RU2776443C2 - Human antibodies against semaphorin 4d - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. An antibody specifically binding to semaphorin-4D (SEMA4D) has been proposed. A conjugate, composition and method for the treatment of diseases associated with SEMA4D pathology, including autoimmune diseases, inflammatory diseases, cancer, neuroinflammatory disorders and neurodegenerative diseases, including antibody, are also proposed. In addition, the invention relates to polynucleotides encoding the specified antibody, expression vectors and host cells for the production of antibodies and conjugates.

EFFECT: group of inventions provides an expansion of the arsenal of tools for the treatment of diseases associated with SEMA4D pathologies.

46 cl, 8 dwg, 4 tbl, 4 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority over U.S. Provisional Application No. 62/501981, filed May 5, 2017, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein that belongs to the semaphorin gene family. SEMA4D is expressed on the cell membrane as a homodimer, but when cells are activated, SEMA4D can be released from the cell membrane by proteolytic cleavage to create sSEMA4D, a soluble form of the protein that is also biologically active. See Suzuki et al., Nature Rev. Immunol. 3:159-167 (2003); Kikutani et al., Nature Immunol. 9:17-23 (2008).

[0003] SEMA4D is highly expressed in lymphoid organs, including the spleen, thymus, and lymph nodes, and in non-lymphoid organs, such as the brain and kidneys. In lymphoid organs, SEMA4D is highly expressed on resting T cells but only weakly expressed on resting B cells and antigen presenting cells (APCs) such as dendritic cells (DCs). However, their expression is activated in these cells after activation of various immunological stimuli. The release of soluble SEMA4D from immune cells is also increased by cell activation.

[0004] SEMA4D has been implicated in some cancers (Ch'ng et al., Cancer 110:164-72 (2007); Campos et al., Oncology Letters 5:1527-35 (2013); Kato et al., Cancer Sci. 102:2029-37 (2011)) and several sources suggest that one of the mechanisms of this influence is the role of SEMA4D in the development of tumor angiogenesis (Conrotto et al., Blood 105:4321-4329 (2005). Basile et al., J Biol Chem 282:34888-34895 (2007) Sierra et al J Exp Med 205:1673 (2008) Zhou et al Angiogenesis 15:391-407 (2012) Tumor growth and metastasis involve a complex process of cross interaction among tumor cells, stroma and immune infiltrate, as well as endothelial cells and vessels. SEMA4D is overexpressed in a wide range of tumor types and is also produced by inflammatory cells recruited to the tumor microenvironment. Recent work indicates that SEMA4D plays an important role in the migration, survival, differentiation, and organization of the various cell types that make up the tumor stroma (Evans et al., Cancer Immunol. Res. 3:689-701 (2015)).

[0005] SEMA4D is involved in neurodegenerative disorders, autoimmune diseases, demyelinating diseases, and cancer. In the central nervous system (CNS), SEMA4D is expressed on, for example, infiltrating immune cells and oligodendrocyte progenitor cells, and its receptors are expressed on, for example, neurons, oligodendrocytes, astrocytes, and endothelial cells (Okuno, T., et al., J Immunol 184:1499-1506 (2010)). SEMA4D may serve as an axonal guidance molecule (Swiercz et al., Neuron 35:51-63 (2002)) and may mediate the development of GABAergic and glutamatergic synapses (Paradis et al., Neuron 53:217-232 (2007)) among other activities.

[0006] SEMA4D has also been shown to play a role in neuronal and oligodendrocyte progenitor cell migration and differentiation, CNS inflammation, and neurodegeneration. For example, SEMA4D-deficient mice are resistant to the development of experimental autoimmune encephalomyelitis (EAE) (Kumanogoh A et al., Immunity 13:621-631 (2000)), and SEMA4D blockade can inhibit microglial activation and neuroinflammation in EAE (Okuno, T., et al., J. Immunol 184:1499-1506 (2010)). Similarly, SEMA4D stimulation of endothelial cells can lead to the production of the pro-inflammatory cytokine IL-8 (Yang, YH et al., PLoS One 6:e25826 (2011)).

[0007] Recent publications have shown the efficacy of anti-SEMA4D binding molecules, eg, antibodies or antigen-binding fragments thereof, in the treatment of both cancer and neuroinflammatory/neurodegenerative diseases. For example, recent phase clinical trials with the anti-SEMA4D VX15/2503 antibody have demonstrated safety and efficacy in the treatment of solid tumors. See, for example, Patnaik, A., et al. Clin. Cancer Res. 22:827-836 (2016) and Southwell AL., et al., Neurobiol. Dis 76:46-56 (2015). However, there remains a need for additional anti-SEMA4D antibodies with modified and/or improved profiles, eg with respect to binding, immunogenicity and/or activity characteristics.

SUMMARY OF THE INVENTION

[0008] The present invention relates to compositions and methods for treating diseases associated with semaphorin-4 B (SEMA4D) pathologies such as autoimmune diseases, inflammatory diseases, cancer, neuroinflammatory diseases, and neurodegenerative disorders. According to aspects of the present invention, illustrated in this description, the proposed antibody or antigennegative fragments that specifically bind to SEMA4D. According to other aspects of the invention provided herein, there is provided a method of treating a subject with an autoimmune disease, inflammatory disease, cancer, neuroinflammatory disease, and neurodegenerative disorder, comprising administering to the subject an effective amount of an antibody, or antigen-binding fragment thereof, that specifically binds to SEMA4D and neutralizes the activity SEMA4D.

[0009] In certain aspects, an antibody or antigen-binding fragment thereof that specifically binds to SEMA4D comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH contains complementarity determining regions (HCDRs) of HCDR1, HCDR2 and HCDR3 containing amino acid sequences identical or identical except for one, two, three or four amino acid substitutions in one, two or three HCDRs SEQ ID NO: 2, SEQ ID NO : 3 and SEQ ID NO: 4, respectively; wherein VL contains complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 containing amino acid sequences identical or identical except for one, two, three or four amino acid substitutions in one, two or three LCDRs SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[00010] In certain aspects, an anti-SEMA4D antibody, or antigen-binding fragment thereof, contains HCDR1, HCDR2, and HCDR3 VH complementarity-determining regions that contain amino acid sequences identical to SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and VL regions defining the complementarity of LCDR1, LCDR2 and LCDR3, which contain amino acid sequences identical to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[00011] In certain aspects, the anti-SEMA4D antibody, or antigen-binding fragment thereof, comprises a VH that contains the framework regions (HFW) of HFW1, HFW2, HFW3, and HFW4, and a VL that contains the framework regions (LFW) of LFW1, LFW2, LFW3, and LFW4 . In certain aspects, the framework regions can be derived from a human antibody or from a non-human antibody.

[00012] In certain aspects, the anti-SEMA4D antibody or antigen-binding fragment thereof comprises a VH that contains the amino acid sequence of SEQ ID NO: 1. In certain aspects, the anti-SEMA4D antibody or antigen-negative fragment thereof contains a VL that contains the amino acid sequence of SEQ ID NO: 5 In certain aspects, the anti-SEMA4D antibody, or antigen-binding fragment thereof, comprises a VH and a VL that comprise the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5, respectively.

[00013] In certain aspects, the VH of an anti-SEMA4D antibody or antigen-binding fragment thereof provided herein contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1. In certain aspects of the VL of the anti-SEMA4D antibody or antigen-binding fragment provided herein contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 5. In certain aspects, the VH may contain an amino acid sequence sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 1; a VL may contain an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 5.

[00014] In certain aspects, an anti-SEMA4D antibody, or fragment thereof, disclosed herein comprises a heavy chain constant region, or fragment thereof, fused to the C-terminus of VH. In certain aspects, the heavy chain constant region, or fragment thereof, is a human heavy chain constant region. In certain aspects, the heavy chain constant region, or fragment thereof, may be an IgG4 constant region. In certain aspects, the heavy chain constant region, or fragment thereof, is a non-human heavy chain constant region. In certain aspects, the heavy chain constant region, or fragment thereof, is a mouse IgG1 constant region. In certain aspects, the anti-SEMA4D antibody, or fragment thereof, comprises VH and VL, with the light chain constant region, or fragment thereof, fused to the C-terminus of VL. In certain aspects, an anti-SEMA4D antibody, or fragment thereof, disclosed herein comprises a light chain constant region, or fragment thereof, fused to the C-terminus of VL. In certain aspects, the light chain constant region is a human light chain constant region, for example, a human lambda or human kappa light chain constant region. In certain aspects, the light chain constant region is a non-human light chain constant region, such as a mouse lambda light chain constant region.

[00015] In certain aspects, an anti-SEMA4D antibody or fragment provided herein can be a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, a single chain Fv (scFv) fragment, or a disulfide-linked fragment Fv(sdFv). In some aspects, the anti-SEMA4D antibody, or fragment thereof, may be multispecific, eg, bispecific.

[00016] In certain aspects, an anti-SEMA4D antibody, or fragment thereof, provided herein can specifically bind to human SEMA4D, mouse SEMA4D, rat SEMA4D, and/or non-human primate SEMA4D, e.g., macaque SEMA4D and/or SEMA4D marmosets. In certain aspects, a SEMA4D antibody or fragment provided herein can bind to SEMA4D, e.g., human SEMA4D, mouse SEMA4D, rat SEMA4D, and/or non-human primate SEMA4D, e.g., cynomolgus monkey SEMA4D and/or marmoset SEMA4D , with an affinity characterized by a dissociation constant KD of not more than 500 nM, 100 nM, 50.0 nM, 40.0 nM, 30.0 nM, 20.0 nM, 10.0 nM, 9.0 nM, 8.0 nM, 7.0 nM, 6.0 nM, 5.0 nM, 4.0 nM, 3.0 nM, 2.0 nM, 1.0 nM, 0.50 nM, 0.10 nM, 0.050 nM, 0.01 nM, 0.005 nM, or 0.001 nM.

[00017] In certain aspects, an anti-SEMA4D antibody, or fragment provided herein, can inhibit the binding of SEMA4D to the SEMA4D receptor, for example, plexin-B1, plexin-B2, CD72, or any combination thereof.

[00018] In certain aspects, an anti-SEMA4D antibody, or fragment thereof, elicits minimal or no immune response to the antibody when administered to a subject.

[00019] In certain aspects, an anti-SEMA4D antibody, or fragment thereof, comprises a heterologous fragment fused or conjugated to it. In some aspects, a heterologous fragment may be a polypeptide, a cytotoxic agent, a therapeutic agent, a prodrug, a lipid, a carbohydrate, a nucleic acid, a detectable label, a polymer, or any combination thereof. In certain aspects, a heterologous moiety may include a binding molecule, an enzyme, a cytokine, a lymphokine, a hormone peptide, or any combination thereof. In certain aspects, a heterologous fragment may include a radionuclide, a biological toxin, an enzymatically active toxin, or any combination thereof. In certain aspects, a heterologous fragment may include an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or any combination thereof.

[00020] In certain aspects, this invention provides a pharmaceutical composition comprising an anti-SEMA4D antibody, or fragment thereof, as provided herein, and a carrier.

[00021] In certain aspects, the invention provides an isolated polynucleotide, or combination of polynucleotides, comprising one or more nucleic acid sequences encoding an anti-SEMA4D antibody, or fragment thereof. In another aspect, the polynucleotide or combination of polynucleotides comprises a nucleic acid sequence encoding a VH, an anti-SEMA4D antibody, or a fragment thereof. In another aspect, the polynucleotide or combination of polynucleotides comprises a nucleic acid sequence encoding a VL, an anti-SEMA4D antibody, or a fragment thereof. In another aspect, the polynucleotide, or combination of polynucleotides, comprises a nucleic acid sequence encoding VH and VL, an anti-SEMA4D antibody, or a fragment thereof. In yet another aspect, the present invention provides a vector containing a polynucleotide that contains one or more nucleic acid sequences encoding an anti-SEMA4D antibody, or a fragment thereof. In another aspect, the present invention provides a host cell containing a vector. In another aspect, the present invention provides a method for producing an anti-SEMA4D antibody, or fragment thereof.

[00022] In a specific aspect of the proposed method, an anti-SEMA4D antibody, or antigen-binding fragment thereof, neutralizes SEMA4D in a human subject.

[00023] In accordance with the aspects illustrated herein, the present invention provides a method of treating a subject with an autoimmune disease or disorder, an inflammatory disease or disorders, cancer, a neuroinflammatory disease or disorder, a neurodegenerative disease or disorder, or any combination thereof, including administering to the subject an effective amount of an antibody, or antigen-binding fragment thereof, that specifically binds to SEMA4D and neutralizes SEMA4D activity. In certain aspects, the neuroinflammatory disease or disorder is multiple sclerosis. In certain aspects, the neurodegenerative disease or disorder is stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, Down's syndrome, ataxia, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), HIV-related cognitive impairment, CNS lupus, moderate cognitive impairment or a combination thereof. In certain aspects, the autoimmune disease or inflammatory disease is arthritis. In certain aspects, the autoimmune disease or inflammatory disease is rheumatoid arthritis.

BRIEF DESCRIPTION OF GRAPHICS / FIGURES

[00024] In FIG. 1A shows a comparison of the binding of MAbD2517 and MAbVX15/2503 (both dissolved in PBS) to human SEMA4D measured by ELISA.

[00025] In FIG. 1B shows a comparison of MAbD2517 and MAbVX15/2503 (both dissolved in PBS) binding to cynomolgus monkey SEMA4D measured by ELISA.

[00026] In FIG. 1C shows a comparison of MAbD2517 and MAbVX15/2503 (both dissolved in PBS) binding to marmoset SEMA4D measured by ELISA.

[00027] In FIG. 1D shows a comparison of the binding of MAbD2517 and MAbVX15/2503 (both dissolved in PBS) to mouse SEMA4D measured by ELISA.

[00028] In FIG. 2A shows the ability of MAbD2517 to block human SEMA4D binding to 293PLXNB1 cells compared to MAbVX15/2503.

[00029] In FIG. 2B shows the ability of MAbD2517 to block cynomolgus monkey SEMA4D binding to 293PLXNB1 cells compared to MAbVX15/2503.

[00030] In FIG. 2C shows the ability of MAbD2517 to block mouse SEMA4D binding to 293PLXNB1 cells compared to MAbVX15/2503.

[00031] In FIG. 2D shows the ability of MAbD2517 to block rat SEMA4D binding to 293PLXNB1 cells compared to MAbVX15/2503.

[00032] In FIG. 3A shows the measurement of tumor volume in Balb/c mice treated with either control mouse IgG1/2B8 or chimeric antibody MAbD2585 (10 mg/kg, PI, weekly X5).

[00033] In FIG. 3B shows the survival time of Balb/c mice treated with either control mouse IgG1/2B8 or chimeric MAbD2585 antibody.

[00034] In FIG. 3C shows the frequency of tumor regressions in Balb/c mice treated with either control mouse IgG1/2B8 or chimeric MAbD2585 antibody.

DETAILED DESCRIPTION

Definitions

[00035] It should be noted that the use of singular definitions does not exclude the possibility of plural definitions, for example, "binding molecule" is one or more binding molecules. Thus, the terms "one or more" and "at least one" may be used interchangeably herein.

[00036] In addition, "and/or" as used herein should be considered as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" used in a phrase such as "A and/or B" in this specification is intended to include "A and B", "A or B", "A (separately)" and "B ( separately)". Likewise, the term "and/or" used in a phrase such as "A, B and/or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separately) and C (separately).

[00037] Unless otherwise indicated, the technical and scientific terms used herein have the same meaning as is generally understood by one of ordinary skill in the art to which this description pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide dictionaries for many of the terms used in this specification to those skilled in the art.

[00038] Units, prefixes and symbols are designated in the form of the international SI system (SI). Numeric ranges include numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in the direction from the amino group to the carboxyl group. The headings provided in this document are not intended to limit the various aspects or aspects of the present invention, which may be a reference to the description as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the description in its entirety.

[00039] All embodiments described with the term "comprises" are similar to those described with the terms "consists of" and/or "consists essentially of".

[00040] Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Nucleotides are also referred to by their common single letter codes.

[00041] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals in which a population of cells is characterized by uncontrolled cell growth. Cancers can be classified, for example, as solid tumors or malignancies, or hematological malignancies or malignancies. Both types can migrate to distant organs as metastases. Solid tumors can be classified, for example, as sarcomas, carcinomas, melanomas or their metastases.

