BR112013019564B1 - ANTIBODIES, EXPRESSION VECTOR, RECOMBINANT PROKYOTIC HOST CELL, PHARMACEUTICAL COMPOSITION, AND, USE OF AN ANTIBODY - Google Patents

Abstract

ANTIBODIES, EXPRESSION VECTOR, RECOMBINANT PROKYOTIC OR EUKARYOTIC HOST CELL, PHARMACEUTICAL COMPOSITION, USE OF AN ANTIBODY, AND, METHODS FOR INDUCING CELL DEATH, INHIBITING GROWTH, AND/OR INHIBITING PROLIFERATION OF A CELL, AND TO TREAT A DISEASE. Isolated human monoclonal antibodies that bind human CD74 and related antibody drug conjugates are disclosed. Pharmaceutical compositions comprising the antibodies or antibody drug conjugates, and therapeutic and diagnostic methods for using the antibodies and/or antibody drug conjugates, are also disclosed.

Description

FIELD OF THE INVENTION

[01] The present invention relates to CD74-specific antibodies and antibody drug conjugates (ADCs) thereof, pharmaceutical compositions of such antibodies or ADCs, and their use in therapeutic applications.

FUNDAMENTALS OF THE INVENTION

[02] The human leukocyte antigen (HLA) class II histocompatibility antigen gamma chain, also called HLA-DR antigen-associated invariant chain, Ia, Ii, and CD74 antigen-associated invariant chain, is a transmembrane protein with a short cytoplasmic tail. The primary function of CD74 is to regulate peptide loading into class II major histocompatibility complex (MHC) heterodimers in intracellular compartments.

[03] Only a small portion of the total cell CD74 content is expressed on the cell surface. The CD74 cell surface is very rapidly internalized both with and without CD74 antibodies bound (Roche PA et al., PNAS 1993; 90: 8581-8585; Hansen H3 et al., Biochem J 1996; 320: 293300; Ong GL et al. al., Immunology 1999;98:296-302). The steady-state level of the CD74 cell surface is therefore very low, ranging in monocytes from a few hundred to a few thousand molecules per cell.

[04] The exact function of CD74 expressed on the cell surface is not known, but studies have documented CD74 as a membrane receptor for the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). Binding of MIF to CD74 activates downstream signaling through the MAPK and Akt pathways and promotes cell proliferation and survival. This interaction is probably also regulated by the presence of CD44, CXCR2 or CXCR4 as coreceptors.

[05] Upregulation of CD74 expression has been observed in many types of cancer, as well as in certain infections and inflammatory conditions. Various formats of a humanized CD74-specific monoclonal antibody, hLL1, have been proposed for the treatment of CD74-positive tumors ( Chang CH et al., Blood 2005; 106: 4308-4314 ; Sapra P et al., Clin Can Res 2005 ; 11: 5257-5264; Stein R et al., Blood 2004; 104: 3705-11; Govindan SV et al. J Nucl Med 2000; 41: 2089-2097; Hertlein E et al., Blood 2010; 116: 2554- 2558; Stein R et al., Clin Cancer Res 2009; 15: 2808-2817; Sharkey RM et al., J Nucl Med 2009; 50: 444-453; Lundberg BB et al., Drug Deliv 2007; 14: 171- 175; Griffiths GL et al., Int J Cancer 1999; 81: 985-992; Griffiths GL et al., Cancer Res 2003; 9: 6567-6571; Ochakovskaya R et al., Clin Cancer Res 2001; 7: 1505- 1510; Shih L et al., Cancer Immunol Immunother; Burton JD et al., Clin Cancer Res 2004; 10: 6606-6611; Lundberg BB et al., J Control Release 2004; 94: 155-161 ).

[06] Although much progress has been made, there remains a need for improved methods of treating serious diseases, for example, improved cancer treatment, based on therapeutic antibodies and ADCs.

SUMMARY OF THE INVENTION

[07] It is an object of the present invention to provide novel CD74-specific monoclonal antibodies and highly specific and effective ADCs of such CD74-specific antibodies. Antibodies or ADCs of the invention exhibit CD74 binding characteristics or other effects on CD74 expressing cells that differ from antibodies described in the art. Particularly, the antibodies are characterized by rapid internalization upon binding to the CD74 antigen, making them suitable for therapeutic applications in the form of ADCs and for other applications where rapid internalization is an advantage. The new ADCs are characterized by a high efficiency in killing tumor cells that express CD74.

[08] Antibodies and corresponding ADCs can be provided in a variety of formats, which include, but are not limited to, antibody fragment and bispecific antibody formats. In preferred embodiments, the antibodies are human.

[09] It is also an object of the present invention to provide ADCs based on such CD74-specific antibodies for medical use, which provides an efficient and selective way of causing cell death of tumor cells.

[010] These and other aspects of the invention are described in more detail below.

Brief Description of Drawings

[011] Figure 1: Amino acid sequences of recombinant CD74 proteins used in the Examples. CD74v1 and -v2, CD74de12-36v1 and -v2, and HisCD74v1 and -v2 correspond to SEQ ID NOs: 1-6, respectively.

[012] Figure 2: Alignment of variable heavy (VH) and variable light (VL) chain sequences of the antibodies of the present invention. The SEQ ID NO of each VH/VL sequence is listed within the parentheses to the right of the sequence. Complementarity determining regions (CDRs) according to the IMGT nomenclature are highlighted as follows: sequences in italics represent CDR1, underlined sequences represent CDR2, and sequences in bold represent CDR3.

[013] Figure 3: Binding of CD74-specific antibodies to the recombinant protein that represents the extracellular domain of variant isoforms 1 and 2 (CD74v1 and CD74v2), determined by ELISA. All human antibodies were produced by HEK-293F cells that transiently cotransfect with the relevant heavy and light chain expression vectors.

[014] Figure 4: Binding of CD74-specific antibodies to cellular CD74 in Raji cells, determined by FACS. All human antibodies were produced by HEK-293F cells that transiently cotransfect with the relevant heavy and light chain expression vectors.

[015] Figure 5: Cross-reactivity of CD74-specific antibodies with cynomolgus CD74. Human tonsils (upper panel) and cynomolgus lymph nodes (lower panel) were stained with CD74-specific antibodies. *: germinal center; Mf: macrophages; #: Mantle zin B cells.

[016] Figure 6: Dose-dependent induction of cell death by CD74-specific antibodies pre-incubated with anti-kappa-ETA'. A representative experiment is shown. Data shown are mean percentages of viability of duplicate pools of cells treated with CD74 HuMab antibodies pre-incubated with anti-kappa-ETA'. Percent viability was calculated as described in Example 14.

[017] Figure 7: Binding of CD74 HuMab antibodies 005 and 006 (A) and 011 (B) and the corresponding ADCs to the extracellular CD74v1 domain recombinant protein, as determined by ELISA. A representative experiment is shown.

[018] Figure 8: Binding of CD74 HuMab antibodies 005 and 006 (A) and 011 (B) and the ADCs corresponding to surface expressed CD74, determined by FACS analysis on Daudi cells. Data shown are mean fluorescence intensities (MFI) calculated from three independent experiments.

[019] Figure 9: Dose-dependent induction of cell death by CD74-specific ADCs. A representative experiment is shown for each of the following cell lines: Daudi (A), Raji (B), M4A4 (C), and NCI-H747 (D) cells. Data shown are percent survivals of duplicate pools of cells treated with CD74-specific ADCs.

[020] Figure 10: In vivo efficacy of CD74-specific ADCs in the therapeutic treatment of Daudi-luc xenografts in SCID mice. Mice with established Daudi-luc tumors were treated with CD74-specific ADCs. Data shown are average bioluminescence imaging (BLI) ± S.E.M. per group (n = 7 mice per group).

[021] Figure 11: In vivo efficacy of CD74-specific ADCs in the therapeutic treatment of Raji-luc xenografts in SCID mice. Mice with established Raji-luc tumors were treated with CD74-specific ADCs. Data shown are average signals of BLI ± S.E.M. per group (n = 7 mice per group).

[022] Figure 12: In vivo efficacy of CD74-specific ADCs in the therapeutic treatment of Raji xenografts in SCID mice. Mice with Raji tumors established s.c. were treated with CD74-specific ADCs. Data shown are mean tumor volumes ± S.E.M. per group (n = 6 mice per group).

[023] Figure 13: In vivo efficacy of anti-CD74 ADCs in the therapeutic treatment of M4A4 xenografts in SCID mice. Mice with established M4A4 tumors were treated with anti-CD74 ADCs. Data shown are mean tumor volumes ± S.E.M. per group (n = 7 mice per group).

[024] Figure 14: Determination of dissociation rates of CD74-specific HuMab antibodies. A representative experiment is shown. Data shown are mean fluorescence intensities (MFI) of triplicate wells of cells incubated with CD74 HuMab antibodies labeled with Alexa Fluor 488® dye, followed by incubation with unlabeled CD74 HuMab antibodies for the indicated time intervals.

[025] Figure 15: Internalization and time-dependent accumulation of anti-CD74 HuMab antibodies. A representative experiment is shown for each cell line. Data shown are mean fluorescence intensities (MFI) of duplicate wells of cells incubated with Alexa-488-labeled anti-CD74 HuMab antibodies. Daudi cells were incubated with Alexa-488-labeled anti-CD74 HuMab antibodies at 4°C (A) or 37°C (B). Raji cells (C) and M4A4 cells (D) were incubated at 37°C.

[026] Figure 16: In vivo efficacy of anti-CD74 HuMab antibodies in the prophylactic treatment of Daudi luc xenografts in SCID mice. Mice were treated with anti-CD74 HuMab antibodies within one hour after intravenous inoculation of Daudi luc tumors. Data shown are average BLI signals ± S.E.M. per group (n = 7 mice per group).

DETAILED DESCRIPTION OF THE INVENTION Definitions

[027] The terms “CD74” and “CD74 antigen” are used interchangeably here. unless otherwise specified, the terms include any of the variants, isoforms and species homologs of human CD74 that are naturally expressed by cells or are expressed in cells transfected with the CD74 gene. At least four human isoforms are known to exist; p43, p41, p35 and pp33 ( Borghese F et al., Expert Opin Ther Targets 2011; 15(3): 237-251 ). These result from the junction of alternative transcript and two translation start sites. p43 (also known as isoform 1, CD74 isoform a, or "long"; see UniProt entry PO4233-1 and NCBI Reference Sequence NP_001020330) contains 296 amino acids, with residues 73-296 forming the extracellular portion. CD74 protein constructs having the extracellular part of isoform 1 are referred to herein as "variant 1" or "CD74v1." p35 (also known as isoform 2, CD74 isoform b or "short"; see Uniprot entry PO4233-2 and NCBI Reference Sequence NP_004346) lacks residues 209-272 of the extracellular part due to alternative splicing. CD74 protein constructs having the extracellular part of isoform 2 are referred to herein as "variant 2" or "CD74v2." p41 and p33 arise from an alternative translation start site (48 base pairs downstream; protein 16 amino acids shorter) leading to variants lacking the endoplasmic reticulum (ER) retention signal that is present within these 16 amino acids, but having an identical extracellular part as p43 and p35, respectively. The sequence of another isoform (known as isoform 3 and isoform c), in which residues 148-160 are substituted and residues 161296 are missing, is provided in NP_001020329. Cynomolgus CD74 homolog sequences are provided, for example, in NCBI Reference Sequence: XP_001099491.2 and NCBI Reference Sequence: XP_002804624.1.

[028] The term “immunoglobulin” refers to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, a pair of low molecular weight light (L) chains and a pair of heavy (H) chains. all four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). In summary, each heavy chain is typically comprised of a heavy chain variable region (herein abbreviated as VH or VH) and a heavy chain constant region (CH or CH). The heavy chain constant region is typically comprised of three domains, CH1, CH2, and CH3. Each light chain is typically comprised of a light chain variable region (herein abbreviated as VL or VL) and a light chain constant region. the light chain constant region is typically comprised of a domain, CL or CL. Typically, the numbering of amino acid residues in the constant region is performed according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and/or shape of structurally defined loops), also called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called matrix regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)).

[029] The term "antibody" or "Ab" in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about an hour, at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about three, four, five, six, seven or more days, etc., or any other relevant functionally defined period (such as a sufficient time to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to antigen and/or sufficient time for the antibody to recruit an effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. Constant regions of antibodies (Abs) can mediate binding of immunoglobulin to tissues or host factors, which include various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of the immune system. complement activation. An antibody can also be multispecific, having specificities for two or more different, typically non-overlapping, epitopes. Examples of multispecific antibodies include bispecific antibodies, diabodies, and similar antibody molecules. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically bind antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-size antibody, for example Fab and F(ab')2 fragments. It is also to be understood that the term antibody, unless otherwise specified, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies, and humanized antibodies. An antibody as generated can have any isotype.

[030] The terms "human antibody", "human Ab" or "HuMab", as used herein, are intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human matrix sequences.

[031] As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system that uses human immunoglobulin sequences, for example, by immunizing a transgenic mouse that carries genes of the human immunoglobulin or by screening a human immunoglobulin gene library, and wherein the selected human antibody is at least 90%, such as at least 95%, for example, at least 96%, such as at least 97%, for example, at least 98%, or such as at least 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, outside of heavy chain CDR3, a human antibody derived from a particular human germline sequence will demonstrate no more than 20 amino acid differences, for example, no more than 10 amino acid differences, such as no more than 9 , 8, 7, 6 or 5, for example no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[032] The terms "monoclonal antibody", "Monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of antibody molecules of unique molecular composition. A monoclonal antibody composition demonstrates a unique binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies that demonstrate a single binding specificity that have variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by a hybridoma that includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene, fused together. to an immortalized cell.

[033] As used herein, "isotype" refers to the class of immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by the heavy chain constant region genes.

[034] The term "full-size antibody" when used herein, refers to an antibody that contains all of the heavy and light chain constant and variable domains that are normally found in an antibody of this isotype.

[035] When used herein, unless contradicted by context, the terms "Fab-branch" or "branch" refer to a heavy chain-light chain pair.

[036] When used herein, unless contradicted by context, the term "Fc region" refers to an antibody region that comprises at least a hinge region, a CH2 domain, and a CH3 domain.

[037] An “antibody deficient in effector function” refers to an antibody that has a significantly reduced or no ability to activate one or more effector mechanisms, such as complement activation or Fc receptor binding. Thus, antibodies deficient in effector function have significantly reduced or no ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). An example of such an antibody is an antibody of the IgG4 isotype or a hinge-stabilized form thereof. Another example is the introduction of mutations in the Fc region that can strongly reduce the interaction with complement proteins and Fc receptors. See, for example, Bolt S et al., Eur J Immunol 1993, 23: 403-411; Oganesyan, Acta Crys. 2008, D64, 700-704; and Shields et al., 313C 2001, 276: 6591-6604.

[038] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for example, lymphocytes (such as B cells and T cells that include cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, mastoids, and granulocytes. , such as neutrophils, eosinophils and basophils. Some effector cells express specific Fc receptors (FcRs) and perform specific immune functions. In some embodiments, an effector cell is capable of inducing ADCC, such as a natural killer cell. For example, monocytes, macrophages, which express FcRs, are involved in the specific killing of target cells and which present antigens to other components of the immune system. In some embodiments, an effector cell can phagocytose a target antigen or target cell. The expression of a particular FcR in an effector cell can be regulated by humoral factors such as cytokines. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell.

[039] In the context of the present invention, an "ADC" refers to an antibody-drug conjugate, in the context of the present invention typically referring to a CD74-specific antibody, which is linked to another moiety as described herein. request.

[040] A “CD74 antibody”, “anti-CD74 antibody”, “CD74 Ab”, “CD74-specific antibody” or “anti-CD74 Ab” is an antibody as described above that specifically binds to the CD74 antigen.

[041] In a preferred embodiment, the antibody of the invention is isolated. An "isolated Ab," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD74 is substantially free of antibodies that specifically bind to CD74). antigens other than CD74). An isolated antibody that specifically binds to an epitope, isoform or variant of human CD74 may, however, be cross-reactive with other related antigens, for example from other species (such as CD74 species homologues). Furthermore, an isolated antibody can be substantially free of other cellular materials and/or chemicals. In one embodiment of the present invention, two or more "isolated" monoclonal antibodies having different antigen binding specificities are combined into a well-defined composition.

[042] When used herein in the context of two or more antibodies, the term “compete with” or “cross-compete with” indicates that the two or more antibodies compete for binding to CD74, e.g., CD74 variants 1, 2 or both. For example, the constructs described in Example 1 can be used in such an assay. In another exemplary type of assay, CD74 is coated onto a plate and allowed to bind to the first antibody, after which the second, labeled antibody is added. If the presence of the first antibody reduces the binding of the second antibody, the antibodies compete. The term "compete with" when used herein is also intended to encompass antibody combinations where one antibody reduces the binding of another antibody, but where no competition is observed when the antibodies are added in reverse order.

[043] The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope can comprise amino acid residues that are directly involved in binding, and other amino acid residues, which are not directly involved in binding, such as amino acid residues that are effectively blocked or specifically covered by the antigen-binding peptide (in other words , the amino acid residue is specifically within the coverage area of the antigen-binding peptide).

[044] As used herein, the term "binding" in the context of binding an antibody to a predetermined antigen or epitope typically is binding with an affinity that corresponds to a KD of about 10-7 M or less, such as from about 10-8M or less, such as about 10-9M or less, about 10-10M or less, or about 10-11M or less when determined, for example, by the technology of surface plasmon resonance (SPR) on a BIAcore 3000 instrument that uses a soluble form of the antigen as the ligand and the antibody as the analyte. Typically, an antibody binds the predetermined antigen with an affinity that corresponds to a KD that is at least ten-fold lower, such as at least 100-fold lower, for example, at least 1,000-fold lower, such as at least 10,000 times lower, e.g. at least 100,000 times lower than your KD for binding to a non-specific antigen (e.g. BSA, casein) that is not identical or closely related to the predetermined antigen. When the antibody's KD is very low (i.e., the antibody has a high affinity), then the KD at which it binds to the antigen is typically at least 10,000 times lower than its KD for a non-specific antigen. .

[045] The term "kd" (s-1), as used herein, refers to the dissociation rate constant of a particular Ab-antigen interaction. Said value is also referred to as the koff value.

[046] The term “ka” (M-1 x s-1), as used herein, refers to the association rate constant of a particular Ab-antigen interaction.

[047] The term “KD” (M), as used herein, refers to the equilibrium dissociation constant of a particular Ab-antigen interaction.

[048] The term "KA" (M-1), as used herein, refers to the equilibrium association constant of a particular Ab-antigen interaction and is obtained by dividing ka by kd.

[049] As used herein, "internalization" when used in the context of a CD74 antibody includes any mechanism by which the antibody is internalized from the cell surface into a cell that expresses CD74. Internalization of an antibody can be evaluated in an indirect assay that measures the effect of an internalized Ab-toxin conjugate or a toxin specifically bound to an antibody by preincubation (such as, for example, the anti-kappa-ETA assay ' of Example 14).

[050] As used herein, the term "inhibits growth" (e.g., referring to cells such as tumor cells) is intended to include any measurable decrease in cell growth when contacted with a CD74 antibody or ADC as compared growth of the same cells not in contact with a CD74 antibody or ADC, for example, inhibition of growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Such a decrease in cell growth can occur by a variety of mechanisms, for example, internalization, antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), death and/or or drug-mediated cell apoptosis.

[051] The present invention also provides antibodies that comprise functional variants of the VL region, VH region, or one or more CDRs of the antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of a CD74 antibody still enables the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95 % or more) of the affinity/avidity and/or specificity/selectivity of the precursor antibody and in some cases such CD74 antibody may be associated with greater affinity, selectivity and/or specificity than that of the precursor Ab.

[052] Such functional variants typically retain significant sequence identity to the precursor Ab. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology = # identical positions / # total positions x 100), taking into account the number of gaps, and the length of each gap, which needs to be entered for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below.

[053] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using an NWSgapdna.CMP matrix and a gap weight of 40 , 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm described by E. Meyers and W. Miller (Computer Appl. Biosci 4, 11-17 (1988)), which was incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, an interval length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970)), which was incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using a Bloss matrix a 62 or a PAM250 array, and an interval weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[054] The sequence of CDR variants may differ from the CDR sequence of precursor antibody sequences through predominantly conservative substitutions; for example, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more more, about 90% or more, (e.g., about 65 to 95%, such as about 92%, 93%, or 94%) of the substitutions in the variant are conservative amino acid residue substitutions.

[055] The sequences of CDR variants may differ from the sequence of the CDRs of precursor antibody sequences through predominantly conservative substitutions; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue substitutions.

[056] The term “stabilized IgG4 antibody” refers to an IgG4 antibody that has been modified to reduce half-molecule exchange (see, for example, international patent application publication W02008145142 or van der Neut Kolfschoten M et al. (2007) Science 14;317(5844) and reference therein.

Classes de Substituição de Resíduos de Aminoácido Conservativos Alternativa

Classificações Físicas e Funcionais Alternativas de Resíduos de Aminoácido

[057] In the context of the present invention, conservative substitutions can be defined by the substitutions within the classes of amino acids reflected in one or more of the following three tables: Amino acid residue classes for conservative substitutions

Alternative Conservative Amino Acid Residue Substitution Classes

Alternative Physical and Functional Classifications of Amino Acid Residues

[058] More groupings of conservative substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[059] Additional groups of amino acids can also be formulated using the principles described, for example, in Creighton (1984) Proteins: Structure and Molecular Properties (2nd Ed. 1993), W.H. Freeman and Company.

[060] Conservation in terms of hydropathic/hydrophilic properties and weight/residue size is also substantially retained in a variant CDR as compared to a CDR of an antibody of the examples (e.g. weight class, hydropathic count, or both, of the sequences is at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more (e.g., about 65 to 99%) retained). For example, conservative residue substitutions can also or alternatively be based on substitution of conservation groups on a strong or weak weight basis, which are known in the art.

[061] Retention of similar residues can also or alternatively be measured by a similarity count, as determined by using a BLAST program (e.g. BLAST 2.2.8 available through NCBI which uses default settings BLOSUM62, Open Range = 11 and Extended Interval = 1). Suitable variants typically exhibit at least about 45%, such as at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 90%, minus about 95%, or more (e.g., about 70 to 99%) similarity to the precursor peptide.

[062] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a loop of circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be linked within the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and in this way are replicated together with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Overall, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably since the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[063] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It is to be understood that such terms are intended to refer not only to the particular individual cell, but also to the progeny of such cell. Because certain modifications may occur in succeeding generations due to mutation or environmental influences, such progeny may, in fact, not be identical to the precursor cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytic cells, and prokaryotic cells such as E. coli.

[064] The term "transfectoma", as used herein, includes recombinant eukaryotic host cells that express the Ab, such as CHO cells, PER.C6, NSO cells, HEK-293 cells, plant cells, or fungal cells, which include cells of yeast.

