ES2359426T3 - ANTIBODIES DIRECTED TO GPNMB AND ITS APPLICATIONS. - Google Patents

Abstract

An isolated human antibody that specifically binds to the possible transmembrane GPNMB protein defined by SEQ ID NO: 289, wherein the antibody comprises a heavy chain CDR1 defined by SEQ ID NO: 22, a heavy chain CDR2 defined by SEQ ID NO: 24, a heavy chain CDR3 defined by SEQ ID NO: 26, a light chain CDR1 defined by SEQ ID NO: 31, a light chain CDR2 defined by SEQ ID NO: 33 and a light chain CDR3 defined by SEQ ID NO: 35.

Description

FIELD OF THE INVENTION

The present invention relates to antibodies with specificity by GPNMB and to uses of said antibodies. In particular, the present invention provides fully human monoclonal antibodies that specifically bind GPNMB and uses thereof. Nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences extending across the framework regions and / or complementarity determining regions are provided. (CDR). The present invention also provides immunoconjugates comprising antibodies directed against GPNMB and methods of using said immunoconjugates. The present invention further provides bispecific antibodies comprising an antibody component directed against GPNMB and a component directed against CD3, and methods of using such bispecific antibodies.

BACKGROUND OF THE INVENTION

GPNMB

A possible transmembrane glycoprotein called "nmb" (registration number X76534 EMBL), hereinafter referred to herein as GPNMB, was identified and described by Weterman et al. (Int J Cancer 60: 73-81, 1995) as differentially expressed in human melanoma cancer cell lines of low metastasis and xenografts compared to a more aggressive melanoma cell line. GPNMB shares 33% identity with the precursor of the melanocyte specific protein pMe117 (Kwon et al., 1991, PNAS 88: 9228-9232). GPNMB has 71% homology with a transmembrane protein associated with dendritic cells, DC-HIL (Shikano et al., 2001 Biol. Chem. 276: 8125-8134). GPNMB is also known as the HGFIN hematopoietic growth factor inducible neurocinin 1 protein (Bandari et al., Reg. Peptides 111: 169-178) and osteoactivin of bone-related genes (Owen et al. Crit Rev Eukaryot Gene Expr 2003, 13 (2-4): 205-220),

An antiserum directed against rabbit GPNMB and two murine hybridomas specific for GPNMB protein has been described (Kuan Chien-Tsun et al. (2003) Proc Am Assoc Cancer Res Annual Meeting 44: 1116-1117).

It was also reported that nmb could reduce the metastatic potential of a melanoma cell line negative for highly metastatic nmb (Weterman, 1995). GPNMB was considered a candidate for marker of glioblastoma tumors after the extraction and identification of the expression through the analysis of public databases (Loging et al., 2000, Genome Research 10: 1393-1402). This gene was found to be overexpressed in lung tumors (U.S. Patent Publication No. US20030064947), in addition to breast, rectal and colon cancers (U.S. Patent Publication No. US2003100720). NCBI SAGE data also show overexpression of this gene in stomach and pancreatic carcinoma. It has been shown that the mouse ortholog is heavily regulated by an NSC line of neurocytoblasts derived from the inactivated TSC2 gene model for tuberous sclerosis complex syndrome (International Publication No. WO 2003/080856).

Antibodies

The antibodies, also known as immunoglobulins, are usually tetrameric glycosylated proteins composed of two light chains (L) (approximately 25 kDa) and two heavy chains (H) (approximately 50-70 kDa). The amino end portion of each chain includes a variable domain of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy end portion of the L and H chain has one and three or four constant domains, respectively, that are primarily responsible for the effector function. There are two types of human L chains classified as kappa and lambda. H chains are classified as mu, delta, gamma, alpha or epsilon based on the amino acid sequence of the constant domain that defines the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. The isotypes can be further divided into subclasses, for example, IgG1, IgG2, IgG3, IgG4.

Immunoglobulins can naturally be produced in vivo by B lymphocytes. Each B cell clone produces an antibody with an antigen receptor that has a unique possible antigen binding structure. The repertoire of antigen receptors, approximately 107 possibilities, exists in vivo before antigen stimulation. This diversity is produced by somatic recombination, that is, the binding of different antibody gene segments. The H chain, the L kappa chain and the L lambda chain of the immunoglobulin are encoded by three separate genetic loci and each locus has multiple copies of at least 3 types of gene segments encoding variable regions (V), constants (C) and binding (J), the heavy chain genes also include a region of diversity (D). The selection of specific V, C and J regions (and D for the heavy chain) from the various available gene segments (45 V heavy chain; 35 V kappa; 23 D heavy chain; 6 J heavy chain; 5 J kappa) generates approximately 1011 possible specificities of germline sequences presented in B cells. The union of those of regions V, C and J can cause the loss or addition of residues in the junctions. The V region of the human antibody L and H chains consists of relatively conserved framework regions (FR) that form a scaffold for three hypervariable regions also known as complementarity determining regions (CDR). From the amino end of both the heavy and light chains, the V domain is composed of FR and CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3. The union of domain V with domain D (heavy chain only) and J adds CDR3-FR4. CDRs are generally responsible for antigen binding.

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The specificity of monoclonal antibodies has made them attractive agents to choose as a target in vivo cancer with the hope of eradicating the disease, while saving normal tissue. The solution that initially used mouse monoclonal antibodies has found limitations on possible efficacy such as immunogenicity; ineffective effector functions and short half-life in vivo. The technologies were developed for: chimeric antibodies that seek to use the variable antigen-binding domains of mouse monoclonal antibodies combined with the constant regions of human antibodies (Boulianne et al. 1984 Nature 313: 643-646; Morrison et al., 1984 PNAS USA 81: 6851-6855); humanized antibodies that grafted complementary antigen-binding (CDR) determining regions of mouse antibodies into human immunoglobulin (Jones et al., 1986 Nature 321: 522-525; Riechmann et al., 1988 Nature 332: 323-327; Verhoeyen et al., 1988 Science 239: 1534-1536; Vaughan et al., 1998 Nature Biotechnol. 16: 535-539); and phage expression libraries of single-chain scFv or Fab antibody fragments (de Haard et al., 1999 J Biol. Chem. 274: 18218-18230; Knappik et al., 2000 J. Mol. Biol. 296: 57-86 ; Sheets et al., 1998 PNAS USA 95: 6157-6162; Vaughan et al., 1994 Nature Biotechnol 14: 309-314, 1996; Griffiths et al. EMBO J. 13: 3245-3260). Additionally, transgenic animals have been developed that have human immunoglobulin genes and non-functional endogenous genes for the immunization and production of fully human monoclonal antibodies (Fishwild et al., 1996 Nature Biotechnol 14: 845-851; Mendez et al., 1997 Nature Genet. 15: 146-156; Nicholson et al., 1999 J. Immunol 163, 6898-6906).

Single-chain antibodies: Single-chain Fv antibodies (scFv) were first described in the late 1980s (Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). A polypeptide linker, which normally ranges in length from 5 to 27 amino acid residues, is used to link the C-terminus of the light chain variable domain (VL) to the N-terminus of the heavy chain variable domain (VH). Alternatively, the linker attaches the C end of VH to the N end of VL. Both forms (VL-VH and VH-VL) have been used successfully in the literature. The most common linker used in the literature is the 15 amino acid linker (Gly4Ser) 3, however there are several other linkers that have been used that include a 25 amino acid linker called 205C (Pantoliano et al., Biochemistry 30: 10117-10125 (1991)). Single chain antibodies are currently in clinical trial; one of the most advanced is h5G1.1 or pexelizumab. This scFv is specific for the human C5 complement and is being used in clinical trials for cardiac patients undergoing cardiopulmonary bypass surgery (Shernan et al., Ann. Thorac Surg. 77: 942-949 (2004)).

Bispecific Antibodies (bi-Ab): An area of mAb research in which considerable progress has been made is in the development of bispecific antibodies (bi-Ab). There are different advantages in the development of therapeutic antibody molecules with dual specificity. For example, bi-Ab can serve as mediators to choose as target immune effector cells such as CTL for unwanted cells (Baeuerle et al., Curr. Opin. Mol. Ther. 5: 413-419 (2003)). In another example, chemically linked bispecific antibodies directed against CD16, CD64 and CD89 gamma Fc receptors were significantly more effective in vitro than conventional IgG antibodies (Peipp and Valerius, Biochem. Soc. Trans. 30: 507-511 (2002)) . One of the challenges in the development of bi-Ab as viable therapeutic agents has been to produce sufficiently large quantities of a stable remainder for clinical applications. Another challenge has been to determine the correct combination of validated targets and the underlying biology that would lead to a therapeutic product. For recent reviews on the difficulties experienced with bi-Ab see (Kontermann, Acta Pharmacol Sin 26: 1-9 (2005); Peipp and Valerius, Soc. Trans. 30: 507-511 (2002)).

Bispecific single chain antibodies (bi-scFv): A notable type of bi-Ab that can be prepared is a bispecific single chain antibody or bi-scFv. For a review on the generation of bi-scFv see (Kipriyanov and Le Gall, Curr Opin Drug Discov Devel 7: 233-242 (2004)). The bi-scFv normally comprise 4 variable domains, 2 heavy (VH) and 2 light (VL), which are derived from 2 different antibodies. The 4 domains are linked together with 3 short linkers ranging in length from 5-27 amino acids. The biological activity of this type of antibody depends on several characteristics related to the construction of the molecule. For example, both the binding sequences between the V domains of antibodies and the order of the 4 V domains of the antibodies (for the 2 antibodies) may vary, in addition to the expression system used; everything can greatly affect the solubility and biological activity of the various resulting products (Kipriyanov et al., J. Mol. Biol. 330: 99-111 (2003); Le Gall et al., Protein Eng. Des. Sel. 17 : 357-366 (2004); Pavlinkova et al., Clin Cancer Res. 5: 2613-1619 (1999)).

Cytotoxic T lymphocytes: Under normal circumstances, T cells are activated when the CD3 / T cell receptor complex (CD3 / TCR) binds to a relevant MHC molecule associated with a specific Ag peptide. The adjustment of CD3 / TCR with MHC produces intracellular signals necessary to trigger an immune response against a pathogen or tumor. Similar signals that produce T cell activation by antibodies that can bind to certain structures of the CD3 / TCR complex can also be achieved. In the literature it has been shown that bi-Ab that recognize both the TCR / CD3 complex and the tumor-associated antigen (TAA) can trigger the CTL activation program in the presence of target cells (Chapoval et al., J. Immunol 155 : 12961303 (1995)).

Recombinant technologies are being used to allow further improvements in the antibody molecules in order to enhance efficacy in vivo. Such technologies provide, for example, optimization of molecular size, affinity, pharmacokinetics, toxicity, specificity, valence, effector functions, direct and indirect armament, combination therapy, and various approaches to prodrugs.

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It would be desirable to have an antibody suitable for the choice as an in vivo target of GPNMB that expresses pathologies and to allow therapeutic efficacy.

SUMMARY OF THE INVENTION

The present invention provides an isolated human antibody that specifically binds to the possible GPNMB transmembrane protein defined by SEQ ID NO: 289, wherein the antibody comprises a heavy chain CDR1 defined by SEQ ID NO: 22, a heavy chain CDR2 defined by SEQ ID NO: 24, a heavy chain CDR3 defined by SEQ ID NO: 26, a light chain CDR1 defined by SEQ ID NO: 31, a light chain CDR2 defined by SEQ ID NO: 33 and a CDR3 of light chain defined by SEQ ID NO: 35. In a preferred embodiment, the antibody comprises the heavy chain variable region as shown in SEQ ID NO: 20. In another preferred embodiment, the antibody comprises a light chain variable region. As shown in SEQ ID NO:

29. Preferably, the antibody is a monoclonal antibody. Most preferably, the antibody is Mab1.15.1.

In one embodiment, the present invention provides antibodies directed against GPNMB of pure IgG1 of the invention having cytotoxic effect for cells expressing GPNMB in excess. In a specific embodiment, the present invention provides the medical use of said antibodies or a composition comprising said antibody directed against GPNMB of pure IgG1 and an immunomodulator (such as, but not limited to, interferons and cytokines).

In another embodiment, the present invention provides immunoconjugates comprising an antibody directed against GPNMB of the invention or a fragment thereof, and a cytotoxic agent. In a specific embodiment, the cytotoxic agent is auristatin E (dolastatin 10) or a derivative thereof. Such immunoconjugates are also provided for medical use.

In one embodiment, the present invention provides bispecific antibodies comprising a component directed against GPNMB of the invention and an antibody component directed against CD3 that allow cytotoxic destruction of target tumor cells by T cells. In another embodiment, the present invention provides antibody Single chain PV of the invention conjugated to a cytotoxic agent. In a specific embodiment, the cytotoxic agent is auristatin E (dolastatin 10) or a derivative thereof. Such bispecific antibodies and conjugated single chain Fv antibodies are also provided for medical use.

Amino acid sequences for human monoclonal antibodies directed against GPNMB of the invention and nucleic acid sequences encoding them are provided.

Compositions comprising antibodies directed against human GPNMB, including therapeutic compositions comprising the same, and uses thereof are provided. Particularly therapeutic immunoconjugates are provided comprising antibodies directed against GPNMB and a cytotoxic or cytostatic agent for treating GPNMB expressing cancers and other disorders related to GPNMB. Dosage guidelines are also provided.

Additional aspects of the disclosure will be set forth in part in the following description, and in part will be obvious from the description, or can be learned by practicing the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Inhibition and complete regression of the tumor growth of SK-MEL-2 xenografts in nude mice after treatment with 2.50 to 20 mg / kg i.v. every 4 days for 4 treatments. The responses of animals that had tumors to reference drugs such as vinblastine (1.7 mg / kg i.v. q4d X4) and paclitaxel (24 mg / kg i.v. q2d X4) are also shown. Control groups are treated with either phosphate buffered saline (PBS) or physiological saline.

Figure 2: Indirect destruction of UACC-62 melanoma cell immunotoxins by antibodies directed against GPNMB

Figure 3: Inhibition of colony formation of UACC-62 cells incubated with antibodies directed against GPNMB conjugated to auristatin E (AE).

Figure 4: Inhibition and complete regression of tumor growth of SK-MEL-2 xenografts in nude mice after treatment with CR011-vcMMAE 5.0 mg / kg i.v. every 4 days for 4 treatments. The lack of responses from animals that had tumors to unconjugated CR011 or free monomethylauristatin E demonstrates that the intact immunoconjugate is essential for antitumor effects.

Figure 5: Reduction and complete regression of SK-MEL-2 xenograft tumor size in nude mice after treatment with 1.25 to 20 mg / kg i.v. every 4 days for 4 treatments. The responses of animals that had tumors to reference drugs such as vinblastine (1.7 mg / kg i.v. q4d X4) and paclitaxel (24 mg / kg i.v. q2d X4) are also shown. Control groups are treated with either phosphate buffered saline (PBS) or physiological saline.

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Figure 6: Serum-time concentration profile of the CR011-vcMMAE antibody after intravenous administration of 1 and 10 mg / kg to athymic mice. Detection was achieved with a sandwich ELISA assay that used the CR011 antigen (CG56972, GPNMB) and antibodies directed against human globulins conjugated to horseradish peroxidase. The results shown are serum concentrations expressed as µg / mL (left x axis) and micromolar concentration (right x axis).

Figure 7: Global responses expressed as percentage cures were recorded for experimental animals treated with 5 different graduated dosage intervals (ie, 0, 1, 4, 8 and 16 days between treatments). The slope of the line is not significantly different from 0 (p <0.2904).

Figure 8: Proportions of complete regressors as a function of the dosage interval and statized by cumulative dose. For each group, n = 6 mice / group. Atymic mice that had established SKMEL-2 tumor implants (day 14, 80 mg) were treated i.v. with CR011-vcMMAE and the incidence of complete regressions is recorded.

Figure 9: Effects of ectopic expression of GPNMB or sensitivity to CR011-vcMMAE. HEK293 cells are transfected with an empty vector (vector) or plasmid containing GPNMB (GPNMB) as described in Materials and procedures. A. Cell lysates are prepared from the transfected HEK293 cells and the expression of GPNMB (upper panel) or actin (lower panel) is determined by immunoblotting. Lane 1: Empty vector transfectants. Lane 2: GPNMB transfectants. B. Flow cytometric analysis of GPNMB expression in cells transfected with empty vector or GPNMB. C. In vitro growth inhibition of CR011-vcMMAE from transfected cells. Cells with various concentrations of CR011-vcMMAE (diamonds: vector or circles: GPNMB) or IgG2vcMMAE (triangles: vector or squares: GPNMB) are treated for 96 hours. After a clonogenic assay, the survival fraction is normalized to the untreated control and is expressed as a percentage of the control using GraphPad Prism graphic software. Each treatment is done in triplicate. A representative graph of two independent experiments is shown.

Figure 10: Effect of GPNMB siRNA on endogenous GPNMB expression and sensitivity to CR011vcMMAE. SK-MEL-2 cells are transfected with 50 nM control siRNA or GPNMB that you choose as the siRNA target. A. Cell lysates are prepared from transfected SK-MEL-2 cells 2 and 4 days post-transfection and the expression of GPNMB (upper panel) or actin (lower panel) is determined by immunoblot. Lane 1: Transfection with empty vector (oligofectamine). Lane 2: Control siRNA transfection. Lane 3: GPNMB siRNA transfection. B. Flow cytometric analysis of GPNMB expression 2 and 4 days after transfection. SK-MEL-2 cells are transfected with empty vector, control siRNA or GPNMB siRNA as indicated in Materials and procedures. C. In vitro growth inhibition of empty vector CR011-vcMMAE (diamonds), control siRNA (circles) or SK-MEL-2 cells transfected with GPNMB siRNA (triangles) is determined by a clonogenic assay as described in Materials and Procedures. The survival fraction is normalized to the untreated control and is expressed as a percentage of the control using GraphPad Prism graphic software. Each treatment is done in triplicate. A representative graph of two independent experiments is shown.

Figure 11: FACS analysis of SK-MEL-2 with isotype control, hybridoma IgG2 (B2), recombinant IgG2 (B19) and recombinant IgG1 (B16) for CG56972 / GPNMB with respect to IgG2 controls (B2, B19) or IgG1 (control, B16).

Figure 12: (A) ADCC mediated by PBMC and mAb (IgG1) of SK-MEL-2 cells. The effector functions of ADCC are measured as described above at 2, 5 and 10 µg / 200 µl using target ratios: effector of 10, 30, 60 and 100 as indicated. (B) PBMC and mAb (IgG2) do not produce ADCC to SK-MEL-2 cells. The effector functions of ADCC are measured as described above at 0, 2, 5 and 10 µg / 200 µl using target ratios: effector of 10, 30, 60 and 100 as indicated.

Figure 13: GPNMB expression in human cancer cell and tissue lines. RTQ-PCR analysis of (A) human brain cancer cell lines or (B) human brain cancer glioma and medulloblastoma biopsies. (C) Analysis of GPNMB expression microarrays in human brain cancer and oligodendroglioma tissues. Tissues or cell lines are collected, mRNA is prepared and RTQ-PCR or CuraChip analyzes are performed as described in Materials and procedures.

Figure 14: FACS analysis of cell surface binding of the CR011 mAb to GPNMB. SK-MEL-2, XF-498, U-118-MG, SNB-78, SF-539 and SF-268 cells are labeled with a saturating concentration (10 µg / ml) of CR011 mAb or control IgG2. Bound mAb is detected by flow cytometry with goat secondary antibody directed against human conjugated PE globulins as described in Materials and procedures. MG: Geometric mean. The SF-268 cell line is a negative GPNMB transcript and is used as a negative control.

Figure 15: Immunoblot analysis of GPNMB expression in human brain cancer cell lines. Cell lysates are resolved in Tris-glycine gels and transferred to membranes. Immunoblot analysis is carried out with a polyclonal antibody to GPNMB followed by enhanced chemiluminescence detection as described in Materials and procedures. The arrowheads indicate the relative mobility of GPNMB p100 and 120 species. The SF-268 cell line is a negative GPNMB transcript and is used as a negative control.

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Figure 16: In vitro growth inhibition of CR011-vcMMAE from astrocytoma / glioblastoma cell growth. XF-498, SNB-78, U-118-MG, SF-539, LOXIMVI and SF-268 cells are incubated with the indicated concentration of CR011-vcMMAE. Cells are also incubated with control PK16.3 mAb (data shown in Table 1) as described in Materials and procedures. Cell growth was determined by clonogenic assay. Surviving colonies are counted and represented using GraphPad Prism graphic software. The experiment is performed in triplicate wells and repeated twice. A representative experiment is shown. IC50s for cell destruction occur in concentrations of ng / ml. The LOXIMVI and SF-268 cell lines are transcribed from negative CG56972 and are used as negative controls.

Figure 17: Development of genetically engineered CR011 antibodies. Four variable domains (V) of antibodies (shown in C for bi-scFv) are derived from the light and heavy chain variable domains (VL and VH) preparing the complete Ig0 antigen binding sites CR011 and directed against CD3. The intermediate linker that joins the 2 individual scFv components together (shown in dashed lines) can play a key role in determining the resulting activity of each scFv component that includes the effective cytolytic activity provided by the T cells. cytotoxic adjusted by the scFv component directed against CD3 of the bi-scFv.

Figure 18: A. ELISA results for CR011 scFv (squares) and CR011 x anti-CD3 bi-scFv (linker set L4-L2-L4) (diamonds). Both genetically engineered CR011 antibodies bound to the GPNMB target. B. Western blot of 2 of the genetically engineered CR011 antibody products (arrows). Clone 16 corresponded with the CHOK1 line expressing scFv of CR011 (monomer), while clone 17 corresponded with the CHOK1 line expressing bi-scFv of CR011 x anti-CD3 (linker set L4-L2-L4) (dimer). Clones 16 and 17 are used to produce genetically engineered antibody products.

Figure 19: Analysis by flow cytometry of the binding of the scFv products of CR011 and bi-scFv of CR011 x anti-CD3 (set of linkers L4-L2-L4) to the native GPNMB protein expressed on the cell surface of target cells Human T cells are used as a source of CD3, while SK-MEL-5 cells are used as a source of GPNMB.

Figure 20: Cytotoxicity analysis showed that purified bi-scFv of CR011 x anti-CD3 (set of Binder L4-L2-L4), but not scFv of CR011, produces the destruction of GP-MB-positive SK-MEL-5 tumor cells by T lymphocytes.

Figure 21: Chemical structure of maleimidocoaproyl-valine-citrulline-monomethyl-auristatin E (vcMMAE).

Figure 22: Disulfides on the CR011 antibody are gently reduced in the presence of TCEP to generate ~ 4 thiols per Ab. Then vcMMAE is added to the antibody solution. Nucleophilic attack of thiolates on maleimide groups produces a stable thioester bond. The resulting conjugate is purified from the mixture.

Figure 23: Reaction of vcMMAE with NAcCys at pH 7.0 and pH 9.0 in the presence or absence of TCEP. 1A: vcMMAE becomes completely the NAcCys adduct after an incubation in phosphate buffer at pH 7. B-E: Aspect of a secondary product in the course of incubation of vcMMAE in borate buffer. F-I: Aspect of secondary products in borate at pH 9 and in the presence of TCEP.

Figure 24: Identification by LC-MS of the secondary product with a retention time of 9.2 min which cannot react with cysteine and, therefore, cannot be conjugated with CR011.

Figure 25: Formation kinetics of NAcCys-vcMMAE and the secondary product (succinimidyl-vcMMAE) after incubation in borate buffer at pH 9.0 in the presence or absence of TCEP.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "antibody" refers to an immunoglobulin or a fragment or derivative thereof and encompasses any polypeptide comprising an antigen binding site, regardless of whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, manipulated and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies" for the purpose of this disclosure, the term "antibody" also includes antibody fragments such as Fab, F (ab ') 2, Fv , scFv, bi-scFv, bi-Ab, Fd, dAb and other antibody fragments that retain antigen binding function, that is, the ability to bind GPNMB specifically. Typically, such fragments would comprise an antigen binding domain.

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As used herein, the terms "antigen binding domain", "antigen binding fragment" and "binding fragment" refer to a part of an antibody molecule comprising amino acids responsible for the specific binding between the antibody and antigen. In cases where an antigen is large, the antigen binding domain can only bind to a part of the antigen. A part of the antigen molecule that is responsible for specific interactions with the antigen binding domain is hereinafter referred to as "epitope" or "antigenic determinant."

An antigen binding domain typically comprises a variable region of the antibody light chain (VL) and a variable region of the antibody heavy chain (VH); however, you don't necessarily have to understand both. For example, a so-called antibody Fd fragment is only made up of a VH domain, but still retains some antigen binding function of the intact antibody.

As used herein, the term "repertoire" refers to a genetically diverse collection of nucleotides derived entirely or partially from sequences encoding expressed immunoglobulins. The sequences are generated by in vivo transposition of, for example, segments V, D and J for H chains and, for example, segment V and J for L chains. Alternatively, the sequences can be generated from a cell line by stimulation in vitro in response to which transposition occurs. Alternatively, part or all of the sequences can be obtained by combining, for example, untranslated V segments with D and J segments by nucleotide synthesis, randomized mutagenesis and other methods, for example, as disclosed in US Pat. nº

5,565,332.

As used herein, the terms "specific interaction" and "specific binding" refer to two molecules that form a complex that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and a low to moderate capacity that differs from non-specific binding that normally has a low affinity with a moderate to high capacity. Normally, binding is considered specific when the affinity constant KA is greater than 106 M-1, or more preferably greater than 108 M-1. If necessary, the non-specific binding can be reduced without substantially affecting the specific binding by varying the binding conditions. Appropriate binding conditions such as antibody concentration, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (eg, serum albumin, milk casein), etc., may be optimized by an expert using routine techniques.

As used herein, the term "as set forth substantially" refers to the fact that the relevant CDR, VH or VL domain of the invention will be or identical to or will only have insubstantial differences in the specified regions (eg, a CDR) whose sequence is exposed. Insubstantial differences include minor changes in amino acids such as 1 or 2 substitutions of any 5 amino acids in the sequence of a specified region.

The term "CR011" refers to a fully human monoclonal antibody that specifically binds GPNMB, hereinafter referred to as mAb 1.15.1 as described herein, or derivatives or antigen-binding fragments thereof.

The term "GPNMB" refers to the possible transmembrane glycoprotein as described above, with essentially the sequence deposited in the registration number X76534 in the EMBL database, and minor variants thereof.

The term "CG56972" refers to the specific cDNA isolated as described in Example 27 (page 113, lines 13-29); its sequence as shown in SEQ ID NO: 289. It corresponds to the possible extracellular domain of an alleged GPNMB splice variant. The translation product of this cDNA was used as an antigen to produce the antibodies of the invention.

As used herein, the term "GPNMB activity" refers to one or more activities associated with GPNMB. To "modulate" the activity of GPNMB is to alter the initial results observed with, and which can be attributed to, GPNMB. To “neutralize” GPNMB is to cancel one or more effects, for example, the activity observed with, and that can be attributed to GPNMB.

As used herein, the term "isolated" refers to a molecule that is substantially free of its natural environment. For example, an isolated protein is substantially free of cellular material or other proteins from the source of cells or tissues from which it is derived. The term "isolated" also refers to preparations in which the isolated protein is pure enough to be administered as a pharmaceutical composition, or at least 7080% (weight / weight) of purity, more preferably at least 80-90% ( weight / weight) of purity, even more preferably 90-95% purity; and most preferably at least 95%, 96%, 97%, 98%, 99% or 100% (weight / weight) of purity.

As used herein, the term "inhibit" or "inhibition of" refers to reducing by a measurable amount, or completely preventing.

As used herein, the term "cytotoxic effect" in reference to the effect of an agent on a cell means destroying the cell. The "cytostatic effect" refers to an inhibition of cell proliferation. A "cytotoxic agent" refers to an agent that has a cytotoxic or cytostatic effect on a cell, thereby reducing or inhibiting the growth of, respectively, cells within a population of cells.

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As used herein, the terms "prevent", "prevent" and "prevention" refer to the inhibition of the development or occurrence of a disorder associated with the expression and / or abnormal activity of GPNMB (eg, cancer) or the prevention of the recurrence, appearance or development of one or more symptoms of a disorder associated with the expression and / or abnormal activity of GPNMB (for example, cancer) in a subject resulting from the administration of a therapy or the administration of a combination of therapies.

As used herein, the term "effective amount" refers to a dosage or amount that is sufficient to reduce the activity of GPNMB to produce symptom improvement in a patient or to achieve a desired biological result.

As used herein, the term "prophylactically effective amount" refers to the amount of a therapy that is sufficient to produce the prevention of the development, recurrence or occurrence of a disorder associated with the expression and / or abnormal activity of GPNMB ( for example, cancer) or one or more symptoms thereof, or to enhance or improve the prophylactic effect (s) of another therapy.

As used herein, a "protocol" includes dosing schedules and dosing guidelines. The protocols in this document are procedures for use and include prophylactic and therapeutic protocols.

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal that includes a non-primate (eg, a cow, pig, horse, cat, dog, rat and mouse) and a primate (for example, a monkey such as a macaque, chimpanzee and a human being), and more preferably a human being.

As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to an agent that can be used in the prevention, treatment, management or improvement of a disorder associated with the expression and / or abnormal activity of GPNMB ( for example, cancer) or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" refers to an antibody that immunospecifically binds to GPNMB. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody that binds immunospecifically to GPNMB.

As used herein, the terms "therapies" and "therapy" may refer to any protocol, procedure and / or agent that may be used in the prevention, treatment, management or improvement of a disorder associated with expression and / or activity. abnormal GPNMB (for example, cancer) or one or more symptoms thereof. In certain embodiments, the terms "therapies" and "therapy" refer to anticancer therapy, biological therapy, supportive therapy and / or other therapies useful in the treatment, management, prevention or improvement of cancer or one or more known symptoms thereof. by an expert in the field such as medical staff.

As used herein, the terms "treat", "treatment" and "treat" refer to the eradication, removal, modification or control of primary, regional or metastatic cancerous tissue, or to the reduction or improvement of progression, severity and / or duration of a disorder associated with the expression and / or abnormal activity of GPNMB, or improvement of one or more symptoms thereof resulting from the administration of one or more therapies. In certain embodiments, such terms in the context of cancer refer to a reduction in the growth of cancer cells, a decrease in the number of cancer cells and / or a reduction in the growth, formation and / or volume of a tumor. In other embodiments, such terms refer to the minimization or delay of the spread of cancer resulting from the administration of one or more therapies to a subject with such a disease. Treatment may include, for example, a decrease in the severity of a symptom, the number of symptoms or the frequency of relapse.

Unless otherwise defined, the scientific and technical terms used for the purpose of the invention described herein must have the meanings that are commonly understood by those skilled in the art. In addition, unless otherwise required by the context, the singular terms must include pluralities and the plural terms must include the singular. Generally, the nomenclatures used for, and techniques of, cell and tissue culture, molecular and chemical biology of proteins and oligo or polynucleotides and hybridization described herein are those well known and commonly used in the art. Conventional techniques are used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (for example, electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications or as commonly performed in the art or as described herein. The above techniques and procedures are generally performed according to conventional procedures well known in the art and as described in various general and more specific references cited and discussed throughout this specification (see, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989). The nomenclatures used with respect to, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry and medical and pharmaceutical chemistry described herein are well known and commonly used in the art. Conventional techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and administration and treatment of patients.

The present invention provides combinations of heavy chain V, D, J and light chain V, J combinations of human germline antibodies that include the nucleotide and amino acid sequence of the FR and CDR regions of the VH and VL domains with specificity by GPNMB.

image7

After exposure to the antigen, those B cells with antigen binding specificity based on germline sequences are activated, proliferated and differentiated to produce immunoglobulins of different isotypes, in addition to experiencing somatic mutation and / or affinity maturation to produce immunoglobulins of greater affinity for the antigen. The present invention provides the nucleotide and amino acid sequence of such affinity-matured FR and CDR regions of the V domain that have GPNMB specificity.

Fab type antibody fragments containing the antigen-binding portion of the antibody molecule may be constituted by the L chain covalently linked by a disulfide bond to a portion of the H chain that has the V domain and the first constant domain . The single chain Fv antibody (scFv) fragment has the variable domain H linked to the variable domain L by a polypeptide linker. The invention provides antibody fragments such as Fab and scFv molecules that have sequences derived from germline V domains or affinity matured antibodies that specifically bind GPNMB.

A bispecific or bifunctional antibody is an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of procedures including hybridoma fusion or Fab 'fragment binding (see, for example, Songsivilai & Lachmann, 1990 Clin. Exp. Immunol. 79: 315-321; Kostelny et al., 1992 J. Immunol. 148: 1547-1553). Bispecific antibodies do not exist in the form of fragments that have a single binding site (for example, Fab, Fab 'and Fv).

It will be appreciated that such bifunctional or bispecific antibodies are contemplated and encompassed by the invention. A bispecific single chain antibody with specificity for GPNMB and for the CD3 antigen in cytotoxic T lymphocytes can be used to direct these T cells towards tumor cells expressing GPNMB and produce apoptosis and tumor eradication. The bispecific scFv constructs for this purpose are described in this document. The scFv components specific to GPNMB may be derived from antibodies directed against GPNMB described herein. In some embodiments, the components of the antibody directed against GPNMB disclosed herein can be used to generate a biologically active scFv directed against GPNMB. The scFv component directed against CD3 of the bispecific therapeutic scFv was derived from a sequence deposited in Genbank (registration number CAE85148). Alternative antibodies known to choose as a CD3 target or other T cell antigens can be similarly effective in the treatment of malignant tumors when coupled with antibodies directed against GPNMB, both in a single stranded skeleton and a complete IgG.