[00042] "Tumor" and "neoplasm" as used herein refer to any mass of tissue that results from overgrowth or proliferation of cells, whether benign (noncancerous) or malignant (cancerous), including precancerous lesions. In certain embodiments, the tumors described herein express the SEMA4D receptor, eg, plexin-B1, plexin-B2, and/or CD72, and/or may express SEMA4D.

[00043] As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chain of two or more amino acids and does not refer to a specific product length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "amino acid chain" or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide" and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications to a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, and with known protecting/blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide can be obtained from a biological source or obtained using recombinant technology, but is not necessarily translated from the designated nucleic acid sequence. It can be created in any way, including by chemical synthesis.

[00044] A polypeptide described herein may have a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more more, 500 or more, 1000 or more, or 2000 or more amino acids. Polypeptides may have a specific three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a specific three-dimensional structure are called folded, and polypeptides that do not have a specific three-dimensional structure, but can take on many different conformations, are called unfolded. As used herein, the term glycoprotein refers to a protein attached to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or nitrogen-containing amino acid side chain, such as serine or asparagine.

[00045] By "isolated" polypeptide or fragment, variant, or derivative thereof is meant a polypeptide that is not found in its natural environment. A specific level of cleaning is not required. For example, an isolated polypeptide may be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as described herein, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable method.

[00046] As used herein, the term "non-naturally occurring polypeptide", or any grammatical variants thereof, is a conditional definition that expressly excludes, but only excludes, those forms of the polypeptide that are, or may be, defined or interpreted by the judge. or an administrative judge, or a judicial body, as "natural".

[00047] Other polypeptides described herein are fragments, derivatives, analogs or variants of the above polypeptides, as well as any combination thereof. The terms "fragment", "variant", "derivative", and "analogue" as used herein include any polypeptide that retains at least some of the properties of the corresponding native polypeptide, antibody, or, for example, binds specifically to an antigen. Polypeptide fragments include, for example, proteolytic fragments as well as deletion fragments, in addition to certain antibody fragments discussed elsewhere herein. Variants, for example, a polypeptide, include the fragments described above, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain aspects, the variants may not be non-natural. Non-natural variants can be obtained using known methods of mutagenesis. Polypeptide variants may contain conservative or non-conservative amino acid substitutions, deletions or insertions. Derivatives of polypeptides that have been modified in such a way as to exhibit additional functions not found in the original polypeptide. Examples include fusion proteins. Polypeptide variants may also be referred to herein as "polypeptide analogs". As used herein, the term "derivative" of a polypeptide may also refer to a particular polypeptide having one or more amino acids chemically derivatized by a functional side group reaction. Also included as "derivatives" are peptides that contain one or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be replaced with proline, 5-hydroxylysine can be replaced with lysine, 3-methylhistidine can be replaced with histidine, homoserine can be replaced with serine, and ornithine can be replaced with lysine.

[00048] A "conservative amino acid substitution" is one in which one amino acid is replaced by another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine , glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine ) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). For example, replacing phenylalanine with tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequence of the polypeptides and antibodies of this invention do not abolish the binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the binding molecule binds. Methods for identifying nucleotide and amino acid conservative substitutions that do not exclude antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999) and Burks et al., Proc Natl Acad Sci USA 94:412-417 (1997)).

[00049] The term "polynucleotide" is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). The polynucleotide may contain a conventional phosphodiester bond or an unconventional bond (eg, an amide bond, eg, peptide nucleic acids (PNAs)). The terms "nucleic acid" or "nucleic acid sequence" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[00050] By "isolated" nucleic acid or polynucleotide is meant any form of nucleic acid or polynucleotide that is separated from its native environment. Thus, for example, a gel-purified polynucleotide or recombinant polynucleotide encoding a polypeptide contained in a vector would be considered "isolated". In addition, a segment of a polynucleotide, such as a PCR product, that has been modified to have restriction sites for cloning is considered "isolated". Other examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or largely) polynucleotides in a non-native solution such as buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides in which the transcript is not one that can be found in nature. Isolated polynucleotides or nucleic acids further include such synthetically produced molecules. In addition, the polynucleotide or nucleic acid may or may contain a regulatory element such as a promoter, a ribosome binding site, or a transcription terminator.

[00051] As used herein, the term "non-naturally occurring polynucleotide" or any grammatical variants thereof, is a conditional definition that expressly excludes, but only excludes, those forms of nucleic acid or polynucleotide that are, or may be, defined or interpreted by a judge or an administrative judge or judicial authority as "natural".

[00052] As used herein, the term "coding region" is the portion of a nucleic acid that consists of codons translated into amino acids. Although a "stop codon" (TAG, TG, or TAA) is not translated into amino acids, it can be considered part of the coding region, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct, eg on a single vector, or in separate polynucleotide constructs, eg on separate (different) vectors. In addition, any vector may contain a single coding region, or may contain two or more coding regions, for example, one vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, the vector, polynucleotide or nucleic acid may contain heterologous coding regions, either fused or not fused to another coding region. Heterologous coding regions include, without limitation, these coding specialized elements or motifs such as a secretory signal peptide or a heterologous functional domain.

[00053] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide containing a nucleic acid that encodes a polypeptide may typically contain a promoter and/or other transcriptional or translational control elements operably linked to one or more coding regions. A functional association where the region encoding a gene product, such as a polypeptide, is linked to one or more regulatory sequences in such a way as to place the expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (e.g., a region encoding a polypeptide and a promoter associated with it) are "operably linked" if induction of promoter function results in transcription of an mRNA encoding the desired gene product, and if the nature of the link between the two DNA fragments does not interfere with the ability of regulatory expression sequences direct the expression of the gene product or does not interfere with the ability of the DNA template to carry out transcription. Thus, a promoter region will be operably linked to a nucleic acid encoding a polypeptide if the promoter was capable of transcribing that nucleic acid. The promoter may be a cell-specific promoter that directs significant transcription of DNA in predetermined cells. Other transcriptional controls other than the promoter, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription.

[00054] A variety of transcriptional control regions are known to those skilled in the art. It includes, without limitation, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (immediate early promoter, in combination with intron-A), simian virus 40 (early promoter), and retroviruses (for example, Rous sarcoma virus). Other areas of transcriptional control include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, promoters induced by interferons or interleukins).

[00055] In addition, various translation controls are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (specifically, the internal ribosome entry site or IRES, also referred to as the Cite sequence).

[00056] In other embodiments, the implementation of the polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA or ribosomal RNA.

[00057] The polynucleotide and nucleic acid coding regions can be linked to additional coding regions that encode secretory or signal peptides that direct secretion of the polypeptide encoded by the polynucleotide, as described herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein as soon as the export of the growing protein chain through the rough endoplasmic reticulum begins. Thus, it is known to those skilled in the art that polypeptides secreted by vertebrate cells may have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce the secreted or "mature" form of the polypeptide. In some embodiments, for example, a native signal peptide, immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of that sequence, is used that retains the ability to direct secretion of the polypeptide that is operably linked to it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader may be replaced with a human tissue plasminogen activator (TPA) or mouse beta-glucuronidase leader.

[00058] Disclosed herein are certain binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Specifically, in relation to a full-length antibody, the term "binding molecule" includes full-length antibodies as well as antigen-binding subunits, fragments, variants, analogs or derivatives of such antibodies, for example, engineered antibody molecules or fragments that bind to the antigen of a similar antibody molecule, but which using a different framework.

[00059] As used herein, the term "binding molecule" refers broadly to a molecule that specifically binds to a receptor, such as an epitope or antigenic determinant. As described later in this description, the binding molecule may contain one or more of the "antigen-binding domains" described in this application. A non-limiting example of a binding molecule is an antibody or fragment thereof that retains antigen-specific binding.

[00060] As used herein, the terms "binding domain" or "antigen binding domain" refer to the binding region of a molecule that is necessary and sufficient for specific binding to an epitope. For example, "Fv", eg, the variable heavy chain and the variable light chain of an antibody, either as two separate polypeptide subunits or as a single chain, is considered a "binding domain". Other binding domains include, without limitation, the variable heavy chain (VHH) of antibodies derived from camelid species or six immunoglobulin complementarity determining regions (CDRs) expressed in the fibronectin framework. A "binding molecule" described herein may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more "antigen binding domains".

[00061] The terms "antibody" and "immunoglobulin" may be used interchangeably herein. An antibody (or fragment, variant, or derivative thereof described herein) contains at least a heavy chain variable region (for camelid species) or at least a heavy chain and light chain variable region. The basic structures of immunoglobulin in vertebrate systems are relatively well understood. See, for example, Harlow et al., Antibodies: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise indicated, the term "antibody" covers everything from a small antigen-binding fragment of an antibody to full-length antibodies, such as IgG antibodies that contain two full heavy chains and two full light chains, to IgA antibodies that contain four full heavy chains and four complete light chains and optionally contain a J chain and/or a secretory component, or IgM antibodies that contain ten or twelve complete heavy chains and ten or twelve complete light chains and optionally contain a J chain.

[00062] As will be discussed in more detail below, the term "immunoglobulin" includes various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4 or α1-α2). The nature of this chain, which defines the "class" of the antibody as IgG, IgM, IgA IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), eg IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , IgA ₂ , etc., are well characterized and are known to have functional specializations. Modified versions of each of these classes and isotypes are readily discernible to one of ordinary skill in the art in light of this disclosure, and are therefore within the scope of this invention.

[00063] Light chains are classified as kappa or lambda (κ, λ). Each class of heavy chain can be linked to either a kappa light chain or a lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other via covalent disulfide bonds or non-covalent bonds when the immunoglobulins are generated by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences start from the N-terminus at the branched ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of some antibodies, such as IgG antibodies, contains two heavy chain subunits and two light chain subunits covalently linked via disulfide bonds to form a "Y" structure, also referred to herein as the "H2L2" structure.

[00064] And light and heavy chains are divided into areas of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be borne in mind that the variable regions of both the variable light (VL) and variable heavy (VH) regions of the chains determine the recognition and specificity of the antigen. Conversely, the light chain (CL) and heavy chain (CH1, CH2 or CH3) constant regions confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, and the like. It is believed that the numbering of the constant regions increases as they become more distal from the antigen binding site or the amino terminus of the antibody. The N-terminal region is the variable region and the C-terminal region is the constant region; CH3 (or CH4 in the case of IgM), and the CL domains actually contain the carboxy-terminus of the heavy and light chain, respectively.

[00065] As noted above, the variable region (ie, "binding domain") allows the binding molecule to selectively recognize and specifically bind epitopes on antigens. That is, a VL region and a VH region, or a subset of complementarity determining regions (CDRs) of a binding molecule, such as an antibody, are combined to form a variable region that defines a three-dimensional antigen binding site. More specifically, the antigen binding site is defined by three CDRs, on each of the VH and VL chains. Some antibodies form larger structures. So, for example, IgA can form a molecule that contains two H2L2 units, a J chain, and a secretory component, all covalently connected via disulfide bonds, and IgM can form a pentameric or hexameric molecule that contains five or six H2L2 units and optionally J chain covalently linked by disulfide bonds.

[00066] The six "complementarity determining regions" or "CDRs" present in the antigen-binding domain of an antibody are short, non-contiguous sequences of amino acids that are specifically arranged to form the binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the amino acids in the binding domain, referred to as "framework" regions, show less intermolecular variability. The framework regions largely adopt the β-sheet conformation and CDRs, form loops connecting and in some cases form part of this β-sheet structure. Thus, the framework regions act to form a framework that positions the CDRs in the correct orientation through interstrand, non-covalent interactions. The binding domain formed by the located CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. The amino acids that make up the CDRs and framework regions, respectively, can be easily identified for any heavy or light chain variable region by one of ordinary skill in the art, as they have been determined in various ways (see, "Sequences of Proteins of Immunological Interest ," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 796:901-917 (1987), which are incorporated herein in their entirety via link).

[00067] In the event that there are two or more definitions of a term that are used and/or accepted in the art, the definition of a term used in this specification includes all such meanings unless expressly stated otherwise. A specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen pooling sites found in the variable region of both heavy and light chain polypeptides. These specific regions have been described, for example, in Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference. The definitions of Kabat and Chothia include an overlap or subset of amino acids when compared against each other. However, the use of a definition (or other definitions known to those skilled in the art) to refer to an antibody CDR or variant is intended to be within the scope of that term, as defined and used herein, unless otherwise indicated. The corresponding amino acids that encompass the CDRs, as defined by each of the above cited references, are shown in Table 1 below by way of comparison. The exact amino acid amounts that a particular CDR spans will vary depending on the sequence and size of the CDR. Those skilled in the art can determine which amino acids a particular CDR typically contains based on the amino acid sequence of an antibody's variable region.

[00068] Immunoglobulin variable regions can also be analyzed using the IMGT information system (www://imgt.cines.fr/) (EVIGT®/V-Quest) to identify variable region segments, including CDRs. See, for example, Brochet, X. et al., Nucl. Acids Res. 36:W503-508 (2008).

[00069] Kabat et al. a numbering system for variable region sequences is also defined that is applicable to any antibody. One skilled in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence, without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). However, unless the use of the Kabat numbering system is explicitly noted, sequential numbering is used for all amino acid sequences in this specification.

[00070] Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof, include, but are not limited to, polyclonal, monoclonal, humanized, human, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') ₂ , Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide linked Fvs (sdFv), fragments containing either the VL or VH region, and the fragments are generated by a Fab expression library. ScFv molecules are known in the art and are described, for example, in US Pat. No. 5,892,019. Immunoglobulin molecules or antibodies covered by this description may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of the immunoglobulin molecule.

[00071] The term "specifically binds" is generally understood to mean that a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and an epitope. By this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen-binding domain, more readily than it would to a random, unbound epitope. The term "specificity" is used herein to qualify the relative affinity by which a particular binding molecule binds to a particular epitope. So, for example, binding molecule "A" can be considered to have a higher specificity for a given epitope than binding molecule "B", or binding molecule "A" can be said to bind to epitope "C" with higher specificity than she does so with the appropriate epitope "D".

[00072] A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof described herein, can be said to bind a target antigen with a dissociation constant (k(off)) less than or equal to 5×10 ^-2 sec ^-1 , 10 ^-2 sec ^-1 , 5×10 ^-3 sec ^-1 , 10 ^-3 sec ^-1 , 5×10 ^-4 sec ^-1 , 10 ^-4 sec ^-1 , 5×10 ^-5 sec ^-1 or 10 ^-5 sec ^-1 5×10 ^-6 sec ^-1 , 10 ^-6 sec ^-1 , 5×10 ^-7 sec ^-1 or 10 ^-7 sec ^-1 .

[00073] A binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof described herein, can be said to bind a target antigen with an association constant (k(on)) greater than or equal to 10 ³ M ^-1 sec ^{- 1} , 5×10 ³ M ^-1 sec ^-1 , 10 ⁴ M ^-1 sec ^-1 , 5×10 ⁴ M ^-1 sec ^-1 , 10 ⁵ M ^-1 sec ^-1 , 5×10 ⁵ M ^-1 sec ^-1 , 10 ⁶ M ^-1 sec ^-1 or 5×10 ⁶ M ^-1 sec ^-1 , or 10 ⁷ M ^-1 sec ^-1 .

[00074] A binding molecule, such as an antibody or fragment, variant, or derivative thereof, can be said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some extent, the binding of the reference antibody or antigen-binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, such as competitive ELISA. The binding molecule can be said to competitively inhibit the binding of the control antibody or antigen-binding fragment thereof to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% .

[00075] As used herein, the term "affinity" refers to the measurement of the binding strength of an individual epitope to one or more binding domains, such as an immunoglobulin molecule. See, for example, Harlow et al., Antibodies: A Laboratory Manual., (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. Used in this description, the term "avidity" refers to the overall stability of the complex between populations of binding domains and antigen. See, for example, Harlow on pages 29-34. Avidity is related both to the affinity of individual binding domains in a population for specific epitopes and to the valence of immunoglobulins and antigen. Thus, for example, an interaction between a divalent monoclonal antibody and an antigen with a highly repetitive epitope structure, such as a polymer, would have high avidity. The interaction between a bivalent monoclonal antibody with a receptor present at high density on the cell surface will also have high avidity.