[065] The term "transgenic non-human animal" refers to a non-human animal having a genome that comprises one or more human heavy and/or light chain transgenes or transchromosomes (integrated or non-integrated within the animal's natural genomic DNA). ) and which is capable of expressing fully human Ab. For example, a transgenic mouse may have a human light chain transgene and a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human CD74 antibodies when immunized with CD74 antigen and/or cells that express CD74. The human heavy chain transgene can be integrated into the mouse chromosomal DNA, as is the case for transgenic mice, e.g. HuMAb® mice, such as HCo7 or HCo12 mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal KM mice as described in WO02/43478. Such transgenic and transchromosomal mice (collectively referred to herein as “transgenic mice”) are capable of producing multiple isotypes of human monoclonal antibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination and switching. of isotype. The non-human, transgenic animal can also be used to produce antibodies against a specific antigen by introducing genes encoding that specific Ab, for example, by operatively linking the genes to a gene that is expressed in the milk of the animal.

[066] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.

[067] An "effective amount" refers to an amount effective, at the dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a CD74 antibody can vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the CD74 antibody to evoke a desired response in the subject. A therapeutically effective amount is also one in which any of the toxic or harmful effects of the antibody or antibody portion are more important than the therapeutically beneficial effects.

[068] An “anti-idiotypic” (Id) antibody is an antibody that recognizes unique determinants generally associated with the antigen-binding site of an Ab.

Other aspects and embodiments of the invention

[069] The invention provides an isolated antibody, such as a human monoclonal antibody, that binds isoforms 1 and 2 of human CD74. The antibody may additionally bind to other CD74 isoforms or species homologs, such as the cynomolgus homolog. In particular, the antibody of the invention efficiently internalizes after binding to CD74 expressed on the surface of a cell, which is advantageous for therapeutic applications of an ADC method. As shown in Examples 19 to 22, CD74 antibody ADCs and vcMMAE or mcMMAF drug combinations effectively reduced the size of tumors in various tumor models in vivo. CD74 ADCs were surprisingly effective despite low surface expression of the CD74 target on tumor cells (Example 22). Furthermore, CD74 antibodies were shown to be effective in preventing tumor growth in an in vivo model of tumor prophylaxis (Example 25).

[070] The antibody may be further characterized by one or more functional properties such that it binds one or more human CD74 variants with high affinity, inhibits MIF binding to CD74, or any combination of the foregoing properties.

[071] In one aspect, the antibody of the invention binds with high affinity to variants 1 and/or 2 of human CD74 or to human cells that naturally express CD74. For example, in one embodiment, antibody (a) binds to the extracellular domain of CD74 variant 1 with an EC50 (apparent affinity) of less than about 500 ng/ml, less than about 400 ng /ml, less than about 350 ng/ml, or less than about 330 ng/ml; b) binds to the extracellular domain of CD74 variant 2 with an EC50 of less than about 400 ng/ml, less than about 300 ng/ml, less than about 250 ng/ml, or less than about 220 ng/ml; or (c) either (a) or (b) when determined as described in Example 11. Also, or alternatively, the antibody can bind CD74 on Raji cells with an EC50 of less than about 400 ng /ml, less than about 300 ng/ml, less than about 250 ng/ml, or less than about 200 ng/ml, when determined as described in Example 12. Also, or alternatively, the antibody can binding to CD74 variants 1, 2, or both with a K D of about 10 -8 M or less, such as about 10 -9 M or less, or about 10 -10 M or less.

[072] In one aspect, the antibody is internalized after binding to CD74 expressed on the surface of a cell. This can be determined according to the assay described in Example 24 which uses fluorescently labeled antibodies, according to the assay described in Example 14 which uses an ADC method which reflects antibody internalization, or which uses a method described in Ong GL et al., Immunology 1999; 98: 296-302; Hansen H3 et al., Biochem J 1996; 320: 293-300; Koch NG et al., J Immunol 1991; 147: 2643-2651; Roche PA et al., PNAS 1993; 90: 8581-8585). The cell may be from a B cell lineage, such as Raji cells, or from another type of tumor cell lineage induced to express high levels of CD74 by IFNY treatment (e.g., HT-29 colonic cancer cells or cells from melanoma SK-MEL-37). In one embodiment, the cell is a Raji cell. In another embodiment, the antibody has an EC50 of less than about 60 ng/ml, less than about 40 ng/ml, less than about 30 ng/ml, or of about 25 ng/ml or less in inducing death of Raji cells in an anti-kappa ETA' assay, when determined as described in Example 14. Alternatively, the antibody has an EC50 of between about 25 and about 60 ng/L of about 25 to 40 ng/ml, or from about 25 to about 30 ng/ml in such an assay.

[073] In one aspect, an antibody of the invention has an EC50 of less than 30 ng/ml, or an EC50 of about 25 ng/ml or less, when determined as described in Example 14.

[074] In one aspect, the antibody is characterized by its rate of dissociation from CD74 antigen, optionally expressed on the surface of a cell. The off-rate can be determined, for example, using a cell assay such as that in Example 23, which typically uses fluorescently (or otherwise) labeled antibodies and determining the off-rate at 0°C. The cell may be from a B cell lineage, such as, for example, Daudi or Raji cells, or another suitable type of tumor cell lineage (e.g., M4A4 cells or NCI-H747 cells). In one embodiment, the cell is a Daudi cell. In one embodiment, the antibody has a dissociation rate in the range of 0.02 to 1.0 min-1, such as from about 0.03 to about 0.30 min-1, such as from 0.03 to about 0.30 min-1. 04 to 0.10 or from 0.15 to 0.30 min-1. In one embodiment, the antibody has a dissociation rate of about 0.07 min -1 . In one embodiment, the antibody has a dissociation rate of about 0.20 or 0.24 min -1 .

[075] The antibody of the invention may also, or alternatively, be characterized by cross-competition with, or binding to the same epitope as, a reference antibody to variant 1, variant 2, or both variants 1 and 2 of human CD74.

[076] An assay that tests for competitive binding of the antibody with a reference antibody may utilize, for example, the extracellular domain of a CD74 variant (e.g., the constructs described in Example 1), cells that express CD74, and /or cell membranes prepared from cells expressing CD74. In an exemplary assay, cells expressing CD74 are pre-incubated with the test antibody at different concentrations ranging from 1 to 100 μg/ml, subsequently incubated with a fluorophore-labeled reference antibody at a concentration of 10 μg/ml. ml. Reference antibody binding is determined using FACS analysis.

[077] In one aspect, the antibody competes for binding to human CD74 variants 1 and 2 with at least one reference antibody selected from (a) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 7 and a VL region comprising the sequence of SEQ ID NO: 23 [005]; (b) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 11 and a VL region comprising the sequence of SEQ ID NO: 26 [006]; (c) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO: 26 [008]; and (d) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 19 and a VL region comprising the sequence of SEQ ID NO: 26 [011].

[078] In separate and specific embodiments, the antibody competes with the antibody of (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), (c) and (d), at least three of (a) to (d), or all of (a), (b), (c) and (d).

[079] In one embodiment, the antibody binds to the same epitope on human CD74 as at least one of the reference antibodies defined in (a), (b), (c), and (d). This can be determined using known techniques for epitope determination, such as, for example, testing for antibody binding to CD74 variants with different point mutations, or phage display techniques (see, for example, Binder et al. , Cancer Res 2007; 67: 3518-3523; Carter 3M et al., Curr Protocols Immunol 2004; Cap 9: Unit 9.4; Hjelm B et al., N Biotechnol 2010; 27: 129-137; Rockberg J et al., Curr Protocols Immunol 2010; Chapter 9: Unit 9.9; Benjamin DC et al., Methods 1996; 9: 508-515).

[080] An antibody or immunoglobulin of the invention may also or alternatively be characterized by sequences comprising specific VH, VL, or CDRs, or specific combinations thereof.

[081] In one aspect, the antibody or immunoglobulin comprises the VH CDR3 region of any one of HuMab-CD74-005, -006, -008, and -011. The invention thus provides an antibody or immunoglobulin that comprises a VH CDR3 that comprises or consists of a sequence selected from SEQ ID NOS: 10, 14, 18, and 22. In one embodiment, the antibody or immunoglobulin comprises SEQ ID NO: 22.

[082] In one aspect, the antibody or immunoglobulin comprises a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 24, AAS and SEQ ID NO: 26 and a) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NOS: 8, 9 and 10 (005); b) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 12, 13 and 14 (006); c) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 16, 17 and 18 (008); d) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 20, 21 and 22 (011), or e) a variant of any of said antibodies or immunoglobulins, wherein said variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in any of said sequences.

[083] In one aspect, the antibody comprises a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 20, 21 and 22 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 24, AAS and 25, with a maximum of 3 amino acid modifications as compared to the original sequences. In one embodiment, the antibody comprises a VH region comprising sequences CDR1, 2 and 3 of SEQ ID NO: 20, 21 and 22 and a VL region comprising sequences CDR1, 2 and 3 of SEQ ID NO: 24 , MS and SEQ ID NO: 25, with at most 1 amino acid modification. In a particular embodiment of (e), the variant comprises an amino acid substitution from residue 7 of the VH CDR2 of (d), such as a conservative amino acid substitution.

[084] In one aspect, the antibody or immunoglobulin comprises a VH having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a sequence of the VH region selected from the group consisting of SEQ ID NO: 7, 11, 15 and 19, or b) at most 20, such as 15, or 10, or 5, 4, 3, 2, or 1 of the amino acid modifications, plus preferably amino acid substitutions, such as conservative amino acid substitutions as compared to a VH region sequence selected from the group consisting of SEQ ID NOs: 7, 11, 15 and 19.

[085] In one aspect, the antibody or immunoglobulin comprises a VL having a) at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a sequence of the VL region selected from the group consisting of: SEQ ID NO: 23 and 26 or b) at most 20, such as 15, or 10, or 5, 4, 3, 2 or 1 of the amino acid modifications, more preferably amino acid substitutions , such as conservative amino acid substitutions as compared to a VL region sequence selected from the group consisting of: SEQ ID NOs: 23 and 26.

[086] In one embodiment of (b), the VL region comprises an amino acid substitution at the position corresponding to residue 36 in SEQ ID NOs: 23 and 26. In SEQ ID NOS: 23 and 26, the amino acid at this position is F and Y, respectively.

[087] In separate and specific aspects, the antibody or immunoglobulin comprises a VH region and a VL region selected from any of the following combinations: a) a VH region comprising the sequence of SEQ ID NO: 7 and a VL region comprising the sequence of SEQ ID NO: 23 (005); b) a VH region comprising the sequence of SEQ ID NO: 11 and a VL region comprising the sequence of SEQ ID NO: 26 (006); c) a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO: 26 (008), d) a VH region comprising the sequence of SEQ ID NO: 19 and a VL region comprising the sequence of SEQ ID NO: 26 (011), and e) a VH region comprising the sequence of SEQ ID NO: 7 and a VL region comprising the sequence of SEQ ID NO: 26 (005/011) ; and f) a variant of any of said antibodies or immunoglobulins, wherein said variant preferably has at most 1, 2 or 3 of the amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in any of said sequences of the VH and/or VL regions.

[088] In one aspect, the invention provides an antibody or immunoglobulin that comprises a VL region comprising the sequence of SEQ ID NO: 26. In one embodiment, the antibody or immunoglobulin comprises the VH CDR3 of SEQ ID NO: 22. In another embodiment, the antibody comprises the VH sequences of CDR1, 2 and 3 of SEQ ID NOS: 20, 21 and 22, respectively.

[089] The antibody of the invention may be characterized by one or more of the functional or structural features of the aspects described above, or by any combination of selected functional and structural features. For example, in one embodiment, the antibody or immunoglobulin of the invention is characterized by any of the following characteristics: a) an EC50 of less than 30 ng/ml, or an EC50 of about 25 ng/ml or less in an anti-kappa ETA' assay, when determined as described in Example 14; b) competing with, or binding the same epitope as, an antibody having the VH and VL sequences of SEQ ID NOS: 19 and 26, respectively; c) a dissociation rate in the range of 0.03 to 0.30 min -1 when determined in accordance with Example 23; d) a VH CDR3 comprising SEQ ID NO: 22; e) a combination of (a) and (b); f) a combination of (a) and (c); g) a combination of (a) and (d) h) a combination of (b) and (c); i) a combination of (b) and (d); j) a combination of (c) and (d); or k) a combination of (a), (b), (c) and (d).

[090] The antibodies of the invention are preferably monoclonal. The monoclonal antibodies of the present invention can be produced for example by the hybridoma method first described by Kohler et al. (Nature 256, 495 (1975)), or can be produced by recombinant DNA methods. Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from murine splenic and B lymph node cells obtained from mice immunized with an antigen of interest, for example, in the form of cells that express an antigen of interest on the surface. , or a nucleic acid encoding an antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from cells expressing antibody from immunized human or non-human mammals such as mice, dogs, primates, etc.

[091] In one embodiment, the antibody of the invention is a human antibody. Human monoclonal antibodies directed against CD74 can be generated using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. Such transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice".

[092] The HuMAb mouse contains a human immunoglobulin gene minisite that encodes unrearranged human heavy (μ and Y) and K light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous μ and K chain sites (Lonberg , N. et al., Nature 368, 856-859 (1994)). Consequently, mice exhibit reduced mouse IgM or k expression and, in response to immunization, introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG,k monoclonal antibodies (Lonberg, N. et al (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365- 93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770 429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

[093] The mice HCo7, HCo12, HCo17 and HCo20 have a JKD disruption in their endogenous light chain genes (kappa; K) (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in its endogenous heavy chain genes (as described in Example 1 of WO 01/14424), and a human kappa light chain transgene KCo5 (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996) )). Additionally, Hco7 mice have a human heavy chain transgene HCo7 (as described in US 5,770,429), mice HCo12 have a human heavy chain transgene HCo12 (as described in Example 2 of WO 01/14424), mice HCo17 have a human heavy chain transgene HCo17 (as described in Example 2 of WO 01/09187) and mice HCo20 have a human heavy chain transgene HCo20. The resulting mice express human immunoglobulin kappa heavy and light chain transgenes in a homozygous background for disruption of endogenous mouse kappa heavy and light chain sites.

[094] In the KM mouse strain, the endogenous mouse kappa light chain gene was homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene was homozygous disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of the chromosome 14 hCF fragment (SC20) as described in WO 02/43478. HCo12-BALB/C mice can be generated by crossing HCo12 with KCo5[J/K]-Balb/C as described in WO 097006.

[095] Splenocyte and lymph node cells from these transgenic mice can be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.

[096] Human monoclonal or polyclonal antibodies of the present invention, or antibodies of the present invention that originate from other species can also be generated transgenically by generating another non-human mammal or plant that is transgenic for both heavy and light chain sequences. of the immunoglobulin of interest and production of the antibody in a form recoverable therefrom. In connection with the production of transgenics in mammals, antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, for example, US 5,827,690, US 5,756,687, US 5,750,172 and US 5,741,957.

[097] In addition, human antibodies of the present invention or antibodies of the present invention from other species can be generated through demonstration-type technologies, which include, without limitation, phage demonstration, retroviral demonstration, ribosomal demonstration, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to further maturation, such as affinity maturation, as such techniques are well known in the art (see, for example, Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith , Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russell et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and US 5,733,743). If demonstration technologies are used to produce antibodies that are not human, such antibodies can be humanized.

[098] The antibody of the invention can be of any isotype. Isotype choice will typically be guided by desired effector functions, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Each of the human light chain constant regions, kappa or lambda, can be used. If desired, a CD74 specific antibody of the present invention can be class switched by known methods. For example, an antibody of the present invention that was originally IgM can be class-switched to an IgG antibody of the present invention. Furthermore, class switching techniques can be used to convert one subclass to another, for example from IgG1 to IgG2. Thus, the effector function of the antibodies of the present invention can be isotype-switched, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one embodiment an antibody of the present invention is an IgG1 antibody, for example an IgG1,K.

[099] In one embodiment, the antibody of the invention is a full-size antibody.

[0100] In one embodiment, the full-size antibody is an IgG1 antibody, such as an IgG1,K antibody.

[0101] In another embodiment, the full-size antibody is an IgG4 antibody.

[0102] In a particular embodiment, the CD74-specific IgG4 antibody is a stabilized IgG4 antibody. Examples of suitable stabilized IgG4 antibody are antibodies in which arginine is at position 409 in a human IgG4 heavy chain constant region, which is indicated in the EU index as in Kabat et al. supra, is substituted with lysine, threonine, methionine, or leucine, preferably lysine (described in WO2006033386) and/or wherein the hinge region comprises a Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO2008145142, which is hereby incorporated by reference in its entirety.

[0103] In one embodiment, the stabilized CD74-specific IgG4 antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group which consists of: Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or a residue selected from the group consisting of: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and where said antibody optionally comprises one or more other substitutions, deletions and/or insertions, but does not comprise a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, human IgG2 or human IgG3.

[0104] In another embodiment, the stabilized CD74-specific IgG4 antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group that consists of: Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or a residue selected from the group consisting of: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and wherein the said antibody optionally comprises one or more other substitutions, deletions and/or insertions and wherein said antibody comprises a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, human IgG2 or human IgG3.

[0105] In another embodiment, the CD74-specific antibody is an antibody of a non-IgG4 type, for example IgG1, IgG2 or IgG3 that has been mutated such that the ability to mediate effector functions such as ADCC has been reduced or even eliminated. Such mutations, for example, were described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol. 75(24): 12161-12168 (2001).

[0106] In one embodiment, the respective isotypes and/or sequences of the two heavy chain constant regions (Fc) are the same. In another embodiment, the respective isotypes and/or sequences of the two heavy chain constant regions (Fc) of a single CD74-specific antibody are different. This is particularly applicable to multispecific, such as bispecific, CD74-specific antibodies, which are described in more detail below.

[0107] In another aspect, the antibody is an antigen-binding fragment. Antibody fragments can be obtained by conventional techniques, such as by fragmenting full-size antibodies or by expressing nucleic acids encoding antibody fragments in recombinant cells (see, for example, Evans et al., J. Immunol. Met. 184, 123-38 (1995)). The fragments can then be tested or screened for properties in the same manner as described herein for full-size antibodies. The following describes exemplary formats for the CD74 specific antigen binding fragments of the invention:

[0108] The F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region. These can be generated, for example, by treating a full-size antibody with pepsin.

[0109] The Fab' or Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains. Fab fragments can be obtained, for example, by treating an IgG antibody with papain. Fab' fragments can be obtained, for example, by reducing the disulfide bonds of an F(ab') 2 fragment using a reducing agent such as dithiothreitol.

[0110] Monovalent antibodies or "antibody half-molecules", which exist in aqueous solutions as a heterodimer of a single light and single heavy chain, described in WO2007059782 (Genmab A/S).

[0111] The Fd fragments, which essentially consist of the VH and CH1 domains.

[0112] The Fv fragments, which consist essentially of the VL and VH domains of a single branch of an antibody and single chain antibodies thereof. Single-chain antibodies (also known as single-chain Fv (scFv) antibodies) are constructs where the VL and VH domains of an Fv fragment are joined, using recombinant methods, by a synthetic linker that enables them to be expressed as a single protein chain in which the pair of VL and VH regions form monovalent molecules (see for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988) ).

[0113] Domain antibodies (also called dAb fragments), essentially consist of a VH domain (see, for example, Ward et al., Nature 341, 544-546 (1989); Holt et al; Trends Biotechnol. 2003 Nov; 21(11): 484-90).

[0114] Other exemplary formats include camelids or nanobodies (see, for example, Revets et al; Expert Opin Biol Ter. 2005 Jan; 5(1): 111-24).

Multispecific antibody formats

[0115] In another embodiment, the invention provides a multispecific antibody comprising a first antigen binding site of a CD74 molecule specific antibody described herein above and at least a second antigen binding site.

[0116] In a particular embodiment, the second antigen binding site is used to recruit a killer mechanism such as, for example, by binding an antigen on a human effector cell or by binding a cytotoxic agent or a second therapeutic agent. Exemplary effector cells include a T cell such as, for example, a cytolytic T cell (CTL), a natural killer (NK) cell, a macrophage, a monocyte, a mastoid, and a granulocyte, such as, for example, a neutrophil, an eosinophil and a basophil. Exemplary effector cell antigens include, but are not limited to, CD1, CD3, CD4, CD8, CD16, CD25, CD28, CD32, CD40, CD64, CD89, FcgRI and HLA-DR. Suitable cytotoxic agents and secondary therapeutic agents are exemplified below, and include toxins (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs.

[0117] In another particular embodiment, the second antigen binding site binds to an antigen on a human B cell, such as, for example, CD19, CD20, CD21, CD22, CD23, CD46, CD80, CD138 and HLA-DR.

[0118] In another particular embodiment, the second antigen binding site binds to a tissue-specific antigen, which promotes the localization of the bispecific antibody to a specific tissue.

[0119] In another particular embodiment, the second antigen binding site binds to an antigen located on the same cell type as the cell that expresses CD74, typically a tumor associated antigen (TAA), but has a specificity of binding different from that of the first antigen binding site. Such multi- or bispecific antibodies can enhance tumor cell binding specificity and/or mesh multiple effector pathways. Exemplary TAAs include carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), α-fetoprotein, CAMEL (CTL-recognized antigen in melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc. ), ganglioside antigens, tyrosinase, gp75, c-Met, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM or a cancer-associated integrin, such as such as α5β3 integrin. Alternatively, the second antigen binding site binds to an epitope other than CD74. The second antigen binding site may alternatively bind an angiogenic factor or other cancer-associated growth factor, such as a vascular endothelial growth factor, a fibroblast growth factor, epidermal growth factor, angiogenin, or a receptor for any of these. of these, particularly receptors associated with cancer progression.

[0120] In another particular embodiment, the second antigen binding site is from a second CD74-specific antibody, such as a CD74-specific antibody of the invention.

[0121] Exemplary formats for the multispecific antibody molecules of the invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to CD74 and the other with a specificity to a second antigen; (ii) a single antibody comprising two different antigen binding regions; (iii) a single chain antibody comprising two different antigen binding regions, for example two scFvs linked in tandem by an extra peptide linker; (iv) a dual variable domain antibody (DVD-Ig), where each light chain and heavy chain contain two variable domains in tandem through a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of two single chain diabodies that results in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody that results in a multivalent molecule; (viii) a so-called “dock and lock” molecule, based on the “dimerization and anchoring domain” in Protein Kinase A, which, when applied to Fabs, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so-called Scorpion molecule, which comprises, for example, two scFvs fused to both termini of a human Fab-branch; and (x) a diabody.

[0122] Another exemplary format for bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known technologies, such as, for example, those known as the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-paired (Amgen) technologies. ), LUZ-Y (Genentech), Strand Exchange Engineered Domain Body (VERDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S).

[0123] In one embodiment, the bispecific antibody is obtained or obtainable via a controlled Fab-branch exchange, typically using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab-branch switching have been described in WO 2008119353 and WO 2011131746 (both by Genmab A/S). In an exemplary method, described in WO 2008119353, a bispecific antibody is formed by exchanging "Fab-branch" or "half-molecule" (swapping a heavy chain and linked light chain) between two monospecific antibodies, both comprising identical CH3 regions. IgG4, in incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences. In another exemplary method, described in WO 2011131746, bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is a CD74 antibody of the present invention: a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is a CD74 antibody of the present invention and the second antibody has a different binding specificity, or vice versa.

