use of an isolated antibody or antigen-binding fragment that specifically binds to semaphorin-4d to

专利摘要:
USE OF BINDING MOLECULES FOR SEMAPHORIN-4D FOR THE MODULATION OF PERMEABILITY OF THE HEMATOENCEPHALIC BARRIER. Provided here are methods for decreasing blood brain barrier permeability in a subject with a neuroinflammatory disorder, comprising administering to the subject an effective amount of an isolated binding molecule, which specifically binds to semaphorin-4D (SEMA4D) or its high affinity Plexin-B1 receptor.
公开号:BR112014008885B1
申请号:R112014008885-3
申请日:2012-10-11
公开日:2021-02-17
发明作者:Ernest S Smith；Maurice Zauderer
申请人:Vaccinex, Inc；
IPC主号:

专利说明:

[0001] [001] The contents of the sequence listing submitted electronically in an ASCII text file (Name: “1843_068PC03_SequenceListing_ascii.txt”; Size: 33,807 bytes; and Creation Date: October 10, 2012) deposited with the request is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION
[0002] [002] Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein (for example, SEQ ID NO: 1 (human); SEQ ID NO: 2 (murine)) that belongs to the semaphorin gene family. SEMA4D is expressed on the cell surface as a homodimer, but in cell activation SEMA4D it can be released from the cell surface through proteolytic cleavage to generate sSEMA4D, a soluble form of the protein, which 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] [003] SEMA4D is expressed at high levels in lymphoid organs, including the spleen, thymus and lymph nodes, and in non-lymphoid organs, such as the brain, heart and kidney. In lymphoid organs, SEMA4D is abundantly expressed in resting T cells, but only weakly expressed in resting B cells and antigen presenting cells (APCs), such as dendritic cells (DCs). Its expression, however, is over-regulated in these cells after activation by various immune stimuli. The release of soluble SEMA4D from immune cells is also increased by cell activation.
[0004] [004] SEMA4D has been implicated in the development of neurodegenerative diseases, autoimmune diseases, demyelinating diseases and certain cancers. Although the role of SEMA4D signaling through its receptors, for example, Plexin-B1, in angiogenesis is well recognized, the effect of SEMA4D signaling on Blood-brain Barrier (BBB) remains unclear. This is important because changes in BBB permeability have a profound influence on brain tissue and function. Therefore, there remains a need for treatments for neuroinflammatory disorders that arise as a result of BBB disruption, and, in particular, therapies that inhibit, suppress, prevent, reverse or reduce BBB disruption. BRIEF SUMMARY OF THE INVENTION
[0005] [005] Methods for using semaphorin-4D binding molecules for modulating permeability of the blood-brain barrier are disclosed herein. Evidence is presented demonstrating that SEAM4D can compromise the integrity of the BBB, thereby increasing its permeability. In accordance with aspects of the invention illustrated here, a method is provided to decrease the blood-brain barrier permeability in a subject having a neuroinflammatory disorder including administering to the subject an effective amount of an isolated binding molecule, which specifically binds to semaphorin -4D (SEMA4D), thus decreasing the blood-brain barrier permeability in the subject.
[0006] [006] According to aspects illustrated here, a method of maintaining or increasing the expression of Claudina-5 is provided in a subject having a neuroinflammatory disorder comprising administering to the subject an effective amount of an isolated binding molecule, which specifically binds to the semaphorin-4D (SEMA4D), in which the binding molecule maintains or increases Claudine-5 expression in the subject.
[0007] [007] According to aspects illustrated here, a method of decreasing the blood-brain barrier permeability in a subject having a neuroinflammatory disorder is provided, comprising administering to the subject an effective amount of an isolated binding molecule, which specifically inhibits interaction of semaphorin 4D (SEMA4D) with a SEMA4D receptor, thus decreasing the blood-brain barrier permeability in the subject.
[0008] [008] According to aspects illustrated here, a method of treating a subject having a neuroinflammatory disorder is provided, comprising administering to the subject an effective amount of an isolated binding molecule, which specifically inhibits the interaction of 4D semaphorin (SEMA4D) with a SEMA4D receptor, in which the binding molecule decreases the permeability of the blood-brain barrier, thus treating the subject.
[0009] [009] According to aspects illustrated here, a method is provided to decrease the blood-brain barrier permeability in a subject having a neuroinflammatory disorder, comprising administering to the subject an effective amount of an isolated binding molecule, which specifically binds to the SEMA4D, in which the binding molecule competitively inhibits a reference monoclonal antibody selected from the group consisting of VX15 / 2503 or 67 from specifically binding to SEMA4D.
[0010] [0010] According to aspects illustrated here, a method of treating a subject having a neuroinflammatory disorder is provided, comprising administering to the subject an effective amount of an isolated binding molecule, which specifically binds to semaphorin-4D (SEMA4D) and an isolated binding molecule, which specifically binds to Plexin-B1, in which the binding molecules to SEMA4D and Plexin-B1 decrease the permeability of the blood-brain barrier, thus treating the subject.
[0011] [0011] According to aspects illustrated here, a method of treating a subject having a neuroinflammatory disorder is provided, comprising administering to the subject an effective amount of a 4D semaphorin interaction inhibitor (SEMA4D) with a SEMA4D receptor, in that the inhibitor decreases the permeability of the blood-brain barrier, thus treating the subject. BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES
[0012] [0012] FIGURE 1: Schematic diagram of the experimental protocol of the dynamic BBB in vitro (“DIV-BBB”) described in the Examples.
[0013] [0013] FIGURE 2: In vitro DIV-BBB model showing BBB integrity measurements as reflected in transendothelial electrical resistance (TEER) in the presence of recombinant SEMA4D (0.05, 0.5, 5 or 50 μg / mL ) and VX15 / 2503 Antibody (“VX15”).
[0014] [0014] FIGURE 3: DIV-BBB in vitro model showing BBB integrity measurements as reflected in transendothelial electrical resistance (TEER) during BBB formation, BBB rupture in the presence of recombinant SEMA4D (0.5, 5 or 50 μg / mL), and the restoration of BBB in the presence of Antibody VX15 / 2503 (“VX15”), but not isotype control (“Iso”).
[0015] [0015] FIGURE 4: DIV-BBB in vitro model showing BBB integrity measurements as reflected in transendothelial electrical resistance (TEER) during BBB formation, BBB rupture in the presence of 0.25, 2.5 , or 25 μg / mL of C35 control antigen (“CTRL”) or 50 μg / mL of recombinant SEMA4D, and the restoration of BBB in the presence of VX15 / 2503 Antibody (“VX15”).
[0016] [0016] FIGURE 5: DIV-BBB model in vitro showing BBB integrity measurements as reflected in transendothelial electrical resistance (TEER) during BBB formation, BBB rupture in the presence of recombinant SEMA4D (50 μg / mL ), and the reestablishment of BBB in the presence of Antibody VX15 / 2503 (“VX15”), anti-Plexin-B1 antibody (“Anti-PLXNB1”), but not isotype control (“Iso”).
[0017] [0017] FIGURE 6: DIV-BBB in vitro model showing BBB integrity measurements as reflected in transendothelial electrical resistance (TEER) during BBB formation, BBB rupture in the presence of activated PBMC (106 / ml) and cessation of flow, and the restoration of the BBB in the presence of Antibody VX15 / 2503 or IgG for Isotype Control.
[0018] [0018] FIGURE 7A-C: Results from the EAE model in vivo showing the integrity of the BBB or loss of it as reflected by immunostaining the penetration of fibrinogen (“Fib. +”) Into brain tissue (left panel 7A and quantification in 7B) and expression of Claudina-5 (“CLN5 +”) as detected by red staining (right panel 7A and quantification in 7C) after treatment with Antibody VX15 / 2503 (“Anti-SEMA4D”) or isotype control (“ Control IgG ”).
[0019] [0019] FIGURE 8: Immunoblot results showing the effect of increasing concentrations of recombinant SEMA4D (1 ng / ml, 10 ng / ml and 100 ng / ml) on the expression of Claudine-5 protein (“CLN-5”) from key tight endothelial junction compared to positive VEGF-A control in primary mouse central nervous system (CNS) endothelial cultures. DETAILED DESCRIPTION OF THE INVENTION I. Definitions
[0020] [0020] It should be noted that the term "one" entity refers to one or more of that entity; for example, "an anti-SEMA4D antibody" is understood to represent one or more anti-SEMA4D antibodies. As such, the terms "one" (or "one"), "one or more" and "at least one" can be used interchangeably here.
[0021] [0021] It should be noted that the terms "blood-brain barrier" and "BBB" are used interchangeably.
[0022] [0022] As used herein, the term "rupture" or "rupture" with respect to BBB, such as "rupture of the blood-brain barrier" or "rupture of the blood-brain barrier" refers to an increase in the permeability of the blood-brain barrier, or, in the case of “DIV-BBB,” a dynamic humanized in vitro model of BBB, a decrease in transendothelial electrical resistance (TEER). McCallister et al., Brain Res. 904: 20-30 (2001); Santaguida et al., Brain Res. 1109: 1-13 (2006); and Cucullo et al., Epilepsy 48: 505 - 16 (2007) have shown that there is a direct (inverse) relationship between TEER and permeability in the DIV-BBB. In addition, an increase in blood-brain barrier permeability or a decrease in electrical resistance may be the result of a decrease in the number, density and / or concentration of endothelial cells present in the BBB; or a change in morphology or interactions between endothelial cells or astrocytes or between endothelial cells and astrocytes that make up the BBB.