[00076] Molecule binding or antigen-binding moiety, variants or derivatives thereof described herein can also be described or defined in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule, such as an antibody or fragment, variant or derivative thereof, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, a binding molecule is cross-reactive if it binds to an epitope other than that which induces its formation. The cross-reactive epitope usually contains many of the same additional structural features as the inducing epitope and, in some cases, may actually fit better than the original.

[00077] A binding molecule, such as an antibody or fragment, variant, or derivative thereof, can also be described or defined in terms of their binding affinity for an antigen. For example, a binding molecule may bind to an antigen with a dissociation constant or K _D of no more than 5×10 ^-2 M, 10 ^-2 M, 5×10 ^-3 M, 10 ^-3 M, 5×10 ^-4 M, 10 ^-4 M, 5×10 ^-5 M, 10 ^-5 M, 5×10 ^-6 M, 10 ^-6 M, 5×10 ^-7 M, 10 ^-7 M, 5×10 ^-8 M, 10 ^-8 M, 5×10 ^-9 M, 10 ^-9 M, 5×10 ^-10 M, 10 ^-10 M, 5×10 ^-11 M, 10 ^-11 M, 5×10 ^-12 M, 10 ^-12 M, 5×10 ^-13 M, 10 ^-13 M, 5×10 ^-14 M, 10 ^-14 M, 5×10 ^-15 M or 10 ^-15 M.

[00078] Antibody fragments, including single chain antibodies or other binding domains, may exist alone or in combination with one or more of the following: a hinge region, CH1, CH2, CH3, or CH4 domains, a J chain, or a secretory component. In addition, antigen-binding fragments are included which may contain any combination of variable region(s) with one or more components from the hinge region, CH1, CH2, CH3, CH4 domains, J chain, or secretory component. The binding molecule, eg antibodies or antigen binding fragments, can be of any animal origin, including birds and mammals. The antibodies can be derived from human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be derived from cartilaginous fish (eg, from sharks). As used herein, the term “human” antibodies includes antibodies having the amino acid sequence of a human immunoglobulin, and includes antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and may in some cases express endogenous immunoglobulins and some not. as described below and, for example, in the US patent. No. 5939598 from Kucherlapati et al.

[00079] As used herein, the term “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain binding molecule, e.g. (eg, upper, middle and/or lower hinge region), CH2 domain, CH3 domain, CH4 domain, or a variant or fragment thereof. For example, binding molecules, such as an antibody or fragment, variant, or derivative thereof, may contain, in addition to the VH region, a CH1 domain; CH1 domain, hinge and CH2 domain; CH1 domain and CH3 domain; CH1 domain, hinge and CH3 domain, or CH1 domain, hinge domain, CH2 domain and CH3 domain. In certain aspects, a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, may comprise, in addition to the VH region, a CH3 domain and a CH4 domain, or a CH3 domain, a CH4 domain, and a J chain. the document may not have parts of the constant region, such as all or part of the CH2 domain. It will be appreciated by any person skilled in the art that these domains (eg, the heavy chain subunit) can be altered such that they differ in amino acid sequence from the original immunoglobulin molecule.

[00080] The heavy chain subunit of a binding molecule, such as an antibody or fragment thereof, may contain domains derived from various immunoglobulin molecules. For example, a polypeptide heavy chain subunit may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain subunit may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain subunit may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[00081] As used herein, the term "light chain subunit" includes amino acid sequences derived from an immunoglobulin light chain. The light chain subunit contains at least one of the VL or CL domains (eg, Cκ or Cλ).

[00082] Binding molecules, such as antibodies or antigen-binding fragments, variants or derivatives thereof, can be described or defined in terms of the epitope(s) or portion(s) of an antigen that they recognize or specifically bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant". The target antigen may contain one epitope or at least two epitopes, and may contain any number of epitopes, depending on the size, conformation, and type of the antigen.

[00083] As previously stated, the subunit structures and three-dimensional configurations of the constant regions of the various classes of immunoglobulins are well known. As used herein, the term "VH region" includes the amino-terminal immunoglobulin heavy chain variable region, and the term "CH1 domain" includes the first (most amino-terminal) domain of the immunoglobulin heavy chain constant region. The CH1 domain is adjacent to the VH region and is amino-terminal to the hinge region of a typical immunoglobulin heavy chain molecule.

[00084] As used herein, the term “CH2 domain” includes the portion of a heavy chain molecule that runs from about amino acid 244 to amino acid 360 of an IgG antibody, for example, using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231 -340), EU numbering system; see Kabat EA et al. op. cit. The CH3 domain starts from the CH2 domain towards the C-terminus of the IgG molecule and contains approximately 108 amino acids. Certain classes of immunoglobulins, such as IgM, additionally contain a CH4 region.

[00085] As used herein, the term "hinge region" includes the heavy chain portion of the molecule that connects the CH1 domain to the CH2 domain. This hinge region contains about 25 amino acids and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently of each other.

[00086] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In certain IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

[00087] As used herein, the term "hybrid antibody" means an antibody in which the immunoreactive region or site is derived or derived from a first species and the constant region (which may be intact, partial, or modified) is derived from a second species. In some embodiments, the target binding region or site will be from a non-human source (eg, mouse or primate) and the constant region is human.

and Heiss, Future Oncol. (5:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). Биспецифическое антитело может также представлять собой диатело.[00088] The terms "multispecific antibody" or "bispecific antibody" refer to an antibody that has binding domains for two or more different epitopes within a single antibody molecule. Other binding molecules in addition to the canonical antibody structure can be designed with dual binding specificity. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed using recombinant means. (

and Heiss, Future Oncol. (5:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). The bispecific antibody may also be a diabody.

[00089] As used herein, the term "engineered antibody" means an antibody in which the variable region of either the heavy and light chains or both has been altered by at least partial substitution of one or more amino acids, either in the CDRs or in the framework regions. In certain aspects, entire CDRs from an antibody of known specificity can be grafted onto the framework regions of a heterologous antibody. Although alternative CDRs may be derived from an antibody of the same class or subclass, even as the antibody from which the framework regions are derived, CDRs may also be derived from an antibody of a different class, such as an antibody from a different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity are grafted onto a human heavy or light chain framework region is referred to herein as a "humanized antibody". In some aspects, not all CDRs are replaced by full CDRs from the donor variable region, and yet the antigen-binding potential of the donor can still be transferred to the recipient variable regions. Taking into account the explanations set forth in, for example, US Pat. .

[00090] As used herein, the term "engineered" includes the manipulation of nucleotide or polypeptide molecules by synthetic means (eg, by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or some combination of these techniques).

[00091] As used herein, the terms "linked", "merged" or "fusion" or other grammatical equivalents may be used interchangeably. These terms refer to the combination of more than two elements or components with each other, by any means, including chemical conjugation or recombinant methods. "Fusion in frame" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF such that the translated reading frame of the original ORFs is maintained. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to the polypeptides encoded by the original ORFs (whose segments are not normally fused in nature.) Although the reading frame is thus produced continuously during the fused segments, the segments may be physically or spatially separated, for example, linker sequences in reading frame. For example, polynucleotides encoding an immunoglobulin variable region CDR may be fused in-frame but separated by a polynucleotide encoding at least one immunoglobulin backbone region or additional CDRs, and the "fused" CDRs are co-translated as part of a continuous polypeptide.

[00092] In the context of polypeptides, "linear sequence" or "sequence" is the order of amino acids in a polypeptide in the direction from the amino terminus to the carboxyl terminus, in which the amino acids that border each other in the sequence are contiguous in the primary structure of the polypeptide. The part of a polypeptide that is "amino-terminal" or "N-terminal" to another part of the polypeptide is that part that is located earlier in the sequential polypeptide chain. Similarly, the part of a polypeptide that is "carboxy-terminal" or "C-terminal" to another part of the polypeptide is that part that is located later in the sequential polypeptide chain. For example, in a typical antibody, the variable region is "N-terminal" to the constant region, and the constant region is "C-terminal" to the variable region.

[00093] The term "expression" as used herein refers to the manner in which a gene produces a biochemical, such as a polypeptide. The process includes any manifestation of the functional presence of a gene in a cell, including, without limitation, gene knockdown, as well as transient expression and stable expression. It includes, without limitation, transcription of a gene into messenger RNA (mRNA) and translation of such mRNAs into polypeptide(s). If the final desired product is biochemical, expression involves the creation of biochemicals and any precursors. The expression of a gene produces a "gene product". As used herein, a gene product may be either a nucleic acid, such as messenger RNA obtained by transcription of a gene, or a polypeptide that is translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation, or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, and the like.

[00094] As used herein, the term "anti-SEMA4D binding molecule" refers to a molecule that specifically binds to SEMA4D, such as an antibody or antigen-binding fragment, variant, or derivative thereof. When specifically referring to a full-length antibody, such as a naturally occurring antibody, the term "anti-SEMA4D antibody" includes full-length antibodies, as well as antigen-binding fragments, as well as antigen-binding fragments, variants, analogs, or derivatives, of such antibodies, for example, natural antibodies or molecules immunoglobulin or engineered antibody molecules or fragments that bind to an antigen in a manner similar to that of antibody molecules.

[00095] Terms such as "treatment" or "treatment process" or "treat" or "alleviate" or "improve" refers to therapeutic measures that cure, slow down, reduce symptoms, and/or stop or slow down the progression of an existing diagnosis pathological condition or disorder. Terms such as prevent, prevent, avoid, contain, etc. refers to prophylactic or preventive measures that prevent the development of an undiagnosed target pathological condition or disorder. Thus, "one in need of treatment" may include those who already have the disorder; tend to have the disorder; and those in whom the disorder is to be prevented. As used herein, phrases such as "subject that would benefit from therapy" and "animals in need of treatment" include subjects, such as mammalian subjects, that would benefit from administration of therapy as described herein.

[00096] The term "therapeutically effective amount" refers to the amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to "treat" a disease or disorder in a subject or mammal.

[00097] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, in particular a mammal, for which a diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, pets, farm animals, and zoo animals, sport animals, or companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and etc.

Description of the target polypeptide - SEMA4D

[00098] As used herein, the terms "semaphorin-4D", "SEMA4D", and "SEMA4D polypeptide" are used interchangeably, as are "SEMA4D" and "Sema4D". In some embodiments, SEMA4D is surface-expressed or secreted by a cell. In another embodiment, SEMA4D is membrane bound. In another embodiment, SEMA4D is soluble, such as sSEMA4D. In another embodiment, SEMA4D may include full-length SEMA4D or a fragment thereof, or a SEMA4D polypeptide variant, wherein the SEMA4D fragment or SEMA4D polypeptide variant retains some or all of the functional properties of full-length SEMA4D.

[00099] The full-length human SEMA4D protein is a transmembrane protein homodimer consisting of two polypeptide chains, 150 kDa in size. SEMA4D belongs to the semaphorin family of cell surface receptors and is also referred to as CD100. Both human and mouse SEMA4D/Sema4D can be proteolytically cleaved from their transmembrane form to create 120 kDa soluble forms, resulting in two isoforms of Sema4D (Kumanogoh et al., J. Cell Science 116:3464 (2003)). Semaphorins are composed of soluble and membrane-bound proteins that were originally identified as axonal guidance factors that play an important role in establishing precise connections between neurons and their respective target. Structurally considered a class IV semaphorin, SEMA4D consists of an amino-terminal signal sequence followed by a characteristic "Sema" domain that contains 17 cysteine residues, an lg-like domain, a lysine-rich stretch sequence, a hydrophobic transmembrane region, and a cytoplasmic tail.

[000100] The SEMA4D polypeptide contains a signal sequence of about 13 amino acids, followed by a semaphorin domain of about 512 amino acids, an immunoglobulin-like (Ig-like) domain of about 65 amino acids, a lysine-rich stretching sequence of 104 amino acids, a hydrophobic transmembrane region of about 19 amino acids and a cytoplasmic tail of 110 amino acids. A consensus site for tyrosine phosphorylation in the cytoplasmic tail supports the predicted association of SEMA4D with tyrosine kinase (Schlossman et al., Eds. (1995) Leucocyte Typing V (Oxford University Press, Oxford).

[000101] SEMA4D is known to have at least three functional receptors, plexin-B1, plexin-B2 and CD72. Plexin-B1 is expressed in non-lymphoid tissues and shows high affinity (1 nM) for the SEMA4D receptor (Tamagnone et al., Cell 99:71-80 (1999)). Stimulation by SEMA4D of plexin-B1 signaling has been shown to induce neuronal growth cone collapse, and induce expansion of collapse and apoptosis of oligodendrocytes (Giraudon et al., J. Immunol. 172:1246-1255 (2004); Giraudon et al., NeuroMolecular Med. 7:207-216 (2005)). After binding to SEMA4D, plexin-B1 signaling mediates R-Ras inactivation, which leads to a decrease in integrin-mediated attachment to the extracellular matrix, as well as RhoA activation, which leads to cell collapse by cytoskeletal reorganization (Kruger et al., Nature Rev. Mol Cell Biol 6:789-800 (2005); Pasterkamp, TRENDS in Cell Biology 15:61-64 (2005)). Plexin-B2 has an intermediate affinity for SEMA4D and a recent report indicates that plexin-B2 is expressed on keratinocytes and activates SEMA4D-positive γδ T cells to promote epithelial repair (Witherden et al., Immunity 37:314-25 (2012 )).

[000102] In lymphoid tissue, CD72 is used as a low affinity (300 nM) SEMA4D receptor (Kumanogoh et al., Immunity 13:621-631 (2000)). B cells and antigen presenting cells (APC) express CD72, and anti-CD72 antibody has many of the same effects as sSEMA4D, such as enhancing CD40-induced B cell responses and CD23 shedding by B cells. CD72 is believed to act as a negative regulator of B cell responses by recruiting the tyrosine phosphatase SHP-1, which can associate with many inhibitory receptors. The interaction of SEMA4D with CD72 results in the dissociation of SHP-1, and the loss of this negative activation signal. SEMA4D has been shown to promote T cell stimulation and B cell aggregation and survival in vitro. The addition of SEMA4D-expressing cells or sSEMA4D enhances CD40-induced B cell proliferation and immunoglobulin production in vitro, and accelerates antibody responses in vivo (Ishida et al., Inter. Immunol. 15:1027-1034 (2003); Kumanogoh and H. Kukutani, Trends in Immunol 22:670-676 (2001)). sSEMA4D enhances CD40-induced DC maturation, including an increase in the activity of costimulatory molecules and increased secretion of IL-12. In addition, sSEMA4D can inhibit immune cell migration, which can be abolished by the addition of mouse anti-SEMA4D blocking antibodies (Elhabazi et al., J. Immunol. 166:4341-4347 (2001); Delaire et al., J. Immunol. 166 :4348-4354 (2001)).

[000103] Sema4D is highly expressed in lymphoid organs, including the spleen, thymus, and lymph nodes, and in non-lymphoid organs, such as the brain, heart, and kidneys. In lymphoid organs, Sema4D is highly expressed on resting T cells but only weakly expressed on resting B cells and antigen presenting cells (APCs) such as dendritic cells (DCs).

[000104] Cellular activation increases the surface expression of SEMA4D as well as the generation of soluble SEMA4D (sSEMA4D). The expression pattern of SEMA4D suggests that it plays an important physiological as well as pathological role in the immune system. SEMA4D has been shown to promote B cell activation, aggregation, and survival; increased CD40-induced proliferation and antibody production; increased antibody response to dependent T-cell antigens; increased T cell proliferation; increased maturation of dendritic cells and the ability to stimulate T cells; and is directly involved in demyelination and axonal degeneration (Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., J. Immunol. 169:1175-1181 (2002) and Watanabe et al., J. Immunol 167:4321-4328 (2001)).

Human anti-SEMA4D antibody

[000105] The present invention provides an anti-SEMA4D binding molecule, e.g., an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof with a heavy chain variable region (VH) and a light chain variable region (VL) associated with, or identical amino acid sequences containing VH and VL SEQ ID NO: 1 and SEQ ID NO: 5, respectively. In certain aspects, the proposed binding molecule, for example, an antibody or antigen-binding fragment, variant, or derivative thereof, is fully human.