[0124] Reducing conditions, for example, can be provided by the addition of a reducing agent, for example selected from 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. Step d) may further comprise restoring the conditions so that they become non-reducing or less-reducing, for example by removing a reducing agent, for example by desalting.

[0125] Preferably, the sequences of the first and second CH3 regions are different, comprising only a few, reasonably conservative, asymmetric mutations such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety. The following are exemplary embodiments of combinations of such asymmetric mutations, optionally wherein one or both of the Fc regions are of the IgG1 isotype.

[0126] In one embodiment, the first Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407, and 409, and the second Fc region has an amino acid at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein the first and second Fc regions are not substituted at the same positions.

[0127] In one embodiment, the first Fc region has an amino acid substitution at position 405, and said second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399 , 407 and 409, optionally 409.

[0128] In one embodiment, the first Fc region has an amino acid substitution at position 409, and said second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399 , 405, and 407, optionally 405 or 368.

[0129] In a particular embodiment, both the first and second of the Fc regions are of the IgG1 isotype, with the first Fc region having a Leu at position 405, and the second Fc region having an Arg at position 409.

Conjugates

[0130] The present invention provides a CD74 specific antibody conjugated to a therapeutic moiety, i.e. a drug. The therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immune stimulator, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as an "antibody drug conjugate" or "ADCs".

[0131] Accordingly, in one aspect, the antibody according to any aspect or embodiment described above is conjugated to a therapeutic moiety. Exemplary therapeutic moieties include a cytotoxic moiety, a radioisotope, a cytokine, and a lytic peptide.

[0132] In one embodiment, the antibody is capable of inducing cytotoxicity in a Raji cell by internalizing the antibody conjugated to or associated with a therapeutic moiety in the Raji cell, for example as described in Example 14 or a similar type of rehearsal. In one embodiment, the antibody induces cytotoxicity by internalization as described in Example 14, with an EC50 value between about 25 ng/ml and about 60 ng/ml, such as between 25 ng/ml and 30 ng/ml, or an EC50 value of less than 60 ng/ml, such as less than 40 ng/ml, or less than 30 ng/ml to induce Raji cell death in an anti-kappa ETA' assay. In another embodiment, an ADC according to the present invention induces cytotoxicity with an EC50 value of less than 10 ng/ml, such as less than 5 ng/ml, less than 1 ng/ml, less than 0 .5 ng/ml or less than 0.1 ng/ml in inducing death of Raji cells or other cells expressing CD74.

[0133] In one embodiment, the antibody is conjugated to a cytotoxic moiety. The cytotoxic moiety, for example, can be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxy anthracine dione; a tubulin inhibitor such as maytansine or an analogue or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1- dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; an antimetabolite such as methotrexatp, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, raquelmycin (CC-1065), or an analogue or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as toxins SLT I, SLT II, SLT IIV, LT, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soy Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin toxins, saporin, modeccin, gelatin, chain abrin A, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor , gelonin, mitogelin, restrictocin, phenomycin, and enomycin; ribonuclease (RNase); DNase I, staphylococcal enterotoxin A; pigweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0134] In one embodiment, the antibody is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Quimioter. 45(12): 3580-3584) and have anticancer (US5663149) and antifungal activity. (Pettit et al., (1998) Antimicrob. Agents and Chemoter. 42: 2961-2965. For example, auristatin E can be reacted with para-acetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other derivatives Typical auristatin agents include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). exemplo, nas Patentes U.S. Nos. 5.635.483, 5.780.588 e 6.214.345 e nas Publicações de pedido de patente internacionais WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 e WO205082023.

[0135] In one embodiment, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or an analogue, derivative or prodrug thereof. Suitable PDBs and PDB derivatives and related technologies are described, for example, in Hartley J.A. et al., Cancer Res 2010; 70(17): 6849-6858; Antonow D. et al., Cancer 3 2008; 14(3): 154-169; Howard P.W. et al., Bioorg Med Chem Lett 2009; 19: 64636466 and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18): 2083-2086.

[0136] In one embodiment, the antibody is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, raquelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E , monomethyl auristatin F, a PDB, or an analogue, derivative, or prodrug of any of these.

[0137] In a particular embodiment, the antibody is conjugated to an anthracycline or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to maytansine or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to calicheamicin or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to duocarmycin or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to raquelmycin (CC-1065) or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to dolastatin 10 or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to dolastatin 15 or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to monomethyl auristatin E or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to monomethyl auristatin F or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to irinotecan or an analogue, derivative or prodrug thereof.

[0138] In one embodiment, a CD74-specific antibody of the invention is conjugated to a nucleic acid or nucleic acid-associated molecule. In such an embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxyribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule ) or an immunostimulatory nucleic acid (e.g., a DNA molecule that contains the immunostimulatory CpG motif). In another embodiment, a CD74-specific antibody of the invention is conjugated to an aptamer or a ribozyme.

[0139] In one embodiment, a CD74-specific antibody of the invention is conjugated, for example, as a fusion protein, to a lytic peptide such as CLIP, Magainin 2, melittin, Cecropin and P18.

[0140] In one embodiment, the antibody is conjugated to a cytokine, such as, for example, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13 , IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN8, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa.

[0141] In one embodiment, the antibody is conjugated to a radioisotope or a chelate that contains radioisotope. For example, the antibody may be conjugated to a chelating linker, for example DOTA, DTPA or tiuxetan, which enables the antibody to be complexed with a radioisotope. The antibody may also or alternatively comprise or may be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. A CD74-specific radiolabeled antibody can be used for both diagnostic and therapeutic purposes. Non-limiting examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, 99TC, 125I, 111In, 131I, 186Re, 213Bi, 225Ac, and 227Th. For therapeutic purposes, a radioisotope that emits beta or alpha particle radiation may for example, 131I, 90Y, 211At, 212Bi, 67Cu, 186Re, 188Re, and 212Pb can be used.

[0142] A therapeutic agent that can be administered in combination with a CD74-specific antibody of the present invention as described elsewhere herein, such as, for example, a chemotherapeutic agent, anti-cancer cytokine, or chemokine, is also a candidate for a moiety. therapy useful for conjugation to an antibody of the present invention.

[0143] A CD74-specific antibody of the present invention may also be chemically modified by covalent conjugation to a polymer to, for example, increase its circulating half-life.

[0144] Exemplary polymers, and methods for linking them to polypeptides, are illustrated, for example, in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG having a molecular weight between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).

[0145] A therapeutic product or other agent can be conjugated directly or indirectly to a CD74-specific antibody of the present invention, according to methods known in the art. An example of indirect conjugation of a second agent is via a spacer moiety for cysteine or lysine residues in the antibody. The therapeutic or other moiety may also or alternatively be conjugated to an N-terminal (amino) or C-terminal (carboxy) residue of a CD74 polypeptide-specific antibody or fragment thereof (e.g., a CD74 H- or L-chain-specific antibody). ) (see, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Conjugated antibody derivatives can also be generated by conjugation to internal residues or sugars, where appropriate. Exemplary methods are also described, for example, in Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Met. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982).

[0146] In one embodiment, a CD74-specific antibody is conjugated to a prodrug molecule via a spacer or linker that can be activated in vivo to a therapeutic drug. For example, the prodrug moiety may be linked to the antibody via a linker through the N- or C-terminus of the peptide or non-peptide drug moiety. After administration, the spacers or linkers are cleaved by enzymes associated with the tumor cell or other specific tumor conditions by which the active drug is formed. Examples of such prodrug and linker technologies are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171 by Syntarga BV, et al. (all incorporated herein by reference). Suitable antibody-prodrug technology and duocarmycin analogues can also be found in U.S. Patent. At the. 6,989,452 (Medarex) (incorporated herein by reference). Suitable prodrug technology for auristatins is described in WO03026577 (Seatte Genetics) and other references on auristatin mentioned above.

[0147] In one embodiment, a CD74-specific antibody is conjugated to a therapeutic or prodrug moiety via a linker sensitive to changes in pH or reducing conditions. Suitable linker technologies are known in the art, and include those described, for example, in Ducry, L and Stump, Bioconjugate Chem. 2010; 21:5-13; Senter P.D., Current Opinion in Chemical Biology 2009; 13: 235-244; and Carter, P.J. and Senter, P.D., The Cancer Journal 2010; 14: 154169.

[0148] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit of the antibody into the intracellular environment. In some embodiments, the linker is cleavable by a cleavable agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveoli). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, which includes, but is not limited to, a lysosomal or an endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug into target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83). : 67-123). In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker (valine-citrulline) or a Phe-Lys linker (phenylalanine-lysine) (see, for example, US6214345, which describes the synthesis of doxorubicin with the Val-Cit linker and different examples of Phe-Lys linkers). Examples of the structures of a Val-Cit linker and a Phe-Lys include, but are not limited to, MC-vc-PAB described below, MC-vc-GABA, MC-Phe-Lys-PAB or MC-PheLys-GABA, where MC or mc is an abbreviation for caproyl maleimido, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate, and GABA is an abbreviation for Y-aminobutyric acid. An advantage of using intracellular proteolytic delivery of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[0149] In another embodiment, the linker unit is not cleavable and the drug is released by antibody degradation (see, for example, US 2005/0238649). Typically, such a linker is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment" in the context of a linker means no more than 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 a sample of antibody-drug compound conjugate are cleaved when the antibody-drug conjugate drug compound is present in an extracellular environment (eg plasma). Whether a binder is not substantially sensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantification of the amount of free drug present in plasma.

em que a linha ondulada indica o sítio de ligação covalente para o ligador.[0150] In a specific embodiment, the CD74-specific antibody is conjugated to MMAE (formula I):

where the wavy line indicates the covalent binding site for the linker.

em que a linha ondulada indica o sítio de ligação covalente para o ligador.[0151] In another specific embodiment, the CD74-specific antibody is conjugated to MMAF (formula II):

where the wavy line indicates the covalent binding site for the linker.

[0152] In a particular embodiment, the linker for MMAE or MMAF is linked to the sulfhydryl groups (free cysteine residues) of the CD74-specific antibody, obtained by the (partial) reduction of the CD74-specific antibody.

em que p indica um número de 1 a 8, S representa um resíduo de cisteína tiol livre do anticorpo específico de CD74, e Ab designa o anticorpo específico de CD74. Em uma forma de realização deste, o ligador- auristatina é vcMMAE. A porção ligadora de medicamento de vcMMAE e os métodos de conjugação são divulgados na WO2004010957, US7659241, US7829531, US7851437 e US 11/833,028 (Seattle Genetics, Inc.), (que são aqui incorporadas por referência), e a porção ligadora de medicamento vcMMAE pode ser ligada aos anticorpos específicos de CD74 nas cisteínas usando um método similar a aqueles aqui divulgados.[0153] In another particular embodiment, the linker-auristatin is MC-vc-PAB-MMAF (also referred to as vcMMAF) or MC-vc-PAB-MMAE (also referred to as vcMMAE (formula III and IV, respectively):

wherein p denotes a number from 1 to 8, S represents a free cysteine thiol residue of the CD74-specific antibody, and Ab denotes the CD74-specific antibody. In one embodiment thereof, the auristatin-linker is vcMMAE. The drug-binding portion of vcMMAE and methods of conjugation are disclosed in WO2004010957 , US7659241 , US7829531 , US7851437 and US 11/833,028 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the drug-binding portion vcMMAE can be linked to CD74-specific antibodies on cysteines using a method similar to those disclosed herein.

em que p indica um número de 1 a 8, S representa um resíduo de cisteína tiol livre do anticorpo específico de CD74, e Ab designa o anticorpo específico de CD74. A porção ligadora de medicamento mcMMAF e métodos de conjugação são divulgados na US7498298, US 11/833,954, e WO2005081711 (Seattle Genetics, Inc.) (que são aqui incorporadas por referência), e a porção ligadora de medicamento mcMMAF pode ser ligada aos anticorpos específicos de CD74 nas cisteínas que usam um método similar a aqueles aqui divulgados.[0154] In another particular embodiment, the linker-conjugate is mcMMAF (formula V):

wherein p denotes a number from 1 to 8, S represents a free cysteine thiol residue of the CD74-specific antibody, and Ab denotes the CD74-specific antibody. The mcMMAF drug-binding moiety and methods of conjugation are disclosed in US7498298, US 11/833,954, and WO2005081711 (Seattle Genetics, Inc.) (which are incorporated herein by reference), and the mcMMAF drug-binding moiety can be linked to antibodies specific CD74 on cysteines using a method similar to those disclosed herein.

[0155] In one aspect, the invention provides a CD74-specific ADC which comprises an antibody binding to the same epitope as an antibody selected from 005, 006, 008 and 011, and a medicament which is auristatin or an analogue, derivative or pro - this medicine. In one embodiment, the EC50 of the ADC binding to the extracellular domain of CD47v1 is lower than about 0.2 µg/ml, such as lower than 0.1 µg/ml, or lower than about 0.1 µg/ml. of 0.05 µg/ml, optionally higher than 0.01 µg/ml, such as higher than 0.02 µg/ml, when determined in an assay as described in Example 16. In one embodiment, CD74-specific ADC induces cell death higher than 70%, 80%, or 90% when measured for Raji, Daudi, or M4A4 cells in an assay as described in Example 18. In one embodiment, the CD74-specific ADC CD74 has an IC50 of less than about 0.5 µg/ml, less than about 0.3 µg/ml, less than about 0.2 µg/ml, or less than about 0.1 μg/ml, and optionally higher than 0.005 μg/ml or about 0.01 μg/ml, in inducing death of Raji, Daudi or M4A4 cells when determined in an assay as described in Example 18. embodiment, the antibody comprises at least the VH CDR3, such as VH CDR1, 2 and 3, optionally VH CDR1, 2 and 3 and VL CDR1, 2 and 3 of 005, described in Table 3. In one embodiment, the antibody comprises at least the VH CDR3, such as VH CDR1, 2 and 3, optionally VH CDR1, 2 and 3 and VL CDR1, 2 and 3 of 006, described in Table 3. In one embodiment, the antibody comprises at least VH CDR3 , such as VH CDR1, 2 and 3, optionally VH CDR1, 2 and 3 and VL CDR1, 2 and 3 of 011, described in Table 3. In one embodiment, the medicament is a monomethyl auristatin derivative, optionally selected from MMAE and MMAF.

[0156] In one aspect, the invention provides a CD74-specific ADC that comprises an antibody comprising the CDR, the VH and/or VL sequences of an antibody selected from the group consisting of 005, 006 and 011, and a drug selected of MMAE and MMAF. In one embodiment, the antibody is 005. In one embodiment, the antibody is 006. In one embodiment, the antibody is 011. In a particular embodiment, the antibody is 005 and the drug is MMAE, optionally vcMMAE. In a particular embodiment, the antibody is 005 and the drug is MMAF, optionally mcMMAF. In a particular embodiment, the antibody is 006 and the drug is MMAE, optionally vcMMAE. In a particular embodiment, the antibody is 006 and the drug is MMAF, optionally mcMMAF. In a particular embodiment, the antibody is 011 and the drug is MMAE, optionally vcMMAE. In a particular embodiment, the antibody is 011 and the drug is MMAF, optionally mcMMAF.

[0157] In specific and separate embodiments, the invention provides the following ADCs specific for CD74: 011-vcMMAE, 006-vcMMAE, 005-vcMMAE, 011-mcMMAF, 006-mcMMAF and 005-mcMMAF.

[0158] Cytostatic drug load is represented by p and is the average number of cytostatic drug portions per antibody in a molecule (also referred to as the drug to antibody ratio, DAR). Cytostatic drug loading can range from 1 to 20 drug portions per antibody and can occur in amino acids with useful functional groups such as, but not limited to, amino or sulfhydryl groups, as in lysine or cysteine.

[0159] Depending on the mode of conjugation, p may be limited by the number of binding sites on the antibody, for example where the binding is a thiol cysteine, i.e. a sulfhydryl group. In general, antibodies do not contain many free and reactive cysteine thiol groups, i.e., sulfhydryl groups, that can be attached to a drug moiety, since most of the cysteine thiol residues in antibodies exist as disulfide bonds. Therefore, in certain embodiments, an antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partially or fully reducing conditions, to generate reactive sulfhydryl groups. In certain embodiments, the drug loading for an ADC of the invention ranges from 1 to about 8, such as from about 2 to 5, such as from about 3 to 5, such as from about 4. A maximum of 8 free sulfhydryl groups may become available after (partial) reduction of the antibody (there are 8 cysteines involved in interchain disulfide bonds).

Expression Constructs

[0160] In further and separate aspects, the invention pertains to nucleic acids encoding a sequence of an antibody of the invention, expression vectors encoding sequences of an antibody of the invention, host cells comprising such expression vectors, to hybridomas that produce antibodies of the invention, and methods of producing an antibody of the invention by culturing such host cells or hybridomas under appropriate conditions whereby the antibody is produced and, optionally, recovered.

[0161] In one embodiment, the invention provides an expression vector comprising a nucleotide sequence encoding one or more amino acid sequences selected from SEQ ID NOS: 7-26. In one embodiment, the expression vector comprises one or more nucleotide sequences encoding one or more of the amino acid sequences selected from the group consisting of SEQ ID NOS: 7, 11, 15, 19, 23 and 26, or any combination of these. In another embodiment, the expression vector comprises a nucleotide sequence encoding any one or more of the VH CDR3 amino acid sequences of SEQ ID NOS: 10, 14, 18, or 22. In another embodiment, the expression vector comprises a nucleotide sequence encoding a VH amino acid sequence selected from SEQ ID NOS: 7, 11, 15 and 19. In another embodiment, the expression vector comprises a nucleotide sequence encoding a VL amino acid sequence selected from SEQ ID NOS: 23 and 26. In another embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of a human antibody light chain, a human antibody heavy chain, or both.

[0162] In a particular embodiment, the expression vector of the invention comprises a nucleotide sequence encoding variants of one or more of the above amino acid sequences, said variants having at most 25 of the amino acid modifications, such as at most 20, such as at most 15, 14, 13, 12 or 11 of the amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions or at least 80% identity to any of said sequences, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97 % identity or 98% identity or 99% identity to any of the aforementioned amino acid sequences.

[0163] An expression vector in the context of the present invention can be any suitable vector, which includes chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a nucleic acid-specific antibody encoding CD74 is comprised of a naked DNA or RNA vector that includes, for example, a linear expression element (as described, for example, in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a packed nucleic acid vector (as described, for example, in US 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC19/18, or pUC118/119, a “mosquito” minimal-sized nucleic acid vector (as described, for example, in Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4- precipitated construct (as described for example in WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0164] In one embodiment, the vector is suitable for the expression of the CD74-specific antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), pET vectors (Novagen, Madison WI) and the like.

[0165] An expression vector may also, or alternatively, be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0166] A nucleic acid and/or vector may also comprise a nucleic acid sequence encoding a secretion/localization sequence, which may target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or within the culture medium. of cell. Such sequences are known in the art, and include secretion or signal leader peptides, organelle-targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), Membrane localization/anchoring sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.

[0167] In an expression vector of the invention, the nucleic acid-specific antibodies encoding CD74 may comprise or be associated with any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE (the skilled artisan will recognize that such terms are indeed descriptors of a degree of gene expression under certain conditions).

[0168] In one embodiment, the expression vector encoding the CD74-specific antibody is positioned on and/or delivered to the host cell or host animal via a viral vector.

[0169] Such expression vectors can be used for the recombinant production of the antibodies of the invention.

[0170] In one aspect, the invention provides a recombinant eukaryotic or prokaryotic host cell that produces the antibody of any aspect or embodiment described herein. Accordingly, the invention provides a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody or immunoglobulin of the invention as defined herein. Examples of host cells include yeast, bacterial and mammalian cells, such as CHO or HEK-293 cells. For example, in one embodiment, the present invention provides a cell that comprises a nucleic acid stably integrated into the cellular genome that comprises a sequence encoding the expression of a CD74-specific antibody of the present invention. In another embodiment, the present invention provides a cell comprising an unintegrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence encoding the expression of a CD74-specific antibody. invention.

[0171] In another aspect, the invention pertains to a hybridoma that produces an antibody of the invention as defined herein. In yet another aspect, the invention pertains to a transgenic non-human animal or plant comprising nucleic acids encoding a human heavy chain and a human light chain, wherein the animal or plant produces an antibody of the invention. The generation of such hybridomas and transgenic animals or plants has been described above, and is further described in the Examples.

[0172] In another aspect, the invention pertains to a method for producing a CD74-specific antibody of the invention, said method comprising the steps of a) culturing a hybridoma or host cell of the invention as described hereinabove, and b) recovering and/or purifying the antibody of the invention from the culture medium, and, optionally, c) preparing a CD74-specific antibody ADC.

[0173] In another aspect, the nucleotide sequence encoding a sequence of an antibody of the invention further encodes a second portion, such as a therapeutic polypeptide. Exemplary therapeutic polypeptides are described elsewhere herein. In one embodiment, the invention relates to a method for producing a CD74-specific antibody fusion protein, said method comprising the steps of a) culturing a host cell which comprises an expression vector comprising such a nucleotide sequence and b) recovering and/or purifying the CD74-specific antibody fusion protein from the culture medium.

pharmaceutical compositions

[0174] In one aspect, the invention provides a pharmaceutical composition comprising an antibody or ADC as defined in any of the above aspects and embodiments, and a pharmaceutically acceptable carrier.

[0175] Pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other of the known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed. ., Mack Publishing Co., Easton, PA, 1995.

[0176] Pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the antibody or antibody conjugate of the present invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the chosen pharmaceutical compound or composition of the present invention (e.g., less than substantial impact (10% or less than relative inhibition, 5% or less relative inhibition, etc.) on antigen binding).

[0177] A pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, detergents (e.g., a non-ionic detergent such as Tween-20 or Tween-80), stabilizers (e.g., sugars or amino acids). protein-free), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.

[0178] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied in order to obtain an amount of the active ingredient that is effective to obtain the desired therapeutic response for a particular patient, composition, and mode of administration. , without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention used, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular compositions used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the art. doctors.

[0179] The pharmaceutical composition may be administered by any suitable route and mode. Suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill in the art.

[0180] In one embodiment, a pharmaceutical composition of the present invention is administered parenterally.

[0181] The terms "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital injection. , intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal and infusion.

[0182] In one embodiment the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0183] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a compound of the present invention. .

[0184] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, colloidal solutions of carboxymethyl cellulose, gum tragacanth, and organic esters injectables, such as ethyl oleate, and/or various tampons. Other carriers are well known in the pharmaceutical arts.

[0185] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is envisaged.

[0186] Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0187] The pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants, for example, (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like ; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0188] The pharmaceutical compositions of the present invention may also comprise isotonicity agents such as sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.

