete reorganization, for example, of the variable or constant region. Changes in the constant region are generally made to improve or alter characteristics such as, for example, complement fixation, cell interaction, and other effector functions. Typically, changes in the variable region are made to improve antigen-binding characteristics, improve the stability of the variable region, or reduce the risk of immunogenicity.

The antigen-binding site of an antibody is that part of the antibody that is sufficient to bind to its antigen. The minimum such region is usually a variable domain or a genetically modified variant thereof. Single domain binding sites can be derived from camel antibodies (see Muyldermans and Lauwereys, J. Mol. Recog. 12: 131-140, 1999; Nguyen et al., EMBO J. 19:921-930, 2000) or from VH domains other species to produce single domain antibodies (dAbs; see Ward et al., Nature 341:544-546, 1989; US Patent No. 6248516 Winter et al.). Typically, the antigen binding site of an antibody contains both a heavy chain variable domain (VH) and a light chain variable domain (VL) that bind to a common epitope. In the context of the present invention, an antibody may contain one or more components in addition to an antigen binding site, for example, another antigen binding site of the antibody (which may bind the same or a different epitope or the same or a different antigen), a peptide linker, an immunoglobulin constant region , immunoglobulin hinge, amphipathic helix (see Pack and Pluckthun, Biochem. 31: 1579-1584, 1992), non-peptide linker, oligonucleotide (see Chaudri et al., FEBS Letters 450:23-26, 1999), cytostatic or cytotoxic medicine and the like, and may represent

- 2 044092 is a monomeric or multimeric protein. Examples of molecules containing an antibody antigen binding site are known in the art and include, for example, Fv, single chain Fv (scFv), Fab, Fab', F(ab')2, F(ab)c, diabodies, minibodies, nanobodies, Fab-scFv fusions, bispecific (scFv)4-IgG and bispecific (scFv)2-Fab. (See, for example, Hie et al., Cancer Res. 56:3055-3061, 1996; Atwell et al., Molecular Immunology 33: 1301-1312, 1996; Carter and Merchant, Curr. Opin. Biotechnol. 8:449 -454, 1997; Zuo et al., Protein Engineering 13:361-367, 2000; and Lu et al., J. Immunol. Methods 267:213-226, 2002.)

The term immunoglobulin refers to a protein consisting of one or more polypeptides substantially encoded by the immunoglobulin gene(s). One form of immunoglobulin constitutes the basic structural unit of native (that is, natural) antibodies in vertebrates. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) are together responsible for antigen binding, and the constant regions are primarily responsible for the effector functions of the antibody. Five classes of immunoglobulin protein (IgG, IgA, IgM, IgD, and IgE) have been identified in higher vertebrates. IgG includes the main class; it generally exists as the second most abundant protein found in plasma. In humans, IgG consists of four subclasses designated IgG1, IgG2, IgG3, and IgG4. Each immunoglobulin heavy chain has a constant region that consists of constant region protein domains (CHI, hinge, CH2 and CH3, IgG3 also contains a CH4 domain) that are essentially invariant for a given subclass within a species. DNA sequences encoding human and non-human immunoglobulin chains are known in the art. (See, for example, Ellison et al., DNA 1: 11-18, 1981; Ellison et al., Nucleic Acids Res. 10:4071-4079, 1982; Kenten et al., Proc. Natl. Acad. Set USA 79:6661-6665, 1982; Seno et al., Nuc. Acids Res. 11:719-726, 1983; Riechmann et al., Nature 332:323327, 1988; Amster et al., Nuc. Acids Res. 8: 2055-2065, 1980; Rusconi and Kohler, Nature 314:330-334, 1985; Boss et al., Nuc. Acids Res. 12:3791-3806, 1984; Bothwell et al., Nature 298:380-382, 1982 ; van der Loo et al., Immuno genetics 42:333-341, 1995; Karlin et al., J. Mol. Evol. 22: 195-208, 1985; Kindsvogel et al., DNA 1:335-343, 1982 ; Breiner et al., Gene 18: 165-174, 1982; Kondo et al., Eur. J. Immunol. 23:245-249, 1993 and GenBank accession number J00228.) For a review of the structure and function of immunoglobulins, see Putnam, The Plasma Proteins, Vol V, Academic Press, Inc., 49-140, 1987 and Padlan, Mol. Immunol. 31: 169-217, 1994. The term immunoglobulin is used herein in its usual meaning, denoting the intact antibody, its constituent chains or fragments of chains, depending on the context.

Full-length immunoglobulin light chains (about 25 kDa or 214 amino acids) are encoded by a variable region gene at the amino terminus (encoding about 110 amino acids) and a kappa or lambda constant region gene at the carboxyl terminus. Full-length immunoglobulin heavy chains (about 50 kDa or 446 amino acids) are encoded by a variable region gene (encoding about 116 amino acids) and a gamma, mu, alpha, delta or epsilon constant region gene (encoding about 330 amino acids), the latter determining the isotype of the antibody, such as IgG, IgM, IgA, IgD or IgE, respectively. In the light and heavy chains, the variable and constant regions are joined by a J region of about 12 or more amino acids, with the heavy chain also containing a D region of about 10 more amino acids. (See generally, Fundamental Immunology (Paul, ed., Raven Press, N.Y., 2nd ed. 1989), Ch. 7).

An immunoglobulin light or heavy chain variable region (also referred to herein as a light chain variable domain (VL domain) or a heavy chain variable domain (VH domain), respectively, consists of a framework region interrupted by three complementarity determining regions or CDRs. The framework regions serve to alignment of CDRs to specifically bind to an epitope of an antigen. Thus, the term CDR refers to the amino acid residues of an antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid assignments for each variable region domain are as defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat has also proposed widely the numbering convention used (Kabat numbering), in which corresponding residues between different heavy chains or between different light chains are assigned the same number. CDRs 1, 2 and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2 and CDR-L3, respectively; CDRs 1, 2 and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2 and CDR-H3, respectively. If indicated, CDR assignment may be according to IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55–77; 2003) instead of Kabat.

Unless the context suggests otherwise, the term monoclonal antibody is not limited to antibodies produced by hybridoma technology. The term monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic or phage clone, and not the method of its preparation. The antibodies described herein are monoclonal antibodies.

The term humanized VH domain or humanized VL domain refers to the VH domain

- 3 044092 or VL of an immunoglobulin containing some or all of the CDRs, wholly or substantially from a non-human donor immunoglobulin (eg, mouse or rat), and variable domain framework sequences wholly or substantially from human immunoglobulin sequences. The non-human immunoglobulin that provides the CDR is called the donor, and the human immunoglobulin that provides the structure is called the acceptor. In some cases, humanized antibodies will retain certain non-human residues within the human variable domain framework regions to improve proper binding characteristics (e.g., mutations in the framework regions may be required to maintain binding affinity when the antibody is humanized).

A humanized antibody is an antibody containing one or both of a humanized VH domain and a humanized VL domain. Immunoglobulin constant region(s) need not be present, but if present they are wholly or substantially human immunoglobulin constant regions.

The CDRs in the humanized antibody are derived substantially from the corresponding CDR in the non-human antibody when at least 60%, at least 85%, at least 90%, at least 95 or 100% of the corresponding residues (as determined by Kabat (or IMGT) ) are identical between the corresponding CDRs. In particular, variations of a humanized VH or VL domain in which the CDRs are derived substantially from a non-human immunoglobulin, the humanized VH or VL domain CDRs have no more than six (e.g., no more than five, no more than four, no more than three, no more than two, or no more than one) amino acid substitutions (preferably conservative substitutions) in all three CDRs compared to the corresponding non-human VH or VL CDRs. The antibody VH or VL variable domain framework sequences, or, if present, immunoglobulin constant region sequences, are derived substantially from the human VH or VL framework sequence, or the human constant region, respectively, when at least about 80% is at least at least 85%, at least 90%, at least 95% or 100% of the corresponding Kabat residues are identical. Therefore, all portions of the humanized antibody, with the exception of the CDRs, are derived entirely or substantially from corresponding portions of naturally occurring human immunoglobulin sequences.

Antibodies are usually provided in isolated form. This means that the antibody typically makes up at least 50% w/w. from enclosing proteins and other contaminants resulting from its preparation or purification, but does not exclude the possibility of combining the antibody with an excess of pharmaceutically acceptable carrier(s) or other carrier medium designed to facilitate its use. Sometimes the antibodies are at least 60%, 70%, 80%, 90%, 95 or 99% w/w. free from interfering proteins and contaminants from manufacturing or refining. Antibodies, including isolated antibodies, can be conjugated to cytotoxic agents and presented as drug-antibody conjugates.

Specific binding of an antibody to its target antigen means an affinity of at least 106, 107, 108, 10 ⁹ or 10 ¹⁰ M ^-1 . Specific binding is detected at a significantly higher magnitude and is distinguishable from nonspecific binding occurring to at least one unrelated target. Specific binding may result from the formation of bonds between specific functional groups or the result of a specific spatial fit (eg, key-lock type), whereas nonspecific binding is usually the result of van der Waals forces.

The term epitope refers to the site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary fold of one or more proteins. Epitopes formed from contiguous amino acids are usually retained when exposed to denaturing solvents, whereas epitopes formed from tertiary stacking are usually lost when exposed to denaturing solvents. An epitope typically includes at least 3 and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to the target antigen. An antibody epitope can also be determined by using X-ray crystallography of the antibody bound to its antigen to identify contact residues.

Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other (provided that such mutations do not result in a global change in the structure of the antigen). Two antibodies have overlapping epitopes if certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

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Competition between antibodies is determined by an assay in which a test antibody inhibits the specific binding of a reference antibody to a common antigen (see, for example, Junghans et al.,

Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of test antibody inhibits binding of the reference antibody.

The competition is assessed according to the format presented in the examples. Antibodies identified by a competition assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance. Antibodies identified by a competition assay also include those that indirectly compete with a reference antibody by causing a conformational change in the target protein, thereby preventing the reference antibody from binding to a different epitope than that bound by the test antibody.

The terms expression unit and expression cassette are used interchangeably herein to refer to a nucleic acid segment encoding a polypeptide of interest and capable of causing the nucleic acid segment to be expressed in a host cell. An expression unit typically contains a transcriptional promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in an operative configuration. In addition to a transcriptional promoter and terminator, the expression unit may further contain other nucleic acid segments, such as, for example, an enhancer or a polyadenylation signal.

As used herein, the term expression vector refers to a nucleic acid molecule, linear or circular, containing one or more expression units. In addition to one or more expression units, the expression vector may also contain additional nucleic acid segments, such as, for example, one or more origins of replication, or one or more selectable markers.

Expression vectors are usually derived from a plasmid or viral DNA, or they may contain elements of both.

In the antibodies or other proteins described herein, reference to amino acid residues corresponding to those specified in SEQ ID NO includes post-translational modifications of such residues.

The term patient includes humans and other mammalian subjects who are receiving either prophylactic or therapeutic treatment.

The term effective amount, in the context of treating an NTB-A-specific disorder by administering an anti-NTB-A antibody as described herein, refers to an amount of such antibody that is sufficient to inhibit the occurrence or reduce the intensity of one or more symptoms of the disorder caused by NTB-A. An effective amount of antibody is administered in an effective manner. The term effective regimen refers to the combination of the amount of antibody administered and the dosage frequency sufficient to provide prophylactic or therapeutic treatment for the disorder.

For purposes of classifying amino acid substitutions as conservative or non-conservative, the following amino acid substitutions are considered conservative substitutions: series substituted with threonine, alanine or asparagine; threonine substituted with proline or serine; asparagine substituted with aspartic acid, histidine or serine; aspartic acid substituted with glutamic acid or asparagine; glutamic acid substituted with glutamine, lysine or aspartic acid; glutamine substituted with arginine, lysine or glutamic acid; histidine substituted with tyrosine or asparagine; arginine substituted with lysine or glutamine; methionine substituted with isoleucine, leucine or valine; isoleucine substituted with leucine, valine or methionine; leucine substituted with valine, isoleucine or methionine; phenylalanine substituted with tyrosine or tryptophan; tyrosine substituted with tryptophan, histidine or phenylalanine; proline substituted with threonine; alanine substituted with serine; lysine substituted with glutamic acid, glutamine or arginine; valine substituted with methionine, isoleucine or leucine; and tryptophan substituted with phenylalanine or tyrosine. Conservative substitutions can also mean substitutions between amino acids of the same class. The classes are as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; group II (neutral hydrophilic side chains): cys, ser, thr; group III (acid side chains): asp, glu; group IV (main side chains): asn, gin, his, lys, arg; group V (residues affecting chain orientation): gly, pro; and group VI (aromatic side chains): tip, tyr, phe.

Two amino acid sequences have 100% amino acid sequence identity if the amino acid residues of the two amino acid sequences are the same when aligned for maximum matching. Sequential comparisons can be performed using standard software such as that included in the LASERGENE bioinformatics suite manufactured by DNASTAR (Madison, WI). other methods

- 5 044092 comparisons of two nucleotide or amino acid sequences by determining the optimal alignment are well known to those skilled in the art. (See, for example, Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997); Wu et al., (eds.), Information Superhighway and Computer Databases of Nucleic Acids and Proteins, in Methods in Gene Biotechnology 123-151 (CRC Press, Inc. 1997); Bishop (ed.), Guide to Human Genome Computing (2nd ed., Academic Press, Inc. 1998).) Two amino acids are thought to sequences have significant sequence identity if two sequences have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to each other.

Percentage sequence identity is determined by antibody sequences maximally aligned using the Kabat numbering convention. After alignment, if the subject region of an antibody (eg, the entire variable domain of a heavy or light chain) is compared to the same region of a reference antibody, then the percent sequence identity between the subject region and the reference antibody is the number of positions occupied by the same amino acid in both the subject region and the reference antibody. and in the reference antibody, divided by the total number of aligned positions of the two regions, omitting gaps, multiplied by 100 to convert to percentages.

Compositions or methods containing one or more elements may contain other elements not specifically listed. For example, a composition that contains an antibody may contain the antibody alone or in combination with other ingredients.

The value range designation includes all integers within or defining the range.

The effector function of an antibody refers to the function caused by the Fc region of the Ig. Such functions may be, for example, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, or complement-dependent cytotoxicity. Such a function may be accomplished, for example, by binding the Fc region to an Fc receptor on an immune cell with phagocytic or lytic activity, or by binding the Fc region to components of the complement system. Typically, the effect(s) mediated by Fc-binding cells or complement components results in inhibition and/or depletion of the cell targeting NTB-A. The Fc regions of antibodies can recruit Fc receptor (FcR)-expressing cells and associate them with antibody-coated target cells. Cells expressing surface FcRs for IgG, including FcyRIII (CD16), FcyRII (CD32), and FcyRIII (CD64), can act as effector cells to kill IgG-coated cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils and eosinophils. The interaction of FcyR with IgG activates antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). ADCC is mediated by CD16+ effector cells through the secretion of membrane pore-forming proteins and proteases, while phagocytosis is mediated by CD32+ and CD64+ effector cells (see Fundamental Immunology, 4thed., Paul ed., Lippincott-Raven, N.Y., 1997, Chapters 3, 17 and 30; Uchida et al., J. Exp. Med. 199: 1659-69, 2004; Akewanlop et al., Cancer Res. 61:4061-65, 2001; Watanabe et al., Breast Cancer Res. Treat. 53: 199- 207, 1999). In addition to ADCC and ADCP, the Fc regions of cell-bound antibodies can also activate the classical complement pathway to cause complement-dependent cytotoxicity (CDC). C1q of the complement system binds to the Fc regions of antibodies when they form complexes with antigens. Binding of C1q to cell-bound antibodies can initiate a cascade of events involving proteolytic activation of C4 and C2 to form C3 convertase. Cleavage of C3 into C3b by C3 convertase allows activation of terminal complement components, including C5b, C6, C7, C8 and C9.

Collectively, these proteins form membrane attack complex pores on antibody-coated cells. These pores disrupt the integrity of the cell membrane, killing the target cell (see Immunobiology, ^6th ed., Janeway et al., Garland Science, NY, 2005, Chapter 2).

The term antibody-dependent cellular cytotoxicity, or ADCC, is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells with lytic immune cells (also called effector cells). Such effector cells include natural killer cells, monocytes/macrophages and neutrophils. Effector cells attach to the Fc region of Ig bound to target cells through their antigen binding sites. Death of the antibody-coated target cell occurs as a result of the activity of effector cells.

The term antibody-dependent cellular phagocytosis or ADCP refers to the process by which antibody-coated cells are internalized, in whole or in part, by phagocytic immune cells (eg, macrophages, neutrophils and dendritic cells) that bind to the Fc region of the Ig.

The term complement-dependent cytotoxicity or CDC refers to a mechanism for inducing cell death in which the Fc region of a target-bound antibody activates a series of enzymatic reactions that culminate in the formation of holes in the target cell membrane.