[000106] In certain aspects, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof provided herein has a VH and VL region containing amino acid sequences that are at least about 80% about 85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99 % or 100% identity to the VH and VL regions of the reference anti-SEMA4D antibody molecule with the VH and VL regions containing the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5, respectively.

[000107] In certain aspects, an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment, variant, or derivative thereof, can inhibit the interaction of SEMA4D with its receptor, eg, plexin-B1, plexin-B2, or CD72. In certain aspects, an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment, variant, or derivative thereof, can inhibit SEMA4D-mediated plexin-B1 signal transduction.

[000108] In certain aspects, anti-SEMA4D binding molecules, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, competitively inhibits a reference antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1 and a light variable region chain (VL) containing the amino acid sequence of SEQ ID NO: 5 from binding to SEMA4D. In some aspects, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, binds to the same SEMA4D epitope as a reference antibody containing a VH containing the amino acid sequence of SEQ ID NO: 1 and a VL containing the amino acid sequence of SEQ ID NO: 5. In certain aspects, the VH of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, contains three complementarity determining regions (CDRs) of HCDR1, HCDR2, and HCDR3, and the VL contains three CDRs, LCDR1, LCDR2, LCDR3, with CDRs, containing the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, except for at least one, two, three, four, five, or six single conservative amino acid substitutions in one or more CDRs. In certain aspects, the CDRs comprise the amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[000109] In certain aspects, the VH of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, contains an amino acid sequence of at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1, a VL of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, contains an amino acid sequence of at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 5. In certain aspects, VH contains the amino acid sequence of SEQ ID NO: 1, and VL contains the amino acid sequence of SEQ ID NO: 5.

[000110] Also provided herein are polypeptides encoding anti-SEMA4D antibodies or antigen-binding fragments, variants or derivatives described herein, polynucleotides encoding such polypeptides, vectors containing such polynucleotides, and host cells containing such vectors or polynucleotides, for use, for example, in the preparation of an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein.

[000111] Suitable biologically active variants of the anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, are provided by this disclosure and can be made and used in accordance with the methods provided herein, or in accordance with methods well known to those skilled in the art. Such variants retain certain desirable binding properties of the reference anti-SEMA4D antibody described herein. Methods for producing antibody variants are generally available in the art.

[000112] Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel, Proc. Natl. Acad. sci. USA 82:488-492 (1985); Kunkel et al., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); US patent No. 4873192; and the links given there; included in this document by reference. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), pp. 345-352, incorporated herein by reference in its entirety. The model of Dayhoff et al. uses a point accepted mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conservative amino acid substitutions. In certain aspects, conservative substitutions are used, such as replacing one amino acid with another having similar properties. Examples of conservative amino acid substitutions described in the PAM 250 matrix model by Dayhoff et al. include, without limitation, Gly↔Ala, Val↔Ile↔Leu, Asp↔Glu, Lys↔Arg, Asn↔Gln, and Phe↔Trp↔Tyr.

[000113] When constructing variants of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment thereof, a polypeptide of interest, modifications made such that the variants still have the desired properties, e.g., the ability to specifically bind to SEMA4D, e.g., human SEMA4D, SEMA4D non-human primate (e.g. monkey, marmoset, and/or SEMA4D rhesus monkey) and/or rodent SEMA4D (e.g. mouse and/or rat SEMA4D), e.g. an antibody or fragment, variant or derivative thereof has the SEMA4D binding, receptor blocking, or neutralizing activity described herein. In certain aspects, mutations made in the DNA encoding the polypeptide variant retain the reading frame and do not create complementary regions that can produce the secondary structure of the mRNA. See European Patent Application Publication No. 75444.

[000114] Methods for measuring the binding specificity and/or activity of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, include, but are not limited to, standard binding assays, including competitive binding assays, assays to monitor immunoglobulin secretion with using T cells or B cells, T cell proliferation assays, apoptosis assays, ELISA assays, and the like. See, for example, such assays described in WO 93/14125; Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., J. Immunol. 169:1175-1181 (2002); Watanabe et al., J. Immunol. 167:4321-4328 (2001); Wang et al., Blood 97:3498-3504 (2001) and Giraudon et al., J. Immunol 172:1246-1255 (2004), all of which are incorporated herein by reference.

[000115] When discussed herein, whether at least any particular polypeptide, including constant regions, CDRs, VH regions, or VL regions, disclosed herein is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% , about 99% or even about 100% identical to another polypeptide, and % identity can be determined using methods and computer programs/software known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm (Smith & Waterman, Adv. Appl. Math. 2:482-489 (1981), using the global alignment algorithm (Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), using similarity search methods (Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988); Altschul et al., Nucl. Acids Res. 25:3389-402 (1997), using computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), typically using default parameters, or manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, Ausubel et al. (eds.), 1994).

[000116] For the purposes of this invention, percent sequence identity can be determined using the Smith-Waterman homology search algorithm using a gap opening penalty of 12 and a gap extension penalty of 2, a BLOSUM matrix of 62. An option may, for example, , differs from a reference anti-SEMA4D antibody (e.g., MAb 2517 provided herein) by only 1 to 15 amino acid residues, only 1 to 10 amino acid residues, such as 6-10, only 5, only 4 , 3, 2 or even 1 amino acid residue.

[000117] In certain aspects, the invention provides an antibody or antigen-binding fragment, variant or derivative thereof that specifically binds to SEMA4D, said antibody or fragment thereof comprising VH and VL. In certain aspects, the VH contains complementarity determining regions (HCDRs) of HCDR1, HCDR2, and HCDR3 containing amino acid sequences that are identical or identical except for one, two, three, four, five, or six amino acid substitutions in one, two, or all three of HCDR in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In certain aspects, HCDR1, HCDR2, and HCDR3 contain identical amino acid sequences to SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. In certain aspects, the VL comprises complementarity determining regions (LCDRs) of LCDR1, LCDR2, and LCDR3 containing amino acid sequences that are identical or identical except for one, two, three, four, five, or six amino acid substitutions in one, two, or all three of LCDR in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In certain aspects, LCDR1, LCDR2 and LCDR3 contain amino acid sequences identical to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In certain aspects, the VH further comprises frame regions (HFW) HFW1, HFW2, HFW3 and HFW4, and the VL further comprises frame regions (LFW) LFW1, LFW2, LFW3 and LFW4. In certain aspects, the framework regions are derived from a human antibody. In certain aspects, for example, in which the antibody is an antibody for use in a non-human model system, the framework regions are derived from a non-human antibody, such as a mouse antibody. In certain aspects, the VH contains the amino acid sequence of SEQ ID NO: 1. In certain aspects, the VL contains the amino acid sequence of SEQ ID NO: 5.

[000118] In certain aspects, this invention provides an antibody, for example, a fully human antibody or antigen-binding fragment, variant or derivative thereof, that specifically binds to semaphorin-4D (SEMA4D) containing VH and VL. In certain aspects, VH contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In certain aspects, VL contains an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In certain aspects, VH contains an amino acid sequence of at least 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1; a VL contains an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In certain aspects, VH contains the amino acid sequence of SEQ ID NO: 1, and VL contains the amino acid sequence of SEQ ID NO: 5. In certain aspects, VH and VL are fully human.

[000119] In certain aspects, an anti-SEMA4D antibody, or antigen-binding fragment, variant or derivative provided herein, can specifically bind to human SEMA4D, rodent SEMA4D, e.g., mouse SEMA4D and/or rat SEMA4D, and/or primate SEMA4D a non-human, such as a SEMA4D macaque and/or a SEMA4D marmoset.

[000120] In certain aspects, the antibody or fragment, variant, or derivative thereof further comprises a heavy chain constant region, or fragment, variant, or derivative thereof, fused to the C-terminus of the VH. The heavy chain constant region or fragment thereof may be derived from any species, but in certain aspects, the heavy chain constant region or fragment, variant or derivative thereof is, or is derived from, a human heavy chain constant region, e.g., IgG1, IgG2, human IgG3, IgG4, IgA, IgE or IgM. In certain aspects, the heavy chain constant region is a human IgG4 constant region, or a fragment, variant, or derivative thereof. In some aspects, for example, when the proposed anti-SEMA4D antibody or fragment thereof is to be used in a non-human model system, the heavy chain constant region or fragment thereof may be a non-human heavy chain constant region, e.g. a mouse heavy chain region, such as a mouse IgG1 constant region.

[000121] In certain aspects, the antibody or fragment, variant, or derivative thereof further comprises a light chain constant region, or fragment, variant, or derivative thereof, fused to the C-terminus of the VL. The light chain constant region or fragment thereof may be derived from any species, but in certain aspects the light chain constant region or fragment thereof is derived from a human light chain constant region, eg, a human kappa or lambda constant region. In certain aspects, the light chain constant region, or a fragment, variant, or derivative thereof, is or is derived from a human lambda light chain constant region. In certain aspects, the light chain constant region is a human lambda constant region. In some aspects, for example, when the proposed anti-SEMA4D antibody or fragment thereof is to be used in a non-human model system, the light chain constant region or fragment thereof may be a non-human light chain constant region, e.g. a mouse light chain region, such as the mouse lambda constant region.

[000122] In certain aspects, the invention provides an anti-SEMA4D antibody fragment comprising the VH and VL regions described above. In certain aspects, the fragment may be, for example, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, a single chain Fv fragment (scFv), or a disulfide-linked Fv fragment (sdFv).

[000123] In certain aspects, an anti-SEMA4D antibody, or fragment, variant, or derivative thereof provided herein may be multispecific, eg, bispecific. In addition to the binding properties of an anti-SEMA4D antibody described herein, a multispecific antibody or fragment thereof provided herein may bind to an additional SEMA4D epitope or may bind to other unbound epitopes.

[000124] In certain aspects, an anti-SEMA4D antibody, or fragment provided herein, can specifically bind to SEMA4D, e.g., human SEMA4D, mouse SEMA4D, rat SEMA4D, and/or non-human primate SEMA4D, e.g., cynomolgus SEMA4D and/or SEMA4D marmoset or any combination thereof with a binding affinity characterized by a KD dissociation constant of no more than 500 nM, 100 nM, 50.0 nM, 40.0 nM, 30.0 nM, 20.0 nM, 10.0 nM , 9.0 nM, 8.0 nM, 7.0 nM, 6.0 nM, 5.0 nM, 4.0 nM, 3.0 nM, 2.0 nM, 1.0 nM, 0.50 nM , 0.10 nM, 0.050 nM, 0.01 nM, 0.005 nM, or 0.001 nM. Methods and instruments for measuring the KD of an anti-SEMA 4D antibody or fragment thereof, such as BIACORE, are well known to those skilled in the art.

[000125] In certain aspects, an anti-SEMA4D antibody or fragment provided herein can inhibit the binding of SEMA4D to the SEMA4D receptor, eg, plexin-B1, plexin-B2, and/or CD72.

[000126] In certain aspects, an anti-SEMA4D antibody or antigen-binding fragment, variant or derivative provided herein is fully human, and elicits minimal or no immune response against the antibody when administered to a human subject. Methods for measuring the immune response against an antibody in a subject are well known in the art. See, for example, Darwish, IA, Int. J Biomed. sci. 2:217-235 (2006).

[000127] The constant region of an anti-SEMA4D antibody can be mutated to alter effector function in a number of ways. For example, see US Patent No. 6,737,056 B1 and US Patent Application Publication No. 2004/0132101 A1, which disclose Fc mutations that optimize binding to antibody Fc receptors.

[000128] In certain aspects, anti-SEMA4D binding molecules, e.g. antibodies or fragments, variants or derivatives thereof provided herein, the Fc portion may be mutated to modulate, e.g. increase or decrease, effector function using methods known in this document. the field of technology. For example, deletion or inactivation (through point mutations or other means) of the constant region domain can decrease Fc receptor binding of the circulating modified antibody, thereby increasing tumor localization. In other instances, constant region modifications consistent with this disclosure modulate complement fixation and thus may increase or decrease serum half-life. Other constant region modifications can be used to modify disulfide bridges or oligosaccharide fragments that allow for increased localization by increasing antigen specificity or antibody flexibility. As a result, physiological profiles, bioavailability, and other biochemical effects of modifications, such as tumor localization, biodistribution, and serum half-life, can be easily measured and calculated using well known immunological methods without undue experimentation.

[000129] Anti-SEMA4D binding molecules, e.g., antibodies or fragments thereof, provided herein include derivatives that are modified, for example, by covalently attaching any type of molecule to the antibody such that the covalent attachment does not interfere with specific binding of the antibody to a cognate epitope . Thus, for example, and not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, and etc. Any of the numerous chemical modifications may be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, and the like. In addition, the derivative may contain one or more non-classical amino acids.

[000130] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, e.g., by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind an anti-SEMA4D polypeptide to block the interaction SEMA4D with its receptor or inhibit, delay or reduce metastasis in a subject, such as a cancer patient).

[000131] For example, one can introduce mutations only in the framework regions or only in the CDR region of an antibody molecule. The introduced mutations may be neutral or silent missense mutations, ie. have little or no effect on the ability of the antibody to bind to the antigen. These types of mutations may be useful for optimizing codons or improving antibody production by hybridomas. Alternatively, non-neutral missense mutations can alter the ability of an antibody to bind an antigen. One of skill in the art will be able to design and test mutant molecules with the desired properties, such as no change in antigen-binding activity or changes in binding activity (eg, improved antigen-binding activity or changes in antibody specificity). Following mutagenesis, the encoded protein can typically be expressed and the functional and/or biological activity of the encoded protein (e.g., the ability to immunospecifically bind at least one epitope of a SEMA4D polypeptide) can be determined using the techniques described herein, or by continually modifying the techniques, known in the art.

[000132] In some aspects, an anti-SEMA4D binding molecule, such as an antibody or fragment thereof provided herein, may comprise at least one optimized complementarity determining region (CDR). By "optimized CDR" is meant that the CDR has been modified and optimized to improve the binding affinity and/or anti-SEMA4D activity that is transferred to the anti-SEMA4D binding molecule containing the optimized CDR. "Anti-SEMA4D activity" or "SEMA4D blocking activity" may include an activity that modulates, for example, reduces, abolishes, reduces or prevents one or more SEMA4D activities. Non-limiting activities of SEMA4D include: B cell activation, aggregation and survival; CD40-induced proliferation and antibody production; antibody response to dependent T-cell antigens; proliferation of T cells or other immune cells; maturation of dendritic cells; demyelination and degeneration of axons; apoptosis of pluripotent neuronal progenitors and/or oligodendrocytes; induction of endothelial cell migration; inhibition of spontaneous migration of monocytes; promoting tumor cell growth or metastasis, binding to plexin-B1 or another cell surface receptor, or any other association activity with soluble SEMA4D or SEMA4D that is expressed on the surface of SEMA4D+ cells. In a particular embodiment, the anti-SEMA4D activity includes the ability to inhibit, delay, or reduce tumor metastasis, either in combination with inhibition, delay, or reduction in primary tumor cell growth and tumor metastasis, or independently of primary tumor cell growth and tumor metastasis. Activity against SEMA4D may also be associated with a reduction in the frequency or severity of diseases associated with the expression of SEMA4D, including, but not limited to, certain cancers, including solid tumors and lymphomas, autoimmune diseases, inflammatory diseases, including inflammatory diseases of the central nervous system (CNS) and peripheral nervous system (PNS), neurodegenerative diseases, transplant rejection, and invasive angiogenesis.

Polynucleotides encoding anti-SEMA4D antibodies

[000133] The invention also provides an isolated polynucleotide, or two or more polynucleotides, comprising one or more nucleic acid sequences encoding an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant or derivative thereof as provided herein.

[000134] In one aspect, the invention provides an isolated polynucleotide or combination of polynucleotides comprising one or more nucleic acid sequences encoding an anti-SEMA4D binding molecule, e.g., an antibody or fragment, variant or derivative thereof as provided herein, or a subunit thereof, for example, a heavy chain subunit or fragment thereof, or a light chain subunit or fragment thereof.