[0189] The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate to the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance shelf life or effectiveness of the pharmaceutical composition. Compounds of the present invention can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, which includes implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biocompatible, biodegradable polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-ortho-esters, and polylactic acid alone or with a wax, or other materials. well known in the art. Methods for preparing such formulations are generally known to those skilled in the art. See for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0190] In one embodiment, the compounds of the present invention can be formulated to ensure proper delivery in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile and post-sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated. Other active or therapeutic compounds may also be incorporated within the compositions.

[0191] Pharmaceutical compositions for injection should typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be an aqueous or non-aqueous solvent or dispersion medium that contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients, for example, as enumerated above, as required, followed by sterilizing microfiltration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other required ingredients, for example those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze drying (lyophilization) which produces a powder of the active ingredient plus any additional desired ingredient from a previously filtered sterile solution of this active ingredient. .

[0192] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilizing microfiltration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze drying (lyophilization) which produces a powder of the active ingredient plus any additional desired ingredient from a previously filtered sterile solution of this ingredient. .

[0193] The pharmaceutical composition of the present invention may contain an antibody or ADC of the present invention, a combination of an antibody or ADC according to the invention with another therapeutic compound, or a combination of compounds of the present invention.

Therapeutic Applications

[0194] In another aspect, the invention pertains to the antibody or ADC of the invention, as defined in any aspect or embodiment herein, for use as a medicament.

[0195] The CD74-specific antibodies of the present invention can be used in the treatment or prevention of disorders involving cells that express CD74. For example, antibodies can be administered to cells in culture, for example, in vitro or ex vivo, or to human subjects, for example, in vivo, to treat or prevent disorders involving cells that express CD74. As used herein, the term "subject" is typically a human who responds to CD74-specific antibody or ADC. Subjects, for example, can include human patients having disorders that can be corrected or ameliorated by modulating CD74 function or by killing the cell, directly or indirectly.

[0196] In one embodiment, the invention provides a method for modulating CD74-associated signaling in a cell that expresses CD74 by contacting the cell with a CD74-specific antibody. A CD74-specific antibody of the invention can, for example, interfere with binding of MIF to CD74, which is a non-limiting example of how an antibody of the invention can modulate CD74-associated signaling.

[0197] In one embodiment, the invention provides a method of killing a cell that expresses CD74 by contacting the cell with a CD74-specific antibody of the invention. Without being bound by theory, antibody-mediated cross-linking or cluster formation (e.g., due to the Fc region of CD74-linked antibodies binding to FcR-expressing cells) of CD74 molecules on the surface of a cell can lead to apoptosis. of the cell.

[0198] In one embodiment, the invention provides a method of killing a cell that expresses CD74 by contacting the cell with a CD74-specific antibody of the invention in the presence of effector cells capable of inducing an Fc-mediated cell effector response such as as a CDC, ADCC, or ADCP response. In this embodiment, the antibody is typically of full size and of an isotype that leads to a CDC or ADCC response, such as, for example, an IgGl.k isotype.

[0199] The CD74-specific antibodies of the invention are characterized by efficient internalization in binding to CD74, making them suitable for an ADC method that uses an ADC as described in any aspect or embodiment described herein.

[0200] Accordingly, in one embodiment, the invention provides a method of killing a cell that expresses CD74 by contacting the cell with an ADC of the invention that requires internalization and trafficking to lysosomes for specific proteolytic cleavage (i.e. cleavable linker) or non-specific (non-cleavable linker) antibody-linker-drug complex. In another embodiment, the invention provides a method of killing a cell that expresses CD74 by contacting the cell with an ADC of the invention wherein the CD74-specific antibody is linked to a therapeutic moiety via a linker that enables the release of the CD74. drug once the ADC is internalized, for example by a change in pH or reducing conditions. Suitable connector technology is known in the art, as described above.

[0201] In another aspect, the present invention provides methods for treating or preventing a disorder involving cells that express CD74 in a subject, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC of the present invention. to an individual in need of it. The method typically involves administering to a subject a CD74 or ADC-specific antibody in an amount effective to treat or prevent the disorder.

[0202] In a particular aspect, a CD74-specific antibody or ADC is administered prophylactically so as to reduce the risk of developing cancer, delay the onset of an event in cancer progression, or reduce the risk of recurrence when a cancer is in remission. and/or a primary tumor has been surgically removed. In the latter case, the CD74-specific antibody, for example, can be administered in conjunction with (i.e., before, during, or after) surgery. Prophylactic administration may also be useful in patients in whom it is difficult to locate a tumor believed to be present due to other biological factors.

[0203] Cells that overexpress CD74, such as cancer cells, are particularly good targets for the CD74-specific antibodies or ADCs of the invention, as more antibodies or ADCs can be bound per cell. Thus, in one aspect, the disorder involving cells expressing CD74 is cancer, i.e., a tumorigenic disorder, such as a disorder characterized by the presence of tumor cells expressing CD74 which includes, for example, disorders where cells are of a solid tumor or hematologic tumor. CD74 expression has been described, for example, in breast cancer (Koretz K et al., Int J Cancer 1989; 44: 816822), colorectal cancer (Cuthbert R3 et al., Eur J Cancer 2009; 45: 16541663), cancer endometrial/cervical cancer (Glew SS et al., Cancer Res 1992; 52: 4009-4016), gastric cancer (Tamori Y et al, Oncol Rep 2005; 14: 873-877), squamous cell carcinoma of the head and neck (SCCHN ) (Han J et al., Head Neck Oncol 2009; 1: 27), lung cancer (McClelland M et al., Am J Pathol 2009; 174: 638-646), glioblastoma (Kitange G3 et al., J Neurooncol 2010 ;100:177-186), malignant lymphoma (Momburg F et al., Int J Cancer 1987;40:598-603), B-cell chronic lymphocytic leukemia (B-CLL) (Narni F et al., Blood 1986; 68:372-377), non-Hodgkin's lymphoma (NHL), monocytoid B-cell lymphoma (MBCL) (Stroup R et al, Hum Pathol 1992; 23:172-177), hairy cell leukemia (HCL) (Spiro RC et al., Leuk Res 1984; 8: 5562), malignant melanoma (Weeraratna AT et al., Oncogene 2004; 23: 22642274), ovarian cancer Aryan (Rangel LB et al., Cancer Biol Ter 2004; 3: 10211027), prostate cancer (Meyer-Siegler KL et al., BMC Cancer 2005; 5: 73), pancreatic cancer (Koide N et al., Clin Cancer Res 2006; 12: 24192426), kidney cancer (Saito T et al., Cancer Lett 1997;115:121-127), thymic epithelial neoplasms (Datta MW et al., Appl Immunohistochem Mol Morphol 2000;8:210-215), malignant fibrous histiosarcomas (Lazova R et al., Cancer 1997 ; 79: 2115-2124), and pituitary adenomas (Rossi ML et al., Tumori 1990; 76: 543-547). CD74 has also been found to be upregulated, for example, in the gastric epithelium during H. pylori infection and ulcerative colitis ( Beswick, World J Gastroenterol. 2009; 15(23): 2855-61 ).

[0204] Exemplary CD74 expressing cells thus include cancer cells such as, for example, NHL cells, multiple myeloma (MM), ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, gastric cancer, colorectal carcinoma and liver cancer .

[0205] In one aspect, the present invention provides methods of treating or preventing a hematologic malignancy, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC of the present invention to a subject in need thereof, and wherein the hematologic malignancy is selected from a lymphoma, myeloma and/or a leukemia. In one embodiment, the hematologic malignancy is selected from the group consisting of malignant lymphoma, B-cell chronic lymphocytic leukemia (B-CLL), burst phase chronic myeloid leukemia (CML), NHL, MM, MBCL, HCL, and T cell lymphoma.

[0206] In one embodiment, the hematologic malignancy is NHL. The CD74 and ADC-specific antibodies of the present invention, for example, can be used in the treatment of both indolent and aggressive forms of NHL. Examples of B cell NHLs include lymphomatoid granulomatosis, follicular lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, primary effusion lymphoma, intravascular large B cell lymphoma, mediastinal large B cell lymphoma, heavy chain diseases (which include y, μ, and α disease), lymphomas induced by therapy with immunosuppressive agents, such as cyclosporine-induced lymphoma, and methotrexate-induced lymphoma. In one embodiment, the hematologic malignancy is multiple myeloma, such as, for example, myeloma light chain disease and monoclonal gammopathy of undetermined significance (MGUS). In other separate and specific embodiments, the hematologic malignancy is malignant lymphoma, B-CLL (such as, for example, small lymphocytic lymphoma; SLL), burst stage CML, MBCL, or HCL. In one embodiment, the hematologic malignancy is a T cell lymphoma, such as, for example, mycosis fungoides, unspecified peripheral T cell lymphomas, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma (ALCL), T cell associated with enteropathy, or hepatosplenic T cell lymphoma. In another embodiment, the hematologic malignancy is Hodgkin's lymphoma. In another embodiment, the hematologic malignancy is Waldenstrom's macroglobulinemia. In one embodiment, the hematologic malignancy is CLL, such as BCLL (e.g., small lymphocytic lymphoma; SLL).

[0207] In one aspect, the present invention provides methods of treating or preventing a solid tumor, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC of the present invention to a subject in need thereof, and wherein the solid tumor is a melanoma, carcinoma, sarcoma, adenoma and/or a glioma. In one embodiment, the cancer is selected from the group consisting of breast cancer (such as, for example, primary or metastatic breast cancer), colorectal cancer, endometrial/cervical cancer, gastric cancer, head and neck cancer (such as , e.g. SCCHN), hepatocellular carcinoma, lung cancer (such as, e.g., small cell lung cancer or non-small cell lung cancer), malignant glioma (such as, e.g., anaplastic astrocytoma and glioblastoma multiforme), melanoma malignant (such as, for example, primary or metastatic melanoma), ovarian cancer (such as, for example, serous, endometrioid, or clear cell adenocarcinoma), pancreatic cancer, prostate cancer, kidney cancer, bladder cancer, thymic cancer ( such as, for example, thymic carcinoma and invasive thymoma), malignant fibrous histiosarcoma, acoustic schvanoma, pituitary adenoma, and a soft tissue tumor.

[0208] In one embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is selected from primary or metastatic breast cancer. In another embodiment, the cancer is pancreatic cancer, such as advanced or metastatic unresectable pancreatic cancer. In another embodiment, the cancer is prostate cancer. In another embodiment, the cancer is gastric cancer. In another embodiment, the cancer is colorectal carcinoma, such as metastatic colorectal carcinoma. In another embodiment, the cancer is hepatocellular carcinoma. In other separate and specific embodiments, the cancer is endometrial/cervical cancer, head and neck cancer, lung cancer, malignant glioma, malignant melanoma, ovarian cancer, kidney cancer, thymic cancer, malignant fibrous histiosarcoma, acoustic schvanoma, pituitary adenoma , or a soft tissue tumor.

[0209] In one aspect, the present invention provides methods of treating or preventing an autoimmune disease, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC of the present invention to a subject in need thereof. In one embodiment, the autoimmune disease is selected from an immune-mediated thrombocytopenia (such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura), dermatomyositis, Sjogren's syndrome, multiple sclerosis, Sydenham's chorea, myasthenia gravis, lupus erythematosus systemic lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigus, diabetes mellitus, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu arthritis, Addison's disease, rheumatoid arthritis, sarcoidosis, ulcerative colitis, erythema multiforme , IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, ubiterative thromboangiitis, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabe dorsalis, giant cell arteritis/p olimialgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.

[0210] In one embodiment, the autoimmune disease is rheumatoid arthritis. In another embodiment, the autoimmune disease is systemic sclerosis. In another embodiment, the autoimmune disease is multiple sclerosis. In another embodiment, the autoimmune disease is an inflammatory bowel disease, such as, for example, Crohn's disease or ulcerative colitis.

[0211] In one embodiment, the invention provides a method of treating any of the disorders of the above aspects and embodiments by administering to a subject in need thereof, a CD74-specific antibody or ADC of any of the aspects. or embodiments above. The invention also pertains to CD74-specific antibodies or ADCs of the invention for use as a therapeutic, for example, in the treatment of cancer or other disorder mentioned herein.

[0212] In one embodiment the selection of patients to be treated with CD74-specific antibodies is based on the level of CD74 expression in a sample, such as a sample that contains tumor cells, or by the detection of tumors that express CD74 using labeled CD74 specific antibodies or antibody fragments, for example those of the invention. Exemplary diagnostic assays for determining CD74 expression using CD74 antibodies or antibody fragment of the invention are described herein.

[0213] Efficient dosages and dosage regimens for the CD74-specific antibody or ADC depend on the disease or condition being treated and can be determined by those skilled in the art.

[0214] A physician having ordinary skill in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the physician may start doses of the CD74-specific antibody used in the pharmaceutical composition at levels lower than those required in order to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is obtained. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest effective dose to produce an effect in accordance with a particular dosage regimen. Such an effective dose will generally depend on the factors described above.

[0215] For example, an "effective amount" for therapeutic use can be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer, for example, can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be assessed by examining the compound's ability to inhibit cell growth or to induce cytotoxicity by in vitro assays known to the skilled artisan. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise improve symptoms in an individual. One of ordinary skill in the art would be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the composition or particular routes of administration selected.

[0216] An exemplary, non-limiting range for a therapeutically effective amount of a CD74-specific antibody of the present invention is from about 0.1 to 100 mg/kg, such as from about 0.1 to 50 mg/kg, for example from about 0.1 to 20 mg/kg, such as from about 0.1 to 10 mg/kg, for example from about 0.5, such as from about 0.3, about 1 , about 3 mg/kg, about 5 mg/kg, or about 8 mg/kg.

[0217] An exemplary, non-limiting range for a therapeutically effective amount of a CD74-specific ADC of the invention is from 0.02 to 100 mg/kg, such as from about 0.02 to 30 mg/kg, such as from from about 0.05 to 10 mg/kg or from 0.1 to 3 mg/kg, for example from about 0.5 to 2 mg/kg.

[0218] Administration, for example, may be intravenous, intramuscular, intraperitoneal, or subcutaneous and, for example, administered close to the target site.

[0219] Dosage regimens in the above methods of treatment and uses are adjusted to provide the desired optimal response (eg, a therapeutic product response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionately reduced or increased as indicated by the requirements of the therapeutic situation.

[0220] In one embodiment, the efficacy-safety window is optimized by decreasing specific toxicity such as, for example, decreasing the drug-antibody ratio (DAR) and/or mixing CD74-specific ADC with antibody unlabeled CD74 specific.

[0221] In one embodiment, the effectiveness of treatment is monitored during therapy, for example at predefined points in time. In one embodiment, effectiveness can be monitored by measuring the level of CD74 in a sample that contains tumor cells, by visualizing the area of disease, or by other diagnostic methods further described herein, for example, by performing a or more PET-CT scans, for example using a labeled CD74-specific antibody, fragment or mini-antibody derived from the CD74-specific antibody of the present invention.

[0222] If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In another embodiment, the CD74-specific antibodies are administered by slow continuous infusion over a long period, such as more than 24 hours, so as to minimize any of the undesired side effects.

[0223] While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition as described above.

[0224] An effective dose of a CD74 specific antibody or ADC of the invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, for example, 8 weeks, 12 weeks or until clinical progression has been established.

[0225] For example, in one embodiment, the CD74-specific antibody or ADC is infused at a weekly dosage between 10 and 500 mg/m2, such as between 200 and 400 mg/m2. Such administration may be repeated, for example, from 1 to 8 times, such as from 3 to 5 times. Administration can be accomplished by continuous infusion over a period of 1 to 24 hours, such as 1 to 12 hours.

[0226] In another embodiment, the CD74-specific antibody or ADC is infused every three weeks at a dosage between 10 and 500 mg/m2, such as between 50 and 200 mg/m2. Such administration may be repeated, for example, from 1 to 8 times, such as from 3 to 5 times. Administration can be accomplished by continuous infusion over a period of 1 to 24 hours, such as 1 to 12 hours.

[0227] In one embodiment, a CD74-specific ADC is administered as a single dose of about 0.1 to 10 mg/kg, such as from about 1 to 3 mg/kg, every week or every third week for up to twelve times, up to eight times, or until clinical progression. Administration can be accomplished by continuous infusion over a period of 1 to 24 hours, such as 1 to 12 hours. Such regimens may be repeated one or more times as needed, for example after 6 months or 12 months. Dosage can be determined or adjusted by measuring the amount of compound of the present invention in the blood on administration, for example, taking a biological sample and using anti-idiotypic antibodies that target the antigen-binding region of CD74-specific antibodies. of the present invention.

[0228] In one embodiment, the CD74-specific antibodies are administered as maintenance therapy, such as, for example, once a week for a period of six months or longer.

[0229] As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of a compound of the present invention in an amount of about 0.1 to 100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0230] Parenteral compositions may be formulated in unit dosage form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically separate units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention is dictated by and directly dependent upon (a) the unique characteristics of the active compound and the particular therapeutic effect to be obtained, and (b) the limitations inherent in the art of combining such active compound. for the treatment of sensitivity in individuals.

combinations

[0231] The invention also provides therapeutic applications where an antibody or ADC of the invention is used in combination with at least one other therapeutic agent relevant to the disease or disorder being treated, as described above. Such administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate.

[0232] Accordingly, the present invention provides methods for treating a disorder involving cells that express CD74 as described above, which method comprises administering a CD74-specific antibody or ADC of the present invention combined with one or more additional therapeutic agents. The present invention also provides the use of a CD74-specific antibody or ADC of the present invention for the preparation of a pharmaceutical composition to be administered with at least one chemotherapeutic agent to treat such a disorder.

[0233] The other therapeutic agent is typically relevant to the disorder being treated. Exemplary therapeutic agents include other anti-cancer antibodies or ADCs, cytotoxic agents, chemotherapeutic agents, anti-angiogenic agents, anti-cancer immunogens, cell cycle controlling/apoptosis-regulating agents, hormone-regulating agents, and other agents described below.

[0234] In one aspect, the other therapeutic agent is at least a second antibody or ADC that binds to another target such as, for example, CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25 , CD30, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, tenascin, HM1,24, or HLA-DR. For example, the second antibody can bind to a B cell antigen, which includes, but is not limited to, CD20, CD19, CD21, CD23, CD38, CD46, CD80, CD138, HLA-DR, CD22, or another epitope. on CD74. In another embodiment, the second antibody binds vascular endothelial growth factor A (VEGF-A). In separate and specific embodiments, the other therapeutic agent is a CD20-specific antibody or a CD138.

[0235] In one embodiment, the CD74 specific antibody or ADC of the invention is for use in combination with a specific therapeutic antibody, such as veltuzumab, bevacizumab (Avastin®), zalutumumab, cetuximab (Erbitux®), panitumumab ( Vectibix®), ofatumumab (Arzerra®), ocrelizumab, zanolimumab, daratumumab, ranibizumab (Lucentis®), Zenapax, Simulect, Remicade, Humira, Tysabri, Xolair, raptiva, nimotuzumab, rituximab and/or trastuzumab (Herceptin®). In one embodiment, the CD74-specific antibody or ADC of the present invention is administered in combination with a CD20-specific antibody such as, for example, veltuzumab, ocrelizumab, or ofatumumab (Arzerra®). In another embodiment, the CD74 specific antibody or ADC of the present invention is administered in combination with bevacizumab (Avastin®).

[0236] In one aspect, the invention provides an antibody or ADC of any of the above aspects or embodiments for treating a disorder involving cells that express CD74, such as cancer, in combination with at least one chemotherapeutic agent.

[0237] In one embodiment, the chemotherapeutic agent is selected from an antimetabolite such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, floxuridine (FudR), 3',5'-O-dioleoyl-FudR, fludarabine , 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine and similar agents.

[0238] In one embodiment, the chemotherapeutic agent is selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine ( DTIC), procarbazine, mitomycin C, and a platinum derivative such as cisplatin, carboplatin, and similar agents.

[0239] In one embodiment, the chemotherapeutic agent is selected from an anti-mitotic agent, such as taxanes, for example, docetaxel, and paclitaxel, and vinca alkaloids, for example, vindesine, vincristine, vinblastine, and vinorelbine.

[0240] In one embodiment, the chemotherapeutic agent is selected from a topoisomerase inhibitor, such as topotecan or irinotecan.

[0241] In one embodiment, the chemotherapeutic agent is selected from a cytostatic drug, such as etoposide and teniposide.

[0242] In one embodiment, the chemotherapeutic agent is selected from a growth factor receptor inhibitor, such as an ErbB1 inhibitor (EGFR) (such as Iressa, erbitux (cetuximab), tarceva and similar agents), a ErbB2 inhibitor (Her2/neu) (such as herceptin and similar agents) and similar agents.

[0243] In one embodiment, the chemotherapeutic agent is selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571), lapatinib, PTK787/ZK222584 and similar agents.

[0244] In one aspect, the present invention provides a method of treating a disorder involving cells that express CD74 in an individual, such as a cancer patient, which method comprises administering a therapeutically effective amount of a specific antibody of CD74 or ADC of the present invention and at least one inhibitor of angiogenesis, neovascularization, and/or other vascularization to an individual in need thereof.

[0245] Examples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar agents), cell migration and proliferation inhibitors endothelial (such as TNP-470, squalamine, 2-methoxyestradiol, combretastatins, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) and similar agents), angiogenic growth factor antagonists (such as ZD6474, SU6668, antibodies against angiogenic agents and/or their receptors (such as VEGF, bFGF, and angiopoietin-1), thalidomide, thalidomide analogues (such as CC5013), Sugen 5416, SU5402 , anti-angiogenic ribozyme (such as angiozyme), interferon α (such as interferon α2a), suramin and similar agents), VEGF-R kinase inhibitors and other angiogenic tyrosine kinase inhibitors (such as SU011248), signal inhibitors endothelial-specific integrin/survival action (such as vitamin and similar agents), copper antagonists/chelators (such as tetrathiomolybdate, captopril and similar agents), carboxyamido-triazole (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E as well as nucleotide molecules that inhibit angiogenesis (such as antisense VEGF cDNA, angiostatin-encoding cDNA, p53-encoding cDNA, and VEGF receptor-2-encoding cDNA disabled) and similar agents.

[0246] Other examples of such inhibitors of angiogenesis, neovascularization, and/or other vascularization are anti-angiogenic heparin derivatives and related molecules (eg, heperinase III), temozolomide, NK4, cyclooxygenase-2 inhibitors, factor 1 inhibitors hypoxia-inducible, anti-angiogenic soy isoflavones, oltipraz, fumaragiline and analogues thereof, somatostatin analogues, pentosan polysulfate, tecogalan sodium, dalteparin, tunstatin, thrombospondin, NM-3, combretastatin, canstatin, avastatin, antibodies against other relevant targets (such as anti-alpha-v/beta-3 integrin and anti-quininostatin mAbs) and similar agents.