[0023] [0023] As used herein, the term “restoration” with respect to BBB, such as “blood-brain barrier restoration” refers to a decrease in blood-brain barrier permeability, or, in the case of DIV-BBB, a model in vitro humanized dynamic of BBB, an increase in transendothelial electrical resistance.
[0024] [0024] As used herein, the term "neuroinflammatory disorder" refers to an inflammatory disorder of the central nervous system (CNS), a neurodegenerative disorder, an autoimmune disorder of the central nervous system, a myelin disorder or a disease that affects oligodendrocytes, or a post-traumatic myelin disorder of the central nervous system. It should be noted that, neuroinflammatory disorders are also often neurodegenerative disorders. However, it is possible for a neurodegenerative disorder to exist in the absence of obvious neuroinflammation. This is the case, for example, in late-stage secondary progressive multiple sclerosis.
[0025] [0025] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of a neuroinflammatory disorder, the therapeutically effective amount of the drug can decrease BBB permeability; reduce, delay or stop an increase in BBB permeability; inhibiting, for example, suppressing, delaying, preventing, stopping or reversing an increased BBB permeability; increase the number, density and / or concentration of endothelial cells present in the BBB; change in the morphology or function of endothelial cells; or a change in the interactions between endothelial cells or astrocytes or between endothelial cells and astrocytes that make up the BBB; relieve to some extent one or more of the symptoms associated with increased BBB permeability, for example, neuroinflammatory disorders; reduce morbidity and mortality; improve the life quality; or a combination of such effects.
[0026] [0026] Terms such as "treat" or "treatment" or "to treat" or "relieve" or "to relieve" refer to both 1) therapeutic measures that heal, slow down, mitigate symptoms of, reverses, and / or interrupts the progression of a condition or pathological disorder diagnosed in relation to 2) prophylactic or preventive measures that prevent and / or delay the development of a target condition or pathological disorder. Thus, those in need of treatment include those already with the disorder; those prone to having the disorder; and those in which the disorder is to be prevented. Beneficial or desired clinical results include, but are not limited to, symptom relief, decrease in disease extent, stabilized (that is, not worsen) disease, delay or delay in disease progression, improvement or palliation of disease status disease, and remission (whether partial or total), whether detectable or not. "Treatment" can also mean prolonging survival, compared to expected survival if you do not receive treatment. Those in need of treatment include those already with the condition or disorder as well as those likely to have the condition or disorder or those, in which the condition or disorder is to be prevented.
[0027] [0027] By "subject" or "individual" or "animal" or "patient" or "mammal", any subject is meant, particularly a mammal subject, for which the diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or companion animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and so on. onwards.
[0028] [0028] As used herein, phrases such as "a subject who would benefit from administration of an anti-SEMA4D antibody" and "an animal in need of treatment" include subjects, such as mammalian subjects, who would benefit from administration of a anti-SEMA4D antibody or other SEMA4D binding molecule used, for example, for the detection of a SEMA4D polypeptide (for example, for a diagnostic procedure) and / or treatment, that is, palliation or prevention of a disease, with an anti-SEMA4D antibody or other SEMA4D binding molecule.
[0029] [0029] A "binding molecule" or "antigen binding molecule" of the present invention refers in its broadest sense to a molecule that specifically binds an antigenic determinant. In one embodiment, the binding molecule specifically binds to SEMA4D, for example, to a transmembrane SEMA4D polypeptide of about 150 kDa or a soluble SEMA4D polypeptide of about 120 kDa (commonly referred to as sSEMA4D). In another embodiment, a binding molecule of the invention is an antibody or a fragment of binding to the antigen thereof. In another embodiment, a binding molecule of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, a binding molecule of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, a binding molecule of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, a binding molecule of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, a binding molecule of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, a binding molecule of the invention comprises at least six CDRs from one or more antibody molecules.
[0030] [0030] The present application is directed to a method of decreasing the blood-brain barrier permeability in a subject having a neuroinflammatory disorder (for example, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, epilepsy, Alzheimer's Disease, Parkinson's Disease, meningitis, cerebral edema, cerebral trauma and stroke), comprising administering to the subject an anti-SEMA4D binding molecule, an anti-PlexinB1 binding molecule or a combination thereof.
[0031] [0031] As used herein, "anti-SEMA4D binding molecule" or "anti-PlexinB1 binding molecule" refers to an antibody or fragment binding to the antigen, variant or derivative thereof. Unless specifically referring to full-length antibodies, such as naturally occurring antibodies, the term "anti-SEMA4D antibody" or "anti-PlexinB1 antibody" encompasses full-length antibodies, as well as antigen-binding fragments, variants , analogs or derivatives of such antibodies, for example, naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind to the antigen in a similar manner to antibody molecules.
[0032] [0032] As used herein, "SEMA4D interaction inhibitor with a SEMA4D receptor" refers to an "anti-SEMA4D binding molecule", an "anti-PlexinB1 binding molecule", as well as an inhibitor molecule small SEMA4D or a SEMA4D receiver.
[0033] [0033] As used herein, "human" or "fully human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin or transgenic animals libraries for one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described below and, for example, in Pat. U.S. № 5,939,598 by Kucherlapati et al. "Human" or "fully human" antibodies also include antibodies comprising at least the variable domain of a heavy chain, or at least the variable domains of a heavy chain and a light chain, where the variable domain (s) ) have the human immunoglobulin variable domain (s) amino acid sequence.
[0034] [0034] "Human" or "fully human" antibodies also include "human" or "fully human" antibodies, as described above, which comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (eg example, the VH regions and / or VL regions) described herein, which the antibodies or their fragments immunospecifically bind to a SEMA4D polypeptide or fragment or its variant. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequence encoding a human anti-SEMA4D antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis that results in amino acid substitutions . Preferably, variants (including derivatives) that encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions in relation to the reference VH region, VHCDR1, VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2 or VLCDR3.
[0035] [0035] In certain embodiments, amino acid substitutions are conservative amino acid substitutions, discussed below. Alternatively, mutations can be introduced randomly over all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (for example, the ability to bind to a SEMA4D polypeptide (e.g., human, murine, or both human and murine SEMA4D). Such variants (or their derivatives) of "human" or "fully human" antibodies can also be referred to as human or fully human antibodies that are "optimized" or "optimized for antigen binding" and include antibodies that have improved antigen affinity .
[0036] [0036] The terms "antibody" and "immunoglobulin" are used interchangeably here. An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain. The basic immunoglobulin structures in vertebrate systems are relatively well understood. See, for example, Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed .; Cold Spring Harbor Laboratory Press).
[0037] [0037] As used herein, the term "immunoglobulin" comprises several broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will assess that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, µ, α, δ, ε) with some subclasses among them (for example, γ1 to γ4). It is the nature of this chain that determines the “class” of the antibody such as IgG, IgM, IgA IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, etc. they are well characterized and are known to confer functional specialization. The modified versions of each of these classes and isotypes are easily discernible to the skilled technician in view of the present disclosure and, consequently, are within the scope of the present invention. All classes of immunoglobulin are clearly within the scope of the present invention, the following debate will generally be directed to the IgG class of immunoglobulin molecules. with respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of approximately 23,000 Dalton molecular weight, and two identical heavy chain polypeptides of 53,000 to 70,000 molecular weight. The four chains are typically joined by disulfide bonds in a "Y" configuration, in which the light chains support the heavy chains starting at the "Y" opening and continuing through the variable region.
[0038] [0038] Light chains are classified as kappa or lambda (ᴋ, λ). Each heavy chain class can be linked with a kappa or lambda light chain. In general, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when immunoglobulins are generated by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences conducted from an N at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
[0039] [0039] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are functionally used. In this regard, it will be assessed that the variable domains of both light (VL or VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the light chain (CL) and heavy chain (CH1, CH2 or CH3) domains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding or amino-terminal site of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminal of the heavy and light chain, respectively.
[0040] [0040] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and the VH domain, or subset of the complementarity determining regions (CDRs) within these variable domains, of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each Y branch. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains. In some cases, for example, certain immunoglobulin molecules derived from camelid species or engineered on the basis of camelid immunoglobulins, a complete immunoglobulin molecule may consist of only heavy chains, with no light chains. See, for example, Hamers-Casterman et al., Nature 363: 446 - 448 (1993).
[0041] [0041] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the domain binding antigen as the antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the amino acids in the antigen-binding domains, referred to as "structure" regions, show less inter-molecular variability. The structure regions, to a large extent, adopt a β-leaf conformation and the CDRs form loops that connect, and in some cases form part of the β-leaf structure. Thus, the structural regions act to form a scaffold that provides to position the CDRs in the correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a complementary surface to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent attachment of the antibody to its cognate epitope. Amino acids comprising CDRs and framework regions, respectively, can be easily identified for any given variable domain of heavy or light chain by a person of ordinary skill in the art, since they have been precisely defined (see, below).
[0042] [0042] In the case where there are two or more definitions of a term that is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region"("CDR") to describe the non-contiguous antigen combination sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al. (1983) US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” and by Chothia and Lesk, J. Mol. Biol. 196: 901 - 917 (1987), which are incorporated herein by reference, where definitions include overlap or subsets of amino acid residues when compared against one another. Nevertheless, the application of both definitions to refer to a CDR of an antibody or its variants is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that span the CDRs as defined by each of the references cited above are shown below in Table 1 as a comparison. The exact numbers of residues that comprise a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR, given the sequence of amino acids in the antibody's variable region.