[000135] In certain aspects, a polynucleotide or combination of polynucleotides provided herein comprises a nucleic acid sequence that encodes the VH of an anti-SEMA4D binding molecule, such as an antibody or fragment, variant, or derivative thereof. In certain aspects, a polynucleotide or combination of polynucleotides provided herein comprises a nucleic acid sequence that encodes the VL of an anti-SEMA4D binding molecule, such as an antibody or fragment, variant, or derivative thereof. In certain aspects, a polynucleotide or combination of polynucleotides provided herein comprises a nucleic acid sequence that encodes for a VH and a nucleic acid sequence that encodes for the VL of an anti-SEMA4D binding molecule, such as an antibody or fragment, variant, or derivative thereof. In certain aspects, a nucleic acid sequence encoding a VH and a nucleic acid sequence encoding a VL are located in the same vector. Such a vector is proposed in this description. In certain aspects, the nucleic acid sequence encoding VH and the nucleic acid sequence encoding VL are located in separate vectors. Such vectors are also provided herein. The vectors proposed in this invention may additionally contain genetic elements that ensure the expression of the antibody or its fragment. Such genetic elements, such as promoters, poly-adenylation sequences, and enhancers, are described elsewhere herein. The invention also provides a host cell comprising a polynucleotide or combination of polynucleotides as provided herein and/or a vector or vectors as provided herein.

[000136] Also provided is a method for producing an anti-SEMA4D antibody or fragment, variant or derivative thereof as provided herein, the method comprising culturing a host cell as provided herein and isolating the antibody or fragment thereof.

[000137] In some aspects, the invention provides an isolated polynucleotide or two or more polynucleotides comprising one or more nucleic acid sequences encoding an anti-SEMA4D antibody or an antigen-binding fragment, variant or derivative, or subunit thereof, wherein the antibody comprises a VH or VL, wherein this VH contains complementarity determining regions (HCDRs) of HCDR1, HCDR2 and HCDR3 containing amino acid sequences identical or identical except for one, two, three or four amino acid substitutions in one, two or three HCDR SEQ ID NO: 2, SEQ ID NO : 3 and SEQ ID NO: 4, respectively; a VL contains complementarity determining regions (LCDR) LCDR1, LCDR2 and LCDR3 containing amino acid sequences that are identical or identical except for one, two, three or four amino acid substitutions in one, two or three LCDR SEQ ID NO: 6, SEQ ID NO : 7 and SEQ ID NO: 8, respectively.

[000138] In certain aspects, the invention provides an isolated polynucleotide or two or more polynucleotides comprising one or more nucleic acid sequences encoding an anti-SEMA4D antibody or an antigen-binding fragment, variant or derivative, or subunit thereof, wherein the antibody comprises VH and VL, wherein this VH contains an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, and VL contains an amino acid sequence at least at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5.

[000139] Any of the polynucleotides described above may further comprise additional nucleic acid sequences encoding, for example, heavy or light chain constant regions or fragments thereof described elsewhere herein, a signal peptide for direct secretion of the encoded polypeptide, or other the heterologous polypeptides described herein. In addition, as described in more detail herein elsewhere, this description includes a composition containing one or more of the polynucleotides described above.

[000140] In one embodiment, this disclosure includes a composition comprising a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a nucleic acid sequence encoding a VH as described herein and a second polynucleotide encoding a VL as described herein.

[000141] This invention also includes fragments of the polynucleotides specified in this document, as described elsewhere. In addition, the present invention provides polynucleotides that encode fusion polypeptides, Fab fragments, and other derivatives as described herein.

[000142] The polynucleotides provided herein can be obtained or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., Bio Techniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides, containing parts of the sequence encoding the antibody, annealing and ligation of these oligonucleotides, and then amplification of the ligated oligonucleotides using PCR.

[000143] Alternatively, a polynucleotide encoding anti-SEMA4D binding molecules, eg, an antibody or antigen-binding fragment, variant or derivative thereof provided herein can be derived from nucleic acid sequences obtained from a suitable source. If a clone containing a polynucleotide encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library derived from or nucleic acid acid, e.g., poly A ⁺ RNA isolated from any tissue or cell expressing an antibody or other anti-SEMA4D antibody, such as hybridoma cells selected as expressing antibodies) by NDP amplification using synthetic primers hybridizing to 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for a particular gene sequence to determine, for example, a cDNA clone from a cDNA library that encodes an anti-SEMA4D antibody or other antibody. The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[000144] Once the nucleotide sequence and corresponding amino acid sequence of an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof has been determined, its nucleotide sequence can be manipulated using methods well known in the art for manipulating the nucleotide sequence, for example, using recombinant DNA techniques. , site-directed mutagenesis, PCR, etc. (See, for example, the methods described in Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, NY, both incorporated herein by reference in their entirety) to generate antibodies having a different amino acid sequence, for example, to create amino acid substitutions, deletions and/or insertions.

[000145] A polynucleotide or combination of polynucleotides encoding an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide or combination of polynucleotides encoding an anti-SEMA4D antibody or antigen-binding fragment, variant or derivative thereof may be composed of single and double stranded DNA, DNA that is a mixture of single and double stranded regions, single and double stranded RNA, and RNA that is is a mixture of single- and double-stranded regions, a hybrid molecule containing DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. In addition, a polynucleotide or combination of polynucleotides encoding an anti-SEMA4D antibody or an antigen-binding fragment, variant or derivative thereof may be composed of three-stranded regions containing RNA or DNA, or both RNA and DNA. A polynucleotide or combination of polynucleotides encoding an anti-SEMA4D antibody or antigen-binding fragment, variant or derivative thereof may also contain one or more modified DNA or RNA bases or backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms.

[000146] An isolated polynucleotide or combination of polynucleotides encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or a light chain portion) can be created by introducing one or more nucleotide substitutions, additions, or deletions to the immunoglobulin nucleotide sequence such that that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. In some aspects, conservative amino acid substitutions may be made at one or more non-essential amino acid residues.

Fusion proteins and antibody conjugates

[000147] As discussed in more detail elsewhere herein, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof provided herein, can optionally be recombinantly fused to a heterologous polypeptide at N- or C- terminally or chemically conjugated (including covalent and non-covalent conjugation) to polypeptides or other heterologous moieties. For example, an anti-SEMA4D antibody can be recombinantly fused or conjugated with molecules used as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, for example, PCT publications WO 92/08495; WO 91/14438; WO 89/12624; US patent No. 5314995 and EP 396387. In some aspects, a heterologous fragment may be a polypeptide, cytotoxic agent, therapeutic agent, prodrug, lipid, carbohydrate, nucleic acid, detectable label, polymer, or any combination thereof. Illustrative heterologous polypeptides include, without limitation, a binding molecule, an enzyme, a cytokine, a lymphokine, a hormone peptide, or any combination thereof. Illustrative cytotoxic agents include, without limitation, a radionuclide, a biological toxin, an enzymatically active toxin, or any combination thereof. Illustrative detectable labels include, without limitation, an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or any combination thereof.

[000148] An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof provided herein may include derivatives that are modified, for example, by covalently attaching any type of fragment to the antibody, such that covalent attachment does not prevent the binding molecule from binding to SEMA4D. For example, and not by way of limitation, antibody derivatives can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. . Any of the numerous chemical modifications may be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, and the like. In addition, the derivative may contain one or more non-classical amino acids.

[000149] An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, as provided herein, may be composed of amino acids linked together by peptide bonds or modified peptide bonds, e.g., peptide isosteres, and may contain amino acids other than the 20 gene-coding amino acids. For example, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be modified by natural processes such as post-translational processing or by chemical modification techniques that are well known in the art. technology. Such modifications are well described in the main texts and in more detailed monographs, as well as in a variety of scientific literature. Modifications can occur anywhere in the anti-SEMA4D binding molecule, including peptide backbones, amino acid side chains, and amino or carboxyl termini, or on moieties such as carbohydrates. In addition, a given anti-SEMA4D binding molecule may contain many types of modifications. Anti-SEMA4D binding molecules can be branched, for example, by ubiquitination, and they can be cyclic, with or without branching. The cyclic, branched, and branched cyclic anti-SEMA4D binding molecule may be the result of post-translational natural processes or may be prepared synthetically. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent attachment, heme residue covalent attachment, nucleotide or nucleotide derivative covalent attachment, lipid or lipid derivative covalent attachment, phosphotidylinositol covalent attachment, crosslinking, cyclization, disulfide bonding, demethylation, covalent crosslinking, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transport mediated sulfation RNA is the attachment of amino acids to a protein, such as arginylation and ubiquitination. (See, for example, Proteins-Structure and Molecular Properties, T. E. Creighton, W. H. Freeman and Company, NY; 2nd ed. (1993); Johnson, ed. (1983) Posttranslational Covalent Modification of Proteins (Academic Press, NY ), pgs. 1-12; Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992)).

[000150] The invention also provides fusion proteins comprising an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, and a heterologous polypeptide. The heterologous polypeptide to which the antibody is fused may be useful for function or useful for targeting cells expressing the anti-SEMA4D polypeptide. An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be fused or conjugated to one or more heterologous polypeptides or other fragments to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art. For example, in one embodiment, PEG or human serum albumin can be fused or conjugated to an anti-SEMA4D binding molecule, as provided herein, to increase its in vivo half-life. See Leong et al., Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002) or Weir et al., Biochem. soc. Transactions 30:512 (2002).

[000151] Moreover, an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be fused to one or more marker sequences, such as a peptide, to facilitate their purification or detection. . In some embodiments, the marker amino acid sequence is a hex-histidine peptide, such as the tag provided in the pQE vector (QIAGEN Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. sci. USA 86:821-824 (1989), for example, hexahistidine provides a convenient purification of the fusion protein. Other peptide tags suitable for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.

[000152] Fusion proteins can be obtained using methods that are well known in the art (see, for example, US patent No. 5116964 and 5225538). The exact site at which the fusion occurs can be chosen empirically to optimize the secretion or binding characteristics of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression.

[000153] An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be used in unconjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve therapeutic properties of the molecule, to facilitate target detection, or to visualize or treat a patient. An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be used in unconjugated form, or may be labeled or conjugated, either before or after purification, or when purification is performed.

[000154] In particular, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be conjugated to therapeutic agents, prodrugs, cytotoxic agents, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents or PEGs.

[000155] Those skilled in the art will appreciate that the conjugates can also be assembled using various methods, depending on the chosen agent with which the conjugation is made. For example, biotin conjugates are prepared, for example, by coupling the polypeptide to an activated biotin ester, such as biotin N-hydroxysuccinimide ester. Similarly, conjugates with a fluorescent marker can be prepared in the presence of a coupling agent, such as those listed herein, or by reaction with an isothiocyanate, such as fluorescein isothiocyanate. Conjugates of an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be prepared in a similar manner.

[000156] The invention further includes an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, conjugated to a diagnostic or therapeutic agent. Such anti-SEMA4D binding molecules can be used diagnostically, eg, to monitor the development or progression of a disease, as part of a clinical testing procedure to, for example, determine the efficacy of a given treatment and/or prophylaxis regimen. For example, detection can be facilitated by linking an anti-SEMA4D binding molecule to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. See, for example, US Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as a diagnostic in accordance with this invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, or ⁹⁹ Tc.

[000157] An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be conjugated to a therapeutic molecule, such as a cytotoxin, a therapeutic agent, or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.

[000158] An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can also be labeled with a detectable label by binding it to a chemiluminescent compound. The presence of a chemiluminescent-labeled anti-SEMA4D binding molecule is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent compounds for labeling are luminol, isoluminol, acridinium aromatic ester, imidazole, acridine salt and oxalic acid ester.

[000159] One way in which an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be detectably labeled by combining it with an enzyme and using the linked product in an enzyme immunoassay ( ELISA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md.; Diagnostic Horizons 2:1-7 (1978); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, Meth Enzymol 73:482-523 (1981); Maggio, ed. (1980) Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa et al., eds. (1981) Enzyme Immunoassay (Kgaku Shoin, Tokyo) An enzyme that is bound to an anti-SEMA4D binding molecule will react with an appropriate substrate, such as a chromogenic substrate, so as to produce a chemical radical that can be detected, for example, spectrophotometrically , fluorimetric eski or by visual means. Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In addition, detection can be carried out using colorimetric methods that use a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the degree of substrate enzymatic reaction compared to similarly prepared standards.

[000160] Detection can also be performed using any of a variety of other immunoassays. For example, by radiolabeling a binding molecule against SEMA4D, the binding molecule can be detected using radioimmunoassay (RIA). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or an autoradiograph.

[000161] An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can also be labeled with fluorescent metals such as ¹⁵² Eu or others from the lanthanide series. These metals can be attached to the binding molecule using metal chelating groups such as diethylene pamine peptaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA).

[000162] Methods for conjugating various fragments to an anti-SEMA4D binding molecule as provided herein are well known to those of ordinary skill in the art.

Expression of antibody polypeptides

[000163] DNA sequences that encode the light and heavy chains of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragments, variants or derivatives thereof, as provided herein, can be generated, either simultaneously or separately, using reverse transcriptase and DNA polymerase according to well known methods. PCR can be initiated with consensus constant region primers or more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, libraries can be screened with consensus primers or larger homologous probes such as mouse constant region probes.

[000164] DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, placed on a restriction map, and sequenced according to standard, well-known methods set forth in detail, for example, in references, associated with recombinant DNA techniques provided herein elsewhere.

[000165] After manipulating the isolated genetic material to provide an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, polynucleotides encoding the binding molecule can be inserted into an expression vector for introduction into one or more host cells that can be used to generate the desired amount of anti-SEMA4D binding molecule.

[000166] Recombinant expression of an anti-SEMA4D antibody, or fragment, derivative, or analog thereof, such as the heavy or light chain of the antibody, requires the construction of one or more expression vectors containing a polynucleotide or combination of polynucleotides that encodes the antibody. Once a polynucleotide or combination of polynucleotides encoding an antibody molecule or an antibody heavy or light chain or portion thereof (e.g. containing a heavy or light chain variable region) has been produced, the vector or vectors for producing the antibody molecule can be produced using recombinant DNA technology with using techniques well known in the art. Thus, this document describes methods for producing a protein by expressing a polynucleotide or a combination of polynucleotides containing antibodies encoding nucleic acid sequences. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding antibodies and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA methods, synthetic methods, and in vivo genetic recombination. The present invention thus provides reproducible vectors comprising nucleic acid sequences encoding anti-SEMA4D binding molecules, for example an antibody as provided herein, or a heavy or light chain thereof, or a heavy or light chain variable region operably linked to promoter. Such vectors may contain a nucleotide sequence encoding an antibody heavy and/or light chain constant region (see, for example, PCT Publication WO 86/05807; PCT Publication WO 89/01036 and US Pat. No. 5,122,464) and the variable region of the antibody may be cloned into a vector such as is suitable for expression of the entire heavy or light chain.

[000167] The term "vector" or "expression vector" is used herein to refer to vectors used in accordance with this invention as a vehicle for ingestion and expression of the desired gene in a host cell. As will be known to those skilled in the art, such vectors may readily be selected from, for example, plasmids, phages, and viruses, such as retroviruses. In general, vectors compatible with this invention will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

[000168] Numerous vector expression systems can be used. For example, one class of vector uses DNA elements that are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus. Others suggest the use of polycistronic systems with internal ribosome binding sites (IRES). In addition, cells in which DNA is integrated into their chromosomes can be selected by introducing one or more markers that select transfected host cells. The marker can confer prototrophy to the auxotrophic host, biocidal resistance (eg antibiotic), or resistance to heavy metals such as copper. Gene selectable markers can either be directly linked to DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

[000169] In certain aspects, the cloned variable regions of the nucleic acid molecule can be inserted into an expression vector along with heavy and light chain constant region genes synthesized as described above. Of course, any expression vector that is capable of causing expression in eukaryotic cells can be used. Examples of suitable vectors include, but are not limited to, pcDNA3, pHCMV/Zeo, pCR3.1, pEF 1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 plasmids. (available from Invitrogen, San Diego, CA) and pCI plasmid (available from Promega, Madison, WI). In general, screening a large number of transformed cells for those expressing respectively high levels of immunoglobulin heavy and light chains is routine experimentation, which can be done, for example, using robotic systems.

[000170] In general, once a vector or DNA sequence encoding a monomeric subunit of an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, has been prepared, as provided herein, the expression vector can be introduced into an appropriate host cell. . Introduction of a plasmid into a host cell can be accomplished by various methods well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, DNA cell fusion, microinjection, and infection with intact virus. See, Ridgway (1988) "Mammalian Expression Vectors" in Vectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.), Chapter 24.2, pp. 470-472. Typically, introduction of the plasmid into the host occurs via electroporation. Host cells carrying the expression construct can be grown under conditions suitable for the production of light chains and heavy chains and can be analyzed for protein synthesis and heavy and/or light chain assembly. Typical assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorting (FACS), immunohistochemistry, and the like.