[0247] In one embodiment, the therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above is an anti-cancer immunogen, such as a cancer antigen/tumor-associated antigen (e.g. example, epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA), tumor associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1 , MDA7, cancer-associated viral vaccines (e.g., human papillomavirus vaccines), tumor-derived heat shock proteins, and similar agents. Various other suitable cancer/tumor-associated antigens known in the art may also or alternatively be Anti-cancer immunogenic peptides also include anti-idiotypic "vaccines" such as anti-idiotypic antibodies BEC2 (Mitumomab), CeaVac and related anti-idiotypic antibodies, anti-idiotypic antibodies s for the MG7 antibody, and other anti-idiotypic anticancer antibodies (see for example, Birebent et al., Vaccine. 21(15), 1601-12 (2003), Li et al., Chin Med J (Engl). 114(9), 962-6 (2001), Schmitt et al., Hybridoma. 13(5), 389-96 (1994), Maloney et al., Hybridoma. 4(3), 191-209 (1985), Raychardhuri et al., J Immunol. 137(5), 1743-9 (1986), Pohl et al., Int J Cancer. 50(6), 958-67 (1992), Bohlen et al., Cytokines Mol Ther. 2(4), 231-8 (1996) and Maruyama, J Immunol Methods. 264(1-2), 121-33 (2002)). Such anti-idiotypic antibodies may optionally be conjugated to a carrier, which may be a carrier for a synthetic (typically inert) molecule, a protein (e.g. keyhole limpet hemocyanin (KLH) (see e.g. Ochi et al., Eur J Immunol. 17(11), 1645-8 (1987)), or a cell (e.g., a red blood cell - see e.g. Wi et al., J Immunol Methods. 122(2), 227-34 ( 1989)).

[0248] In one embodiment, the therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above is a cytokine, chemokine, or cytokine/chemokine combination with cancer growth inhibitory properties. . Examples of suitable cytokines and growth factors include the ligands IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα (eg INFα2b), IFNβ, GM-CSF, CD40L, Flt3, stem cell factor, ancestim , and TNFα. Suitable chemokines may include Glu-Leu-Arg (ELR) negative chemokines such as IP-10, MCP-3, MIG, and SDF-1α from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins. These and other methods or uses involving nucleic acids encoding naturally occurring peptide herein may alternatively or additionally be accomplished by "gene activation" and homologous recombination gene upregulation techniques, such as those described in US 5,968. 502, US 6,063,630 and US 6,187,305 and EP 0505500.

[0249] In one embodiment, the therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above may be a cell cycle control/apoptosis regulator (or "regulatory agent") . A cell cycle control/regulator of apoptosis can include molecules that target and modulate cell cycle control/regulators of apoptosis such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxy-staurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such such as BIBR1532, SOT-095, GRN163 and compositions described for example in US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis ligand-2 (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and Bcl-2 of anti-sense.

[0250] In one embodiment, the therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above is a hormone-regulating agent, such as agents useful for anti-androgen therapy and anti-estrogenic. Examples of such hormone regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/stinyl, an antiandrogen (such as flutamind/eulexin), a progestin (such as hydroxy-progesterone caproate). , medroxyprogesterone/provera, megestrol acepate/megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone releasing hormone (and analogues thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazol/arimidex, aminoglutethimide/citraden, exemestane), a hormone inhibitor (such as octreotide/sandostatin) and similar agents.

[0251] In one embodiment, the therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above is an anti-anergic agent (e.g., small molecule compounds, proteins, glycoproteins, or antibodies that break tolerance to tumor and cancer antigens). Examples of such compounds are molecules that block CTLA-4 activity, such as MDX-010 (ipilimumab, Yervoy®) ( Phan et al., PNAS USA 100, 8372 (2003 )).

[0252] In one embodiment, the therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above is a nucleic acid or vector that contains a tumor suppressor gene such as an adenovirus deficient in replication encoding human recombinant wild-type p53/SCH58500, etc.; antisense nucleic acids targeted to oncogenes, mutated, or dysregulated genes; or siRNA targeted to mutated or dysregulated genes. Examples of tumor suppressor targets include, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC.

[0253] In one embodiment, a therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above is an anti-cancer nucleic acid. Exemplary anti-cancer nucleic acids include genasense (augmerosen/G3139), LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS 112989), LE-AON/LEraf-AON (liposome encapsulated c-raf antisense oligonucleotide / ISIS-5132), MG98, and other antisense nucleic acids that target PKCα, clusterin, IGFBPs, protein kinase A, cyclin D1, or Bcl-2.

[0254] In one embodiment, the therapeutic agent for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above is an anticancer inhibitory RNA molecule (see, for example, Lin et al., Curr Cancer Drug Targets 1(3), 241-7 (2001), Erratum in: Curr Cancer Drug Targets 3(3), 237 (2003), Lima et al., Cancer Gene Ther 11(5), 309 -16 (2004), Grzmil et al., Int J Oncol. 4(1), 97-105 (2004), Collis et al., Int 3 Radiat Oncol Biol Phys. 57(Supl 2), 5144 (2003 ), Yang et al., Oncogene 22(36), 5694-701 (2003 ) and Zhang et al., Biochem Biophys Res Commun 303(4), 1169-78 (2003 ) ).

[0255] The compositions and combination administration methods of the present invention also include the administration of nucleic acid vaccines, such as naked DNA vaccines encoding such cancer antigens/tumor associated antigens (see, for example, the US 5,589,466, US 5,593,972, US 5,703,057, US 5,879,687, US 6,235,523, and US 6,387,888). In one embodiment, the combination administration method and/or combination composition comprises an autologous vaccine composition. In one embodiment, the combination composition and/or method of administering the combination comprises a whole cell vaccine or cell that expresses cytokine (e.g., a fibroblast that expresses recombinant IL-2, dendritic cell that expresses recombinant cytokine, and the like) (see, for example, Kowalczyk et al., Acta Biochim Pol. 50(3), 613-24 (2003), Reilly et al., Methods Mol Med. 69, 233-57 (2002 ) and Tirapu et al. al., Curr Gene Ther. 2(1), 79-89 (2002) Another example of such an autologous cell method that may be useful in combination methods of the present invention is the MyVax® Personalized Immunotherapy method (previously alluded to as GTOP-99) (Genitope Corporation - Redwood City, CA, USA).

[0256] In one embodiment, a CD74-specific antibody or ADC according to the invention is combined or co-administered with a virus, viral proteins, or the like. Replication-deficient viruses, which are generally capable of one or only a few rounds of replication in vivo, and which are targeted to tumor cells, for example, can be useful components of such compositions and methods. Such viral agents may comprise or be associated with nucleic acids encoding immunostimulants, such as GM-CSF and/or IL-2. Both naturally occurring oncolytic and such recombinant oncolytic viruses (e.g., HSV-1 virus, reovirus, replication-deficient and replication-sensitive adenovirus, etc.) can be useful components of such methods and compositions. Accordingly, in one embodiment, the present invention provides combination compositions and combination administration methods wherein a CD74-specific antibody is combined or co-administered with an oncolytic virus. Examples of such viruses include oncolytic adenoviruses and herpes viruses, which may or may not be modified viruses (see, for example, Shah et al., J Neurooncol. 65(3), 203-26 (2003), Stiles et al. , Surgery 134(2), 357-64 (2003), Sunarmura et al., Pancreas. 28(3), 3269 (2004), Teshigahara et al., J Surg Oncol. 85(1), 42-7 ( 2004), Varghese et al., Cancer Gene Ther 9(12), 967-78 (2002), Wildner et al., Cancer Res 59(2), 410-3 (1999), Yamanaka, Int J Oncol. 24(4), 919-23 (2004) and Zwiebel et al., Semin Oncol 28(4), 336-43 (2001).

[0257] The combination compositions and combination administration methods of the present invention may also involve "whole cell" and "adoptive" immunotherapy methods. For example, such methods may comprise infusion or reinfusion of immune system cells (e.g., tumor-infiltrating lymphocytes (TILs), such as CD4+ and/or CD8+ T cells (e.g., T cells expanded with tumor-specific antigens). tumor and/or genetic enhancements), antibody-expressing B cells or other antibody-producing or presenting cells, dendritic cells (e.g., dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loaded dendritic cells), anti-tumor NK cells, called hybrid cells, or combinations thereof. Cell lysates may also be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in such respects include Canvaxin®, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens dispersed from cancer cells, and mixtures thereof (see, for example, Bystryn et al. ., Clinical Can cer Research Vol. 7, 1882-1887, July 2001), optionally blended with adjuvants such as alum, can also be components in such combination methods and compositions.

[0258] In one embodiment, a CD74-specific antibody or ADC is released to a patient in combination with the request for an internal vaccination method. Internal vaccination refers to induced tumor or cancer cell death, such as drug-induced or radiation-induced cell death, cryo-ablation or radiofrequency induced cell death of tumor cells, in a patient, which typically leads to evoking an immune response directed at (i) the tumor cells as a whole or (ii) parts of the tumor cells that include (a) secreted proteins, glycoproteins or other products, (b) membrane-associated proteins or glycoproteins or others components associated with or inserted into membranes, and/or (c) intracellular proteins or other intracellular components. An immune response induced by internal vaccination can be humoral (i.e., antibody-mediated by complement) or cell-mediated (e.g., the development and/or increase of endogenous cytotoxic T lymphocytes that recognize the internally dead tumor cells or parts thereof) . In addition to radiotherapy, non-limiting examples of drugs and agents that can be used to induce said tumor cell death and internal vaccination are conventional chemotherapeutic agents, cell cycle inhibitors, anti-angiogenesis drugs, monoclonal antibodies, apoptosis-inducing agents, and signal transduction inhibitors.

[0259] Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above are differentiation-inducing agents, retinoic acid analogues (such as all-trans retinoic acid, β-cis retinoic acid and similar agents), vitamin D analogues (such as seocalcitol and similar agents), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRalpha, PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and similar agents.

[0260] Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above are cathepsin B, dehydrogenase activity of cathepsin D modulators, glutathione-S-transferase (such as glutacylcysteine synthetase and lactate dehydrogenase), and similar agents.

[0261] Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with a CD74-specific antibody to treat the disorders as described above are estramustine and epirubicin.

[0262] Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with a CD74-specific antibody to treat the disorders as described above are one of the HSP90 inhibitors such as 17-(Allylamino)-17- desmethoxygeldanamycin, antibodies directed against a tumor antigen such as PSA, CA125, KSA, etc., integrins such as β integrin, VCAM inhibitors and similar agents.

[0263] Examples of other anti-cancer agents that may be relevant as therapeutic agents for use in combination with a CD74-specific antibody or ADC to treat the disorders as described above are calcineurin inhibitors (such as valspodar, PSC 833 and other MDR-1 or p-glycoprotein inhibitors), TOR inhibitors (such as sirolimus, everolimus and rapamycin) and inhibitors of “lymphocytic resident” mechanisms (such as FTY720), and agents with effects on cell signaling such as inhibitors of adhesion molecule (eg anti-LFA, etc.).

[0264] In one embodiment, a CD74-specific antibody or ADC may be administered in connection with the release of one or more agents that promote access of the CD74-specific antibody or combination composition to the interior of a tumor. Such methods, for example, can be carried out in association with the release of a relaxin, which is capable of relaxing a tumor (see, for example, US 6,719,977). In one embodiment, a CD74-specific antibody or ADC of the present invention can be linked to a cell-penetrating peptide (CPP). Cell-penetrating peptides and related peptides (such as engendered cell-penetrating antibodies) are described, for example, in Zhao et al., J Immunol Methods. 254(1-2), 137-45 (2001), Hong et al., Cancer Res. 60(23), 6551-6 (2000). Lindgren et al., Biochem J. 377(Pt 1), 6976 (2004), Buerger et al., J Cancer Res Clin Oncol. 129(12), 669-75 (2003), Pooga et al., FASEB J. 12(1), 67-77 (1998) and Tseng et al., Mol Pharmacol. 62(4), 864-72 (2002).

[0265] In yet another embodiment, the CD74-specific antibody or ADC is administered in conjunction with an agent that upregulates CD74, such as, for example, IFNY or inactivated H. Pylori.

[0266] In one embodiment, the present invention provides a method of treating a disorder involving cells that express CD74 in a subject, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC and at least one anti-inflammatory, immunosuppressive and/or immunomodulatory agent to an individual in need thereof.

[0267] In one embodiment such an anti-inflammatory agent may be selected from aspirin and other salicylates, Cox-2 inhibitors (such as rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam , ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL-6R antibodies, anti-IL-8 antibodies (e.g. antibodies described in WO2004058797, such as 10F8), anti-IL-15 antibodies (e.g. e.g. antibodies described in WO03017935 and WO2004076620 ), anti-IL-15 receptor Abs, anti-CD4 antibodies (e.g. zanolimumab), anti-CD11a antibodies (e.g. efalizumab), anti-alpha-4/integrin antibodies beta-1 (VLA4) (eg natalizumab), CTLA4-Ig for the treatment of inflammatory diseases, prednisolone, prednisone, disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidine synthesis inhibitors (such as such as leflunomide), acts IL-1 receptor blocking agents (such as anakinra), TNF-α blocking agents (such as etanercept, infliximab, and adalimumab) and similar agents.

[0268] In one embodiment, such an immunosuppressive and/or immunomodulatory agent may be selected from cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids such as prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide , mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), thymopentin, thymosin-α and similar agents.

[0269] In one embodiment, such an immunosuppressive and/or immunomodulatory agent may be selected from immunosuppressive Abs, such as antibody binding to IL-2 receptor p75, antibodies against CD25 (e.g., those described in WO2004045512 such as such as AB1, AB7, AB11, and AB12), antibodies against thymocyte globulin, or binding of antibodies to, for example, MHC, CD2, CD3 (such as, for example, OKT3), CD4, CD7, CD28, B7, CD40, CD45 , IFNy, TNF-α, IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11α, or CD58, or antibody binding to their respective receptor(s) or ligand( s).

[0270] In one embodiment, such an immunosuppressive and/or immunomodulatory agent may be selected from soluble IL-15R, IL-10, B7 molecules (B7-1, B7-2, variants thereof, and fragments thereof), ICOS , and OX40, an inhibitor of a negative T cell regulator (such as an antibody against CTLA4) and similar agents.

[0271] In one embodiment, the present invention provides a method of treating a disorder involving cells that express CD74 in a subject, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC and a anti-C3b(i) antibody to an individual in need thereof.

[0272] In one embodiment, a therapeutic agent for use in combination with CD74-specific antibodies or ADCs to treat the disorders as described above may be selected from histone deacetylase inhibitors (e.g., phenyl butyrate) and/or DNA repair agents (eg, DNA repair enzymes and related compositions such as dimericin).

[0273] The methods of the present invention for treating a disorder as described above which comprises administering a therapeutically effective amount of a CD74 or ADC-specific antibody may also comprise targeted photodynamic anti-cancer therapy (e.g., laser anti-cancer therapy - which optionally can be practiced with the use of photosensitizing agent, see, for example, Zhang et al., J Control Release. 93(2), 141-50 (2003)), anticancer therapies with sound wave and shock wave (see for example, Kambe et al., Hum Cell. 10(1), 87-94 (1997)), and/or nutraceutical anticancer therapy (see for example, Roudebush et al., Vet Clin North Am Small Anim Pract. 34( 1), 249-69, viii (2004) and Rafi, Nutrition. 20(1), 78-82 (2004) Likewise, a CD74-specific antibody can be used for the preparation of a pharmaceutical composition to treat a disorder as described above to be administered with targeted photodynamic anticancer therapy (e.g., anti-cancer laser therapy - which optionally may be practiced using a photosensitizing agent), anti-cancer therapies with sound wave and shock wave, and/or anti-cancer nutraceutical therapy.

[0274] In one embodiment, the present invention provides a method of treating a disorder involving cells that express CD74 in a subject, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC of the present invention. invention, and radiotherapy to an individual in need thereof.

[0275] In one embodiment, the present invention provides a method of treating or preventing cancer, which method comprises administering a therapeutically effective amount of a CD74-specific antibody or ADC of the present invention, and radiotherapy to a subject in need for this.

[0276] In one embodiment, the present invention provides the use of a CD74-specific antibody or ADC of the present invention, for the preparation of a pharmaceutical composition for treating cancer to be administered in combination with radiotherapy.

[0277] Radiotherapy may comprise radiation or the associated administration of radiopharmaceuticals to a patient. The radiation source may be external or internal to the patient being treated (radiation treatment, for example, may be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used in the practice of such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123 , iodine-131, and indium-111.

[0278] In another embodiment, the present invention provides a method of treating or preventing cancer, which method comprises administering to a subject in need thereof a therapeutically effective amount of a CD74-specific antibody or ADC of the present invention. , in combination with surgery.

[0279] As described above, a pharmaceutical composition of the present invention may be administered in combination therapy, i.e., combined with one or more agents relevant to the disease or condition to be treated as separate pharmaceutical compositions or with a compound of the present invention. co-formulated with one or more additional therapeutic agents as described above. Such combination therapies may require lower dosages of the compound of the present invention and/or the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

[0280] In one embodiment, the other therapeutic agent for a particular therapeutic use is selected from the following: - A CD20-specific antibody, particularly for the treatment of a hematologic malignancy such as, for example, B-CLL or lymphoma follicular; - A CD138 specific antibody, particularly for the treatment of a hematological malignancy such as, for example, myeloma; - A CD38-specific antibody, particularly for the treatment of a hematological malignancy such as, for example, myeloma or CLL; - Melphalan (or melphalan hydrochloride) for the treatment of a haematological malignancy such as, for example, myeloma; - An anti-VEGF-A antibody such as, for example, bevacizumab, particularly for the treatment of a cancer such as, for example, breast cancer; - Lenalidomide or bortezomib, particularly for the treatment of a haematological malignancy such as, for example, myeloma; - Fluorouracil or genticabine, particularly for the treatment of a cancer such as, for example, pancreatic cancer; - Irinotecan, particularly for the treatment of cancer such as, for example, colorectal cancer; and - Cisplatin or another platinum derivative, particularly for the treatment of a cancer such as, for example, SCCHN.

Applications in Diagnostics

[0281] The CD74-specific antibodies of the invention may also be used for diagnostic purposes, which uses a composition comprising a CD74-specific antibody as described herein. Accordingly, the invention provides diagnostic methods and compositions that use the CD74-specific antibodies described herein. Such methods and compositions may be used purely for diagnostic purposes, such as detecting or identifying a disease involving cells that express CD74, as well as monitoring the progress of therapeutic treatments, monitoring disease progression, assessing the situation after treatment, monitor for disease recurrence, assess the risk of developing a disease, and the like.

[0282] In one aspect, the CD74-specific antibodies of the present invention are used ex vivo, such as in diagnosing a disease in which cells expressing CD74 are indicative of disease or involved in pathogenesis, by detecting CD74 levels or levels of cells expressing CD74 on their cell surface in a sample taken from a patient. This can be achieved, for example, by contacting the sample to be tested, optionally together with a control sample, with the CD74-specific antibody under conditions that allow the antibody to bind to CD74. Complex formation can then be detected (eg using an ELISA). When using a control sample along with the test sample, the level of CD74-specific antibody or CD74-CD74 complex-specific antibody is analyzed in both samples and a statistically significant higher level of CD74-specific antibody or CD74-CD74 complex-specific antibody in the test sample indicates a higher level of CD74 in the test sample compared to the control sample.

[0283] Examples of conventional immunoassays in which CD74-specific antibodies of the present invention can be used include, without limitation, ELISA, RIA, FACS, plasmon resonance assays, chromatographic assays, tissue immunohistochemistry, Western blot, and /or immunoprecipitation.

[0284] In one embodiment, the invention relates to a method for detecting the presence of CD74 antigen, or a cell that expresses CD74, in a sample comprising:

[0285] - contacting the sample with a CD74-specific antibody of the invention under conditions that enable the CD74-specific antibody to bind to CD74 in the sample; and

[0286] - Analyze whether a complex was formed. Typically, the sample is a biological sample.

[0287] In one embodiment, the sample is a tissue sample known or suspected to contain CD74 antigen and/or cells that express CD74. For example, in situ detection of CD74 expression can be accomplished by removing a histological specimen from a patient, and providing the antibody of the present invention to such a specimen. The antibody can be provided by applying or overlaying the antibody on the specimen, which is then detected using suitable means. It is then possible to determine not only the presence of CD74 or CD74 expressing cells, but also the distribution of CD74 or CD74 expressing cells in the tissue examined (e.g. in the context of assessing the spread of cancer cells). Using the present invention, those of ordinary skill will readily appreciate that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[0288] In the above assays, the CD74-specific antibody can be labeled with a detectable substance enabling the CD74-bound antibody to be detected. Alternatively, the bound (primary) CD74-specific antibody can be detected by a secondary antibody that is labeled with a detectable substance and that binds to the primary antibody.

[0289] The level of CD74 in a sample can also be estimated by a competition immunoassay using CD74 standards labeled with a detectable substance and an unlabeled CD74-specific antibody. In this type of assay, the biological sample, the labeled CD74 standard(s), and the CD74-specific antibody are combined, and the amount of the labeled CD74 standard bound to the unlabeled CD74-specific antibody is determined. . The amount of CD74 in the biological sample is inversely proportional to the amount of labeled CD74 standard bound to the CD74-specific antibody.

[0290] Suitable labels for CD74-specific antibody, secondary and/or standard CD74 antibody used in in vitro diagnostic techniques include, without limitation, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride and phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive materials include 125I, 131I, 35S and 3H.

[0291] In one aspect, the CD74-specific antibodies of the invention are used in in vivo imaging of tissues that express CD74 such as tumors. For in vivo methods, antibody fragments such as, for example, (Fab')2, Fab and Fab' fragments, are particularly advantageous because of their rapid distribution kinetics.

[0292] In vivo imaging can be performed by any suitable technique. For example, a CD74-specific antibody (such as, for example, a fragment) labeled with 99Tc, 131I, 111In or another isotope that emits gamma rays can be used to image the accumulation or distribution of CD74-specific antibody in tissues. expressing CD74 such as tumors with a gamma scintillation camera (eg, an Elscint Apex 409ECT device), typically using a low-energy, high-resolution collimator or an all-purpose low-energy collimator. Alternatively, labeling with 89Zr, 76Br, 18F or another positron-emitting radionuclide can be used to image CD74-specific antibody or antibody fragment distribution in tumors using positron emission tomography (PET). Images obtained using such techniques can be used to assess the biodistribution of CD74 in a patient, mammal, or tissue, for example, in the context of using CD74 as a biomarker for the presence of cancer cells. Variations on this technique may include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques. Conventional immunoscintigraphy methods and principles are described, for example, in Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum Press 1988), Chase, "Medical Applications of Radioisotopes," in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro et al., (eds.), pp. 624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies," in Biotechnology And Pharmacy 227-49, Pezzuto et al., (eds.) (Chapman & Hall 1993). (Chapman & Hall 1993). Furthermore, such images can also, or alternatively, serve as the basis for surgical techniques to remove tumors. Furthermore, such in vivo imaging techniques may make it possible to identify and localize a tumor in a situation where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.) can be identified by traditional analytical techniques. All of these methods are features of the present invention.