[0043] [0043] Kabat et al. also defined a numbering system for variable domain strings that is applicable to any antibody. A person of ordinary skill in the art can unequivocally assign this “Kabat numbering” system to any variable domain sequence, without relying on any experimental data other than the sequence itself. As used here, the “Kabat numbering” refers to the numbering system presented by Kabat et al. (1983) U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest.” Unless otherwise specified, references to the numbering of specific amino acid residue positions in an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative of the present invention are in accordance with the Kabat numbering system .
[0044] [0044] Antibodies or antigen-binding fragments, variants, or derivatives of the invention include, but are not limited to, polyclonal, monoclonal, multi-specific and bispecific, wherein at least one branch is specific for anti-SEMA4D antibodies , human, humanized, primatized or chimeric, single chain antibodies, epitope-binding fragments, for example, Fab, Fab 'and F (ab') 2, Fd, Fvs, Single chain Fvs (scFv), Fvs bound to disulfide (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to anti-SEMA4D antibodies disclosed herein ). ScFv molecules are known in the art and are described, for example, in U.S. Pat. U.S. No. 5,892,019. The immunoglobulin or antibody molecules of the invention can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, etc. .), or subclass of immunoglobulin molecule.
[0045] [0045] As used herein, the term "heavy chain moiety" includes amino acid sequence derived from an immunoglobulin heavy chain. In certain embodiments, a polypeptide comprising a heavy chain portion comprises at least one of: a VH domain, a CH1 domain, a hinge domain (e.g., upper, middle, and / or lower hinge region), a CH2 domain, CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the invention can comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the invention comprises a chain of polypeptides comprising a CH3 domain. In addition, a binding polypeptide for use in the invention may lack at least a portion of a CH2 domain (for example, all or part of a CH2 domain). As presented above, it will be understood by a person of ordinary skill in the art that these domains (e.g., the heavy chain moieties) can be modified, such that they vary in amino acid sequence from a naturally occurring immunoglobulin molecule.
[0046] [0046] In certain anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein, the heavy chain portions of a polypeptide chain of a multimer are identical to those in a second polypeptide chain of the multimer. Alternatively, the monomers containing the heavy chain portion of the invention are not identical. For example, each monomer can comprise a different target binding site, forming, for example, a bispecific antibody.
[0047] [0047] The heavy chain portions of a binding molecule for use in the methods disclosed herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion may comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion may comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
[0048] [0048] As used herein, the term "light chain moiety" includes amino acid sequence derived from an immunoglobulin light chain, for example, a kappa or lambda light chain. Preferably, the light chain portion comprises at least one of a VL or CL domain.
[0049] [0049] Anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein can be described or specified in terms of the epitope (s) or portion (s) of an antigen, for example, a target polypeptide disclosed herein (e.g., SEMA4D) that they recognize or specifically bind to. The portion of a target polypeptide that specifically interacts with the antigen-binding domain of an antibody is an "epitope," or an "antigenic determinant". A target polypeptide can comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen. In addition, it should be noted that an "epitope" in a target polypeptide can be or can include elements of non-polypeptides, for example, an epitope can include a carbohydrate side chain.
[0050] [0050] The minimum size of a peptide or polypeptide epitope for an antibody is considered to be about four to five amino acids. Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, amino acids comprising an epitope need not be contiguous, and in some cases, may not even be in the same peptide chain. A peptide or polypeptide epitope recognized by anti-SEMA4D antibodies of the present invention can contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous SEMA4D amino acids.
[0051] [0051] By "specifically binding," it is generally understood that an antibody binds to an epitope that, through its antigen-binding domain, and that binding involves a complementarity between the antigen-binding domain and the epitope . According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, through its antigen-binding domain more easily than it would bind to a random unrelated epitope. The term "specificity" is used here to describe the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" can be considered to have greater specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with greater specificity than has for the related “D” epitope.
[0052] [0052] By "preferentially binds", it is understood that the antibody specifically binds to an epitope more easily than it would bind to a related, similar, homologous or analogous epitope. Thus, an antibody that “binds preferentially” to a particular epitope would be more likely to bind to that epitope than to a related epitope, although such an antibody may cross-react with the related epitope.
[0053] [0053] By way of non-limiting example, an antibody can be considered to bind to a first epitope, preferably it binds to said first epitope with a dissociation constant (KD) that is less than the KD of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind to a first antigen, preferably it binds to the first epitope with an affinity that is at least an order of magnitude less than the KD of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind to a first epitope, preferably it binds to the first epitope with an affinity that is at least two orders of magnitude less than the antibody KD to the second epitope.
[0054] [0054] In another non-limiting example, an antibody can be considered to bind a first epitope, preferentially binds to the first epitope with a dissociation constant (k (off)) that is less than ak (off) of the antibody for the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope, preferably it binds to the first epitope with an affinity that is at least an order of magnitude less than the k (off) of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind to a first epitope, preferably it binds to the first epitope with an affinity that is at least two orders of magnitude less than the k (off) of the antibody to the second epitope . An antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind to a target polypeptide disclosed herein (e.g., SEMA4D, e.g., human, murine, or both human and murine SEMA4D) or a fragment or its variant with a dissociation constant (k (off)) less than or equal to 5 X 10-2 s-1, 10-2 s-1, 5 X 10-3 s-1 or 10-3 s -1. More preferably, an antibody of the invention can be said to bind to a target polypeptide disclosed herein (for example, SEMA4D, for example, human, murine, or both human and murine SEMA4D) or a fragment or variant thereof with a dissociation constant (k (off)) less than or equal to 5 X 10-4 s-1, 10-4 s-1, 5 X 10-5 s-1, or 10-5 s-1, 5 X 10- 6 s-1, 10-6 s-1, 5 X 10-7 s-1 or 10-7 s-1.
[0055] An antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind to a target polypeptide disclosed herein (e.g., SEMA4D, e.g., human, murine, or both human and SEMA4D) murine) or a fragment or its variant with an association constant (k (on)) greater than or equal to 103 M-1 s-1, 5 X 103 M-1 s-1, 104 M-1 s-1 or 5 X 104 M-1 s-1. More preferably, an antibody of the invention can be said to bind to a target polypeptide disclosed herein (for example, SEMA4D, for example, human, murine, or both human and murine SEMA4D) or a fragment or variant thereof with a association constant (k (on)) greater than or equal to 105 M-1 s-1, 5 X 105 M-1 s-1, 106 M-1 s-1, or 5 X 106 M-1 s-1 or 107 M-1 s-1.
[0056] [0056] An antibody is said to competitively inhibit the binding of a reference antibody to a particular epitope if it preferentially binds to that epitope insofar as it blocks, to some degree, the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competitive ELISA assays. An antibody can be said to competitively inhibit the binding of the reference antibody to a particular epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[0057] [0057] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope to the CDR of an immunoglobulin molecule. See, for example, Harlow et al. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) Pages 27 - 28. As used herein, the term "avidity" refers to the overall stability of the complex between an immunoglobulin population and an antigen, that is, the strength of the functional combination of an immunoglobulin mixture with the antigen. See, for example, Harlow on pages 29 - 34. Avidity is related both to the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also to the valences of immunoglobulins and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be highly avid.
[0058] [0058] Anti-SEMA4D antibodies or antigen-binding fragments, variants, or derivatives of the invention can also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody, specific for an antigen, to react with a second antigen; a measure of affinity between two different antigenic substances. Thus, a cross-reactive antibody binds to an epitope except one that induces its formation. The cross-reactive epitope generally contains any of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.
[0059] [0059] For example, certain antibodies have some degree of cross-reactivity, in which, when related, they bind, but are not identical, for example, epitopes with at least 95% identity, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% (as calculated using methods known in the art and described herein) a a reference epitope. An antibody can be considered to have little or no cross-reactivity if it does not bind to epitopes with identity less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70% , less than 65%, less than 60%, less than 55%, and less than 50% (as calculated using methods known in the art and described herein) to a reference epitope. An antibody can be considered "highly specific" for a certain epitope, if it does not bind to any other analog, orthologist, or homologue to that epitope.
[0060] [0060] Anti-SEMA4D binding molecules, for example, antibodies or antigen-binding fragments, variants or derivatives thereof, of the invention can also be described or specified in terms of their binding affinity to a polypeptide of the invention, for example , SEMA4D, for example, human, murine, or both human and murine SEMA4D. Preferred binding affinities include those with a dissociation constant or Kd less than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M, 10-4 M, 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 10-6 M, 5 x 10-7 M, 10-7 M, 5 x 10-8 M, 10-8 M, 5 x 10-9 M, 10-9 M, 5 x 10-10 M, 10-10 M, 5 x 10-11 M, 10-11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M. In certain embodiments, the anti-SEMA4D binding molecule, for example For example, an antibody or antigen-binding fragment thereof, of the invention binds to human SEMA4D with a Kd of about 5 x 10-9 to about 6 x 10-9. In another embodiment, the anti-SEMA4D binding molecule, for example, an antibody or binding fragment with the antigen thereof, of the invention binds to murine SEMA4D with a Kd of about 1 x 10-9 a about 2 x 10-9.
[0061] [0061] As used herein, the term "chimeric antibody" will be retained to mean any antibody, in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified according to the present invention) is obtained from a second species. In preferred embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.