[000171] An expression vector can be transfected into a host cell using conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein ,. Thus, the invention includes host cells comprising a polynucleotide or combination of polynucleotides encoding an antibody as provided herein, or a heavy or light chain thereof, operably linked to a promoter, eg, a heterologous promoter. In some embodiments, for the expression of double chain antibodies, vectors encoding both heavy and light chains can be co-expressed into a host cell to express and assemble the entire binding molecule, as detailed below.

[000172] As used herein, the term "host cell" refers to cells that carry vectors constructed using recombinant DNA techniques and encoding at least one heterologous polynucleotide. In descriptions of processes for isolating antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody, unless explicitly stated otherwise. In other words, recovery of a polypeptide from "cells" can mean either from centrifuged pelleted whole cells or from a cell culture containing both medium and suspended cells.

[000173] A variety of host vector expression systems can be used to express an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein. Such host expression systems are vehicles by which coding sequences of interest can be generated and then purified, but are also cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule provided herein in situ. . These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences, yeast (e.g., Saccharomyces , Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculoviruses) containing sequences encoding antibodies; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing sequences encoding antibodies; or mammalian cell systems (eg, COS, CHO, BLK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the mammalian cell genome (eg, metallothionein promoter) or from mammalian viruses (eg, adenovirus late promoter; 7 ,5K vaccinia virus promoter or human cytomegalovirus immediate early promoter). In certain aspects, bacterial cells such as Escherichia coli or eukaryotic cells, especially for the expression of recombinant whole antibody molecules, can be used to express the recombinant antibody molecule. For example, mammalian cells such as the Chinese Hamster Ovary (CHO) cell, when combined with a vector, provide an efficient antibody expression system (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8: 2 (1990)).

[000174] The host cell line used to express the protein may be derived from a mammal. Examples of a host cell line include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11 (Chinese Hamster Ovary Lines minus DHFR), HeLa (Human Cervical Cancer), CVI (Monkey Kidney Lineage), COS (derived from CVI with SV40 T antigen), VERY, BHK (newborn hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblasts) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney lineage), SP2/O (mouse myeloma), P3X63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes) and 293 (human kidney). Host cell lines are generally available from commercial suppliers, the American Cell Culture Collection, or from the published literature.

[000175] In addition, a host cell strain can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the particular desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important to the function of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and handling of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.

[000176] Stable expression can be used for long-term, high yield production of recombinant proteins. Thus, for example, cell lines can be constructed that stably express antibody molecules. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA under the control of appropriate expression control elements (e.g., promoter, enhancer, transcription termination sequence, polyadenylation site, etc.) and a selectable marker. After the introduction of foreign DNA, the engineered cells can grow for 1-2 days in an enriched medium, and then switch to selective media. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow into foci, which in turn can be cloned and propagated into cell lines. This method can be successfully used to construct cell lines that stably express an antibody molecule.

[000177] Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel (1987) "The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning" (Academic Press, NY) Vol 3. When a marker in an antibody expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will result in an increase in the copy number of the marker gene.Because the enhanced region is associated with the antibody gene, antibody production will also be increased (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[000178] In vitro production allows scaling up to produce large numbers of desired polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, such as in an airlift reactor or a continuously stirred reactor, or immobilized or trapped cell cultures, such as in hollow fibers, microcapsules, on agarose microbeads, or ceramic cartridges. If necessary and/or desired, solutions of the polypeptides may be purified by conventional chromatographic techniques, e.g., size exclusion filtration, ion exchange chromatography, DEAE-cellulose chromatography, or (immuno-)affinity chromatography, e.g., after biosynthesis of the synthetic hinge region of the polypeptide or before or after the HIC chromatography step described herein.

[000179] Nucleic acid molecules encoding an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can also be expressed in non-mammalian cells such as insect cells, bacteria or yeast, or plants. Bacteria that readily take up nucleic acids include members of Enterobacteriaceae such as strains of Escherichia coli or Salmonella; and Bacillaceae such as Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. It should also be borne in mind that when expressed in bacteria, heterologous polypeptides, as a rule, become part of inclusion bodies. Inclusion bodies can be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits can then self-assemble into tetravalent antibodies (WO 02/096948 A2).

[000180] In bacterial systems, a number of expression vectors may advantageously be selected depending on the intended use for the antibody molecule to be expressed. Thus, for example, when a large amount of such a protein must be obtained to create pharmaceutical compositions of the antibody molecule, vectors may be desirable that direct the expression of high levels of fusion protein products that are easy to purify.

[000181] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although a number of other strains are also commonly available, such as Pichia pastoris.

[000182] In the insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector for the expression of foreign genes. The virus grows in the cells of Spodoptera frugiperda. An antibody coding sequence can be cloned individually into non-essential regions (eg polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg polyhedrin promoter).

[000183] Once a SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, has been recombinantly expressed, it can be purified by any method known in the art to purify the immunoglobulin molecule. , for example, by chromatography (eg, ion exchange, affinity, in particular, affinity for protein A, and column chromatography by size), centrifugation, differential solubility, or any other standard method for purifying proteins. Alternatively, methods for increasing the affinity of antibodies as provided herein are disclosed in US Patent Application Publication No. 20020123057 A1.

Therapeutic Therapeutic Anti-SEMA4D Antibodies

[000184] The present invention relates to methods of using an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, for the treatment of subjects having a disease or disorder associated with the pathology of SEMA4D.

[000185] The following discussion relates to methods for diagnosing and treating various diseases and disorders with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, e.g., capable of specifically binding SEMA4D, e.g., SEMA4D human, mouse or human and mouse, and having the neutralizing activity of SEMA4D.

[000186] In one aspect, the invention provides a method for neutralizing SEMA4D in a human, comprising administering to a human subject an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein. In certain aspects, a human subject in need of treatment for an autoimmune disease or disorder, an inflammatory disease or disorder, cancer, a neuroinflammatory disease or disorder, a neurodegenerative disease or disorder, or any combination thereof. In certain aspects, the neuroinflammatory disease or disorder is multiple sclerosis. In certain aspects, the neurodegenerative disease or disorder is stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, Down syndrome, ataxia, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), HIV-related cognitive impairment, CNS lupus, moderate cognitive impairment or a combination thereof. In certain aspects, the autoimmune disease or inflammatory disease is arthritis, such as rheumatoid arthritis, atherosclerosis (see, for example, PCT Publication No. WO 2015/054628, which is incorporated herein by reference in its entirety), or an osteodegenerative disease, such as osteoporosis (see, for example, US patent No. 9447191, which is incorporated into this description by reference in its entirety).

[000187] In one aspect, treatment comprises administering or administering an anti-SEMA4D binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof, as provided herein, to a subject, or administering or administering to an isolated tissue or cell line from a subject, wherein the subject has a disease, a symptom of a disease, or a predisposition to the disease. In another aspect, treatment also includes administering or administering a pharmaceutical composition comprising an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, to a subject, or into an isolated tissue or cell line from a subject, having a disease, a symptom of a disease, or a predisposition to a disease.

[000188] In one aspect, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be useful in the treatment of cancer, e.g., in the treatment of various malignant and benign tumors. By "antitumor activity" is meant a reduction in the rate of proliferation of malignant cells or accumulation, and therefore a reduction in the rate of growth of an existing tumor or tumor that occurs during therapy and/or the destruction of existing neoplastic cells (tumors) or newly formed tumor cells, and therefore , reduction in overall tumor size during therapy. Thus, for example, therapy with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can elicit a physiological response, e.g., contraction of blood vessels or migration of CTL to the tumor microenvironment. Methods for treating cancer with an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be found, for example, in PCT Publication No. WO 2014/209802 [combination immunotherapy], which is incorporated herein. description as reference in full.

[000189] In one aspect, the invention provides an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, for use as a drug, in particular for use in treatment or prevention cancer or for use in a precancerous condition or lesion.

[000190] In one aspect, an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be useful in the treatment of an autoimmune disease or inflammatory disease. In one aspect, an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may be useful in the treatment of a neuroinflammatory or neurodegenerative disease. Methods for treating a neuroinflammatory or neurodegenerative disease can be found, for example, in PCT Publication No. WO 2013/055922 [BBB], PCT Publication No. WO 2015/061330 [HD] and PCT Publication No. WO 2013/170221 [neurogenesis/stroke], content which are incorporated by reference in their entirety.

[000191] According to the methods provided herein, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used to promote a positive therapeutic response to a cancer cell person. The term "positive therapeutic response" in relation to the treatment of cancer means a positive effect on the disease in combination with the antitumor activity of these binding molecules, for example, antibodies or its fragment and/or improvement of symptoms associated with the disease. That is, there may be an antiproliferative effect, prevention of further tumor growth, reduction in tumor size, reduction in tumor vasculature, reduction in the number of cancer cells, and/or reduction in one or more symptoms associated with the disease. For example, an improvement in a disease can be characterized as a complete response. The term "complete response" refers to the absence of clinically detectable diseases with the normalization of any previously abnormal radiological studies, bone marrow and cerebrospinal fluid (CSF). Alternatively, improvement in disease may be classified as a partial response. By "partial response" is meant a reduction of at least 50% of all measurable tumor burdens (i.e., the number of tumor cells present in the subject) in the absence of new lesions and persisting for at least one month. This answer only applies to measurable tumors.

[000192] An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may also find use in the treatment of inflammatory diseases and deficiencies or disorders of the immune system. Inflammatory diseases are characterized by inflammation and tissue destruction, or a combination of the two. By "anti-inflammatory activity" is meant the reduction or prevention of inflammation. "Inflammatory disease" includes any inflammatory immune-mediated process in which the trigger event or target of the immune response includes non-self antigen(s), including, for example, alloantigens, xenoantigens, viral antigens, bacterial antigens, unknown antigens, or allergens. In one embodiment, the inflammatory disease is an inflammatory disease of the peripheral or central nervous system. In another embodiment, the inflammatory disease is an inflammatory disease of the joints.

[000193] In addition, the term "inflammatory disease(s)" includes "autoimmune disease(s)". As used herein, the term "autoimmune" is generally understood to encompass immune-mediated inflammatory processes associated with "self" antigens. In autoimmune diseases, self antigen(s) cause the host's immune response. An autoimmune disease may be the result of an inappropriate immune response directed against the self antigen (self antigen), which is a deviation from the normal state of self-tolerance. In general, an antibody (in particular, but not exclusively, an IgG antibody), acting as a cytotoxic molecule or as immune complexes, is a major mediator of various autoimmune diseases, many of which can be debilitating or life-threatening.

[000194] In one embodiment, an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used to treat multiple sclerosis (MS). MS, also known as multiple sclerosis or encephalomyelitis disseminata, is an autoimmune disease in which the immune system attacks the central nervous system, resulting in demyelination. The name multiple sclerosis refers to the scars (sclerosis, also referred to as plaques or lesions) that form in the nervous system. PC lesions typically involve areas of white matter close to the cerebellar ventricles, the brainstem, basal ganglia and spinal cord, and the optic nerve. PC leads to the destruction of oligodendrocytes, the cells responsible for building and maintaining the myelin sheath. PC leads to thinning or complete loss of myelin and, with the development of the disease, to transection of axons.

[000195] Neurological symptoms can vary with MS, and the disease often progresses to physical and mental disability. PC takes many forms, with new symptoms occurring either in discrete attacks (relapsing form) or slowly accumulating over time (progressive forms). Between attacks, symptoms may go away completely, but permanent neurological damage often occurs, especially as the disease progresses.

[000196] Neutralization of SEMA4D using an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used to reduce the severity of MS through several different mechanisms, e.g., anti-SEMA4D monoclonal antibodies can block immune maturation and activation of SEMA4D to reduce the rate of relapse by reducing secondary immune responses to CNS antigens, and monoclonal antibodies against SEMA4D can block the action of soluble SEMA4D in mediating oligodendrocyte apoptosis in the CNS, which can reduce the severity of the disease by reducing demyelination.

[000197] In one aspect, an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used to treat arthritis. Arthritis is an inflammatory joint disease that can be caused by an autoimmune condition in which the immune system attacks the joints. In certain embodiments, the arthritis is selected from the group consisting of osteoarthritis, gouty arthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, infectious arthritis, inflammatory arthritis, septic arthritis, degenerative arthritis, arthritis mutilans, and Lyme arthritis. In one embodiment, the arthritis is rheumatoid arthritis (RA).

[000198] The present invention relates to methods for treating or preventing arthritis by administering to a subject the anti-SEMA4D binding molecule provided herein. The methods provided herein can reduce pain, swelling, or stiffness associated with arthritis, such as rheumatoid arthritis. The present invention also provides methods for improving joint performance, function and health. In certain embodiments of this disclosure, treatment results in a decrease in arthritis severity score, a decrease in arthritis severity/area under the curve, a decrease in arthritis-related histopathology parameters (inflammation, pannus, cartilage damage, and bone damage), a decrease in serum levels of arachidonic acid, or a decrease in antibodies against collagen. In certain embodiments, useful or desired clinical results include, but are not limited to, relief of symptoms associated with arthritis; arthritis prevention; delayed onset of arthritis; reducing the incidence of arthritis in the population; reduction in degree from conditions associated with arthritis; stabilization (eg, non-aggravation) status of a pathological condition, disorder or disease associated with arthritis; delaying the onset or retardation of a condition, disorder or disease associated with the progression of arthritis; improvement in a condition, disorder or condition, remission (partial or complete) of a condition, disorder or disease associated with arthritis, whether detectable or undetectable; or an increase or improvement in a condition, disorder or disease associated with arthritis.

[000199] The methods provided herein can be used to treat people who have arthritis or who are at risk of developing arthritis. Thus, in some embodiments, the invention provides a method of treating a subject having normal joints, borderline arthrosis, or severely arthritic joints, the method comprising administering an anti-SEMA4D binding molecule as provided herein to the subject described herein. In some embodiments, the methods provided herein can be used to treat chronic arthritis for the remainder of a subject's life.

[000200] According to the methods provided herein, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used to promote a positive therapeutic response to treatment. or the prevention of an autoimmune disease and/or an inflammatory disease. The term "positive therapeutic response" in relation to an autoimmune disease and/or inflammatory disease means alleviation of the disease in combination with anti-inflammatory activity, anti-angiogenic activity, anti-apoptotic activity, or the like of these antibodies, and/or alleviation of symptoms associated with the disease. That is, anti-proliferative effects, prevention of further spread of SEMA4D-expressing cells, reduction of the inflammatory response, including, without limitation, reduced secretion of pro-inflammatory cytokines, adhesion molecules, proteases, immunoglobulins (in cases where the cell carrying SEMA4D is B -cell), combinations thereof, and the like, increasing the production of anti-inflammatory proteins, reducing the number of self-reactive cells, increasing immune tolerance, inhibiting the survival of self-reactive cells, reducing apoptosis, reducing endothelial cell migration, increasing spontaneous migration of monocytes, reducing and/or alleviating one or more symptoms mediated by stimulation of sSEMA4D or SEMA4D-expressing cells. Such positive therapeutic responses are not limited to routes of administration and may include administration to the donor, to the donor tissue (such as, for example, organ perfusion), to the host, any combination thereof, and the like.

[000201] Clinical response can be assessed using screening methods such as magnetic resonance imaging (MRI), x-ray imaging, computed tomography (CT), flow cytometry analysis or fluorescence activated cell sorting (FACS), histology, macropathology, and chemical analysis of blood, including, without limitation, changes detected using ELISA, RIA, chromatography, and the like. In addition to these positive therapeutic responses, a subject being treated with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, may benefit from improving the symptoms associated with the disease.

[000202] An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used in combination with at least one other cancer therapy, including, without limitation, surgery or surgical procedure; immunomodulatory therapy, radiation therapy; chemotherapy, optionally in combination with autogenous bone marrow transplantation, or other cancer therapy; wherein the complementary cancer therapy is administered before, during, or after therapy with an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment thereof. Thus, when the combined treatments include the administration of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment thereof, as provided herein, in combination with the administration of another therapeutic agent, as in chemotherapy, radiation therapy, other anticancer antibody therapies, small molecule based cancer therapy or vaccine/immunotherapy based cancer therapy, the methods provided herein include co-administration using separate drugs or a single pharmaceutical formulation, or sequential administration in any order.