Human GPNMB binding antibodies may include constant H or L domains that include kappa or lambda L constant regions, or any constant isotype H domain. A human antibody with GPNMB binding specificity may contain germline sequences such as heavy chain V regions: VH1-2 (SEQ ID NO: 308), VH2-5 (SEQ ID NO: 360), VH3-11 (SEQ ID NO: 361), VH3-21 (SEQ ID NO: 362), VH3-30 (SEQ ID NO: 363), VH3-33 (SEQ ID NO: 364), VH4-31 (SEQ ID NO: 365), VH4-59 (SEQ ID NO: 366) or VH5-51 (SEQ ID NO: 367); heavy chain D region: D1-20 (amino acid sequences translated by SEQ ID NO: 375), D1-26 (amino acid sequences translated by SEQ ID NO: 376), D3-10 (amino acid sequences translated by SEQ ID NO: 377), D3-16 (amino acid sequences translated by SEQ ID NO: 378), D3-22 (amino acid sequences translated by SEQ ID NO: 379), D3-9 (amino acid sequences translated by SEQ ID NO: 380), D4-17 (amino acid sequences translated by SEQ ID NO: 381), D5-24 (amino acid sequences translated by SEQ ID NO: 382), D613 (amino acid sequences translated by SEQ ID NO: 383) or D6 -19 (amino acid sequences translated by SEQ ID NO: 384); heavy chain region J: JH3b (SEQ ID NO: 385), JH4b (SEQ ID NO: 386), JH5b (SEQ ID NO: 387) or JH6b (SEQ ID NO: 388); Kappa A2 light chain regions V (SEQ ID NO: 373), A3 (SEQ ID NO: 371), A20 (SEQ ID NO: 370), A27 (SEQ ID NO: 369), A30 (SEQ ID NO: 374 ), L2 (SEQ ID NO: 372) or O1 (SEQ ID NO: 368); and region J JK1 (SEQ ID NO: 389), JK2 (SEQ ID NO: 390), JK3 (SEQ ID NO: 391), JK4 (SEQ ID NO: 392) or JK5 (SEQ ID NO: 393). (Generally, see Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. 1987 and 1991; see also Chothia & Lesk 1987 J. Mol. Biol. 196: 901-917; Chothia et al. 1989 Nature 342 : 878-883). Human antibodies with GPNMB binding specificity can be combined with germ line regions as shown in Table 1.

image8

TABLE 1: Combinations of germline regions of antibodies directed against human GPNMB.

Ab

Vh D JH VL JL

1.10.2

 VH4-59 D6-19 JH4b A3 JK5

1.15.1

 VH4-31 D-20 JH4b L2 JK1

1.2.2

 VH2-5 D3-16 JH4b O1 JK5

1.7.1

 VH4-31 D1-20 JH4b L2 JK1

2.10.2

 VH3-30 D3-10 JH6b A3 JK5

2.15.1

 VH3-33 D4-17 JH4b TO 20 JK4

2.16.1

 VH3-11 D6-13 JH3b L2 JK3

2.17.1

 VH1-2 D6-19 JH5b A2 JK4

2.21.2

 VH3-21 D1-26 JH4b TO 20 JK5

2.22.1

 VH4-31 D3-22 JH6b A30 JK1

2.24.1

 VH5-51 D5-24 JH4b A27 JK1

2.3.1

 VH1-2 D3-10 JH4b A2 JK4

2.7.1

VH3-33 D3-10 JH4b TO 20 JK4

2.8.1

 VH2-5 D3-9 JH4b O1 JK4

The isolated antibody may have a heavy chain variable region polypeptide comprising a

5 amino acid sequence selected from the group consisting of SEQ ID NO: 2, 20, 38, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 253, 256, 260, 265 , 270, 274, 277, 281 and 285. Such amino acid sequences may be encoded by the nucleotide sequences selected from the group consisting of SEQ ID NO: 1, 19, 37, 55, 73, 91, 109, 127, 145, 163, 181, 199, 217 and 235. An isolated antibody that specifically binds GPNMB and has a light chain variable region polypeptide comprising a sequence is also disclosed.

10 amino acids selected from the group consisting of SEQ ID NO: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227 and 245. Such amino acid sequences may be encoded by nucleotide sequences selected from the group consisting of SEQ ID NO: 10, 28, 46, 64, 82, 100, 118, 136, 154, 172, 190, 208, 226 and 244. An isolated antibody is also disclosed that specifically binds GPNMB and has a heavy chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID

15 NO: 2, 20, 38, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 253, 256, 260, 265, 270, 274, 277, 281 and 285 and have a light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227 and 245. Antibodies directed against GPNMB may comprise at least one CDR of any of the CDR H or L SEQ ID NO polypeptide sequences: 4, 6, 8, 13, 15, 17, 22, 24, 26, 31, 33, 35 , 40, 42, 44, 49, 51, 53, 58, 60, 62, 67, 69, 71, 76, 78, 80, 85, 87,

20 89, 94, 96, 98, 103, 105, 107, 112, 114, 116, 121, 123, 125, 130, 132, 134, 139, 141, 143, 148, 150, 152, 157, 159, 161 , 166, 168, 170, 175, 177, 179, 184, 186, 188, 193, 195, 197, 202, 204, 206, 211, 213, 215, 220, 222, 224, 229, 231, 233, 238 , 240, 242, 247, 249, 251, 254, 257, 261, 266, 271, 278, 282, 286, 255, 258, 262, 267, 272, 275, 279, 283, 287, 259, 263, 264 , 268, 269, 273, 276, 280, 284 and 288.

In a particular embodiment, the antibody directed against human GPNMB is Mab1.15.1. This antibody and 25 other antibodies directed against GPNMB described herein have amino acid sequences and nucleic acid sequences encoding them identified in this application as shown in Tables 2A-2D.

image9

TABLE 2A Nucleotide (DNA) and amino acid (AA) antibody sequences TABLE 2B: Nucleotide (DNA) and amino acid (AA) antibody sequences TABLE 2C: Nucleotide (DNA) and amino acid (AA) antibody sequences 2D TABLE: Nucleotide (DNA) and amino acid (AA) antibody sequences

Gene segment

1.10.2  1.15.1 1.2.2 1.7.1

H variable DNA

SEQ ID NO: 1 SEQ ID NO: 19 SEQ ID NO: 37 SEQ ID NO: 55

AA variable H

SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: 38 SEQ ID NO: 56

H FR1

SEQ ID NO: 3 SEQ ID NO: 21 SEQ ID NO: 39 SEQ ID NO: 57

H CDR1

SEQ ID NO: 4 SEQ ID NO: 22 SEQ ID NO: 40 SEQ ID NO: 58

H FR2

SEQ ID NO: 5 SEQ ID NO: 23 SEQ ID NO: 41 SEQ ID NO: 59

H CDR2

SEQ ID NO: 6 SEQ ID NO: 24 SEQ ID NO: 42 SEQ ID NO: 60

H FR3

SEQ ID NO: 7 SEQ ID NO: 25 SEQ ID NO: 43 SEQ ID NO: 61

H CDR3

SEQ ID NO: 8 SEQ ID NO: 26 SEQ ID NO: 44 SEQ ID NO: 62

H FR4

SEQ ID NO: 9 SEQ ID NO: 27 SEQ ID NO: 45 SEQ ID NO: 63

Variable DNA L

SEQ ID NO: 10 SEQ ID NO: 28 SEQ ID NO: 46 SEQ ID NO: 64

AA variable L

SEQ ID NO: 11 SEQ ID NO: 29 SEQ ID NO: 47 SEQ ID NO: 65

L FR1

SEQ ID NO: 12 SEQ ID NO: 30 SEQ ID NO: 48 SEQ ID NO: 66

L CDR1

SEQ ID NO: 13 SEQ ID NO: 31 SEQ ID NO: 49 SEQ ID NO: 67

L FR2

SEQ ID NO: 14 SEQ ID NO: 32 SEQ ID NO: 50 SEQ ID NO: 68

L CDR2

SEQ ID NO: 15 SEQ ID NO: 33 SEQ ID NO: 51 SEQ ID NO: 69

L FR3

SEQ ID NO: 16 SEQ ID NO: 34 SEQ ID NO: 52 SEQ ID NO: 70

L CDR3

SEQ ID NO: 17 SEQ ID NO: 35 SEQ ID NO: 53 SEQ ID NO: 71

L FR4

SEQ ID NO: 18 SEQ ID NO: 36 SEQ ID NO: 54 SEQ ID NO: 72

image10

Gene segment

2.10.2  2.15.1 2.16.1 2.17.1

H variable DNA

SEQ ID NO: 73 SEQ ID NO: 91 SEQ ID NO: 109 SEQ ID NO: 127

AA variable H

SEQ ID NO: 74 SEQ ID NO: 92 SEQ ID NO: 110 SEQ ID NO: 128

H FR1

SEQ ID NO: 75 SEQ ID NO: 93 SEQ ID NO: 111 SEQ ID NO: 129

H CDR1

SEQ ID NO: 76 SEQ ID NO: 94 SEQ ID NO: 112 SEQ ID NO: 130

H FR2

SEQ ID NO: 77 SEQ ID NO: 95 SEQ ID NO: 113 SEQ ID NO: 131

H CDR2

SEQ ID NO: 78 SEQ ID NO: 96 SEQ ID NO: 114 SEQ ID NO: 132

H FR3

SEQ ID NO: 79 SEQ ID NO: 97 SEQ ID NO: 115 SEQ ID NO: 133

H CDR3

SEQ ID NO: 80 SEQ ID NO: 98 SEQ ID NO: 116 SEQ ID NO: 134

H FR4

SEQ ID NO: 81 SEQ ID NO: 99 SEQ ID NO: 117 SEQ ID NO: 135

Variable DNA L

SEQ ID NO: 82 SEQ ID NO: 100 SEQ ID NO: 118 SEQ ID NO: 136

AA variable L

SEQ ID NO: 83 SEQ ID NO: 101 SEQ ID NO: 119 SEQ ID NO: 137

L FR1

SEQ ID NO: 84 SEQ ID NO: 102 SEQ ID NO: 120 SEQ ID NO: 138

L CDR1

SEQ ID NO: 85 SEQ ID NO: 103 SEQ ID NO: 121 SEQ ID NO: 139

L FR2

SEQ ID NO: 86 SEQ ID NO: 104 SEQ ID NO: 122 SEQ ID NO: 140

L CDR2

SEQ ID NO: 87 SEQ ID NO: 105 SEQ ID NO: 123 SEQ ID NO: 141

L FR3

SEQ ID NO: 88 SEQ ID NO: 106 SEQ ID NO: 124 SEQ ID NO: 142

L CDR3

SEQ ID NO: 89 SEQ ID NO: 107 SEQ ID NO: 125 SEQ ID NO: 143

L FR4

SEQ ID NO: 90 SEQ ID NO: 108 SEQ ID NO: 126 SEQ ID NO: 144

image11

Gene segment

2.21.2  2.22.1 2.24.1 2.3.1

H variable DNA

SEQ ID NO: 145 SEQ ID NO: 163 SEQ ID NO: 181 SEQ ID NO: 199

AA variable H

SEQ ID NO: 146 SEQ ID NO: 164 SEQ ID NO: 182 SEQ ID NO: 200

H FR1

SEQ ID NO: 147 SEQ ID NO: 165 SEQ ID NO: 183 SEQ ID NO: 201

H CDR1

SEQ ID NO: 148 SEQ ID NO: 166 SEQ ID NO: 184 SEQ ID NO: 202

H FR2

SEQ ID NO: 149 SEQ ID NO: 167 SEQ ID NO: 185 SEQ ID NO: 203

H CDR2

SEQ ID NO: 150 SEQ ID NO: 168 SEQ ID NO: 186 SEQ ID NO: 204

H FR3

SEQ ID NO: 151 SEQ ID NO: 169 SEQ ID NO: 187 SEQ ID NO: 205

H CDR3

SEQ ID NO: 152 SEQ ID NO: 170 SEQ ID NO: 188 SEQ ID NO: 206

H FR4

SEQ ID NO: 153 SEQ ID NO: 171 SEQ ID NO: 189 SEQ ID NO: 207

Variable DNA L

SEQ ID NO: 154 SEQ ID NO: 172 SEQ ID NO: 190 SEQ ID NO: 208

AA variable L

SEQ ID NO: 155 SEQ ID NO: 173 SEQ ID NO: 191 SEQ ID NO: 209

L FR1

SEQ ID NO: 156 SEQ ID NO: 174 SEQ ID NO: 192 SEQ ID NO: 210

L CDR1

SEQ ID NO: 157 SEQ ID NO: 175 SEQ ID NO: 193 SEQ ID NO: 211

L FR2

SEQ ID NO: 158 SEQ ID NO: 176 SEQ ID NO: 194 SEQ ID NO: 212

L CDR2

SEQ ID NO: 159 SEQ ID NO: 177 SEQ ID NO: 195 SEQ ID NO: 213

L FR3

SEQ ID NO: 160 SEQ ID NO: 178 SEQ ID NO: 196 SEQ ID NO: 214

L CDR3

SEQ ID NO: 161 SEQ ID NO: 179 SEQ ID NO: 197 SEQ ID NO: 215

L FR4

SEQ ID NO: 162 SEQ ID NO: 180 SEQ ID NO: 198 SEQ ID NO: 216

image12

Gene segment

2.7.1 2.8.1

H variable DNA

SEQ ID NO: 217 SEQ ID NO: 235

AA variable H

SEQ ID NO: 218 SEQ ID NO: 236

H FR1

SEQ ID NO: 219 SEQ ID NO: 237

H CDR1

SEQ ID NO: 220 SEQ ID NO: 238

H FR2

SEQ ID N0: 221 SEQ ID NO: 239

H CDR2

SEQ ID NO: 222 SEQ ID NO: 240

H FR3

SEQ ID NO: 223 SEQ ID NO: 241

H CDR3

SEQ ID NO: 224 SEQ ID NO: 242

H FR4

SEQ ID NO: 225 SEQ ID NO: 243

Variable DNA L

SEQ ID NO: 226 SEQ ID NO: 244

AA variable L

SEQ ID NO: 227 SEQ ID NO: 245

L FR1

SEQ ID NO: 228 SEQ ID NO: 246

L CDR1

SEQ ID NO: 229 SEQ ID NO: 247

L FR2

SEQ ID NO: 230 SEQ ID NO: 248

L CDR2

SEQ ID NO: 231 SEQ ID NO: 249

L FR3

SEQ ID NO: 232 SEQ ID NO: 250

L CDR3

SEQ ID NO: 233 SEQ ID NO: 251

L FR4

SEQ ID NO: 234 SEQ ID NO: 252

Antibodies directed against GPNMB derived from VH4-31: In a particular embodiment, the human GPNMB binding antibodies of the invention comprise the VH4-31 germline heavy chain region V or are derived therefrom and have a sequence of amino acids of formula: X1SGPGLVKPSQX2LSLTCTVSGGSISSX3X4YX5WX6WJRX7HPGKGLEWIGYIYYSGX8TYX9NPSLKS RVX10ISVDTSKNQFSLX11LSSVTAADTAVYYCAR In which: X1 is E or Q;

10 X2 is T or N; X3 is A, F or G; X4 is N or G; X5 is Y or F; X6 is T or S;

15 X7 is Q or H; X8 is S or N; X9 is C, S or Y;

image13

X10 is I or T; X11 is K or T; (SEQ ID NO: 253). SEQ ID NO: 253 can be combined with D3-22 or D1-20. In addition the combination of SEQ ID NO: 253 with D3

22 or D1-20 is combined with JH6b or JH4b and in specific embodiments, after affinity maturation, these human GPNMB binding antibodies, for example Mab1.15.1, Mab1.7.1 and Mab2.22.1, have SEQ amino acid sequences ID NO: 20, 56 and 164 and can be encoded by the nucleotide sequences of SEQ ID NO: 19, 55 and 163.

In addition, in particular embodiments, the H-chain CDR1 sequences are the VH4-31 CDR of the germ line or affinity-matched sequences of the formula: CDR1: GGSIS SX3X4YX5WX6 Where: X3 is A, F or G ; X4 is N or G; X5 is Y or F; X6 is T or S; (SEQ ID NO: 254). An antibody directed against GPNMB may comprise a CDR1 sequence selected from the following: SEQ ID NO: 22, 58, 166. In particular embodiments, the CDR2 sequences of the H chain are the VR4-31 CDR of the germ line or affinity matured sequences thereof of formula: CDR2: YIYYSGX8TYX9NPSLKS Where: X8 is S or N; X9 is C, S or Y; (SEQ ID NO: 255). An antibody directed against GPNMB may comprise a CDR2 sequence selected from the following: SEQ ID NO: 24, 60 and 168.

The CDR3 sequence of the H chain may be a combination of D3-22, JH6b having SEQ ID NO: 170. Alternatively, the CDR3 sequence of the H chain may be a combination of D1-20, JH4b having SEQ ID NO: 26 or 62.

Antibodies directed against GPNMB derived from VH1-2:

Human GPNMB-binding antibodies may comprise a heavy chain region V of the VH-2 germ line or are derived therefrom and include an amino acid sequence of the formula: QLVQSGAEVKKPGASVKVSCKA GYTFT GX1YMH WVRQX2PGQGLEWMG W1NPNSXGTQ3DQXXGTQ3DQXXGTQ3DVXXG4QKDKDXD05XD05GXD

TSISTX6YMELSRLRSDDTAVYYCAR

In which: X1 is Y or F;

X2 is A or T;

X3 is N or Y;

X4 is A or V;

X5 is D or G;

X6 is A or V;

(SEQ ID NO: 256).

SEQ ID NO: 256 can be combined with D3-10 or D6-19.

In addition, the combination of SEQ ID NO: 256 with D3-10 or D6-19 is combined with JH4b or JH5b and after affinity maturation, these human GPNMB binding antibodies, for example mAb 2.3.1 and mAb 2.17. 1, have amino acid sequences: SEQ ID NO: 128 and 200 and can be encoded by the nucleotide sequences of SEQ ID NO: 127 and 199.

image14

In addition, the H-chain CDR1 sequences may be the germline VH1-2 CDR or affinity-matched sequences of the same formula:

CDR1: GYTPTGX1YMH Where: X1 is Y or F, (SEQ ID NO: 257). An antibody directed against GPNMB may comprise a CDR1 sequence selected from SEQ ID

NO: 130 and 202.

In the H-chain CDR2 sequences it can be the VH1-2 CDR of the germ line or affinity-matched sequences of the same formula:

 CDR2: WiNPNSGGTX3YX4QKFQX5 Where: X3 is N or Y; X4 is A or V; X5 is D or G (SEQ ID NO: 258). An antibody directed against GPNMB may comprise a CDR2 sequence selected from SEQ ID

NO: 132 and 204.

The CDR3 sequences of the H chain are combinations of D3-10, JH4b of the germ line or affinity-matched sequences thereof having the amino acid sequence of the formula:

 CDR3: X1X2X3GSGSX4X5 Where: X1 is Y or D; X2 is Y or F; X3 is Y or F; X4 is Y or L; X5 is Y or L (SEQ ID NO: 259). An antibody directed against GPNMB may comprise a CDR3 sequence selected from SEQ ID

NO: 134 and 206.

Antibodies directed against GPNMB derived from VH2-5: Human GPNMB binding antibodies may comprise a VH2-5 germline heavy chain region V or are derived therefrom and include an amino acid sequence of the formula: ITLKESGPTLVX1PTQTLTLTCTFS GFSLSX1X3GX4GVG WIRQPDDXX5KDYXDKLX -SPSLX8SRLTITK

DTSKNQVVLX9X10TNMDPVDTATYYCAH

In which: X1 is K or T;

X2 is T or A;

X3 is S or G;

X4 is M or V;

X5 is D or E;

X6 is A or T;

X7 is R or H;

X8 is K or R;

image15

X9 is T or R; X10 is M or 1; (SEQ ID NO: 260). SEQ ID NO: 260 can be combined with D3-9 or D3-16 and can also be combined with JH4b. After

Affinity maturation, these GPNMB-binding human antibodies, for example, mAb 2.8.1 and mAb 1.2.2, have amino acid sequences SEQ ID NO: 38 and 236 and can be encoded by the nucleotide sequences of SEQ ID NO : 37 and 235.

In addition, the H-chain CDR1 sequences may be the VH2-5 CDR of the germ line or affinity-matched sequences of the same formula: CDR1: GFSLS X2X3GX4GVG Where: X2 is T or A; X3 is S or G; X4 is M or V; (SEQ ID NO: 261). An antibody directed against GPNMB may comprise a CDR1 sequence selected from SEQ ID NO: 40 and 238. The CDR2 sequences of the H chain may be the VR2-5 germline CDR2 or affinity matured sequences thereof. formula: CDR2: LIYWNODKX7YSPSLX8S Where: X7 is R or H; X8 is K or R; (SEQ ID NO: 262). An antibody directed against GPNMB may comprise a CDR2 sequence selected from SEQ ID NO: 42 and 240. The CDR3 sequences of the H chain may be combinations of D3-9, JH4b of the germ line or affinity matured sequences thereof. and include an amino acid sequence of the formula: CDR3: X1YDILTGX2X3 Where: X1 is Y or H; X2 is Y or F; and X3 is Y or N (SEQ ID NO: 263). An antibody directed against GPNMB comprises an amino acid CDR3 sequence SEQ ID NO: 242. The CDR3 sequences of the H chain may be combinations of D3-16, JH4b of the germ line or affinity matured sequences thereof and include an amino acid sequence of the formula: CDR3: YDYX1WGS Where: X1 is V or D (SEQ ID NO: 264). An antibody directed against GPNMB comprises an amino acid CDR3 sequence SEQ ID NO: 44. Antibodies directed against GPNMB derived from VH3-33: Human GPNMB binding antibodies can comprise a heavy chain region V of the germline VH3-33 or are derived therefrom and have an amino acid sequence of the formula: QVQLX1X2SGGGVVQPGRSLRLSCAASGFTFX3X4YGX5H WVRQAPGKGLEWVA YIWX6DGX7N-KYY ADSYKG RFTISRDNSKNTLILQMNSLRAEDXBA VYXA, XBAV1, EVX, VVXX,

image16

X2 is E or Q; X3 is S or N; X4 is S or N; X5 is M or 1; X6 is Y or F; X7 is S or R; X8 is T or A; X9 is R or K (SEQ ID NO: 265). SEQ ID NO: 265 can be combined with D3-10 or D4-17 and also with JH4b. After maturation by

affinity, these GPNMB binding human antibodies, for example mAb 2.7.1 and mAb 2.15.1, have amino acid sequences: SEQ ID NO: 92 and 218 and can be encoded by the nucleotide sequences of SEQ ID NO: 91 and 217. In addition, the H-chain CDR1 sequences may be the germline VH3-33 CDR or affinity-matched sequences thereof of the formula: CDR1: GFTFX3X4YGX5H

In which: X3 is S or N; X4 is S or N; X5 is M or I; (SEQ ID NO: 266). An antibody directed against GPNMB can comprise an amino acid CDR1 sequence

selected from SEQ ID NO: 94 and 220.

The CDR2 sequences of the H chain may be the CDR2 of VH3-33 of the germ line or affinity matured sequences thereof of the formula:

 CDR2: VIWX6DOX7NKYYADSVKG Where: X6 is Y or F; X7 is S or R; (SEQ ID NO: 267). An antibody directed against GPNMB may comprise a CDR2 sequence selected from SEQ ID

NO: 96 and 222.

The CDR3 sequences of the H chain may be combinations of D3-10, JH4b or affinity matured sequences thereof and include an amino acid sequence of the formula:

 CDR3: YYYGSGX1 In which: X1 is S or L (SEQ ID NO: 268). The antibody directed against GPNMB 2.7.1 disclosed herein has a CDR3 sequence of

amino acids SEQ ID NO: 224.

The CDR3 sequences of the H chain can be combinations of D4-17, JH4b or affinity matured sequences thereof and include an amino acid sequence of the formula:

 CDR3: DYGDX1 In which: X is Y or S (SEQ ID NO: 269). The antibody directed against GPNMB 2.15.1 disclosed herein has a CDR3 sequence of

image17

amino acids SEQ ID NO: 98. Antibodies directed against GPNMB derived from VH3-11: Human GPNMB binding antibodies may comprise a heavy chain region V of the VH3-11 germ line or are derived therefrom and have a sequence of formula amino acids: QVQLVESGGGLVKPGGSLRLSCAAS GFTFS X1YX2MX3 WIRQAPGKGLEWVS YISX4SGSX5X6-X7YADSVKG

RFTX8SRADNKNSLILQMNSLRAEDTAVYYCAR

Where: X1 is D or S;

X2 is S or Y;

X3 is S or T;

X4 is S or I;

X5 is T or I;

X6 is T or I;

X7 is Y or H;

X8 is I or M;

(SEQ ID NO: 270).

SEQ ID NO: 270 can be combined with D6-13 and also with JH3b. After affinity maturation,

these human GPNMB binding antibodies, for example mAb 2.16.1, have an amino acid sequence SEQ ID NO: 110 and can be encoded by the nucleotide sequence of SEQ ID NO: 109. In addition, the H-chain CDR1 sequences may be the VH3-11 CDR1 of the germ line or affinity-matched sequences thereof of the formula: CDR1: GFTFS X1YX2MX3 Where: X1 is D or S; X2 is S or Y; X3 is S or T; (SEQ ID NO: 271). The antibody directed against GPNMB can comprise an amino acid CDR1 sequence SEQ ID NO: 112. The CDR2 sequences of the H chain may be the CDR2 of VH3-11 of the germ line or affinity-matched sequences of the same formula: CDR2: YISX4SGSX5X6X7YADSVIKG Where: X4 is S or I; X5 is T or I; X6 is T or I; X7 is Y or H; (SEQ ID NO: 272). An antibody directed against GPNMB may comprise a CDR2 sequence SEQ ID NO: 114. The CDR3 sequences of the H chain may be combinations of D6-13, JH3b or affinity matured sequences thereof and include an amino acid sequence of the formula: CDR3: X1X2AAAG--AFDI In which: X1 is G or D; X2 is I or G; (SEQ ID NO: 273).

image18

The antibody directed against GPNMB 2.16.1 disclosed herein has an amino acid CDR3 sequence SEQ ID NO: 116. Antibodies directed against GPNMB derived from VH3-21: Human GPNMB binding antibodies may comprise a VH3-21 germline heavy chain region V or are derived therefrom and have an amino acid sequence of formula: X1VQLX2X3SGGGLVKPGGSLRX4 SCAAS GFTFS SYSMN WVRQAPGKGLEWVS X5ISS SSSYI-YYADSVKG RFTISRADNKNSLILQMNSLRAEDTAVYYCAR Where: X1 is E or Q; X2 is V or E; X3 is E or Q; X4 is F or L; X5 is S or F; (SEQ ID NO: 274).

SEQ ID NO: 274 can be combined with D1-26 and also with JH4b. After affinity maturation, these GPNMB binding human antibodies, for example mAb 2.21.1, can have an amino acid sequence SEQ ID NO: 146 and can be encoded by the nucleotide sequence of SEQ ID NO: 145.

In addition, the CDR1 sequences of the H chain can be the CDR1 of VH3-21 of the germ line, SEQ ID NO: 148 or affinity matured sequences thereof. The CDR2 sequences of the H chain can be the CDR2 of VH3-21 of the germ line or affinity matured sequences of the same formula: CDR2: X5ISS SSSYIYYADSVKG Where: X5 is S or F; (SEQ ID NO: 275). An antibody directed against GPNMB may comprise an amino acid CDR2 sequence SEQ ID NO: 150. The CDR3 sequences of the H chain can be combinations of D1-26, JH4b or affinity matured sequences thereof and include an amino acid sequence of the formula: CDR3: X1X2VGAT-FDY Where: X1 is G or D; X2 is 1 or W; (SEQ ID NO: 276). The antibody directed against GPNMB 2.21.1 disclosed herein has an amino acid CDR3 sequence SEQ ID NO: 152. Antibodies directed against GPNMB derived from VH3-30: Human GPNMB binding antibodies may comprise a VH3-30 germline heavy chain region V or are derived therefrom and include an amino acid sequence of formula: QLVESGGGVVQPGRSLRLSCAAS GFX1FS SYGMH WVRQAPGKGLEWVA VISYDGX2NKYYAD-SVKG RFTISRDNSKNTLILQMNSLRAEDTAVYYCAK Where: X1 is T or A; X2 is S or N; (SEQ ID NO: 277).

SEQ ID NO: 277 can be combined with D3-10 and also with JH6b. After affinity maturation, these GPNMB-binding human antibodies, for example mAb 2.10.2, can have an amino acid sequence SEQ ID NO: 74 and can be encoded by the nucleotide sequence of SEQ ID NO: 73.

In addition, the CDR1 sequences of the H chain may be the CDR1 of VH3-30 of the germ line or

image19

affinity matured sequences thereof having an amino acid sequence of formula:

 GFX1FS SYGMH

In which: X1 is T or A;

(SEQ ID NO: 278).

An antibody directed against GPNMB may comprise a CDR1 sequence SEQ ID NO: 76.

The CDR2 sequences of the H chain may be the CDR2 of VH3-30 of the germ line or sequences

matured by affinity of the same formula:

 CDR2: VISYDGX2NKYYADSVKG

In which: X2 is S or N;

(SEQ ID NO: 279).

An antibody directed against GPNMB may comprise a CDR2 sequence of amino acids SEQ ID

NO: 78.

The CDR3 sequence of the H chain may be combinations of D3-10, JH6b or affinity matured sequences thereof and include an amino acid sequence of the formula:

 CDR3: X1X2X3VRGX4X5X6 Where: X1 is I or D; X2 is T or L; X3 is M or V; X4 is V or I; X5 is I or R; X6 is I or G; (SEQ ID NO: 280). The antibody directed against GPNMB 2.10.2 disclosed herein has a CDR3 sequence of

amino acids SEQ ID NO: 80. Antibodies directed against GPNMB derived from VH4-59: Human GPNMB binding antibodies may comprise a VH4-59 germline heavy chain region V or are derived therefrom and include an amino acid sequence of the formula: QVQLQESOPGLVKPSETLSLTCTVS GX1SIS X2YYWSWIRQPPGKGLEWIGQPPGKGLE YX3YYSGSTNYN

PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

Where: X1 is G or D;

X2 is S or N;

X3 is I or F;

(SEQ I D NO: 281).

SEQ ID NO: 281 can be combined with D6-19 and also with JH4b. After affinity maturation,

these human GPNMB binding antibodies, for example mAb 1.10.24, can have an amino acid sequence SEQ ID NO: 2 and can be encoded by the nucleotide sequence of SEQ ID NO: 1. In addition, the H-chain CDR1 sequences may be the VH4-59 CDR1 of the germ line or affinity-matched sequences having an amino acid sequence of the formula: GX1SIS X2YYWS Where: X1 is G or D ; X2 is S or N; (SEQ ID NO: 282). An antibody directed against GPNMB may comprise a CDR1 sequence SEQ ID NO: 4.

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The CDR2 sequences of the H chain may be the CDR2 of VH4-59 of the germ line or affinity matured sequences of the same formula: CDR2: YX3YYSGSTNYNPSLKS Where: X3 is I or F; (SEQ ID NO: 283). An antibody directed against GPNMB may comprise an amino acid CDR2 sequence SEQ ID NO: 6. The CDR3 sequences of the H chain may be combinations of D6-19, JH4b or affinity matured sequences thereof and include an amino acid sequence of the formula: CDR3: X1X2 GW --- DY Where: X1 is S or D; X2 is S or R; (SEQ ID NO: 284). The antibody directed against GPNMB 1.10.2 described herein has an amino acid CDR3 sequence SEQ ID NO: 8. Antibodies directed against GPNMB derived from VH5-51: Human GPNMB binding antibodies may comprise a VH-51 germline heavy chain region V or are derived therefrom and include an amino acid sequence of formula: QLVQSGAEVKKPGESLKISCX1GS GYX2FTX3YWIG WVRQMPG X4IYPX5DSDTRYS-PSFQG QVTISADKSISTAILQWSSLKASDTAX6YYCAR Where: X1 is K or Q; X2 is S or I; X3 is S or N; X4 is I or V; X5 is G or D; X6 is M or I; (SEQ ID NO: 285).

SEQ ID NO: 285 can be combined with D5-24 and also with JH4b. After affinity maturation, these GPNMB-binding human antibodies, for example mAb 2.24.1, can have an amino acid sequence SEQ ID NO: 182 and can be encoded by the nucleotide sequence of SEQ ID NO: 181.

In addition, the H-chain CDR1 sequences may be the VH-51 CDR1 of the germ line or affinity-matched sequences having an amino acid sequence of the formula: GYX2FT X3YWIG Where: X2 is S or 1 ; X3 is S or N; (SEQ ID NO: 286). An antibody directed against GPNMB may comprise a CDR1 sequence SEQ ID NO: 184. The CDR2 sequences of the H chain may be the VR5-51 CDR2 of the germ line or affinity matured sequences of the same formula: CDR2: X4IYPX5DSDTRYSPSFQG Where: X4 is I or V; X5 is G or D; (SEQ ID NO: 287).

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An antibody directed against GPNMB may comprise an amino acid CDR2 sequence SEQ ID NO: 186.

The CDR3 sequences of the H chain may be combinations of D5-24, JH4b or affinity matured sequences thereof and include an amino acid sequence of the formula:

 CDR3: X1WLQX2 - FDY

In which: X1 is R or K;

X2 is L or H;

(SEQ ID NO: 288).

The antibody directed against GPNMB 2.24.1 disclosed herein has an amino acid CDR3 sequence SEQ ID NO: 188.

The antibodies of the invention bind to an epitope of CG56972 (SEQ ID NO: 289), preferably within the mature GPNMB sequence and more preferably within the extracellular domain (ECD) of GPNMB.

The antibodies of the invention bind GPNMB with an affinity of 10-6 to 10-11, preferably with an affinity of 10-7 or higher and even more preferably 10-8 or higher. In a preferred embodiment, the antibodies described herein bind GPNMB with very high affinities (Kd), for example, a human antibody that can bind GPNMB with a Kd less than, but is not limited to, 10-7, 10-8, 10-9, 10-10, 10-11, 10-12, 10-13 or 10-14 M, or any interval or value within it. The affinity and / or avidity measurements can be measured by KinExA® and / or BIACORE® as described in this document. In particular embodiments, the antibodies of the invention bind GPNMB with Kd ranging from 50 to 150 pM.

Epitope mapping and analysis of secondary and tertiary structures can be carried out to identify specific 3D structures assumed by the disclosed antibodies and their complexes with antigens (see, for example, Epitope Mapping Protocols, ed. Morris, Humana Press, 1996) . Such procedures include, but are not limited to, X-ray crystallography (Biochem. Exp. Biol., 11: 7-13, 1974) and computer modeling of virtual representations of currently disclosed antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

In addition, the specific part of the protein immunogen recognized by the antibody can be determined by testing the reactivity of the antibody with respect to parts of the protein, for example, one half of the N-terminus and the C-terminus. The resulting reactive fragment can then be further dissected, Smaller portions of the immunogen are tested consecutively with the antibody until the minimum reactive peptide is defined. Alternatively, the binding specificity, that is, the epitope, of antibodies directed against GPNMB of the invention can be determined by subjecting the GPNMB immunogen to SDS-PAGE both in the absence and in the presence of a reducing agent and analyzed by immunoblot. Epitope mapping can also be performed using SELDI. SELDI ProteinChip® (LumiCyte) matrices were used to define protein-protein interaction sites. The GPNMB protein antigen or fragments thereof can be specifically captured by covalently immobilized antibodies on the surface of the ProteinCHIP matrix. Bound antigens can be detected by a laser induced desorption procedure and analyzed directly to determine their mass.