[0293] In vivo imaging and other diagnostic methods provided by the present invention are particularly useful in detecting micrometastases in a human patient (e.g., a patient not previously diagnosed with cancer or a patient in a period of recovery/remission). of a cancer).

[0294] In one embodiment, the present invention provides an in vivo imaging method wherein a CD74-specific antibody of the present invention is conjugated to a radio-opaque agent that promotes detection, the conjugated antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. Through this technique and any other diagnostic method provided herein, the present invention provides a method for screening for the presence of disease-related cells in a human patient or a biological sample taken from a human patient and/or for assessing the distribution of CD74-specific antibody prior to CD74-specific ADC therapy.

[0295] For diagnostic imaging, radioisotopes can be linked to a CD74-specific antibody directly or indirectly through the use of an intermediate functional group. Useful intermediate functional groups include chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example, US 5,057,313).

[0296] In addition to radioisotopes and radio-opaque agents, diagnostic methods can be performed using CD74-specific antibodies that are conjugated to dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, and agents. (eg, paramagnetic ions) for magnetic resonance imaging (MRI) (see, for example, US Pat. No. 6,331,175, which describes MRI techniques and the preparation of antibodies conjugated to an agent which enhances MRI). Such diagnostic/detection agents may be selected from agents for use in MRI, and fluorescent compounds. In order to load a CD74-specific antibody with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which a multitude of chelating groups are attached for ion binding. Such a tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which chelating groups can be attached such as, for example, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and similar known groups. be useful for this purpose. Chelates can be bound to CD74-specific antibodies using standard chemistries.

[0297] Thus, the present invention provides a diagnostic CD74-specific antibody, wherein the CD74-specific antibody is conjugated to a contrast agent (such as for magnetic resonance imaging, computed tomography, or enhancement agent). contrast agent) or a radionuclide which may be, for example, a gamma, beta, alpha, electron Auger, or positron emitting isotope.

[0298] In another aspect, the invention relates to a kit for detecting the presence of CD74 antigen or a cell expressing CD74, in a sample, comprising: - A CD74-specific antibody or ADC of the invention; and - Instructions for using the kit.

[0299] In one embodiment, the present invention provides a kit for the diagnosis of cancer that comprises a container comprising a CD74-specific Ab, and one or more reagents for detecting binding of CD74-specific antibody to CD74. Reagents may include, for example, fluorescent labels, enzyme labels, or other detectable labels. Reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, where enzymatic reactions produce a product that can be visualized. In one embodiment, the present invention provides a diagnostic kit comprising one or more CD74-specific Abs of the present invention in labeled or unlabeled form in suitable container(s), reagents for the incubations for an assay. indirect, and substrates or derivatizing agents for detection in such an assay, depending on the nature of the label. Control reagent(s) and instructions for use may also be included.

[0300] Diagnostic kits may also be provided for use with a CD74-specific Ab, such as a conjugated/labeled CD74-specific Ab, for detecting the presence of CD74 in a tissue sample or host. In such diagnostic kits, as well as in kits for the therapeutic uses described elsewhere herein, a CD74-specific antibody typically can be provided in a lyophilized form in a container, alone or in conjunction with additional antibodies specific to a target cell or cell. peptide. Typically, a pharmaceutically acceptable carrier (e.g., an inert diluent) and/or components thereof, such as a Tris, phosphate, or carbonate buffer, stabilizers, preservatives, biocides, inert proteins, e.g., serum albumin, or the like, are also included (typically in a separate container for mixing) and additional reagents (also typically in separate container(s)). In certain kits, a secondary antibody capable of binding to CD74-specific Ab, which is typically present in a separate container, is also included. The second antibody is typically conjugated to a label and formulated in a similar manner to the CD74-specific antibody of the present invention. Using the methods described above and elsewhere herein, CD74-specific antibodies can be used to define subsets of cancer/tumor cells and characterize such cells and related tumor tissues.

Anti-Idiotypic Antibodies

[0301] In another aspect, the invention pertains to an anti-idiotypic antibody that binds a CD74-specific antibody of the invention as described herein.

[0302] An anti-idiotypic antibody (Id) is an antibody that recognizes unique determinants generally associated with the antigen-binding site of an antibody. An anti-Id antibody can be prepared by immunizing an animal of the same species and genetic type as the source of a CD74-specific monoclonal antibody with the monoclonal antibody to which an anti-Id is being prepared. The immunized animal typically can recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). Such antibodies are described, for example, in US 4,699,880. Such antibodies are further features of the present invention.

[0303] An anti-Id antibody can also be used as an “immunogen” to induce an immune response in another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id antibody can be epitopically identical to the original mAb, which induced the anti-Id antibody. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones that express antibodies of identical specificity. Anti-Id antibodies can be varied (thus producing anti-Id antibody variants) and/or derivatized by any suitable technique, such as those described elsewhere herein with respect to the CD74-specific antibodies of the present invention. For example, a monoclonal anti-Id antibody can be linked to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice. Sera from these mice will typically contain anti-anti-Id antibodies that have similar, if not identical, binding properties to a CD74-specific parent antibody/precursor.

[0304] The present invention is further illustrated by the following examples which should not be interpreted as limiting.

EXAMPLES Example 1 Construction of expression vectors CD74v1 and -v2, His-CD74v1 and -v2 and CD74de12-36v1 and -v2

[0305] The sequences encoding human CD74 variant 1 (CD74v1) (identical to Genbank sequence NP 001020330) and human CD74 variant 2 (CD74v2) (identical to Genbank sequence AAV383110330) were synthetically fabricated and fully codon optimized (GeneArt, Regensburg, Germany). The constructs were cloned into the mammalian expression vector pEE13.4 (Lonza Biologics, Slough, UK). These constructs were named pEE13,4CD74v1 and pEE13,4CD74v2. To enhance the level of CD74 expression on the cell surface, the cytoplasmic ER retention signal (aa2-36) was removed as described (Khalil H et al., J Cell Sci 2005; 118:4679-4687). For this purpose, new constructions were made by amplifying the CD74 encoding regions of pEE13,4CD74v1 and pEE13,4CD74v2, and removing the aa2-36 encoding regions in the process. These PCR fragments were recloned into pEE13,4 and fully sequenced to confirm the accuracy of the new constructs. These expression vectors were named pEE13,4CD74v1de12-36 and pEE13,4CD74v2de12-36.

[0306] The coding regions for the extracellular domains of CD74v1 (aa 73-296) and -v2 (aa 73-232) were amplified by PCR from pEE13,4CD74v1 and pEE13,4CD74v2, introducing the coding region for a hexameric N-terminal His label. The PCR fragments were cloned into the mammalian expression vector pEE12.4 (Lonza Biologics) which contains the coding region for an efficient signal peptide (HMM38 [Barash S et al., Biochem Biophys Res Commun 2002; 294: 835842). The expression vectors were fully sequenced and named pEE12,4SPHisCD74v1 and pEE12,4SPHisCD74v2. The resulting proteins were named HisCD74v1 and HisCD74v2.

[0307] The protein sequences of the CD74 variants are shown in Figure 1.

Example 2 Transient expression in HEK-293F cells and in CHO-S cells

[0308] Freestile® 293-F cells (a subclone HEK-293 adapted for suspension growth and chemically defined Freestile medium; HEK-293F) were obtained from Invitrogen and transfected with pEE13,4CD74v1, pEE13,4CD74v2, pEE13,4CD74de12- 36v1, pEE13,4CD74de12-36v2, pEE12,4SPHisCD74v1 or pEE12,4SPHisCD74v2, using 293fectin (Invitrogen) according to the manufacturer's instructions.

[0309] Cell culture supernatants, in the case of pEE12,4SPHisCD74v1 or pEE12,4CD74v2, were harvested and soluble HisCD74v1 or HisCD74v2 were purified by metal affinity chromatography as described below.

[0310] In the case of pEE13,4CD74v1, pEE13,4CD74v2, pEE13,4CD74v1de12-36 or pEE13,4CD74v2de12-36, cells were harvested 1 to 2 days after transfection and used in subsequent assays. These cells were named TH2013-CD74v1, TH2013-CD74v2, TH2013-CD74v1de12-36 and TH2013-CD74v2de12-36.

[0311] A suspension-adapted CHO-K1SV cell line (CHO-S, Invitrogen) was transfected with pEE13,4CD74v1, pEE13,4CD74v2, pEE13,4CD74v1de12-36 or pEE13,4CD74v2de12-36, according to the manufacturer's protocol using the CHO-Max reagent (Invitrogen). Transfected CHO-S cells were harvested 1 to 2 days after transfection and used in subsequent assays. These cells were named TC2013-CD74v1, TC2013-CD74v2, TC2013-CD74v1de12-36 and TC2013-CD74v2de12-36.

[0312] In the case of antibody expression, the appropriate heavy chain and light chain vectors as described in Example 9 were co-expressed in HEK-293F cells as described above.

Example 3 Stable expression in NSO cells

[0313] Plasmid pEE13,4CD74v1de12-36 was transfected into NSO cells (Lonza Biologics). Cells were selected for stable expression vector integration by culturing in glutamine-free cell culture medium in the presence of 25 μM methylsulfoximine (MSX) as described (Bebbington CR et al., Biotechnology (N Y) 1992; 10: 169-175). Cells expressing CD74 were pooled and used as a semi-stable population or individual stable clones were selected and used. These cells were named N2013de1-v1-012.

Example 4 Purification of His-labeled CD74

[0314] HisCD74v1 and HisCD74v2 were expressed on HEK-293F cells. The His tag on the proteins allowed purification with immobilized metal affinity chromatography. In this process, a chelator fixed on the chromatographic resin is loaded with Co2+ cations. The supernatant containing CD74ECDHis is incubated with the resin in batch mode (ie solution). The His-tagged protein binds strongly to the resin beads, whereas the other proteins present in the culture supernatant do not bind strongly. After incubation, the beads are recovered from the supernatant and packed in a column. The column is washed to remove weakly bound proteins. Tightly bound CD74ECDHis proteins are then eluted with a buffer containing imidazole, which competes with the binding of His to Co2+. The eluent is removed from the protein by buffer exchange in a desalting column.

Example 5 Immunization procedure of transgenic mice

[0315] Antibodies HuMab-CD74-005, -006, -008 and -011 were derived from immunizations of HCo17 HuMAb mice (human monoclonal antibody; Medarex Inc., San Jose, CA, USA) that have four genetic modifications. These mice were made transgenic for the human Ig heavy chain and human kappa Ig light chain and doubly silenced for the mouse heavy chain and mouse kappa light chain sites. These disruptions prevent the expression of any antibody that is completely murine. Different strains were used; HCo12, HCo12-BALB/c, HCo17 and HCo20. These differ in the number of human VH (variable region heavy chain) and VL (variable region light chain) genes. HCo12-BALB/c mice were derived by crossing with KCo5-BALB/c mice (kappa light chain transgenic).

[0316] Six different immunogens were used for the immunizations: TH2013-CD74v1de12-36, TH2013-CD74v2de12-36, N2013de1-v1-012, SU-DHL-4 cells (human B cell lymphoma cell line) and HisCD74v1 or HisCD74v2 bound to the carrier protein KLH (Californian keyhole limpet hemocyanin). Mice were immunized every two weeks, alternating with 5 x 10 6 cells or with 15 μg of protein. Eight immunizations were performed in total, four intraperitoneal (IP) and four subcutaneous (SC).

[0317] Antibodies -005, -006 and -008 were obtained from the immunization of an HCo17 mouse with 5 x 10 6 TH2013-CD74v1de12-36 IP cells, alternated with 15 μg of HisCD74v2 SC. The first immunization was performed IP, with cells in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA), the following immunizations in incomplete Freund's adjuvant (IFA) (protein, SC) or in PBS (cells, IP ).

[0318] Antibody -011 was obtained from the immunization of an HCo17 mouse with 5 x 10 6 TH2013-CD74v1de12-36 IP cells, alternated with 15 μg of HisCD74v1 SC. The first immunization was performed with protein in CFA (IP), the following immunizations in IFA (protein, SC) or PBS (cells, IP).

[0319] When serum titers were found to be sufficient (serum dilution of 1/50 or lower finding positive in the antigen-specific screening assay as described in Example 6 in at least two sequential biweekly screening events), the mice were additionally boosted twice intravenously (IV) with 10 µg of HisCD74 protein in 100 µl of PBS, four and three days before fusion.

Example 6 Homogeneous antigen-specific screening assay

[0320] Mouse hybridoma sera and supernatants were analyzed in a high throughput screening (HTS) Fluorometric Micro Volume Assay Technology (FMAT assay; Applied Biosystems, Foster City, CA, USA) for the presence of anti-antibody CD74s . In this assay, TC2013-CD74v1de12-36 and TC2013-CD74v2de12-36 cells were used to detect human anti-antibody CD74s. Wild-type CHO-S cells were used to measure non-specific binding. The samples were added to the cells allowing binding to CD74. Subsequently, HuMab binding was detected using a fluorescent conjugate (goat anti-human IgG-Cy5; Jackson Immunoresearch). Anti-mouse human antibody CD74 (Becton Dickinson; IgG2a, K; clone M-B741), labeled with Alexa-647 as described below, was used as a positive control and mouse chrompure (Jackson Immunoresearch) labeled with Alexa- 647 was used as a negative control.

[0321] Antibodies were labeled with Alexa Fluor® 647 Dye (Molecular Probes), hereinafter “Alexa-647”, using the following procedure:

[0322] An antibody solution of 1 mg/ml IgG was prepared in 0.1 M sodium carbonate buffer pH 9.0 (NaHCO3, Riedel de Haen, cat. no. 31437). Alexa-647 was freshly prepared by adding 100 μL of DMSO (Sigma, cat. no. D2438) and 900 μL of 0.1 M sodium carbonate buffer pH 9.0 to a vial (Alexa Fluor® 647 acid carboxylic acid, succinimidyl ether (1 mg/vial), Molecular Probes, Leiden, The Netherlands, Cat No. A-20006). A five-fold molar excess of Alexa-647, calculated as indicated below, was added to the IgG solution and incubated, with rotation, in the dark at room temperature for 1 hour. After labeling, unbound Alexa-647 was removed, using a PD-10 column (Amersham Biosciences, cat. no. 17-0851-01), with Tris buffer pH 8.0 (50 mM Tris [Trisma base, Sigma, cat. no. T-6066]; 100 mM NaCl [Riedel de Haen, cat. no. 31437]; 0.01% sodium azide [NaN3, Riedel de Haen, cat. no. 13412]). The amount of Alexa-647 to be added to the IgG solution was calculated using the formula: Volume of Alexa-647 to be added (in μL) = (IgG conc (mg/ml)/MW IgG (Da) * ratio * volume * MW Alexa-647 * 1,000 MW IgG = 150,000 Da; ratio is the molar excess of Alexa-647 to be used; volume is the volume of the sample to be labeled (in ml); MW Alexa-647 = 1250 Da.

[0323] Protein concentration (IgG) and degree of labeling (D.O.L.) were determined by measuring the OD 280 nm and 650 nm on an Ultrospec 2100 Pro (Amersham Biosciences). The IgG concentration (mg/ml) was calculated using the formula:

[0324] IgG concentration = [A280 - (0.03 * A65o)]/IgG extinction coefficient.

[0325] D.O.L. was calculated using the formula: D.O.L. = A650/239,000/[A280 - (0.03 * A650)/(IgG extinction coefficient * MW IgG)].

[0326] 239,000 is the extinction coefficient of Alexa-647 at Amax in cm-1M-1; 0.03 is the correction factor (A280 of free dye/Amax of free dye) (both supplied by the manufacturer).

[0327] Bovine serum albumin (BSA; Sigma, cat. no. A 2934) was added from a 10% (w/v) solution to a final concentration of 0.1% (w/v) and antibodies labeled were stored at 5°C.

[0328] In a few fusion screens, in addition to anti-human IgG-Cy5,5 to detect human antibodies, a Cy5,5-labeled conjugate of anti-mouse IgG was used to detect specific chimeric antibodies. Samples were screened using an Applied Biosystems 8200 Cell Detection System (8200 CDS) and 'fluorescence counts x' were used as readouts.

Example 7 HuMab hybridoma generation

[0329] HuMAb mice developing sufficient antigen-specific titer (defined as above) were sacrificed and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Fusion of splenocytes and lymph node cells to a mouse myeloma cell line was performed by electrofusion using a CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), essentially according to the manufacturer's instructions. The fused cells were seeded in fusion medium containing 10% Clone I Fetal Bovine serum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5 U/ml penicillin, 0.5 U/ml streptomycin (Cambrex), 50 μM 2-mercaptoethanol (Invitrogen), 600 ng/ml interleukin 6 (IL-6) (Strathmann), 1 x HAT (Sigma) and 0.5 mg/ml kanamycin (Invitrogen) in HyQ mADCF-Mab (Perbio). After ten days, the supernatant was harvested and the cells were refreshed with harvest medium, which contains 10% Clone I Fetal Bovine serum, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 600 ng/ml IL-6 and 1x proHT (Cambrex) in HyQ mADCF-Mab. Hybridoma culture supernatants were screened by primary FMAT screening assays on TC2013-CD74v1de12-36 cells and TC2013-CD74v2de12-36 cells to detect hybridomas that produce human (or chimeric) anti-CD74 antibodies as described above. The top 60 primary wells were seeded on semi-solid medium made from 40% CloneMedia (Genetix, Hampshire, UK) and 60% 2x HyQ complete medium (Hyclone, Waltham, USA). From each primary well, two wells of a black Genetix 6 well plate were seeded. From each pool, 33 sub-clones were chosen, using the ClonePix system (Genetix). Subclones were chosen in harvest medium. After seven days, the subclone supernatants were screened once more for CD74-specific human IgG binding and the human IgG concentration was measured using Octet (Fortebio, Menlo Park, USA). From each primary pool, the best sub-clone was expanded in expansion medium containing only 600 ng/ml IL-6, 0.5 U/ml penicillin, 0.5 U/ml streptomycin and 1 x proHT. The sub-clones were expanded from a 96-well plate well to a 24-well plate well to four 24-well plate wells to six 6-well plate wells for Hyperflask (small-scale production). The supernatants from the hypervials were screened for CD74-specific human IgG binding.

[0330] The clones derived by this process were designated PC2013.

Example 8 Mass Spectrometry of Purified Antibodies

[0331] Small 0.8 ml aliquots of supernatant containing 6-reservoir or Hypervial stage antibody were purified using PhyTip columns containing Protein G resin (PhyNexus Inc., San Jose, USA) on a Scyclone ALH workstation 3000 (Caliper Lifesciences, Hopkinton, USA). PhyTip columns were used according to the manufacturer's instructions, but the buffers were replaced. PBS (B. Braun, Medical B.V., Oss, The Netherlands) was used as the Binding Buffer and 0.1 M Glycine-HCl pH 2.7 (Fluka Riedel-de Haen, Buchs, Germany) was used as the Elution Buffer. After purification, samples were neutralized with 2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, The Netherlands). Alternatively, in some cases larger volumes of culture supernatant were purified using Protein A affinity column chromatography.

[0332] After purification, the samples were placed on a 384-well plate (Waters, 100 μl square reservoir plate, art* 186002631). Samples were deglycosylated with N-glycosidase F (Roche cat no 11365177001) at 37°C overnight. DTT (15 mg/ml) was added (1 μL/well) and incubated at 37°C for 1 h. Samples (5 or 6 μL) were desalted in an Acquity UPLC® (Waters, Milford, USA) with a BEH300 C18 column, 1.7 μm, 2.1 x 50 mm at 60°C. MQ water and LC-MS grade acetonitrile (Biosolve, cat no 01204101, Valkenswaard, The Netherlands), both with 0.1% formic acid (Fluka, cat no 56302, Buchs, Germany), were used as Eluents A and B. Time-of-flight electrospray ionization mass spectra were recorded in-line on a microOTOF® mass spectrometer (Bruker, Bremen, Germany) operating in positive ion mode. Prior to analysis, a range of 900-3000 m/z was calibrated with ES tuning mix (Agilent Technologies, Santa Clara, USA). Mass spectra were deconvoluted with DataAnalysis® v3.4 software (Bruker) using the Maximal Entropy algorithm which searches for molecular weights between 5 and 80 kDa.

[0333] After deconvolution the resulting heavy and light chain masses for all samples were compared in order to discover duplicate antibodies. In comparing the heavy chains the possible presence of C-terminal lysine variants was taken into account. This resulted in a list of unique antibodies, where unique is defined as a unique combination of heavy and light chains. In the case where duplicate antibodies were found, the results of other tests were used to decide which material would be used to continue the experiments.

[0334] Mass spectrometry analysis of the heavy and light chain molecular weights of 41 anti-CD74 hybridomas yielded 18 unique antibodies (unique heavy chain/light chain combination).

Example 9 Sequence analysis of CD74-specific HuMab variable domains and cloning into expression vectors

[0335] Total RNA of HuMab anti-CD74 antibodies was prepared from 5 x 10 6 hybridoma cells and 5'-RACE complementary DNA (cDNA) was prepared from 100 ng of total RNA using the amplification kit of SMART RACE cDNA (Clontech), according to the manufacturer's instructions. Regions encoding VH (heavy chain variable region) and VL (light chain variable region) were amplified by PCR. The amplified PCR products of antibodies 006, 008 and 011 were cloned into the pCR-Blunt II-TOPO vector (Invitrogen) using the Zero Blunt PCR Cloning Kit (Invitrogen).

[0336] The 005 antibody amplified VH and VL PCR products were cloned into pcDNA3,3 vectors (Invitrogen) encoding G1f and Kappa constant domains. For each HuMab, 16 VL clones and 8 VH clones were sequenced. Clones with heavy and light chain mass predicted according to the mass of hybridoma-derived material of the same antibody (as determined by mass spectrometry) were selected for further study and expression.

[0337] The resulting sequences are shown in the Sequence Listing and Figure 2 here.

Tabela 2: Homologias de Cadeia Leve

Tabela 3: Referências à listagem de sequências:

[0338] Table 1 and Table 2 (below) give an overview of the antibody sequence information and the most homologous germline sequences. Table 1: Heavy Chain Homologies

Table 2: Light Chain Homologies

Table 3: Sequence listing references:

Example 10 Antibody purification

[0339] The culture supernatant was filtered through 0.2 μm dead-end filters, loaded into columns with 5 ml of Protein A (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. The eluate was immediately neutralized with 2M Tris-HCl, pH 9 and dialyzed to 12.6 mM NaH 2 PO 4 , 140 mM NaCl, pH 7.4 (B. Braun), overnight. After dialysis, samples were sterile filtered on 0.2 μm dead-end filters. Purity was determined by SDS-PAGE and concentration was measured by nephelometry and absorbance at 280 nm. Purified antibodies were aliquoted and stored at -80°C. Once thawed, aliquots of purified antibody were kept at 4°C. Mass spectrometry was performed to identify the molecular mass of the heavy antibody and the light chains expressed by the hybridomas as described in Example 9.