[000203] An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used in combination with any known therapies for the treatment of autoimmune and inflammatory diseases, including any agent or combination agents known to be useful, or which have been used or are currently being used, for the treatment of autoimmune and inflammatory diseases. Thus, when combined therapies involve administering an anti-SEMA4D binding molecule in combination with administering another therapeutic agent, the methods provided herein include co-administration using separate drugs or a single pharmaceutical formulation, or sequential administration in any order. In some embodiments, the anti-SEMA4D antibodies described herein are administered in combination with immunosuppressive or anti-inflammatory drugs, wherein the antibody and therapeutic agent(s) may be administered sequentially, in any order, or simultaneously (i.e., in parallel or in the same period of time).

[000204] In certain aspects, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used alone or in combination with immunosuppressants for the treatment and/or prevention of rheumatoid arthritis. As discussed above, the efficacy of a treatment may be assessed using any means and includes, but is not limited to, efficacy as measured by clinical responses as defined by the American College of Rheumatology Criteria, the European Rheumatism Criteria League, or some other criterion. See, for example, Felson et al., Arthritis. Rheum. 38:727-35 (1995) and van Gestel et al., Arthritis Rheum. 39:34-40 (1996).

[000205] In other aspects, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used alone or in combination with immunosuppressants for the treatment and/or prevention of multiple sclerosis.

[000206] Another aspect of this invention is the use of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, for diagnostic monitoring of tissue protein levels, as part of a clinical trial procedure, e.g., to determine the effectiveness of a given treatment regimen. For example, detection can be facilitated by binding the antibody to the substance to be detected. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ³⁵ S, or ³ H.

VIII. Pharmaceutical Compositions and Routes of Administration

[000207] Methods for preparing and administering an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, to a subject in need thereof are well known or can be easily determined by those skilled in the art. The route of administration of the anti-SEMA4D binding molecule may be, for example, oral, parenteral, inhalation or topical. The term parenteral as used herein includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these forms of administration are considered to be within the scope of this description, an example of an administration form would be an injection solution, in particular for intravenous or intra-arterial injection or intravenous drip. Generally, a suitable injectable pharmaceutical composition may contain a buffer (eg, an acetate, phosphate, or citrate buffer), a surfactant (eg, polysorbate), optionally a stabilizing agent (eg, human albumin), and the like. However, in other methods consistent with those set forth herein, an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be delivered directly to the site of an unfavorable condition, e.g., into a solid tumor, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[000208] An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be administered in a pharmaceutically effective amount in vivo for the treatment of SEMA4D-mediated diseases such as cancer, autoimmune diseases, inflammatory diseases, including inflammatory diseases of the central nervous system (CNS) and peripheral nervous system (PNS), neurodegenerative diseases and invasive angiogenesis. In this regard, it should be understood that the disclosed binding molecules can be formulated in such a way as to facilitate administration and promote the stability of the active agent. In certain aspects, the pharmaceutical compositions provided herein may include a pharmaceutically acceptable, non-toxic, sterile carrier such as saline, non-toxic buffers, preservatives, and the like. For the purposes of this application, a pharmaceutically effective amount of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, conjugated or unconjugated, means an amount sufficient to achieve effective binding to the target and to achieve the effect , for example, to alleviate the symptoms of a disease or disorder, or to detect a substance or cell.

[000209] Pharmaceutical compositions used herein include pharmaceutically acceptable carriers including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, dihydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, substances based on celluloses, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, polyethylene glycol and lanolin.

[000210] Formulations for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffer media. Pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, for example 0.05 M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases, and the like.

[000211] In some aspects, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and must be fluid to the extent that it fits freely into a syringe. It must be stable under the conditions of manufacture and storage and can be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Fluidity can be maintained, for example, through the use of a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Suitable compositions for use in therapeutic methods disclosed herein are described in Remington Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[000212] Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some aspects, isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride, may be included in the composition. Prolonged absorption of injectable compositions can be accomplished by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[000213] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, alone or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of the ingredients listed in this description, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods may include vacuum drying and freeze drying, which provides a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution. Formulations for injection are processed, dispensed into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions in accordance with methods known in the art. In addition, the preparations may be packaged and sold in the form of a kit, such as that described in US patent application No. 09/259337. Such articles may have labels or inserts indicating that the compositions associated therewith are useful in treating a patient suffering from or predisposed to a disease or disorder.

[000214] Parenteral compositions can be a single bolus dose, an infusion, or a loading bolus followed by a maintenance dose. These compositions may be administered at certain fixed or variable intervals, such as once a day or "on demand".

[000215] Some pharmaceutical compositions provided herein may be administered orally in a suitable dosage form, including, for example, capsules, tablets, aqueous suspensions or solutions. Some pharmaceutical compositions may also be administered by nasal spray or inhalation. Such compositions may be formulated as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

[000216] The amount of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, that can be combined with carrier materials to form a single dosage form, will vary depending on the patient being treated and specific route of administration. The composition can be administered as a single dose, multiple doses, or over a set period of time in an infusion. The dosage regimen may also be adjusted to provide the optimal desired response (eg, therapeutic or prophylactic response).

[000217] An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be administered to a human or other animal in accordance with the aforementioned treatments in an amount sufficient to produce a therapeutic effect. An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be administered to a human or other animal in a conventional dosage form prepared by combining the antibody with a conventional pharmaceutically acceptable carrier or diluent as known in the art. methods. One of skill in the art will appreciate that the form and nature of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well known variables.

[000218] By "therapeutically effective dose or amount" or "effective amount" is meant the amount of an anti-SEMA4D binding molecule, e.g., an antibody variant or antigen-binding fragment or derivative thereof, as provided herein, which, when administered, results in a positive therapeutic response in regarding the treatment of a patient with a disease to be treated.

[000219] Therapeutically effective dosages of the compositions as provided herein for the treatment of SEMA4D-mediated diseases such as cancer; autoimmune diseases such as arthritis, multiple sclerosis, inflammatory diseases, including inflammatory diseases of the central nervous system (CNS) and peripheral nervous system (PNS); neurodegenerative diseases and invasive angiogenesis varies depending on many different factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Doses of treatment may be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[000220] The amount of anti-SEMA4D binding molecule, eg, antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, to be administered is readily determined by one of ordinary skill in the art without undue experimentation. Factors affecting the route of administration and the appropriate amount of the anti-SEMA4D binding molecule include, but are not limited to, disease severity, disease history, and age, height, weight, health, and physical condition of the individual being treated. Likewise, the amount of anti-SEMA4D binding molecule to be administered will depend on the route of administration and whether the subject will receive a single dose or multiple doses of the agent.

[000221] The invention also provides the use of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, in the manufacture of a medicament for the treatment of an autoimmune disease and/or inflammatory disease, including, for example, arthritis, disseminated sclerosis, inflammatory diseases of the CNS and PNS, neurodegenerative diseases or cancer.

[000222] The invention also provides the use of an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, in the manufacture of a medicament for the treatment of an autoimmune disease and/or an inflammatory disease, including, for example, arthritis, disseminated sclerosis, inflammatory diseases of the CNS and PNS, neurodegenerative diseases, or cancer, wherein the drug is administered to a subject who has been previously treated with at least one other therapy. By "pre-treated" or "pre-treatment" is meant that the subject has received one or more other therapies (eg, been treated with at least one other cancer therapy) prior to taking the drug containing the anti-SEMA4D binding molecule. "Pre-treated" or "pre-treatment" means that subjects who have been treated with at least one other therapy for 2 years, for 18 months, for 1 year, for 6 months, for 2 months, for 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days or even within 1 day prior to the start of treatment with a drug containing binding molecules against SEMA4D, for example, an antibody or its antigen-binding fragment, variant, or derivative, as presented in this document. It is not necessary for the subject to be a pre-treatment responder or pre-treatment responder. Thus, a subject who is taking a drug containing an anti-SEMA4D binding molecule might be a responder, or might not respond (e.g., the cancer was refractory) to pre-treatment with pre-treatment, or to one or more of pre-treatments where pre-treatments, including multiple therapies. Examples of other cancer therapies for which a subject may receive pre-treatment prior to receiving a drug containing an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, include, without limitation, operations; radiation therapy; chemotherapy, optionally in combination with autogenous bone marrow transplantation; other monoclonal antibody anticancer therapies, small molecule cancer therapies, or vaccine/immunotherapy based cancer therapies, steroid therapies; other cancer therapies; or any combination of them.

IX. Diagnostics

[000223] The present invention further provides a diagnostic method useful in diagnosing a SEMA4D-mediated disease such as certain cancers, autoimmune diseases, inflammatory diseases including, for example, arthritis, multiple sclerosis, inflammatory diseases of the central nervous system (CNS ) and the peripheral nervous system (PNS), neurodegenerative diseases, and invasive angiogenesis, which involves measuring the expression level of the SEMA4D protein or transcript in tissue or other cells or body fluid from an individual and comparing the measured expression level with a standard expression level of SEMA4D in normal tissue or fluid organism, whereby an increase in the level of expression compared to the standard is an indicator of the disorder.

[000224] An anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be used to analyze the levels of SEMA4D protein in a biological sample using classical immunohistological methods known to those of skill in the art ( for example, see Jalkanen, et al., J. Cell Biol 101:976-985 (1985); Jalkanen et al., J. Cell Biol 105:3087-3096 (1987)). Other antibody-based methods used to detect expression of the SEMA4D protein include immunological, such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable assays are described in more detail elsewhere herein.

[000225] By "assessing the level of expression of a SEMA4D polypeptide" is meant a qualitative or quantitative measurement or assessment of the level of a SEMA4D polypeptide in a first biological sample, either directly (for example, by determining or assessing the absolute level of a protein) or relatively (for example, by comparing with a disease associated polypeptide level in the second biological sample). In some aspects, the level of expression of the SEMA4D polypeptide in the first biological sample can be measured or estimated, and compared to a standard level of the SEMA4D polypeptide, the second biological sample is taken as a standard, obtained from an individual without a disorder, or it is determined by averaging levels from a population of individuals without the disorder. As will be appreciated in the art, once the "standard" level of the SEMA4D polypeptide is known, it can be reused as a standard for comparison.

[000226] The term "biological sample" refers to any biological sample obtained from an individual, cell line, tissue culture or other source of cells potentially expressing SEMA4D. Methods for obtaining tissue and body fluid biopsies from mammals are well known in the art.

X. Immunological tests

[000227] An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be assayed for immunospecific binding by any method known in the art. Immunological assays that can be used include, but are not limited to, competitive and non-competitive assays using methods such as Western blotting, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation, precipitation reactions, diffuse gel precipitation reactions, immunodiffuse assays, agglutinations, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, but are not limited to these. Such assays are common and well known in the art (see, for example, Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, which is incorporated herein description by reference in full). Typical immunoassays are briefly described below (but are not intended to be limiting).

[000228] An anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can also be used histologically, as in immunofluorescence, immunoelectron microscopy, or non-immunological assays, to detect SEMA4D protein in situ or conserved variants or peptide fragments. In situ detection can be performed by taking a histological sample from a patient and applying to it a labeled anti-SEMA4D antibody or antigen-binding fragment thereof, for example by applying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the SEMA4D protein, or conserved variants or peptide fragments, but also its distribution in the tissue under study. With the present invention, it will be readily appreciated by one of ordinary skill in the art that any of a wide range of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[000229] The binding activity of a given batch of an anti-SEMA4D binding molecule, eg, an antibody or antigen-binding fragment, variant, or derivative thereof, as provided herein, can be determined according to well-known methods. Those skilled in the art will be able to determine the operating and optimal assay conditions for each determination using routine experimentation.

[000230] There are many methods available for measuring the affinity of an antigen-antibody interaction, but relatively few for determining rate constants. Most methods rely on any antibody or antigen labeling, which inevitably complicates routine measurements and introduces uncertainties in the measured values.

[000231] Surface plasmon resonance (SPR) performed on BIACORE® offers a number of advantages over traditional methods for measuring the affinity of antigen-antibody interactions: (i) no need to label either antibody or antigen; (ii) the antibodies do not have to be purified in advance, the cell culture supernatant can be used directly; (iii) real-time measurements allow a quick semi-quantitative comparison of various monoclonal antibody interactions, which are included and sufficient for many evaluation purposes; (iv) the biospecific surface can be reconstructed so that a number of different monoclonal antibodies can be easily compared under the same conditions; (v) analytical procedures are fully automated and extensive series of measurements can be performed without user intervention. BIAapplications Handbook, version AB (reprinted 1998), BIACORE® code No. BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE® code No. BR-1001-84. SPR-based binding studies require that one member of the binding pair be immobilized on the surface of the sensor. The immobilized binding partner is referred to as the ligand. The binding partner in solution is called the analyte. In some cases, the ligand is attached indirectly to the surface through binding to another immobilized molecule, which is called a capture molecule. The SPR response reflects the change in mass concentration on the surface of the detector when the analytes bind or dissociate.

[000232] Based on the PPR, real-time BIACORE® measurements allow monitoring of interactions directly as they occur. The method is well suited for determining kinetic parameters. Comparative affinity rankings are easy to do and both kinetics and affinity constants can be derived from sensorgram data.

[000233] When the analyte is passed discretely across the surface of the ligand, the resulting sensogram can be divided into three main steps: (i) association of the analyte with the ligand during sample injection; (ii) an equilibrium or steady state during sample injection, where the level of analyte binding is balanced by dissociations of the complex; (iii) dissociation of the analyte from the surface during the flow of the buffer.

[000234] The association and dissociation phases provide information about the kinetics of the analyte-ligand interaction (k _a and k _d , complex formation and dissociation rates, k _d /k _a =K _D ). The equilibrium phase contains information about the affinity of the interaction between the analyte-ligand (K _D ).

[000235] The BIAevaluation software provides comprehensive tools for curve fitting using both numerical integration and global fit algorithms. With appropriate data analysis, the individual rates and affinity constant for the interaction can be derived from a simple BIACORE® assay. The range of measurable affinity according to this technology is very wide, ranging from mM to pM.

[000236] Epitope specificity is an important characteristic of a monoclonal antibody. Epitope mapping with BIACORE®, unlike conventional methods using radioimmunoassay, ELISA, or other surface adsorption methods, does not require labeling or purified antibodies, and also allows multi-site specificity tests to be performed using sequences from multiple monoclonal antibodies. In addition, a large number of analyzes can be processed automatically.

[000237] Pairwise binding experiments test the ability of two MAbs to simultaneously bind to the same antigen. MAbs directed against individual epitopes will bind independently of each other, while MAbs directed against identical or closely related epitopes will prevent each other from binding. These BIACORE® binding experiments are easy to perform.

[000238] For example, a capture molecule can be used to bind the first Mab followed by the addition of the antigen and the second MAb in series. The sensorgrams will show: (1) how many antigens bind to the first Mab, (2) to what extent the second MAb binds to the surface-coupled antigen, (3) if the second MAb does not bind, whether the paired test order will change the results.

[000239] Peptide inhibition is another technique used for epitope mapping. This method can complement pairwise antibody assays and can combine functional epitopes along structural lines when the primary antigen sequence is known. Peptides or antigen fragments were tested for inhibition of binding of various MAbs to the immobilized antigen. Peptides that interfere with the binding of a given MAb are considered structurally related to the epitope defined by the given MAb.

[000240] The present invention uses, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are known to those skilled in the art. Such methods are described in detail in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. US patent No. 4683195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide to Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[000241] General principles for designing antibodies are set forth in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). The general principles of protein engineering are set forth in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). The general principles of antibodies and antibody-hapten binding are set out in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.) and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y. ). In addition, standard methods in the field of immunology known in the art and not specifically described may be those disclosed in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

[000242] Standard works establishing general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY (1982)); Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyzes (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) Kuby Immunology (4th ed.; W.H. Freeman and Co., NY); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[000243] All references above, as well as all references cited in this description, are incorporated into this description by reference in their entirety.

[000244] The following examples are offered by way of illustration and not limitation.