The epitope recognized by the antibodies directed against GPNMB described herein can be determined by exposing the ProteinCHIP matrix to a random combinatorial library of 12-mer peptides expressed in filamentous phage (New England Biolabs). The phage bound to the antibody is eluted and then amplified and passed through additional binding and amplification cycles to enrich the whole in favor of the binding sequences. After three or four rounds, the individual binding clones are further tested for binding by phage ELISA assays performed on antibody coated wells and characterized by DNA sequencing specific to positive clones.

Derivatives

This disclosure also provides a method for obtaining an antibody specific for GPNMB. The CDRs in such antibodies are not limited to the specific sequences of the variable domains H and L identified in Table 1 and may include variants of these sequences that retain the ability to specifically bind GPNMB. An expert can derive such variants from the sequences listed in Table 1 using techniques well known in the art. For example, amino acid substitutions, deletions or additions can be made in the FR and / or in the CDR. While changes in FR are normally designed to improve the stability and immunogenicity of the antibody, changes in CDRs are normally designed to increase the affinity of the antibody for its target. FR variants also include naturally occurring immunoglobulin allotypes. Such changes that increase affinity can be determined empirically by routine techniques that involve altering the CDR and testing the affinity of the antibody for its target. For example, conservative amino acid substitutions can be made within any one of the disclosed CDRs. Various alterations can be made according to the procedures described in the art (Antibody Engineering, 2nd ed., Oxford University Press, ed. Borrebaeck, 1995). These include, but are not limited to, nucleotide sequences that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a "silent" change. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs (see Table 3). In addition, any native residue in the polypeptide can also be substituted with alanine (Acta Physiol. Scant. Suppl. 643: 55-67, 1998; Adv. Biophys. 35: 1-24, 1998).

image22

TABLE 3. Amino acid substitutions

Original AA Residue

Possible substitutions  Preferred replacement

Wing (A)

Val, Leu, Ile Val

Arg (R)

Lys, Gln, Asn Lys

Asn (N)

Gln Gln

Asp (D)

Glu Glu

Cys (C)

It will be the Be

Gln (Q)

Asn Asn

Gly (G)

Pro, wing To

His (H)

Asn, Gln, Lys, Arg Arg

Ile (I)

Leu, Val, Met, Ala, Phe, Norleucine Leu

Leu (L)

Norleucine, Ile, Val, Met, Ala, Phe Ile

Lys (K)

Arg, 1,4-diaminobutyric acid, Gln, Asn Arg

Met (M)

Leu, Phe, Ile Leu

Phe (F)

Leu, Val, Ile, Ala, Tyr Leu

Pro (P)

Gly wing Gly

To be

Thr, Ala, Cys Thr

Thr (T)

Be Be

Trp (W)

Tyr, Phe Tyr

Tyr (Y)

Trp, Phe, Thr, Being Phe

Val (V)

Ile, Met, Leu, Phe, Ala, Norleucine Leu

10

Antibody derivatives and analogs of the invention can be produced by various techniques well known in the art including recombinant and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany).

15 Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity by forming protein complexes, (4) alter binding affinities and (4) confer or modify other physicochemical or functional properties of such analogs. The analogs may include various muteins of a sequence other than the naturally occurring peptide sequence. For example, individual or multiple amino acid substitutions can be made (preferably

20 conservative amino acid substitutions) in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain (s) that form intermolecular contacts). A conservative amino acid substitution should not substantially change the structural characteristics of the original sequence (for example, a substitution amino acid should not tend to break a helix that occurs in the original sequence,

image23

or disturb other types of secondary structure that characterize the original sequence). Examples of secondary and tertiary polypeptide structures recognized in the art are described in the art (eg, Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)).

A method of preparing a variable domain H which is a variant amino acid sequence of a variable domain H of the invention comprises a step of adding, deleting, replacing or inserting one or more amino acids in the amino acid sequence of the currently disclosed variable domain H , optionally combining the variable domain H thus provided with one or more variable domains L, and testing the variable domain H

or combination of variable H / variable L or combinations for specific binding to GPNMB or and, optionally, testing the ability of such antigen binding domain to modulate GPNMB activity. The variable domain L may have an amino acid sequence that is identical or is substantially as set forth in Table 1.

An analogous procedure can be used in which one or more variant sequences of a variable domain L disclosed herein are combined with one or more variable domains H.

Another aspect of the disclosure provides a method of preparing antigen-binding fragment that specifically binds GPNMB. The method comprises: (a) providing a starting repertoire of nucleic acids encoding a variable domain H that includes either a CDR3 to be substituted or lacks a coding region of CDR3; (b) combining the repertoire with a donor nucleic acid encoding an amino acid sequence as set forth substantially herein for a variable CDR3 H so that the donor nucleic acid is inserted into the CDR3 region in the repertoire so that it is provided a repertoire of nucleic acid products encoding a variable domain H; (c) express the nucleic acids of the product repertoire; (d) select a specific binding fragment for GPNMB; and (e) recovering the specific binding fragment or nucleic acid that encodes it.

Again, an analogous method can be used in which a variable CDR3 L of the invention is combined with a nucleic acid repertoire encoding a variable domain L, which includes either a CDR3 to be substituted or lacks a coding region of CDR3. The donor nucleic acid may be selected from nucleic acids encoding an amino acid sequence as set forth substantially in SEQ ID NO: 2, 20, 38, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 253, 256, 260, 265, 270, 274, 277, 281, 285, 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227 and 245. A sequence encoding a CDR of the invention (e.g., CDR3) can be introduced into a repertoire of variable domains that lack the respective CDR (e.g., CDR3) using recombinant DNA technology, for example, using the methodology described by Marks et al. . (Bio / Technology (1992) 10: 779-783). In particular, consensus primers directed toward or adjacent to the 5 'end of the variable domain area can be used in conjunction with consensus primers for the third structural region of human H variable genes to provide a repertoire of variable H domains that lack a CDR3. The repertoire can be combined with a CDR3 of a particular antibody. Using analogous techniques, sequences derived from CDR3 can be shuffled with repertoires of variable H or L variable domains that lack a CDR3, and the full H or variable L domains shuffled combined with a related variable L or H variable domain to prepare antibodies GPNMB specific. The repertoire can then be expressed in a suitable host system such as the phage display system as described in WO92 / 01047 so that suitable antigen-binding fragments can be selected.

Shuffling techniques or similar combinatorics can be used (eg, Stemmer, Nature (1994) 370: 389-391). Novel variable regions H or variable L variables can be generated that carry one or more sequences derived from the sequences disclosed herein using random mutagenesis of one or more selected H variable genes and / or L variables such as error-prone PCR (Proc. Nat Acad. Sci. USA (1992) 89: 3576-3580). Another procedure that can be used is to direct mutagenesis to CDR of variable H or L variable genes (Proc. Nat. Acad. Sci. USA (1994) 91: 3809-3813; J. Mol. Biol. (1996) 263: 551- 567). Similarly, one or more, or all three CDRs can be grafted onto a repertoire of variable H or L variable domains, which are then screened for a specific antigen-binding fragment by GPNMB.

A portion of an immunoglobulin variable domain will comprise at least one of the CDRs as set forth substantially herein and, optionally, intervening structural regions as set forth herein. The portion may include at least about 50% of one or both of FR1 and FR4, 50% being 50% of the C end of FR1 and 50% of the N end of FR4. Additional wastes at the N or C-terminus of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, the construction of antibodies by recombinant DNA techniques can result in the introduction of N or C end residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to bind variable domains to other protein sequences that include constant regions of immunoglobulin heavy chain, other variable domains (e.g., in the production of diabody), or proteinaceous tags as discussed below. in greater detail.

Although the antibodies illustrated in the examples comprise a pair of "pairing" of variable H and L variable domains, an expert will recognize that alternative antibodies may comprise antigen-binding fragments that only contain a single CDR of either the variable domain L or either the H variable. Any one of the single-chain specific binding domains can be used to screen complementary domains that can form a two-domain specific antigen-binding fragment that can, for example, bind GPNMB. Screening can be carried out by phage expression screening methods using the so-called hierarchical dual combinatorial solution disclosed in WO92 / 01047 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones that encodes the other chain (L

image24

or H), and the resulting double-stranded specific binding domain is selected according to phage expression techniques as described.

The antibodies directed against GPNMB described herein can be linked to another functional molecule, for example, another peptide or protein (albumin, another antibody, etc.), toxin, radioisotope, cytotoxic agents.

or cytostatics. For example, antibodies can be ligated by chemical crosslinking or by recombinant procedures. The antibodies can also be linked to one of a variety of non-proteinaceous polymers, for example, polyethylene glycol, polypropylene glycol or polyoxyalkylenes in the manner set forth in US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulation half-life. Polymers and exemplary methods for joining them are also shown in US Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546.

The disclosed antibodies can also be altered to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate moieties can be deleted and / or one or more glycosylation sites added to the original antibody. The addition of glycosylation sites to currently disclosed antibodies can be accomplished by altering the amino acid sequence to contain consensus sequences of glycosylation sites known in the art. Another means of increasing the number of carbohydrate residues in the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody (WO 87/05330; CRC Crit. Rev. Biochem., 22: 359-306 , 1981). The removal of any remaining carbohydrate from the antibodies can be carried out chemically or enzymatically (Arch. Biochem. Biophys., 259: 52,1987; Anal. Biochem., 118: 131, 1981; Meth. Enzymol., 138 : 350, 1987). Antibodies can also be labeled with a detectable or functional label. Detectable labels include radiolabels such as 131I or 99Tc, which can also bind antibodies using conventional chemistry. Detectable brands also include enzyme brands such as horseradish peroxidase or alkaline phosphatase. The detectable labels additionally include chemical moieties such as biotin, which can be detected by binding to a specific related detectable moiety, for example, labeled avidin.

The valence of antibodies can be tailored to affect affinity and avidity, retention time at binding sites (see, for example, Am H. Patol, 2002 160: 1597-1608; J. Med. Chem. 2002 45: 2250-2259; Br. J. Cancer 2002 86: 1401-1410; Biomol. Eng. 2001 18: 95-108; Int J. Cancer 2002 100: 367-374).

Multiple specificity (bifunctional) binding reagents can be designed based on the GPNMB specific sequences of the invention (Biomol. Eng. 2001 18: 31-40). For example, a bispecific or bifunctional antibody is an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of procedures including hybridoma fusion or ligation of Fab 'fragments (Clin. Exp. Immunol. 1990, 79: 315-321; J. Immunol. 199,2148: 1547-1553). Such bispecific antibodies can be generated comprising a specificity by GPNMB and a second specificity by a second molecule using techniques that are well known (Immunol Methods 1994,4: 72-81; Wright and Harris, above; Traunecker et al. 1992 Int. J. Cancer (Suppl.) 7: 51-52). Bispecific antibodies prepared in this way selectively destroy cells expressing GPNMB.

Antibodies in which the CDR sequences differ only insubstantially from those set forth in SEQ ID NOs: 22, 24, 26, 31, 33, 35 and the formulas: 254, 255 are encompassed within the scope of this invention. Normally, an amino acid is substituted by a related amino acid that has similar, hydrophobic or stereochemical loading characteristics. Such substitutions would be within the skills of an expert. Unlike CDRs, more substantial changes can be made in FRs without adversely affecting the binding properties of an antibody. Changes in FRs include, but are not limited to, handling certain residues of structural regions that are important for antigen contact or for stabilizing the binding site, for example, changing the class or subclass of the constant region, changing residues specific amino acids that can alter effector function such as binding to the Fc receptor (US Patent Nos. 5,624,821; 5,648,260; Lund et al. (1991) J. Immun. 147: 2657-2662; Morgan et al. (1995) Immunology 86: 319-324) or change the species from which the constant region is derived.

One skilled in the art will appreciate that the derivatives and modifications described above are not exhaustive, and that many other modifications would be obvious to an expert in view of the teachings of the present disclosure.

Nucleic acids, cloning and expression systems

The present disclosure further provides isolated nucleic acids encoding the disclosed antibodies. Nucleic acids can comprise DNA or RNA and can be completely or partially synthetic or recombinant. The reference to a nucleotide sequence as set forth herein encompasses a DNA molecule with the specified sequence, and encompasses an RNA molecule with the specified sequence in which U is substituted by T, unless the context requires it to another way.

image25

The nucleic acids provided herein comprise a coding sequence for a CDR, a variable domain H and / or a variable domain L disclosed herein.

The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes comprising at least one nucleic acid encoding a CDR, a variable domain H and / or a variable domain L disclosed herein.

The disclosure further provides a host cell comprising one or more constructs as before.

Nucleic acids encoding any CDR (CDR1, CDR2, CDR3 of both the variable domain H and L), variable domain H or variable L are also provided, in addition to methods for preparing the encoded products. The process comprises expressing the encoded product from the encoded nucleic acid. Expression can be achieved by culturing recombinant host cells containing nucleic acid under appropriate conditions. After expression production, a variable domain H or variable L, or specific binding member, can be isolated and / or purified using any suitable technique, then used as appropriate.

Antigen-binding fragments, variable domains H and / or L variables and encoding nucleic acid molecules and vectors can be isolated and / or purified from their natural environment in substantially pure or homogeneous form or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function.

Systems for cloning and expressing a polypeptide in a variety of different host cells are well known in the art including cells suitable for producing antibodies (Gene Expression Systems, Academic Press. Eds. Fernandez et al., 1999). Briefly, suitable host cells include bacteria, plant cells, mammalian cells and yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, newborn hamster kidney cells, NS0 mouse myeloma cells, and many others. A common bacterial host is

E. coli. Any protein expression system compatible with the invention can be used to produce the disclosed antibodies. Suitable expression systems also include transgenic animals (Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999).

Suitable vectors may be chosen or constructed to contain appropriate regulatory sequences that include promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids or virals, for example, phage, or phagemid, as appropriate (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989). Many known techniques and protocols for nucleic acid manipulation are known in the art, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, (Current Protocols in Molecular Biology, 2nd edition, eds. Ausubel et al., John Wiley & Sons, 1992).

The invention also provides a host cell comprising a nucleic acid as disclosed herein. A method is also provided comprising introducing such nucleic acid into a host cell. The introduction can employ any available technique. For eukaryotic cells, suitable techniques may include transfection with calcium phosphate, DEAE-dextran, electroporation, liposome-mediated transfection and transduction using retroviruses or other viruses, for example, variolovaccine or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include transformation with calcium chloride, electroporation and transfection using bacteriophage. The introduction of nucleic acid into cells can be continued to cause or allow the expression of nucleic acid, for example, by culturing host cells under conditions for gene expression.

Immunoconjugates

In another aspect, the antibodies of the invention can be used as the target agent of choice for the administration of another therapeutic agent or a cytotoxic agent for a cell expressing GPNMB. An antibody directed against GPNMB coupled to a therapeutic agent or a cytotoxic agent for use as a medicament is an aspect of the invention.

Antibodies directed against GPNMB are conjugated to a therapeutic agent, such as a cytotoxic compound, so that the resulting immunoconjugate exerts a cytotoxic or cytostatic effect on a cell expressing GPNMB. Particularly suitable residues for antibody conjugation are chemotherapeutic agents, prodrug converting enzymes or toxins. For example, an antibody directed against GPNMB can be conjugated with a cytotoxic agent such as a chemotherapeutic agent (see below) or a toxin (for example, abrin, ricin A, pseudomonas exotoxin or diphtheria toxin). Alternatively, the antibody directed against GPNMB can be conjugated with a prodrug converting enzyme. The prodrug converting enzyme can be recombinantly fused with the antibody or derivative thereof or chemically conjugated thereto using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, βglucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.

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Any agent that exerts a therapeutic effect on cells expressing GPNMB can be used as an agent for conjugation to an antibody directed against GPNMB of the invention. Useful classes of cytotoxic agents include, for example, antitubulin agents, auristatins, minor groove binding ligands of DNA, inhibitors of DNA replication, alkylating agents (e.g., platinum complexes such as cis-platinum, mono (platinum) , bis (platinum) and trinuclear and carboplatin platinum complexes), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated purimidines, ionophores, lexithropsins, nitrosoureas, platinum, preforming compounds, purimetains, purimetains, purimetains radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.

The therapeutic agent may be a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (eg, auristatin E, AFP, MMAF, MMAE), DNA minor groove binding ligands (eg, enediines and lexitropsins), cuocarmycins, taxanes (eg, paclitaxel and docetaxel ), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, equinomycin, combretastatin, netropsin, epothilone A and B, stramustine, cryptofisin, cemadotine discoins, cemadotine, cemadotine, cemadotine, cemadotine, cemadotine Eleuterobin and mitoxantrone.

In a specific embodiment, the cytotoxic or cytostatic agent is auristatin E (dolastatin 10) or a derivative thereof (for example, an ester formed between auristatin E and a keto acid). Other typical auristatin derivatives include AFP, MMAR and MMAE. The synthesis and structure of auristatin E and its derivatives are described in U.S. Patent Application Publication. No. 20030083263; documents PCT / US03 / 24209; PCT / US02 / 13435; and U.S. patents No. 6,323,315; 6,239,104; 6.034065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

In a specific embodiment, the antibody directed against GPNMB 1.15.1 was coupled to monomethylauristatin E by the intracellular peptide linker protease-sensitive valine-citrulline (vcMMAE). The procedures for preparing the immunoconjugate can be found in Doronina S.O. et al., 2003 Nature Biotechnology 21 (7): 778-794.

Techniques for conjugating therapeutic agents to proteins, and in particular, antibodies are known in the art (see, for example, Arnon et al., 1985 in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. Eds., Alan

R. Liss, Inc., 1985; Hellstrom et al., 1987 in Controlled Drug Delivery, Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987; Thorpe 1985, in Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al. eds., EDITOR, 1985; Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. eds., Academic Press 1985; and Thorpe et al., 1982, Immunol. Rev. 62: 119-58).

In certain embodiments of the invention, antibodies directed against GPNMB that bind to cells expressing GPNMB are internalized and accumulate in the cell. Thus, antibody immunoconjugates directed against GPNMB accumulate in cells expressing GPNMB. Normally, if the immunoconjugate of antibodies directed against GPNMB is internalized, the agent is preferentially active. Alternatively, immunoconjugates of antibodies directed against GPNMB are not internalized and the drug is effective in reducing or inhibiting cells expressing GPNMB by binding to the cell membrane. The therapeutic agent can be conjugated in a way that reduces its activity, unless it is separated by cleavage of the antibody (for example, by hydrolysis or by a cleavage agent). In this case, the agent can bind to the antibody or derivative thereof with a cleavable linker that is sensitive to cleavage in the intracellular environment of the target, but is not substantially sensitive to the extracellular environment, such that the conjugate is cleaved from the antibody or derivative thereof when internalized by the cell expressing GPNMB (for example, in the endosomal environment or, for example, by virtue of pH sensitivity or protease sensitivity, in the lysosomal environment or in a caveola).

A therapeutic agent of the immunoconjugate can be charged with respect to the plasma membrane (for example, polarized or net charge with respect to the plasma membrane), thus minimizing further the ability of the agent to cross the plasma membrane once it has been internalized by a cell.

The immunoconjugate of antibodies directed against GPNMB may comprise a binding region between the therapeutic agent and the antibody. The linker can be cleavable under intracellular conditions so that cleavage of the linker releases the therapeutic agent of the antibody into the intracellular environment. The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme that includes, but is not limited to, a lysosomal or endosomal protease. Frequently, the peptidyl linker is at least two amino acids in length or at least three amino acids in length. The cleavage agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze derivatives of dipeptide drugs causing the release of the active drug within target cells (see Dubowchik and Walker, 1999 Pharm. Therapeutics 83: 67-123). Other binders are described, for example, in US Pat. No. 6,214,345.

Binding agents that may be pH sensitive can frequently be hydrolyzed under acidic conditions such as those found in the lysosome (see, for example, US Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999 Pharm. Therapeutics 83: 67-123; Neville et al., 1989 Biol. Chem. 264: 14653-14661). Such linkers are relatively stable under neutral pH conditions such as those in the blood, but they are unstable at pH below 5.5 or 5.0, the lysosome pH. The linkers can be cleaved under reducing conditions (e.g., a disulfide linker) (see, for example, Thorpe et al., 1987 Cancer Res. 47: 5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimmagery and Therapy of Cancer, CW Vogel ed, Oxford U. Press, 1987; U.S. Patent No. 4,880,935). The linker may be a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3 (10): 1299 -1304) or a 3'-N-amide analogue (Lau et al., 1995, Bioorg-Med-Chem, 3 (10): 1305-1312).

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Prophylactic and therapeutic uses of the present invention

The antibodies of the invention can be either agonists or GPNMB antagonists, depending on the methods of their use. Antibodies can be used to prevent, diagnose or treat medical disorders in a subject, especially in humans. The antibodies of the invention can also be used to isolate GPNMB or cells expressing GPNMB. In addition, antibodies can be used to treat a subject at risk of or susceptible to a disorder or who has a disorder associated with the expression or function of abnormal GPNMB. The antibodies of the invention can be used to detect GPNMB in such subjects.

The present invention provides a composition that can choose as target cells expressing GPNMB for medical use. In particular, the composition is used for the treatment and / or prevention of a disease.

or disorder associated with overexpression of GPNMB and / or hyperproliferative disorders, particularly hyperproliferative cell disease such as cancer. In one embodiment, the composition comprises an immunoconjugate comprising an antibody of the invention and a cytotoxic agent. In another embodiment, the composition comprises a naked IgG1 antibody of the invention and one or more immunomodulators. In yet another embodiment, the composition comprises a single chain (anti-GPNMB) Fv antibody conjugated to a cytotoxic agent, or a bispecific antibody having an antibody component directed against single chain GPNMB and an antibody component directed against CD3.

In a preferred embodiment, the hyperproliferative cell disease is cancer. More preferably, the cancer is melanoma, or a cancer of the CNS system such as astrocytoma, glioblastoma, medulloblastoma, or neoplastic meningitis.

The present invention provides antibodies directed against GPNMB for the treatment or prevention of a disorder characterized by or associated with the expression and / or abnormal activity of GPNMB or a symptom thereof. The treatment or prevention may comprise the use of one or more antibodies of the invention, or one or more immunoconjugates comprising one or more antibodies of the invention. The treatment or prevention may further comprise the use of one or more therapies (for example, one or more prophylactic or therapeutic agents) other than the antibodies of the invention.

The prophylactic or therapeutic agents of the combination therapies of the invention can be administered sequentially or simultaneously. In a specific embodiment, the combination therapies of the invention comprise an effective amount of one or more antibodies of the invention and an effective amount of at least one other therapy (eg, prophylactic or therapeutic agent) having a mechanism of action other than said antibodies. In certain embodiments, the combination therapies of the present invention improve the prophylactic or therapeutic effect of one or more antibodies of the invention by working together with the antibodies to have an additive or synergistic effect. In certain embodiments, the combination therapies of the present invention reduce the side effects associated with the therapies (eg, prophylactic or therapeutic agents).

The prophylactic or therapeutic agents of the combination therapies can be administered to a subject, preferably a human subject, in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered simultaneously to a subject in separate pharmaceutical compositions. Prophylactic or therapeutic agents can be administered to a subject by the same or different routes of administration.

In a specific embodiment, a pharmaceutical composition comprising one or more antibodies of the invention described herein is administered to a subject, preferably a human being, to prevent and / or treat a disorder characterized by or associated with the expression and / or abnormal GPNMB activity or a symptom thereof. According to the invention, the pharmaceutical compositions of the invention may also comprise one or more therapies (for example, prophylactic or therapeutic agents) other than the antibodies of the invention.

The antibodies of the invention can also be used to detect the presence of GPNMB in biological samples (in diagnostic procedures or use as an efficacy marker). The amount of GPNMB detected may be related to the level of GPNMB expression which, in turn, is related to the disease, tumor type, burden or tumor status using procedures known in the art (see, for example, the recommendations of the AAPS Ligand Binding Assay Bioanalytical Focus Group (LBABFG) Pharm Res. 2003 Nov; 20 (11): 1885-900). Detection methods that employ antibodies are well known in the art and include, for example, ELISA, radioimmunoassay, immunoblot, Western blot, IHC, immunofluorescence, immunoprecipitation. Antibodies can be provided in a diagnostic kit that incorporates one or more of these techniques to detect GPNMB. Such a kit may contain other components, packages, instructions or other material to aid in the detection of the protein. In a specific embodiment, the antibodies of the invention are conjugated to a radioactive isotope and injected into a subject to detect cells that overexpress GPNMB.

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If the antibodies are intended for diagnostic purposes, it may be desirable to modify them, for example, with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope

or an enzyme). If desired, the antibodies of the invention can be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, reagents with electron density, enzymes and ligands having specific binding components. Enzymes are normally detected by their activity. For example, horseradish peroxidase can be detected by its ability to convert tetramethylbenzidine (TMB) into a blue pigment, quantifiable with a spectrophotometer. For detection, suitable binding components include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand pairs known in the art. Other permutations and possibilities will be readily apparent to those skilled in the art, and are considered as equivalent within the scope of the present invention.

The antibodies of the invention can be used in screening methods to identify effective GPNMB inhibitors as therapeutic agents. In such a screening assay, a first binding mixture is formed by combining GPNMB and an antibody of the invention; and the amount of binding in the first bonding mixture (M0) is measured. A second binding mixture is also formed by combining GPNMB, the antibody and the compound or agent to be screened, and the amount of binding of the second binding mixture (M1) is measured. A compound to be tested may be another antibody directed against GPNMB. The binding amounts in the first and second binding mixture are then compared, for example, by calculating the M1 / M0 ratio. It is considered that the compound or agent can modulate the responses associated to GPNMB if a decrease in the binding of the second binding mixture with respect to the first binding mixture is observed. The formulation and optimization of the binding mixtures is within the skill level in the art, such binding mixtures may also contain buffers and salts necessary to enhance or optimize the binding, and additional control assays may be included in the test of screening of the invention. Thus, compounds found that reduce antibody-GPNMB binding at least about 10% (i.e., M1 / M0 <0.9), preferably more than about 30%, can be identified and then, if desired, screened for the second time for the ability to improve a disorder in other animal trials or models as described below. The intensity of the binding between GPNMB and an antibody can be measured using, for example, an enzyme-linked immunoadsorption assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance-based technology (eg, Biacore), all techniques being well known in the art.

Then, the compound can be tested in vitro as described in the examples, below.

Dosage and frequency of administration

The amount of prophylactic or therapeutic agent or a composition of the invention that will be effective in the prevention and / or treatment of a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB can be determined by conventional clinical procedures. For example, the dosage of the composition that will be effective in the treatment and / or prevention of cancer can be determined by administering the composition to an animal model. In addition, in vitro tests may optionally be used to help identify optimal dosage ranges. Preliminary doses, such as, for example, are determined according to animal tests and the escalation of dosages for human administration is performed according to the practices accepted in the art. Toxicity and therapeutic efficacy can be determined by conventional pharmaceutical procedures in cell cultures or experimental animals. Data obtained from cell culture assays or animal studies can be used in the formulation of a dosage range for use in humans. Therapeutically effective dosages achieved in an animal model can be converted for use in another animal, which includes humans, using conversion factors known in the art (see, for example, Freineich et al. (1966) Cancer Chemother. Reports, 50 ( 4): 219-244).

The selection of the preferred effective dose can be determined (for example, by clinical trials) by an expert based on the consideration of several factors that will be known to a person skilled in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, sex, immune status and other factors known to the expert to reflect the accuracy of the pharmaceutical compositions administered. A person skilled in the art can select appropriate guidelines considering such factors and following, for example, dosages reported in the literature and recommended in the vademecum (59th ed., 2005).

The precise dose to be used in the formulation will also depend on the route of administration and the severity of the cancer, and should be decided according to the doctor's judgment and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro test systems or animal models.

For other cancer therapeutic agents administered to a patient, typical doses of various cancer therapeutic agents are known in the art. Given the invention, certain preferred embodiments will encompass the administration of minor dosages in combination treatment guidelines to the dosages recommended for administration of single agents.

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In a specific embodiment, the dosage of an antibody or an immunoconjugate comprising an antibody of the invention administered to prevent and / or treat a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB (eg, cancer) in a patient is 30 mg / kg or less, 25 mg / kg or less, 20 mg / kg or less, 15 mg / kg or less, preferably 12 mg / kg or less, 11 mg / kg or less, 10 mg / kg or less, 9 mg / kg or less, 8 mg / kg or less, 7 mg / kg or less, 6 mg / kg or less, 5 mg / kg or less, 4 mg / kg or less, 3 mg / kg or less , 2 mg / kg or less, or 1 mg / kg or less of the patient's body weight. In another embodiment, the dosage of an antibody or an immunoconjugate of the invention administered to prevent and / or treat a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB (eg, cancer) in a patient is a unit dose. from about 0.01 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 8 mg / kg, about 0.1 mg / kg at about 7 mg / kg, about 0.1 mg / kg at about 6 mg / kg, about 0.1 mg / kg at about 5 mg / kg, about 0.1 mg / kg at about 4 mg / kg, preferably , approximately 0.1 mg / kg to approximately 3 mg / kg, approximately 0.2 mg / kg to 3 mg / kg, approximately 0.3 mg / kg to approximately 3 mg / kg, approximately 0.4 mg / kg a approximately 3 mg / kg, approximately 0.6 mg / kg to approximately 3 mg / kg, approximately 0.8 mg / kg to approximately 3 mg / kg, approximately 0.1 mg / kg to 2 mg / kg, approximately 0.1 mg / kg to 1 mg / kg. In certain embodiments, the dosage of an antibody or an immunoconjugate comprising an antibody of the invention administered to prevent and / or treat a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB (eg, cancer) in a patient it is a unit dose of approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.4 mg / kg, approximately 0.6 mg / kg, approximately 0.8 mg / kg, approximately 1.1 mg / kg, or about 1 mg / kg.

In certain embodiments, a subject is administered one or more doses of an effective amount of one or more antibodies or immunoconjugates of the invention to prevent and / or treat a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB, wherein the dose of an effective amount of said antibodies, immunoconjugates, compositions or combination therapies reduces and / or inhibits the proliferation of cancer cells by at least 20% to 25%, preferably at least 25% to 30%, at less than 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% at 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, at least 80 to 85%, at least 85% to 90%, at least 90% to 95%, or at least 95% to 98% with respect to a control such as PBS in an in vitro and / or in vivo assay well known in the art.

In other embodiments, a subject is administered one or more doses of an effective amount of one or more antibodies or immunoconjugates of the invention to prevent and / or treat a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB, in which the dose of an effective amount achieves a serum titer of at least 0.1 µg / mL, at least 0.5 µg / mL, at least 1 µg / mL, at least 2 µg / mL, at least 5 µg / mL, at least 6 µg / mL, at least 10 µg / mL, at least 15 µg / mL, at least 20 µg / mL, at least 25 µg / mL, at least 50 µg / mL, at least 100 µg / mL, at least 125 µg / mL, at least 150 µg / mL, at least 175 µg / mL, at least 200 µg / mL, at least 225 µg / mL, at least 250 µg / mL, at least 275 µg / mL, at least 300 µg / mL, at least 325 µg / mL, at least 350 µg / mL, at least 375 µg / mL, or at least 400 µg / mL of the antibodies of the invention. In still other embodiments, a subject is administered a dose of an effective amount of one or more antibodies or immunoconjugates of the invention to achieve a serum titer of at least 0.1 µg / mL, at least 0.5 µg / mL, at least 1 µg / mL, at least 2 µg / mL, at least 5 µg / mL, at least 6 µg / mL, at least 10 µg / mL, at least 15 µg / mL, at least 20 µg / mL, at least 25 µg / mL, at least 50 µg / mL, at least 100 µg / mL, at least 125 µg / mL, at least 150 µg / mL, at least 175 µg / mL, at least 200 µg / mL , at least 225 µg / mL, at least 250 µg / mL, at least 275 µg / mL, at least 300 µg / mL, at least 325 µg / mL, at least 350 µg / mL, at least 375 µg / mL, or at least 400 µg / mL of the antibodies, and a subsequent dose of an effective amount of one or more antibodies or immunoconjugates of the invention is administered to maintain a serum titer of at least 0.1 µg / mL, at least 0 , 5 µg / mL, at least 1 µg / mL, at least 2 µg / mL, at least 5 µg / mL, at least 6 µg / mL, at least 10 µg / mL , at least 15 µg / mL, at least 20 µg / mL, at least 25 µg / mL, at least 50 µg / mL, at least 100 µg / mL, at least 125 µg / mL, at least 150 µg / mL, at least 175 µg / mL, at least 200 µg / mL, at least 225 µg / mL, at least 250 µg / mL, at least 275 µg / mL, at least 300 µg / mL, at least 325 µg / mL, at minus 350 µg / mL, at least 375 µg / mL, or at least 400 µg / mL. According to these embodiments, a subject may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more subsequent doses.

In a specific embodiment, the immunoconjugate antibodies of the invention are used in a unit dose of at least 0.01 mg / kg, at least 0.1 mg / kg, at least 0.2 mg / kg, at least 0.4 mg / kg, at least 0.6 mg / kg, at least 0.8 mg / kg, at least 1 mg / kg, or at least 1.1 mg / kg of one or more antibodies or immunoconjugates of the invention. In another embodiment, the antibodies or immunoconjugates of the invention are used in a unit dose of at least 0.01 mg / kg, at least 0.1 mg / kg, at least 0.2 mg / kg, at least 0.4 mg / kg, at least 0.6 mg / kg, at least 0.8 mg / kg, at least 1 mg / kg, or at least 1.1 mg / kg of one or more antibodies or immunoconjugates of the invention once every 7 days, preferably, once every 10 days, once every 12 days, once every 14 days, once every 16 days, once every 18 days, once every three weeks, or once a month. In a preferred embodiment, an immunoconjugate of the present invention is used at a unit dose of about 0.1 mg / kg, about 0.2 mg / kg, about 0.4 mg / kg, about 0.6 mg / kg, about 0.8 mg / kg, about 1.1 mg / kg, or about 1 mg / kg once every 10 to 20 days with 2 to 4 cycles.