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Typically, antigen-antibody complexes, such as those located on antibody-coated target cells, bind and activate the complement component C1q, which in turn activates the complement cascade leading to the death of the target cell. Activation of complement may also result in the deposition of complement components onto the surface of the target cell, which facilitate

ADCC by binding complement receptors (eg, CR3) on leukocytes.

The cytotoxic effect refers to the depletion, elimination and/or death of the target cell. A cytotoxic agent refers to an agent that has a cytotoxic effect on a cell. Cytotoxic agents can be conjugated to the antibody or administered in combination with the antibody.

The cytostatic effect refers to the inhibition of cell proliferation. A cytostatic agent refers to an agent that has a cytostatic effect on a cell, thereby inhibiting the growth and/or division of a specific subset of cells. Cytostatic agents can be conjugated to an antibody or administered in combination with an antibody.

The term pharmaceutically acceptable means approved or approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans. The term pharmaceutically compatible ingredient refers to a pharmaceutically acceptable diluent, adjuvant, excipient or carrier with which the anti-NTB-A antibody is admixed.

The phrase pharmaceutically acceptable salt refers to pharmaceutically acceptable organic or inorganic salts. Examples of salts include the following salts: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sucrose, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e. 1,1'methylene-bis-(2 hydroxy-3- naphthoate)). The pharmaceutically acceptable salt may have the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge in the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In the case where multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

Unless otherwise clear from the context, when a value is expressed as being about X or approximately X, the stated value of X will be considered accurate to ±10%.

Glycosylation depends on the host cell used to express the antibody. Because the cell type used to express recombinant antibodies as potential therapeutics is rarely the native cell type, significant changes in the glycosylation pattern of antibodies can occur between recombinantly expressed antibodies in non-native cells and antibodies of the same primary heavy and light chain sequences that are expressed in their native cells. Rodent-derived mammalian cell lines (such as SP2/0, CHO or BHK) are capable of producing glycosylation that has some similarities to human glycosylation. However, some human components may be absent (for example, 2,6-linked sialylation), and a number of other components not typically found in humans may be present, such as terminal sialic acids, which do not normally exist in human cells (NeuGc, for example) or terminal galactose bound to another galactose, usually absent in human cells (Gal-Gal structures). Recombinant IgG expressed in CHO cells is generally less galactosylated compared to recombinant immunoglobulins expressed in mouse myeloma cells. Accordingly, recombinant IgG produced in CHO cells may contain higher levels of GO glycans compared to rIgG produced in mouse myeloma cell lines.

The glycosylated structure of antibodies can be analyzed using conventional carbohydrate analysis techniques, including lectin chromatography, NMR, mass spectrometry, HPLC, gel permeation chromatography, monosaccharide compositional analysis, sequential enzymatic digestion, and high performance anion exchange chromatography with pulsed amperometric detection, which uses anion exchange chromatography high pH for charge-based separation of oligosaccharides. Methods for releasing oligosaccharides for analytical purposes include enzymatic treatment (usually performed using peptide-N-glycosidase F/endo-beta-galactosidase), elimination using harsh alkaline conditions to release primarily O-linked structures, and chemical methods using anhydrous hydrazine to release of both N- and O-linked oligosaccharides.

Thus, the glycosylation pattern of a recombinantly expressed antibody may be characteristic of the cell type in which expression is performed (e.g., CHO), and distinguished by any of the above methods from other cell types, in particular cells of other species, such as

- 7 044092 mouse and man.

Solvates in the context of the invention are those forms of the compounds of the invention that form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are one specific form of solvate in which coordination occurs with water. Preferred solvates in the context of this invention are hydrates.

Detailed Description of the Invention

I. General information

The present invention provides, among other things, 20F3 antibodies that specifically bind to NTB-A, as well as chimeric, venous and humanized forms thereof. Also provided are antibodies that compete for binding with 20F3 antibodies or bind to the same epitope as the 20F3 antibody. The antibodies are useful, for example, for the treatment and diagnosis of various types of NTBA-expressing cancers, as well as for the detection of NTB-A (eg, detecting the expression of NTB-A in cells). Also provided are methods for such treatment, diagnosis and detection of NTB-A using antibodies of the invention, including naked antibodies and conjugated antibodies.

II. Target molecules

Unless otherwise stated, NTB-A means human NTB-A. The representative human sequence is designated SEQ ID NO: I (Swiss Prot Q96DU3). Four splice variant isoforms are known. The mature extracellular region is limited by residues 22-226 of Q96DU3. The shorter extracellular domain in isoform 4 has a C2 domain and two ITSM motifs and lacks residues 18–121, which include the immunoglobulin domain.

Unless the context otherwise indicates, reference to NTB-A means at least the extracellular domain of a protein consistent with a complete protein other than the cleavable signal peptide (amino acids 1-21 of Q96DU3).

Cynomolgus NTB-A (SEQ ID NO: 2) (cyno-NTB-A) has 84.6% amino acid identity and 93.3% amino acid similarity to human NTB-A (SEQ ID NO: 1).

III. Antibodies

A. Binding Specificity and Functional Properties

This disclosure relates, among other things, to the mouse antibody designated 20F3 and to chimeric, venated and humanized forms of the 20F3 antibody, as well as antibodies that compete with the 20F3 antibody for binding to NTB-A and antibodies that bind to the same epitope as the 20F3 antibody. Antibody 20F3 binds to the canonical form of human NTB-A (SEQ ID NO: 1), human isoform 4 and cynomolgus NTB-A. These binding characteristics suggest that its epitope lies outside the NTB-A segment missing from isoform 4 (within residues 128–226) and an epitope that is completely or substantially conserved between the human and cynomolgus forms of NTB-A.

The humanized antibody HDLD 20F3 has a Kd for human NTB-A isoform 1 on Ramos cells of about 2 nM. Other humanized forms of the 20F3 antibody preferably have a Kd that is substantially the same or within 2, 3, or 5 times that of the HDLD 20F3 antibody. The humanized HDLD antibody has an EC50 for human NTB-A on Ramos cells of about 12 nM in an antigen binding competition assay. Some humanized antibodies have an EC50 within 2, 3 or 6% of the HDLD of the humanized 20F3 antibody.

Preferred humanized 20F3 antibodies bind to the same epitope and/or compete with mouse 20F3 for binding to human NTB-A. Here, as elsewhere in this application, EC ₅₀ and Kd can be measured in accordance with the methods of the examples.

B. Humanized antibodies

A humanized antibody is a genetically modified antibody in which CDRs from a non-human donor antibody are grafted onto human antibody acceptor sequences (see, for example, Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205 and Foote , US 6881557). The acceptor antibody sequences may be, for example, a mature human antibody sequence, a composition of such sequences, a consensus sequence of a human antibody sequence, or a germline region sequence. Human acceptor sequences can be selected for high sequence identity in variable region frameworks with donor sequences to match canonical shapes between acceptor and donor CDRs, among other criteria. Suitable acceptor sequences for 20F3 heavy chains include IGHV7-4-1-IGHJ6. Thus, a humanized antibody is an antibody having CDRs derived wholly or substantially from donor antibodies and variable region and constant region framework sequences, if present, derived wholly or substantially from human antibody sequences. Likewise, a humanized heavy chain typically has all three CDRs derived entirely or substantially from the donor antibody heavy chain and the heavy chain variable region framework sequence, and the heavy chain constant region, if present, derived substantially from the heavy chain variable region framework

- 8 044092 people and constant region sequences. Likewise, a humanized light chain typically has all three CDRs derived entirely or substantially from the donor antibody light chain and the light chain variable region framework sequence, and the light chain constant region, if present, derived substantially from the human light chain variable region framework and constant region sequences. CDRs in a humanized antibody are derived substantially from the corresponding CDR in a non-human antibody when at least 80%, 85%, 90%, 95% or 100% of the corresponding residues (as determined by Kabat) are identical between the corresponding CDRs. Antibody variable chain or antibody constant region framework sequences are substantially derived from a human variable region or human constant region framework sequence, respectively, when at least 80%, 85%, 90%, 95% or 100% of the corresponding residues are defined according to Kabat, are identical.

Although humanized antibodies often contain all six CDRs (preferably defined by Kabat or IMGT®) from a mouse antibody, they can also be created with fewer CDRs (e.g., at least 3, 4, or 5) CDRs from a mouse antibody (e.g. Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164: 1432-1441, 2000).

Certain amino acids from the human variable region framework residues may be selected for replacement depending on their potential effect on CDR conformation and/or antigen binding. Investigation of such possible influences involves modeling, studying the characteristics of amino acids at specific sites, or empirically observing the effects of substitution or mutagenesis of certain amino acids.

For example, when an amino acid differs in a mouse variable region framework residue from a selected human variable region framework residue, the human framework amino acid may be replaced by an equivalent mouse antibody framework amino acid when it is reasonable to expect that the amino acid:

(1) directly non-covalently binds antigen, (2) is adjacent to the CDR region, (3) otherwise interacts with the CDR region (eg, is within about 6 A of the CDR region);

(4) mediates interactions between the heavy and light chains or (5) results from a somatic mutation in the mouse chain.

(6) is a glycosylation site.

Skeleton residues of classes (1)–(3) are sometimes alternately referred to as canonical and vernier residues. Framework residues that define a canonical class of CDR donor loops that define the conformation of a CDR loop are sometimes called canonical residues (Chothia and Lesk, J. Mol. Biol. 196, 901-917 (1987), Thornton & Martin J. Mol. Biol., 263 , 800-815, 1996). The layer of scaffolding residues that maintain antigen-binding loop conformations, playing a role in fine-tuning the fit of the antibody to the antigen, is sometimes called vernier residues (Foote & Winter, 1992, J Mol Bio. 224, 487-499).

This invention provides the 20F3 antibody and its humanized, chimeric and vernier forms. The present invention provides humanized forms of the mouse 20F3 antibody containing five typical humanized mature heavy chain variable regions (HA, HB, HC, HD and HE) (SEQ ID NO: 5-9) and four typical humanized light chains (LA, LB, LC and LD) (SEQ ID NO: 1518), which can be combined in different permutations with adequate binding. Of these permutations, HDLD is preferred because it has the best combination of binding properties and an acceptable number of back mutations. HDLD also retained binding to cyno-NTB-A. HDLD also has good yield in cell culture and shows little aggregation. Other combinations with EC50 within mouse factor 20F3 and smaller back mutations than HDLD include HCLB, HCLD, HDLB and HELB.

The present invention provides humanized forms of mouse 20F3 antibody, wherein the antibody contains 3 heavy chain CDRs of SEQ ID NO:3 and 3 light chain CDRs of SEQ ID NO:13, wherein the CDRs are designated by Kabat. The present invention also provides humanized forms of the mouse 20F3 antibody, wherein the antibody contains the 3 heavy chain CDRs of SEQ ID NO: 3 and the 3 light chain CDRs of SEQ ID NO: 13, wherein the CDRs are defined by IMGT. Kabat heavy chain CDR as set forth in SEQ ID NO: 10-12, and Kabat light chain CDR as set forth in SEQ ID NO: 19-21. IMGT® heavy chain CDR as set forth in SEQ ID NO: 22-24, and IMGT® light chain CDR as set forth in SEQ ID NO: 25-27.

The present invention provides humanized forms of the mouse 20F3 antibody, in which the light chain framework region is derived from the human germline donor sequence vhIGRV3-11 and the hIGRJ4 exon, and the heavy chain is derived from the human germline donor sequence hIGHV7-4-1 and the hIGHJ6 exon. Accordingly, the humanized antibodies described in

- 9 044092 herein can be humanized using the human germline donor sequence hIGKV3-11 and the hIGKJ4 exon for the light chain variable region and the human germline donor sequence IGHV7-4-1 and the hIGHJ6 exon for the heavy chain variable region. CDRs can be defined by Kabat or IMGT.

This invention provides humanized HDLD antibody variants wherein the humanized mature heavy chain variable region exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, and the mature humanized light chain variable region exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:18. Preferably, such antibodies retain some or all of the back mutations in HDLD. In some antibodies, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 of the following variable region framework positions are occupied as indicated: H2 occupies I, H44 occupies D, H46 occupies K, H73 occupies K, H76 occupies N, L1 occupies Q, L5 occupies S, L21 occupies M, L46 occupies P, L47 occupies W, L58 occupies V, L71 occupies Y; numbering is carried out according to the Kabat numbering system. The CDR regions of such humanized antibodies are preferably substantially identical to the HDLD CDR regions defined by Kabat, which are the same as that of the mouse donor antibody. In one embodiment, the humanized antibody comprises a heavy chain containing 3 CDRs of SEQ ID NO: 8 and variable region framework sequences with an identity equal to at least 95% of the variable region framework sequence of SEQ ID NO: 8. In another embodiment, the humanized the antibody contains a light chain containing 3 CDRs of SEQ ID NO:18 and variable region framework sequences with an identity equal to at least 95% of the variable region framework sequence of SEQ ID NO:18.

This invention provides humanized HDLD antibody variants wherein the humanized mature heavy chain variable region exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8 and the mature humanized light chain variable region exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. Some antibodies have the following framework positions variable region occupies as indicated: H2 occupies I, H38 occupies R or K, H44 occupies D or G, H46 occupies K or E, H68 occupies V or A, H73 occupies K, H76 occupies N or S, H91 occupies Y or F , LI occupies Q or E, L5 occupies S or T, L21 occupies M or L, L46 occupies P, L47 occupies W, L58 occupies V or I, and L71 occupies Y; numbering is carried out according to the Kabat numbering system. The CDR regions of such humanized antibodies are preferably substantially identical to the HDLD CDR regions defined by Kabat, which are the same as that of the mouse donor antibody. In one embodiment, the humanized antibody comprises a heavy chain containing 3 CDRs of SEQ ID NO: 8 and variable region framework sequences with an identity equal to at least 95% of the variable region framework sequence of SEQ ID NO: 8. In another embodiment, the humanized the antibody contains a light chain containing 3 CDRs of SEQ ID NO: 18 and variable region framework sequences with an identity equal to at least 95% of the variable region framework sequence of SEQ ID NO: 18.

Since the humanized 20F3 antibodies exhibit any deviation from the exemplary humanized HDLD antibody, one possibility for such additional variation is additional back mutations in the variable region framework sequences. Any or all of the positions backmutated in other examples of humanized mature heavy or light chain variable regions may be implemented (ie, 1, 2, or all 3 of H38 occupies K, H68 occupies A, H91 occupies F). However, such additional back mutations are not preferred as they do not generally improve affinity and introducing more murine residues may result in an increased risk of immunogenicity. Options include HALA, HALB, HALC, HALD, HBLA, HBLB, HBLC, HBLD, HCLA, HCLB, HCLC, HCLD, HDLA, HDLB, HDLC, HELA, HELB, HELC and HELD.

Another possible change is to replace some residues in the mouse antibody CDR with corresponding residues of human CDR sequences, typically from human CDR acceptor sequences used in the construction of typical humanized antibodies. In some antibodies, only a portion of the CDR is needed to maintain binding in a humanized antibody, namely a subset of CDR residues required for binding, called SDRs. CDR residues that do not contact the antigen and are not included in the SDR can be identified based on previous studies using molecular modeling and/or empirically. In such humanized antibodies, at positions that lack one or more donor CDR residues, the amino acid occupying the position may be the amino acid occupying the corresponding position in the sequence of the acceptor antibody. The number of such acceptor substitutions for donor amino acids contained in the CDR reflects a balance of competing considerations. Such substitutions are potentially beneficial for reducing mouse amino acids

- 10 044092 in a humanized antibody and, as a result, reducing potential immunogenicity. However, substitutions can also cause changes in affinity, and significant decreases in affinity are preferably avoided.

Although other non-preferred amino acid substitutions may be made, for example, at non-CDR-contacting framework residues, or even at some potential CDR-contacting amino acid residues within the CDR. Often, substitutions made in humanized sequence variants are conservative relative to the HDLD amino acid substitutions. Preferably, substitutions relative to HDLD (whether conservative or not) do not significantly affect the binding affinity or activity of the humanized mAb, that is, its ability to bind human NTB-A and inhibit the growth of cancer cells.

Variants typically differ from the mature HLD heavy and light chain variable region sequences by a small number (eg, typically no more than 1, 2, 3, 5, or 10 in the mature light chain or heavy chain variable region, or both) substitutions, deletions, or insertions. Antibodies can be assayed for specific binding by conventional methods, including immunoassays, which include assay systems using techniques such as Western blotting, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation assay, precipitin assay, gel assay. diffusion precipitins, immunoradiometric assays, fluorescence immunoassays, protein A immunoassays, and complement fixation tests. Such an assay is routine and well known in the art (see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The binding saturation protocol in the examples can be used to determine a specific binding.