Example 11 Binding of CD74-specific HuMab antibodies to the recombinant extracellular domain of two CD74 isoforms, determined by ELISA, and to cellular CD74 in Raji cells, determined by FACS

[0340] Binding of anti-CD74 HuMab antibodies to two isoforms of CD74 was measured by ELISA (CD74 coated recombinant extracellular domain) and to cellular CD74 on Raji cells (ATCC, Manassas, VA) by FACS analysis.

[0341] ELISA plates (Greiner BioOne) were coated overnight at 4°C with 2 µg/ml, 100 µl per well, recombinant CD74v1 or CD74v2 in PBS (B. Braun Melsungen AG). The sequences and production of the isoforms were described above. The ELISA wells were washed three times with PBS containing 0.05% Tween-20 (PBST), emptied, and blocked with 1% (w/v) fraction V of BSA (Roche) in PBS at room temperature for 1 h under agitation (300 rpm), and emptied. Subsequently, 100 μL of anti-CD74 HuMab antibodies were added in serial dilutions in 0.2 % (w/v) fraction V of BSA in PBST (assay buffer) and incubated under shaking at room temperature for 90 min. ELISA plates were washed three times with PBST, emptied, and bound HuMab antibodies were detected using HRP-conjugated goat anti-human IgG (100 μL; 1:5,000; goat anti-human IgG Affinipure Peroxidase, Fragment Specific F(ab')2 [min X Bov,Hrs,Ms Sr Prot]; Jackson Immunoresearch) in assay buffer and incubated under shaking at room temperature for 90 min. Plates were washed three times with PBST, emptied, and incubated with 100 µL ABTS solution (50 ml ABTS buffer [Roche] and one ABTS tablet [50 mg; Roche]). After incubation in the dark at room temperature for 30 min, the reaction was stopped by the addition of 100 μL per reservoir of oxalic acid (2 % [w/v]). Plates were measured at OD 405 nm on an ELISA reader (Biotek Instruments, EL808 Absorbance Microplate Reader).

[0342] For FACS analysis, 105 cells in 100 μL of FACS buffer (PBS supplemented with 0.1% BSA and 0.02% sodium azide) were seeded per well in 96 well round bottom plates . The cells were spun (1200 rpm, 4°C, 5 min) and the supernatant was discarded. Serially diluted anti-CD74 HuMab antibodies were added (100 μL) and incubated on ice for 1 h. Cells were washed with FACS buffer, supernatants were discarded, and 100 µl of R-Phycoerythrin-labeled goat anti-human IgG (R-Phycoerythrin AffiniPure F(ab')2 Frag Gt Anti-Human IgG, Fcy Frag Spec [min X Bov,Hrs,Ms Sr Prot]; Jackson Immunoresearch), diluted 1:100 in FACS buffer, were added. After 1 h on ice (in the dark), cells were washed once in FACS buffer, the supernatant was discarded, and specific binding of HuMab antibodies was detected by flow cytometry on a FACS Corner II (BD Biosciences).

[0343] Isotype Ab IgG1-b12 control was used as a negative control. Binding curves were analyzed using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).

[0344] Figure 3 shows that HuMab-CD74-006 and -011 bound with high affinity (EC50 values between 210 and 344 ng/ml) to both isoforms of the CD74 extracellular domain. HuMab-CD74-008 bound with intermediate affinity (EC50 between 759 and 1391 ng/ml) to both isoforms.

[0345] Figure 4 shows that HuMab-CD74-006 and -011 also bound with high affinity (EC50 between 150 and 200 ng/ml) to cellular CD74 expressed by Raji cells. HuMab-CD74-008 and -005 replaced cellular CD74 with intermediate affinity (EC50 values could not be determined because maximum binding was not reached).

Os valores EC50 estão em ng/ml. nd - não puderam ser calculados. nt - não testados.[0346] Table 4 shows EC50 values of CD74-specific HuMab antibodies for binding to the extracellular domain of CD74v1 and CD74v2 by ELISA and to cellular CD74 by FACS on Raji cells. Table 4: Overview of EC50 values for binding of CD74-specific HuMab antibodies to the extracellular domain of CD74v1 and CD74v2, as determined by ELISA, and to cellular CD74 on Raji cells, as determined by FACS. All HuMab antibodies were produced by HEK293F cells transiently co-transfecting with the relevant heavy and light chain expression vectors (as described above)

EC50 values are in ng/ml. nd - could not be calculated. nt - not tested.

Example 12 Cross-reactivity of anti-CD74 HuMab antibodies to cynomolgus monkey tissues

[0347] The ability of CD74-specific HuMab antibodies to bind to cynomolgus CD74 was tested by immunohistochemistry. Immunohistochemistry with anti-CD74 HuMab antibodies was performed on frozen tissue from human tonsils and cynomolgus lymph node, with predicted CD74 expression on B lymphocytes (follicular) and macrophages. Frozen tissue sections were cut (4 to 6 μm thick) and fixed in acetone. HuMab antibodies were complexed with fluorescein isothiocyanate (FITC) by incubation with goat anti-human IgG (Fc)-FITC (Fab) (Protos) (1:1 ratio with Humab). Prior to staining with HuMab (1 μg/ml), tissues were blocked for endogenous biotin, peroxidase (PO) and immunoglobulins. The HuMab-Fab-FITC complex was detected by subsequent incubations with rabbit anti-FITC (Invitrogen) (diluted 1:1000) and PO-conjugated goat anti-rabbit IgG (Powervision, [rb IgG]-PO; undiluted) . PO activity was visualized with amino-ethyl-carbazole (AEC) as substrate, resulting in a red color, and nuclei were visualized with hematoxylin (blue). Fabric dyes were examined with a light microscope (Axioskop-2 plus), converted to digital images by an Axiocam camera and stored as digital images.

[0348] Figure 5 shows that HuMab-CD74-006 and -011 showed cross-reactivity with cynomolgus CD74, as shown by staining of macrophages and follicular B cells (staining for isotype control is negative). The degree of cross-reactivity with cynomolgus CD74 was lower for HuMab-CD74-006 than for -011, as shown by the less intense staining of cynomolgus tissue when compared to human tissue.

Example 13 ADCC and CDC induction

[0349] The induction of ADCC by CD74-specific HuMab antibodies was tested in a 51Cr release assay. Briefly, Raji cells were labeled with 100 μCi 51Cr and used as target cells. Peripheral blood mononuclear cells, isolated from white cell layers, were used as effector cells. Target cells were pre-incubated with anti-CD74 HuMab antibodies (room temperature, 30 min) and effector cells were added, resulting in an effector-to-target ratio of 100:1, and incubated at 37°C, 5% CO2 overnight. The release of 51Cr in the supernatant was measured in a gamma counter. No significant induction of ADCC by anti-CD74 HuMab antibodies was detected.

[0350] The induction of CDC by anti-CD74 HuMab antibodies was tested using the propidium iodide method. Briefly, Raji cells were pre-incubated with anti-CD74 HuMab antibodies (room temperature, 15 min) and normal human serum was added, to a final concentration of 20%, and incubated at 37°C, 5% CO2 per 45 min. The plates were placed on ice to stop the reaction. Propidium iodide was added and cells were analyzed by FACS analysis. No significant induction of CDC by anti-CD74 HuMab antibodies was detected.

Example 14 Antibody-mediated internalization and cell killing by anti-CD74 HuMab antibodies in an anti-kappa ETA' assay

[0351] To assess the suitability of anti-CD74 HuMab antibodies for an antibody-drug conjugate method, a generic in vitro cell-based death assay using kappa-targeted pseudomonas exotoxin A (ETA' anti-kappa) was developed. In this assay, a construct consisting of a high affinity antibody kappa light chain domain antibody and a truncated form of the pseudomonas exotoxin A plus a KDEL retention motif was used. On internalization, the anti-kappa-ETA' domain antibody construct undergoes proteolysis and reduction of the disulfide bond, which separates the catalytic domain and the binding domain. The catalytic domain is believed to be transported from the Golgi system to the endoplasmic reticulum via the KDEL retention motif, and subsequently translocated to the cytosol where it inhibits protein synthesis and induces apoptosis (Kreitman RI BioDrugs 2009; 23( 1): 1-13).

[0352] Antibody-mediated internalization and toxin cell killing was tested for different anti-CD74 HuMab antibodies with Raji cells. The number of CD74 molecules expressed on the surface of the Raji cell was estimated to be 104 molecules per cell, using the QIFIKIT® method (Dako, Glostrup, Denmark). 104 cells per well in cell culture medium were seeded in 96 well tissue culture plates (Greiner Bio-a). The plates were incubated at 37°C for 1 h to allow the cells to sediment. To identify the anti-CD74 HuMab antibodies that allow for internalization and killing by the toxin, a fixed concentration (1 μg/ml final concentration in the reservoirs) of anti-kappa-ETA', which did not induce non-specific cell death in the absence of antibody, was pre-incubated for 30 min with a titrated amount of anti-CD74 HuMab antibodies before addition to the cells. After three days, the amount of viable cells was quantified with Alamar Blue (BioSource International, San Francisco, US), added in 10 μl per well according to the manufacturer's instructions. After incubation at 37°C for 4 h, fluorescence was monitored using the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamar Blue settings. An isotype control antibody (IgG1-b12), pre-incubated with anticapa-ETA', was used as a negative control. Staurosporine (Sigma-Aldrich) was used as a control to determine the background signal and added to the cells at a final concentration of 1 μg/ml.

[0353] Percent viability was calculated as follows: (FLtreated - FLbackground) / (FLcontrol -FLbackground) X 100%. FLcontrol = fluorescence from untreated reservoirs FLbackground = fluorescence from reservoirs treated with staurosporine.

[0354] Figure 6 and Table 5 show that all anti-CD74 HuMab antibodies pre-incubated with anti-kappa-ETA' were able to kill Raji cells in a dose-dependent manner. HuMab-CD74-006, -011, -005 and -008 pre-incubated with anti-kappa-ETA' induced efficient killing (EC50 between 25 and 250 μg/ml and minimum percent viability allowed to be between 0 and 15). Control IgG1-b12 mAb pre-incubated with ETA' anticapa did not induce cell death. Table 5 - Overview of EC50 values and percentages of cell viability left after treatment of Raji cells with anti-CD74 HuMab antibodies pre-incubated with anti-kappa-ETA'. Data shown are EC50 values (in μg/ml) and minimum percentages of viability of Raji cells treated with anti-CD74 HuMab antibodies pre-incubated with anti-ETA', measured in a representative experiment.

Example 15 Preparation of CD74-specific ADCs

[0355] HuMab-CD74-005, HuMab-CD74-006, HuMab-CD74-011 and IgG1-b12 negative control were transiently produced in HEK-293F cells. The antibodies were purified by Protein A chromatography according to standard procedures, finally yielding approximately 263 mg of purified HuMab-CD74-005, 165 mg of HuMab-CD74-006 and 720 mg of HuMab-CD74-011. The amount of antibody conjugate obtained is shown in Table 6. The drug-linker vcMMAE or mcMMAF was alkylated to the cysteines of the reduced antibodies according to procedures described in the literature ( Sun et al. (2005) Bioconjugate Chem. 16: 1282- 1290; McDonagh et al., (2006) Protein Eng. Design Sel. 19: 299-307; Alley et al., (2008) Bioconjugate Chem. 19: 759-765 ). The reaction was quenched by the addition of an excess of N-acetylcysteine. Any residual unconjugated drug was removed by diafiltration and the final CD74-specific antibody drug conjugates were formulated in PBS.

Tabela 7 - Análise de conjugados de medicamento de anticorpo

* DAR não pôde ser designado por causa da co-elução de picos[0356] The CD74-specific antibody drug conjugates were subsequently analyzed for concentration (by absorbance at 280 nm), drug to antibody ratio ('DAR') by reverse phase chromatography (RP-HMLC) and hydrophobic interaction (HIC), amount of unconjugated drug (by reversed-phase chromatography), percent aggregation (by size exclusion chromatography, SEC-HMLC), and endotoxin levels (by Amebocyte Lysate endotoxin test). Limulus (LAL)). The results are shown in Table 7. Table 6 - Quantity of ADC obtained

Table 7 - Analysis of antibody drug conjugates

* DAR could not be assigned because of co-eluting peaks

Example 16 Binding of anti-CD74 ADCs to the recombinant extracellular domain of CD74v1, determined by ELISA

[0357] Binding of CD74-specific ADCs to CD74 was measured by ELISA (CD74v1 coated recombinant extracellular domain) and compared to binding of unconjugated CD74-specific HuMab antibodies.

[0358] ELISA plates (Greiner BioOne) were coated with 2 µg/ml, 100 µl per well, of recombinant CD74ECDHis in PBS (B. Braun Melsungen AG) at 4°C overnight. The ELISA plates were emptied and blocked with 200 μl/reservoir of PBS containing 0.05% Tween-20 (PBST) under agitation (300 rpm) at room temperature for 1 hour, washed three times with 300 μl of PBST and emptied.

[0359] Subsequently, 100 μl of unconjugated anti-CD74 ADCs or CD74-specific HuMab antibodies were added in serial dilutions in PBST and incubated under shaking at room temperature for 2 hours. ELISA plates were washed with PBST and emptied. Bound anti-CD74 ADCs and unconjugated HuMab antibodies were detected by adding HRP-conjugated mouse anti-human IgG1 (100 μl; 0.015 μg/ml; Sanquin; # M1328) in PBST and incubating under shaking at room temperature for 2 hours. Plates were washed with PBST, emptied and incubated with 100 µl ABTS solution (50 ml ABTS buffer [Roche] and one ABTS tablet [50 mg; Roche]). After incubation in the dark under agitation at room temperature for 30 min, the reaction was stopped by incubating with 100 μl per reservoir of oxalic acid (2 % [w/v]; Riedel de Haen) in the dark under agitation for 10 min. . Plates were measured at OD 405 nm on an ELISA reader (Biotek Instruments, EL808 Absorbance Microplate Reader).

[0360] IgG1-b12, an antibody binding to an unrelated antigen, was used as a negative control (both unconjugated as well as in ADC format). Binding curves were analyzed by nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).

[0361] All anti-CD74 HuMab antibodies and ADCs bound within a similar range to the CD74v1 extracellular domain in an ELISA (EC50 values between 0.02 and 0.04 μg/ml), as demonstrated by the binding curves in Figure 7 Table 8 shows EC50 values of CD74-specific HuMab antibodies and ADCs for binding to the extracellular domain of CD74. Table 8 - Overview of EC50 values for binding of CD74-specific HuMab antibodies and ADCs to the extracellular domain of CD74v1, as determined by ELISA. EC50 values are in μg/ml. Data shown are average EC50 values calculated from four independent experiments.

Example 17 Binding of CD74-specific ADCs to surface-expressed CD74 as determined by FACS analysis on Daudi cells

[0362] Binding of anti-CD74 ADCs to surface-expressed CD74 was measured by FACS analysis on Daudi cells and compared to binding of unconjugated anti-CD74 HuMab antibodies.

[0363] 1 x 10 5 Daudi cells in 100 µl of PBS containing 0.1% bovine serum albumin (BSA) (Roche, cat. no. 10735086001) and 0.02% sodium azide (Sigma-Aldrich, cat. No. 13412) (FACS buffer) were seeded per well into 96 well round bottom plates (Greiner bio-one, cat. no. 650101). Cells were spun (1200 rpm, 4°C, 3 min) and the supernatant was discarded. Serially diluted anti-CD74 HuMab antibodies or ADCs were added (100 μl) and incubated on ice for 30 min. Cells were washed twice with 150 µl of FACS buffer and 100 µl of rabbit anti-human IgG-FITC (cat. nr. F0185, Dako), diluted 1:100 in FACS buffer, was added. After 30 min on ice (in the dark), cells were washed twice in 150 μl of FACS buffer and specific binding of HuMab antibodies and ADCs were detected by flow cytometry on a FACS Canto II (BD Biosciences).

[0364] The IgG1-b12 isotype control antibody, an antibody binding to an unrelated antigen, was used as a negative control (both unconjugated and as an ADC). Binding curves were analyzed using nonlinear regression (non-sigmoidal dose response with variable slope) using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).

Tabela 10 - Vista geral de intensidades de fluorescência média a 10 μg/ml de anticorpos HuMab específicos de CD74 e ADCs, determinadas pela análise de FACS em células Daudi. Os dados mostrados são valores médios de MFI máximos como medidos a 10 μg/ml de HuMab-CD74 mAbs e ADCs. Os valores médios de MFI máximos foram calculados a partir de três experimentos independentes.

[0365] Figure 8 shows binding curves and Tables 9 and 10 show EC50 values and mean maximum fluorescence intensities for binding to CD74 expressed on the surface of anti-CD74 HuMab antibodies and ADCs. All but one of the surface-bound conjugated anti-CD74 HuMab antibodies expressed CD74 on Daudi cells with similar affinity to the corresponding unconjugated HuMab antibodies. The vcMMAE conjugate of HuMab-CD74-005 bound with higher affinity (lower EC50 value) than the unconjugated HuMab. HuMab-CD74-005 and its mcMMAF conjugate bound with lower affinity than HuMab-CD74-006 and -011 and their conjugates. Maximum binding was lower for vcMMAE-conjugated HuMab-CD74-006 and -011 than for the corresponding unconjugated HuMab antibodies. Table 9 - Overview of EC50 values for binding of CD74-specific HuMab antibodies and ADCs to surface-expressed CD74, determined by FACS analysis on Daudi cells. EC50 values are in μg/ml. Data shown are average EC50 values calculated from three independent experiments.

Table 10 - Overview of mean fluorescence intensities at 10 μg/ml of CD74-specific HuMab antibodies and ADCs, determined by FACS analysis on Daudi cells. Data shown are mean maximum MFI values as measured at 10 µg/ml of HuMab-CD74 mAbs and ADCs. Mean maximum MFI values were calculated from three independent experiments.

Example 18 Antibody-mediated internalization and cell death by anti-CD74 ADCs in an in vitro death assay

[0366] To determine the ability of anti-CD74 ADCs to induce cytotoxicity, an in vitro cell-based death assay was performed.

[0367] Cell death from four cell lines was tested for different anti-CD74 ADCs. All cell lines were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA): Raji (cat. no. CCL-86), Daudi (cat. no. CCL-213), M4A4 (cat. no. CRL-2914; derived from the human cell line MDA MB 435) and NCI-H747 cells (cat. no. CCL-252, derived from colorectal adenocarcinoma metastasis). Cells were seeded at optimal concentration (Raji: 1 x 104 cells/well; Daudi: 1 x 103 cells/well, M4A4: 2 x 103 cells/well, NCI-H747 3 x 103 cells/well) in 100 μl of medium of cell culture (for Daudi and Raji; RPMI 1640 [Lonza, cat. no. BE12-115F] supplemented with 10% Cosmic Calf Serum [Perbio Science Nederland B.V., cat. no. SH30087.04], 2 mM of L-glutamine [Lonza, cat. no. BE17-605F] and 1 mM Sodium Pyruvate [Lonza, cat. no. BE13-115E]; for NCI-H747: RPMI 1640 supplemented with 10% Cosmic Calf Serum, 1 mM Sodium Pyruvate, 0.15% Sodium Bicarbonate [Lonza, Cat. No. BE17-613E] and 0.5% Glucose [Sigma, Cat. No. G8769]; and for M4A4: DMEM [Lonza] , Cat No. BE12-709F] supplemented with 10% Cosmic Calf Serum) in 96-well tissue culture plates (Greiner Bio-one) and allowed to adhere. Serial dilutions of anti-CD74 ADCs were added and incubated at 37°C for three (Raji, Daudi) or five (M4A4, NCI-H747) days. The amount of viable cells was quantified with Alamar Blue (cat. nr. DAL1100, BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with default Alamar Blue settings. IgG1-b12 (an antibody binding to an unrelated antigen) ADCs were used as negative controls. Staurosporine (Sigma, #S6942) was used to induce maximal cell death. The amount of CD74 molecules in the cell lines was determined by QIFIKIT® (Dako, Glostrup, Denmark), according to the manufacturer's instructions, using mouse anti-CD74 IgG1 (By2 clone; Santa Cruz, cat. no. SC- 20062) and isotype control antibody (CLB, cat. no. M1415). Both antibodies were used at a concentration of 10 μg/ml. Raji and Daudi cells were determined to express 20,000; and M4A4 cells ~11,000 CD74 molecules on the cell surface.

a) não pôde ser calculada visto que os níveis de platô da curva não foram atingidos.[0368] Figure 9 and Table 11 show that all anti-CD74 ADCs were able to kill Raji, Daudi and M4A4 cells in a dose dependent manner. IC50 values for all conjugates were about 5- to 12-fold higher in M4A4 cells (i.e. lower efficacy), which express about six-fold lower levels of CD74. NCI-H747 cells were killed only at the highest dose of ADCs tested (10 μg/ml). For HuMab-CD74-006 and -011 the mcMMAF conjugates were slightly more efficient in inducing the death of Daudi and Raji cells than the vcMMEA conjugates (-006: in Daudi cells three times lower and in Raji cells five times lower IC50; -011: in Daudi cells two times lower and in Raji cells four times lower IC50). Table 11 - Overview of IC50 values and percentages of cell death induced by anti-CD74 ADCs. Data shown are mean IC50 values (in μg/ml) and mean maximum percent kill (at a concentration of 10 μg/ml) of the indicated cell lines treated with anti-CD74 ADCs. Data were calculated from three independent experiments. The percentage of cell death (% of death) was calculated as follows: (MFI untreated -MFI anti-CD74 treated with ADC) / (MFI untreated - MFI treated with stauroporine) X 100%.

a) could not be calculated since the plateau levels of the curve were not reached.

Example 19 Therapeutic treatment of Daudi tumor xenografts in SCID mice with CD74-specific ADCs

[0369] The in vivo efficacy of HuMab-CD74-011 ADCs was determined in established intravenous (i.v.) Daudi (Burkitt's lymphoma) xenograft tumors in SCID mice.

[0370] Daudi cells were transfected by electroporation with luciferase gWIZ (Aldevron, Fargo, ND, USA) and pPur vector (BD Biosciences, Alfen a/d Rijn, The Netherlands) in a ratio of 4:1. After 48 h, puromycin was added to select a stably transfected clone (Daudi-luc). Daudi-luc #1E3 cells were cultured in RPMI supplemented with 10% cosmic calf serum (cat. no. SH30087.04, Hyclone), 1% penicillin/streptomycin (cat. no. DE17-603, Cambrex, Germany) , 1% sodium pyruvate and 1 µg/ml puromycin (cat. no. P-8833, Sigma, Zwijndrecht, The Netherlands). 2.5 x 10 6 Daudiluc tumor cells in 100 µl PBS were injected i.v. in the tail vein of female SCID mice. Mice were imaged directly after tumor inoculation, followed by imaging at weekly intervals starting on day 14. For imaging, mice were anesthetized using isoflurane, followed by intraperitoneal (i.p.) administration of 2.5 mg of D-luciferin (acid form, cat. no. BT11-1000; Biothema, Haninge, Sweden) in 200 µl of 10 mg/ml TRIS (cat. no. T60666-1 kg, Sigma). Bioluminescence imaging (BLI), from the back side (dorsal view), started 10 min after D-luciferin administration, 5 min exposure time, in a Biospace Imager. Black and white images were taken for anatomical reference. Mice were treated twice weekly with 60 μg (~3 mg/kg) of HuMab-CD74-011 and control antibody (IgG1-b12), both as ADC and as unconjugated IgG1, in 100 μl of PBS from the day 21 after tumor inoculation, four times in total. Prior to treatment, mice were divided into groups of seven mice each, each group having equal mean BLI signals and equal variances.