The treatment or prevention of a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB may comprise: (a) administering to a subject in need thereof one or more doses of a prophylactic or therapeutically effective amount of one or more antibodies or immunoconjugates of the invention; and (b) monitoring the plasma level / concentration of said antibody or antibodies administered in said subject after administration of a certain number of doses of said antibody or antibodies. Furthermore, preferably, said certain number of doses is 1, 2, 3, 4, 5, 6, 7 or 8 doses of a prophylactic or therapeutically effective amount of one or more antibodies or immunoconjugates of the invention.

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The treatment or prevention of a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB may comprise: (a) administering to a subject in need thereof a dose of at least 0.1 mg / kg (preferably at least at least 0.2 mg / kg, at least 0.4 mg / kg, at least 0.6 mg / kg, at least 0.8 mg / kg, at least 1 mg / kg, or at least 1.1 mg / kg) of one or more antibodies or immunoconjugates of the invention; and (b) administering one or more subsequent doses to said subject when the plasma level of the antibody or antibodies administered in said subject is less than 0.1 µg / mL, preferably less than 0.25 µg / mL, less than 0, 5 µg / mL, less than 0.75 µg / mL,

or less than 1 µg / mL. The treatment or prevention of a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB may comprise: (a) administering to a subject in need thereof one or more doses of at least 0.1 mg / kg (preferably at least 0.2 mg / kg, at least 0.4 mg / kg, at least 0.6 mg / kg, at least 0.8 mg / kg, at least 1 mg / kg, or at least 1.1 mg / kg) of one or more antibodies of the invention; (b) monitoring the plasma level of the antibody or antibodies administered of the invention in said subject after administration of a certain number of doses; and (c) administering a subsequent dose of the antibody or antibodies of the invention when the plasma level of the antibody or antibodies administered in said subject is less than 0.1 µg / ml, preferably less than 0.25 µg / mL, less than 0.5 µg / mL, less than 0.75 µg / mL, or less than 1 µg / mL. Preferably, said certain number of doses is 1, 2, 3, 4, 5, 6, 7 or 8 doses of an effective amount of one or more antibodies or immunoconjugates of the invention.

Therapies (for example, prophylactic or therapeutic agents) other than antibodies or immunoconjugates of the invention that have been or are currently being used to prevent and / or treat a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB can be administered. in combination with one or more antibodies or immunoconjugates of the invention to treat and / or prevent a disorder associated with or characterized by the abnormal expression and / or activity of GPNMB. Preferably, the dosages of prophylactic or therapeutic agents used in the combination therapies of the invention are lower than those that have been or are currently being used to prevent and / or treat a disorder associated with or characterized by abnormal expression and / or activity of GPNMB

Therapies (for example, prophylactic or therapeutic agents) can be administered separated less than 5 minutes, separated less than 30 minutes, separated 1 hour, separated approximately 1 hour, separated approximately 1 to approximately 2 hours, separated approximately 2 hours to approximately 3 hours , separated approximately 3 hours to approximately 4 hours, separated approximately 4 hours to approximately 5 hours, separated approximately 5 hours to approximately 6 hours, separated approximately 6 hours to approximately 7 hours, separated approximately 7 hours to approximately 8 hours, separated approximately 8 hours to about 9 hours, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours apart at 36 hours, separated 36 hours at 48 hours, separated 48 hours to 52 hours, separated 52 hours to 60 hours, separated 60 hours to 72 hours, separated 72 hours to 84 hours, separated 84 hours to 96 hours, or separated 96 hours to 120 hours. Preferably, two or more therapies are administered within the same visit of the patient.

One or more antibodies of the invention and one or more therapies (for example, prophylactic or therapeutic agents) can be administered cyclically. Cyclic therapy involves the administration of a first therapy (for example, a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (for example, a second prophylactic or therapeutic agent) during a period of time, optionally followed by the administration of a third therapy (for example, prophylactic or therapeutic agent) for a period of time, etc., and repeating this sequential administration, that is, the cycle in order to reduce the development of resistance to one of the therapies to avoid or reduce the side effects of one of the therapies, and / or to improve the effectiveness of the therapies.

Pharmaceutical compositions and administration procedures

The disclosure provides compositions comprising antibodies directed against GPNMB. Such compositions may be suitable for pharmaceutical use and administration to patients. The compositions typically comprise one or more antibodies of the present invention and a pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable excipient" includes each and every solvent, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents and absorption retarding agents and the like, which are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds that provide complementary, additional or enhanced therapeutic functions. Pharmaceutical compositions may also be included in a container, container or dispenser together with instructions for administration.

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A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. The procedures for performing the administration are known to those skilled in the art. Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous or transdermal. It may also be possible to obtain compositions that can be administered topically or orally,

or that can be transmitted through mucous membranes.

The solutions or suspensions used for intradermal or subcutaneous application usually include one

or more of the following components: a sterile diluent such as water for injection, saline solution, nonvolatile oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabenos; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Such preparations may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the point where there is easy syringeability. It must be stable under the conditions of manufacture and storage and must be preserved from the contaminating action of microorganisms such as bacteria and fungi. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars; Polyalcohols such as mannitol, sorbitol and sodium chloride in the composition. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerin, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. Prolonged absorption of the injectable compositions can be caused by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral administration, the antibodies can be combined with excipients and used in the form of tablets, troches or capsules. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following components, or compounds of a similar nature; a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a slide such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, appropriate penetrants are used in the formulation to permeate the barrier. Such penetrants are generally known in the art and include, for example, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be carried out, for example, using lozenges, nasal sprays, inhalers or suppositories. For example, in the case of antibodies comprising the Fc portion, the compositions can be transmitted through the mucous membranes to the intestine, mouth or lungs (for example, by the route mediated by the FcRn receptors as described in the patent of U.S. 6,030,613). For transdermal administration, the active compounds can be formulated in ointments, balms, gels or creams as is generally known in the art. For administration by inhalation, the antibodies may be administered in the form of an aerosol spray of a pressure vessel or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

In certain embodiments, currently disclosed antibodies are prepared with vehicles that will protect the compound from rapid removal from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. The procedures for the preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions containing the currently disclosed antibodies can also be used as pharmaceutically acceptable vehicles. These can be prepared according to procedures known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It may be advantageous to formulate oral or parenteral compositions in a unit dosage form to facilitate administration and dosage uniformity. The term "unit dosage form" as used herein refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit containing a predetermined amount calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.

image32

The toxicity and therapeutic efficacy of the composition of the invention can be determined by conventional pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically dose effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions that have large therapeutic indices are preferred.

For any composition used in the present invention, the therapeutically effective dose can be estimated initially from assays in cell cultures. Examples of suitable bioassays include DNA replication assays, clonogenic assays and other assays, for example, are described in the examples. Data obtained from cell culture assays and animal studies can be used in the formulation of a dosage range for use in humans. A dose can be formulated in animal models to achieve a range of circulating plasma concentration that includes the IC50 (ie, the concentration of the antibody that achieves a 50% inhibition of symptoms). Plasma circulating levels can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage is preferably within a range of circulating concentrations with little or no toxicity. The dosage may vary depending on the pharmaceutical form used and the route of administration used.

Antibodies can be modified to become immunotoxins using techniques that are well known in the art (Vitetta 1993, Immunol Today 14: 252; U.S. Patent No. 5,194,594). Cytotoxic immunoconjugates are known in the art and have been used as therapeutic agents. Such immunoconjugates may use, for example, maitansinoids (US 6,441,163), tubulin polymerization inhibitor, auristatin (Mohammad et al., 1999 Int. J. Oracol 15 (2): 367-72; Doronina et al. , 2003 Nature Biotechnology 21 (7): 778-784), dolastatin derivatives (Ogawa et al., 2001 Toxicol Lett. 121 (2): 97-106) 21 (3) 778-784), Mylotarg® (Wyeth Laboratories , Philadelphia, PA); maitansinoids (DM1), taxane or mertansin (ImmunoGen Inc.).

Immunoradiopharmaceuticals that use antibodies directed against GPNMB can be prepared using techniques that are well known in the art (Junghans et al. In Cancer Chemotherapy and Biotherapy 655686 (2nd edition, Chafner and Longo, eds., Lippincott Raven (1996); patents U.S. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471 and 5,697,902) Each of the radiolabeled immunotoxins and antibody molecules selectively destroy cells that express GPNMB Radiolabels are known in the art and have been used for diagnostic or therapeutic radioimmunoconjugates Examples of radiolabels include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y , 99Tc, 111In, 125I, 131I, 177Lu, 105Rh, rhenium 186, rhenium 188, samarium 153, copper 64, scandium 47) For example, radionuclides that have been used in clinical diagnosis guided by radioimmunoconjugates include, but n or are limited to: 131I, 125I, 123I, 99Tc, 67Ga, in addition to 111In. Antibodies have also been labeled with a variety of radionuclides for possible use in targeted immunotherapy (see Peirersz et al., 1987). These radionuclides include, for example, 188Re and 186Re, in addition to 90Y, and to a lesser extent 199Au and 67Cu. I- (131) (see, for example, U.S. Patent No. 5,460,785). Radiotherapeutic chelators and chelating conjugates are known in the art (U.S. 4,831,175, 5,099,069, 5,246,692, 5,286,850 and 5,124,471).

EXAMPLES

The following examples, which include the experiments performed and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the present invention.

Example 1: Immunogen

The recombinant human GPNMB clone CG56972 (SEQ ID NO: 289), specifically the extracellular domain (ECD), was prepared for use as an immunogen. Generally, the cDNA encoding GPNMB ECD with a V5-HIS label of the C-terminus was transfected into HEK 293 cells, expressed and purified using cation exchange chromatography with POROS HS 50 (Applied Biosystems, Foster City, CA) . The sample was eluted with 1M NaCl at a pH of 5.5, followed by metal affinity chromatography (5 mL of Pharmacia metal chelate). The sample was eluted against a linear gradient of 10-500 mM imidazole for 10 VC (column volume). Dialysis was produced using 20 mM Tris / 50 mM NaCl at pH 7.4 (2 L x 2). Then, the sample was filtered through a 0.22 µm filter.

Example 2: Immunization

A preferred method for generating fully human antibodies uses XenoMouse® strains of mice that have been genomanipulated to contain fragments of the germline configuration of 245 kb and 190 kb in size of the human heavy chain locus and the kappa light chain locus (Green et al. 1994 Nature Genetics 7: 13-21; Mendez et al. 1997 Nature Genetics 15: 146-156; Green and Jakobovits, 1998 J. Exp. Med. 188: 483495; US Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181 and 5,939,598.) In an alternative solution, the minilocus solution, an exogenous Ig locus is mimicked by including pieces (individual genes) of the locus of Ig. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a constant mu region and a second constant region (preferably a gamma constant region) are formed in a construct for insertion into an animal (Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al. (1994), Taylor et al. (1994) and Tuaillon et al., (1995), Fishwild et al., (1996); U.S. Patent Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, 6,255,458,

image33

5

10

fifteen

twenty

25

30

35

40

5,591,669, 6,023,010, 5,612,205, 5,721,367, 5,789,215, 5,643,763, 5,981,175). It is understood that enoκ XenoMouse® can be used to generate antibodies directed against GPNMB using V lambda regions. Such antibodies are within the scope of the invention.

Immunization

As an antigen, the GPNMB-V5His immunogen was used (as prepared in Example 1). Monoclonal antibodies against GPNMB were developed by sequentially immunizing XenoMouse® mice (strain XMG2 of XenoMouse®), Abgenix, Inc. Fremont, CA. XenoMouse® animals were immunized via the plantar pad pathway for all injections. The total volume of each injection was 50 µl per mouse, 25 µl per plantar pad.

For cohort 1 (10 XMG2 mice), the initial immunization was with 10 µg of GPNMB-V5H mixed 1: 1 (v / v) with 100 µg alum gel ("Adju-Phos": phosphate gel adjuvant aluminum, Superfos BIOSECTOR ™ a / s, distributed by EM Sergent Pulp and Chemical Co., Clifton, NJ, Cat. No. 1452-250) per mouse. The five subsequent reinforcements were made with 5 µg of GPNMB-V5His mixed 1: 1 (v / v) with 100 µg alum gel in pyrogen-free D-PBS. The seventh reinforcement consisted of 5 µg of GPNMB-V5His mixed 1: 1 (v / v) with TITERMAX GOLD® (Sigma; Cat. No. T2684). The eighth injection consisted of 5 µg of GPNMB-V5His mixed 1: 1 v / v with 100 µg alum gel. A final booster was made with 5 µg of GPNMB-V5His in pyrogen-free DPBS, without adjuvant. XenoMouse® mice were immunized on days 0, 3, 6, 10, 14, 17, 23 and 27 for this protocol and the fusions were performed on day 31. Bleeding was done by the retro-orbital bleeding procedure in the 21st day after the sixth reinforcement.

For cohort 2 (10 XMG2 mice), the initial immunization was with 10 µg GPNMB-V5His mixed 1: 1 (v / v) with 100 µg alum gel per mouse. The two subsequent reinforcements were made with 5 µg GPNMB-V5His mixed 1: 1 (v / v) with 100 µg alumina gel in pyrogen-free D-PBS. The fourth reinforcement consisted of 5 µg of GPNMB-V5His mixed 1: 1 (v / v) with TITERMAX GOLD® (Sigma; Cat. No. T2684). The next fifth to seventh injections consisted of 5 µg of GPNMB-V5His mixed 1: 1 v / v with 100 µg alum gel. The eighth injection and the final booster was made with 5 µg of GPNMB-V5His in pyrogen-free DPBS, without adjuvant. XenoMouse® mice were immunized on days 0, 3, 7, 11, 14, 17, 22, 25 and 74 for this protocol and fusions were performed on day 78. Bleeding was done by the retrograde bleeding procedure. orbital on day 21 after the sixth reinforcement.

Injection in the plantar pads was performed using the following protocol using only the ventral surface of both legs of the hind limbs. A solution was injected under the skin without crossing the muscle tissue using a 1/2 ml insulin syringe with a 28 or 30 x 1/2 ”needle attached. The mouse to be injected was grabbed by the loose fur along its neck and loin so that it was immobilized and thus turned so that the ventral side was accessible. The hind limb was grasped and the needle (bevelled side up) was inserted into the ankle, passing just below the skin until the needle reached the leg. The needle was inserted along the outer length of the hind leg gently to avoid the vein located behind the inner side of the leg. Once the tip of the needle reached the leg, the solution was slowly injected until resistance was lacking or the designated volume had been dispensed. Then, the needle was removed and the second hind leg was injected in the same way.

The following Table 4 provides the immunization schedule for the 2 groups of mice.

Table 4: GPNMB antigen immunization program: GPNMB soluble at 0.43 mg / ml

Diana

 Group No. Immunization mode  No. of mice Antigen  1st injection 2nd reinforcement

GPNMB

one Plantar pad 10 GPNMB soluble 10 ug / mouse of alum gel Day 0 5 ug / mouse of alum gel Day 3

3rd

4th 5th 6th Bleeding  7th 8th Fusion

reinforcement

reinforcement

reinforcement

reinforcement

reinforcement

reinforcement

5 ug / alum gel mouse Day 6

5 ug / mouse of alum gel Day 10 5 ug / alum gel mouse Day 14 5 ug / alum gel mouse Day 17 Day 21 5 ug / mouse of Titermax Gold Day 23 10 ug / mouse of D-PBS Day 27 Day 31

image34

Diana

Group No. Immunization mode  No. of mice Antigen 1st injection 2nd reinforcement

GPNMB

2 Plantar pad  10 GPNMB soluble 10 ug / mouse of alum gel Day 0 5 ug / mouse of alum gel Day 3

3rd

4th 5th 6th Bleeding  7th 8th 9th Fusion

reinforcement

reinforcement

reinforcement

reinforcement

reinforcement

reinforcement

reinforcement

5 ug / alum gel mouse Day 7

5 ug / mouse of Titermax Gold Day 11 5 ug / alum gel mouse Day 14 5 ug / alum gel mouse Day 17 Day 21 5 ug / mouse of alum gel Day 22 10 ug / mouse of D-PBS Day 25 10 ug / mouse of D-PBS Day 100 Day 104

Selection of animals for collection by title

Antibody titers directed against GPNMB in the serum of immunized XenoMouse® mice are

5 determined by ELISA. Briefly, three sets of ELISAs were established. GPNMB (+ NMB) at 1 µg / mL, GPNMB (NMB) at 1 µg / mL and NMB at 1 µg / mL were coated on 96-well plates of Costar Labcoat Universal Binding polystyrene (Corning, Acton, MA) overnight at 4 ° C in antigen coating buffer (0.1 M carbonate buffer, pH 9.6, NaHCO3 (PM 84) 8.4 g / L). The next day, the plates were washed three times with wash buffer (0.05% Tween 20 in 1 x PBS) using a Biotek plate washer. Then, the plates were blocked with 200 µl / well of

10 blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% thimerosal in 1 x PBS) and incubated at room temperature for 1 h. After blocking for one hour, the plates were washed three times with wash buffer using a Biotek plate washer. Sera from both XenoMouse® mice immunized with GPNMB and untreated XenoMouse® animals were titrated in 0.5% BSA / PBS buffer at 1: 3 dilutions in duplicate from an initial 1: 100 solution. The last well was left blank. These plates were incubated at room temperature

15 for 2 h, and then the plates were washed three times with wash buffer using a Biotek plate washer. A goat antibody directed against human IgG conjugated with horseradish peroxidase (HRP, Pierce, Rockford, IL) specific to Fc at a final concentration of 1 µg / mL was added and incubated for 1 hour at room temperature. The plates were washed three times with wash buffer using a Biotek plate washer. After washing, the plates were revealed with the addition of TMB chromogenic substrate (BioFx BSTP-0100-01) for 10-20 min or until

20 negative control wells will begin to show color. Then, the ELISA was stopped by adding stop solution (650 nM stop reagent for TMB (BioFx BSTP-0100-01) reconstituted with 100 ml of H2O per bottle). The specific titer of each XenoMouse® animal was determined from the optical density at 650 nm and is shown in Tables 2 and 3 below. The value of the title is the reciprocal of the highest dilution of sera with a DO reading double that of the noise. Therefore, the higher the number, the greater the immune response

25 humoral to GPNMB. The results are provided in Table 5.

Table 5: Serum titres directed against XENOMOUSE® GPNMB

Group 1 mice, fusion on day 21 after 6 injections.

Mouse ID

Reactivity to GPNMB Reactivity to GPNMB (+ GPNMB) Reactivity to GPNMB

Titles using hIgG

Titles using hIgG

Titles using hIgG

1-1

 20,000 5,000 225

1-2

 5,000 800 200

1-3

 35,000 7,500 225

1-4

 75,000 22,000 225

1-5

 8,000 2,000 325

image35

1-6

 6,000 800 1800

1-7

 22,000 7,500 225

1-8

 6,000 2,000 200

1-9

 7,000 2,000 75

1-10

 22,000 7,500 200

1-NC1

 <100 <100 <100

1-NC2

 <100 <100 <100

Group 2 mice, bleeding on day 21 after 6 injections

Mouse ID

Reactivity to GPNMB (-GPNMB) Reactivity to GPNMB (+ GPNMB) Reactivity to GPNMB

Titles using hIgG

Titles using hIgG

Titles using hIgG

2-1

 100,000 2,600 fifty

2-2

 8,000 2,600 fifty

2-3

 15,000 4,000 fifty

2-4

 7,000 2,200 75

2-5

 22,000 6,500 250

2-6

 60,000 22,000 60

2-7

 19,000 7,000 fifty

2-8

 5,000 1,200 fifty

2-9

 16,000 3,500 110

2-10

 12,000 5,000 110

2-NC1

 <100 <100 <100

2-NC2

 <100 <100 <100

Serums directed against GPNMB pooled from immunized animals were also evaluated by FACS for reactivity to UACC-62, SF539, SKMEL5, U87MG and LOX1 MVI cell lines. The pooled sera were tested at 1:10, 1: 100 and 1: 500 compared to serum directed against IL13 (control) and pre-bred diluted at 1:10, 1: 100

5 (control).

Example 3: Antibodies

Hybridoma cell lines were generated from immunized mice that demonstrated having titers directed against GPNMB using conventional techniques (see Mendez et al., 1997, Nat Genet. 15: 146-156).

Immunized mice were sacrificed by cervical dislocation, and collected and collected.

10 lymph nodes of each cohort. The lymphoid cells were dissociated by milling in DMEM to release the cells from the tissues, and the cells were suspended in DMEM. The cells were counted and 0.9 ml of DMEM per 100 million lymphocytes were added to the cell pellet to gently resuspend the cells, but completely. Using 100 µl of CD90 + magnetic beads per 100 million cells, the cells were labeled by incubating the cells with the magnetic beads at 4 ° C for 15 minutes. The magnetically labeled cell suspension it contained

15 to 108 positive cells (or up to 2x109 total cells) were loaded onto an LS + column and the column was washed with DMEM. The total effluent was collected as the negative fraction for CD90 (most of these cells were expected to be B cells).

image36

The fusion was performed by mixing the enriched B cells washed from before and non-secreting myeloma cells P3X63Ag8.653 purchased from ATCC, cat. CRL 1580 (Kearney et al., J. Immunol. 123, 1979, 1548-1550) at a ratio of 1: 1. The cell mixture was gently suspended by centrifugation at 800 g. After complete removal of the supernatant, the cells were treated with 2-4 mL of Pronase solution (CalBiochem, cat. No. 53702; 0.5 mg / ml in PBS) for no more than 2 minutes. 3-5 ml of SBF was then added to stop the enzymatic activity and the suspension was adjusted to 40 ml of total volume using Electro cell fusion solution, ECFS (0.3 M sucrose, Sigma, cat. No. S7903, acetate of 0.1 mM magnesium, Sigma, Cat. No. M2545, 0.1 mM calcium acetate, Sigma, Cat. No. C4705). The supernatant was removed after centrifugation and the cells were resuspended in 40 ml of ECFS. This washing step was repeated and the cells were resuspended again in ECFS at a concentration of 2x106 cells / ml.

Electro cell fusion was performed using a fusion generator, model ECM2001, Genetronic, Inc., San Diego, CA. The size of the fusion chamber used was 2.0 mL, using the parameters of the Abgenix, Inc. instrument optimal for ECF.

After ECF, the cell suspension was gently removed from the fusion chamber under sterile conditions and transferred to a sterile tube containing the same volume of hybridoma culture medium (DMEM (JRH Biosciences), 15% SBF ( Hyclone) supplemented with L-glutamine, pen / strep, OPI (oxaloacetate, pyruvate, bovine insulin) (all from Sigma) and IL-6 (Boehringer Mannheim)). The cells were incubated for 15-30 minutes at 37 ° C and then centrifuged at 400 g (1000 rpm [but, in which rotor ?, otherwise remove the rpm]) for five minutes. Cells were gently resuspended in a small volume of hybridoma selection medium (hybridoma culture medium supplemented with 0.5 x HA (Sigma, Cat. No. A9666)) and the volume was appropriately adjusted with more medium selection hybridomas based on a final seeding of 5x106 total B cells per 96-well plate and 200 µl per well. The cells were mixed gently and pipetted into 96-well plates and allowed to grow. On day 7 or 10, half of the medium was removed and the cells were fed again with hybridoma selection medium.

After 14 days of culture, hybridoma supernatants were screened for monoclonal antibodies specific for GPNMB. In primary screening, ELISA plates (Fisher, cat. No. 12-565-136) were coated with 50 µL / well of GPNMB (1 µg / mL) in coating buffer (0.1 M carbonate buffer, pH 9.6, NaHCO3 8.4 g / L), then incubated at 4 ° C overnight. After incubation, the plates were washed with wash buffer (0.05% Tween 20 in PBS) three times. 200 µL / well of blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% thimerosal in 1 x PBS) was added and the plates were incubated at room temperature for 1 h. After incubation, the plates were washed with wash buffer three times. Aliquots (50 µl / well) of hybridoma supernatants and positive and negative controls were added and the plates were incubated at room temperature for 2 h. The positive control used was serum of the XenoMouse® mouse immunized with relevant GPNMB and the negative control was serum of the relevant strain immunized with KLH of the XenoMouse® mouse. After incubation, the plates were washed three times with wash buffer. 100 µL / well of goat detection antibody directed against huIgGfc-HRP (Caltag, Cat. No. H10507, using a dilution concentration 1: 2000) was added and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed three times with wash buffer. 100 µl / well of TMB (BioFX Lab. Cat. No. TMSK-0100-01) was added and the plates were allowed to develop for approximately 10 minutes (until the negative control wells barely began to show color). Then 50 µl / well stop solution (TMB stop solution (BioFX Lab. Cat. No. STPR-0100-01) was added and the plates were read in an ELISA plate reader at a wavelength of 450 nm.

The old culture supernatants of the hybridoma cell growth wells based on the primary screening were completely removed and the IL-1b positive hybridoma cells were suspended in new hybridoma culture medium and transferred to 24-well plates. After 2 days in culture, these supernatants were ready for secondary confirmation screening. In the secondary confirmation screening, the positives in the first screening were screened in GPNMB binding ELISA described as above, and two sets of detection system for the secondary confirmation ELISA, a set for the detection of hIgG, a set for the detection of the human Ig kappa light chain (goat antibody directed against hIg kappa-HRP, Southern Biotechnology, cat. no. 2060-05) in order to demonstrate the completely human composition for both heavy and light chains. The two sets of ELISA procedures were identical to the previous descriptions, except that the three different detection antibodies were used separately. All positive successes of the ELISA assay of secondary confirmation were screened in a counter for immunogen binding by ELISA in order to exclude those that cross-reacted with IL-1a. ELISA plates (Fisher, cat. No. 12565-136) were coated with 50 µL / well of irrelevant V5His fusion protein, 1 ug / mL in coating buffer (0.1 M carbonate buffer, pH 9.6, NaHCO3 8.4 g / l), then incubated at 4 ° C overnight. The rest of the procedures were identical to the previous descriptions. There are 33 monoclonal antibodies specific for completely human GPNMB that were generated.

Hybridoma supernatants were screened for GPNMB binding by ELISA as described above in Example 2. The results are shown in Table 6.

image37

Table 6. Activity directed against GPNMB of hybridomas.

3 µg / mL

1 µg / mL 333 ng / mL 111 ng / mL 37 ng / mL 12.3 ng / mL

Average OD

Average OD

Average OD

Average OD

Average OD

Average OD

1.2.2

 0.763 0.499 0.356  0.199  0.094  0.049

1.7.3

 1,003 0.871 0.760  0.451  0.239  0.094

1.15.1

 1,159 1,051 0.902  0.701  0.381  0.168

1.16.2

 0.036 0.015 0.010  0.008  0.008  0.007

2-3

 1,282 1,204 0.963  0.713  0.359  0.179

2-6

 1,254 1,295 1,092  0.875  0.443  0.183

2-7

 0.827 0.719 0.680  0.494  0.308  0.156

2-8

 0.921 0.635 0.229  0.109  0.056  0.028

2-10

 1,095 1,066 0.849  0.583  0.272  0.132

2-15

 0.601 0.568 0.578  0.395  0.246  0.127

2-16

 0.359 0.173 0.068  0.032  0.017  0.011

2-17

 0.053 0.019 0.010  0.009  0.008  0.011

2-22

 0.714 0.707 0.538  0.355  0.171  0.068

2-24

 0.060 0.042 0.028  0.023  0.016  0.017

Isotype control

0.009 0.008 0.009  0.009  0.009  0.011

Irrelevant antibody

0.009  0.008 0.012  0.013

Secondary Ab Control

0.011

Anti-V5 Ab control

3,066

Certain hybridoma cell supernatants (29) for GPNMB binding were analyzed by the BiaCore® 2000 biosensor equipped with a CM5 quality sensor chip for research. A 1:25 dilution of 5 cell supernatant was passed through a protein A surface for 5 min followed by surface washing for 10 min. GPNMB was subsequently injected for 90 s on the surface at a concentration of 880 nM followed by dissociation. The double reference binding data were obtained by subtracting the signal from a control flow cell and subtracting the initial deviation of an injected buffer just before the antigen injection. The GPNMB binding data for each mAb was normalized to the amount of mAb captured on each surface. Corrected responses of normalized deviation were also measured. The sensograms were adjusted to a simple kinetic model

1: 1. The results are shown in Table 7. Sixteen of the cell supernatants contained mAb that significantly bound GPNMB, and three mAb, 15.1, 15.2 and 15.3 showed a strong binding to GPNMB.

image38

Table 7

Sample

Kd (nM) ka (M-1s-1) kd (s-1) Expression level

15.1

 52 16524 8.55E-04 means, medium

15.3

 59 13417 7.97E-04 means, medium

15.2

 61 12635 7.70E-04 tall

2.2

 96 9257 8.90E-04 means, medium

10.2

 118 3955 4.66E-04 low

7.3

 121 9648 1.17E-03 means, medium

7.1

 122 11842 1.44E-03 means, medium

7.2

 141 9356 1.32E-03 tall

10.3

 147 3626 5.32E-04 low

10.1

 209 4235 8.85E-04 low

8.2

 242 7555 1.83E-03 means, medium

8.3

 264 6551 1.73E-03 low

8.1

 329 6830 2.25E-03 means, medium

12.3

 407 1549 6.31 E-04 means, medium

12.2

 435 1280 5.57E-04 means, medium

12.1

 630 1587 1.00E-03 low

1.1

 > 1000 <1500 nd tall

1.2

 > 1000 <1500 nd tall

1.3

 > 1000 <1500 nd means, medium

2.1

 > 1000 <1500 nd means, medium

5.1

 > 1000 <1500 nd means, medium

5.2

 > 1000 <1500 nd means, medium

5.3

 > 1000 <1500 nd means, medium

9.1

 > 1000 <1500 nd means, medium

9.2

 > 1000 <1500 nd low

9.3

 > 1000 <1500 nd low

11.1

 > 1000 <1500 nd low

11.2

 > 1000 <1500 nd low

11.3

 > 1000 <1500 nd low

image39

Example 4: Antibody Clustering

Certain antibodies described in this document were pooled according to the protocol described in U.S. Patent Application Publication. No. 20030157730. Pearls conjugated with MxhIgG are prepared for coupling to the primary antibody. The volume of supernatant needed is calculated using the following formula: (n + 10) x 50 µL (in the

5 that n = total number of samples on the plate). If the concentration is known, 0.5 µg / mL is used. The pearl pool is gently removed with vortex, then diluted in the supernatant at a concentration of 2500 of each pearl per well or 0.5x105 / ml and incubated on a shaker in the dark at RT overnight, or 2 hours if it is at a known concentration of 0.5 µg / mL. After aspiration, 50 µL of each bead is added to each well of the filter plate, then washed once by adding 100 µL / well of wash buffer and aspirated. The antigen and the controls

10 are added to the filter plate at 50 µL / well, then covered and allowed to incubate in the dark for 1 hour on the stirrer. After a washing step an unknown secondary antibody is added at 50 µL / well using the same dilution (or concentration if known) as used for the primary antibody. Then, the plates are incubated in the dark for 2 hours at RT on the stirrer followed by a washing step. Then 50 µL / well of biotinylated mxhIgG diluted 1: 500 is added and allowed to incubate in the dark for 1 hour on the stirrer at RT. After

A wash step is added 50 µL / well of streptavidin-PE at 1: 1000 and allowed to incubate in the dark for 15 minutes in the stirrer at RT. After a washing step, each well is resuspended in 80 µL of blocking buffer and read using Luminex. The results show that monoclonal antibodies belong to different clusters. Competitive antibody binding of different clusters supports antibody specificity for similar or adjacent epitopes. Non-competitive binding supports the specificity of antibodies by unique epitopes.

20 Three clusters were created to further test the binding of six antibodies directed against GPNMB. Cluster 1 included GPNMB antibodies (1.2.1), (1.10.1) and (2.22.1). Cluster 2 included GPNMB antibodies (2.3.1) and (1.15.1) and cluster 3 included GPNMB antibody (2.10.1). The results of the clustering tests are given below in Tables 8 and 9.

image40

Table 8 Table 9

BB

 1.1 1.2 1.3 1.5 1.7 1.8 1.9 1.11 1.12  1.13  1.15 xV5

BB

0 16 58 24 6 25 14 9 8 9 7 fifteen 32

1.1

-16 0 57 16  -29  3. 4 9 -35 -9 -7 -24 35 28

1.2

-42 -16 0 -60 -89 -49 -81 -75 -73 -65 -81 -43 Four. Five

1.3

-eleven -33 8 0 -75 -40 -49 171 -29 -33 -67 -73 -fifteen

1.5

25 35 64 60 0 twenty 10 24  17  27  12 -8 61

1.7

-one 76 65 twenty -8 0 -8 4 4 6 -3 -3 95

1.8

-7 29 Four. Five 35 -3 -7 0 4 -one 0 -6 3 52

1.9

-5 18 47 -7 -10 3 4 0 4 5 -5 -one 17

1.11

18 40 60 29 -eleven one fifteen 16 0 8 5 -2. 3 48

1.12

-10  26 43 27 -5 3 -12 -4 -12 0 -9 -13 57

1.13

one 30 40 27 2 9 2 10  eleven  17 0 -13 59

1.15

-19 91 79 71 fifteen twenty-one 8 12  10  fifteen  13 0 89

xV5

41 134 239 46 5 443 230 -one 70  257 24  535 0

I

II III IV V SAW VII VIII IX

1.1

1.2 1.3 1.5  1.7  1.8 1.9 1.15  xV5

1.13

1.11

1.12

image41

1.1

 1.2 1.3 1.5 1.7 1.8 1.9 1.11 1.12 1.13 1.15 xV5 BB

1.1

0 72 39 -36  49 8 -14 -3 18  -14 35 28 -2

1.2

10 0 -60 -103 -46 -64 -76 -71 -69 -83 -74 44 -46

1.3

-49 -9 0 -111 -88 -78 281 -66 -57 -93 -115 -89 -33

1.5

61 106 77 0 13 28 17 twenty 40  2 -3 87 19

1.7

94 77 51 -25  0 -9 -3 12 4 -4 -17  96 17

1.8

42 71 74 -24 2 0 -9 one -one -12 -5 61 4

1.9

14 74 28 -24  6 4 0 3 5 -13 8 16 -17

1.11

59 66 77 -twenty  3 -5 13 0 eleven  -9 -5 92 twenty-one

1.12

84 67 61 -36 -12 -8 -6 -4 0 -16 -3. 4 95 12

1.13

74 93 49 -12 22 12 2. 3 twenty-one 19  0 twenty  98 55

1.15

127 90 51 -9 17 12 19 19 twenty-one  5 0 125 59

xV5

189 330 22 14  611 376 -17 113 445  44 750 0 100

BB

25 73 65 3 3. 4 2. 3 14  19 22  13 39 44 0

I

II III IV V SAW VII VIII

Cut = 100

1.1 1.2 1.3 1.5  1.7 1.9 1.15 xV5

1.8

1.11

1.12

1.13

I

II III IV V SAW VII VIII  IX

Cut = 90

1.1 1.2 1.3 1.5  1.7 1.8  1.9 1.15 xV5

1.13

1.11

1.12

Example 5: GPNMB Immunohistochemical (IHC) Analysis

Monoclonal antibodies directed against GPNMB were evaluated for reactivity with specimens of

5 frozen and fixed fabrics. Tissue sections (5 µm) of formalin fixed and paraffin embedded tissue samples were cut and rehydrated by incubations in xylene and a series in graduated ethanol ending in PBS. The endogenous peroxidase activity was inactivated in a 3% solution of hydrogen peroxide in methanol.