Any of the antibodies may be selected to have the same or overlapping epitope specificity as a typical antibody such as mouse 20F3 antibody, by competitive binding assay or otherwise. Antibody competition is assessed by the ability of a test antibody to compete with a reference antibody, in this case mouse -20F3, for specific binding to human NTB-A. A typical mouse 20F3 antibody has the mature heavy and light chain variable regions of SEQ ID NOs: 3 and 13, and is of the IgG1 kappa isotype. Preferred antibodies have the same epitope specificity as antibody 20F3. Those skilled in the art are able to identify the epitope bound by an antibody using a variety of techniques. For example, an array-based oligopeptide scan or Pepscan assay uses a library of oligopeptide sequences from overlapping and non-overlapping segments of a target antigen and tests their ability to bind the antibody of interest. See, for example, Geysen et al., PNAS 81:3998-4002 (1984). Non-linear epitopes can be identified using, for example, CLIPS™ technology, a variation of array-based oligopeptide scanning. See, for example, Timmerman et al., Open Vaccine J. 2:56-67 (2009). The antigen protein can also be mutagenized and then used to assess binding to the antibody of interest. Protein systematic site-directed mutagenesis can be used, or a library of mutations can be generated and used to screen for antibody binding. Mutation libraries can be purchased, for example, from Integral Molecular. MS based on hydrogen/deuterium amide exchange can be used to identify epitopes. Antigens of interest are placed in deuterated water and labeled with deuterons. The protein is then digested with a protease and the resulting peptide fragments are subjected to mass spectrometric analysis. The antigen is also assessed in the presence of the antibody, and differences in the labeling of the peptide fragments indicate the binding regions of the antibody.

IV. Selecting a constant region

Anti-NTBA antibodies containing a VH and/or VL domain may be bound to at least a portion of an immunoglobulin constant region (eg, a human immunoglobulin constant region). For example, some anti-NTB-A antibodies comprise first and second polypeptide chains, wherein the first polypeptide chain comprises a VH domain, as described herein, linked to at least a portion of an immunoglobulin heavy chain constant region, and the second polypeptide chain comprises a VL domain, as described herein. described herein, associated with at least a portion of the constant region of an immunoglobulin light chain. Typically, the VH or VL domain is linked amino-terminally to the immunoglobulin constant region or part thereof. In particular, variations of an antibody containing first and second polypeptide chains, wherein the first and second polypeptide chains have a domain structure corresponding to the heavy and light chains of an intact native antibody, for example, the first polypeptide (heavy) chain having a domain structure in the direction from the amino terminus to the carboxyl terminus a VH-CH1-hinge-CH2-CH3 structure and a second polypeptide (light) chain having a VL-CL domain structure in the direction from the amino terminus to the carboxyl end.

Some anti-NTB-A antibodies are single-chain antibodies containing a VH domain, a VL domain, and at least a portion of an immunoglobulin constant region (eg, a heavy chain constant region without the CH1 domain) linked within a single polypeptide chain. For example, the VH and VL domains can be designed as a single-chain Fv (scFv) in the VH/VL orientation

- 11 044092 or VL/VH (amino terminus/carboxyl terminus), wherein the scFv is linked (typically from the amino terminus) to a heavy chain constant region, such as, for example, a constant region containing CH2 and

CH3, but without the CH1 domain. ScFv is typically linked to the constant region via a linker, such as, for example, a linker derived from an immunoglobulin hinge region.

The choice of constant region may depend in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis, and/or complement-dependent cytotoxicity is required. For example, human IgG1 and IgG3 isotopes have strong complement-dependent cytotoxicity, human IgG2 has weak complement-dependent cytotoxicity, and human IgG4 has no complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant regions may be lambda or kappa. Typical heavy and light chain constant regions are shown in SEQ ID NOS: 27, 28 and 29. Antibodies can be expressed, for example, as tetramers containing two light and two heavy chains, as separate heavy chains, light chains such as Fab , Fab', F(ab') ₂ and Fv or as single chain antibodies in which the heavy and light chain variable domains are linked through a spacer. In addition, constant regions can be mutated if necessary. In some aspects, the mutant form of the natural human constant region will reduce binding to the Fcy receptor relative to the natural human constant region.

Human constant regions exhibit allotypic variation and isoallotypic variation between different individuals, that is, constant regions may differ between individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that the serum recognizing the isoallotype binds to a non-polymorphic region of one or more other isotypes.

One or more amino acids at the amino or carboxyl terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be absent or derivatized in proportion or in all molecules. Substitutions can be made in constant regions to decrease or increase effector function, such as complement-mediated cytotoxicity or ADCC (see, for example, Winter et al., US Pat. No. 5,624,821; Tso et al., US Pat. No. 5,834,597; and Lazar et al. , Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong the half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004).

A typical substitution involves an amino acid substitution of a native amino acid with a cysteine residue introduced at amino acid position 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332, preferably the S239C mutation in the human IgG1 isotype (US 20100158909; Fc region numbering corresponds to EU index). In some aspects, the presence of an additional cysteine residue allows the formation of interchain disulfide bonds. This formation of an interchain disulfide bond may cause steric mismatch, thereby reducing the affinity of the Fc-FcyR region binding interaction. Cysteine residue(s) introduced adjacent to or directly into the Fc region of the IgG constant region may also serve as sites for conjugation with therapeutic agents (i.e., binding of cytotoxic drugs using thiol specific reagents such as maleimide derivatives of drugs ). The presence of a therapeutic agent causes steric mismatch, thereby further reducing the affinity of the Fc-FcyR region binding interaction. Other substitutions at any of positions 234, 235, 236 and/or 237 reduce the affinity for Fcy receptors, in particular the FcyRI receptor (see, for example, US 6624821, US 5624821).

The in vivo half-life of an antibody may also influence its effector functions. The half-life of an antibody can be increased or decreased to alter its therapeutic activity. FcRn is a receptor that is structurally similar to MHC (major histocompatibility complex) class I antigen, which associates non-covalently with 2-microglobulin. FcRn regulates IgG catabolism and transcytosis through tissues (Ghetie and Ward, Annu. Rev. Immunol. 18:739766, 2000; Ghetie and Ward, Immunol. Res. 25:97-113, 2002). The IgG-FcRn interaction occurs at pH 6.0 (intracellular vesicle pH) but not at pH 7.4 (blood pH); this interaction allows the return of IgG back into the bloodstream (Ghetie and Ward, 2000, supra; Ghetie and Ward, 2002, supra). The region of human IgG1 involved in FcRn binding has been mapped (Shields et al., J. Biol. Chem. 276:6591-604, 2001). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., supra). IgG1 molecules containing these substitutions have a longer serum half-life. Therefore, these modified IgG1 molecules can perform their effector functions and hence exert their therapeutic efficacy over a longer period of time compared to unmodified IgG1. Other typical substitutions to increase FcRn binding include Gin at position 250 and/or Leu at position 428. EU numbering is used for all positions in the constant region.

- 12 044092

Oligosaccharides covalently attached to the conserved Asn297 are involved in the ability of the Fc region of IgG to bind FcyR (Lund et al., J. Immunol. 157:4963-69, 1996; Wright and Morrison, Trends Biotechnol. 15:26-31, 1997). Modification of this glycoform to IgG can significantly improve IgG-mediated ADCC. Addition of bisecting modifications of N-acetylglucosamine (Umana et al., Nat. Biotechnol. 17: 176-180, 1999; Davies et al., Biotech. Bioeng. 74:288-94, 2001) to this glycoform or removal of fucose (Shields et al., Biotech. Bioeng. 74:288-94, 2001) to this glycoform or removal of fucose al., J. Biol. Chem. 277:26733-40, 2002; Shinkawa et al., J. Biol. Chem. 278:6591-604, 2003; Niwa et al., Cancer Res. 64:2127-33, 2004) from this glycoform are two examples of IgG Fc modifications that improve binding between IgG Fc and FcyR, thereby enhancing Ig-mediated ADCC activity.

Systemic replacement of solvent-accessible amino acids in the Fc region of human IgG1 has generated IgG variants with altered FcyR binding affinities (Shields et al., J. Biol. Chem. 276:6591-604, 2001). Compared with parental IgG1, a subset of these variants containing substitutions at Thr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 to Ala show an increase in both FcyR binding affinity and ADCC activity (Shields et al., 2001, supra; Okazaki et al., J. Mol. Biol. 336:1239-49, 2004).

The complement fixation activity of antibodies (both Clq binding and CDC activity) can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., J. Immunol. 166:2571-2575, 2001). The same substitutions in the human IgG2 backbone can convert an antibody isotype that binds poorly to Clq and has greatly reduced complement activation activity into one that can bind Clq and mediate CDC (Idusogie et al., supra). Several other methods have also been applied to improve the complement fixation activity of antibodies. For example, transplantation of the 18 amino acid carboxyl terminus of IgM onto the carboxyl terminus of IgG significantly enhances their CDC activity. This is observed even with IgG4, which usually does not exhibit CDC activity (Smith et al., J. Immunol. 154:2226-36, 1995). Also, by replacing Ser444 located close to the carboxyl terminus of the IgG1 heavy chain with Cys-induced tail-to-tail dimerization of IgG1 with a 200-fold increase in CDC activity compared to monomeric IgG1 (Shopes et al., J. Immunol. 148:2918-22, 1992 ). In addition, a bispecific diabody design with Clq specificity also provides CDC activity (Kontermann et al., Nat. Biotech. 15:629-31, 1997).

Complement activity can be reduced by mutating at least one of the heavy chain amino acid residues 318, 320 and 322 with a residue having a different side chain, such as Ala. Other alkyl-substituted nonionic residues such as Gly, He, Leu or Val, or aromatic non-polar residues such as Phe, Tyr, Trp and Pro instead of any of the three residues also reduce or eliminate Clq binding. Ser, Thr, Cys and Met can be used on residues 320 and 322, but not 318, to reduce or eliminate Clq binding activity. Substitution of a polar residue at residue 318 (Glu) may alter, but not eliminate, Clq binding activity. Substitution of residue 297 (Asn) with Ala removes lytic activity, but only slightly reduces (about three times weaker) the affinity for Clq. This change disrupts the glycosylation site and the presence of carbohydrates, which is required for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combination thereof also reduce Clq binding: D270A, K322A, P329A and P311S (see WO 06/036291).

A reference to a human constant region includes a constant region with any naturally occurring allotype or any permutation of residues occupying polymorphic positions in natural allotypes. In addition, up to 1, 2, 5 or 10 mutations relative to the natural human constant region, such as those listed above, may be present to reduce Fc gamma receptor binding or increase FcRn binding.

V. Nucleic acids and methods of production

The present invention also provides nucleic acids encoding any of the VH and/or VL domains described above, including polypeptides containing VH and/or VL domains associated with additional polypeptide segments, such as, for example, polypeptide segments corresponding to an immunoglobulin constant region . Typically, the nucleic acids also encode a signal peptide fused at the amino terminus to a mature polypeptide containing VH and/or VL domains. Coding sequences on nucleic acids may be in operative association with regulatory sequences to promote expression of coding sequences such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The nucleic acids may occur in isolated form or may be cloned into one or more vectors. Nucleic acids can be synthesized, for example, by solid phase synthesis or PCR with overlapping oligonucleotides. Nucleic acids encoding both the VH and VL domains (e.g., in the context of antibodies containing separate heavy and light chains) may be joined as a single contiguous nucleic acid, e.g., within an expression vector, or may be separate, e.g., each cloned into its own expression vector.

- 13 044092

Antibodies against NTB-A are typically produced by recombinant expression of one or more nucleic acids encoding one or more antibody chains. Recombinant polynucleotide constructs typically contain an expression control sequence operably linked to the coding sequences of one or more polypeptide chains containing VH and/or VL domains, including naturally occurring or heterologous promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for expression of nucleotide sequences at high levels and for collection and purification of cross-reacting antibodies.

To express antibodies containing first and second polypeptide chains (eg, heavy and light chains), the two polypeptide chains can be co-expressed from separate vectors in a host cell to express the entire antibody molecule. Alternatively, two polypeptide chains can be co-expressed from separate expression units in the same vector in a host cell to express the entire antibody molecule.

Mammalian cells are the preferred host for the expression of nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of useful host cell lines capable of secreting intact heterologous proteins have been developed in the art and include CHO cell lines (eg, DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody producing myelomas, including Sp2/0 and NS0. Preferably the cells are not human cells.

Expression vectors for these cells may contain expression control sequences such as origin of replication, promoter, enhancer (Queen et al., Immunol. Rev. 89:49, 1986) and necessary information processing sites such as ribosome binding sites, splice sites RNA, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus and the like. See Co et al., J. Immunol. 148:1149, 1992.

Once expressed, antibodies can be purified according to standard methods in the art, including HPLC purification, column chromatography, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)) .

VI. Antibody drug conjugates

Antibodies against NTB-A can be conjugated to cytotoxic or cytostatic moieties to form antibody drug conjugates (ADCs). Particularly useful moieties for conjugation with antibodies are cytotoxic agents (eg, chemotherapeutic agents), prodrug-converting enzymes, radioactive isotopes or compounds, or toxins (these moieties are collectively referred to as a therapeutic agent). For example, an anti-NTB-A antibody may be conjugated to a cytotoxic agent, such as a chemotherapeutic agent, or toxin (eg, a cytostatic or cytocidal agent such as, for example, abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin). Examples of useful classes of cytotoxic agents include, for example, DNA minor groove binders, DNA alkylating agents and tubulin inhibitors. Typical cytotoxic agents include, for example, auristatins, camptothecins, calicheamicins, duocarmycins, etoposides, maytansinoids (eg, DM1, DM2, DM3, DM4), taxanes, benzodiazepines (eg, pyrrole[1,4]benzodiazepines, indolinobenzodiazepines and oxazolidinobenzodiazepines, including pyrrole [1,4]benzodiazepine dimers, indolinobenzodiazepine dimers and oxazolidinobenzodiazepine dimers) and bindweed alkaloids.

The anti-NTB-A antibody can be conjugated to an enzyme that promotes the conversion of the prodrug. The prodrug conversion enzyme can be recombinantly fused to or chemically conjugated to the antibody using known methods. Typical prodrug conversion enzymes are carboxypeptidase G2, betaglucuronidase, penicillin V-amidase, penicillin G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

Techniques for conjugating therapeutic agents to proteins and, in particular, antibodies are well known. (See, for example, Alley et al., Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14(3): 154-169.) The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (eg by hydrolysis, proteolytic degradation or a cleavage agent). In some aspects, the therapeutic agent is attached to an antibody with a cleavable linker that is sensitive to cleavage in the intracellular environment of the NTB-A-expressing cancer cell, but is not significantly sensitive to the extracellular environment, such that the conjugate is cleaved from the antibody when it is internalized by NTB-A. A-expressing cancer cell (for example, in the endosomal or, for example, due to pH sensitivity or protease sensitivity, in the lysosomal environment or in the caveolar environment). In some aspects, the therapeutic agent can also be attached to the antibody with a non-cleavable linker.

Typically, an ADC contains a linker region between a cytotoxic or cytostatic agent and an anti-NTB-A antibody. As noted above, typically, the linker may be cleavable under intracellular conditions such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (eg, a lysosome or endosome or caveola). The linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Degrading agents may include cathepsins B and D and plasmin (see, for example, Dubowchik and Walker, Pharm. Therapeutics 83:67123, 1999). The most typical are peptidyl linkers, which are cleaved by enzymes that are present in cells expressing NTB-A. For example, a peptidyl linker may be used that is cleaved by the thiol-dependent protease cathepsin-B, which is expressed at high levels in cancer tissue (eg, a linker containing a Phe-Leu or Val-Cit peptide).

The cleavable linker may be pH sensitive, that is, sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzed under acidic conditions. For example, an acid-labile linker that is hydrolyzed in the lysosome (eg, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) may be used. (See, for example, US Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999; Neville et al., Biol. Chem. 264: 14653-14661, 1989.) Such linkers relatively stable under neutral pH conditions, such as in the blood, but unstable below pH 5.5 or 5.0, the approximate pH of the lysosome.

Other linkers are cleaved under alkaline conditions (eg disulfide linker). Disulfide linkers include those that can be formed using SATA (Nsuccinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (Nsuccinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methylalpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. (See, for example, Thorpe et al., Cancer Res. 47:5924-5931, 1987; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987 See also US Patent No. 4880935.)

The linker may also be a malonate linker (Johnson et al., Anticancer Res. 15: 1387-93, 1995), a maleimidobenzoyl linker (Lau et al., Bioorg-Med-Chem. 3: 1299-1304, 1995), or 3N -amide analog (Lau et al., Bioorg-Med-Chem. 3: 1305-12, 1995).

The linker may also be a non-cleavable linker, such as a maleimidoalkylene or maleimide aryl linker, which is directly attached to the therapeutic agent and released by proteolytic degradation of the antibody.

Typically, the linker is not significantly sensitive to the extracellular environment, meaning no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5% no more than about 3 % or no more than about 1% of the linkers in an ADC sample are cleaved when the ADC is present in the extracellular environment (eg, plasma). Whether a linker is substantially insensitive to the extracellular environment or not can be determined, for example, by plasma-independent incubation of both (a) the ADC (ADC sample) and (b) an equal molar amount of unconjugated antibody or therapeutic agent (control sample) in over a specified period of time (eg, 2, 4, 8, 16 or 24 hours), and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample with that present in a control sample, measured, for example, using high performance liquid chromatography.