[0062] [0062] As used herein, the term "engineered antibody" refers to an antibody, where the variable domain in the heavy or light chain or both is altered by at least a partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, partial replacement of the region of the structure and change of the sequence. Although CDRs can be derived from an antibody of the same class or subclass as the antibody from which the framework regions are derived, it is considered that CDRs will be derived from an antibody of different class and, preferably, of an antibody from a different species. An engineered antibody, in which one or more "donor" CDRs from a non-human antibody of known specificity is inserted into a region of human heavy or light chain structure is referred to herein as a "humanized antibody". It may not be necessary to replace all CDRs with complete CDRs from the donor variable domain to transfer the antigen-binding capacity from one variable domain to another. Once again, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site.
[0063] [0063] It is also recognized that the structure regions within the variable domain in a heavy or light chain, or both, of a humanized antibody can only comprise residues of human origin, in which case these structure regions of the humanized antibody are referred to as “completely human structure regions” (for example, MAb VX15 / 2503, disclosed in U.S. Patent Application Publication № US 2010/0285036 A1 as MAb 2503, incorporated herein by reference in its entirety). Alternatively, one or more residues of the donor variable domain structure region (s) can be engineered within the corresponding position of the human domain region (s) of a variable domain in a heavy chain or take, or both, of a humanized antibody if necessary to maintain proper binding or enhance the binding to the SEMA4D antigen. A region of human structure that has been engineered in this way would comprise a mixture of human and donor structure waste, and is referred to here as a "region of partially human structure".
[0064] [0064] For example, the humanization of an anti-SEMA4D antibody can essentially be performed according to the method of Winter and co-workers (Jones et al., Nature 321: 522 - 525 (1986); Riechmann et al., Nature 332 : 323 - 327 (1988); Verhoeyen et al., Science 239: 1534 - 1536 (1988)), replacing the rodent or mutant rodent CDRs or CDR sequences with the corresponding sequences of a human anti-SEMA4D antibody. See, also Pat. U.S. No. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; incorporated herein by reference. The resulting humanized antiSEMA4D antibody would comprise at least one rodent or mutant rodent CDR within the fully human framework regions of the heavy and / or light chain variable domain of the humanized antibody. In some cases, residues within the framework regions of one or more variable domains of the humanized anti-SEMA4D antibody are replaced by corresponding non-human residues (eg, rodent) (see, for example, U.S. Pat. No. 5,585,089 ; 5,693,761; 5,693,762; and 6,180,370), in which case the resulting humanized anti-SEMA4D antibody would comprise partially human framework regions within the variable domain of the heavy and / or light chain.
[0065] [0065] In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the performance of the antibody (for example, to obtain the desired affinity). In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody, optionally, will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see, Jones et al., Nature 331: 522 - 525 (1986); Riechmann et al., Nature 332: 323 - 329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593 - 596 (1992); incorporated herein by reference. Consequently, such "humanized" antibodies can include antibodies, in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies, in which some CDR residues and possibly some structure residues are replaced by residues from sites analogous to rodent antibodies. See, for example, Pat. U.S. No. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See, also Pat. U.S. No. 6,180,370, and International Publication No. WO 01/27160, where humanized antibodies and techniques for producing humanized antibodies having improved affinity for a predetermined antigen are disclosed. II. Blood-brain barrier (“BBB”)
[0066] [0066] The blood-brain barrier (BBB) is an active interface between circulating blood and the central nervous system (CNS). BBB restricts the free circulation of different substances between the two areas and plays a crucial role in maintaining CNS homeostasis. The BBB has both a barrier function and a carrier function. As a barrier, BBB restricts the circulation of cells and potentially toxic or harmful substances from the blood to the brain. As a carrier, on the other hand, BBB is responsible for transporting nutrients to the brain and removing metabolites.
[0067] [0067] The BBB is mainly composed of three components: endothelial cells, astrocytes and pericytes. Endothelial cells form a continuous layer that covers the inner surface of capillaries and blood vessels in the brain. (Ransohoff et al., “Three or More Routes for Leukocyte Migration Into the Central Nervous System”, Nature Rev. Immun. 3: 569 - 581 (2003). Endothelial cells are located adjacent to the basement membrane, which consists mainly of collagen IV, fibronectin, laminin and proteoglycans, and are interconnected by tight junctions that form a belt-like structure in the apical region of cells.The endothelial cells restrict the diffusion of microscopic objects (eg, bacteria) and large or hydrophilic molecules in the brain parenchyma and cerebrospinal fluid (CSF), while allowing the diffusion of small hydrophobic molecules (O2, hormones, CO2) .The barrier cells actively transport metabolic products, such as glucose through the barrier with specific proteins.
[0068] [0068] The endothelial cells that form the capillaries of the brain are different from those found in other tissues of the body. Endothelial cells in brain capillaries are assembled by tight intercellular junctions that form a continuous wall against passive diffusion of molecules from blood to the brain and other parts of the CNS (including cerebrospinal fluid, CSF). These cells are also different in that they have some pinocytic vesicles which in other tissues allow for a little selective transport through the capillary wall. Also deficient, are gaps or continuous channels that operate between cells that would allow unrestricted passage.
[0069] [0069] In addition to endothelial cells, BBB is also composed of pericytes and astrocytes. Pericytes are located within the basement membrane, interact with endothelial cells and play an important role in regulating endothelial proliferation, angiogenesis and inflammatory processes. Astrocytes are characteristic star-shaped glial cells in the brain and spinal cord and are the most abundant cells in the human brain. They perform many functions, including biochemical support of endothelial cells that form the blood-brain barrier, supplying nutrients to the tissue of the nervous system, maintaining balance of extracellular ions, and a role in the repair and healing process of the brain and spinal cord after traumatic injuries.
[0070] [0070] The blood-brain barrier functions to ensure that the brain environment is constantly controlled. Levels of various substances in the blood, such as hormones, amino acids, and ions, are subject to frequent small fluctuations that can be triggered by activities, such as eating and exercising (Goldstein et al., “The Blood-Brain Barrier”, Scientific American 255: 74 - 83 (1986); Pardridge, "Receptor-Mediated Peptide Transport Through the Blood-Brain Barrier", Endocrin. Rev. 7: 314 - 330 (1986)). If the brain has not been protected by the blood-brain barrier from these variations in serum composition, the result may be uncontrolled neural activity.
[0071] [0071] The isolation of the brain from the bloodstream is not complete. If this were the case, the brain would be unable to function properly due to a lack of nutrients and because of the need to exchange chemicals with the rest of the body. The presence of specific transport systems within capillary endothelial cells ensures that the brain receives, in a controlled manner, all of the compounds necessary for normal growth and functioning. In many cases, these transport systems consist of membrane-associated proteins, which selectively bind to and transport certain molecules across barrier membranes. These carrier proteins are known as solute transporters.
[0072] [0072] Although the BBB serves to protect the brain and central nervous system from damage from molecules and external cells, the molecules and external cells can often pass through the BBB and, in limited numbers, may even be beneficial, such as for immunological surveillance of the CNS. However, when highly active cells, such as, for example, B cells, T cells, leukocytes and macrophages, cross the BBB in excess and reach the brain, they can cause damage to the brain. Patients suffering from edema, brain trauma, stroke and multiple sclerosis, for example, presenting a BBB rupture.
[0073] [0073] The effect of BBB on various neuroinflammatory disorders has been studied. (Zlokovic BV, “The Blood-Brain in Health and Chronic Neurodegenerative Disorders”, Neuron 57: 178 - 201 (2008); Zhong Z et al., “ALS-causing SOD1 mutants generate vascular changes prior to motor neuron degeneration” Nature Neuroscience 11 (4): 420 - 422 (2008); Hawkins BT et al., “The Blood-Brain Barrier / neurovascular Unit in Health and Disease”, Pharmacological Rev 57 (2): 173 - 185 (2005); Oby E et al., "The BloodBrain Barrier and Epilepsy", Epilepsy 47 (11); 1761 - 1774 (2006)). In addition, there is growing evidence that inflammation and the blood-brain barrier (BBB) (Banks and Erickson, 2010; Lochhead et al., 2010) are involved in the pathogenesis of neurological diseases, such as meningitis (van der et al., 2004) , cerebral edema (Stamatovic et al., 2006), Alzheimer's disease (Kalaria, 1992), Parkinson's disease (Westin, JE, et. al., “Endothelial Proliferation and Increased Blood-Brain Barrier Permeability in the Basal Ganglia in a Rat Model of 3,4-Dihydroxyphenyl-L-Alanine-Induced Dyskinesia ”, The Journal of Neuroscience 26 (37): 9448 - 9461 (2006)) and multiple sclerosis (Minagar and Alexander, 2003).
[0074] [0074] In the case of multiple sclerosis, for example, it was shown using Magnetic Resonance Imaging ("MRI"), that when a person is experiencing an "attack" of MS, the BBB was broken into a part of the brain or spinal cord spinal cord, allowing T lymphocytes to pass through and attack the myelin that protects and insoles neurons in the central nervous system in both the brain and spinal cord. (Zlokovic 2008; Waubant E., “Biomarkers indicative of blood – brain barrier disruption in multiple sclerosis.” Disease Markers 22 (4): 235-44 (2006)).
[0075] [0075] Meningitis, on the other hand, occurs when there is an inflammation of the membranes that surround the brain and spinal cord (these membranes are known as meninges). When meninges are inflamed, the blood-brain barrier can be broken, allowing both inflammatory cells and various substances (including toxins or antibiotics) to enter the brain. (Beam, TR Jr., et al. (December 1977). “Blood, brain, and cerebrospinal fluid concentrations of several antibiotics in rabbits with intact and inflamed meninges.” Antimicrobial Agents and Chemotherapy 12 (6): 710-6) .