Tissue sections were blocked in blocking buffer (5% BSA (Sigma), 1% goat serum (Jackson Immunolabs, West Grove, PA) in PBS) for 1 hour. Primary and secondary antibodies were precomplexed in 5% BSA and 1% goat serum in PBS for 1 hour at 37 ° C at an approximately 10: 1 molar ratio of antibody directed against GPNMB or control IgG with respect to antibody of goat

image42

directed against secondary biotinylated human IgG (Jackson Immunolabs). The complexes were blocked with a 1: 2000 dilution of human serum and incubated again for 1 hour at 37 ° C. Tissue sections were incubated with GPNMB directed antibody or isotype control antibody complexes diluted in blocking buffer for 1 hour. Sections were washed in 3 changes of PBS for 5 to 10 minutes each and incubated with

5 a 1: 200 dilution of horseradish peroxidase conjugated to streptavidin (Jackson Immunolabs) in blocking buffer for 30 minutes and then washed as before. The antibody was detected using the DAB reagent (Vector labs). The sections were counterstained in hematoxylin (Fisher Scientific) and dehydrated by alcohol and xylene and covered on the coverslip with Permount (Fisher Scientific).

MAbs directed against GPNMB 2.22.1 and 2.22.2 were used to stain micromatrices of normal human tissue

10 and tumor (IMPATH, Los Angeles, CA). Positive staining was observed in carcinomas of the lung, ovary, renal, esophagus and head and neck, squamous cell carcinoma, melanomas and specimens of normal skin. Melanoma and lung carcinomas showed the highest staining intensities with subcellular staining located in the membrane and cytoplasm. The mAb directed against GPNMB 2.10.2 also stained primary melanoma.

Staining of antibodies directed against GPNMB of melanoma tissue matrices showed a large proportion of melanoma cases that stained positively as shown in Tables 10 and 11.

Table 10: mAb melanoma staining intensity directed against GPNMB

Staining Intensity *

No. of samples %

0

10 17

one

one

2

1-2

 2 3

1-3

 eleven 19

2

9 fifteen

2-3

 17 29

3

9 fifteen

Total

 n = 59 100

On a scale of 0 (no staining) to 3 (strong staining)

Table 11: mAb staining frequency directed against GPNMB

Tumor reactivity in% *

No. of samples %

0-24

 18 31

25-49

 6 10

50-74

 7 12

75-100

 28 47

n = 59

100%

* tumor cells in% that have positive staining

The antibody directed against GPNMB stained 10 of 14 samples of lung squamous cell carcinoma (SCC) in a general oncology tissue microarray and 24 of 60 in a specific SCC matrix were positive.

image43

Example 6: FACS analysis of the binding of antibodies directed against GPNMB to melanoma cell lines

The specificity of antibodies directed against GPNMB by GPNMB protein bound to the cell membrane expressed by the UACC-62 melanoma cancer cell line was analyzed by FACS analysis. As a negative control, a renal cancer cell line, TK10, which did not express GPNMB antigen was used. As the negative control, the antibody of the same isotype K16.3 was used. The cells were washed twice with PBS (free of Ca and Mg), incubated with Versene at 37 ° C until the cells were detached, counted and aliquots of 1 million cells per test tube were taken. The cells were then washed twice and resuspended in ice cold FACS buffer (0.01 M HEPES, 0.15 M NaCl, 0.1% NaN3 and 4% SBF). Primary antibody at 1 µg / mL was added to the cells.

10 The cells were incubated on ice for 30 min, washed 2-3 times and resuspended in 1 ml of ice cold FACS buffer. Goat antibody directed against human immunoglobulin conjugated to R-PE (Jackson ImmunoResearch Laboratory) was added at 1: 100 dilution and the cells were incubated on ice for 30 min. After washing 3 times with 1 ml of cold FACS buffer on ice, the cells were fixed with 0.5-1 mL of 1% formaldehyde in PBS and analyzed by flow cytometry.

The results expressed as geometric mean ratios are summarized in Table 12 and show UACC-62 cells, but not TK10 cells, which express highly CR011 protein on the cell surface that was detected by antibodies 2.10.2; 2.22.1 and 1.15.1.

Table 12: Ratio of geometric means of antibody staining

directed against GPNMB (with respect to pK16)

A. Antibody

B. UACC-62 cells C. TK10 cells

D. 2.10.2

E. 2.60 F. 1.10

G. 2.22.1

H. 4.46 I. 1.10

J. 1.2.2

K. 1.24 L. 0.98

M. 1.15.1

N. 7.89 O. 0.96

P. 2.6.2

Q. 1.90 R. 1.70

twenty

To examine the relative expression of the GPNMB antigen between melanoma cell lines, the mAb 1.15.1 antibody was used to measure a panel of 15 melanoma cell lines by FACS analysis. As shown in Table 13, 80% (12/15) of the cell lines showed GPNMB antigen expression. The SK-MEL-2 cell line demonstrated the highest ratio of geometric means between the cell lines tested.

25 Table 13: Relationship of geometric means of antibody staining directed against GPNMB of melanoma cell lines

Cellphone line

Ratio of geometric means

(with respect to the isotype)

SK-MEL-2

 16.5

M14

16.1

MEWO

 14.1

WM-266-4

 13.6

HEMNLP

 10.2

G361

 8

HT144

7.4

UACC-257

 7

image44

RPMI-7951

 6

SK-MEL-5

 5.7

UACC-62

 5.5

A2058

4.1

SK-MEL-24

 1.9

WM115

 1.3

LOXIMVI

one

Example 7: FACS analysis of mAb binding directed against GPNMB to lymphoma and leukemia

To determine the relative expression of GPNMB on the surface of hematopoietic malignant cells, cell lines derived from various lymphomas and leukemia were incubated with antibody directed against GPNMB and analyzed by FACS. Cells derived from lymphoma or leukemia were washed twice with ice cold FACS buffer and resuspended at 1 million cells per test tube. The 1.15.1 mAb antibody at 1 µg / ml was added to the cells and the cells were incubated on ice for 30 min. Then, the cells were washed 2-3 times and resuspended in 1 ml of ice cold FACS buffer. Goat anti-human antibody conjugated with R-PE at 1: 100 dilution was added and the cells were incubated on ice for 30 min. The cells were washed 3 times with 1 ml of 10 cold FACS buffer on ice, fixed with 0.5-1 ml of 1% formaldehyde in PBS and analyzed by flow cytometry.

Approximately half of the cell lines examined, which were derived from both myeloid lineages

as lymphoids, they showed expression on the cell surface of GPNMB (Table 14). The U937 cell line demonstrated the

greater ratio of geometric means between the cell lines tested.

Table 14: Relationship of geometric means of antibody staining directed against GPNMB of lymphoma cells and leukemia

Cellphone line

Ratio of geometric means

U937

 (histiocytic, monocytic lymphoma) 17.3

Jurkat

(acute T-cell leukemia) 14.7

MR

(Anaplastic large T-cell lymphoma, ALCL) 7.1

KG-1

(acute myelogenous leukemia) 6.9

MOLT-4

(acute lymphoblastic T-cell leukemia) 6.2

THP-1

(acute monocytic leukemia) 6.1

MV4-11

 (myelomonocytic leukemia) 1.9

AML-193

(acute monocytic leukemia) 1.8

HUT-78

(T cell lymphoma) 1.5

CCRF-CEM

(acute lymphoblastic T-cell leukemia) 10.9

Karpas 299

(ALCL) 10.7

SU-DHL-1

 (ALCL) 4.8

SU-DHL-4

(B cell lymphoma) 1.8

ML-2

(acute myelomonocytic leukemia) 2.1

H H

(cutaneous T-cell leukemia) one

image45

SUP-M2

 (ALCL) 4.8

PL-21

(AML) 12

OF THE

 (ALCL) 7.9

SIG-M5

(acute monocytic leukemia) 2.9

K562

(Chronic myelogenous leukemia) 2.8

KG1a

(acute myelogenous leukemia) 2.7

HL-60

(acute promyelocytic leukemia) 2.3

WSU-NH2

(B cell lymphoma) one

EOL-1

(AML) one

HUT-102

(T cell lymphoma) one

5

10

fifteen

twenty

25

30

35

Example 8: GPNMB protein detection by IP and Western blot analysis

The cells were washed twice with PBS (free of Ca and Mg), incubated with Versene at 37 ° C until the cells were detached, counted and lysed in lysis buffer (0.15 M NaCl, Tris-HCl 0, 02 M, 10% glycerin, 1% NP-40, 0.01 M EDTA) and protease inhibitors containing pancreas extract, Pronase, thermolysin, chymotrypsin and papain (Roche, Germany) for 30 min on ice. Supernatants were collected and protein concentrations were determined by the BCA protein assay kit (Pierce, USA). Primary antibody was added to the cell lysates and incubated on ice for 3 h followed by the addition of G-agarose protein (Amersham, USA) for 2 h. Immunoprecipitated proteins were washed, boiled in sample buffer and resolved by 4-20% gels. For immunoblotting, the proteins were transferred to PVDF membranes (Invitrogen, USA) and probed with antibody directed against GPNMB (0.5 µg / ml) followed by goat antibody directed against human IgG conjugated with HRP (Jackson ImmunoResearch Laboratory) at dilution 1: 4000. Immunocomplexes were detected with Western ECL transfer detection reagents (Amersham, USA).

Western blot analysis showed that antibodies directed against GPNMB immunoprecipitated the GPNMB protein expressed in cell lysates of UACC-62, SK-MEL-5 and SK-MEL-2 cell lines. The results coincide with the cell surface expression determined by FACS analysis.

Example 9: Indirect destruction of cells mediated by antibodies directed against GPNMB

UACC-62, a cell line that expresses the GPNMB antigen, and TK10, a cell line that does not express, were seeded on flat-bottom 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ, USA). .) at a density of 3000 cells per well. Once the cells reached ~ 25% confluence, 100 ng / well of secondary antibody-toxin conjugate (goat antibody directed against IgGsaporin; Advanced Targeting Systems, San Diego, USA, HUM-ZAP; from cat. IT-22). The mAbs directed against GPNMB 2.10.2, 2.22.1, 1.15.1 or the isotype control mAb (PK16.3) were added to each well at a final concentration of 10 or 50 ng / ml. A monoclonal antibody directed against EGFR (MS-269-PABX, NeoMarkers, Fremont, CA, USA) was used as a positive primary antibody control. The 5FU chemotherapy reagent at 600 µM was used as a positive reagent control. On day 5, the cells were trypsinized, transferred to 6-well tissue culture plates and incubated at 37 ° C. The plates were examined daily and between 8-10 days, all plates were stained with Giemsa and the colonies were counted.

The percentage viability of UACC-62 positive for GPNMB after treatment is shown in Figure

2. The 5-FU chemotherapy reagent induced complete destruction, while the addition of the secondary antibody conjugated to the saporin toxin alone or in combination with the PK16.3 isotype control antibody had no effect on cell growth for both lines. cell phones. Both UACC-62 and TK10 cell lines express the EGFR protein and the addition of EGFR specific antibody at 50 ng / mL and the secondary antibody-toxin conjugate produced complete destruction of the UACC-62 and TK10 cells. At the same dose, the three GPNMB specific antibodies, 2.10.2, 2.22.1 and 1.15.1, induced more than 70% of the destruction of UACC-62 cells. Antibodies directed against GPNMB 2.10.2 and 2.22.1 induced less than 5% and 1.15.1 less than 24% cell death in GPKMB-negative TK10 cells.

image46

5

10

fifteen

twenty

25

30

35

Example 10: Destruction of cells by antibodies directed against GPNMB conjugated to auristatin E (AE)

UACC-62 and TK10 cells were seeded in flat-bottom 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ, USA). On day 2 or when the cells reach ~ 25% confluence, various concentrations (1 to 1000 ng / mL) of unconjugated antibodies and conjugated with auristatin E (Seattle Genetics, Bothell, WA, USA) were added to the cells. UU.), Including isotype control, EGFR (NeoMarkers MS-269-PABX, Fremont, CA, USA), 2.22.1 or 2.10.2. The mAb 2.3.1 was chosen for isotype control in this study because it does not bind to cells expressing GPNMB as demonstrated by FACS analysis. A monoclonal antibody generated against the EGF receptor was used to demonstrate specific destruction mediated by antibody conjugated to AE. On day 5, the cells were trypsinized, transferred to 6-well tissue culture plates and incubated at 37 ° C. Plates were examined daily. On days 8-10, all the plates were stained with Giemsa and the colonies were counted on the plates.

The percentage viability in UACC-62 positive cells and GPKMB negative TK10 cells is presented in Figure 3. The results indicate that the unconjugated 2.6.2 immunoconjugate and AE conjugate had no effect on the growth of both UACC cells. -62 as TK10. However, both UACC-62 and TK10 cell lines were susceptible to cell destruction mediated by the AE-EGFR immunoconjugate in a dose-dependent manner with more than 95% cell death at 1000 ng / ml. At the same dose, both 2.22.1-AE and 2.10.2-AE immunoconjugates induced approximately 75% of UACC62 cell death when compared to isotype control. The response to cell destruction was dose dependent. The survival of GPKMB-negative TK10 cells was not affected by immunoconjugates 2.22.1-AE or 2.10.2-AE at the same dose range. These results demonstrate the specific and cytotoxic effects of antibodies directed against GPNMB conjugated to AE on antigen-expressing cells.

Example 11: Melanoma cells susceptible to destruction by immunoconjugate mAb 1.15.1-AE

Melanoma cell lines were seeded in flat-bottom 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ, USA). On day 2 or when the cells reach ~ 25% confluence, various concentrations of 1.15.1 without conjugate and conjugated with auristatin E. were added to the cells. In this study mAb 2.6.2-AE was also used as a control of conjugated isotype. On day 5, the cells were trypsinized, transferred to 6-well tissue culture plates and incubated at 37 ° C. Plates were examined daily. On days 8-10, all the plates were stained with Giemsa and the colonies were counted on the plates.

The IC50 of destruction mediated by 1.15.1-AE on positive and negative GPNMB cells is presented in Table 15. 1.15.1 unconjugated and 2.6.2 conjugated with AE had no effect on the growth of all cell lines of melanoma tested. However, the SK-MEL-2, WM-266-4, G361, UACC-257, UACC-62, RPMI-7951 and SK-MEL-5 cell lines were susceptible to destruction mediated by 1.15.1-AE in a dose dependent mode. SK-MEL-2 demonstrated the lowest IC50 in this study (Table 15). These results show the specific and cytotoxic effects of 1.15.1 conjugated with AE on most melanoma cells expressing GPNMB.

Table 15: Relationship of geometric means and IC50 values of destruction by 1.15.1-AE of melanoma cells

Melanoma

Ratio of geometric means Clonogenic assay with 1.15.1-AE

Cellphone line

(with respect to the isotype) IC50 in ng / ml (pM)

SK-MEL-2

 16.5 111 (750)

M14

 16.1 Inconclusive

MEWO

 14.1 Inconclusive

WM-266-4

 13.6 345 (2300)

HEMNLP

 10.2 Inconclusive

G361

 8 1053 (6500)

HT144

7.4 Inconclusive

image47

UACC-257

 7 825 (5500)

RPMI-7951

 6 972 (6000)

SK-MEL-5

 5.7 237 (1600)

UACC-62

 5.5 697 (4300)

A2058

4.1 Without effect

SK-MEL-24

1.9 No data

WM115

1.3 No data

LOXIMVI

one Without effect

Example 12: Destruction by mAb 1.15.1-AE of lymphoma and leukemia cell lines

Lymphoma or leukemia cell lines were mixed with methylcellulose base medium (R&D Systems, USA) and at various concentrations of unconjugated 1.15.1 antibody and conjugated with auristatin E before seeding in

5 6-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ, USA). MAb 2.6.2AE was also included as a conjugated isotype control in this study because it does not bind to cells expressing GPNMB. The plates were incubated at 37 ° C and examined daily. On days 14-18 the colonies were counted on the plates.

The IC50 of cell destruction induced by 1.15.1-AE on antigen-expressing cells is presented in Table 16. 1.15.1 unconjugated and the immunoconjugate of 2.6.2 conjugated to AE had no effect on 10 growth of all hematopoietic cell lines positive for antigen. However, as presented in Table 16, U937, SR and THP-1 cell lines derived from either myeloid or lymphoid lineage were susceptible to destruction mediated by 1.15.1-AE in a dose-dependent manner with values of IC50 ranging from 207 ng / ml (1.4 nM) to 340 ng / ml (2.4 nM). These results show the specific and cytotoxic effects of immunoconjugate 1.15.1-AE on the hematopoietic malignant cell lines that express the GPNMB antigen.

15 Table 16: Relations of geometric means and IC50 values of the destruction by 1.15.1-AE of lymphoma and leukemia cells

Clonogenic assay with 1.15.1-AE

Cellphone line

Ratio of geometric means IC50 in ng / mL (pM)

U937

 (histiocytic, monocytic lymphoma) 17.3  340 (2400)

Jurkat

(acute T-cell leukemia) 14.7 No effect (repeat)

MR

(Anaplastic large T-cell lymphoma, ALCL) 7.1  296 (2000)

KG-1

(acute myelogenous leukemia) 6.9 No growth

MOLT-4

(acute lymphoblastic T-cell leukemia) 6.2 No effect (repeat)

THP-1

(acute monocytic leukemia) 6.1 207 (1400)

MV4-11

(myelomonocytic leukemia) 1.9 ND

AML-193

(acute monocytic leukemia) 1.8 ND

HUT-78

(T cell lymphoma) 1.5 ND

image48

CCRF-CEM

(acute lymphoblastic T-cell leukemia) 10.9 No growth

Karpas 299

(ALCL) 10.7 Inconclusive

SU-DHL-1

(ALCL) 4.8 Without effect

SU-DHL-4

(B cell lymphoma) 1.8 ND

ML-2

 (acute myelomonocytic leukemia) 2.1 ND

H H

(cutaneous T-cell leukemia) one ND

SUP-M2

(ALCL) 4.8 No growth

PL-21

(AML) 12 Without effect

OF THE

(ALCL) 7.9 Without effect

SIG-M5

(acute monocytic leukemia) 2.9 ND

K562

(chronic myelogenous leukemia) 2.8 ND

KG1a

(acute myelogenous leukemia) 2.7 ND

HL-60

 (acute promyelocytic leukemia) 2.3 ND

WSU-NH2

(B cell lymphoma) one ND

EOL-1

(AML) one ND

HUT-102

(T cell lymphoma) one ND

* ND: without doing

Example 13: CR011-vcMMAE inhibits the growth of human SK-MEL-2 melanoma xenografts leading to complete regression of melanoma tumor established in athymic mice (study N-386)

The N-386 study was conducted to evaluate the potency and therapeutic efficacy of the antibody-drug conjugate, 5 CR011-vcMMAE, against the human SK-MEL-2 melanoma xenograft established in athymic mice.

Materials and procedures:

Experimental animals: Five to 6 weeks old athymic mice (female CD-1 nu / nu) used for xenografts of human tumors were obtained from Harlan Laboratories (Indianapolis, IN). The animals were housed in specific pathogen-free conditions according to the guidelines of the International Association for

10 evaluation and accreditation of laboratory animal care (AAALAC International). Experimental animals were provided with granulated food and water at will and kept in a room with conditioned ventilation (HVAC), temperature (22º ± 2ºC), relative humidity (55% ± 15%) and photoperiod (12 h). All studies were carried out with protocols for the care and use of approved institutional animals.

Xenograft models of human melanoma. Tumor inhibitory activity of the immunoconjugate

CR011-MMAE was measured in an anti-tumor xenograft model using nude mice according to published procedures (see Geran et al., Cancer Chemother. Rep. 3: 1-104 (1972)). Briefly, experimental animals were implanted subcutaneously by trocar small fragments of a human melanoma (60-125 mg) removed from donors of atymic mouse tumor. When the tumors were established (10-20 days), the animals formed pairs in groups (n = 6 mice / group) and the treatment was administered by intravenous injection (vein of the

20 tail).

SK-MEL-2 human melanoma (ATCC HTB-68 #) was derived from a metastatic site (thigh skin) of a 60-year-old Caucasian male with malignant melanoma, and SK-MEL-5 human melanoma (No. ATCC HTB-70) was derived from a metastatic site (axillary lymph node) of a 24-year-old female with malignant melanoma (see Fogh et al., J. Natl. Cancer Inst. 59: 221-226 (1977)) . Both cell lines were obtained from the American Type Culture Collection.

image49

The effects of the treatment were monitored by repeated measurements of the tumor through 2 diameters with Vernier calipers; Tumor size (in mg) was calculated using a standard formula, (W2 x L) / 2, assuming a specific gravity of 1.0. Tumor size and body weights were evaluated twice a week. Mice were examined daily, however, dying animals were humanely sacrificed if clinical indications of excessive pain or discomfort were observed (i.e., prostration, hunched posture, paralysis / paresis, distended abdomen, ulcers, abscesses, seizures and / or hemorrhages ). Animals with tumors exceeding 2,000 mg were removed from the study and sacrificed humanely.

Xenograft studies in the athymic mouse have been shown to effectively demonstrate antitumor effects for a variety of agents that have subsequently shown to have activity against clinical cancer (Johnson et al., Br J Cancer 84: 1424-1431 (2001)).

Results:

Antitumor effects in vivo against SK-MEL-2 melanoma. Based on the potency and cytotoxicity of CR011-vcMMAE against cells expressing GPNMB in vitro, antitumor effects were examined in vivo.

The effects of intravenous treatment with CR011-vcMMAE on the growth of subcutaneous human SK-MEL-2 melanoma are shown in Figure 1. After implanting fragments of the SK-MEL-2 tumor and that the tumors had been established (day 17 , 61 mg), treatment began with intravenous administration of: CR011vcMMAE (0.625 - 20 mg / kg iv, every 4 days for a total of 4 treatments (i.e., q4d X4); saline controls and saline solution buffered with phosphate (iv, q4d X4); and two known antitumor reference agents, vinblastine sulfate (iv, 1.7 mg / kg, q4d X4) and paclitaxel (iv, 24 mg / kg, q2d X4). The reference agents were administered at the maximum tolerated dose (DMT) determined in previous studies.

Tumors in animals treated with saline or PBS grew progressively until the tumor mass reached 2,000 mg, at which time the animals were removed from the study and sacrificed humanely. SK-MEL-2 tumors had a high "acceptance" rate in immunosuppressed hosts (97%) and a low spontaneous regression rate (3%) (Dykes et al., Development of human tumor xenograft models for in vivo evaluation of new antitumor drugs, in Immunodeficient mice in Oncology, vol. 42 (Fiebig HH and Berger DPe eds), page 1-22, Contrib. Oncol. Basel, Karger (1992)).

Vinblastine produced a very slight, but not significant, antitumor effect (P ≤ 0.20); In this and other tumor models (for example, SK-MEL-5), vinblastine produces a noticeable inhibition of tumor growth, but is only occasionally significant. However, paclitaxel showed significant inhibition of tumor growth and tumor stasis (i.e., 100% growth inhibition) for approximately 2 weeks after starting treatment (P ≤ 0.0077).

The antitumor effects of CR011-vcMMAE administered i.v. mice that had SK-MEL-2 were notable. At 20, 10, 5 or 2.5 mg / kg, the tumors rapidly decreased in size for most experimental animals; Significant treatment effects were observed and 4 days after treatment began (P ≤ 0.014). Tumors that experienced a complete setback did not grow again during the observation period (> 200 days).

The animals in this study showed no abnormal effects of the treatment on the macroscopic examination. Body weight determinations twice a week showed neither observable nor statistically significant effects of treatment with CR011-vcMMAE on body weight or weight gain.

Conclusions:

CR011-vcMMAE produces substantial, dose-dependent and reproducible antitumor effects that begin as tumor growth inhibition, but that soon lead to the complete regression of established human melanoma xenografts; Regressions are long lasting and tumor growth has not been observed after satisfactory therapy.

Example 14: Sequencing of antibodies and their corresponding DNA

Heavy chain sequences and kappa transcripts derived from mAbs directed against human GPNMB were obtained from hybridomas by direct sequencing of PCR products generated from poly (A +) RNA. PCR products were also cloned into pCRII using a TA cloning kit (Invitrogen) and both chains were sequenced using Prism dye terminator sequencing kits and an ABI 377 sequencing instrument. Each PCR reaction used a mixture of primers 5 'direction provided in Table 17 below.

image50

Table 17: Used primers

Vh

cacc ATG GAC TGG (C) ACC TGG AGG ATC SEQ ID NO: 290

Vh

cacc ATG GAC TGG ACC TGG AGA (C) ATC SEQ ID NO: 291

Vh

cacc ATG GAC TGG ACC TGG AGG GTC SEQ ID NO: 292

Vh

cacc ATG GAC TGG ATT TGG AGG ATC SEQ ID NO: 293

Vh

cacc ATG GAC ACA CTT TGC TC (A) C AC SEQ ID NO: 294

Vh

cacc ATG GAA (G) TTG GGG CTG AGC TGG SEQ ID NO: 295

Vh

cacc ATG GAG TTG (T) GGA CTG AGC TGG SEQ ID NO: 296

Vh

cacc ATG GAG TTT GGG CTG (T) AGC TGG SEQ ID NO: 297

Vh

cacc ATG GAA CTG GGG CTC CGC TGG SEQ ID NO: 298

Vh

cacc ATG GAG TTG GGG CTG TGC TGG SEQ ID NO: 299

Vh

cacc ATG GAG TTT TGG CTG AGC TGG SEQ ID NO: 300

Vh

cacc ATG ACG GAG TTT GGG CTG AGC SEQ ID NO: 301

Vh

cacc ATG AAA (G) CAC CTG TGG TTC TTC SEQ ID NO: 302

Vh

cacc ATG AAA CAT CTG TGG TTC TTC SEQ ID NO: 303

Vh

cacc ATG GGG TCA ACC GCC ATC CTC SEQ ID NO: 304

Vh

cacc ATG TCT GTC TCC TTC CTC ATC TTC SEQ ID NO: 305

VK

ATG GGG TCC CAG GTT CAC CTC SEQ ID NO: 306

VK

ATG TTG CCA TCA CAA CTC ATT G SEQ ID NO: 307

All sequences were analyzed by alignments with the "V BASE sequence directory" (Tomlinson et al., MRC Center for Protein Engineering, Cambridge, UK) using the 5 MACVECTOR® and GENEWORKS ™ software programs.

Example 15: Structural analysis of antibodies directed against GPNMB

The variable heavy chains and the variable light chains for the antibodies shown in Table 17 were sequenced to determine their DNA and protein sequences. 10 Antibody -1.10.2 Heavy chain variable region Nucleotide sequence

image51

image52

Amino acid sequence

image53

REGION

SEQUENCE AA WASTE * SEQ ID NO:

FRI

QVQLQESGPGLVKPCONJUNTOLSLTCTVS 1-25 SEQ ID NO: 3

CDR1

GDSISNYYWS 26-35 SEQ ID NO: 4

FR2

WIRQPPGKGLEWIG 36-49 SEQ ID NO: 5

CDR2

YFYYSGSTNYNPSLKS 50-65 SEQ ID NO: 6

FR3

RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 66-97 SEQ ID NO: 7

CDR3

DRGWADY 98-104 SEQ ID NO: 8

FR4

WGQGTLVTVSSA 105-116 SEQ ID NO: 9

* AA waste of SEQ ID NO: 2

Variable light chain region Nucleotide sequence

image52

Amino acid sequence Antibody -1.15.1 Heavy chain variable region Nucleotide sequence

image52

image54

TABLE 19. Domains of region V of the light chain of 1.10.2.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

EIVLTQSPGTLSLSPGERATLSC 1-23 SEQ ID NO: 12

CDR1

RTSQSISSSILA 24-35 SEQ ID NO: 13

FR2

WYQQKPGQVPRLLIY 36-50 SEQ ID NO: 14

CDR2

GASSRAT 51-57 SEQ ID NO: 15

FR3

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 58-89 SEQ ID NO: 16

CDR3

QQYGSSIT 90-97 SEQ ID NO: 17

FR4

FGQGTRLEIKR 98-108 SEQ ID NO: 18

* AA waste of SEQ ID NO: 11

image52

Amino acid sequence

image52

TABLE 20. Domains of region V of the heavy chain of 1.15.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLQESGPGLVKPSQTLSLTCTVSGGSIS 1-30 SEQ ID NO: 21

CDR1

SFNYYWS 31-37 SEQ ID NO: 22

FR2

WIRHHPGKGLEWJG 38-51 SEQ ID NO: 23

CDR2

YIYYSGSTYSNPSLKS 52-67 SEQ ID NO: 24

FR3

RVTISVDTSKNQFSLTLSSVTAADTAVYYCAR 68-99 SEQ ID NO: 25

CDR3

GYNWNYFDY 100-108 SEQ ID NO: 26

FR4

WGQGTLVTVSSA 109-120 SEQ ID NO: 27

* AA waste of SEQ ID NO: 20

image55

Variable light chain region Nucleotide sequence

image52

Amino acid sequence

image56

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

EIVMTQSPATLSVSPGERATLSC 1-23 SEQ ID NO: 30

CDR1

RASQSVDNNLV 24-34 SEQ ID NO: 31

FR2

WYQQKPGQAPRLLIY 35-49 SEQ ID NO: 32

CDR2

GASTRAT 50-56 SEQ ID NO: 33

FR3

GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 57-88 SEQ ID NO: 34

CDR3

QQYNNWPPWT 89-98 SEQ ID NO: 35

FR4

FGQGTKVEIKR 99-109 SEQ ID NO: 36

* AA waste of SEQ ID NO: 29

Antibody -1.2.2 Heavy chain variable region

Nucleotide sequence

image52

Amino acid sequence Light chain variable region Nucleotide sequence

image52

image57

TABLE 22. Domains of region V of the heavy chain of 1.2.2.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

ITLKESGPTLVKPTQTLTLTCTFS 1-24 SEQ ID NO: 39

CDR1

GFSLSAGGVGVG 25-36 SEQ ID NO: 40

FR2

WIRQPPGKALEWLA 37-50 SEQ ID NO: 41

CDR2

LIYWNDDKRYSPSLRS 51-66 SEQ ID NO: 42

FR3

RLTITKDTSKNQVVLTITNMDPVDTATYYCAH 67-98 SEQ ID NO: 43

CDR3

SHYDYDWGSYFDY 99-111 SEQ ID NO: 44

FR4

WGQGTLVTVSSA 112-123 SEQ ID NO: 45

* AA waste of SEQ ID NO: 38

image58

Amino acid sequence

image59

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIVMTQTPLSLPVTPGEPASISC 1-23 SEQ ID NO: 48

CDR1

RSSQSLLDSDDGNTILD 24-40 SEQ ID NO: 49

FR2

WILQKPGQSPQLLIY 41-55 SEQ ID NO: 50

CDR2

TLSYRAS 56-62 SEQ ID NO: 51

FR3

GVPDRFSGSGSGTDFTLNISRVEAEDVGVYYC 63-94 SEQ ID NO: 52

CDR3

MQRIEFPIT 95-103 SEQ ID NO: 53

FR4

FGQGTRLEIKR 104-114 SEQ ID NO: 54

* AA waste of SEQ ID NO: 47

image60

Antibody -1.7.1 Heavy chain variable region Nucleotide sequence

image52

Amino acid sequence

image61

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLQESGPGLVKPSQTLSLTCTV 1-25 SEQ ID NO: 57

CDR1

GGSISSANYYWT 26-37 SEQ ID NO: 58

FR2

WIRQHPGKGLEWIG 38-51 SEQ ID NO: 59

CDR2

YIYYSGSTYCNPSLKS 52-67 SEQ ID NO: 60

FR3

RVIISVDTSKNQFSLKLSSVTAADTAVYYCAR 68-99 SEQ ID NO: 61

CDR3

GYNWNYFDY 100-108 SEQ ID NO: 62

FR4

WGQGTLVTVSSA 109-120 SEQ ID NO: 63

* AA waste of SEQ ID NO: 56

Variable light chain region 10 Nucleotide sequence

image52

Amino acid sequence Antibody -2.10.2 Heavy chain variable region Nucleotide sequence

image52

image62

TABLE 25. Domains of region V of the light chain of 1.7.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIVMTQSPATLSVSPGERATLSC 1-23 SEQ ID NO: 66

CDR1

RASQSVSSNLA 24-34 SEQ ID NO: 67

FR2

WYQERPGQAPRLLIY 35-49 SEQ ID NO: 68

CDR2

GASTRAT 50-56 SEQ ID NO: 69

FR3

GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 57-88 SEQ ID NO: 70

CDR3

QQYNKWPPWT 89-98 SEQ ID NO: 71

FR4

FGQGTKVEIER 99-109 SEQ ID NO: 72

* AA waste of SEQ ID NO: 65

image52

Amino acid sequence

image52

image63

TABLE 26. 2.10.2 Domains of region V of the heavy chain.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QLVESGGGVVQPGRSLRLSCAAS 1-23 SEQ ID NO: 75

CDR1

GFAFSSYGMH 24-33 SEQ ID NO: 76

FR2

WVRQAPGKGLEWVA 34-47 SEQ ID NO: 77

CDR2

VISYDGNNKYYADSVKG 48-64 SEQ ID NO: 78

FR3

RFT1SRDNSKNTLILQMNSLRAEDTAVYYCAR 65-96 SEQ ID NO: 79

CDR3

DLVVRGRGYYYYFGMDV 97-114 SEQ ID NO: 80

FR4

WGQGTTVTVSSA 115-126 SEQ ID NO: 81

* AA waste of SEQ ID NO: 74

Variable light chain region Nucleotide sequence

image58

Amino acid sequence

image64

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIVMTQSPLSLPVTPGEPASISC 1-23 SEQ ID NO: 84