[0371] Figure 10 shows that both HuMab-CD74-011-vcMMAE and -mcMMAF were effective in reducing the size of Daudiluc i.v. tumors. As shown in Figure 10, there was an apparent trend towards greater inhibition of tumor growth. high in the case of unconjugated HuMab-CD74-011 when compared to the control antibody group, although the differences were not significant.

Example 20 Therapeutic treatment of Raji tumor xenografts in SCID mice with anti-CD74 ADCs

[0372] The in vivo efficacy of anti-CD74 ADCs was also determined in an i.v. Raji xenograft tumor model. in SCID mice.

[0373] Raji cells were transfected by electroporation with gWIZ luciferase (Aldevron, Fargo, ND, USA) and pPur vector (BD Biosciences, Alfen a/d Rijn, The Netherlands) in a 4:1 ratio. After 48 h, puromycin was added to select a stably transfected clone (Raji-luc). Raji-luc #2D1 cells were cultured in RPMI supplemented with 10% cosmic calf serum (cat. no. SH30087.04, Hyclone), 1% penicillin/streptomycin (cat. no. DE17-603, Cambrex, Germany ), 1 % sodium pyruvate and 1 μg/ml puromycin (cat. no. P-8833, Sigma, Zwijndrecht, The Netherlands). 2.5 x 10 6 Raji-luc tumor cells in 100 µl PBS were injected i.v. in the tail vein of female SCID mice. Mice were imaged directly after tumor inoculation, followed by imaging twice weekly from day 7 onwards. For imaging, mice were anesthetized using isoflurane, followed by i.p. of 2.5 mg of D-luciferin (acid form, cat. no. BT11-1000; Biothema, Haninge, Sweden) in 200 µl of 10 mg/ml TRIS (cat. no. T60666-1kg, Sigma). Bioluminescence imaging (BLI) of the back (dorsal view) began 10 min after D-luciferin administration, 5 min exposure time, in a Biospace Imager. The black and white images were taken for anatomical reference. Mice were treated twice a week with 60 μg (~3 mg/kg) of HuMab-CD74-011 or control antibody (IgG1-b12), either as ADC or as unconjugated IgG, in 100 μl of PBS at from day 11 after tumor inoculation, four times in total. Prior to treatment, mice were divided into groups of seven mice each, each group having equal mean BLI signals and equal variances.

[0374] Figure 11 shows that both HuMab-CD74-011-vcMMAE and -mcMMAF eliminated virtually all Raji-luc tumors. As shown in Figure 11, there was an apparent trend towards higher tumor growth inhibition in the case of unconjugated HuMab-CD74-011 when compared to the control antibody group, although the differences were not significant.

Example 21 Therapeutic treatment of Raji tumor xenografts in SCID mice with anti-CD74 ADCs

[0375] The in vivo efficacy of anti-CD74 ADCs was also determined in established subcutaneous (s.c.) Raji xenograft tumors (Burkitt's lymphoma) in SCID mice.

[0376] 5 x 10 6 Raji-luc #2D1 tumor cells (obtained as described in Example 20) in 200 µl PBS were injected s.c. in the right flank of female SCID mice, followed by two injections with anti-CD74 ADCs or controls (IgG1-b12; both as ADC and as unconjugated IgG1), one when tumor sizes averaged ~400 mm3, on day 17 , and the other four days later, on day 21 (by injection 60 μg/mouse, -3 mg/kg, in 100 μl, intraperitoneally). Prior to the first treatment, tumor-growing mice were divided into groups with equal tumor volume distribution. Tumor volume was determined at least twice a week. Tumor volumes (mm 3 ) were calculated from gauge measurements (MLEXX) as: 0.52 x (length) x (width) 2 .

[0377] Figure 12 shows that all anti-CD74 ADCs effectively reduced the size of s.c. established Raji-luc tumors. Tumors in IgG1-b12 treated mice, both as ADC and unconjugated, continued to grow.

Example 22 Therapeutic treatment of M4A4 tumor xenografts in SCID mice with anti-CD74 ADCs

[0378] The in vivo efficacy of anti-CD74 ADCs was also determined in established subcutaneous (s.c.) M4A4 xenograft tumors in SCID mice. M4A4 melanoma cells (cat. no. CRL-2914; American Tissue Culture Collection, ATCC; derived from the human MDA-MB-435 cell line) were cultured in DMEM (cat. no. BE12-709F, Cambrex, Germany) which contains 10% cosmic calf serum (cat. no. SH30087.04, Hyclone, The Netherlands) and 1% penicillin/streptomycin (cat. no. DE17-603, Cambrex, Germany). 107 M4A4 tumor cells in 200 µl PBS were injected s.c. in the right flank of female SCID mice, followed by four injections with anti-CD74 ADCs or controls (IgG1-b12; both as ADC and as unconjugated IgG1), starting when tumor sizes were ~200 mm3: day 11, day 14 , day 18 and day 21 (by injection 60 μg/mouse, - 3 mg/kg, in 100 μl, intraperitoneally). Before the first treatment, mice were divided into groups with equal mean tumor volume and equal variation in tumor volume. Tumor volume was determined at least twice a week. Tumor volumes (mm3) were calculated from gauge measurements (MLEXX) as: 0.52 x (length) x (width)2.

[0379] Figure 13 shows that while all anti-CD74 ADCs inhibited tumor growth of established M4A4 tumors s.c., the vcMMAE conjugate strongly reduced tumor size. Compared to unconjugated IgG1-b12, IgG1-b12 ADCs slightly inhibited tumor growth.

Example 23 Determination of the dissociation rate of anti-CD74 HuMab antibodies in Daudi cells

[0380] This Example describes the determination of dissociation rates of anti-CD74 HuMab antibodies on binding to Daudi cells.

[0381] Antibodies were labeled with the Alexa Fluor® 488 dye (Molecular Probes), hereinafter “Alexa-488”, using the following procedure:

[0382] An antibody solution of 1 mg/ml IgG was prepared in 0.1 M sodium carbonate buffer pH 9.0 (NaHCO3, Riedel de Haen, cat. no. 31437). Alexa-488 was freshly prepared by adding 100 μl of DMSO (Sigma, cat. no. D2438) to a vial (Alexa Fluor® 488 carboxylic acid, succinimidyl ether (1 mg/vial), Molecular Probes, Leiden, The Netherlands Baixos, Cat. No. A20000). A 25-fold molar excess of Alexa-488, calculated as indicated below, was added to the IgG solution and incubated, with rotation, in the dark at room temperature for 1 hour. After labeling, unbound Alexa-488 was removed, using a PD-10 column (Amersham Biosciences, cat. no. 17-0851-01), with Tris buffer pH 8.0 (50 mM Tris [Trizma base, Sigma , cat. no. T-6066]; 100 mM NaCl [Riedel de Haen, cat. no. 31437]; 0.01% sodium azide [NaN3, Riedel de Haen, cat. no. 13412]). The amount of Alexa-488 to be added to the IgG solution was calculated using the formula:

[0383] Volume of Alexa-488 to be added (in μl) = (conc of IgG (mg/ml)/MW of IgG (Da) * ratio * volume * MW of Alexa-488 * 100.

[0384] MW of IgG = 150,000 Da; the reason is the molar excess of Alexa-488 to use; volume is the volume of the sample to be labeled (in ml); MW Alexa-488 = 643 Da.

[0385] Protein concentration (IgG) and degree of labeling (D.O.L.) were determined by measuring the OD 280 nm and 495 nm on an Ultrospec 2100 Pro (Amersham Biosciences). IgG concentration (mg/ml) was calculated using the formula: IgG concentration = [A280 - (0.11 * A495)]/IgG extinction coefficient. D.O.L. was calculated using the formula: D.O.L. = A495/71,000/[A280 - (0.11 * A495)/(IgG extinction coefficient * IgG MW)]. 71,000 is the extinction coefficient of Alexa-488 at Amax in cm-1M-1; 0.11 is the correction factor (A280 free dye/Amax free dye) (both supplied by the manufacturer).

[0386] Bovine serum albumin (BSA; Sigma, cat. no. A 2934) was added from a 10% (w/v) solution to a final concentration of 0.1% (w/v) and labeled antibodies were stored at 5°C.

[0387] Daudi cells were incubated with Alexa-488-labeled anti-CD74 HuMab antibodies. Daudi cells were washed twice with ice-cold PBS. 10 5 cells per well in ice-cold FACS buffer were seeded in 96 well round bottom tissue culture plates (Greiner Bio-one). 0.5 µg/ml (HuMab-CD74-006 and -011; final concentration) or 1 µg/ml (HuMab-CD74-008; final concentration) of Alexa-488-labeled anti-CD74 HuMab was added in FACS buffer. ice cold. After incubation on ice for 30 min, 50 µg/ml (HuMab-CD74-006 and -011; final concentration) or 100 µg/ml (HuMab-CD74-008; final concentration) of unlabeled anti-CD74 HuMab was added. and incubated on ice for different time intervals ranging from 15 to 180 min. Total incubation time with unlabeled antibody is indicated below in the graphs. To determine maximal binding, Daudi cells were incubated with Alexa-488-labeled HuMab antibodies on ice for 30 min. As a negative control, cells were incubated with IgG1-b12 isotype control antibody (0.5 µg/ml final concentration), followed by unlabeled IgG1-b12 (50 µg/ml final concentration). After antibody incubation, cells were washed once in FACS buffer and Alexa-labeled HuMab anti-CD74 antibodies were detected by flow cytometry on a FACS Corner II (BD Biosciences).

[0388] Figure 14 shows that the dissociation rates of HuMab-CD74-006 and -008 measured at 0°C were very rapid (half of the bound Alexa-488-labeled antibodies were replaced with unlabeled antibodies within ~3 and ~4 min at 0°C, the K values were 0.24 and 0.20 min-1; [K = 1<off]), whereas the dissociation rate of -011 measured at 0°C was a slightly slower (half of the Alexa 488-bound labeled antibodies were replaced with unlabeled antibodies within ~10 min at 0°C, the K value was 0.07 min-1).

Example 24 Internalization and accumulation of anti-CD74 HuMab antibodies

[0389] To determine whether anti-CD74 HuMab antibodies are suitable for an antibody-drug conjugate method, antibody internalization and accumulation were studied by FACS analysis after incubation of different anti-CD74 HuMab antibodies with Daudi cells. 10 5 cells per well in cell culture medium were seeded in 96 well round bottom tissue culture plates (Greiner Bio-one). 10 µg/ml (final concentration) of Alexa-488-labeled anti-CD74 HuMab antibodies were added at different time points and incubated at 4°C (to measure binding to the CD74 expressed cell surface) or 37°C ( to measure binding and internalization). The total incubation time with antibody is indicated below in the graph. Internalization and accumulation at 37°C were also tested using Raji and M4A4 cells with a final concentration of 3 µg/ml of Alexa-488-labeled anti-CD74 HuMab antibodies. After incubation with antibody, cells were placed on ice and plates were washed twice with FACS buffer. Cell-associated labeled antibodies were detected by flow cytometry on a FACS Canto II (BD Biosciences).

[0390] Figure 15A shows that only low levels of Alexa-488-labeled HuMab anti-CD74 antibody binding to the Daudi cell surface were detected after incubation at 4°C at any time point. Therefore, the observed fluorescence intensities measured after incubation at 37°C represent internalized antibody. Figure 15B shows that all tested anti-CD74 HuMab antibodies were internalized, but with different efficacies. Internalization was faster for HuMab-CD74-011, slower for HuMab-CD74-006 and slower for HuMab-CD74-008. The same was observed for internalization and accumulation in Raji cells (15C) and M4A4 cells (15D).

Example 25 Prophylactic treatment of Daudi tumor xenografts in SCID mice with anti-CD74 HuMab antibodies

[0391] The in vivo efficacy of anti-CD74 HuMab antibodies was determined in an intravenous (i.v.) Daudi tumor xenograft tumor model in SCID mice. Daudi cells were transfected by electroporation with gWIZ luciferase (Aldevron, Fargo, ND, USA) and pPur vector (BD Biosciences, Alfen a/d Rijn, The Netherlands) in a 4:1 ratio. After 48 h, puromycin was added to select a stably transfected clone (Daudi-luc). Daudi luc #1E3 cells were cultured in RPMI supplemented with 10% cosmic calf serum (cat. no. SH30087.04, Hyclone), 1% penicillin/streptomycin (cat. no. DE17 603, Cambrex, Germany), 1 % sodium pyruvate and 1 μg/ml puromycin (cat. no. P 8833, Sigma, Zwijndrecht, The Netherlands). 2.5 x 10 6 Daudi luc tumor cells in 100 µl PBS were injected i.v. in the tail vein of female SCID mice (7 mice per group). Mice were treated on the day of tumor inoculation with 100 µg (~5 mg/kg) of HuMab-CD74-005, -006 or -011 or control antibody (IgG1 b12), in 200 µl of PBS, intraperitoneally (i.p. ). Mice were imaged directly after tumor inoculation, followed by imaging at weekly intervals starting on day 14. For imaging, mice were anesthetized using isoflurane, followed by i.p. of 2.5 mg of D-luciferin (acid form, cat. no. BT11 1000; Biothema, Haninge, Sweden) in 200 µl of 10 mg/ml TRIS (cat. no. T60666-1kg, Sigma). Bioluminescence imaging (BLI) of the back (dorsal view) began 10 min after D-luciferin administration, 5 min exposure time, in a Biospace Imager. The black and white images were taken for anatomical reference.

[0392] Figure 16 shows that all tested anti-CD74 HuMab antibodies almost completely prevented the growth of Daudi luc i.v.

EQUIVALENTS

[0393] Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims that follow. Any and all combinations of embodiments disclosed in the dependent claims are also considered to be within the scope of the invention.

Claims (35)

1. An isolated antibody, characterized in that it binds to human CD74 variants 1 and 2 and comprises a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 24, SEQ ID NO: 32 and SEQ ID NO: 25, and (a) a VH region comprising the sequences of CDRs 1, 2 and 3 of SEQ ID NOs: 20, 21 and 22 [011]; (b) a VH region comprising the sequences of CDRs 1, 2 and 3 of SEQ ID NOs: 8, 9 and 10 [005]; (c) a VH region comprising the sequences of CDRs 1, 2 and 3 of SEQ ID NOs: 12, 13 and 14 [006]. 2. An antibody according to claim 1, characterized in that (a) it binds to the extracellular domain of CD74 variant 1 with an EC50 of less than 500 ng/ml, less than 400 ng/ml, less than than 350 ng/ml, or less than 330 ng/ml; (b) binds to the extracellular domain of CD74 variant 2 with an EC50 of less than 400 ng/ml, less than 300 ng/ml, less than 250 ng/ml, or less than 220 ng/ml; (c) both of (a) and (b); or (d) binds to CD74 on Raji cells with an EC50 of less than 400 ng/ml, less than 300 ng/ml, less than 250 ng/ml, or less than 200 ng/ml when binding CD74 variant 1 extracellular domain and CD74 variant 2 extracellular domain binding are determined by ELISA, and CD74 binding on Raji cells is determined by FACS analysis. 3. Antibody according to any one of claims 1 and 2, characterized in that it binds to cynomolgus CD74. 4. Antibody according to any one of claims 1 to 3, characterized in that it is internalized after binding to CD74 expressed on the surface of a cell. An antibody according to any one of claims 1 to 4, characterized in that it has an EC50 of less than 60 ng/ml, less than 40 ng/ml, less than 30 ng/ml, or of 25 ng/ml or less in inducing death of Raji cells in an in vitro cell-based death assay using kappa-directed pseudomonas exotoxin. 6. Antibody according to any one of claims 1 to 5, characterized in that it has a dissociation rate at 0°C of 0.02 to 1.0 min-1, from 0.03 to 0.30 min-1. 1, from 0.04 to 0.10 or from 0.15 to 0.30 min-1. An antibody according to any one of claims 1 to 6, characterized in that it comprises a VH region having a VH region sequence selected from the group consisting of SEQ ID NOS: 7, 11 and 19. 8. Antibody according to any one of claims 1 to 7, characterized in that it comprises a VL having a VL region sequence selected from the group consisting of: SEQ ID NOS: 23 and 26. 9. Antibody, characterized in that it binds to CD74 variants 1 and 2 and comprises: (a) a VH region comprising the sequence of SEQ ID NO: 19 and a VL region comprising the sequence of SEQ ID NO: 26 [011]; (b) a VH region comprising the sequence of SEQ ID NO: 7 and a VL region comprising the sequence of SEQ ID NO: 23 [005]; or (c) a VH region comprising the sequence of SEQ ID NO: 11 and a VL region comprising the sequence of SEQ ID NO: 26 [006]. 10. Antibody according to any one of claims 1 to 9, characterized in that it is a human monoclonal antibody. 11. Antibody according to any one of claims 1 to 10, characterized in that it has a selected isotype of IgG1 and IgG4. 12. Antibody according to any one of claims 1 to 11, characterized in that it is conjugated to a therapeutic moiety. 13. Antibody according to claim 12, characterized in that it is conjugated to the therapeutic moiety through a linker linked to sulfhydryl residues in the antibody, obtained at least by partial reduction of the antibody. 14. Antibody according to claims 12 and 13, characterized in that the therapeutic moiety is a cytotoxic moiety, a radioisotope, a chemotherapeutic agent, a lytic peptide or a cytokine. 15. Antibody according to claim 14, characterized in that it is conjugated to a cytotoxic moiety. 16. Antibody according to claim 15, characterized in that the cytotoxic moiety is selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxy anthracine dione; maytansine or an analogue or derivative thereof; an auristatin or a functional peptide analogue or derivative thereof such as monomethyl auristatin E (MMAE) or F (MMAF); dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1- dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; an antimetabolite such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, raquelmycin (CC-1065), or an analogue or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as toxins SLT I, SLT II, SLT IIV, LT, C3, Shiga toxin, whooping cough toxin, tetanus toxin, Bowman-Birk soy protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modeccin, gelatin, abrin A chain , modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, the toxins gelonin, mitogelin, restrictocin, phenomycin, and enomycin; ribonuclease (RNase); DNase I, staphylococcal enterotoxin A; pigweed antiviral protein; diphtheria toxin; and Pseudomonas endotoxin. 17. Antibody according to claim 15, characterized in that it is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, a pyrrolo[2,1-c][1,4]-benzodiazepine, maytansine, calicheamicin , duocarmycin, raquelmycin (CC-1065), dolastatin 10 or 15, irinotecan, monomethyl auristatin E and monomethyl auristatin F, or an analogue, derivative, or prodrug of any of these. 18. Antibody according to claim 15, characterized in that the cytotoxic portion is an auristatin or a functional peptide analogue or derivative thereof, optionally conjugated to the antibody via a linker linked to one or more cysteine residues in the antibody . 19. Antibody according to claim 14, characterized in that it is conjugated to a cytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNy, GM-CSF, CD40L, binding Flt3, stem cell factor, ancestim, and TNFα. 20. Antibody according to any one of claims 1 to 19, characterized in that it is a multispecific antibody, comprising a first antigen binding region of an antibody as defined in any one of claims 1 to 19, and at least a second antigen binding region having a different binding specificity. 21. Antibody according to claim 20, characterized in that it is a bispecific antibody, optionally wherein the second antigen binding region has binding specificity for an antigen on a human effector cell, such as a human T cell. 22. Antibody according to any one of claims 20 and 21, characterized in that it is a bispecific antibody in which the first antigen binding region is linked to a first Fc region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409, and the second antigen binding region is linked to a second Fc region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409, and the first and second Fc regions are not substituted at the same positions. 23. Expression vector, characterized in that it comprises a nucleotide sequence as defined in any one of SEQ ID NOs: 27, 28, 29, 30 or 31, or degenerate sequences thereof that respectively encode the same amino acid sequences of the SEQ ID NOS: 7, 11, 19, 23 and 26, or any combination thereof. 24. Expression vector according to claim 23, characterized in that it further comprises a nucleotide sequence that encodes the constant region of a human antibody light chain, a human antibody heavy chain, or both. 25. Recombinant prokaryotic host cell, characterized in that it produces the antibody as defined in any one of claims 1 to 14 or 16. 26. Pharmaceutical composition, characterized in that it comprises the antibody as defined in any one of claims 1 to 22 and a pharmaceutically acceptable carrier. 27. Antibody according to any one of claims 1 to 22, characterized in that it is for use as a medicine. 28. Antibody according to any one of claims 1 to 22, characterized in that it is for use in the treatment of cancer. 29. An antibody according to any one of claims 1 to 22, characterized in that it is for use in cancer prophylaxis, wherein the cancer prophylaxis comprises one or more of reducing the risk of developing cancer, reducing the risk for the progression of cancer, and/or reduce the risk of recurrence of a cancer in relapse. 30. Antibody for the use according to any one of claims 27 to 29, characterized in that the cancer is selected from the group consisting of breast cancer, colorectal cancer, endometrial/cervical cancer, gastric cancer, cancer of the head and neck, lung cancer, malignant glioma, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, liver cancer, thymus cancer, malignant fibrous histiosarcoma, acoustic schvanoma, pituitary adenoma, and an adenoma, or the group consisting malignant lymphoma, chronic B-cell lymphocytic leukemia (BCLL), burst phase chronic myeloid leukemia (CML), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), monocytoid B-cell lymphoma (MBCL), hair cell (HCL), and T cell lymphoma. 31. Antibody for use according to claim 27, characterized in that it is for use in the treatment of an autoimmune disease. 32. Antibody, characterized in that it is for the use according to any one of claims 27 to 31 in combination with at least one other therapeutic agent. 33. Antibody for the use according to claim 33, characterized in that at least one other therapeutic agent is selected from a second antibody or ADC; a chemotherapeutic agent; an inhibitor of angiogenesis, neovascularization, and/or other vascularization; an anticancer immunogen; a cytokine or chemokine; a regulator of cell cycle control or apoptosis; a hormone-regulating agent; an anti-anergic agent; a nucleic acid or vector that contains a tumor suppressor gene; an anti-cancer nucleic acid; a virus or viral proteins; immune system cells; a differentiation-inducing agent; an agent that upregulates CD74; and an anti-inflammatory, immunosuppressive and/or immunomodulatory agent; or a combination of any of these. 34. Antibody for the use according to claim 33, characterized in that at least one therapeutic agent is selected from a CD20-specific antibody, a CD138-specific antibody, a CD38-specific antibody, an anti-VEGF- A, melphalanan, lenalidomide, bortezomib, fluorouracil, genticabine, irinotecan, cisμlatin, or a derivative or analogue thereof. 35. Use of an antibody as defined in any one of claims 1 to 22, characterized in that it is for the manufacture of a medicament for the treatment of cancer.

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