[0076] [0076] Similarly, in the case of Parkinson's disease (PD), it has been suggested that the absorption or metabolism of putative PD toxins, and their defective elimination throughout the BBB, due to the low activity of the transporter P-glycoprtein (P-gp ), an ATP-dependent efflux pump that mediates the rapid removal of ingested toxic lipophilic metabolites, may play a role in the pathogenesis of PD (Kortekaas, R., Leenders, KL, van Oostrom, JC, Vaalburg, W., Bart, J., Willemsen, AT, and Hendrikse, NH Blood-brain barrier dysfunction in parkinsonian midbrain in vivo. Ann. Neurol. 57, 176 - 179, 2005). Neuroinflammation also appears to be a ubiquitous discovery in patients with PD and experimental models of PD. Phagocyte activation, increased synthesis and release of pro-inflammatory cytokines, complement activation, activation of microglia, and release of reactive oxygen species (ROS) have been described (Whitton, PS Inflammation as a causative factor in the aetiology of Parkinson's disease Br. J. Pharmacol. 150, 963-976, 2007).
[0077] [0077] In epilepsy, studies have implicated the failure of the blood-brain barrier function to trigger chronic or acute seizures due to certain interactions between a protein, albumin and common blood astrocytes. These results suggest that acute seizures are a result of BBB disruption by artificial or inflammatory mechanisms. (Oby, E; et al. (2006). “The Blood – Brain Barrier and Epilepsy” (PDF). Epilepsy 47 (11): 1761 - 1774).
[0078] [0078] In patients with Alzheimer's Disease (AD), the evidence points to the disruption of the blood-brain barrier in allowing blood plasma containing beta-amyloid (Aβ) to enter the brain via RAGE, a major influx transporter for Aβ across BBB. Studies have shown that the interaction of Aβ / RAGE results in transcitosis of circulating Aβ throughout the BBB in the brain parenchyma and its connection to neurons, endothelial activation mediated by NF-kB resulting in the secretion of pro-inflammatory cytokines, the expression of molecules adhesion, and the generation of endothelin-1, which suppresses CBF (Cerebral Blood Flow). In addition, it has been shown that the Aβ / RAGE interaction contributes to neuronal death by producing oxidative damage to neurons that express RAGE and activating microglia. (Zlokovic, B.V. The Blood-Brain Barrier in Health and Chronic Neurodegenerative Disorders. Neuron 57, 178 - 201, 2008). Deficient Aβ efflux outside the brain parenchyma and into the microvasculature through the BBB has also been found in the adjustment of AD pathogenesis and has been attributed, in part, to impaired low density lipoprotein receptor (LRP1) protein 1 function. LRP1 is an abluminal BBB membrane protein that binds and carries structural combinators other than Aβ (Deane et al., “LRP / amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms.” Neuron 43, 333 - 344, 2004 ). Exposure of Aβ changes the expression patterns of the tight junction protein cell surface, including claudin-5 and ZO-2, in brain microvascular endothelial cells to the cytoplasm (Marco et al., “Amyloid β-peptide 1-42 alters tight junction protein distribution and expression in brain microvessel endothelial cells ”. Neurosci. Lett. 401, 219 - 224, 2006), and severely compromises the transendothelial electrical resistance (TEER) of monolayers of these cells (Gonzalez-Velasquez et al.,“ Soluble aggregates of the amyloid-beta protein selectively stimulate permeability in human brain microvascular endothelial monolayers. ”J. Neurochem. 107, 466 - 477, 2008).
[0079] [0079] In amyotrophic lateral sclerosis (ALS), studies have suggested that rupture of the BBB can result in the escape of serum proteins that interact with motor neurons to produce ROS (Reactive Oxygen Species) and initiate an autoimmune response, causing demyelination, disruption of neuronal transmission, and cell death. (Zlokovic 2008).
[0080] [0080] A recent study suggests that the weakening of the BBB may result from a disturbance in endothelial cells mediated through its VEGFA receptor. (Argaw AT et al., “VEGF-mediated disruption of endothelial CLN-5 promotes bloodbrain barrier breakdown”, PNAS 106 (6): 1977 - 1982 (2009)). According to this study, VEGF-A, which is derived from astrocytes, targets and disrupts the expression of both claudin-5 (CLN-5) and ocludine (OCLN) of endothelial transmembrane tight junction protein. As an expression of both CLN-5 and OCLN decreases, the BBB rupture increases.
[0081] [0081] As shown in the present examples, another possible mechanism for the weakening of the BBB is as a result of disturbance of endothelial cells through the high affinity Plexin-B1 receptor (1 nM) for SEMA4D. Plexin-B1 can be expressed by endothelial cells. In the presence of SEMA4-D, endothelial cells can undergo a transformation that alters the morphology or function of endothelial cells, as well as causing a weakening of the BBB, for example, by modifying tight junctions. This weakening of the BBB can, in turn, increase the permeability of the BBB to cells and molecules and allow such cells and molecules to enter and alter the activity of the brain and central nervous system. The addition of anti-SEMA4D or anti-Plexin-B1, therefore, can prevent endothelial cells from undergoing a transformation and reduce the weakening of the BBB. III. Description of Target Polypeptide
[0082] [0082] As used herein, the terms "semaphorin-4D," "SEMA4D" and "SEMA4D polypeptide" are used interchangeably, as are "SEMA4D" and "Sema4D." In certain embodiments, SEMA4D is expressed on the surface of or secreted by a cell. In another embodiment, SEMA4D is attached to the membrane. In other embodiments, SEMA4D is soluble, for example, sSEMA4D. In other embodiments, SEMA4D may include a full-length SEMA4D or fragment thereof, or a SEMA4D variant polypeptide, wherein the SEMA4D fragment or SEMA4D variant polypeptide retains some or all of the functional properties of SEMA4D from full size.
[0083] [0083] The full-length human SEMA4D protein is a homodimeric transmembrane protein consisting of two 150 kDa polypeptide chains. SEMA4D belongs to the cell surface receptor semaphorin family and is also referred to as CD100. SEMA4D / Sema4D both human and mouse are proteolytically cleaved from their transmembrane form to generate soluble forms of 120-kDa, indicating the existence of two isoforms of Sema4D (Kumanogoh et al., J. Cell Science 116 (7): 3464 (2003 )). Semaphorins include soluble and membrane-bound proteins that were originally defined as axonal orientation factors during development that play an important role in establishing precise connections between neurons and their appropriate target. Structurally considered, a full-size class IV, SEMA4D semaphorin includes an amino-terminal signal sequence followed by a characteristic 'Sema' domain, which contains 17 conserved cysteine residues, an Ig-type domain, a lysine-rich stretch, a hydrophobic transmembrane region, and a cytoplasmic tail.
[0084] [0084] Each SEMA4D polypeptide chain includes a signal sequence of about 13 amino acids followed by a semaphorin domain of about 512 amino acids, an immunoglobulin-like domain (Ig type) of about 65 amino acids, a rich stretch in 104 amino acid lysine, 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 a tyrosine kinase (Schlossman, et al., Eds. (1995) Leucocyte Typing V (Oxford University Press, Oxford)).
[0085] [0085] SEMA4D is known to have at least two receivers. One of the receptors, Plexin-B1, is expressed in non-lymphoid tissues and has been shown to be a high affinity (1 nM) receptor for SEMA4D (Tamagnone et al., Cell 99: 71 - 80 (1999)). SEMA4D stimulation of Plexin-B1 signaling has been shown to induce collapse of the neuron growth cone, and induce collapse of oligodendrocyte process extension and apoptosis (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, leading to a decrease in integrin-mediated binding to the extracellular matrix, as well as RhoA activation, leading to cytoskeleton reorganization and cell migration. See, Kruger et al., Nature Rev. Mol. Cell Biol. 6: 789 - 800 (2005); Pasterkamp, TRENDS in Cell Biology 15: 61-64 (2005)).
[0086] [0086] In lymphoid tissues, CD72 is used as a SEMA4D receptor (Kumanogoh et al., Immunity 13: 621 - 631 (2000) with low affinity (300 nM)). B cells and APCs express CD72, and anti-CD72 antibodies have many of the same effects as sSEMA4D, such as increased CD40-induced B cell responses and CD23 B cell shedding. CD72 is considered to act as a negative regulator of B cell responses by recruiting 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 in vitro survival. The addition of cells expressing SEMA4D or sSEMA4D increases 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 increases CD40-induced maturation of DCs, including supraregulation of costimulatory molecules and increased IL-12 secretion. In addition, sSEMA4D can inhibit the migration of immune cells, which can be reversed by the addition of anti-SEMA4D blocking antibodies (Elhabazi et al., J. Immunol. 166: 4341 - 4347 (2001); Delaire et al., J. Immunol 166: 4348 - 4354 (2001)).
[0087] [0087] Sema4D is expressed at high levels in lymphoid organs, including the spleen, thymus and lymph nodes, and in non-lymphoid organs, such as the brain, heart and kidney. In lymphoid organs, Sema4D is abundantly expressed in remaining T cells, but only weakly expressed in remaining B cells and antigen presenting cells (APCs), such as dendritic cells (DCs). Cellular activation increases the expression of the SEMA4D surface, as well as the generation of soluble SEMA4D (sSEMA4D).