CDR1

RSSQSLLHSNGINILD 24-39 SEQ ID NO: 85

FR2

WILQKPGQSPQLLIY 40-54 SEQ ID NO: 86

CDR2

LGSNRAS 55-61 SEQ ID NO: 87

FR3

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 62-93 SEQ ID NO: 88

CDR3

MQGLQTPIT 94-102 SEQ ID NO: 89

FR4

FGQGTRLEIKR 103-113 SEQ ID NO: 90

* AA waste of SEQ ID NO: 83

image65

Antibody - 2.15.1 Heavy chain variable region Nucleotide sequence

image52

Amino acid sequence

image66

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLVESGGGVVQPGRSLRLSCAAS 1-25 SEQ ID NO: 93

CDR1

GFTFSNYGIH 26-35 SEQ ID NO: 94

FR2

WVRQAPGKGLEWVA 36-49 SEQ ID NO: 95

CDR2

VIWFDGRNKYYADSVKG 50-66 SEQ ID NO: 96

FR3

RFTISRDNSKNTLILQMNSLRAEDAAVYYCAR 67-98 SEQ ID NO: 97

CDR3

DPFDYGDSFFDY 99-110 SEQ ID NO: 98

FR4

WGQGTLVTVSSA 111-122 SEQ ID NO: 99

* AA waste of SEQ ID NO: 92

Variable light chain region 10 Nucleotide sequence

image52

Amino acid sequence Antibody -2.16.1 Heavy chain variable region Nucleotide sequence

image67

TABLE 29. Domains of region V of the light chain of 2.15.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

LTQSPSSLSASVRDRVTITC 1-20 SEQ ID NO: 102

CDR1

RASQDISNILA 21-31 SEQ ID NO: 103

FR2

WYQQKPGKVPNLLIY 32-46 SEQ ID NO: 104

CDR2

AASTLQ 47-52 SEQ ID NO: 105

FR3

GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC 53-84 SEQ ID NO: 106

CDR3

QKYNSAPLT 85-93 SEQ ID NO: 107

FR4

FGGGTKVEIKR 94-104 SEQ ID NO: 108

* AA waste of SEQ ID NO: 101

image52

Amino acid sequence

image68

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLVESGGGLVKPGGSLRLSCAAS 1-25 SEQ ID NO: 111 I

CDR1

GFTFSOYYMT 26-35 SEQ ID NO: 112

FR2

WIRQAPGKGLEWVS 36-49 SEQ ID NO: 13

CDR2

YISISGSITHYADSVKG 50-66 SEQ ID NO: 114

FR3

RFTMSRADNKNSLILQMNSLRAEDTAVYYCAR 67-98 SEQ ID NO: 115

CDR3

DGAAAGTDAFDI 99-110 SEQ ID NO: 116

FR4

WGHGTKVTVSSA 111-122 SEQ ID NO: 117

* AA waste of SEQ ID NO: 110

image69

Variable light chain region Nucleotide sequence

image52

Amino acid sequence

image52

TABLE 31. Domains of region V of the light chain of 2.16.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

EIVMTQSPATLSVSPGDRATLSC 1-23 SEQ ID NO: 120

CDR1

RASQNVSSNLA 24-34 SEQ ID NO: 121

FR2

WYQQKPGQAPRLLIF 35-49 SEQ ID NO: 122

CDR2

GASTRAT 50-56 SEQ ID NO: 123

FR3

GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 57-88 SEQ ID NO: 124

CDR3

QQYHYWPT 89-96 SEQ ID NO: 125

FR4

FGPGTKVDIKR 97-107 SEQ ID NO: 126

* AA waste of SEQ ID NO: 119

Antibody -2.17.1

10 Heavy chain variable region Nucleotide sequence

image52

Amino acid sequence Light chain variable region Nucleotide sequence

image52

image70

TABLE 32. Domains of region V of the heavy chain of 2.17.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QLVQSGAEVKKPGASVKVSCKAS 1-23 SEQ ID NO: 129

CDR1

GYTFTGFYMH 24-33 SEQ ID NO: 130

FR2

WVRQTPGQGLEWMG 34-47 SEQ ID NO: 131

CDR2

WINPNSGGTYYVQKFQG 48-64 SEQ ID NO: 132

FR3

RVTMTRDTSISTVYMELSRLRSDDTAVYYCAR 65-96 SEQ ID NO: 133

CDR3

DGYSSGEDWFDP 97-108 SEQ ID NO: 134

FR4

WGQGTLVTVSSA 109-120 SEQ ID NO: 135

* AA waste of SEQ ID NO: 128

image58

Amino acid sequence

image71

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIVMTQTPLSLSVTPGQPASISC 1-23 SEQ ID NO: 138

CDR1

KSSQSLLHSGGKTILY 24-39 SEQ ID NO: 139

FR2

WILQRPGQPPQLLIY 40-54 SEQ ID NO: 40

CDR2

EVSNRFS 55-61 SEQ ID NO: 141

FR3

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 62-93 SEQ ID NO: 142

CDR3

MQSIHLPLT 94-102 SEQ ID NO: 143

FR4

FGGGTKVEIKR 103-113 SEQ ID NO: 144

* AA waste of SEQ ID NO: 137

image72

Antibody -2.21.1 Heavy chain variable region Nucleotide sequence

image52

Amino acid sequence

image73

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLEQSGGGLVKPGGSLRFSCAAS 1-25 SEQ ID NO: 147

CDR1

GFTFSSYSMN 26-35 SEQ ID NO: 148

FR2

WVRQAPGKGLEWVS 36-49 SEQ ID NO: 149

CDR2

FISSSSSYIYYADSVKG 50-66 SEQ ID NO: 150

FR3

RFTISRADNKNSLILQMNSLRAEDTAVYYCAR 67-98 SEQ ID NO: 151

CDR3

EDWVGATFDY 99-108 SEQ ID NO: 152

FR4

WGQGTLVTVSSA 109-120 SEQ ID NO: 153

* AA waste of SEQ ID NO: 146

Variable light chain region 10 Nucleotide sequence

image52

Amino acid sequence Antibody -2.22.1 Heavy chain variable region Nucleotide sequence

image52

image74

TABLE 35. Domains of region V of the light chain of 2.21.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIQLTQSPSSLSASVGDRVTITC 1-23 SEQ ID NO: 156

CDR1

RASQGIRNILA 24-34 SEQ ID NO: 157

FR2

WYQQKPGKVPKLLIY 35-49 SEQ ID NO: 158

CDR2

AASALKL 50-56 SEQ ID NO: 159

FR3

GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC 57-88 SEQ ID NO: 160

CDR3

QKYNSAPIT 89-97 SEQ ID NO: 161

FR4

FGQGTRLDIKR 98-108 SEQ ID NO: 162

* AA waste of SEQ ID NO: 155

image58

Amino acid sequence

image75

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLEQSGPGLVKPSQNLSLTCTVS 1-25 SEQ ID NO: 165

CDR1

GGSISSGGYFWS 26-37 SEQ ID NO: 166

FR2

WIRQHPGKGLEWIG 38-51 SEQ ID NO: 167

CDR2

YIYYSGNTYYNPSLKS 52-67 SEQ ID NO: 168

FR3

RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 68-99 SEQ ID NO: 169

CDR3

DYYYDTSGFSYRYDWYYGMDV 100-120 SEQ ID NO: 170

FR4

WGQGTTVTVSSA 121-132 SEQ ID NO: 171

* AA waste of SEQ ID NO: 164

image76

Variable light chain region Nucleotide sequence

image52

Amino acid sequence

image77

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIQLTQSPSSLSASVGDRVTITC 1-23 SEQ ID NO: 174

CDR1

PASQGIRNDLG 24-34 SEQ ID NO: 175

FR2

WYQQKPGKAPKRLIY 35-49 SEQ ID NO: 176

CDR2

AASSLQN 50-56 SEQ ID NO: 177

FR3

GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC 57-88 SEQ ID NO: 178

CDR3

LQHNTYPA 89-97 SEQ ID NO: 179

FR4

FGQGTKVEIKR 98-108 SEQ ID NO: 180

* AA waste of SEQ ID NO: 173

Antibody - 2.24.1

Variable heavy chain region 10 Nucleotide sequence

image52

Amino acid sequence

image52

image78

TABLE 38. Domains of region V of the heavy chain of 2.24.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QLVQSGAEVKKPGESLKISCQGS 1-23 SEQ ID NO: 183

CDR1

GYIFTNYWIG 24-33 SEQ ID NO: 184

FR2

WVRQMPGKGLEWMG 34-47 SEQ ID NO: 185

CDR2

VIYPDDSDTRYSPSFQG 48-64 SEQ ID NO: 186

FR3

QVTISADKSISTAILQWSSLKASDTAIYYCAR 65-96 SEQ ID NO: 187

CDR3

QKWLQHPFDY 97-106 SEQ ID NO: 188

FR4

WGQGTLVTVSSA 107-118 SEQ ID NO: 189

* AA waste of SEQ ID NO: 182

Variable light chain region

Nucleotide sequence

image58

Amino acid sequence

image52

TABLE 39. Domains of region V of the light chain of 2.24.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

EIVLTQSPGTLSLSPGERVTLSC 1-23 SEQ ID NO: 192

CDR1

RASQSVSSRILA 24-35 SEQ ID NO: 193

FR2

WYQQKPGQAPRLLIY 36-50 SEQ ID NO: 194

CDR2

GASSRAT 51-57 SEQ ID NO: 195

FR3

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYY 58-88 SEQ ID NO: 196

CDR3

QQYGSSPRT 89-97 SEQ ID NO: 197

FR4

FGQGTKVEIKR 98-109 SEQ ID NO: 198

* AA waste of SEQ ID NO: 191

image79

Antibody -2.3.1 Heavy chain variable region Nucleotide sequence

image52

Amino acid sequence

image80

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLVQSGAEVKKPGASVKVSCKAS 1-25 SEQ ID NO: 201

CDR1

GYTFTGYYMH 26-35 SEQ ID NO: 202

FR2

WVRQAPGQGLEWMG 36-49 SEQ ID NO: 203

CDR2

WINPNSGGTNYAQKFQD 50-66 SEQ ID NO: 204

FR3

RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR 67-98 SEQ ID NO: 205

CDR3

DFFGSGSLLYFDY 99-111 SEQ ID NO: 206

FR4

WGQGTLVTVSSA 112-123 SEQ ID NO: 207

* AA waste of SEQ ID NO: 200

Variable light chain region 10 Nucleotide sequence

image52

Amino acid sequence Antibody -2.6.1 Heavy chain variable region Nucleotide sequence

image52

image81

TABLE 41. Domains of region V of the light chain of 2.3.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIVMTQTPLSLSVTPGQPASISC 1-23 SEQ ID NO: 210

CDR1

KSSQSLLHSGGKTILY 24-39 SEQ ID NO: 211

FR2

WILQRPGQPPQLLIY 40-54 SEQ ID NO: 212

CDR2

EVSNRFS 55-61 SEQ ID NO: 213

FR3

GVPDRFSGSGSGTDFTLICISRVEAEDVGVYYC 62-93 SEQ ID NO: 214

CDR3

MQSIHLPLT 94-102 SEQ ID NO: 215

FR4

FGGGTKVEIKR 103-113 SEQ ID NO: 216

* AA waste of SEQ ID NO: 209

image52

Amino acid sequence

image82

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLVQSGAEVKKPGASVKVSCKAS 1-25 311

CDR1

 GYTFTGYYMH 26-35 312

FR2

WVRQAPGQGLEWMG 36-49 313

CDR2

 WINPNSGGTNYAQKFQD 50-66 314

FR3

RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR 67-98 315

CDR3

 DFFGSGSLLYFDY 99-112 316

FR4

WGQGTLVTVSSA 113-124 317

* AA waste of SEQ ID NO: 310

image83

Variable light chain region

Nucleotide sequence

image52

Amino acid sequence

image52

TABLE 43. Domains of region V of the light chain of 2.6.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIVMTQTPLSLSVTPGQPASISC 1-23 320

CDR1

 KSSQSLLHSGGKTILY 24-39 321

FR2

WILQRPGQPPQLLIY 40-54 322

CDR2

 EVSNRFS 55-61 323

FR3

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 62-93 324

CDR3

 MQSIHLPLT 94-102 325

FR4

FGGGTKVEIKR 103-113 326

* AA waste of SEQ ID NO: 319

Antibody -2.7.1

10 Heavy chain variable region Nucleotide sequence

image52

Amino acid sequence Light chain variable region Nucleotide sequence

image52

image84

TABLE 44. Domains of region V of the heavy chain of 2.7.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QVQLEQSGGGVVQPGRSLRLSCAAS 1-25 SEQ ID NO: 219

CDR1

GFTFNNYGMH 26-35 SEQ ID NO: 220

FR2

WVRQAPGKGLEWVA 36-49 SEQ ID NO: 221

CDR2

VIWYDGSIVKYYADSVKG 50-66 SEQ ID NO: 222

FR3

RFTISRDNSKNTLILQMNSLRAEDTAVYYCAK 67-98 SEQ ID NO: 223

CDR3

DEEYYYVSGLDY 99-110 SEQ ID NO: 224

FR4

WGQGTLVTVSSA 111-122 SEQ ID NO: 225

* AA waste of SEQ ID NO: 218

image58

Amino acid sequence

image52

TABLE 45. Domains of region V of the light chain of 2.7.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

LTQSPSSLSASVRDRVTITC 1-20 SEQ ID NO: 228

CDR1

RASQDISNILA 21-31 SEQ ID NO: 229

FR2

WYQQKPGICVPNLLIY 32-46 SEQ ID NO: 230

CDR2

AASTLQ 47-52 SEQ ID NO: 231

FR3

GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC 53-84 SEQ ID NO: 232

CDR3

QKYNSAPLT 85-93 SEQ ID NO: 233

FR4

FGGGTKVEIKR 94-104 SEQ ID NO: 234

* AA waste of SEQ ID NO: 227

image85

Antibody - 2.8.1 Heavy chain variable region Nucleotide sequence

image52

Amino acid sequence

image86

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

QITLKESGPTLVTPTQTLTLTCTFS 1-25 SEQ ID NO: 237

CDR1

GFSLSTGGMGVG 26-37 SEQ ID NO: 238

FR2

WIRQPPGKALDWLT 38-51 SEQ ID NO: 239

CDR2

LIYWNDDKHYSPSLKS 52-67 SEQ ID NO: 240

FR3

RLTITKDTSKNQVVLRMTNMDPVDTATYYCAH 68-99 SEQ ID NO: 241

CDR3

LHYDILTGFNFDY 100-112 SEQ ID NO: 242

FR4

WGQGTLVTVSSA 113-124 SEQ ID NO: 243

* AA waste of SEQ ID NO: 236

Variable light chain region 10 Nucleotide sequence

image52

Amino acid sequence

image52

image87

5

10

fifteen

twenty

25

30

35

TABLE 47. Domains of region V of the light chain of 2.8.1.

REGION

SEQUENCE AA WASTE * SEQ ID

FRI

DIVMTQTPLSLPVTPGEPASISC 1-23 SEQ ID NO: 246

CDR1

RSSQSLLDSDDGNTILD 24-40 SEQ ID NO: 247

FR2

WILQKPGQSPQLLIY 41-55 SEQ ID NO: 248

CDR2

TLSYRAS 56-62 SEQ ID NO: 249

FR3

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 63-94 SEQ ID NO: 250

CDR3

MQRIEFPLT 95-103 SEQ ID NO: 251

FR4

FGGGTKVEIKR 103-114 SEQ ID NO: 252

* AA waste of SEQ ID NO: 245

Example 16: Use of antibodies directed against GPNMB as a diagnostic agent

GPNMB antigen detection in a sample:

A protocol for an enzyme-linked immunosorbent assay (ELISA) for the detection of GPNMB antigen in a sample is given below. In the assay, the wells of a microtiter plate, such as a 96-well microtiter plate or a 384-well microtiter plate, are adsorbed for several hours with a first fully human monoclonal antibody directed against GPNMB. The immobilized antibody acts as a capture antibody for any of the GPNMB that may be present in a test sample. The wells are rinsed and treated with a blocking agent such as milk protein or albumin to prevent non-specific adsorption of the analyte.

Subsequently, the wells are treated with a test sample suspected of containing the GPNMB antigen, or with a solution containing a standard amount of GPNMB antigen. Such a sample may be, for example, a serum sample that is suspected to have circulating GPNMB levels that are considered to be diagnoses of a pathology.

After clarifying the test sample or standard, the wells are treated with a second antibody directed against fully human monoclonal GPNMB that is labeled by conjugation with biotin. The antibody directed against labeled GPNMB acts as a detection antibody. After clearing the excess of the second antibody, the wells are treated with horseradish peroxidase (HRP) conjugated with avidin and a suitable chromogenic substrate. The concentration of antigen in the test samples is determined by comparing with a standard curve developed from the standard samples.

This ELISA assay provides a highly specific and very sensitive assay for the detection of GPNMB antigen in a test sample.

Determination of GPNMB antigen concentration in patients:

A sandwich ELISA can also be used to quantify levels of GPNMB in human serum. The 2 antibodies directed against fully human monoclonal GPNMB used in the sandwich ELISA recognize different epitopes on the GPNMB molecule. The ELISA is performed as follows: ELISA plates (Fisher) are coated with 50 µl of capture antibody directed against GPNMB in coating buffer (0.1 M NaHCO3, pH 9.6) at a concentration of 2 µg / mL . After incubation at 4 ° C overnight, the plates are treated with 200 µl of blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% thimerosal in PBS) for 1 h at 25 ° C . The plates are washed (3x) using 0.05% Tween 20 in PBS (wash buffer, WB). Normal or patient sera (Clinomics, Bioreclaimation) are diluted in blocking buffer containing 50% human serum. Plates are incubated with serum samples overnight at 4 ° C, washed with WB and then incubated with 100 µl / well of antibody directed against biotinylated GPNMB for 1 h at 25 ° C. After washing, the plates are incubated with HRP-streptavidin for 15 min, washed as before and then treated with 100 µl / well of o-phenylenediamine in H2O2 (Sigma developing solution) for color generation. The reaction is stopped with 50 µl / well of H2SO4 (2 M) and analyzed using an ELISA plate reader at 492 nm. The GPNMB antigen concentration in serum samples is calculated by comparing with dilutions of purified GPNMB antigen using a four parameter curve fit program.

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Staging of cancer in a patient:

It will be appreciated that based on the results set forth and treated in the previous diagnostic examples it is possible to stage a cancer in a subject based on the GPNMB antigen expression levels. For a given type of cancer (for example, melanoma) blood samples are taken from the diagnosed subjects as being at various stages in the progression of the disease, and / or at various times in the therapeutic treatment of cancer. The concentration of GPNMB antigen present in blood samples is determined using a procedure that specifically determines the amount of antigen that is present. Such a procedure includes an ELISA procedure such as the procedure described in the previous diagnostic examples. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of antigen concentrations is designated that can be considered characteristic of each phase.

In order to stage the progression of cancer in a subject under study, or to characterize the subject's response to a therapy development, a blood sample is taken from the subject and the concentration of GPNMB antigen present in the sample is determined . The concentration thus obtained is used to identify in what concentration range the value is. The interval thus identified is related to a stage of progression or a stage of therapy identified in the various populations of diagnosed subjects, thus providing a stage in the subject under study.

Example 17: Diagnosis of cancer with antibodies directed against GPNMB

A subject that is suspected of having an ovarian cancer tumor is identified and a sample of tumor tissue suspected for the assay is removed. Then, the excised tissue is contacted with antibodies directed against GPNMB that have a colorimetric label. A determination is made as to whether antibodies directed against GPNMB specifically bind to the excised tissue. Binding is indicative of cancerous tissue, while the absence of binding is indicative of non-cancerous tissue. Therefore, the patient's condition is diagnosed to facilitate subsequent testing, counseling and / or treatment.

Example 18: Cancer treatment with antibodies directed against GPNMB

The choice of GPNMB as a target in tumor cells is useful for treating a subject at risk of or suffering from cancer. Such a subject would benefit from treatment with an antibody directed against GPNMB of the present invention. Normally, the antibodies are administered in an outpatient clinic by weekly administration of approximately 0.1-1.0 mg / kg of dose by slow intravenous (IV) infusion. The appropriate therapeutically effective dose of an antibody is selected by a practical physician and would range from about 1 µg / kg to 20 mg / kg, from 1 µg / kg to 10 mg / kg, from 1 µg / kg to 1 mg / kg, from 10 µg / kg to 1 mg / kg, from 10 µg / kg to 100 µg / kg, from 100 µg / kg to 1 mg / kg and from 500 µg / kg to 5 mg / kg.

Antibodies are also used to prevent and / or reduce the severity and / or disease symptoms associated with disorders related to GPNMB.

To test the clinical efficacy of antibodies in humans, individuals with cancer, particularly, but not limited to, ovarian, lung or colon carcinoma, are identified and randomized in treatment groups. Treatment groups include a group that is not treated with antibodies and groups treated with different doses of antibody directed against GPNMB. Individuals are eventually followed and individuals receiving antibody treatment have an improvement in their condition.

Example 19: The specificity of the antitumor effects of CR011-vcMMAE (CR011-ONC-1)

The study was conducted to determine the antitumor effects of the constituent components of the antibody-drug conjugate and its formulation and to refer these effects to the antitumor effects of the intact immunoconjugate.

Results:

SK-ME-2 melanoma fragments were implanted by trocar in mice and, after tumors were established, treatment with CR011-vcMMAE and various components was tested to demonstrate the specificity of antitumor effects of this agent. Control groups, dosed with either phosphate buffered saline (vehicle) or immunoconjugate preparation excipients (3% DMSO, sucrose, phosphate medium) regularly increased tumor size to a maximum of 2,000 mg , at which time they were removed from the study. No apparent or statistically significant antitumor effects were observed. However, treatment with CR011-vcMMAE (at 5 mg / kg / treatment, q4d x4) produced measurable inhibition after the first 2 doses. Tumor growth inhibition continued until no appreciable tumor was detected in 6 of the experimental animals (Figure 4). In preliminary studies, tumor regression was complete and was not followed by tumor regrowth despite prolonged observation periods (up to 200 days).

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Conclusions:

The regressions produced by the immunoconjugate were not due to the individual components of the immunoconjugate or the formulation components of that immunoconjugate. This is demonstrated by the lack of inhibition of tumor growth after treatment with CR011 antibody alone (group 3) or free monomethylauristatin E (group 4), in which the doses applied were identical to those contained in the intact immunoconjugate. In addition, the lack of antitumor effects observed with free MMAE suggests that the antitumor effects of MMAE as a result of low release of the antibody-drug conjugate cannot explain the anti-tumor effects of the immunoconjugate. It has been shown that the release of MMAE from the antibody-MMAE conjugates is a very slow procedure in vivo (T½β = 6.0 days in the case of antibody immunoconjugate directed against CD30auristatin E (Sanderson et al., Clin. Cancer Res. 11: 843-852 (2005)) and would provide plasma or serum concentrations that would be considerably lower than the "bolus" doses used in this study, which were ineffective in slowing the growth of human melanoma xenografts.

Example 20: CR011-vcMMAE inhibits the growth of SK-MEL-5 human melanoma xenografts leading to complete regression of established melanoma tumors in athymic mice (CR011-ONC-3)

This study was conducted to evaluate the potency and therapeutic efficacy of the antibody-drug conjugate, CR011-vcMMAE, against a second established human melanoma model, the SK-MEL-5 xenograft.

Results:

Although they are of unrelated origin, SK-MEL-5 expresses GPNMB on the surface of the cell membrane and is destroyed by CR011-vcMMAE in vitro. In this study, the anti-tumor effects of the immunoconjugate of CR011 were examined, together with the PBS and saline vehicles, and the reference agents vinblastine and paclitaxel. Similar to the SK-MEL-2 tumor, vinblastine produced a perceptible, but not significant, inhibition of tumor growth (P ≤ 0.21) when compared to saline and PBS control groups (Figure 5). However, shortly after the start of paclitaxel treatment, a significant tumor growth inhibition (P ≤ 0.039) was observed on day 3 after starting treatment, and this antitumor effect continued, producing 100% growth inhibition ( stasis). The responses of experimental animals that carried SK-MEL-5 to vinblastine and paclitaxel were ephemeral. After cessation of treatment at the maximum tolerated doses, the tumors resumed rapid progressive growth. A long-term tumor-free survivor occurred in the paclitaxel group and spontaneous regression occurred in the saline treated group.

Substantial tumor growth inhibition, in addition to tumor growth retardation and complete regressions produced in animals that had SK-MEL-5 tumors after treatment with CR011-vcMMAE, and these effects were dose related. At 10 mg / kg / treatment significant antitumor effects were already noted 7 days (the equivalent of 2 treatments) after starting treatment when compared with saline (P ≤ 0.0096), and already 10 days after starting treatment when compared to controls treated with PBS (P = 0.039). In a dose-related mode, CR011-vcMMAE produced a delay in tumor growth that led to complete regressions of established xenografts of the SK-MEL-5 melanoma (see table insert to Figure 5 for proportions of animals with complete regressions). Complete regressions occurred at doses of CR011-vcMMAE of 2.5 mg / kg / treatment, but not at 1.25 mg / kg / treatment.

As in previous studies, there was no indication of immunoconjugate toxicity in treated animals as evidenced by the mortality of effects on body weight or weight gain.

Conclusions:

CR011-vcMMAE exerts substantial dose-dependent antitumor effects against established xenografts of human melanoma SK-MEL-5. After only one or two treatments, a significant inhibition of tumor growth is observed, leading to long-term tumor-free survivors. Complete regressions occurred at doses of ≥ 2.5 mg / kg i.v., q4d X4.

Example 21: Pharmacokinetics of CR011-vcMMAE (CR011-PK-1A)

The purpose of this study was to determine the stability of CR011-vcMMAE in vivo after intravenous injection, the expected route of clinical administration.

Materials and procedures.

The CR011 antibody component of CR011-vcMMAE was measured by a sandwich-linked enzyme-linked immunosorbent assay (ELISA) in which serum was added to the wells of microtiter plates coated with the related antigen (GPNMB, CG56972-03) for CR011 antibody, and the amount of human antibody was detected with an anti-globulin conjugated to the signal generator (horseradish peroxidase).

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5

10

fifteen

twenty

25

30

35

Results:

Pharmacokinetics The persistence of compound availability for the antibody component of CR011-vcMMAE was examined in a pharmacokinetic study in athymic mice (study CR011-PK-1, Figure 6). The serum-time concentration profile for the antibody-drug conjugate was determined in nude mice after intravenous administration of CR011-vcMMAE and the results are presented in Figure 6. The athymic mice that received 1 or 10 mg / kg intravenously showed serum concentrations proportional to the dose with respect to the full duration of sampling times (42 days). The concentration-time pattern was biphasic. However, the initial phase (α) was smaller since it contributed to <2% of total ABC. However, the compound disappeared very slowly from the peripheral blood (T½β = 10.3 days) with serum concentrations of 1 µg / ml and 10 µg / ml that remained in the blood for 6 weeks after dosing.

The approximate calculations of the pharmacokinetic parameters for CR011-vcMMAE are presented in the Table

48. A parameter is of interest. The volume of distribution in the steady state (Vss) is very low, approaching the theoretical minimum; This suggests that the compound is not distributed outside the extravascular space. The distribution pattern, in addition to the phase of elimination β for CR011-vcMMAE, has a good relationship with values obtained for antibodies in general (see Reviews of Mahmood and Green, Clin. Pharmacokinet 44: 331-347 (2005); or Lobo et al. J. Pharm. Sci. 93: 2645-2668 (2004)) and are related to values obtained for an antibody-auristatin E immunoconjugate with comparable drug load (Hamblett et al., Clin. Cancer Res. 10: 7063-7070 (2004)).

Table 48. PK parameters of CR011-vcMMAE after intravenous administration.

Parameter

Units 1 mg / kg 10 mg / kg

TO

µg / ml 8.97 74.6

B

µg / ml 9.82 113

Alpha

 1 hour 0.179 0.0812

Beta

 1 hour 0.00269 0.00281

ABC

 h * µg / ml 3712 41210

Half-life alpha

h 3.88 8,531

Beta half-life

h 258 247

Volume

ml / kg 53.2 53.2

Cmx

 µg / ml 18.8 188

Cl

ml / h / kg 0.269 0.243

MRT

 h 368 348

Vss

 ml / kg 99.0 84.5

Abbreviations: A: Pre-exponential constant for the alpha phase; Alpha: Exponential velocity constant for the alpha phase; ABC: Total area under the curve from 0 to infinity; B: Pre-exponential constant for the beta phase; Beta: Exponential velocity constant for the beta phase; Cl: Total or systemic elimination; Cmax: Maximum concentration observed; MRT: Average residence time; Volume: Volume of the central compartment; Vss: Volume of distribution in the steady state.

The approximate calculations for the pharmacokinetic parameters are presented in Table 48. A parameter is of interest. The volume of distribution in the steady state (Vss) approximates the theoretical minimum. These data suggest that the compound was not distributed outside the extravascular space. Together these data are considered to have a good relationship with the data in other immunoconjugates carrying the cytotoxic moiety -vcMMAE (see Hamblett et al., Clin. Cancer Res. 10: 7063-7070 (2004)).

Conclusions:

The antibody-drug conjugate CR011-vcMMAE has a serum concentration profile that favors sufficient continuous exposure for the disturbance and eradication of melanoma xenografts. The immunoconjugate after administration i.v. It has a long enough half-life to guarantee the exposure of tumor cells for prolonged periods (T½β = 10.3 days), and may not require frequent dosing. The durability of CR011-vcMMAE in vivo (eg, nude mice) is comparable to that of other auristatin E immunoconjugates.

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Example 22: Program dependence on the antitumor effects of CR011-vcMMAE (CR011-ONC-1)

The purpose of this study was to determine the extent to which the curative antitumor effects of the CR011 antibody-drug conjugate depend on the dosage schedule and, if possible, determine the optimal dosage range for this xenograft model.

Materials and procedures:

The protocol for this study is presented in Table 49. To test the hypothesis that curative antitumor effects are influenced by the dosing schedule, the antitumor effects of CR011-vcMMAE are

10 measured at 5 different dosing intervals (i.e. 0, 1, 4, 8 and 16 days between treatments) and for each dosing interval 3 dosage levels were used (i.e. cumulative doses of 2, 8 and 32 mg / kg); for each group, n = 6 athymic mice.

Note well: please note that although the 5 sets of groups in this experiment (for example, groups 5, 6 and 7 represent a set and received 32, 8 and 2 mg / kg of cumulative doses, respectively) they received

15 the same cumulative doses, the first set that received the “bolus dose” is different from the other 4 sets. The Cmax for each group in the "bolus" set was probably four times higher than the Cmax for the other 4 sets (see the section on pharmacokinetics for dose linearity after iv administration), since the 4 sets of groups They received 4 treatments, while the first set received only one “bolus” treatment (see column 7, Table 49 below).

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TABLE 49. Protocol for the study of the dosage interval (CR011-PHM-2).

Group

 ROA Dose Treatment (mg / kg) Pattern Dosage interval (days) No. of treatments (n) Cumulative doses (mg / kg)

one

Phosphate buffered saline solution Bolus 0  one N.A.

2

CR011-AE i.v. 32 Bolus 0 one 32

3

CR011-AE i.v. 8 Bolus 0 one 8

4

CR011-AE i.v. 2 Bolus 0 one 2

5

CR011-AE i.v. 8 qd x4 one 4 32

6

CR011-AE i.v. 2 qd x4 one 4 8

7

CR011-AE i.v. 0.5 qd x4 one 4 2

8

CR011-AE i.v. 8 q4d x4 4 4 32

9

CR011-AE i.v. 2 q4d x4 4 4 8

10

CR011-AE i.v. 0.5 q4d x4 4 4 2

eleven

CR011-AE i.v. 8 q8d x4 8 4 32

12

CR011-AE i.v. 2 q8d x4 8 4 8

13

CR011-AE i.v. 0.5 q8d x4 8 4 2

14

CR011-AE i.v. 8 q16d x4 16 4 32

fifteen

CR011-AE i.v. 2 q16d x4 16 4 8

16

CR011-AE i.v. 0.5 q16d x4  16 4 2

17

Excipients i.v. N.A. q16d x4 16 4 N.A.

Results:

For this study, the frequency of complete regressions with long-term tumor-free survivors was determined empirically after 5 different dosing intervals (i.e. 0, 1, 4, 8 and 16 days between treatments). The overall responses for each set of groups, in which a set is defined as 3 groups of graduated dosage levels, but a dosage interval (groups 5, 6 and 7 represent 1 set, all being treated with a dosage interval of 1 day), are shown in Figure 7. The overall responses for experimental animals that respond to CR011-vcMMAE seem to suggest that bolus dosing and 1 day and 4 day intervals provide a very slight advantage to the proportion of cures in comparison with longer intervals such as 8 days and 16 days between doses. However, this effect was not significant (P <0.2904). Therefore, the data suggests that the antitumor effects of CR011-vcMMAE in the SK-MEL-2 model do not depend on the program. This conclusion is reinforced by the fact that experimental animals in the bolus assembly (groups 2, 3 and 4) that were exposed to plasma concentrations of approximately four times higher than any of the other groups showed no higher percentage of cured subjects. .

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The original design of this study was extended to include an examination of the effects of various dosage levels. For a joint box, a group of animals received a cumulative dose of 8 mg / kg which, from previous studies using a 4-day dosing interval, provided consistent therapeutic effects that led to tumor-free animals in the long term. In addition, cumulative doses of 2 mg / kg and 32 mg / kg were used.

The effects of dosage levels, together with various dosage ranges, are presented in Figure 8. The athymic mice that received a cumulative dose of 32 mg / kg showed complete regressions in 100% of each group, regardless of the dosage range. ; that is, a cumulative dose of 32 mg / kg is independent of the program and represents a dose that is well above that for complete regressions in 100% of experimental animals (5 groups of 6 animals / group = 30 experimental animals ). Animals that received 8 mg / kg of cumulative dose showed no dependence on the program and showed almost the same proportions of complete regressions (ie 28/30 = 93%); the experimental animals that received 2 mg / kg (cumulative dose), which was recognized in preliminary studies that was below the threshold for cures (using a standardized guideline of q4d X4), appeared to be program dependent, although this was not significant and produced a much lower proportion of complete regressors (i.e. 13%).

Conclusions:

Data from the dosing interval study suggest that SK-MEL-2 melanoma xenograft responses do not depend on the CR011-vcMMAE administration program. Although no advantage could be shown for dosing or bolus guidelines with low dosage intervals, it is suggested that under a certain cumulative dose there may be some advantage to combining multiple treatments is a single bolus dose.