The linker may also facilitate cellular internalization. The linker may facilitate cellular internalization when conjugated to a therapeutic agent (ie, in the linker medium of a therapeutic agent ADC or ADC derivative as described herein). Alternatively, the linker may promote cellular internalization when conjugated to both a therapeutic agent and an anti-NTB-A antibody (ie, in an ADC medium as described herein).

The anti-NTB-A antibody can be conjugated to the linker via the antibody heteroatom. These heteroatoms may be present on the antibody in its natural state or may be introduced into the antibody. In some aspects, the anti-NTB-A antibody will be conjugated to a linker via the nitrogen atom of a lysine residue. In other aspects, the anti-NTB-A antibody will be conjugated to a linker via the sulfur atom of a cysteine residue. Methods for conjugating a linker and drug linker to antibodies are known in the art.

Typical antibody drug conjugates include auristatin-based antibody drug conjugates, which means that the drug moiety is an auristatin preparation. Auristatins, which bind tubulin, have been shown to influence microtubule dynamics and nuclear and cellular division and have antitumor activity. Typically, the auristatin drug-antibody conjugate contains a linker between the auristatin drug and the anti-NTB-A antibody. The linker may be, for example, a cleavable linker (eg, a peptidyl linker) or a non-cleavable linker (eg, a linker released by antibody degradation). Auristatins include MMAF and MMAE. The synthesis and structure of typical auristatins are described in US Publication Nos. 7659241, 7498298, 2009-0111756, 2009-0018086 and 7968687, each of which is incorporated herein by reference in its entirety and for all purposes.

Other exemplary drug-antibody conjugates include maytansinoid antibody drug conjugates, which means that the drug moiety is a maytansinoid drug, and benzodiazepine antibody drug conjugates, which means that the drug moiety is a benzodiazepine (e.g., pyrrole[ dimers 1,4]benzodiazepine, indolinobenzodiazepine dimers and oxazolidinobenzodiazepine dimers).

The present inventors have found that an ADC targeting humanized NTB-A containing a PBD linker drug is particularly effective.

The preferred PBD for use in this invention is represented by Formula I. The preferred stereochemistry of the drug component of the PBD is represented by Formula 1a

or a pharmaceutically acceptable salt, solvate or solvate of salt; where index n is 1 or 3.

The PBD dimer of Formula I (or a pharmaceutically acceptable salt, solvate or salt solvate thereof) is typically linked to an antibody via a linker unit, LU. The linker unit is used to release the PBD dimer of formula I (or a pharmaceutical salt, solvate or solvate of a salt thereof) at a target site (eg, inside a cancer cell). The PBD-linker drug compound for use in the present invention is represented by formula II below (preferred stereochemistry as shown in IIa), where LU represents the linker unit. The linker unit may be, for example, a cleavable peptide linker unit (e.g., a valinalanine peptide-containing linker) or a cleavable disulfide linker unit

or a pharmaceutically acceptable salt, solvate or solvate of salt; where index n is 1 or 3.

A preferred PBD linker drug compound for use in the present invention is represented by Formula III below:

’QUe or a pharmaceutically acceptable salt, solvate or solvate of salt; where index n is equal to 1 or 3, and index m is equal to an integer 2-5.

The preferred stereochemistry of the drug linker drug PBD component is represented by Formula IIIa below

ΌΜβ (Sha)

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The preferred stereochemistry of the drug component PBD and the drug linker components of SGD-1910 is represented by Formula IIIb below

The PBD linker drug is conjugated to the 20F3 antibody, including vernier, chimeric and humanized forms, to produce a drug-antibody conjugate targeting NTB-A. For example, the antibody may be conjugated to a linker drug of Formula II or Formula III. A typical NTB-A type target antibody-drug conjugate is shown below in Formulas IV, IVa and IVb

or a pharmaceutically acceptable salt, solvate or solvate of salt; where index n is equal to 1 or 3, index m is equal to the integer 2-5, and index p is equal to the integer 1-4.

The present invention provides compositions, including pharmaceutical compositions, containing anti-NTBA antibody drug conjugates, including those specifically described herein. The compositions typically include a population of drug-antibody conjugate molecules.

Drug load - p

Referring to drug-antibody conjugates targeting NTB-A having formulas IV, IVa and IVb, the index p represents the drug loading of the antibody molecule (the number of drug molecules attached to the antibody molecule) and is an integer value. In a composition containing a population of drug-antibody conjugate molecules, the average drug load (eg, the average number of drug-antibody molecules in the population) is an important quality attribute because it determines the amount of drug that can be delivered to the target cells. The average drug load can be an integer or non-integer value, but is typically a non-integer value.

The heterogeneity of the drug-antibody conjugate composition will in some aspects depend on the conjugation technology used to conjugate the drug molecules to the antibody molecules. For example, in some aspects, the conjugation technology used to conjugate drug-linker molecules to antibody molecules will result in a drug-antibody conjugate composition that is heterogeneous with respect to the distribution of drug-linker molecules on the antibodies and/or with respect to the amount of drugs -linkers on antibody molecules (for example, when conjugating through interchain disulfides using non-site-specific technology). For example, in some aspects, the conjugation technology used to conjugate drug-linker molecules to antibody molecules will result in a drug-antibody conjugate composition that is substantially homogeneous with respect to the distribution of drug-linker molecules on the ligand molecules and/or with respect to the amount linker drugs on antibody molecules (for example, using site-specific conjugation technology). As in

- 17 044092 using site-specific and non-site-specific methods there may also be a small percentage of unconjugated antibody molecules. The percentage of unconjugated antibody molecules is included in the average drug load.

In preferred aspects of the present invention, the average drug loading, when it comes to a composition comprising a drug-antibody conjugate population, is from about 2 to about 14, preferably from about 2 to about 10. For PBD-antibody drug conjugates, such as exemplified herein, a particularly preferred average drug load is about 2. In some aspects, the actual drug load for individual antibody molecules in the drug-antibody conjugate population is from 1 to 4, from 1 to 3, or from 1 to 2 at a predominant drug load of 2. In preferred aspects, an average drug load of 2 is achieved using site-specific conjugation techniques (eg, modified cysteines introduced into the antibody at position 239, according to the EU base numbering system).

In other aspects of the present invention, the average drug load attributable to a composition comprising a PBD drug-antibody conjugate population is about 3 or about 4, and the actual drug load for individual antibody molecules in the drug-antibody conjugate population is 1-6 , 1-5, 1-4 or 1-3.

The average number of drug-linker units per ligand unit in the conjugation reaction preparation can be characterized by conventional methods such as mass spectroscopy, ELISA, HIC (hydrophobic interactive chromatography) and HPLC. In addition, the quantitative distribution of ligand-linker-drug conjugates relative to p can be determined. In some cases, by means such as reverse phase HPLC or electrophoresis, it is possible to achieve the separation, purification and characterization of homogeneous ligand-linker drug conjugates, in which p is a certain value, from a ligand linker-drug conjugate with other drug load values.

VII. Applications

The anti-NTBA antibodies described herein, as naked antibodies or as drug-antibody conjugates, can be used in methods of treating a disease or disorder associated with a cell expressing NTB-A.

For example, the anti-NTBA antibodies described herein, as naked antibodies or as drug-antibody conjugates, can be used to treat cancers expressing NTB-A. Some of these cancers exhibit detectable levels of NTB-A, measured either at the protein level (for example, using an immunoassay using one of the typical antibodies) or at the mRNA level. Some of these cancers exhibit elevated levels of NTB-A relative to tissue of the same type that is not affected by cancer, preferably from the same patient. The approximate treatable level of NTB-A in cancer cells is 5,000-150,000 NTB-A molecules per cell, although higher or lower levels can be treated. Optionally, the level of NTB-A in the cancer is measured before treatment.

Examples of cancers associated with NTB-A expression and amenable to treatment with naked antibodies or antibody-drug conjugates described herein include hematologic malignancies, including B cell, T cell, and NK cell malignancies. In one embodiment of the invention, naked antibodies or drug-antibody conjugates described herein bind receptors on NK cells, causing cytolytic activity and proliferation, which stimulates the antitumor activity of the patient's immune system. In some treatments, the patient suffers from a cancer that is multiple myeloma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), T-cell leukemia, T-cell or B-cell lymphoma, such as, for example, non-Hodgkin's lymphoma (NHL). ), or myeloma-related malignancies such as primary amyloidosis, Waldenström's macroglobulinemia, or high-risk MGUS (monoclonal gammopathy of undetermined significance). The treatment can be applied to patients with primary or metastatic tumors of these types. The treatment may also be used for patients who are not suitable for traditional treatments or who relapse after responding to such treatments.

The anti-NTBA antibodies described herein, as naked antibodies or as drug-antibody conjugates, can be used to treat autoimmune diseases and inflammatory diseases. Diseases and disorders treatable by the present methods include those associated with B cells, such as those characterized by excessive B cells, B cell overactivity, or B cell dysfunction. These diseases include inflammatory diseases and autoimmune disease. Typical diseases treatable by the present methods include rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, asthma, allergies, celiac disease, graft-versus-host disease, and graft rejection.

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The present anti-NTB-A antibodies, as naked antibodies or antibody conjugates, can be used as monotherapy or combination therapy, for example, standard of care for the treatment of such diseases and/or disorders. Accordingly, methods of treating cancer include administering to a patient in need thereof an effective amount of a naked antibody or an antibody-drug conjugate as described herein, as monotherapy or in combination with an additional anticancer agent or other agent to alleviate the symptoms of cancer. Methods of treating an autoimmune disease include administering to a patient in need thereof an effective amount of a naked antibody or a drug-antibody conjugate as described herein, as monotherapy or in combination with an additional therapeutic agent to treat the autoimmune disease. Methods of treating an inflammatory disease include administering to a patient in need thereof an effective amount of a naked antibody or a drug-antibody conjugate as described herein, as monotherapy or in combination with an additional therapeutic agent to treat the inflammatory disease.

A typical combination therapy is carfilzomib (eg, KYPROLIS®), a proteasome inhibitor used to treat multiple myeloma (see Siegel DS et al., phase 2 study of single-agent carfilzomib (PX-171-003-A1) in patients with relapsed and refractory multiple myeloma. Blood 2012;120:2817-2825). Carfilzomib can be given as an intravenous (IV) infusion. In one embodiment, carfilzomib is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Carfilzomib can also be administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention and an additional agent. Carfilzomib has been combined with various additional agents for the treatment of multiple myeloma. For example, carfilzomib was combined with lenalidomide and dexamethasone (see Stewart KA et al., Carfilzomib, lenalidomide, and dexamethasone for relapsed multiple myeloma. N Engl. J Med. 2015; 372: 142-152). In one embodiment, carfilzomib is administered in combination therapy with lenalidomide, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the invention. In another embodiment, carfilzomib is administered in combination therapy with lenalidomide, dexamethasone, and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with lenalidomide, dexamethasone and h20F3ec-1910(2) of this invention.

Carfilzomib was also combined with dexamethasone (see Dimopoulos MD et al., Carfilzomib and dexamethasone versus bortezomib and dexamethasone for patients with relapsed or refractory multiple myeloma (ENDEAVOR): a randomized, phase 3, open-label, multicentre study. Lancet Oncology 2016 ; 17:27-38). In one embodiment, carfilzomib is administered in combination therapy with dexamethasone and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with dexamethasone and h20F3ec1910(2) of the present invention.

Carfilzomib has also been combined with panobinostat (see Berdeja JG et al., Phase I/II study of the combination of panobinostat and carfilzomib in patients with relapsed/refractory multiple myeloma. Haematologica 2015; 100:670-676). In one embodiment, carfilzomib is administered in combination therapy with panobinostat and an anti-NTB-A antibody-drug conjugate of the invention. In another embodiment, carfilzomib is administered in combination therapy with panobinostat and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with panobinostat and h20F3ec-1910(2) of the present invention.

Carfilzomib has also been combined with pomalidomide and dexamethasone (see Shah J et al., Carfilzomib, pomalidomide, and dexamethasone (CPD) in patients with relapsed and/or refractory multiple myeloma. Blood 2015; 126: 2284-2290). In one embodiment, carfilzomib is administered in combination therapy with pomalidomide, dexamethasone and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with pomalidomide, dexamethasone and a humanized anti-20F3 antibody drug conjugate of the present invention. In another embodiment, carfilzomib is administered in combination therapy with pomalidomide, dexamethasone and h20F3ec-1910(2) of this invention.

Another typical agent for combination therapy is daratumumab (eg, DAR

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ZALEX™), a human monoclonal antibody that binds CD38 (a glycoprotein highly expressed on many myeloma cells). Daratumumab can be administered to patients by intravenous infusion for the treatment of multiple myeloma (see Lokhorst HM et al., Targeting CD38 with daratumumab monotherapy in multiple myeloma. N Engl J Med 2015; 373:1207-1219). In one embodiment, daratumumab is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, daratumumab is administered in combination therapy with a humanized anti-20F3 antibody drug conjugate of the present invention. In another embodiment, daratumumab is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Daratumumab can also be administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention and an additional agent.

Daratumumab has been combined with various additional agents for the treatment of multiple myeloma. For example, daratumumab was combined with bortezomib and lenalidomide (see Phipps C et al., Daratumumab and its potential in the treatment of multiple myeloma: overview of the preclinical and clinical development. Ther Adv Hematol 2015; 6: 120-127). In one embodiment, daratumumab is administered in combination therapy with bortezomib, lenalidomide and an anti-NTB antibody-drug conjugate of the present invention. In another embodiment, daratumumab is administered in combination therapy with bortezomib, lenalidomide and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, daratumumab is administered in combination therapy with bortezomib, lenalidomide and h20F3ec-1910(2) of the present invention.

Daratumumab has also been combined with bortezomib and dexamethasone (see Phipps C et al.). In one embodiment, daratumumab is administered in combination therapy with bortezomib, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, daratumumab is administered in combination therapy with bortezomib, dexamethasone, and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, daratumumab is administered in combination therapy with bortezomib, dexamethasone and h20F3ec-1910(2) of the present invention.

Another typical combination therapy is elotuzumab (eg, EMPLICITI™), a monoclonal antibody that binds CD319 or the signaling lymphocyte activation molecule F7 (SLAMF7), a cellular marker of malignant multiple myeloma. Elotuzumab can be administered to patients by intravenous infusion for the treatment of multiple myeloma (see Zonder JA et al., A phase 1, multicenter, open-label, dose escalation study of elotuzumab in patients with advanced multiple myeloma. Blood 2012; 120: 552-559 ). In one embodiment, elotuzumab is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, elotuzumab is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, elotuzumab is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Elotuzumab can also be administered in combination therapy with an anti-NTBA antibody-drug conjugate of the present invention and an additional agent. Elotuzumab has been combined with various additional agents for the treatment of multiple myeloma. For example, elotuzumab has been combined with lenalidomide and dexamethasone (see Lonial S et al., Elotuzumab therapy for relapsed or refractory multiple myeloma. N Engl J Med 2015; 373:621-631). In one embodiment, elotuzumab is administered in combination therapy with lenalidomide, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, elotuzumab is administered in combination therapy with lenalidomide, dexamethasone, and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, elotuzumab is administered in combination therapy with lenalidomide, dexamethasone and h20F3ec-1910(2) of this invention.

Another typical agent for combination therapy is lenalidomide (eg, REVLIMID®), an immunomodulatory agent prescribed to patients for the treatment of multiple myeloma (see Richardson PG, A randomized phase 2 study of lenalidomide therapy for patients with relapsed or relapsed and refractory multiple myeloma. Blood 2006, 108: 3458-3464). Lenalidomide can be supplied in the form of a capsule, pill or tablet for oral administration. In one embodiment, lenalidomide is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Lenalidomide can also be administered in combination therapy with an anti-antibody conjugate.

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NTB-A is the drug of this invention and an additional agent. Lenalidomide has been combined with various additional agents for the treatment of multiple myeloma. For example, lenalidomide was combined with bortezomib and dexamethasone (see Richardson PG et al., Lenalidomide, bortezomib, and dexamethasone combination therapy in patients with newly diagnosed multiple myeloma. Blood 2010; 116:679–686). In one embodiment, lenalidomide is administered in combination therapy with bortezomib, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with bortezomib, dexamethasone, and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with bortezomib, dexamethasone and h20F3ec-1910(2) of this invention.

Lenalidomide has also been combined with carfilzomib and dexamethasone (see Stewart KA et al.). In one embodiment, lenalidomide is administered in combination therapy with carfilzomib, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with carfilzomib, dexamethasone, and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with carfilzomib, dexamethasone and h20F3ec-1910(2) of this invention.

Lenalidomide has also been combined with daratumumab and bortezomib (see Phipps C et al.). In one embodiment, lenalidomide is administered in combination therapy with daratumumab, bortezomib and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with daratumumab, bortezomib and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with daratumumab, bortezomib and h20F3ec-1910(2) of the present invention.