[0088] [0088] The SEMA4D expression pattern suggests that it plays an important physiological role, as well as a pathological role in the immune system. SEMA4D has been shown to promote B cell activation, aggregation and survival; enhancing CD40-induced proliferation and antibody production; enhancing the antibody response to T cell-dependent antigens; increase the proliferation of T cells; enhance the 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)).
[0089] [0089] SEMA4D of knockout mice (SEMA4D - / -) provided additional evidence that SEMA4D plays an important role in both humoral and cellular immune responses. There are no known major abnormalities of non-lymphoid tissues in SEMA4D - / - of mice. The SEMA4D - / - dendritic cells (DCs) of mice have little allo-stimulatory capacity and show defects in the expression of co-stimulatory molecules, which can be rescued by the addition of sSEMA4D. SEMA4D-deficient (SEMA4D - / -) mice are unable to develop experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide, because myelin oligodendrocyte glycoprotein specific T cells are poorly generated in the absence of SEMA4D (Kumanogo. , J Immunol 169: 1175 - 1181 (2002)). A significant amount of soluble SEMA4D is also detected in the serum of MRL / lpr mice prone to autoimmunity (model of systemic autoimmune diseases, such as SLE), but not in normal mice. In addition, sSEMA4D levels correlate with autoantibody levels and increase with age (Wang et al., Blood 97: 3498 - 3504 (2001)). Soluble SEMA4D has also been shown to accumulate in the cerebrospinal fluid and serum of patients with demyelinating disease, and sSEMA4D induces apoptosis of human pluripotent neural precursors (Dev cells), and both inhibits process extension and induces rat oligodendrocyte apoptosis in vitro ( Giraudon et al., J Immunol 172 (2): 1246 - 1255 (2004)). This apoptosis was blocked by an anti-SEMA4D Mab. IV. Anti-SEMA4D antibodies
[0090] [0090] Antibodies that bind to SEMA4D have been described in the art. See, for example, Publ. US №s 2008/0219971 A1, US 2010/0285036 A1, and US 2006/0233793 A1, International Patent Applications WO 93/14125, WO 2008/100995, and WO 2010/129917, and Herold et al., Int. Immunol . 7 (1): 1 - 8 (1995), each of which is incorporated herein in its entirety as a reference.
[0091] [0091] The present application generally relates to a method of decreasing the permeability of the blood-brain barrier in a subject, for example, a human patient, having a neuro-inflammatory disorder, for example, an inflammatory disorder or neurodegenerative disorder of the CNS, comprising the administration of an antibody that specifically binds to SEMA4D, or a fragment of binding to the antigen, variant, or derivative thereof. In certain embodiments, the antibody blocks the interaction of SEMA4D with one or more of its receptors, for example, Plexin-B1. Anti-SEMA4D antibodies having these properties can be used in the methods provided herein. Antibodies that can be used include, but are not limited to, MAbs VX15 / 2503, 67, and 76 and antigen-binding fragments, variants, or derivatives thereof, which are fully described in US 2010/0285036 A1. Additional antibodies that can be used in the methods provided herein include antibodies BD16 and BB18 described in US 2006/0233793 A1, as well as antigen-binding fragments, variants, or derivatives thereof; or any of MAb 301, MAb 1893, MAb 657, MAb 1807, MAb 1656, MAb 1808, Mab 59, MAb 2191, MAb 2274, MAb 2275, MAb 2276, MAb 2277, MAb 2278, MAb 2279, MAb 2280, MAb 2281, MAb 2282, MAb 2283, MAb 2284, and MAb 2285, as well as any fragments, variants or derivatives thereof, as described in US 2008/0219971 A1. In certain embodiments, an anti-SEMA4D antibody for use in the methods provided herein binds to human, murine, or both human and murine SEMA4D. Also useful are antibodies that bind to the same epitope as any of the aforementioned antibodies and / or antibodies that competitively inhibit any of the aforementioned antibodies from binding to SEMA4D.
[0092] [0092] In certain embodiments, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided here has an amino acid sequence that is at least about 80%, about 85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% sequence identity to the amino acid sequence for a reference anti-SEMA4D antibody molecule, for example, that described above. In another embodiment, the binding molecule participates at least about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to a reference antibody.
[0093] [0093] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin heavy chain variable domain ( VH domain), where at least one of the VH domain CDRs has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97 %, about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 9 or 10.
[0094] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin heavy chain variable domain ( VH domain), where at least one of the VH domain CDRs has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97 %, about 98%, about 99%, or identical to SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
[0095] [0095] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin heavy chain variable domain ( VH domain), where at least one of the VH domain CDRs has an identical amino acid sequence, except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
[0096] [0096] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of a VH domain that has an amino acid sequence which is at least 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 100% identical to SEQ ID NO: 9 or SEQ ID NO: 10, wherein the anti-SEMA4D antibody comprising the specifically encoded VH domain, preferably, or competitively if turn on SEMA4D.
[0097] [0097] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin light chain variable domain (domain VL), where at least one of the CDRs in the VL domain has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97% , about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 17 or 18.
[0098] [0098] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin light chain variable domain (domain VL), where at least one of the CDRs in the VL domain has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97% , about 98%, about 99%, or identical to SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
[0099] [0099] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin light chain variable domain (domain VL), where at least one of the VR domain CDRs has an identical amino acid sequence, except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
[0100] [00100] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of a VL domain that has an amino acid sequence which is at least 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 100% identical to SEQ ID NO: 17 or SEQ ID NO: 18, wherein the anti-SEMA4D antibody comprising the specifically encoded VL domain, preferably, or competitively if turn on SEMA4D.
[0101] [00101] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin heavy chain variable domain ( VH domain) and an immunoglobulin light chain variable domain (VL domain), where at least one of the CDRs in the VH domain has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 9 or 10 and at least one of the CDRs in the VL domain has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 17 or 18.
[0102] [00102] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin heavy chain variable domain ( VH domain) and an immunoglobulin light chain variable domain (VL domain), where at least one of the VH domain CDRs has an identical amino acid sequence, except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, the SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and where at least one of the VR domain CDRs has an identical amino acid sequence, except for 1, 2, 3, 4, or 5 amino acid substitutions conservative, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
[0103] [00103] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative useful in the methods provided herein comprises, consists essentially of, or consists of a VH domain that has an amino acid sequence which is at least 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 100% identical to SEQ ID NO: 9 or SEQ ID NO: 10, and a VL domain that has an amino acid sequence that is at least 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 100% identical to SEQ ID NO: 17 or SEQ ID NO: 18, wherein the anti-SEMA4D antibody comprising the specifically encoded VH and VL domains, preferably, or competitively binds to SEMA4D.
[0104] [00104] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative of this useful in the methods provided here comprises, consists essentially of, or consists of the three CDRs of the VL domain and three CDRs of the VH domain of MAb VX15 / 2503, 67, or 76, which are fully described in US 2010/0285036 A1. In some embodiments, the anti-SEMA4D antibody useful in the methods provided here comprises MAb VX15 / 2503 or 67.
[0105] [00105] Also included for use in the methods provided here are polypeptides encoding anti-SEMA4D antibodies, or antigen binding fragments, variants, or derivatives thereof as described herein, polynucleotides encoding such polypeptides, vectors comprising such polynucleotides, and cells hosts comprising such vectors or polynucleotides, all to produce anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof for use in the methods described herein.
[0106] [00106] Suitable biologically active variants of the antiSEMA4D antibodies of the invention can be used in the methods of the present invention. Such variants will retain the desired binding properties of the precursor anti-SEMA4D antibody. Methods for making antibody variants are generally available in the art.
[0107] [00107] Methods for mutagenesis and alterations of nucleotide sequences 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.); Pat. U.S. No. 4,873,192; and the references cited therein; incorporated herein by reference. Guidance as to appropriate amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found in the model by Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), pages 345 to 352, hereby incorporated by reference in their entirety. The model by Dayhoff et al. uses the Point Accepted Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conservative amino acid substitutions. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred. Examples of conservative amino acid substitutions as shown by the PAM 250 matrix of the model by Dayhoff et al. include, but are not limited to, Gly↔Ala, Val↔Ile↔Leu, Asp↔Glu, Lys↔Arg, Asn↔Gln, and Phe↔Trp↔Tyr.
[0108] [00108] In the construction of variants of the anti-SEMA4D binding molecule, for example, an antibody or antigen-binding fragment thereof, polypeptides of interest, modifications are made such that variants continue to have the desired properties, for example, being capable of specifically binding to a SEMA4D, for example, human, murine, or both human and murine SEMA4D, for example, expressed on the surface of or secreted by a cell and having SEMA4D blocking activity, as described herein. Obviously, any mutations made to the DNA encoding the variant polypeptide should not place the sequence outside the reading frame and preferably will not create complementary regions that can produce secondary mRNA structure. See EP Patent Application Publication № 75,444.
[0109] [00109] Methods for measuring the anti-SEMA4D binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof, binding specificity includes, but is not limited to, standard competitive binding assays, assays to monitor immunoglobulin secretion by T cells or B cells, T cell proliferation assays, apoptosis assays, ELISA assays, and the like. See, for example, such assays disclosed 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 (2): 1246 - 1255 (2004), all of which are incorporated herein by reference.