Example 23: GPNMB transcript expression in human melanoma

It was recently shown that GPNMB was expressed in glioblastoma and mediated in vitro and in vivo invasiveness of tumor cells derived from glioblastoma (see, for example, Loging et al., Genome Res. 10: 1393-1402 (2000); and Rich et al., J. Biol. Chem. 278: 15951-15975 (2003)). To confirm and extend these findings to additional types of cancer, the inventors examined the expression of the GPNMB transcript in human cancer cell lines and tissues.

Material and procedures:

Total RNA was isolated using the RNeasy kit with a DNase digestion stage (Qiagen Inc., Valencia CA). RT-PCR was performed using the one-stage RT-PCR kit (Qiagen) as follows. RT: 50 ° C for 45 min and 95 ° C for 15 min for 1 cycle. PCR: 1 min at 95 ° C, 1 min at 50 ° C and 2 min at 72 ° C for 30 cycles with final extension for 10 min at 72 ° C. The products were separated on a 2% agarose gel / 0.33% low melting agarose and visualized by staining with ethidium bromide. The integrity of each RNA sample was verified by RT-PCR with primers designed to amplify GAPDH. The specific primers (5'-3 ') used were:

GPNMB: Direct-GAATTCAGAGTTAAACCTTGAG (SEQ ID NO: 327)

Inverse-CAGGAATCTGATCTGTTACCAC (SEQ ID NO: 328)

MART-1: Direct-CTGACCCTACAAGATGCCAAGAG (SEQ ID NO: 329)

Inverse-ATCATGCATTGCAACATTTATTGATGGAG (SEQ ID NO: 330)

Tyrosinase: Direct-TTGGCAGATTGTCTGTAGCC (SEQ ID NO: 331)

Inverse-AGGCATTGTGCATGCTGCTT (SEQ ID NO: 332)

pMEL-17: Direct-TATTGAAAGTGCCGAGATCC (SEQ ID NO: 333)

Inverse-TGCAAGGACCACAGCCATC (SEQ ID NO: 334)

RTQ-PCR analysis was performed with an ABI Prism 7700 sequence detection system using TaqMan reagents (PE Applied Biosystems, Foster City, CA). Equal amounts of normalized RNA were used as template in PCR reactions for 40 cycles with GPNMB specific primers to obtain threshold cycle (CT) values. The following primers (5'-3 ') were used:

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Direct-TCAATGGAACCTTCAGCCTTA (SEQ ID NO: 335) Inverse-GAAGGGGTGGGTTTTGAAG (SEQ ID NO: 336) Probe-TET-CTCACTGTGAAAGCTGCAGCACCAG-TAMRA (SEQ ID NO: 337)

Result: 5 The expression analysis of the inventors' transcript indicated that GPNMB was strongly expressed in a high percentage of human metastatic melanoma samples. Using RTQ-PCR it was found that GPNMB was highly expressed (CT <27.0) in 5/7 melanoma cell lines and in 5/5 melanoma clinical specimens examined (Table 50). In contrast, GPNMB was not expressed in a renal carcinoma cell line, TK-10, which was used as a negative control in the inventors' experiments. 10 Table 50: GPNMB transcript expression in human melanoma cell lines and clinical specimens

Sample details Expression *

Cell lines

UACC-62

Melanoma met. 21.2

M14

Met melanoma, amelanotic 22.2

SK-MEL-5

Met melanoma, axillary ganglion 22.9

SK-MEL-28

Melanoma met., Skin 24.1

WM-266-4

Melanoma met., Skin 24.5

A-375

Melanoma met., Skin 29.0

LOXIMVI

Met melanoma, amelanotic 30.9

TK-10

Renal cell carcinoma 40.0

Clinical specimens

No. 1

Melanoma met. 26.6

No. 2

Melanoma 26.4

No. 3

Melanoma 26.9

No. 4

Melanoma met. 24.1

No. 5

Melanoma met. 25.3

* Threshold cycle (CT) values of the RTQ-PCR analysis. Met: Metastatic.

To extend these results, the inventors investigated the expression of the GPNMB transcript in a panel of 17 melanoma cell lines by semi-quantitative RT-PCR (Table 51). The results show that the

GPNMB transcript is highly expressed in 15/17 melanoma cell lines, is weakly expressed in 1/17 melanoma cell lines (A-375) and is not detectable in 1/17 melanoma cell lines (LOXIMVI) or in the TK-10 control.

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Table 51. Analysis by RT-PCR

Expression*

Cellphone line

Annotation GPNMB MART-1 Tyrosinase pMel-17

M14

Met melanoma, amelanotic +++ +++ +++ +++

SK-MEL-5

Met melanoma, axillary ganglion +++ +++ +++ +++

SK-MEL-28

Melanoma met., Skin +++ +++ +++ +++

WM-266-4

Melanoma met., Skin +++ +++ +++ +++

SK-MEL-2

Melanoma met., Skin +++ +++ +++ +++

UACC-257

Melanoma met. +++ +++ +++ +++

A2058

Met melanoma, lymph node +++ +++ +++ +++

G361

Melanoma met., Skin +++ +++ +++ +++

HT-144

Melanoma met., Skin +++ +++ +++ +++

MEWO

Met melanoma, lymph node +++ +++ +++ +++

SK-MEL-3

Met melanoma, lymph node +++ +++ +++ +++

MALME-3M

 Melanoma met. +++ +++ +++ +++

UACC-62

Melanoma met. +++ +++ +++ -

SK-MEL-24

Met melanoma, lymph node +++ - +++ -

RPMI-7951

Met melanoma, lymph node +++ - + -

A-375

Melanoma met., Skin + - - -

LOXIMVI

Met melanoma, amelanotic - - - -

TK-10

Renal cell carcinoma - - - -

* Analysis by RT-PCR: Strongly (+++), weakly (+) or undetectable (-). Met: Metastatic.

In addition, comparing the expression of the GPNMB transcript with transcripts of known melanoma / melanocyte associated genes (MART-1, tyrosinase and pMEL-17) in the melanoma cell lines (Table 51) demonstrated a strong expression of MART-1, tyrosinase and pMEL-17 on 13/17, 14/17 and 12/17 melanoma cell lines, respectively. Notably, 12/17 samples co-expressed high levels of GPNMB and the three genes associated with melanoma / melanocyte. Both LOXIMVI and TK-10 cell lines that had undetectable GPNMB expression also lacked the three melanoma / melanocyte associated genes examined.

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Example 24: The growth inhibitory activity of CR011-vcMMAE depends on the expression of GPNMB

Material and procedures:

Flow cytometry: The quantitative analysis of GPNMB expression on the cell surface of cell lines was determined by flow cytometry. Approximately 1 x 10 6 cells were collected, washed and incubated with a saturating amount (10 µg / mL) of either CR011 or control antibody of the same isotype in staining buffer containing PBS (pH 7.4), SBF at 4 % and 0.1% NaN3 for 30 min on ice, followed by washing and staining with goat antibody directed against human IgG conjugated to R-phycoerythrin (PE) (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) at 1: 100 for 30 min on ice. The cells were fixed in 1% paraformaldehyde / PBS and examined on a Becton Dickinson FACSCalibur flow cytometer. Data analysis was performed with Becton Dickinson Cell Quest version 3.3 software and the geometric mean fluorescence intensity ratio (GMR) was determined for each cell type.

The internalization of the antibodies bound to the cell surface was evaluated by a modified flow cytometry procedure. In summary, cell suspensions were labeled with 10 µg / ml of unconjugated CR011 or conjugated with MMAE for 30 min on ice. After washing the cells, the incubation was changed at 37 ° C for 1 h to allow internalization of bound antibodies. Cells that remained on the ice (bound to the total surface) or that were incubated at 37 ° C (internalized) were stained with goat antibody directed against human IgG conjugated to PE at 1: 100 for 30 min to detect surface-preserved CR011 mobile. The labeled cells were analyzed by flow cytometry as described above. The percentage of internalized antibody was determined using GMR and the following formula:

Internalized percentage = United to the total area (4ºC) - linked to the

total area (37 ° C) / United to total area (4 ° C) x 100

Immunoprecipitation and immunoblot analysis: Cells were harvested and lysed on ice for 30 min in lysis buffer containing 1% NP-40, 0.15 M NaCl, 0.02 M Tris-HCl, 10% glycerin, EDTA 0.01 M and complete mixture of protease inhibitor (Roche Molecular Biochemicals, Indianapolis, IN). Supernatants were collected and protein concentration was determined with the BCA protein assay kit (Pierce, Rockford, IL). For immunoprecipitation, 2 µg of primary antibody was added to 0.5-1 mg of total cell lysates and incubated at 4 ° C for 3 h, followed by incubation with A-agarose protein (Amersham Biosciences, Uppsala, Sweden) on ice during 2 h. The agarose beads were washed in ice cold TBST (PBS with 0.1% Tween 20). The immunoprecipitates were recovered from the supernatants after boiling in Laemmli sample buffer and centrifugation.

For immunoblot analysis, total cell lysates (50 µg) or immunoprecipitates were resolved under a reducing condition in 4-20% Tris-glycine gels (Invitrogen) and electrophoretically transferred to 0.45 µm PVDF membranes ( Invitrogen). The membranes were blocked with 3% BSA (Sigma, St. Louis, MO) in TBST for 3 h and probed with rabbit polyclonal antibody directed against GPNMB (1: 1000) for 3 h. Secondary goat antibody directed against peroxidase-conjugated rabbit IgG (H + L) (Jackson ImmunoResearch Labs) was added and incubated for 30 min. The membranes were washed in TBST and subjected to enhanced chemiluminescence (Amersham) following the manufacturer's protocol.

Clonogenic assays: The growth inhibitory activity of CR011-vcMMAE was determined by clonogenic assay. Cells were seeded in 96-well plates and allowed to coat overnight. Unconjugated CR011, free MMAE, CR011-vcMMAE or vcMMAE conjugated antibody of the same isotype at various concentrations were added to subconfluent cell cultures and incubated for 4 days at 37 ° C. Then, the cells were transferred to 6-well plates and allowed to form colonies. The colonies were stained with Giemsa staining (Sigma) and counted. Survivor cell fractions were calculated based on the ratio of the treated sample and the untreated control. The results were expressed as a percentage of the control using GraphPad Prism version 4 software. IC50 was defined as the concentration that produces a 50% reduction in colony formation compared to untreated control cultures.

Results:

To demonstrate that the growth inhibitory activity of CR011-vcMMAE depends on GPNMB expression, the full-length GPNMB protein was expressed ectopically in HEK293 cells. Immunoblot analysis (Figure 9A) and FACS (Figure 9B) confirmed that GPNMB was expressed in cells transfected with GPNMB / plasmid. Treatment of cells with CR011-vcMMAE, followed by a clonogenic assay, demonstrated that HEK293 cells expressing GPNMB were more sensitive to growth inhibition mediated by CR011vcMMAE than control cells lacking GPNMB expression (Figure 9C).

To further verify the inventors' findings, SK-MEL-2 cells expressing GPNMB were transfected with siRNA to specifically inhibit endogenous expression of GPNMB. Immunoblot and FACS analyzes performed 2 and 4 days after transfection demonstrated that the levels of the total GPNMB protein (Figure 10A) and superficial GPNMB (Figure 10B) were significantly reduced in SKMEL-2 cells after transfection when compared with control transfectants. The amount of GPNMB expression was reduced for at least 7 days after transfection. Treatment of these cells with CR011vcMMAE showed that SK-MEL-2 cells were less sensitive to the growth inhibitory activity of CR011vcMMAE after siRNA-mediated GPNMB silencing (Figure 10C). Taken together, these data indicate that the growth inhibitory activity of CR011-vcMMAE required the expression of GPNMB from the cell surface.

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Example 25: Cell cycle arrest and apoptosis induction by CR011-vcMMAE

To evaluate the mechanism of CR011-vcMMAE growth inhibition, the cell cycle analysis was performed.

Material and procedures:

The effects of the CR011-vcMMAE cell cycle were evaluated after treating cells in full growth medium for 24 or 48 h. Briefly, the cells were pulsed at the indicated times with 30 µM of bromodeoxyuridine (BrdU, Sigma) for 30 min, collected, fixed and permeabilized in methanol. Nascent DNA synthesis was detected by staining with antibromodeoxyuridine-FITC (BD Biosciences, San Jose, CA). Total DNA content was detected using propidium iodide (PI, Sigma). For apoptosis analysis, cells were treated as before and labeled with annexin V-FITC followed by exclusion with propidium iodide using annex I kit for detection of annexin V-FITC apoptosis (BD PharMingen, San Diego, CA) according to the manufacturer's protocols. Flow cytometry (as described in the previous example) was used to test both cell cycle and apoptosis studies.

Results:

SK-MEL-2 positive cells or GPKMB-negative TK-10 control cells were treated with CR011vcMMAE for various durations, followed by bromodeoxyuridine for 30 minutes to detect nascent DNA synthesis and finally propidium iodide to detect Total DNA content DNA synthesis and cell cycle progression were determined by flow cytometry (Table 52).

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Table 52. Analysis of the cell cycle of cells treated with CR011-vcMMAE

Treatment (ng / ml)% of G1% of phase S% of G2 / M% of sub-G1

SK-MEL-2

24 hours Untreated 55.2 30.0 9.9 0.5 CR011 (1000) 63.6 25.2 6.4 0.5 IgG2-vcMMAE (1000) 65.9 21.8 5.8 0.8 CR011-vcMMAE (100) 56.0 26.9 12.4 0.2 CR011-vcMMAE (1000) 43.7 20.0 28.5 1.1

TK-10

24 hours Untreated 39.7 43.7 7.0 0.5 CR011 (1000) 42.0 39.8 6.3 0.3 IgG2-vcMMAE (1000) 42.8 40.2 5.9 0.3

CR011-vcMMAE (100) 51.1 35.1 4.5 0.7 CR011-vcMMAE (1000) 52.6 34.2 3.9 0.8

The cell cycle analysis was carried out by flow cytometry and the percentages of cells in each phase of the cell cycle were determined by the CellQuest software (Becton Dickinson).

Exposure of GPNMB positive cells at 1000 ng / mL of CR011-vcMMAE, but not at the control of the IgG2-vcMMAE isotype for 24 h, produced a decrease in the percentage (10%) of cells in G1 and S phase and a

5 percentage increase (18.6%) of cells in G2 / M when compared to untreated cells. In contrast, CR011vcMMAE did not affect the cycling of GPNMB negative cells. 48 h after treatment, CR011-vcMMAE further reduced the percentage (11%) of cells in G1 and S phase and increased the percentage (24%) of cells in G2 / M.

The increase in the sub-G1 population after treatment with CR011-vcMMAE suggested the appearance of apoptosis. To investigate this possibility, apoptosis analysis was performed using annexin V surface attachment and

10 loss of exclusion of propidium iodide (PI). The results of the inventors showed that 1000 ng / ml of CR011vcMMAE induced apoptosis specifically in cells expressing GPNMB as indicated by an 11% increase in monoteined cells (annexin V + / PI-) after 48 h of treatment with CR011-vcMMAE (Table 53).

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5

10

fifteen

Table 53. Induction of apoptosis in human melanoma cells by CR011-vcMMAE

An V + / PI-% of an V- / PI +% of an V + / PI +% of an V- / PI-% Treatment (ng / ml) LR UL UR LL

SK-MEL-2 48 hours Without treatment 1.23 1.23 94.37 3.16 CR011 (1000) 0.36 0.45 94.45 4.74 IgG2-vcMMAE (1000) 0.17 0.51 95 , 93 3.39 CR011-vcMMAE (100) 0.30 0.40 89.93 9.37 CR011-vcMMAE (1000) 2.08 2.02 82.08 13.83

TK-10 48 hours Without treatment 0.54 0.66 96.92 1.87 CR011 (1000) 0.83 0.34 98.27 0.55 IgG2-vcMMAE (1000) 0.62 0.95 97.09 1.33 CR011-vcMMAE (100) 0.71 0.57 97.72 1.00 CR011-vcMMAE (1000) 0.86 0.83 97.75 0.56

The apoptosis analysis was carried out by flow cytometry and the percentages of cells in the quadrants UL (upper left), UR (upper right), LL (lower left) and LR (lower right) were determined by the CellQuest software ( Becton Dickinson). An V: annex V-FITC and PI: propidium iodide.

In addition, an increase in dyed cells (annexin V + / PI +) after treatment with CR011vcMMAE indicated that the CR011 immunoconjugate potentiated cell death. These results suggest together that CR011-vcMMAE selectively induced G2 / M cell cycle arrest followed by apoptotic cell death.

Example 26: CR011: A purely completely human IgG1 for use in melanoma therapy by exploiting the antibody-dependent cell cytotoxicity mechanism (ADCC)

Fully human monoclonal antibodies (mAb) -IgG2 were generated for CG56972 / GPNMB, an antigen found predominantly on the surface of melanoma and brain tumor cells. The naked IgG2 mAb of CR011 (mAb 1.15) had no effect on cells expressing CG56972 both in vitro and in vivo. Therefore, the inventors examined whether the change of isotype from an IgG2 to an IgG1 could allow the mAb to destroy human melanoma cells by ADCC effector functions.

Briefly, to change CR011 of an IgG2 antibody to IgG1 the constant region encoding the double stranded IgG1 DNA (allotype Gm (f)) was synthesized and the constant region of IgG2 was replaced by the constant region of IgG1 using the PCR solution by overlap. The sequences are described below:

image100

5

10

fifteen

twenty

25

30

Mature heavy chain of mAb CR011 1.15.1 (IgG2)

image52

Mature heavy chain of mAb CR011 1.15.1 (IgG1)

cell surface and bind to the IgG2 of CR011. As shown in Figure 11, both IgG1 and IgG2 mAbs produced comparable FACS shifts in SK-MEL-2 cells compared to isotype control mAbs (Figure 11), which indicates that both isotypes bind to CG56972 / GPNMB with comparable saturation densities and affinities.

The inventors then examined whether the IgG1 mAb of CR011 could induce ADCC in SK-MEL-2 cells in culture in the presence of human PBMC. Human PBMCs were isolated from whole blood using a Ficoll plate. Briefly, in a 50 mL tube 15 mL of PBS was added to 20 mL of whole blood that was below 10 mL of Ficoll plate and the tube was centrifuged at 2000 rpm. Mononuclear cells were collected from the interface and washed 3 times with PBS. The ADCC assay was carried out in a 96-well plate using a Perkin-Elmer cytolysis fluorescence assay (DELFIA EuTDA cytotoxic assay). The procedure is based on loading target cells with a fluorescence enhancer ligand (BATDA, bis (acetoxymethyl) terpyridine dicarboxylate). The hydrophilic ligand quickly penetrates the membrane. Within the cell, the ester bonds hydrolyze to form a hydrophilic ligand (TDA, terpyridine dicarboxylic acid) that can no longer pass through the membrane. After cytolysis, the ligand is released and introduced into the europium solution. The europium and ligand form a highly fluorescent and stable chelate (EuTDA). Fluorescence intensity is recorded using excitation and emission wavelengths as ex = 340 nm and em = 613 nm, respectively.

The antibody-mediated cell-mediated cytotoxity on SK-MEL-2 cells was tested in the presence of PBMC and monoclonal antibody CR011 using effector ratios: target of 10, 30, 60 and 100 and various concentrations of IgG1 or IgG2 mAb against CG56972 / GPNMB (2, 5, 10 µg / 200 µl). The data of the inventors showed that between 30 and 100 times of PBMC, mAb IgG1 produced the cytolysis of SK-MEL-2 cells in a dose-dependent manner (Figure 12A), while mAb IgG2 showed no cytolysis (Figure 12B). Therefore, the inventors concluded that the IgG1 mAb of CR011 for CG56972 / GPNMB can destroy melanoma cells expressing CG56972 / GPNMB in vitro and possibly human melanoma in vivo by effector functions of ADCC. CR011 IgG1 mAb may also be useful in combination with immune effector cytokines that could provide some clinical benefit in metastatic melanoma such as high dose IL-2, interferon gamma or TNF-alpha. CR011 can also be used to treat melanoma in combination with vaccine immunotherapy, immunomodulators such as MDX-010, radiotherapy and / or chemotherapy.

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Example 27: Treatment of astrocytoma, glioblastoma, medulloblastoma and other CNS tumors

Astrocytoma / glioblastoma is a highly drug resistant neoplasm that represents significant unmet medical needs. The researchers identified CG56972 as a human gene (also known as GPNMB) that is highly expressed in these cancer tissues and human cancer cell lines. GPNMB is a type I transmembrane protein that possibly participates in vesicular traffic with a very limited expression pattern in the human brain. The inventors generated fully human monoclonal antibodies against CG56972, the extracellular domain of GPNMB (SEQ ID NO: 289). The inventive guide monoclonal antibody, designated CR011-vcMMAE, was biochemically characterized and tested for therapeutic activity against cell lines derived from human brain tumors of astrocytoma, glioblastoma, medulloblastoma or neuroectodermal origin.

Transcript expression analysis demonstrated highly elevated GPNMB mRNA in brain tumors derived from astrocytoma, glioblastomas, medulloblastoma and tumors of neuroectodermal origin with limited low expression in the normal brain. CR011 was linked by FACS analysis to superficial GPNMB on brain cancer cell lines. The CR011 mAbs transferred the predicted 100 and 120 kDa gene products by Western. Clonogenic assays demonstrated that CR011-vcMMAE mAbs inhibited the growth of brain cancer cell lines.

Material and procedures:

Cell lines and culture conditions: All human cell lines, SK-MEL-2, XF-498, SNB-78, U118-MG, SF-539, H79MG, D392-MG, D534-MG, SK-N-SH , U-251, SF-295, D450-MG, U87MG, SF-268, T98G and SW1783 were obtained from the American Type Crop Collection (Manassas, VA) or purchased from NCI (Bethesda, MD). The cells were maintained in DMEM or RPMI (Invitrogen, Carlsbad, CA) containing 10% SBF (Gemini Bio-Products, Woodland, CA) and penicillin-streptomycin.

Quantitative real-time PCR (RTQ-PCR): Total RNA was isolated using the RNeasy kit with a DNase digestion stage (Qiagen Inc., Valencia). RNA samples were derived from commercially obtained normal human tissues (Clontech, Palo Alto, CA; Invitrogen, Carlsbad, CA) or cultured cell lines according to specifications. RNA was collected and PCR was performed as previously described (Shimkets RA et al. Nat Biotechnol., 1999. 17-8: 798-803) using TaqMan® reagents (PE Applied Biosystems, Foster City, CA). RNAs were normalized using human β-actin and TaqMan® probes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) according to the manufacturer's instructions. Equal amounts of normalized RNA were used as templates in PCR reactions with specific GPNMB reagents to obtain threshold cycle (CT) values. For the graphical representation, the numbers of CT became relative expression with respect to the sample that presented the highest level of expression. RTQ-PCR analysis was performed with an ABI Prism 7700 sequence detection system using TaqMan reagents (PE Applied Biosystems, Foster City, CA). The following primers (5'-3 ') were used:

Direct-TCAATGGAACCTTCAGCCTTA (SEQ ID NO: 338)

Inverse-GAAGGGGTGGGTTTTGAAG (SEQ ID NO: 339)

Probe-TET-CTCACTGTGAAAGCTGCAGCACCAG-TAMRA (SEQ ID NO: 340)

CuraChip ™: Trizol tissues were lysed. Biotin-labeled cDNA was prepared using 15 mg of total RNA with poly (T) primers. Gene expression was evaluated by hybridization to the patented CuraChip microarray (CuraGen, New Haven, CT) of 11,000 oligonucleotide probes. Slides were hybridized for 15 h at 30 ° C with constant rotation, washed for 30 min at room temperature (RT), incubated in streptavidin solution (4 ° C, 30 min), washed three times for 15 min at RT, incubated in buffer of detection conjugated with Cy3 (4 ° C, 30 min) and washed three times for 15 min at RT. The slides were screened (GMS 418 Scanner, Genetic Microsystems, Woburn, MA) and analyzed using the IMAGENE software (BioDiscovery, Marina del Rey, CA). The data underwent standardization at the 90th centile and the expression of the GPNMB gene was analyzed in comparison with that of the GAPDH maintenance gene. The oligonucleotide sequence used to detect GPNMB is 5'TGATCAGTAAGGATTTCACCTCTGTTTGTA (SEQ ID NO: 341). The oligonucleotide sequence used to detect GAPDH is 5'-ACCTTGTCATGTACCATCAATAAAGTACCC (SEQ ID NO: 342), corresponding to pb 1243-1272 of the GAPDH transcript (registration no. NM_002046).

Flow cytometry: Quantitative analysis of GPNMB expression on the surface of cell lines was determined by flow cytometry (FACS). Approximately 1 x 10 6 cells were collected, washed and incubated with a saturating amount (10 µg / mL) of either CR011 or control antibody of the same isotype in staining buffer containing PBS (pH 7.4), SBF at 4 % and 0.1% NaN3 for 30 min on ice, followed by washing and staining with goat antibody directed against human IgG conjugated to R-phycoerythrin (PE) (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) at 1: 100 for 30 min on ice. The cells were fixed in 1% paraformaldehyde / PBS and examined on a Becton Dickinson FACSCalibur flow cytometer. Data analysis was performed with Becton Dickinson Cell Quest version 3.3 software and the ratio of geometric mean fluorescence intensity (GMR) was determined for each cell type.

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Immunoblot analysis: SK-MEL-2, XF-498, SNB-78, U-118-MG, SF-539, H79MG, D392-MG, D534-MG, SK-N-SH, U-251 cells were collected , SF-295, D450-MG, U87MG, SF-268, T98G and SW-1783 and lysed on ice for 30 min in lysis buffer containing 1% NP-40, 0.15 M NaCl, Tris-HCl 0.02 M, 10% glycerin, 0.01 M EDTA and complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). Supernatants were collected and protein concentration was determined with the BCA protein assay kit (Pierce, Rockford, IL). For immunoblot analysis, 40 ul of total cell lysate from a well of confluent cells collected from a 6-well Falcon tissue culture plate was boiled in Laemmli sample buffer, centrifuged and resolved under reducing condition in gels of 4-20% Tris-glycine (Invitrogen). The gels were electrophoretically transferred to 0.45 µm PVDF membranes (Invitrogen). The membranes were blocked with 3% BSA (Sigma, St. Louis, MO) in TBST for 3 h and probed with goat polyclonal IgG directed against GPNMB (R&D Systems; 1 µg / ml, total 10 µg)) for 3 h Secondary antibody directed against peroxidase-conjugated goat globulin (Jackson ImmunoResearch Labs) was added and incubated for 30 min. The membranes were washed in TBST and subjected to enhanced chemiluminescence (Amersham) following the manufacturer's protocol.

Clonogenic assays: The growth inhibitory activity of CR011-vcMMAE was determined by clonogenic assay. Cells were seeded in 96-well plates and allowed to coat overnight. CR011-vcMMAE or monoclonal antibody of the same isotype at various concentrations was added to subconfluent cell cultures and incubated for 4 days at 37 ° C. Then, the cells were transferred to 6 well plates and allowed to form colonies. The colonies were stained with Giemsa staining (Sigma) and counted. Survivor cell fractions were calculated based on the ratio of the treated sample and the untreated control. The results were expressed as a percentage of control using GraphPad Prism version 4 software. IC50 was defined as the concentration that produces a 50% reduction in colony formation compared to untreated control cultures.

Results:

one.

 GPNMB transcript expression in astrocytoma, glioblastoma, medulloblastoma and human tumors of neuroectodermal origin.

The inventors examined the expression of GPNMB transcripts in human cancer cell and tissue lines (Figures 13 A and B). The transcript expression analysis of the inventors indicated that GPNMB was strongly expressed in all (15/15) lines of human brain cancer cells tested (Figure 13A). Using RTQPCR it was found that GPNMB was expressed in cells of origin of mixed glioblastoma / astrocytoma, glioblastoma / gliomas, astrocytomas and metastatic neuroblastomas. Most of the brain tumor or CNS cell lines showed a high level expression with CT <27. It was found important that GPNMB is highly expressed (CT <27.0) in XF-498, U-118-MG cells , SNB-78 and SF-539. As shown in Figure 13B, GPNMB was also expressed at high levels in 4/5 biopsies of human glioma and 1/4 biopsies of human medulloblastoma. Using microarray analysis of a homemade chip containing a large panel of human genes (Figure 13C), GPNMB was found to be highly expressed in 5/9 brain cancers of astrocytoma or glioblastoma origin, in addition to 4/9 oligodendrogliomas . The inventors' analysis of these tumor expression profiles showed that the GPNMB messenger was detected to a much lesser degree in normal brain tissues. These data are also related to the immunohistochemical data of the inventors who demonstrated the lack of staining of CR011 in normal human brain cells that include neurons and glia cells. Taken together, these data demonstrate that GPNMB transcript is expressed at highly elevated amounts in brain cancer and oligodendroglioma cell lines and specimens isolated from human tumors.

2.

 Generation of fully human CR011 monoclonal antibodies for CG56972 / GPNMB

It is predicted that the GPNMB protein is a transmembrane type I glycoprotein. The highly elevated expression of GPNMB transcripts and the possible localization on the cell surface of this protein in human cancer samples encouraged the inventors to generate monoclonal antibodies (mAb) as a possible therapeutic agent of cancer. Thus, the inventors cloned the extracellular domain of human GPNMB (ECD; aa 23-480). Sequencing of the cloned cDNA revealed the presence of a 36 nt insert in the frame, probably due to an alternative splice at the limit of exon 6/7, which added an additional 12 aa (ATTLKSYDSNTP) (SEQ ID NO: 343) after residue 339 of the published GPNMB protein sequence. The inventors verified the authenticity of the 36 nt insertion by RT-PCR. The cDNA was then expressed in human HEK293 cells. The resulting protein was collected, purified from conditioned media and used as an immunogen to generate fully human mAbs against CG56972-ECD. Following immunization of XenoMouse® mAbs were generated that specifically recognized the CG56972-ECD protein by ELISA. The inventor guide mAb, designated 1.15 or CR011 against purified CG56972-ECD, which presented a 52 nM Kd against purified CG56972-ECD protein, was selected for in-depth characterization and will be the center of the rest of this example.

3.

 CR011 1.15 mAb detection of GPNMB protein expression in human brain cancers

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The inventors additionally used CR011 monoclonal antibodies to examine surface expression of GPNMB protein in a variety of brain cancer cell lines by flow cytometry (Figure 14 and Table 54). FACS analyzes showed that the XF-498, U-118-MG, SNB78 and SF-539 brain cancer cell lines, all positive for GPNMB transcript expression, showed superficial staining with CR011 monoclonal antibodies of at least 1.5 times higher than the noise of the isotype control mAb (Figure 14). The SF-268 cell line, which was the weakest positive (CT> 32) for GPNMB transcript expression (Figure 13C and Table 54), showed minimal surface staining as expected approximately 1.5 times above of the noise of the control mAb. The SK-MEL-2 melanoma cell line of the inventors, which is the positive control of the inventors for the expression of GPNMB, showed a strong staining of the cell surface.

To investigate the expression of the GPNMB protein in the panel of the inventors of brain cancer cell lines, the total cell lysates were collected, resolved by SDS-PAGE, transferred to membrane filters and subjected to analysis of immunoblotting with a GPNMB polyclonal antibody. As shown in Figure 15, the GPNMB polyclonal antibody detected two protein species that are glycosylation products other than GPNMB of approximately 100 and 120 kDa from various brain cancer cell lines that have been shown to express transcripts of GPNMB (Figure 13A). GPNMB protein was highly expressed in XF-498, SNB-78 and H79-MG and SF-539 cells. Both the p100 and p120 species were detected to a lesser extent in U118-MG, U251, D534-MG and D450-MG. Few or no GPNMB proteins were detected in the GPNMB transcript cell line that weakly expresses, SF-268. A control IgG2 antibody of the same isotype did not immunoprecipitate GPNMB from any of the cell lines examined. All these data are related to the expression on the cell surface of the GPNMB protein of the molecular weights predicted in brain cancer.

4. In vitro growth inhibition of astrocytoma / glioblastoma cell lines with CR011-vcMMAE.

GPNMB has a very limited expression pattern in human tissue. In preliminary studies, CR011 did not inhibit the growth of cancer cell lines expressing GPNMB when used directly (data not shown). As GPNMB is a cell surface molecule in cancers and brain melanoma, and since CR011 was internalized after incubation with cells expressing GPNMB, the inventors evaluated whether CR011 would inhibit the growth of cancer cells when combined with a secondary antibody conjugated to protein synthesis inhibitors (saporin). The results of the inventors indicated that CR011 could specifically inhibit the growth of cancer cells expressing GPNMB (data not shown). Thus, the inventors conjugated CR011 directly with the cytotoxic drug monomethylaurostatin E (MMAE) by means of a binding of valinacitrulline (vc) cleavable by highly stable but intracellular protease. The resulting antibody-drug conjugate was called CR011-vcMMAE.

To investigate whether CR011-vcMMAE specifically inhibited the growth of brain cancer cells, clonogenic assays were performed to assess cell viability after treatment with CR011-vcMMAE. As shown in Figure 16 and Table 54, the results of the inventors indicated that cells expressing GPNMB were sensitive to growth inhibition produced by CR011-vvMMAE, but not cells that poorly expressed this antigen (see SF- 268 and LOXIMVI) at vcMMAE concentrations below 3 µg / mL. Surprisingly, CR011-vcMMAE possessed IC50 of approximately 215, 450, 1250 and 1050 ng / mL in XF498, SNB-78, U-118-MG and SF-539 cells (Figure 16 and Table 54). In these experiments, IC50s were related to cell surface density as measured by FACS analysis. In contrast, the human control IgG2-vcMMAE antibody failed to inhibit the growth of any of the cell lines examined at concentrations of up to 3 µg / ml (Table 54) with IC50 exceeding 1.5 or 4.5 µg / ml ( Table 54).