Lenalidomide has also been combined with elotuzumab and dexamethasone (see Lonial S et al., Elotuzumab therapy for relapsed or refractory multiple myeloma. N Engl J Med 2015; 373:621-631). In one embodiment, lenalidomide is administered in combination therapy with elotuzumab, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with elotuzumab, dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, lenalidomide is administered in combination therapy with elotuzumab, dexamethasone and h20F3ec-1910(2) of this invention.

Another typical combination therapy is bortezomib (eg, VELCADE®), a proteasome inhibitor prescribed to patients for the treatment of multiple myeloma and mantle cell lymphoma (see Richardson PG et al., A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 2003;348:2609-2617). Bortezomib can be given to patients by intravenous injection. In one embodiment, bortezomib is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Bortezomib can also be administered in combination therapy with an anti-NTBA antibody-drug conjugate of the present invention and an additional agent. Bortezomib has been combined with various additional agents for the treatment of multiple myeloma. For example, bortezomib has been combined with thalidomide and dexamethasone (see Kapoor P et al., Bortezomib combination therapy in multiple myeloma. Semin Hematol 2012; 3:228-242). In one embodiment, bortezomib is administered in combination therapy with thalidomide, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the invention. In another embodiment, bortezomib is administered in combination therapy with thalidomide, dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with thalidomide, dexamethasone and h20F3ec-1910(2) of this invention.

Bortezomib has also been combined with dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide and etoposide (see Kapoor P et al.,). In one embodiment, bortezomib is administered in combination therapy with dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide, etoposide, and an anti-NTB-A antibody-drug conjugate of the invention. In another embodiment, bortezomib is administered in combination therapy with dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide, etoposide and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide, etoposide and h20F3ec-1910(2) of the present invention.

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Bortezomib has also been combined with daratumumab and lenalidomide (see Phipps C et al.). In one embodiment, bortezomib is administered in combination therapy with daratumumab, lenalidomide and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with daratumumab, lenalidomide and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with daratumumab, lenalidomide and h20F3ec-1910(2) of the present invention.

Bortezomib is also combined with lenalidomide and dexamethasone (see Richardson PG et al., 2010). In one embodiment, bortezomib is administered in combination therapy with lenalidomide, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with lenalidomide, dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with lenalidomide, dexamethasone and h20F3ec-1910(2) of this invention.

Bortezomib has also been combined with panobinostat and dexamethasone (see Richardson P et al., PANORAMA 2: panobinostat in combination with bortezomib and dexamethasone in patients with relapsed and bortezomib-refractory myeloma. Blood 2013: 122:2331-2337). In one embodiment, bortezomib is administered in combination therapy with panobinostat, dexamethasone, and an anti-NTBA antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with panobinostat, dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, bortezomib is administered in combination therapy with panobinostat, dexamethasone and h20F3ec1910(2) of this invention.

Another typical combination therapy is dexamethasone (eg, DECADRON®), a corticosteroid used to treat cancer (including multiple myeloma, leukemia and lymphoma), inflammation, allergies and nausea. Dexamethasone can be administered as a tablet, pill or capsule for oral administration or by intravenous infusion. In one embodiment, dexamethasone is administered in combination therapy with an anti-NTBA antibody-drug conjugate of the present invention. In another embodiment, dexamethasone is administered in combination therapy with a humanized anti-20F3 antibody drug conjugate of the present invention. In another embodiment, dexamethasone is administered in combination therapy with h20F3ec-1910(2) of the present invention. Dexamethasone can also be administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention and an additional agent.

Another typical combination therapy is cyclophosphamide (eg, CYTOXAN®), an alkylating agent used to treat cancer (including, but not limited to, multiple myeloma, acute myelocytic leukemia, Hodgkin and non-Hodgkin lymphoma, breast cancer and lung cancer). Cyclophosphamide can be administered by injection, infusion as a tablet, pill or capsule for oral administration, or by injection into a muscle, peritoneum or pleura. In one embodiment, cyclophosphamide is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, cyclophosphamide is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, cyclophosphamide is administered in combination therapy with h20F3ec-1910(2) of the present invention. Cyclophosphamide can also be administered in combination therapy with an anti-NTBA antibody-drug conjugate of the present invention and an additional agent.

Another typical combination therapy is melphalan, an alkylating agent used to treat cancer (including multiple myeloma and ovarian cancer). Melphalan can be given orally, as an injection, or as an infusion. In one embodiment, melphalan is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, melphalan is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, melphalan is administered in combination therapy with h20F3ec-1910(2) of the present invention. Melphalan can also be administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention and an additional agent.

Another typical combination therapy is pomalidomide (eg, POMALYST®), an immunomodulatory agent used to treat multiple myeloma. Pomalidomide can be administered in the form of a capsule, pill or tablet for oral administration. In one embodiment, pomalidomide is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, pomalidomide is administered in combination therapy with a humanized anti-20F3-22044092 antibody drug conjugate of the present invention. In another embodiment, pomalidomide is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Pomalidomide can also be administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention and an additional agent. Pomalidomide has been combined with various additional agents for the treatment of multiple myeloma. Pomalidomide was combined with dexamethasone (see Richardson P et al., Pomalidomide alone or in combination with low-dose dexamethasone in relapsed and refractory multiple myeloma: a randomized phase 2 study. Blood 2014; 123: 1826-1832). In one embodiment, pomalidomide is administered in combination therapy with dexamethasone and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, pomalidomide is administered in combination therapy with dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, pomalidomide is administered in combination therapy with dexamethasone and h20F3ec-1910(2) of this invention.

Pomalidomide has also been combined with carfilzomib and dexamethasone (see Shah J et al., Carfilzomib, pomalidomide, and dexamethasone (CPD) in patients with relapsed and/or refractory multiple myeloma. Blood 2015; 126: 2284-2290). In one embodiment, pomalidomide is administered in combination therapy with carfilzomib, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, pomalidomide is administered in combination therapy with carfilzomib, dexamethasone and a humanized anti-20F3 antibody drug conjugate of the present invention. In another embodiment, pomalidomide is administered in combination therapy with carfilzomib, dexamethasone and h20F3ec-1910(2) of this invention.

Another typical combination therapy is panobinostat (eg, FARYDAK®), a histone deacetylase (HDAC) inhibitor used to treat cancer (including multiple myeloma) (see Wolf JL et al., A phase II study of oral panobinostat (LBH589) in adult patients with advanced refractory multiple myeloma. ASH Annual Meeting Abstracts, 2008). Panobinostat can be administered in the form of a pill, capsule, or tablet for oral administration. In one embodiment, panobinostat is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, panobinostat is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, panobinostat is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Panobinostat can also be administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention and an additional agent. Panobinostat has been combined with various additional agents for the treatment of multiple myeloma. For example, panobinostat was combined with carfilzomib (see Berdeja JG et al.). In one embodiment, panobinostat is administered in combination therapy with carfilzomib and an anti-NTBA antibody-drug conjugate of the present invention. In another embodiment, panobinostat is administered in combination therapy with carfilzomib and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, panobinostat is administered in combination therapy with carfilzomib and h20F3ec-1910(2) of the present invention.

Panobinostat has also been combined with bortezomib and dexamethasone (see Richardson P et al., 2013). In one embodiment, panobinostat is administered in combination therapy with bortezomib, dexamethasone, and an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, panobinostat is administered in combination therapy with bortezomib, dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, panobinostat is administered in combination therapy with bortezomib, dexamethasone and h20F3ec-1910(2) of this invention.

Another typical combination therapy is ixazomib (NINLARO®), a proteasome inhibitor used to treat cancer (including multiple myeloma). Ixazomib can be administered orally. In one embodiment, ixazomib is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, ixazomib is administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, ixazomib is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Ixazomib can also be administered in combination therapy with an anti-NTBA antibody-drug conjugate of the present invention and an additional agent. Ixazomib has been combined with various additional agents for the treatment of multiple myeloma. For example, ixazomib was combined with lenalidomide and dexamethasone (see Moreau P et al., Ixazomib, an investigational oral proteasome inhibitor, in combination with lenalidomide and dexamethasone, significantly extends progression-free survival for patients with relapsed and/or refractory multiple myeloma: the phase 3 tourmaline-MMl

- 23 044092 study. ASH Annual Meeting Abstracts, 2015). In one embodiment, ixazomib is administered in combination therapy with lenalidomide, dexamethasone, and an anti-NTB antibody-drug conjugate of the present invention. In another embodiment, ixazomib is administered in combination therapy with lenalidomide, dexamethasone and a humanized anti-20F3 antibody-drug conjugate of the present invention. In another embodiment, ixazomib is administered in combination therapy with lenalidomide, dexamethasone and h20F3ec-1910(2) of this invention.

Other typical agents for combination therapy (especially in the treatment of non-Hodgkin's lymphoma) include anti-CD20 antibodies (including rituximab, ibritumomab tiuxetan, tositumomab, ofatumumab, veltuzumab and obinutuzumab), anti-CD52 antibodies (including alemtuzumab), anti-PD1 antibodies (including nivolumab, pidilizumab and pembrolizumab), anti-PDL1 antibodies (including durvalumab and atezolizumab), brentuximab vedotin, bendamustine and bortezomib. In one embodiment, anti-CD20 antibody, anti-CD52 antibody, anti-PD1 antibody, anti-PD1 antibody, brentuximab vedotin, bendamustine, and bortezomib are administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In one embodiment, anti-CD20 antibody, anti-CD52 antibody, anti-PD1 antibody, anti-PD1 antibody, brentuximab vedotin, bendamustine, and bortezomib are administered in combination therapy with a humanized anti-20F3 antibody-drug conjugate of the present invention. In one embodiment, anti-CD20 antibody, anti-CD52 antibody, anti-PD1 antibody, anti-PD1 antibody, brentuximab vedotin, bendamustine and bortezomib are administered in combination therapy with h20F3ec1910(2) of the present invention.

In addition, other combination therapy agents include chemotherapy regimens such as CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); CVP (cyclophosphamide, vincristine and prednisone); RCVP (rituximab+CVP); RCHOP (rituximab+CHOP); RCHP (rituximab, cyclophosphamide, doxorubicin and prednisone): RICE (rituximab + ifosamide, carboplatin, etoposide); RDHAP, (rituximab + dexamethasone, cytarabine, cisplatin); RESHAP (rituximab+etoposide, methylprednisolone, cytarabine, cisplatin); R-BENDA (rituximab and bendamustine), RGDP (rituximab, gemcitabine, dexamethasone, cisplatin). In one embodiment, one of CHOP, CVP, RCVP, RCHOP, RCHP, RICE, RDHAP, RESHAP, R-BENDA, and RGDP is administered in combination therapy with an anti-NTB-A antibody-drug conjugate of the present invention. In another embodiment, one of CHOP, CVP, RCVP, RCHOP, RCHP, RICE, RDHAP, RESHAP, R-BENDA and RGDP is administered in combination therapy with a humanized anti-20F3 antibody drug conjugate of the present invention. In another embodiment, one of CHOP, CVP, RCVP, RCHOP, RCHP, RICE, RDHAP, RESHAP, R-BENDA and RGDP is administered in combination therapy with h20F3ec-1910(2) of the present invention.

Anti-NTB-A antibodies, either as naked antibodies or as drug-antibody conjugates, are administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and/or improves at least one sign or symptom of cancer. In some cases, therapeutic efficacy may be observed in an individual patient compared to historical controls or past experience in the same patient. In other cases, therapeutic efficacy may be demonstrated in preclinical or clinical trials in a population of treated patients compared with a control population of untreated patients.

Typical doses for naked anti-NTB-A antibody are 0.1 to 50 mg/kg of patient body weight, more typically 1 to 30 mg/kg, 1 to 20 mg/kg, 1 to 15 mg/kg, from 1 to 12 mg/kg, or from 1 to 10 mg/kg, or from 2 to 30 mg/kg, from 2 to 20 mg/kg, from 2 to 15 mg/kg, from 2 to 12 mg/kg, or 2 to 10 mg/kg, or 3 to 30 mg/kg, 3 to 20 mg/kg, 3 to 15 mg/kg, 3 to 12 mg/kg, or 3 to 10 mg/kg.

Typical doses for anti-NTBA antibody-drug conjugates are 1.0 μg/kg to about 10 mg/kg, 1.0 μg/kg to about 5 mg/kg, 1.0 μg/kg to about 5 mg/kg, from about 1.0 µg/kg to about 1.0 mg/kg, from about 10 µg/kg to about 3 mg/kg, from about 10 µg/kg to about 2 mg/kg, from about 1 .0 µg/kg to 1.0 mg/kg or from about 1.0 µg/kg to 500.0 µg/kg, or from about 0 µg/kg to 80.0, 100.0, or 200.0 µg /kg.

Typical doses for PBD conjugates targeting NTB-A are generally from about 1.0 to 1.0 mg/kg, or from about 1.0 to 500.0 μg/kg, or from about 0 to 80.0. 100.0 or 200.0 mcg/kg, although alternative doses are provided.

Administration is usually carried out parenterally. The injection can also be localized directly to the tumor. Preferred is administration into the systemic circulation by intravenous or subcutaneous administration. Intravenous administration can be carried out, for example, by infusion over a period such as 30-90 minutes or by a single bolus injection.

The frequency of administration depends on the half-life of the antibody or conjugate in the circulation, the condition of the patient, and the route of administration, among other factors. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in

- 24 044092 standing patient or progression of cancer treatment. The typical frequency of intravenous administration is twice weekly and quarterly during the continuous course of treatment, although higher or lower dosing frequencies are also possible. Other typical frequencies for intravenous administration range from between weekly to three times every four weeks for a continuous course of treatment, although more or less frequent dosing is possible. For subcutaneous administration, typical dosing frequency is daily to monthly, although more or less frequent dosing is possible.

The number of doses administered depends, in part, on the nature of the disorder (eg, whether acute or chronic symptoms are present) and the response of the disorder to treatment. For acute disorders or acute exacerbations of a chronic disorder, 1 to 10 doses are often sufficient. Sometimes a single bolus dose, not necessarily in a divided form, is sufficient for an acute disorder or an acute exacerbation of a chronic disorder. Treatment may be repeated for recurrence of the acute disorder or acute exacerbation. For chronic disorders, the antibody can be administered at regular intervals, for example, weekly, biweekly, monthly, quarterly, every six months for at least 1, 5 or 10 years or for the life of the patient.

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP (GMP) conditions. Pharmaceutical compositions may be presented in unit dosage form (ie, unit dose). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or excipients. The composition depends on the chosen route of administration. For injection, antibodies can be mixed in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain auxiliary substances such as suspending, stabilizing and/or dispersing agents. Alternatively, the drug-antibody conjugates may be in lyophilized form for reconstitution with a suitable vehicle, such as pyrogen-free sterile water, prior to use. The concentration of the antibody in the liquid composition may be, for example, 1-100 mg/ml, such as 10 mg/ml.

Treatment with naked antibodies or antibody drug conjugates described herein can be combined with chemotherapy, radiation therapy, stem cell treatment, or other treatments effective against the disorder being treated. Suitable classes of other agents that can be administered with an anti-NTB-A antibody or drug-antibody conjugate include, for example, antibodies to other receptors expressed on cancer cells, anti-tubulin agents (eg, auristatins), DNA minor groove binding agents , DNA replication inhibitors, alkylating agents (eg, platinum complexes such as ds-platinum, mono(platinum), bis(platinum) and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides , fluorinated pyrimidines, ionophores, lexitropsins, nitrosourea derivatives, platinones, preforming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids and the like.

Treatment with naked antibodies or an anti-NTB-A antibody-drug conjugate, optionally in combination with any of the other agents or regimens described above, alone or as an anti-NTB-A antibody-drug conjugate, may increase median progression-free or overall survival survival time of patients having cancer expressing NTB-A (eg, multiple myeloma, AML, NHL (non-Hodgkin's lymphoma)), especially when recurrent or persistent, is at least 30 or 40%, but preferably 50%, from 60 to 70% or even 100% or more compared to the same treatment (eg chemotherapy) but without the anti-NTB-A antibody alone or as a conjugate. In addition, or alternatively, treatment (eg, standard chemotherapy), including an anti-NTB-A conjugate, may increase the complete response rate, partial response rate, or objective response rate (complete+partial) of patients who have NTB-A- expressing the cancer by at least 30 or 40%, but preferably by 50%, 60% to 70%, or even 100% compared to the same treatment (eg, chemotherapy) but without the anti-NTB-A antibody.

Typically, in a clinical trial (eg, phase II, phase II-III, or phase III trials), the above increases the median progression-free survival and/or response rate of patients treated with standard therapy plus anti-NTB-A antibodies compared with a control group of patients receiving standard therapy alone (or plus placebo) are statistically significant, for example at the p=0.05 or 0.01 or even 0.001 level. Complete and partial response rates are determined by objective criteria commonly used in clinical trials for the treatment of cancer, such as those listed or adopted by the National

- 25 044092 by the Cancer Institute and/or the Food and Drug Administration.