[0110] [00110] When debated here whether any particular polypeptide, including the constant regions, CDRs, VH domains, or VL domains 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 about 100% identical to another polypeptide, the% identity can be determined using methods and computer programs / software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482 - 489, to find the best homology segment between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, certainly, such that the percentage of identity is calculated on the natural size of the reference polypeptide sequence and which ranges in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
[0111] [00111] For the purposes of the present invention, the percentage of sequence identity can be determined using the Smith-Waterman homology search algorithm using a related range search with an opening penalty for gaps of 12 and an extension penalty for gaps of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is shown in Smith and Waterman (1981) Adv. Appl. Math. 2: 482-489. A variant may, for example, differ from a reference anti-SEMA4D antibody (for example, MAb VX15 / 2503, 67 or 76) by only 1 to 15 amino acid residues, only 1 to 10 amino acid residues, such as 6 to 10, only 5, only 4, 3, 2, or even 1 amino acid residue.
[0112] [00112] The constant region of an anti-SEMA4D antibody can be mutated to alter the effector function in several ways. For example, see Pat. No. 6,737,056B1 and U.S. Patent Application Publication No. 2004 / 0132101A1, which disclose Fc mutations that optimize antibody binding to Fc receptors.
[0113] [00113] In certain anti-SEMA4D antibodies or fragments, variants or derivatives thereof useful in the methods provided here, the Fc portion can be mutated to decrease effector function using techniques known in the art. For example, suppression or inactivation (via point mutations or other means) of a constant region domain can reduce the binding of the circulating modified antibody Fc receptor thereby increasing the tumor location. In other cases, modifications of the constant region compatible with the present invention moderate the complement binding and thus reduce the serum half-life. Still other modifications of the constant region can be used to modify disulfide bonds or portions of oligosaccharides that take into account the enhanced location due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor location, biodistribution and serum half-life, can easily be measured and quantified using well-known immunological techniques without undue experimentation. Anti-SEMA4D antibodies for use in the methods provided here include derivatives that are modified, for example, by covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivation by known protecting / blocking groups, proteolytic cleavage, binding to a ligand cell or other protein, etc. Any of the numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. In addition, the derivative may contain one or more non-classical amino acids.
[0114] [00114] A "conservative amino acid substitution" is one in which the 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 (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and side chains aromatic (eg tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly over all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that maintain the activity (for example, the ability to bind an anti-SEMA4D polypeptide, to block the interaction of SEMA4D with its receptor, or to decrease BBB permeability in a subject, for example, a patient with a neuroinflammatory disorder).
[0115] [00115] For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations can be silent or neutral sense-shifting mutations, that is, they have none, or have little, effect on an antibody's ability to bind to the antigen. These types of mutations can be useful to optimize the use of the codon, or to improve the antibody production of a hybridoma. Alternatively, non-neutral sense-shifting mutations can alter an antibody's ability to bind to the antigen. A person of skill in the art would be able to design and test mutant molecules with desired properties such as no change in antigen binding activity or change in binding activity (for example, improvements in antigen binding activity or change in antibody specificity ). Following mutagenesis, the encoded protein can be routinely expressed and the functional and / or biological activity of the encoded protein, (for example, ability to immunospecifically bind at least one epitope to a SEMA4D polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.
[0116] [00116] In certain embodiments, anti-SEMA4D antibodies for use in the methods provided here comprise at least one optimized complementarity determining region (CDR). By "optimized CDR" it is intended that the CDR be modified and optimized to improve the binding affinity and / or anti-SEMA4D activity that is communicated to an anti-SEMA4D antibody comprising the optimized CDR. "Anti-SEMA4D activity" or "SEMA4D blocking activity" may include activity that modulates one or more of the following activities associated with SEMA4D: B cell activation, aggregation and survival; CD40-induced proliferation and antibody production; antibody response to T cell dependent antigens; proliferation of T cells or other immune cells; maturation of dendritic cell; demyelination and axonal degeneration; apoptosis of pluripotent neural precursors and / or oligodendrocytes; induction of endothelial cell migration; inhibition of spontaneous monocyte migration; binding to Plexin-B1 from the cell surface or other receptor, or any other activity associated with soluble SEMA4D or SEMA4D that is expressed on the surface of SEMA4D + cells. Anti-SEMA4D activity can also be attributed to a decrease in the incidence or severity of diseases associated with SEMA4D expression or overexpression, including, but not necessarily limited to, neuroinflammatory diseases including inflammatory diseases of the central nervous system (CNS) and nervous system peripheral (PNS).
[0117] [00117] Examples of antibodies optimized based on murine anti-SEMA4D MAbs BD16 and BB18 have been described in Publ. US № 2008/0219971 A1, International Patent Application WO 93/14125 and Herold et al., Int. Immunol. 7 (1): 1 - 8 (1995), each of which is incorporated herein as a reference in its entirety. The modifications may involve the replacement of amino acid residues within the CDR such that an anti-SEMA4D antibody maintains specificity for the SEMA4D antigen and has improved binding affinity and / or improved anti-SEMA4D activity. V. Treatment Methods Using Anti-SEMA4D and Anti-Plexin B1 Therapeutic Antibodies
[0118] [00118] Methods of the invention are directed to the use of an inhibitor of SEMA4D interaction with a SEMA4D receptor, for example, antiSEMA4D binding molecules, anti-Plexin B1 binding molecules, or a combination of these, for example, antibodies, including fragments of binding to the antigen, variants, and derivatives thereof, to decrease the permeability of the blood-brain barrier in a subject having a neuroinflammatory disorder. In certain embodiments, the neuroinflammatory disorder is, for example, Multiple sclerosis, Amyotrophic lateral sclerosis, epilepsy, Alzheimer's disease, Parkinson's disease, meningitis, cerebral edema, brain trauma, or stroke. In certain embodiments, endothelial cells express a SEMA4D receptor; and in certain embodiments, the receptor is Plexin-B1. Although the following debate concerns the administration of an anti-SEMA4D antibody, an anti-Plexin B1 antibody, and a combination thereof, the methods described here are also applicable to the antigen-binding fragments, variants, and derivatives of these anti-SEMA4D antibodies or anti-Plexin B1 which maintain the desired properties of the anti-SEMA4D or anti-Plexin B1 antibodies of the invention, for example, capable of specifically binding SEMA4D, for example, human, mouse, or human and mouse SEMA4D, having neutralizing activity of SEMA4D, and / or blocking the interaction of SEMA-4D with its receptor, for example, Plexin-B1.
[0119] [00119] In one embodiment, treatment includes the application or administration of an anti-SEMA4D binding molecule, an antiPlexin B1 binding molecule, or combination thereof, for example, an antibody or binding fragment with the antigen thereof as described here to a patient, where the patient has, or is at risk of developing a neuroinflammatory disorder. In another embodiment, treatment is also intended to include the application or administration of a pharmaceutical composition comprising the anti-SEMA4D binding molecule, the anti-Plexin B1 binding molecule, or a combination thereof, for example, an antibody or fragment of binding with the antigen of the same to a patient, where the patient has, or is at risk of developing a neuroinflammatory disorder. It should be assessed that due to the interaction of SEMA4D with a receptor on endothelial cells, it is expected that the application or administration of an anti-SEMA4D binding molecule, an anti-Plexin B1 binding molecule, or a combination of these will occur on the blood side of the blood-brain barrier. By administering an anti-SEMA4D binding molecule, an anti-Plexin B1 binding molecule, or combinations thereof via a route that exposes it to the blood side, for example, including, but not limited to, intravenous administration, the binding molecule anti-SEMA4D, anti-Plexin B1 binding molecules, or combinations thereof will be allowed to inhibit the interaction of SEMA4D with the SEMA4D receptor that is expressed by endothelial cells.
[0120] [00120] Anti-SEMA4D binding molecules, anti-Plexin B1 binding molecules, or a combination thereof, for example, antibodies or binding fragments thereof as described herein are useful for the treatment of various neuroinflammatory disorders. In some embodiments, the treatment of a neuroinflammatory disorder is intended to include a reduction, or decrease, in the permeability of the BBB. In other embodiments, the treatment of a neuroinflammatory disorder is intended to include an increase in the resistivity of the BBB. In other embodiments, the treatment of a neuroinflammatory disorder is intended to include an increase in the number, density and / or concentration of endothelial cells present in the BBB. In other embodiments, the treatment of a neuroinflammatory disorder is intended to include a change in morphology or function or endothelial cells, or in the interactions between endothelial cells or astrocytes or between endothelial cells and astrocytes that form the BBB.
[0121] [00121] In one embodiment, the invention relates to the use of anti-SEMA4D binding molecules, anti-Plexin B1 binding molecules, or a combination thereof, for example, antibodies or antigen binding fragments, variants, or derivatives thereof, as a medicine, in particular for use in the treatment or prophylaxis of neuroinflammatory disorders to inhibit, reduce, prevent, or minimize a rupture in the BBB, or an increase in the permeability of the BBB.