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Table 54. Summary of RTQ-PCR, FACS and inhibition of in vitro growth of human cancer cell lines with mAb CR011

Cellphone line

Description CT values CR011 offset times IC50 of CR011-AE (ng / ml) IC50 of IgG2-AE (ng / ml)

SK-MEL-2

Melanoma ND 13.1, 21.4, 17.8 303 > 1500

XF-498

 Glioblastoma +++ 10, 9.5 216 > 1500

SNB-78

 Astrocytoma +++ 8.6 449 > 1500

U-118-MG

Glioblastoma / astrocytoma +++  7.4 1254 > 1500

SF-539

 Glioblastoma + 5.4 1030 > 4500

H79MG

 Glioblastoma / astrocytoma ND  4.7, 3.9 ND ND

D392-MG

Glioblastoma ND 3.1 ND ND

D534-MG

Glioblastoma ND 2.3 ND ND

SK-N-SH

 Neuroblastoma + 2 ND ND

U-251

 Glioblastoma +++ 1.9 ND ND

SF-295

 Glioblastoma ++ 1.8 ND ND

D450-MG

Glioblastoma ND 1.6 ND ND

U87MG

 Glioblastoma / astrocytoma ++ 1.5 ND ND

SF-268

 Glioblastoma / astrocytoma +  1.5 > 1500 > 4500

T98G

Glioblastoma + 1.3 ND ND

SW 1783

Astrocytoma + 1.1 ND ND

aCR011-vcMMAE (mAb 1.15): CT values were determined by RTQ-PCR as described in Materials and procedures. The geometric mean ratios (GMR) were determined by flow cytometry analysis. The antibody-drug cytotoxity (ADC) or cell destruction was determined by clonogenic assay as described. b The IC50 value is the mean and SD of a representative clonogenic assay with each experiment performed in triplicate wells. ND: Not done.

Conclusion:

These data indicate that CR011-vcMMAE can be a highly potent and selective agent for the treatment of astrocytoma / glioblastoma and its metastases, in addition to medulloblastoma brain tumors and neuroectodermal origin. CR011-vcMMAE may also be useful for the treatment of melanoma metastases from

brain and other brain neoplasms such as neoplastic meningitis.

Example 28: Genetically manipulated antibodies derived from CR011

The bi-scFv CR011 (see Figure 17) of this work were designed to bind to a CD3 epitope of the T-cell receptor 10 in cytotoxic human T lymphocytes and, at the same time, to choose diseased cells expressing GPNMB as target. the net result of facilitating lysis or destruction of diseased cells.

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The VL and VH domains of mAb CR011, clone 1.15, were used in the construction of 3 CR011 based on genetically engineered antibodies:

(one)

Single-chain antibody CR011 (scFv CR011)

(2)

bispecific single chain antibody (bi-scFv) CR011 x anti-CD3, linker set L4-L2-L4

(3)

bispecific single chain antibody (bi-scFv) CR011 x anti-CD3, linker set L4-L4-L4

The components of the scFv CR011 protein were: signal peptide-VL (CR011) -linker 4-VH (CR011) -Brand Flag. The components of the bi-scFv CR011 x anti-CD3 protein (set of linkers L4-L2-L4) were: signal peptide-VL (CR011) -binder 4-VH (CR011) -binder 2-VH (anti-CD3) -linker 4-VL (anti-CD3) -Brand Flag. The components of the bi-scFv CR011 x anti-CD3 protein (set of linkers L4-L4-L4) were: signal peptide-VL (CR011) -binder 4-VH (CR011) -binder 4-VH (anti-CD3) -linker 4-VL (anti-CD3) -Brand Flag.

The various DNA components set forth above were used to generate the three genetically engineered CR011 antibody products. The DNA components were synthesized by Blue Heron and cloned into commercially available plasmid vectors by methods familiar to those skilled in the art. Then, these plasmids were used in PCR to combine the components indicated in the 3 previous examples to generate insertions of genetically engineered antibodies for expression vectors. In the examples of host expression systems that implement this invention described below, the inventors have used mammalian CHOK1 cells for the expression vectors of CR011, but the expression is not limited to these cells; It will be recognized by those skilled in the art that the genetically engineered CR011 antibodies of this invention can be expressed using other vectors, systems and cells that include, but are not limited to: pET vectors, inducible and constitutive promoters, and hosts may include E. coli, Bacillus species, yeast that includes Pichia pastoris or insect cells. Other expression hosts may also include various species of transgenic plants and animals such as goats.

SP (signal peptide): The inventors incorporated a signal peptide in their constructs in order to express products that will be secreted. The signal peptide that was used for the expression of CHO cells was derived from an immunoglobulin light chain leader peptide (Jirik et al., 1986), or from the CR002 antibody (CuraGen).

Order of bi-scFv components: The order of the variable antibody domains was fixed in both bi-scFv constructs as follows: VL1-L-VH1-L-VH2-L-VL2. Each of the 4 V domains was linked by a linker segment, L. VL1 and VH1 represent the immunoglobulin light and heavy chain variable domains, respectively, of CR011, and VH2 and VL2 represent the heavy and light chain variable domains. of immunoglobulin, respectively, of an antibody directed against CD3 that was used for both bi-scFv constructs. Other orders of the V domains can also be used for the 2 scFv components as recognized by those skilled in the art, and the products evaluated for biological activity.

Brand: The inventors used the 8-amino acid Flag brand at the C-terminus of genetically engineered CR011 antibodies to facilitate detection and purification of products (Hickman et al., 2000).

scFv directed against CD3: The sequences of the VL and VH components of the antibody directed against CD3 used to generate the bi-scFv constructs can be found in the NCBI database under registration number CAES5148 (Lutterbuese et al.)

Linkers used in constructs: The sequence of L2, a 5-amino acid short linker that binds the 2 monomeric scFv components together in bi-scFv CR011 x anti-CD3 (linker set L4-L2-L4) is G4S (Mack et al. ., nineteen ninety five). L4 is a 25 amino acid linker based on linker 205C (Denzin et al., 1991): LSADDAKKDAAKKDDAKKDDAKKDL (SEQ ID NO: 344) and is used in both the bi-scFv CR011 species to bind the VL and VH of CR011 as the VH and VL directed against CD3. In the case of the bi-scFv CR011 x anti-CD3 (linker set L4-L4-L4), L4 is also used to bind the 2 monomeric scFv components together. For scFv CR011, linker L4 was used to link the two variable domains together.

1. DNA plasmid constructs for the expression of scFv CR011 and bi-scFv CR011 x anti-CD3 species

Flag brand of scFv CR011: The PCR amplification product to generate the expression construct for scFv CR011 was generated from a synthetic DNA template (Blue Heron) using primers F1 / R1 followed by PCR nested with the pair of primers Nested F1 / R1 (Table 55) and Pfu Turbo DNA polymerase (Stratagene, cat. No. 600322) according to the manufacturer's instructions. A Salt I / EcoR I PCR fragment encoding the sc0v CR011 cassette was cloned into the corresponding restriction sites of the pCTN vector (CuraGen Corporation, mammalian expression vector) using the Fast-Link DNA ligation kit (Epicenter, Cat. No. LK11025).

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Table 55

Name

Sequence

F1 primer

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R1 primer

image52

Primer

nested F1

F2 primer

image52

R2 primer

image52

Primer

nested F2

F3 primer

image52

R3 primer

image52

Primer

nested F3

Flag brand of the binder set (L4-L2-L4) of bi-scFv CR011 x anti-CD3: The PCR amplification product for the bi-scFv CR011 x anti-CD3 that has the binder set (L4-L2- L4) was generated from a

5 synthetic DNA template (Blue Heron) using primers F2 / R2 followed by nested PCR with the pair of nested F2 / R2 primers (see Table 55 for oligonucleotide sequences) and Pfu Turbo DNA polymerase (Stratagene, cat. 600322) according to the manufacturer's instructions. The Salt I / EcoR I PCR fragment having the coding sequence for bi-scFv CR011 x anti-CD3 (L4-L2-L4) was cloned into the corresponding sites of the pCTN vector using the Fast-Link DNA ligation kit ( Epicenter, Cat. No. LK11025).

10 Flag brand of the binder set (L4-L4-L4) of bi-scFv CR011 x anti-CD3: The PCR amplification product for bi-scFv CR011 x anti-CD3 that has the binder set (L4-L4- L4) was generated from a synthetic DNA template (Blue Heron) using primers F3 / R3 followed by nested PCR with the pair of nested F3 / R3 primers (Table 55) and Pfu Turbo DNA polymerase (Stratagene, cat. 600322) according to the manufacturer's instructions. The Nru I / Xho I PCR fragment having the coding sequence for bi-scFv CR011 x anti-CD3 (set

15 of linkers L4-L4-L4) was cloned into the corresponding sites of the expression vector pEE14.4FL2 (Lonza Biologics plc, 228 Bath Road, Slough, Berkshire SL1 4Dx, UK) using the Fast-Link DNA ligation kit ( Epicenter, Cat. No. LK11025).

The DNA sequences of the 3 inserts of the above expression constructs were verified by sequencing both chains of the relevant DNA products.

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2. Protein production of genetically engineered CR011 antibodies in CHOK1 cells

Adherent Chinese hamster ovary cells (CHOK1) (ATCC catalog number CCL-61) were cultured in DMEM medium (Invitrogen, cat. No. 10564-011) supplemented with 10% fetal bovine serum (Gemini, cat no. 100106), complement of GS (JRH Biosciences, cat. No. 58672-100M) and 50 mg / l of gentamicin (Invitrogen, no.

5 cat 15750078).

CHOK1 cells were transfected with FuGENE 6 reagent (Roche, cat. No. 1815075) according to the manufacturer's instructions. Expression and secretion was verified by Western blotting performed approximately 48 hours after transfections. The selection of stable secreted scFv CR011 and bi-scFv CR011 x bi-scFv lines (set of L4-L2-L4 linkers) was performed in selection medium A (Table 56),

10 while the selection of a stable secreted anti-CD3 bi-scFv x-scFv line (linker set L4-L4L4) was performed in selection medium B (Table 57).

Table 56

CHOK1 selection medium A (Adherent)

Seller Product No. Description

DMEM-Glutamine Free

JRH Biosciences 51435-1000M Glutamine free medium for GS SystemTM (DMEM / highly modified)

10% dialyzed SBF (heat inactivated at 56 ° C for 30 minutes)

JRH Biosciences 12117-500M Fetal bovine serum, dialyzed (500 ml)

1X GS complement

JRH Biosciences 58672-100M GS complement (50X)

50 mg / L gentamicin

Invitrogen 15750078 Solution of gentamicin reagent (50 mg / ml), liquid

1 mg / mL of G418

Invitrogen 10131027  Geneticin (G418)

Table 57

CHOK1 selection medium B (Adherent)

Seller Product No. Description

DMEM-Glutamine Free

JRH Biosciences 51435-1000M Glutamine free medium for GS SystemTM (DMEM / highly modified)

10% dialyzed SBF (heat inactivated at 56 ° C for 30 minutes)

JRH Biosciences 12117-500M Fetal bovine serum, dialyzed (500 ml)

1X GS complement

JRH Biosciences 58672-100M GS complement (50X)

50 mg / L gentamicin

Invitrogen 15750078 Solution of gentamicin reagent (50 mg / ml), liquid

25 µM MSX

Sigma M 5379 L-Methionine Sulfoximin (MSX)

fifteen

In each case, 8 of 96 CHOK1 clones of scFv CR011 and bi-scFv CR011 x anti-CD3 (set of linkers L4-L2-L4) that were secreting products were expanded and archived. The best stable clones that secreted products in each case were adapted for suspension culture in oscillating flasks with selection medium C (Table 58) at 37 ° C and 5% CO2. Protein production for scFv GR011 and bi-scFv CR011 x CD3

20 (set of linkers L4-L2-L4) was carried out in 4 L of selection medium D (Table 59) at 30 ° C and 5% CO2.

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Table 58

Large-scale selection medium C (suspension) of CHOK1

Seller  Product number Description

Ex-Cell 302

JRH Biosciences 14324-1000M Ex-Cell 302 CHO serum-free medium without L-glutamine (1000 mL)

5% SBF

JRH Biosciences 12117-500M Fetal bovine serum, dialyzed (500 mL)

GS complement

JRH Biosciences 58672-100M GS complement (50X)

HT complement

Invitrogen 11067030 HT complement (100X)

1 mg / mL of G418

Invitrogen 10131027  Geneticin (G418)

Table 59

Large-scale selection medium C (suspension) of CHOK1

Seller  Product number Description

Ex-Cell 302 + Ex-Gell CD CHO (1: 1)

JRH Biosciences 14324-1000M Ex-Cell 302 CHO serum-free medium without L-glutamine (1000 mL)

JRH Biosciences

14360-1000M CD CHO medium (1000 mL)

5% SBF

JRH Biosciences 12117-500M Fetal bovine serum, dialyzed (500 mL)

GS complement

JRH Biosciences 58672-100M GS complement (50X)

HT complement

Invitrogen 11067030 HT complement (100X)

1 mg / mL of G418

Invitrogen 10131027  Geneticin (G418)

5 Only one of two hundred CHOK1 clones of bi-scFv CR011 x anti-CD3 (linker set L4-L4-L4) was found to produce a secreted product; This clone was expanded and archived. Protein production for clone CR011 x anti-CD3 (set of linkers L4-L4-L4) was carried out using a cell manufacturing apparatus (Nunc, cat. No. 164327) in selection medium B (Table 57 ), 1 mM sodium butyrate (Sigma, cat. no. B5887) at 37 ° C and 10% CO2.

10 3. Protein purification of the CR011 manipulated antibodies

Protein purification for Flag of scFv CR011 and Flag of bi-scFv CR011 x anti-CD3 (set of linkers L4-L2-L4) was carried out in three chromatography stages that included affinity, ion exchange and exclusion chromatography. of size. For the purification of Flag protein from bi-scFv CR011 x anti-CD3 (linker set L4-L4-L4) affinity and size exclusion chromatographs were used.

15 Affinity chromatography was performed using anti-FLAG M2 affinity gel (Sigma, Cat. No. A2220-25 ml) according to the manufacturer's instructions on a BioCAD 700E instrument (Applied Biosystems). Ion exchange chromatography was performed on a MonoQ 5/50 GL column (Amersham, cat. No. 17-5166-01) using 20 mM Tris-HCI at pH 7.5 as equilibrium buffer and gradient elution with NaCl 0-1 M. The size exclusion chromatography was performed using a Superdex 75/10/300 GL column (Amersham, cat. No. 17-5174-01) following the

20 protocols of the manufacturer in the instrument of liquid chromatography BioCAD 700E (Applied Biosystems).

The approximate yields of 1 l of CHOK1 conditioning medium were:

(one)

scFv CR011: 1.0 mg

(2)

bi-scFv CR011 x anti-CD3 (linker set L4-L2-L4): 0.5 mg

(3)

bi-scFv CR011 x anti-CD3 (linker set L4-L4-L4): 1.5 mg

The amino acid sequence of the N-terminus of the purified proteins was determined by Edman degradation using methods known to those skilled in the art. The sequence of the first five amino acids was: EIVMT in each case (the mature N-terminus of the VL CR011 protein), which indicates that precise processing by the signal peptidase had occurred to give a soluble secreted product of the predicted sequence and size .

image110

The DNA and amino acid sequences of the 3 manipulated CR011 products are provided below.

SEQ ID for scFv CR011 - (VL-L4-VH) Flag. The human kappa light chain signal peptide was used as

It is described in Kabat et al. 45 CLL-CL). There was a FLAG mark included at the end C. The Kozak sequence

CCACC was included in the 5 'PCR primer.

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SEQ ID for bi-scFv CR011 x anti-CD3 (linker set L4-L2-L4) - The signal peptide of the

Kappa human light chain as described in Kabat et al. 45 CLL-CL). There was a FLAG brand included in the

end C.

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SEQ ID for bi-scFv CR011 x anti-CD3 (linker set L4-L4-L4) - The signal peptide of CR002 was used. There was a FLAG mark included on the C end.

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4. Test of the 3 antibodies manipulated CR011 by ELISA, flow cytometry and determination of cytotoxicity:

ELISA: Binding of genetically engineered CR011 antibodies to purified recombinant GPNMB (2 µg / mL) was measured using coated plates overnight at 4 ° C. Then, the plates were blocked and washed. Be

5 added various dilutions of the genetically engineered CR011 antibodies to the wells. The plates were incubated for 1 h and washed. MAb directed against FLAG M2 conjugated with HRP (Sigma, St. Louis, MO) was added to the wells for 1 h, washed and the reaction was developed with TMB substrate reagent as described by the manufacturer (Pharmingen, San Jose , CA).

The product binding of scFv CR011 and bi-scFv CR011 x anti-CD3 (linker set L4-L2-L4) to

GPNMB was first confirmed using ELISA as shown in Figure 18. Plates were coated with human GPNMB protein labeled His and V5. The coated plates were incubated with both supernatants containing biscFv CR011 x anti-CD3 and purified scFv monomer CR011. Binding of recombinant mAbs (both monomer and dimer) was detected using mAb directed against FLAG M2 conjugated to HRP (Sigma). As can be seen in Figure 18, both species of antibody directed against GPNMB described bind to the recombinant GPNMB protein indicating that the specificity and binding activity of the antibody directed against GPNMB manipulated using the procedures described in this example was preserved.

image113

Flow cytometry: The binding of genetically engineered CR011 antibodies to native proteins was analyzed by FACS. Briefly, human T-cells and SK-MEL-5 cells were incubated with both scFv CR011 and biscFv CR011 x anti-CD3 (set of linkers L4-L2-L4) (5 µg / sample / 100 µl) with subsequent staining with anti mAb -FLAG of mouse (Sigma) and F (ab ') 2 of goat anti-mouse Ig conjugated to PE. (Jackson ImmunoResearch, West Grove, PA). Ten thousand events were collected and analyzed on a FACSCalibur instrument (Becton Dickinson, Mountain View, CA).

To confirm the binding of scFv CR011 and bi-scFv CR011 x anti-CD3 products (set of L4L2-L4 linkers) to the native GPNMB protein expressed on the cell surface, the inventors used SK-MEL-5 cells that naturally expressed GPNMB . To verify the binding of bi-scFv to human CD3 molecules, the inventors used purified human T cells. As a positive control the inventors used mAb directed against native CD3-PE and CR011 conjugated to PE. ScFv CR011 binding was detected using mAb directed against FLAG M2 with subsequent staining with antibody directed against mouse IgG conjugated to PE, while for the detection of CR011 mAb binding the inventors used antibody directed against human IgG-PE. The mAb directed against control CD3 bound to T cells and the mAb directed against control GPNMB was bound to SK-MEL-5 tumor cells. The inventors found that only bi-scFv CR011 x anti-CD3 (set of linkers L4-L2-L4) stained T cells; scFv monomer CR011 did not bind to CD3 positive T cells as expected (see Figure 19). Binding to SK-MEL-5 cells by both scFv CR011 monomer and by bi-scFv CR011 x anti-CD3 (linker set L4-L2-L4) was present at a low level (Figure 19).

Cytotoxicity: The ability of bi-scFv CR011 x anti-CD3 (set of L4-L2-L4 linkers) to redirect human T lymphocytes to destroy relevant human tumor cells was measured by flow cytometry. Tumor cells were labeled with the PKH2 green fluorescent linker kit (Sigma) and washed. The purified T cells were grown overnight with PKH2 labeled tumor cells in the presence or absence of purified bi-scFv. The death of GPNMB positive tumor cells was measured by incorporation of propidium iodide (PI).

To evaluate the ability of the bi-scFv CR011 x anti-CD3 product (set of L4-L2-L4 linkers) to increase T-cell mediated destruction of GPNMB positive cells, the inventors performed a cytotoxic test. The purified T cells were cultured overnight with PK-2 labeled SK-MEL-5 tumor cells (GPNMB positive) in the presence of various doses of sc0v CR011 and bi-scFv CR011 x antiCD3 products (set of linkers L4-L2- L4) purified.

Conclusion:

Bi-scFv CR011 x anti-CD3 (set of linkers L4-L2-L4) significantly increased the destruction of SK-MEL-5 tumor cells by T lymphocytes (Figure 20). In contrast, the addition of scFv directed against monospecific GPNMB did not increase the destruction of SK-MEL-5 tumors. In addition, cytotoxicity was not observed when tumor cells were cultured with bi-scfv CR011 x anti-CD3 (set of linkers L4-L2-L4) without T lymphocytes (Figure 20). These data indicate that bi-scFv CR011 x anti-CD3 (set of linkers L4-L2-L4) provided sufficient binding by bridges between T cells and SK-MEL-5 cells to induce cell death and that both components of this bispecific antibody CR011 manipulated were biologically active. Thus, the bi-scFv CR011 x antiCD3 antibody (L4-L2-L4 linker pool) genetically engineered of the present invention can be used as a therapeutic agent to treat diseases such as melanoma and other cancers in which levels of GPNMB and T cells present are regulated by increment.

Other cytotoxicity analysis procedures including fluorescence and chromium release assay can be used to demonstrate the usefulness of bi-scFv CR011 x anti-CD3 (linker set L4-L2-L4) in the treatment of tumors. Other linkers can also be used to link the two scFv monomer components, as in the CR011 x anti-CD3 molecule (linker set L4-L4-L4) described above.

Example 29: Optimized production procedure of CR011-vcMMAE

CR011-AE is an antibody-drug conjugate composed of the fully human GPNMB directed antibody (CG56972) CR011 conjugated to the auristatin E toxin by means of a cleavable protease linker. The toxin to antibody ratio is approximately 4.0, but may vary between 3.5 and 4.2. Therefore, while the GR011 antibody is IgG2 it is possible to add up to 12 toxin molecules per antibody molecule using free thiols as a reactive site.

The structure of maleimidocaproyl-valine-citrulline-monomethyl-auristatin E (vcMMAE) is shown in Figure

twenty-one.

Conjugation: A procedure for generating the active substance that is constituted by the mAb CR011 attached to vcMMAE. An overview of the conjugation procedure is summarized in Figure 22.

Briefly, the completely human CR011 antibody conjugation procedure consists of the following 4 steps. 1) Buffer exchange and removal of sucrose by diafiltration, 2) reduction of disulfides, 3) conjugation to vcMMAE and finally, 4) purification of CR011-vcMMAE conjugated by diafiltration. There are several assays throughout the procedure, that is, trials in the process that include Ellman assay and protein concentration determination. At the end of the procedure, the active ingredient, that is, the conjugate, is analyzed for the drug ratio to antibody, free drug content and protein concentration.

image114

Diafiltration of the block antibody: The block antibody originally formulated in phosphate at pH 7 10% sucrose was changed from buffer in conjugation buffer (borate to pH 9.0 - NaCl) by diafiltration for 10

5.5 mg / mL and filtered through a set fingers filters
׽ diavolumes. At the end of the diafiltration, CR011 was diluted to

which consisted of 1.2 and 0.22 µm. Buffer change is required because sucrose interferes with the reduction. In addition, a high pH improves the solubility of CR011.

CR011 Reduction - General Considerations: CR011 is produced as an IgG2 isotype product and contains 6 disulfide bridges in the hinge region. These disulfides can be reduced under mild conditions to give rise to 12 cysteine residues. Therefore, it is possible to bind a maximum of 12 vcMMAE drug molecules per antibody. For the procedure, however, the block antibody can only be partially reduced because the objective is to generate conjugates with an average of 4 vcMMAE molecules. The reason for this is twofold. First, it expands the therapeutic window by decreasing the possible systemic toxicity associated with MMAE. Second, it is difficult and sometimes impossible to produce fully charged conjugates with low aggregation due to the greatly reduced solubility conferred by the hydrophobic drug.

Procedure: Tris- (carboxyethyl) -phosphine or TCEP in the 4: 1 molar ratio (TCEP: mAb) was added to CR011 at a concentration of ~ 5.5 mg / ml in the double-walled reactor equipped with a stirrer assembly at 90 rpm The reaction was allowed to proceed for 3 hours at 37 ° C in the presence of 1 mM EDTA. In the end, the Ellman test was used to determine the amount of free thiols. Normally they were 4.2 thiols per antibody. Then, the reactor was cooled to 4 ° C.

Conjugation of CR011 - General considerations: The TCEP was not fully consumed during the reduction. The remaining TCEP could react with vcMMAE. However, this false side reaction was slower compared to the conjugation reaction and can be mitigated by adding an excess of vcMMAE. The advantage of TCEP compared to DTT is that it does not require the removal of the excess reducing agent.

Procedure: vcMMAE was dissolved in DMSO and a 20% molar excess was added to the reduced CR011 mAb. The reaction was allowed to proceed for 1 hour. The final concentration of DMSO is 4% (v / v). DMSO played a dual purpose in the procedure. It is required to solubilize the drug and helps solubilize the conjugate. At the end of the conjugation N-acetylcysteine was added to extinguish any unreacted drug.

Purification of CR011-vcMMAE: The temperature in the reactor was brought to room temperature. A 40% sucrose stock solution was used to adjust the final sucrose concentration to 10% (weight / volume) followed by a pH adjustment using 300 mM-HCl histidine buffer at pH 5.0 to a final pH of 6, 0. Then, the conjugate was purified by diafiltration in 20 mM histidine buffer at pH 6.0-10% sucrose (weight / volume) and using 10 diavolumes. At the end of the diafiltration, the conjugate was concentrated at ~ 7 mg / mL and filtered through a set of three filters consisting of 1.2, 0.45 and finally 0.22 µm.

Formulation of CR011-vcMMAE: The conjugate was formulated by adding Tween 20 to a final concentration of 0.02% and diluting up to 6 mg / mL (± 10%) using formulation buffer (20 mM histidine at pH 6.0, sucrose at 10%, 0.02% Tween 20). Then, the conjugate was stored at 4 ° C until it was assembled if more than one batch (also known as staging time) had been prepared. After the meeting, the final concentration was adjusted to 5.0 mg / mL (± 5%) and the active substance was stored frozen.

1. Pre-conjugation of UF / DF: Sucrose Removal

The experiments were performed to monitor the sucrose removal rate during UF / DF by Ellman test and estimate the diavolume necessary to achieve the highest ratio of SH to Ab.

It was found that it is desirable to perform at least 6 diavolumes in order to remove sucrose to a level that does not prevent the reduction of CR011. To ensure robustness, at least 10 diavolumes should be used during the procedure.

2. The effect of the percentage of DMSO on aggregation in the conjugation reaction

Experiments were performed to determine the effect of DMSO on the conjugation reaction on: (1) aggregation; and (2) drug molar ratio: Ab (ie completeness of conjugation).

The percentage of aggregates in the reaction with 12% DMSO was found to be lower than in 15% DMSO, 4.4 and 3.0%, respectively. The formulation in pH 9.0 buffer versus pH 7.0 buffer had no effect on aggregation or yield, provided that 10% sucrose was included in the formulation. The percentage of aggregates in the reactions with 10%, 8%, 6% and 4% (v / v) DMSO were 2.7. 1.7. 1.0 and 0.5%, respectively. This suggests that CR011 and CR011-AE were very susceptible to aggregation when a higher percentage of DMSO is present.

image115

5

10

fifteen

twenty

25

30

35

40

The four conjugation reactions produced a final molar ratio of 4.0 drugs / Ab, suggesting that the four reactions were completed. The safety margins for the percentage of DMSO in the conjugation reaction are 4-6%. This predicted to give an aggregation level of 1% or less.

5. Investigation of the side reaction during the conjugation of CR011 to vcMMAE

Experiments were performed to: (1) investigate the degree and kinetics of the secondary reaction in which the maleimide drug becomes an unreacted byproduct producing incomplete conjugation and low drug loading; (2) determine factors that influence the secondary reaction; and (3) determine whether the old vcMMAE batch (SGD1006-0-04) differed in reactivity compared to the new batch (SGD1006-0-06).

The reactions (100 µl) containing vcMMAE at 30 µM final concentration were incubated in borate buffer at pH 9.0 both in the absence and in the presence of a double molar excess of TCEP (with respect to vcMMAE). The reactions were quenched at 0, 2, 7 or 15 min with excess NAcCys. The control constituted by vcMMAE in phosphate buffer at pH 7.0 was extinguished at the time 15 min. Chromatograms are shown in Figure 23.

In phosphate buffer at pH 7 15 min and in borate buffer at pH 9.0 0 min after drug addition, a single product extinguished with Cys (rt = 9.0 min) is formed (see A and B). In borate buffer at pH 9.0 a non-reactive by-product (rt = 9.2 min) is formed in a time-dependent mode (B, C, D and E). In borate buffer and in the presence of TCEP (such as CR011 conjugation conditions), the formation of the non-reactive product is catalyzed resulting in> 90% conversion of maleimide to succinimide after only 2 min incubation (F to I) . Both the old vcMMAE lot (SGD-1006-0-06) and the new lot (SGD-1006-0-04) had similar reactivity towards high pH and TCEP, as well as similar kinetics.

Figure 24 shows the identification by LC-MS of the non-reactive product as succinimidyl-vcMMAE (rt = 9.2 min, m / z = 1318). Figure 25 shows the relative kinetics of succinimide formation in the presence or absence of TCEP.

Conclusions

The secondary product is a result of the conjugation performed at pH 9 instead of at pH 7.4 (PBS). The formation of the secondary product is enhanced in the presence of TCEP. The main stable by-product has been identified by EM-CL as succinimidyl-vcMMAE. It remains to identify minority and less stable secondary products. Both vcMMAE batches behaved similarly.

6. Expiration of the secondary reaction during the conjugation of CR011 to vcMMAE

Experiments were performed to investigate whether the secondary reaction can be overcome by providing a large excess of the drug for conjugation.

Several ways were proposed to suppress the secondary reaction: (1) perform conjugation at a lower pH, for example 8.5 instead of 9.0 (high risk due to the reduced solubility of CR011); (2) remove excess TCEP by UF / DF (not practical); and (3) raise the excess of initially added (practical) vcMMAE.

100 mg of CR011 that was previously changed from buffer in 50 mM borate - 50 mM NaCl was reduced with TCEP to generate 4.35 free thiols per Ab. The reaction was divided into two halves. For the first half of 50 mg, a 10% excess of vcMMAE based on 1 drug per thiol ratio was added. For the second half an excess of 20% was used. The conjugates were purified by UF / DF in 10 mM histidine pH 6/10% sucrose solution. The results are summarized in Table 60.

Table 60. Preparation of CR011-vcMMAE conjugates using various vcMMAE excesses based on 1 drug per thiol ratio. The drug ratios with respect to Ab were determined by RP-HPLC.

Excess of vcMMAE,%

10

 twenty

SH by Ab

4.35  4.35

Relationship of drug with respect to Ab (in the reaction)

3.9 4.1

Relationship of drug with respect to Ab (in the final product)

3.7 4.0

image116

Conclusions

A conjugation of 100 mg was compared using 10% versus 20% excess vcMMAE. The greater excess of vcMMAE provided a drug ratio with respect to Ab closer to the expected value and, therefore, has been determined to be optimal.

The above description and examples detail certain preferred embodiments of the antibodies and describe the best mode contemplated by the inventors. However, it will be appreciated that no matter how detailed the foregoing may appear in the text, the methods of preparation and use of the antibodies described herein can be practiced in many ways and the invention is defined by the appended claims as provided by the European Patent Convention. The descriptive report previously written is considered

10 sufficient to allow a person skilled in the art to implement the embodiments described in this document.

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Claims (31)

R E I V I N D I C AC I O N E S

one.

An isolated human antibody that specifically binds to the possible transmembrane GPNMB protein defined by SEQ ID NO: 289, wherein the antibody comprises a heavy chain CDR1 defined by SEQ ID NO: 22, a heavy chain CDR2 defined by SEQ ID NO: 24, a heavy chain CDR3 defined by SEQ ID NO: 26, a light chain CDR1 defined by SEQ ID NO: 31, a light chain CDR2 defined by SEQ ID NO: 33 and a light chain CDR3 defined by SEQ ID NO: 35.

2.

The antibody of claim 1, wherein the heavy chain variable region is defined by SEQ ID NO:

twenty.

3.

The antibody of claim 1 or claim 2, wherein the variable region of the light chain is defined by SEQ ID NO: 29.

Four.

The antibody of any previous claim wherein the antibody is a monoclonal antibody.

5.

The antibody of any previous claim wherein the antibody specifically binds GPNMB with an affinity constant greater than 107 M-1.

6.

The antibody of any prior claim comprising the germline region VH4-3 1.

7.

The antibody of claim 7 further comprising the L2 germ line region.

8.

The antibody of any one of claims 1 to 5 comprising a region derived from the VH4-31 germline region.

9.

The antibody of claim 8 further comprising a region derived from the germline region L2.

10.

The antibody of any of claims 1-9, wherein said antibody is an IgG1 antibody.

eleven.

An immunoconjugate comprising the antibody of any of claims 1 to 4 and a cytotoxic agent.

12.

The immunoconjugate of claim 11, wherein the cytotoxic agent is auristatin E or dolastatin 10 or a derivative thereof.

13.

A pharmaceutical composition comprising the immunoconjugate of claim 11.

14.

A pharmaceutical composition comprising the antibody of claim 10, and an immunomodulator.

fifteen.

An isolated nucleic acid encoding the antibody of any one of claims 1 to 4.

16.

An expression vector comprising the nucleic acid of claim 15.

17.

A host cell comprising the vector of claim 16.

18.

The host cell of claim 17, wherein the host cell is an E. coli bacteria, a Chinese hamster ovary cell, a HeLa cell or an NSO cell.

19.

The nucleic acid of claim 15, wherein the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 19.

twenty.

A single chain Fv antibody comprising a VL domain of a human monoclonal antibody directed against GPNMB of claim 1 or claim 3 linked to a VH domain of said antibody directed against GPNMB.

twenty-one.

The single chain Fv antibody of claim 20 further comprising a VH domain of an antibody directed against CD3 linked to a VL domain of said antibody directed against CD3.

22

An immunoconjugate comprising a single chain Fv antibody of claim 20 and a cytotoxic agent.

2. 3.

The single chain Fv antibody of claim 20 or 21 further comprising a signal peptide or a Flag tag.

24.

The single chain Fv antibody of claim 20 comprising the amino acid sequence of SEQ ID NO: 355.

25.

The single chain Fv antibody of claim 21 comprising the amino acid sequence of SEQ ID NO: 357 or SEQ ID NO: 359.

26.

The antibody of claim 10, the immunoconjugate of claims 11, 12 or 22 or the single chain PV antibody of claim 20 for use as a medicament.

27.

The antibody of claim 10, the immunoconjugate of claims 11, 12 or 22 or the single chain PV antibody of claim 20 for the treatment of melanoma or a neoplasm of the CNS system.

28.

The antibody, immunoconjugate or single chain Fv antibody of claim 27, wherein said CNS system neoplasm is astrocytoma, glioblastoma, medulloblastoma or neoplastic meningitis.

image 1 29. The antibody, immunoconjugate or single chain Fv antibody of claim 26, wherein the medicament is for the treatment of a human patient. 30. The immunoconjugate of claim 26, wherein the medicament is used as a unit dose between 0.1 mg / kg and 10 mg / kg, with 2 to 4 administrations. 31. The immunoconjugate of claim 30, wherein the unit dose is between 0.1 mg / kg and 2 mg / kg. 10 32. The immunoconjugate of claim 31, wherein the unit dose is approximately 1 mg / kg.