In other applications, the anti-NTB-A antibodies disclosed herein may be used to detect NTB-A in the context of clinical diagnosis or treatment, or in research. Expression of NTB-A in cancer indicates that the cancer is treatable with the antibodies of this invention. Antibodies can also be sold as research reagents for laboratory studies in detecting NTB-A-bearing cells and their response to various stimuli. In such applications, the anti-NTB-A antibody may be labeled with a fluorescent molecule, spin-labeled molecule, enzyme, or radioisotope and may be provided as a kit with all necessary reagents to perform the NTB-A assay. Antibodies can also be used to purify NTB-A, for example using affinity chromatography.

All patent applications, other publications, accession numbers and the like mentioned above or below are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual claim had been specifically and individually indicated to be incorporated by links. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number on the effective filing date of this application is implied. The effective filing date means the earlier of the actual filing date or the filing date of the priority application related to the accession number, as applicable. Likewise, if different versions of a publication are published at different times, the version most recently published on the effective filing date of the application is implied unless otherwise noted. Any function, step, element, embodiment or aspect of the invention may be used in combination with any other unless specifically stated otherwise. Although the present invention has been described in more detail by way of illustration and example for the sake of clarity and understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

Examples

The cell lines described in the following examples were maintained in culture in accordance with the conditions established by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ) or as it is otherwise known.

Example 1: Antibody selection

Lymphocytes collected from the spleen and lymph nodes of NTB-A antibody-producing mice were fused with myeloma cells. The confluent cells were reconstituted overnight in hybridoma growth medium. After recovery, cells were centrifuged and then plated on semisolid media. The hybridomas were incubated and IgG hybridoma clones were collected. Anti-20F3 antibody was one of the few antibodies that showed strong cytotoxicity as an ADC and bound to cynomolgus NTB-A.

Example 2: Construction of Humanized Antibodies

Humanized antibodies were derived from mouse 20F3 antibody. Five humanized heavy chains (HA-HE) and four humanized light chains (LA-LD) were designed to include back mutations at different positions. In some cases, back mutations will match the mouse germline, but in other cases they will not (as is the case with somatic mutations). Humanized heavy and light chains were paired. See fig. 1 and 2 for sequence alignment and table. 1-4.

Table 1. Humanizing mutations in 20F3 heavy chain variants

Option _VH Exon V _H acceptor sequence Donor framework residues hV _H A VH7-4-1 None hV _H B VH7-4-1 H2 and H73 hV _H C VH7-4-1 H2, H44, H73 and H76 hV _H D VH7-4-1 H2, H44, H46, H73, H76 hV _H E VH7-4-1 H2, H38, H44, H46, H68, H73, H76 and H91

Table 2. Humanizing mutations in 20F3 light chain variants

Option Vl Acceptor sequence of exon V _L Donor framework residues hV _L A VK3-11 None hV _L B VK3-11 L46, L47, L71 hV _L C VK3-11 LI, L5, L46, L47, L71 hV _L D VK3-11 LI, L21,L5, L46, L47, L58,L71

- 26 044092

Table 3. Specific mutations in 20F3 heavy chain variants

Option N2 NZN N44 N46 N6K N73 N76N91

NV Г· R С В V К» S¥

NS FRD ^:!; E V K ⁴ GS'¥

Hl) V R GG K V K* FV-Y

NOT L K* 1U K- L* K- N' ^: F* *Remains of the mouse *Remains of the mouse

Table 4. Specific mutations in 20F3 light chain variants

Option LI 15 L21 146 1.47 L5H L7I I.A E 1 1. I. 1. I F LB E T L P ^: w 1 Y EU Q ^: · s.* I. P*W'·· 1 Y'·' LD S·'· NT· P* _W - V- Υ ^Λ

*Mouse remains

Example 3: EC50 and Kd measurements

To determine EC50, three-point dose titrations of unlabeled humanized anti-human NTB-A antibodies were mixed with a constant concentration (5 nM final) of mouse 20F3 antibody conjugated to Alexa Fluor 647. Antigen positive Ramos cells were seeded at 1 × 10 ⁵ cells per well in a 96-well plate with V-bottom (Thermo Scientific, Rochester, NY). Fifty-fold serial dilutions of antibodies were prepared in FACs buffer (PBS+2% fetal bovine serum) and added to cells in two parallel assays. Antibody solutions were incubated with cells for 1 hour on ice, without access to light. Cells were washed twice with FACs buffer and analyzed on an LSRII flow cytometer (BD Biosciences, San Jose, CA). EC50 values were determined using GraphPad Prism software (La Jolla, CA). The results are presented below in Table 5.

Table 5. Determination of EC50 binding for various permutations of humanized heavy and light chains of humanized 20F3 compared to mouse 20F3

EC50 (nM)

H.A. HB HC HD H.E. L.A. 14? 71 25 IS 31' from 12 b 5 3 4 L.C. ......27..... 12 8 6 10 ' LD 12 4 5 3 2

The EC50 for 20F3 mouse is 2 nM.

Dose titration of anti-human NTB-A antibody conjugated to Alexa Fluor 647 (h20F3_HDLD-AF647) was used to generate binding saturation curves. Antigen positive Ramos cells were seeded at 1 × ¹⁰⁵ cells per well in a 96-well V-bottom plate (Thermo Scientific, Rochester, NY). Threefold serial dilutions of antibodies at a concentration of 2X were prepared in FACs buffer (PBS+2% fetal bovine serum) and added to the cells in two parallel assays. Antibody solutions were incubated with cells for 1 hour on ice, without access to light. Cells were washed twice with FACs buffer and analyzed on an LSRII flow cytometer (BD Biosciences, San Jose, CA). KD values were determined using GraphPad Prism software (La Jolla, CA). Anti-human NTB-A h20F3_HDLD and chimeric 20F3 antibodies were tested for binding to 293-F17 cells overexpressing NTB-A. 293-F17 Cynomolgus/NTB-A Cynomolgus. Cells were seeded at ^5x105 cells/well in FACS buffer (PBS+2% fetal bovine serum+0.02% azide) and incubated with 20 μg/ml antibody for 30 min on ice. Cells were washed twice and stained with 30 mg/ml goat anti-human IgG-PE (Jackson ImmunoResearch, West Grove, PA) for 30 min on ice and protected from light. Cells were again washed twice with FACs buffer. Stained cells were analyzed on a Calibur FACs flow cytometer (BD Biosciences, San Jose, CA). The results are presented in the table below. 6.

- 27 044092

Table 6. Saturation binding for humanized 20F3 antibody variant HDLD compared to chimeric 20F3

Antibody Ramos Tying (cynomolgus) Kd (nM) Chimeric 1, 68 Positive HDLD 2.2 Positive

Example 4. Effector function

Complement Dependent Cytotoxicity (CDC): Normal human T cells (All Cells, Alameda, CA) or cancer cell lines (ATCC, Manassas, VA) were labeled with 5 μM Sytox Green (Life Technologies, Grand Island, NY ), and were then linked to serial titration sequences of h20F3 anti-human NTB-A antibody or PBD-dimer ADC (0.0250 μg/ml) in RPMI1640+1020% human serum (Complement Technology, Tyler, TX or Solomon Park Research Laboratories , Kirkland, Washington). Cells were incubated at 37°C, 5% CO ₂ for 2 h, and fluorescence from lysed cells was measured using an Envision plate reader (Perkin Elmer, Waltham, MA). Data were expressed as the percentage of maximum cell lysis determined by a positive control of 1% cells treated with Triton X-100 (Sigma, St. Louis, MO).

Three types of cells were used for the analysis: normal human T cells, WIL2-S cells and Raji cells. The expression of the corresponding receptors for each cell type is presented in table. 7 and is indicated as the number of receptors per cell.

Table 7. Receptor expression

Cell type NTB-A CD52 CD20 T cells 6900 115200 0 WIL2S 19900 34200 502700 Raj i 39900 29200 394100

As can be seen from Table 8 below, the humanized IgG1 antibody 20F3-HDLDwt and the EC variant (antibodies with cysteine at position 239 bearing the designation EC) do not have CDC activity against normal resting T lymphocytes and the WIL2-S and Raji cell lines. Similar results were observed for ADC h20F3ec-1910(2).

Table 8. CDC activity for humanized antibodies 20F3 and ADC compared to Campat and Rituximab

Cell type Maximum % specific cell lysis h20F3wt h20F3ec h20F3ec- 1910(2) Kampat (CD52) Rituxim ab (CD20) Normal T cells person 1.3+2.8 2.2+1.8 2.1+3.0 108+5.2 Not tested WIL2-S cell line 0.0+0.0 0.14+0.2 0.3+0.0 Don't test dug 66+1.4 Raj 1 cell line 2.1 +0.6 3.3+0.7 0.0+0.0 Don't test dug 45+5.7

Antibody-dependent cellular cytotoxicity (ADCC)

Activity Assay: ADCC cytotoxic activity was measured using chromium-51 release, in which effector natural killer (NK) cells kill (lyse) target cells by binding to an antibody or ADC on the target cells. WIL2-S cells were labeled with chromium-51, bound to antibody (0.1 ng/ml - 10 μg/ml) and then incubated with NK cells at an effector cell:target cell ratio of 10:1 for 4 hours at 37 °C, 5% CO ₂ . The supernatant was removed onto a filter plate, and the amount of chromium-51 was measured using TopCount (Perkin Elmer, Waltham, MA). Data were expressed as the percentage of maximum specific cell lysis determined by the positive control of 1% cells treated with Triton X-100 (Sigma, St. Louis, MO).

A flow cytometry test was used to measure ADCC activity on normal human T cells. PKH2-labeled normal human T cell targets were bound to antibody titers (0.1 ng/ml - 10 μg/ml) and then incubated with effector NK cells at an effector cell:target cell ratio of 10: 1 for 4 hours at 37°C, 5% CO2. Viability was determined by 7-AAD incorporation measured on a FACsCalibur flow cytometer (BD Biosciences, San Jose, CA).

As can be seen from table. 9 below, the humanized antibody 20F3-HDLDwt and the ec variant have low to moderate ADCC activity. The PBD dimer ADC h20F3ec-1910 (2) demonstrated decreased ADCC activity relative to the unconjugated h20F3ec antibody. Receptor numbers are disclosed in table. 7.

Table 9. ADCC activity for humanized 20F3 compared to Campat and Rituximab

Cell type Maximum % specific cell lysis h20F3wt h20F3ec h20F3ec- 1910(2) Kampat (CD52) Rituxim ab (CD20) Normal Human T cells 38+0.8 4 0+0.6 19+1.0 60+3.5 Not tested WIL2-S cell line 21+0.6 25+2.4 16+3.3 Not tested 42+6.0

To assess NTB-A-mediated ADCP activity, normal T lymphocytes or tumor cells were labeled with the red fluorescent membrane dye, PKH26, then labeled with antibody or ADC in a 5-point, 10-fold dilution series starting at 2 μg/ml. Cells were then incubated for 30 min on ice and washed twice with PBS. The cells were mixed with monocyte-derived macrophages for one hour at 37°C. The cell mixture was then stained by conjugation with Alexa Fluor 488-conjugated mouse anti-CDHb to label macrophages, and analyzed by flow cytometry to detect PKH26+CD11b+ dual-isotope-labeled fluorescent cells. Phagocytic activity was calculated as the quotient of (percentage of PKH26+CD11b+ cells) divided by (percentage of CD11b+ cells) multiplied by one hundred.

As can be seen from table. 10 below, the humanized antibody 20F3-HDLDwt and the ec variant have ADCP activity moderately lower than the control antibodies rituximab and campate in cancer cell lines. Similar results were observed for the ADC h20F3ec-1910(2).

Table 10. ADCP activity for humanized 20F3 compared to Campat and Rituximab

Cell type Maximum % specific cell lysis h20F3wt IgGl h20F3ec h20F3ec- 1910 (2) Kampat (CD52) Rituxim ab (CD20) Normal T cells person 42 33 37 57 Not tested WIL2-S cell line 45 45 45 Not tested 56 Line cells Raji 48 44 45 Not tested 57

Example 5: ADC Internalization

Multiple myeloma cell lines (MM.1R or U-266) were bound to saturating concentrations of ADC (10 μg/ml), washed with medium, and incubated at 37 or 4°C. Time points were noted at which samples were stained with labeled goat anti-human IgG-PE antibody (Jackson ImmunoResearch, West Grove, PA) and fixed in 1% PFA/PBS. After all time points were noted, mean fluorescence intensity was measured on a Fibs Calibur flow cytometer (BD Biosciences, San Jose, CA). The PBD dimer anti-NTB-A ADC h20F3ec-1910 (2) was rapidly internalized into MM.1R cells, with 80% of the ADC internalized within 4 h of incubation at 37°C. Only 20% of h20F3ec-1910(2) ADC was internalized after 4 h when cells were maintained at 4°C.

The cell surface and subcellular localization of ADC in MM.1R cells was also detected using a mouse anti-idiotypic antibody specific for the h20F3ec antibody. ADC was allowed to bind MM.1R cells for 30 min at 4°C and ADC was found to be rapidly internalized into lysosomes.

- 29 044092

Example 6 ADC Cytotoxicity on MM Cancer Cell Lines

Humanized antibodies (HDLD) were tested as PBD and auristatin drug-antibody conjugates. The antimitotic agent monomethyl auristatin E (MMAE) was conjugated to an anti-NTB-A mAb via a cleavable cathepsin valine-citrulline (vc)-liner. A second auristatin, Auristatin-2, was conjugated to anti-NTB-A mAb via a peptide linker. For PBD conjugates, the drug linker SGD-1910 was conjugated to the anti-NTBA antibody through the thiol group of a cysteine residue introduced at position 239 of the IgG1 antibody chain (numbered according to the EU numbering system), and the average drug load was approximately 2 drug molecules per antibody. Antibodies with cysteine at position 239 are designated ec. Preparation was as described in WO 2011/130613 using the anti-NTBA antibodies described herein. ADCs were serially diluted 3-fold in media to generate a 10-point dose curve (1000 ng/mL - 0.05081 ng/mL) and applied to multiple myeloma cells cultured in 96-well assay plates. Cell lines were treated with anti-NTB ADC in four parallel trials and incubated for 96 h at 37°C, 5% CO ₂ . Cells were assayed for viability using a Glo fluorescent cytotoxicity cell titration assay (Promega) and data collected using an Envision plate reader (Perkin Elmer). Dose-response curves and IC50 values were calculated using GraphPad Prism software. The results are presented in tables 11 and 12.

The mechanism of action on MM.1R cells was also analyzed. MM.1R cells were treated with ADC, and activation of ATR and ATM kinases was quantified by Western blotting with phosphoepitope-specific antibodies. ADC-activated ATM/ATR levels are almost equivalent to the free PBD dimer preparation, thus ADC activates the DNA damage response in MM cells.

Table 11. IC50 values of various humanized anti-20F3 drug conjugates in multiple myeloma cell lines (IC50 values in ng/ml, hIgG-non-binding negative control antibody)

Antibody Linker drug Drug load Multiple myeloma cell lines (number of NTB-A receptor/cell) E.J.M. (9800) MM.1R (21000) MM.1S (14800) U2-66 (18300) LP-1 (0) h20F3 vcMMAE 4 162 696 >1000 22 >1000 h20F3 Auristatin-2 8 22 6 7 4 >1000 h20F3ec PBD dimer 2 >1000 1 1 8 >1000 hlgG vcMMAE 4 >1000 >1000 >1000 >1000 >1000 hlgG Auristatin-2 8 >1000 >1000 >1000 >1000 >1000 hlgG PBD dimer 2 >1000 >1000 >1000 >1000 >1000

Table 12. IC50 values of various humanized anti-20F3 antibody:drug conjugates in NHL and AML cell lines (IC50 values in ng/ml, hIgG non-binding negative control antibody)

Antibody Linker drug Drug load NHL and AML cell lines (number of NTB-A receptor/cell) Ramos (24500) CA46 (15000) HEL92.1. 7 (10200) HNT-34 (57300) h20F3 vcMMAE 4 eleven 44 >1000 19 h20F3 Auristatin-2 8 2 3 455 2 h20F3ec PBD dimer 2 0.8 0.9 2 0.5 hlgG vcMMAE 4 >1000 >1000 >1000 >1000 hlgG Auristatin-2 8 >1000 >1000 >1000 >1000 hlgGec PBD dimer 2 >1000 >1000 >1000 >1000

Example 7 ADC Cytotoxicity on Human Lymphocytes

To evaluate the cytotoxic effects of anti-NTB-A ADCs on resting human T and B lymphocytes, purified cells were seeded into black 96-well plates for analysis. Cells were then treated with anti-NTB-A ADC starting at 10 μg/ml, titrated 5-fold for a total of 8 dilution points. For free drug treatments, cells were dosed with either MMAE starting at 1000 nM or free PBD dimer starting at 100 nM and titrated 5-fold for a total of 8 dilution points. The plates were incubated at 37°C, 5% CO ₂ for 96 hours, then allowed to equilibrate to room temperature. An equal volume was added to each well.