Examples

Example 1 Generation of fully human anti-SEMA4D monoclonal antibody MAb D2517

[000245] The fully human anti-SEMA4D monoclonal antibody D2517 was generated using the following method. Fully human antibody libraries were generated in vaccinia virus and screened for SEMA4D binding according to the methods described in US Patent Application Publication No. 2013-0288927-A1, which is incorporated herein by reference in its entirety. A total of 96 antibodies were grouped into six different epitope groups by ELISA testing. Competitive binding of human antibodies to native human SEMA4D on Jurkat cells was assessed using mouse antibodies mAb76 and mAb67 and control mouse IgG. mAb76 and mAb67 bind to known human SEMA4D epitopes, and competition with either of these is indicative of functionality. Jurkat cells were pre-incubated for 30 minutes on ice with mAb76, mAb67 or control mouse IgG at 5 μg/ml in 100 μl and 200,000 cells in FACS buffer (1X PBS + 0.05% BSA + 2 mM EDTA). This provided saturation of mouse antibody binding to block epitope binding. Test antibodies and control human antibodies were pre-incubated at 1 μg/ml on ice for 30 minutes with a secondary goat-Dylight 649 anti-human Fc antibody reagent (Jackson Immunoresearch 496-170). After incubation of Jurkat cells with mouse IgG antibodies, cells were washed and incubated with test antibodies/secondary complex mixture for 30 minutes on ice. The cells were then washed twice with 200 μl of FACS buffer and resuspended in a fixative solution of 250 μl of PBS/1% BSA and 0.5% paraformaldehyde with PI. After resuspension, cells were analyzed by flow cytometry on FACS CANTO II. The negative PI population was gated and the Dylight649 shift was recorded and evaluated. Percent binding inhibition was calculated in comparison to the binding of human antibodies to Jurkat cells pre-incubated with irrelevant mouse IgG.

[000246] MAb C2305 was selected for further characterization because of its ability to cross-block previously characterized mouse anti-SEMA4D antibodies D67-2 and 76-1. See, for example, US patent No. 8496938, the description of which is incorporated into this description by reference in its entirety.

[000247] The polynucleotide encoding VH MAb C2305 was cloned into a mammalian expression vector that contained the gamma-4 human heavy chain constant region coding sequence, creating a full-length heavy chain. The polynucleotide encoding VL MAb C2305 was cloned into a mammalian expression vector that contained the lambda constant region coding sequence, creating a full-length light chain. Expression vectors containing a heavy chain and a light chain were co-transfected into CHO-S cells. The monoclonal antibody that was obtained was secreted from the cells and collected after a 3-6 day expression period. The resulting MAb was purified by protein A chromatography and characterized. Using flow cytometry and ELISA, it was demonstrated that the resulting fully human MAb (MAb C2305) is specific for SEMA4D, and can compete with mouse MAb 67-2 for binding to SEMA4D. The functional activity of MAb C2305 was further evaluated in a receptor blocking assay according to the method described in Example 3 below. Receptor blocking was observed, but at a lower level than the humanized MAb 2503 (US Pat. No. 8,496,938, also referred to as VX15/2503), which was used as a comparator.

[000248] MAb C2305 was fully sequenced and then modified to improve affinity. The heavy chain complementarity determining region 3 (HCDR3) was subjected to standard site-directed mutagenesis techniques and a new fully human light chain variable region (VL) was identified from the light chain library according to the methods described in U.S. Patent Application Publication No. 2013-0288927 -A1. Of these changes, an affinity improved Mab, MAb D2517, was chosen as the lead antibody. MAb D2517 was cloned, harvested, and expressed as a fully human IgG4 lambda light chain antibody. The VH, VL and CDR sequences of MAb D2517 are shown in Table 2.

Example 2 Binding characterization of MAb D2517

[000249] MAb D2517 binding characteristics were determined using the BIACORE assay, as follows. The antibodies, as well as the VX15/2503 comparator antibody, were both diluted in PBS, captured on a goat anti-human IgGFc probe at low density, and then marmoset SEMA4D-His was passed through the captured antibody over an antigen concentration range of 0-25 nM. The results are shown in Table 3. The affinity of MAb D2517 for SEMA4D was similar to that of VX15/2503.

[000250] The ability of MAb D2517 to bind to human, mouse, marmoset and macaque SEMA4D was determined by ELISA using the following method. Nunc MaxiSorp C-well ELISA plates were coated overnight with 100 μl/well of CD100-His dissolved in 1X PBS. After overnight incubation, the plates were washed and then blocked with 200 μl/well PBS + 0.5% BSA + 0.025% Tween 20 for at least 1 hour at room temperature. After additional washing, 100 μl/well diluted MAbs (100 ng/ml) were added to each well of the ELISA plate, which was incubated at room temperature for 2 hours and then washed. Next, 100 μl/well 1:10,000 goat anti-human Fc (Jackson cat. no. 109-035-098 lot 118460) was added to each well, and the ELISA plate was incubated at room temperature for 1 hour. The plate was then washed and assayed with 100 μl/well TMB substrate for 15 minutes. The detection reaction was stopped with 100 μl/well of 2N sulfuric acid and the plate was read using a plate reader at a wavelength of 450-570 nm.

[000251]. The results are presented in Fig. 1A (human SEMA4D), FIG. 1B (SEMA4D marmosets), FIG. 1C (SEMA4D monkey) and FIG. 1D (SEMA4D mice).

Example 3 MAb D2517 Blocks SEMA4D Binding to Its Receptor

[000252] The ability of MAbD2517 to block SEMA4D derived from various species for binding to its receptor, plexin B1, was evaluated as follows.

[000253] MAbD2517 and MAbVX15/2503 were diluted in acetate buffer. The antibodies were compared in a 3-fold dilution series for their ability to block SEMA4D-His (human, macaque, mouse, or rat) SEMA4D complexes from binding to 293PLXNB1 cells. All antibodies diluted from 1.5 μg/ml to 88 ng/ml in a 96-well plate and combined 1:1 with 0.8 μg/ml of the corresponding SEMA4D-His were incubated overnight at 4°C.

[000254] The following day, antibody/SEMA4D-His complexes or controls were added to 2.5×10 ⁵ 293PLXNB1 cells in a 96-well plate and binding was performed for 30 minutes at 4°C. After washing, cells were stained with anti-6xHis-APC (30 min, 4°C), washed and analyzed by flow cytometry on FACS Canto II. The results are presented in Fig. 2A-2D. EC50 values were calculated from the curves and are shown in Table 4.

Example 4 Testing the Mouse Chimeric Equivalent of MAbD2517 in a Tumor Model

[000255] The VH and VL amino acid sequences (SEQ ID NO: 1 and SEQ ID NO: 5) of MAb D2517 were inserted into vectors expressing mouse IgG1 and lambda constant regions to generate chimeric MAb D2585 antibody. Antibodies were expressed in CHO-S cells and purified as described in Example 1.

[000256] Balb/C female mice 6-8 weeks of age (n=12) were inoculated with 30,000 Tubo.A5 mammary tumor cells subcutaneously into mammary adipose tissue. Treatment with control mouse IgG1/2B8.1E7 antibody or chimeric anti-SEMA4D antibody MAbD2585 was started 7 days after inoculation (10 mg/kg, IP (ip), weekly X5). Tumors were measured with a caliper 2x/week starting 11 days after implantation. Animals were sacrificed when the tumor volume reached 800 mm ³ .

[000257] Tumor growth was measured with a compass and the measurements were used to calculate tumor volume using the formula (w ² ×l)/2 where the tumor has w = width, whichever is smaller, and l = length in mm. Mean tumor volume and Kaplan-Meier survival curves, defined as time to endpoint where tumor volume = 800 mm ³ , are shown in FIG. 3A and 3B, respectively. Statistical analysis was performed using two-way analysis of variance (ANOVA) for mean tumor volume (p<0.05), and log Rank analysis for survival (p<.0.1), respectively, which were statistically significant.

[000258] Tumor regression rates in the Tubo tumor model were also measured and shown in FIG. 3C. Regression is the absence of palpable tumor, defined as tumor measurement <50 mm ³ for at least two consecutive measurements. Treatment with the chimeric MAbD2585 antibody increased the number of regressions in Tubo bearing mice. The number of regressions in the group of mice treated with the chimeric MAbD2585 antibody was statistically significant compared to control Ig (p=0.01) as determined by Fisher's exact test.

[000259] The spirit and scope of this invention should not be limited by any of the illustrative embodiments described above, but should only be defined in accordance with the following claims and their equivalents.

Claims (46)

1. An antibody or antigen-binding fragment thereof that specifically binds to semaphorin-4D (SEMA4D), containing a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH contains complementarity determining regions (HCDRs) of HCDR1, HCDR2, and HCDR3 containing amino acid sequences identical to SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and the VL contains complementarity determining regions (LCDRs) of LCDR1, LCDR2 and LCDR3 containing amino acid sequences identical to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. 2. The antibody or its fragment according to claim 1, characterized in that the VH additionally contains the framework regions (HFW) HFW1, HFW2, HFW3 and HFW4, while the VL additionally contains the framework regions (LFW) LFW1, LFW2, LFW3 and LFW4. 3. An antibody or fragment thereof according to claim 2, characterized in that said framework regions originate from a human antibody. 4. An antibody or fragment thereof according to claim 2, characterized in that said framework regions originate from an antibody that is not a human antibody. 5. An antibody or fragment according to any one of paragraphs. 1-3, characterized in that VH contains the amino acid sequence of SEQ ID NO: 1. 6. An antibody or fragment according to any one of paragraphs. 1-3, characterized in that VL contains the amino acid sequence of SEQ ID NO: 5. 7. An antibody or fragment according to any one of paragraphs. 1-3, characterized in that VH contains the amino acid sequence of SEQ ID NO: 1, and VL contains the amino acid sequence of SEQ ID NO: 5. 8. An antibody or fragment according to any one of paragraphs. 1–7, additionally containing the heavy chain constant region or its fragment fused to the C-terminus of VH. 9. An antibody or fragment thereof according to claim 8, wherein said heavy chain constant region or fragment thereof is a heavy chain constant region of a human antibody. 10. An antibody or fragment thereof according to claim 9, characterized in that said heavy chain constant region or fragment thereof is a human IgG4 constant region. 11. An antibody or fragment thereof according to claim 8, wherein said heavy chain constant region or fragment thereof is a non-human heavy chain heavy chain constant region. 12. An antibody or fragment thereof according to claim 11, characterized in that said heavy chain constant region or fragment thereof is a mouse IgG1 constant region. 13. An antibody or fragment according to any one of paragraphs. 1–12, additionally containing a light chain constant region or its fragment fused to the C-terminus of VL. 14. An antibody or fragment thereof according to claim 13, wherein said light chain constant region or fragment thereof is a light chain constant region of a human antibody. 15. An antibody or fragment thereof according to claim 14, characterized in that said light chain constant region or fragment thereof is a light chain constant region of a human lambda antibody. 16. An antibody or fragment thereof according to claim 13, characterized in that said light chain constant region or fragment thereof is a light chain constant region that is not a light chain of a human antibody. 17. An antibody or fragment thereof according to claim 16, characterized in that said light chain constant region or fragment thereof is a mouse lambda antibody light chain constant region. 18. An antibody or fragment according to any one of paragraphs. 1-17, characterized in that said fragment is a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment, an Fd fragment, a single-chain Fv fragment (scFv) or a disulfide-linked Fv fragment (sdFv). 19. An antibody or fragment according to any one of paragraphs. 1-18, characterized in that the specified antibody or its fragment is able to specifically bind to two or more different epitopes. 20. An antibody or fragment thereof according to any one of paragraphs. 1-19 that can specifically bind to human SEMA4D, mouse SEMA4D, rat SEMA4D, or non-human primate SEMA4D. 21. An antibody or fragment thereof according to claim 20, wherein said non-human primate SEMA4D is cynomolgus monkey SEMA4D or marmoset SEMA4D. 22. An antibody or fragment thereof according to claim 20 or 21 that specifically binds to SEMA4D with an affinity characterized by a dissociation constant KD of not more than 500 nM, 100 nM, 50.0 nM, 40.0 nM, 30.0 nM, 20 .0 nM, 10.0 nM, 9.0 nM, 8.0 nM, 7.0 nM, 6.0 nM, 5.0 nM, 4.0 nM, 3.0 nM, 2.0 nM, 1 .0 nM, 0.50 nM, 0.10 nM, 0.050 nM, 0.01 nM, 0.005 nM, or 0.001 nM; and wherein SEMA4D is human SEMA4D, mouse SEMA4D, cynomolgus SEMA4D, marmoset SEMA4D, or any combination thereof. 23. An antibody or fragment according to any one of paragraphs. 1-22, which can inhibit the binding of SEMA4D to the SEMA4D receptor. 24. An antibody or fragment thereof according to claim 23, characterized in that said SEMA4D receptor is plexin-B1, plexin-B2, CD72, or a combination thereof. 25. An antibody or fragment according to any one of paragraphs. 1-24 that elicit minimal or no immune response to the antibody when administered to a human subject. 26. A conjugate capable of specifically binding to semaphorin-4D (SEMA4D), consisting of an antibody or fragment according to any one of paragraphs. 1–25 and a heterologous fragment. 27. The conjugate of claim 26, wherein said heterologous fragment comprises a polypeptide, a cytotoxic agent, a therapeutic agent, a prodrug, a lipid, a carbohydrate, a nucleic acid, a detectable label, a polymer, or any combination thereof. 28. The conjugate of claim 27, wherein said polypeptide comprises a binding molecule, an enzyme, a cytokine, a lymphokine, a hormonal peptide, or any combination thereof. 29. The conjugate of claim 27, wherein said cytotoxic agent comprises a radionuclide, a biological toxin, an enzymatically active toxin, or any combination thereof. 30. The conjugate of claim 27, wherein said detectable label includes an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or any combination thereof. 31. Composition containing an antibody or fragment according to any one of paragraphs. 1-25 or a conjugate according to any one of paragraphs. 26-30 and vehicle, to neutralize SEMA4D in a subject. 32. Polynucleotide containing one or more nucleic acid sequences encoding an antibody or fragment according to any one of claims. 1–25. 33. A polynucleotide according to claim 32 containing a nucleic acid sequence encoding the VH of an antibody or fragment thereof. 34. A polynucleotide according to claim 32 containing a nucleic acid sequence encoding the VL of an antibody or fragment thereof. 35. The combination of polynucleotides to obtain an antibody or fragment according to any one of paragraphs. 1-25 containing a polynucleotide that has a nucleic acid sequence encoding the heavy chain of an antibody or fragment according to any one of paragraphs. 1-25, and a polynucleotide that has a nucleic acid sequence encoding a light chain of an antibody or fragment according to any one of paragraphs. 1–25. 36. An expression vector containing a polynucleotide according to claim 32 or a combination of polynucleotides according to claim 35. 37. A combination of expression vectors containing the combination polynucleotides of claim 35. 38. The host cell to obtain an antibody or fragment according to any one of paragraphs. 1-25 containing a polynucleotide according to paragraph 32, or a combination of polynucleotides according to paragraph 35, or a vector according to paragraph 36, or a combination of vectors according to paragraph 37. 39. The host cell to obtain the conjugate according to any one of paragraphs. 26–30 containing a polynucleotide according to paragraph 32, or a combination of polynucleotides according to paragraph 35, or a vector according to paragraph 36, or a combination of vectors according to paragraph 37. 40. The method of obtaining an antibody or fragment according to any one of paragraphs. 1-25, including culturing the host cell according to claim 38 and isolating the specified antibody or its fragment. 41. A method for neutralizing SEMA4D in a human subject, comprising administering to said human subject an antibody or fragment thereof according to any one of paragraphs. 1-25, the conjugate according to any one of paragraphs. 26-30, or compositions according to item 31. 42. The method of claim 41, wherein said human subject is in need of treatment for an autoimmune disease or disorder, an inflammatory disease or disorder, cancer, a neuroinflammatory disease or disorder, a neurodegenerative disease or disorder, or any combination thereof. 43. The method of claim 42, wherein said neuroinflammatory disease or disorder is multiple sclerosis. 44. The method of claim 42, wherein said neurodegenerative disease or disorder is stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, Down's syndrome, ataxia, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), HIV-related cognitive impairment, CNS lupus, mild cognitive impairment, or a combination thereof. 45. The method of claim 42 wherein said autoimmune disease or inflammatory disease is arthritis. 46. The method of claim 45 wherein said autoimmune disease or inflammatory disease is rheumatoid arthritis.

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