- 30 044092 Cell-Titer Glo reagent and the plate was incubated for a further 30 minutes at room temperature. The plates were then read on a Perkin Elmer Envision plate reader. Dose-response curves and IC50 values were calculated using GraphPad Prism software. The results are presented in table. 13.

Table 13. IC50 values of chimeric antibody 20F3 and free drug for resting human T and B lymphocytes

Donor Cell type NTB-A (number of receptors) c20F31910 (2) (ng/ml) c20F3- vcMMAE(4) (ng/ml) Available MMAE (nM) Free PBD dimer (nM) 1 B cell 6811 1468 >1000 3 1 2 B cell 11037 1270 >1000 2 1 3 B cell 8771 3421 >1000 5 3 4 T cell 5547 >1000 >1000 >1000 28 5 T cell 7979 >1000 >1000 >1000 25 6 T cell 5522 >1000 >1000 >1000 27

Resting human T cells were not affected by PBD dimers or the aristatin chimeric ADC 20F3 in a 96-hour cytotoxic assay. The free PBD dimer was cytotoxic to resting T lymphocytes, indicating that they are sensitive to this DNA-damaging drug. Therefore, cell surface levels of NTB-A are too low for ADC C20F3-1910 (2) to internalize sufficient amounts of PBD drug to kill resting T cells. In contrast, neither the free drug MMAE nor the ADC C20F3-vcMMAE (4) was effective against resting T cells. The remaining human B cells were sensitive to both free drug medium MMAE and PBD dimer. However, ADC C20F3-1910 (2) has a cytotoxic effect on resting B lymphocytes only at high ADC concentrations (>1000 ng/ml), while ADC C20F3-vcMMAE (4) has no cytotoxic effect. Therefore, cell surface levels of NTB-A are too low on B cells to mediate potent cytotoxic effects by PBD dimer or auristatin C20F3.

Example 8. In vivo studies of MM xenograft

NSG (NOD SCID gamma; NOD.Cg-Prkdc ^sCid n2rg ^tm1Wjl /SzJ) mice were implanted with 1 million MM.1R cells per animal intravenously to generate a disseminated multiple myeloma model. Five days after tumor cell implantation, n=10 mice per treatment group received one intraperitoneal injection of the PBD ADC h2F3ec-1910 (2) or the non-binding control PBD ADC hIgGec-1910 (2). PBD ADC dose levels were 333, 111, and 37 μg/kg. Mice with elevated tumor burden were sacrificed when symptoms of hindlimb paralysis, cranial tumor, and/or morbidity appeared. As shown in FIG. 3, PBD dimer ADC HDLD h20F3ec-1910 (2) induced long-term complete responses in 10/10 mice at all doses (single dose), while control mice carrying the no-binding dose of PBD ADC all died due to disease at 80th day of the study. A statistically significant difference between groups (P < 0.0001 Mantel-Cox test) of ADC h20F3ec-1910(2) (37 μg/kg) versus control ADC hIgGec-1910(2) (333 μg/kg) was achieved.

NSG (NOD SCID gamma; NOD.Cg-Prkdc ^scid n2rg ^tm1Wjl /SzJ) mice were implanted with 5 million U266 cells per animal intravenously to generate a disseminated multiple myeloma model. Five days after tumor cell implantation, n=10 mice per treatment group received one intraperitoneal injection of the PBD ADC h2F3ec-1910 (2) or the non-binding control PBD ADC hIgGec-1910 (2). PBD ADC dose levels were 111, 56, and 28 μg/kg. Mice with elevated tumor burden were sacrificed when symptoms of hindlimb paralysis, cranial tumor, and/or morbidity appeared. As shown in FIG. 4, PBD dimer ADC HDLD h20F3ec-1910 (2) induced long-term complete responses in 9/10 mice at 111 μg/kg (single dose). At lower doses (28 and 56 μg/kg) h20F3ec-1910 (2) there was a significant (P < 0.0001 min Mantel-Cox test) delay in disease onset versus control PBD ADC hIgGec-1910 (2) (111 μg/kg) .

NSG (NOD SCID gamma; NOD.Cg-Prkdc ^scid Il2rg ^to1Wjl /SzJ) mice were implanted with 10 million EJM cells per animal intravenously to generate a disseminated multiple myeloma model. Five days after tumor cell implantation, n=10 mice per treatment group received one intraperitoneal injection of the PBD ADC h2F3ec-1910 (2) or the non-binding control PBD ADC hIgGec-1910 (2). PBD ADC dose levels were 333, 111, and 37 μg/kg. Mice with elevated tumor burden were sacrificed when symptoms of hindlimb paralysis, cranial tumor, and/or morbidity appeared. As shown in FIG. 5, PBD dimer ADC HDLD h20F3ec1910 (2) induced long-term complete responses in 8/10 mice at 333 μg/kg (single dose). At lower doses (37 and 111 μg/kg) h20F3ec-1910 (2) there was a significant (P < 0.0001 min Mantel-Cox test) delay in disease onset versus the control PBD ADC hIgGec-1910 (2) (333 μg/kg). kg).

- 31 044092

SCID (CB-n/Sz-Prkdc ^scld ) mice were implanted with 5 million HNT-34 acute myeloid leukemia cells per animal subcutaneously. When the mean tumor volume reached 100 mm ³ , n=10 mice per treatment group received one intraperitoneal injection of the PBD ADC h2F3ec-1910 (2) or the non-binding control PBD ADC hIgGec-1910 (2). PBD ADC dose levels were 333, 111, and 37 μg/kg. Individual mice were sacrificed when the subcutaneous HNT-34 tumor volume reached 1000 mm ³ . As shown in FIG. 6, PBD dimer ADC HDLD h20F3ec-1910 (2) induced long-term complete responses in all mice at doses of 333 and 111 μg/kg. At a lower dose of 37 μg/kg, h20F3ec-1910 (2) produced a strong delay in tumor formation and 2/10 durable complete responses. The control PBD ADC hIgGec-1910 (2) produced 0/10 complete responses at 333 μg/kg.

NSG (NOD SCID gamma; NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ) mice were implanted with 1 million MM.1R cells per animal intravenously to generate a disseminated multiple myeloma model. Five days after tumor cell implantation, n=10 mice per treatment group received one intraperitoneal injection of the ADC h20F3-Auristatin2(8) or the non-binding control ADC hIgG-Auristatin2(8). Auristatin2 ADC dose levels were 3.0 and 1.0 mg/kg. Mice with elevated tumor burden were sacrificed when symptoms of hindlimb paralysis, cranial tumor, and/or morbidity appeared. As shown in FIG. 7, HDLD ADC h20F3-Auristatin2(8) caused a significant delay in tumor formation (P<0.0001 Mantel-Cox test) at doses of 3.0 and 1.0 mg/kg versus control ADC hIgG-Auristatin2(8) (3.0 mg/kg) in the dispersed model MM.1R.

NSG (NOD SCID gamma; NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ) mice were implanted with 5 million U266 cells per animal intravenously to generate a disseminated multiple myeloma model. Five days after tumor cell implantation, n=10 mice per treatment group received one intraperitoneal injection of either the anti-NTB-A ADC h20F3-vcMMAE(4), the ADC h20F3Auristatin2(8), or the non-binding control ADC hIgG-vcMMAE(4) and hIgG -Auristatin2(8). ADC dose levels were 3.0 and 1.0 mg/kg. Mice with elevated tumor burden were sacrificed when symptoms of hindlimb paralysis, cranial tumor, and/or morbidity appeared. As shown in FIG. 8 ADC h20F3-vcMMAE(4) induced durable complete responses in 8/10 mice at 3.0 μg/kg. The ADC h20F3-Auristatin2(8) produced 10/10 durable complete responses at doses of 1.0 and 3.0 mg/kg. The ADC NTB-A Auristatin2 had greater antitumor activity than the ADC VCMMAE (4), especially at a dose of 1.0 mg/kg.

NSG (NOD SCID gamma; NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ) mice were implanted with 10 million EJM cells per animal intravenously to generate a disseminated multiple myeloma model. Five days after tumor cell implantation, n=10 mice per treatment group received one intraperitoneal injection of either the anti-NTB-A ADC h20F3-vcMMAE(4), the ADC h20F3Auristatin2(8), or the non-binding control ADC hIgG-vcMMAE(4) and hIgG -Auristatin2(8). ADC dose levels were 3.0 and 1.0 mg/kg. Mice with elevated tumor burden were sacrificed when symptoms of hindlimb paralysis, cranial tumor, and/or morbidity appeared. As shown in FIG. 9, HDLD ADC h20F3-vcMMAE(4) caused a significant (P=0.0002 Mantel-Cox test) delay in tumor formation compared with control ADC hIgG-vcMMAE(4) at a dose of 3.0 mg/kg. The ADC h20F3-Auristatin2(8) produced 2/10 durable complete responses at doses of 1.0 mg/kg and 3.0 mg/kg, respectively. ADC NTB-A Auristatin2 had significantly greater antitumor activity than ADC vcMMAE(4) at doses of 1.0 mg/kg (P=0.0002 Mantel-Cox test) and 3.0 mg/kg (P<0.0001 test Mantel-Cox).

SCID (SV-n/Sz-Prkdc ^sCld ) mice were implanted with 5 million HNT-34 acute myeloid leukemia cells per animal subcutaneously. When the mean tumor volume reached 100 mm ³ , n=10 mice per treatment group received one intraperitoneal injection of either anti-NTB-A ADC h20F3vcMMAE(4), ADC h20F3-Auristatin2(8), or the non-binding control ADC hIgG-vcMMAE(4) and hIgG-Auristatin2(8). ADC dose levels were 3.0 and 1.0 mg/kg. Individual mice were sacrificed when the subcutaneous HNT-34 tumor volume reached 1000 mm ³ . As shown in FIG. 10, both the HDLD ADC vcMMAE(4) and Auristatin2(8) h20F3 were highly active in the subcutaneous HNT-34 AML model. The ADC h20F3-vcMMAE(4) induced durable complete responses in 4/10 or 9/10 mice at 1.0 or 3.0 μg/kg, respectively. The ADC h20F3-Auristatin2(8) produced 3/10 or 9/10 durable complete responses at doses of 1.0 and 3.0 mg/kg, respectively.

Example 9 In Vivo Non-Hodgkin's Lymphoma Xenograft Studies

SCID (CB-n/Sz-Prkdc ^scid ) mice were implanted with 5 million Raji non-Hodgkin's lymphoma cells per animal subcutaneously. When the mean tumor volume reached 100 mm ³ , n=6 mice per treatment group received one intraperitoneal injection of PBD ADC h2F3ec-1910 (2) or control PBD ADC hIgGec-1910 (2). PBD ADC dose levels were 100, 50, and 25 μg/kg. Individual mice were sacrificed when the subcutaneous volume of the Raji tumor reached 1000 mm ³ . As shown in FIG. 11, PBD dimer ADC HDLD h20F3ec-1910 (2) induced long-term complete responses in all mice at 100 μg/kg. In addition, the PBD ADC h20F3ec-1910 (2) induced robust complete responses in 4/6 mice at a dose of 50 μg/kg, while tumor growth inhibition was observed at a dose of 25 μg/kg. Control PBD ADC

- 32 044092 hIgGec-1910 (2) had no antitumor activity at 100 μg/kg and caused 0/6 complete responses.

SCID (CB-n/Sz-Prkdc ^scld ) mice were implanted with 5 million WSU-DLCL2 non-Hodgkin lymphoma cells per animal subcutaneously. When the mean tumor volume reached 100 mm ³ , n=8 mice per treatment group received one intraperitoneal injection of PBD ADC h2F3ec-1910 (2) or control PBD ADC hIgGec-1910 (2). PBD ADC dose levels were 100, 50, and 25 μg/kg. Individual mice were sacrificed when the subcutaneous WSU-DLCL2 tumor volume reached 1,000 mm ³ . As shown in FIG. 12, PBD dimer ADC HDLD h20F3ec-1910 (2) induced long-term complete responses in 2/8 mice at 100 μg/kg. At lower doses of 25 and 50 μg/kg, the PBD ADC h20F3ec-1910 (2) caused measurable delays in tumor growth relative to untreated mice. The control PBD ADC hIgGec-1910(2) had no antitumor activity at 100 μg/kg and produced 0/8 complete responses.

List of sequences

SEQ ID NO: 1 is the amino acid sequence of human NTB-A.

MLWLFQSLLFVFCFGPGNWSQSSLTPLMVNGILGESVTLPLEFPAGEKVNFITWLFNE TSLAFIVPHETKSPEIHVTNPKQGKRLNFTQSYSLQLSNLKMEDTGSYRAQISTKTSAKLSSYT LRILRQLRNIQVTNHSQLFQNMTCELHLTCSVEDADDNVSFRWEALGNTLSSQPNLTVSWDPRI SSEQDYTCIAEN AVSNLSFSVSAQKLCEDVKIQYTDTKMILFMVSGICIVFGFIILLLLVLRKR RDSLSLSTQRTQGPAESARNLEYVSVSPTNNTVYASVTHSNRETEIWTPRENDTITIYSTINHS KESKPTFSRATALDNW

SEQ ID NO: 2 is the amino acid sequence of cynomolgus NTB-A.

MLWLFQSLLFVFCFGPGNLVSQSSSTPLMVNGVLGESVILPLELSAGEMIASITWLCNG TSLAFIEPSETKSPNIRVTHPKQRKRLNFTQSYSLKLSNLEMEDTGSYSAQITTETSVKLSSYT LRIFRQLRSIQVNNYSQLFQNRTCEIHLTCSVEDADDNVSFRWEALGSTLSSEPNITTSWDPRI SGEQDYTCIAENAVSN LSFSVSAQKLCGDVKIQYTDTKMILFWFGICIVTGFIIMLLLVLRKR RDSLPLSTQRTQGPAEPAGNIEYVSVSPVNNTVYASVTHSNRETEISTPIKNATVTIYSTVNHS KESKPTFSRATALDNW

SEQ ID NO: 3 is the amino acid sequence of the mouse 20F3 mature heavy chain variable region.

QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKDLKWMGWINTYSGEPR

YADDFKGRFAFSLEKSANTAYLQINNLKNEDMATYFCARDYGRWYFDVWGTGTTVTVSS

SEQ ID NO: 4 is the amino acid sequence of the humanized 20F3 mature heavy chain variable region without backmutations and with a mouse CDR, as determined by Kabat.

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYSGEPR

YADD FKGRFVFS LDT SVS TAYLQIS S LKAEDTAVYYCARDYGRWY FDVWGQGT TVTVS S

SEQ ID NO: 5 is the amino acid sequence of the humanized 20F3 HA mature heavy chain variable region.

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYSGEPR

YADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYGRWYFDVWGQGTTVTVSS

SEQ ID NO: 6 is the amino acid sequence of the mature heavy chain variable region of the humanized 20F3 NV.

QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYSGEPR

YADDFKGRFVFSLDKSVSTAYLQISSLKAEDTAVYYCARDYGRWYFDVWGQGTTVTVSS

SEQ ID NO: 7 is the amino acid sequence of the mature heavy chain variable region of the humanized 20F3 HC.

QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQDLEWMGWINTYSGEPR

YADDFKGRFVFSLDKSVNTAYLQISSLKAEDTAVYYCARDYGRWYFDVWGQGTTVTVSS

SEQ ID NO: 8 is the amino acid sequence of the humanized 20F3 HD mature heavy chain variable region.

QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQDLKWMGWINTYSGEPR

YADDFKGRFVFSLDKSVNTAYLQISSLKAEDTAVYYCARDYGRWYFDVWGQGTTVTVSS

SEQ ID NO: 9 is the amino acid sequence of the humanized 20F3 HE mature heavy chain variable region.

QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQDLKWMGWINTYSGEPR

YADDFKGRFAFSLDKSVNTAYLQISSLKAEDTAVYFCARDYGRWYFDVWGQGTTVTVSS

SEQ ID NO: 10 is the amino acid sequence of mouse 20F3 and humanized 20F3 CDR-H1 as determined by Kabat.

NYGMN

SEQ ID NO: 11 is the amino acid sequence of mouse 20F3 CDR-H2 and humanized 20F3 as determined by Kabat.

WINTYSGEPRYADDFKG

SEQ ID NO: 12 is the amino acid sequence of mouse 20F3 and humanized 20F3 CDR-H3 as determined by Kabat.

DYGRWYFDV

SEQ ID NO: 13 is the amino acid sequence of the mouse 20F3 mature light chain variable region.

QIVLSQSPAILSASPGEKVTMTCRASSSVSHMHWYQQKPGSSPKPWIYATSNLASGVPA

RFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSTPRTFGGGTKLEIKR

SEQ ID NO: 14 is the amino acid sequence of the mature heavy chain variable region of humanized 20F3 without back mutations and with a mouse CDR, as determined by Kabat.

EIVLTQSPATLSLSPGERATLSCRASSSVSHMHWYQQKPGQAPRLLIYATSNLASGIPA

RFSGSGSGTDFTL TISSLEPEDFAVYYCQQWSSTPRTFGGGTKVEIKR

SEQ ID NO: 15 is the amino acid sequence of the humanized 20F3 LA mature light chain variable region.

EIVLTQSPATLSLSPGERATLSCRASSSVSHMHWYQQKPGQAPRLLIYATSNLASGIPA

RFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSTPRTFGGGTKVEIKR

SEQ ID NO: 16 is the amino acid sequence of the humanized 20F3 LB mature light chain variable region.

EIVLTQSPATLSLSPGERATLSCRASSSVSHMHWYQQKPGQAPRPWIYATSNLASGIPA

RFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSTPRTFGGGTKVEIKR

SEQ ID NO: 17 is the amino acid sequence of the humanized 20F3 LC mature light chain variable region.

QIVLSQSPATLSLSPGERATLSCRASSSVSHMHWYQQKPGQAPRPWIYATSNLASGIPA

RFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSTPRTFGGGTKVEIKR

SEQ ID NO: 18 is the amino acid sequence of the mature light chain variable region of the humanized 20F3 LD.

QIVLSQSPATLSLSPGERATMSCRASSSVSHMHWYQQKPGQAPRPWIYATSNLASGVPA

RFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSTPRTFGGGTKVEIKR

SEQ ID NO: 19 is the amino acid sequence of mouse 20F3 and humanized 20F3 CDR-L1 as determined by Kabat.

RASSSVSH MN

SEQ ID NO: 20 is the amino acid sequence of mouse 20F3 and humanized 20F3 CDR-L2 as determined by Kabat.

ATS N LAS

SEQ ID NO: 21 is the amino acid sequence of mouse 20F3 and humanized 20F3 CDR-L3 as determined by Kabat.

QQWSSTPRT

SEQ ID NO: 22 is the amino acid sequence of mouse 20F3 CDR-H1 as determined by IMGT.

GYTFTNYG

SEQ ID NO: 23 is the amino acid sequence of mouse 20F3 CDR-H2 as determined by IMGT.

INTYSGEP

SEQ ID NO: 24 is the amino acid sequence of mouse 20F3 CDR-H3 as determined by IMGT.

ARDYGRWYFDV

SEQ ID NO: 25 is the amino acid sequence of mouse 20F3 CDR-L1 as determined by IMGT.

SSVSH

The amino acid sequence of mouse 20F3 CDR-L2 determined by IMGT is:

ATS

SEQ ID NO: 26 is the amino acid sequence of mouse 20F3 CDR-L3 as determined by IMGT.

QQWSSTPRT

SEQ ID NO: 27 is the amino acid sequence of the human light chain constant region.

-

Claims (59)

TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 28 is the amino acid sequence of the natural human heavy chain constant region. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG SEQ ID NO: 29 is the amino acid sequence of the human heavy chain variant constant region (S239C). ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG CLAIM 1. An antibody that specifically binds to human NTB-A protein, wherein the antibody comprises a mature heavy chain variable region and a mature light chain variable region, wherein: (a) the mature heavy chain variable region contains three heavy chain complementarity determining regions (CDRs) according to Kabat numbering: Heavy chain CDR1 consisting of SEQ ID NO: 10, Heavy chain CDR2 consisting of SEQ ID NO: 11, Heavy chain CDR3 consisting of SEQ ID NO: 12; and the mature light chain variable region contains three light chain CDRs according to Kabat numbering: CDR1 light chain consisting of SEQ ID NO: 19, CDR2 light chain consisting of SEQ ID NO: 20 and CDR3 light chain consisting of SEQ ID NO: 21; or (b) the mature heavy chain variable region contains three heavy chain CDRs CDRs according to IMGT numbering: Heavy chain CDR1 consisting of SEQ ID NO: 22, Heavy chain CDR2 consisting of SEQ ID NO: 23, and Heavy chain CDR3 consisting of SEQ ID NO: 24; and the mature light chain variable region contains three light chain CDRs according to IMGT numbering: CDR1 light chain consisting of SEQ ID NO:25, Light chain CDR2, consisting of the amino acid sequence ATS, and Light chain CDR3 consisting of SEQ ID NO:26. 2. The antibody of claim 1, wherein the antibody is a humanized, chimeric or venous antibody. 3. The antibody of claim 1, wherein the antibody is a humanized antibody. 4. An antibody according to any one of claims 1 to 3, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 8 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 18. 5. An antibody according to any one of claims 1 to 3, wherein the mature heavy chain variable region contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, and the mature light chain variable region contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 18. 6. The antibody of claim 4, wherein the mature heavy chain variable region contains an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 8, and the mature light chain variable region contains an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 8, and the mature light chain variable region contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 8. identical to the sequence of SEQ ID NO: 18, and - 35 044092 wherein the amino acids of the mature heavy chain variable region and SEQ ID NO: 8 and the amino acids of the mature light chain variable region and SEQ ID NO: 18 differ only by conservative substitutions. 7. An antibody according to any one of claims 1 to 3, wherein the mature heavy chain variable region contains an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 8, and the mature light chain variable region contains an amino acid sequence that is at least at least 95% identical to the sequence of SEQ ID NO: 18. 8. An antibody according to any one of claims 1 to 3, wherein the mature heavy chain variable region contains an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 8, and the mature light chain variable region contains an amino acid sequence that is at least is at least 95% identical to the sequence of SEQ ID NO: 18, and wherein the amino acids of the mature heavy chain variable region and SEQ ID NO: 8 and the amino acids of the mature light chain variable region and SEQ ID NO: 18 differ only by conservative substitutions. 9. Antibody according to any one of claims 3-8, in which H2 is occupied by I or V, H38 is occupied by R or K, H44 is occupied by D or G, H46 is occupied by K or E, H68 is occupied by V or A, H73 is occupied by K or T, H76 occupied by N or S, H91 occupied by Y or F, L1 occupied by Q or E, L5 occupied by S or T, L21 occupied by M or L, L46 occupied by P or L, L47 occupied by W or L, L58 occupied by V or I and L71 occupied Y or F; according to Kabat numbering. 10. Antibody according to any one of claims 3-8, in which the following positions in the variable region framework are occupied: H2 is occupied by I, H38 is occupied by R, H44 is occupied by D, H46 is occupied by K, H68 is occupied by V, H73 is occupied by K, H76 is occupied by N, H91 occupied by Y, L1 occupied by Q, L5 occupied by S, L21 occupied by M, L46 occupied by P, L47 occupied by W, L58 occupied by V, L71 occupied by Y according to Kabat numbering. 11. An antibody according to any one of claims 3 to 8, wherein the following positions in the variable region framework are occupied as indicated: H2 occupied by I, H44 occupied by D, H46 occupied by K, H73 occupied by K, H76 occupied by N, L1 occupied by Q, L5 occupied by S, L21 occupied by M, L46 occupied by P, L47 occupied by W, L58 occupied by V, L71 occupied by Y according to Kabat numbering. 12. The antibody of claim 1, wherein the mature heavy chain and the mature light chain comprise amino acid sequences selected from the group consisting of, respectively; SEQ ID NO: 5 and SEQ ID NO: 5 and SEQ ID NO: 5 and SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 6 and SEQ ID NO: 6 and SEQ ID NO: 6 and SEQ ID NO: 7 and SEQ ID NO: 7 and SEQ ID NO: 7 and SEQ ID NO: 7 and SEQ ID NO: 8 and SEQ ID NO: 8 and SEQ ID NO: 8 and SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 9 and SEQ ID NO: 9 and SEQ ID NO: 9 and SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; respectively; accordingly and accordingly. 13. The antibody according to any one of claims 1 to 12, wherein the mature heavy chain variable region is fused to the heavy chain constant region and the mature light chain variable region is fused to the light chain constant region. 14. The antibody of claim 13, wherein the heavy chain constant region is a mutant form of the natural human constant region and wherein the heavy chain constant region has reduced binding to the Fc-gamma receptor compared to the natural human constant region. 15. The antibody of claim 13, wherein the heavy chain constant region is of the IgG1 isotype. 16. The antibody of claim 13, wherein the heavy chain constant region has the amino acid sequence comprising SEQ ID NO: 28 or SEQ ID NO: 29, and the light chain constant region has the amino acid sequence comprising SEQ ID NO: 27. 17. An antibody that specifically binds to the human NTB-A protein, wherein the antibody comprises a mature heavy chain variable region and a mature light chain variable region, wherein: (a) the mature heavy chain variable region contains three heavy chain CDRs according to number - 36 044092 tions Kabat: A heavy chain CDR1 consisting of SEQ ID NO: 10 or SEQ ID NO: 10 with one conservative substitution, A heavy chain CDR2 consisting of SEQ ID NO: 11 or SEQ ID NO: 11 with one conservative substitution, and Heavy chain CDR3 consisting of SEQ ID NO: 12 or SEQ ID NO: 12 with one conservative substitution; and the mature light chain variable region contains three light chain CDRs according to Kabat numbering: Light chain CDR1 consisting of SEQ ID NO: 19 or SEQ ID NO: 19 with one conservative substitution, A light chain CDR2 consisting of SEQ ID NO: 20 or SEQ ID NO: 20 with one conservative substitution, and Light chain CDR3 consisting of SEQ ID NO: 21 or SEQ ID NO: 21 with one conservative substitution; or (b) the heavy chain variable region contains three heavy chain CDRs according to IMGT numbering: Heavy chain CDR1 consisting of SEQ ID NO: 22 or SEQ ID NO: 22 with one conservative substitution, A heavy chain CDR2 consisting of SEQ ID NO: 23 or SEQ ID NO: 23 with one conservative substitution, and Heavy chain CDR3 consisting of SEQ ID NO: 24 or SEQ ID NO: 24 with one conservative substitution; and the mature light chain variable region contains three light chain CDRs according to IMGT numbering: Light chain CDR1 consisting of SEQ ID NO: 25 or SEQ ID NO: 25 with one conservative substitution, Light chain CDR2, consisting of the sequence ATS or ATS with one conservative substitution, and Light chain CDR3 consisting of SEQ ID NO: 26 or SEQ ID NO: 26 with one conservative substitution. 18. An antibody-drug conjugate containing an antibody according to any one of claims 1 to 17 and a cytotoxic or cytostatic agent, where the antibody is conjugated with a cytotoxic or cytostatic agent. 19. The antibody-drug conjugate of claim 18, wherein the cytotoxic agent is a maytansinoid, auristatin, pyrrole[1,4]benzodiazepine, indolinebenzodiazepine or oxazolidinebenzodiazepine. 20. The antibody-drug conjugate of claim 18, wherein the cytotoxic agent is auristatin. 21. The antibody-drug conjugate of claim 20, wherein the cytotoxic agent is MMAE. 22. The antibody-drug conjugate of claim 20, wherein the cytotoxic agent is MMAF. 23. The antibody-drug conjugate of claim 20, wherein the cytotoxic agent is auristatin-2. 24. The antibody-drug conjugate according to claim 19, where the cytotoxic agent is where index n is 1 or 3. 25. Anti-NTB-A antibody-drug conjugate with formula where index n is 1 or 3; - 37 044092 LU is a cleavable linker unit; Ab is an antibody according to claim 17; index p is equal to an integer 1-4. 26. Antibody-drug conjugate according to claim 24 with the formula where index n is 1 or 3; index m is 2-5; Ab is an antibody according to any one of claims 1-17; index p is equal to an integer 1-4. 27. Antibody-drug conjugate according to claim 26 with the formula 28. Antibody-drug conjugate according to claim 26 with formula 29. The antibody-drug conjugate according to any one of claims 26-28, where n is 1. 30. Antibody-drug conjugate according to any one of claims 26-28, where n is 3. 31. Antibody-drug conjugate according to any one of claims 22-26, where m is equal to 5. 32. The antibody-drug conjugate according to any one of claims 26-28, where the attachment to the Ab occurs through the sulfur atom of the modified cysteine residue of the Ab. 33. The antibody-drug conjugate according to any one of claims 26 to 32, wherein the attachment to the Ab occurs through a sulfur atom or a modified cysteine residue at position 239 of the heavy chain constant region in accordance with the EU numbering system. 34. The antibody-drug conjugate according to any one of claims 25 to 33, wherein p is equal to 2. 35. An antibody-drug conjugate composition for the treatment of an NTB-A-related disease, comprising a population of anti-NTB-A antibody-drug conjugate molecules of the formula where index n is 1-3; LU is a cleavable linker unit; Ab is an antibody according to any one of claims 1-17; index p is equal to an integer 1-4; where the average drug load in the composition is about 2. 36. The composition according to claim 35, containing a population of anti-NTB-A antibody-drug conjugate molecules with the formula - 38 044092 where index n is 1-3; index m is 2-5; Ab is an antibody according to any one of claims 1-17; index p is equal to an integer 1-4; where the average drug load in the composition is about 2. 37. The composition according to claim 36, in which the molecules of the antibody-drug conjugate have the formula 38. The composition according to claim 36, where the molecules of the antibody-drug conjugate have the formula 39. Composition according to any one of paragraphs 36-38, in which n is equal to 1. 40. Composition according to any one of claims 36-38, in which n is 3. 41. Composition according to any one of claims 36-40, in which n is 5. 42. The composition according to any one of claims 36-41, where the attachment to Ab occurs through the sulfur atom of the modified cysteine residue of Ab. 43. The composition according to any one of claims 36 to 41, wherein the attachment to the Ab is linked through a sulfur atom or a modified cysteine residue at position 239 of the heavy chain constant region in accordance with the EU numbering system. 44. A pharmaceutical composition containing a population of anti-NTB-A antibody-drug conjugate molecules and a pharmaceutically acceptable carrier, wherein the anti-NTB-A antibody-drug conjugate molecules have the formula where index n is 1-3; index m is 2-5; Ab is an antibody according to any one of claims 1-17; index p is equal to an integer 1-4; where the average drug load in the composition is about 2. 45. The pharmaceutical composition according to claim 44, in which the molecules of the antibody-drug conjugate have the formula 46. The pharmaceutical composition according to claim 44, in which the molecules of the antibody-drug conjugate have the formula 47. Pharmaceutical composition according to any one of claims 44-46, in which n is 1. - 39 044092 48. Pharmaceutical composition according to any one of claims 44-46, in which n is 3. 49. Pharmaceutical composition according to any one of claims 44-48, in which n is 5. 50. Pharmaceutical composition according to any one of claims 44-49, where the attachment to Ab occurs through the sulfur atom of the modified cysteine residue of Ab. 51. The pharmaceutical composition according to any one of claims 44 to 50, wherein the attachment to the Ab occurs through a sulfur atom or a modified cysteine residue at position 239 of the heavy chain constant region in accordance with the EU numbering system. 52. A pharmaceutical composition containing a population of anti-NTB-A antibody-drug conjugate molecules with the formula where index n is 1 -3; LU is a cleavable linker unit; An Ab is an antibody comprising a mature heavy chain comprising a heavy chain variable region having the amino acid sequence SEQ ID NO: 8 and a heavy chain constant region having the amino acid sequence SEQ ID NO: 29, and a light chain comprising a light chain variable region having the amino acid sequence SEQ ID NO: 18 and light chain constant region with amino acid sequence SEQ ID NO: 27; and the index p is equal to the integer 1-4; where the average drug load in the composition is about 2. 53. A method of treating a cancer patient whose cells express NTB-A, comprising administering to the patient a composition according to any one of claims 35 to 52 in an effective manner. 54. A method of treating a patient with a hematologic malignancy that expresses NTB-A, comprising administering to the patient in an effective manner a composition containing a population of anti-NTB-A antibody-drug conjugate molecules of the formula where index n is 1-3; LU is a cleavable linker unit; An Ab is an antibody comprising three heavy chain CDRs of the sequence SEQ ID NO: 3 and three light chain CDRs of the sequence SEQ ID NO: 13, where the CDRs are as defined by Kabat or IMGT numbering; and the index p is equal to the integer 1-4; where the average drug load in the composition is about 2. 55. A method of treating a patient with a hematologic malignancy that expresses NTB-A, comprising administering to the patient an effective amount of an anti-NTB-A antibody-drug conjugate according to any one of claims 26 to 34. 56. The method according to any one of claims 53 to 55, wherein the cancer is multiple myeloma, non-Hodgkin's lymphoma, acute myeloid leukemia, chronic lymphocytic leukemia, Waldenström's macroglobulinemia or monoclonal gammopathy of unknown etiology (MGUS). 57. The method according to any one of claims 53-55, in which the cancer is T-cell lymphoma. 58. A method of treating a patient with an NTB-A-related autoimmune disease, comprising administering to the patient a composition according to any one of claims 35 to 52 in an effective regimen. 59. A method of treating a patient with an NTB-A-related autoimmune disease, comprising administering to the patient an effective amount of an anti-NTB-A antibody-drug conjugate according to any one of claims 18 to 34. -