[0122] [00122] According to the methods of the present invention, at least one anti-SEMA4D binding molecule or anti-Plexin B1 binding molecule, for example, an antibody or binding fragment with the antigen, variant, or derivative thereof, as defined anywhere here can be used to promote a positive therapeutic response with respect to neuroinflammatory disorder. A "positive therapeutic response" with respect to neuroinflammatory disorder is intended to include an improvement in the disease in association with the anti-inflammatory activity, anti-apoptotic activity, or the like, of these antibodies, and / or an improvement in the symptoms associated with the disease. . That is, an antiproliferative effect, the prevention of further cell proliferation that expresses SEMA4D, a reduction in the inflammatory response, including, but not limited to reduced secretion of inflammatory cytokines, adhesion molecules, proteases, immunoglobulins (in examples where the carrier cell SEMA4D is a B cell), combinations of these, and the like, increased production of anti-inflammatory proteins, a reduction in the number of autoreactive cells, an increase in immune tolerance, inhibition of autoreactive cell survival, reduction in apoptosis, reduction in migration of endothelial cell, increase in spontaneous monocyte migration, reduction in and / or a decrease in one or more symptoms mediated by the stimulation of sSEMA4D or cells that express SEMA4D can be observed. Such positive therapeutic responses are not limited to the route of administration and may comprise administration to the donor, donor tissue (such as for example organ perfusion), the host, any combination thereof, and the like. In particular, the methods provided here are aimed at inhibiting, preventing, reducing, alleviating, or decreasing the development of a neuroinflammatory disorder in a patient. Thus, for example, an improvement in the disease can be characterized as an absence of clinically observable symptoms, a decrease in BBB permeability, an increase in the number, density or concentration of endothelial cells present in the BBB, a change in cell morphology or function endothelial cells, or a change in the interactions between endothelial cells and pericytes or astrocytes or between endothelial cells, pericytes and astrocytes that form the BBB.
[0123] [00123] Changes in BBB permeability can be measured using in vitro models. In certain embodiments, a dynamic in vitro DIV-BBB model can be used. Cucullo et al. presented a model of DIV-BBB composed of normal adult human brain microvascular endothelial cells and adult human astrocytes to study how hemodynamic changes and systemic inflammation affect the integrity of the brain's microvasculature. Specifically, this model uses a cartridge, or hollow tube, to represent the blood-brain barrier with the inside of the cartridge representing the blood side of the blood-brain barrier and the outside of the cartridge representing the brain side of the blood-brain barrier. The inside of the cartridge is lined with adult human brain microvascular endothelial cells and the outside is lined with adult human astrocytes. As a blood-brain barrier modifying agent, such as SEMA4D, is introduced into the cartridge lumen, the electrical current between the inside and outside of the tube is monitored using the Transendothelial Electrical Resistance Measurement, described below. One embodiment of this model has the innovation of having transcapillary micro-holes to allow the traffic of the transendothelial cell between the vascular compartment and the parenchyma. A detailed description of the in vitro DIV-BBB model and the derivation and culture of human microvascular endothelial cells and adult astrocytes used can be found in, for example, Cucullo et al., Brain Research. 951 243 - 254 (2002); and Cucullo et al., Journal of Cerebral Blood Flow & Metabolism. 2: 767 - 77 (2011). It should be assessed that persons skilled in the art will recognize that other BBB models have been described and usefully used to study the role of BBB in the disease in the prior art and that the present disclosure should not be limited to any particular model.
[0124] [00124] BBB permeability can be monitored using Transendothelial Electrical Resistance Measurement (TEER). TEER is used to monitor BBB integrity in real time, which has been shown to correlate with BBB permeability. The TEER system uses electronic multiplexing to measure multiple cartridges in rapid succession and assesses the integrity and viability of tissue culture bilayers quickly and safely (Cucullo et al., 2002; Cucullo et al., 2010; Santaguida et al, 2006 ). In operation, the system applies an excitation voltage (0.06 V) through the excitation electrodes inserted in each cartridge in the luminous and extraluminal compartments. A microcontroller computes the resistivity and capacitance (per cm2) of the barrier based on physical parameters. Capacitance values are calculated by comparing the voltage and current waveforms. The peak-to-peak delay of the two waveforms is proportional to the capacitance value, which is expressed as arc voltage. TEER can be measured from the initial configuration throughout the course of each experiment.
[0125] [00125] Anti-SEMA4D binding molecules, anti-Plexin B1 binding molecules, or a combination thereof, for example, antibodies or binding fragment with the antigens, variants, or derivatives thereof, may be used in combination with at least one or plus other treatments for neuroinflammatory disorders; where additional therapy is administered before, during, or subsequent to the anti-SEMA4D binding molecule, anti-Plexin B1 binding molecules, or a combination thereof, for example, antibody or binding fragment with the antigen, variant, or derivative thereof, therapy. Thus, where combined therapies comprise the administration of an anti-SEMA4D binding molecule, anti-Plexin B1 binding molecules, or a combination thereof, for example, an antibody or binding fragment with the antigen, variant, or derivative thereof, in In combination with the administration of another therapeutic agent, the methods of the invention encompass co-administration, using separate formulations or a single pharmaceutical formulation, with simultaneous or consecutive administration in any order. SAW. Pharmaceutical Compositions and Administration Methods
[0126] [00126] Methods of preparing and administering antiSEMA4D binding molecules, anti-Plexin B1 binding molecules, or a combination thereof, for example, antibodies, or antigen binding fragments, variants, or derivatives thereof to a subject in need of these are well known to or are easily determined by those skilled in the art. The route of administration of the anti-SEMA4D binding molecule, the anti-Plexin B1 binding molecule, or combination thereof, for example, antibody, or antigen-binding fragment, variant, or derivative thereof, may be, for example, oral , parenteral, by inhalation or topical. The term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these forms of administration are clearly considered to be within the scope of the invention, an example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. A pharmaceutical composition suitable for injection may comprise a buffer (for example, acetate, phosphate or citrate buffer), a surfactant (for example, polysorbate), optionally a stabilizing agent (for example, human albumin), etc. However, in other methods compatible with the teachings here, anti-SEMA4D binding molecules, anti-Plexin B1 binding molecules, or a combination thereof, for example, antibodies, or antigen-binding fragments, variants, or derivatives thereof can be released directly to the site of the adverse cell population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
[0127] [00127] As discussed here, anti-SEMA4D binding molecules, anti-Plexin B1 binding molecules, or a combination thereof, for example, antibodies, or antigen binding fragments, variants, or derivatives thereof, can be administered in a pharmaceutically amount effective for the in vivo treatment of neuroinflammatory disorders. In this regard, it will be assessed that the disclosed binding molecules can be formulated so as to facilitate administration and promote the stability of the active agent. In certain embodiments, pharmaceutical compositions according to the present invention comprise a sterile, non-toxic, pharmaceutically acceptable carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the present application, a pharmaceutically effective amount of an anti-SEMA4D binding molecule, an anti-Plexin B1 binding molecule, or combination thereof, for example, an antibody, or antigen-binding fragment, variant, or derivative of these, it must be maintained to mean an amount sufficient to obtain effective binding to a target and to obtain a benefit, for example, to decrease the permeability of BBB in a patient with a neuroinflammatory disorder.
[0128] The pharmaceutical compositions used in this invention comprise pharmaceutically acceptable carriers, including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid , potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.
[0129] [00129] Preparations for parenteral administration include 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 buffered medium. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01 to 0.1 M phosphate buffer and preferably 0.05 M or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
[0130] [00130] More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and must be fluid as the capacity for easy syringe injection exists. It must be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington’s Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).
[0131] [00131] The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be caused by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
[0132] [00132] In any case, sterile injectable solutions can be prepared by incorporating an active compound (for example, an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative of these, alone or in combination with others active agents) in the required amount in an appropriate solvent with one or a combination of ingredients listed here, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle, which contains a basic dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which produces a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Injection preparations are processed, filled in containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. In addition, the preparations can be packaged and sold in the form of a kit. Such articles of manufacture may have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to, a disease or disorder.
[0133] [00133] Parenteral formulations can be a single bolus dose, an infusion or a bolus loading dose followed with a maintenance dose. These compositions can be administered at fixed intervals or specific variables, for example, once a day, or on an "as needed" basis.
[0134] [00134] Certain pharmaceutical compositions used in this invention can be administered orally in an acceptable dosage form including, for example, capsules, tablets, suspensions or aqueous solutions. Certain pharmaceutical compositions can also be administered by nasal spray or inhalation. Such compositions can be prepared as solutions in saline solution, using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other conventional solubilizing or dispersing agents.
[0135] [00135] The amount of an anti-SEMA4D binding molecule, an anti-Plexin B1 binding molecule, or combination thereof, for example, antibody, or fragment, variant, or derivative thereof, to be combined with the carrier materials to produce a single dosage form will vary depending on the treated host and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens can also be adjusted to provide the ideal desired response (for example, a therapeutic or prophylactic response).
[0136] [00136] According to the scope of the present disclosure, antiSEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof can be administered to a human or other animal according to the treatment methods mentioned above in a sufficient amount to produce a therapeutic effect. Anti-SEMA4D antibodies, or antigen-binding fragments, variants or derivatives thereof can be administered to such a human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent from according to known techniques. It will be recognized by a person of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent are dictated by the amount of active ingredient with which they must be combined, the route of administration and other well-known variables. Those skilled in the art will further evaluate that a cocktail comprising one or more species of anti-SEMA4D binding molecules, anti-Plexin B1 binding molecules, or combinations thereof, for example, antibodies, or antigen binding fragments, variants, or derivatives of these, of the invention can be used.
[0137] [00137] By "therapeutically effective dose or amount" or "effective amount" is meant an amount of anti-SEMA4D binding molecule, anti-Plexin B1 binding molecule, or a combination thereof, for example, antibody or binding fragment with antigen, variant, or derivative thereof, which when administered causes a positive therapeutic response with respect to the treatment of a patient with a disease to be treated, for example, a decrease in BBB permeability, an increase in BBB resistivity, an increase in number, density or concentration of endothelial cells present in the BBB, a change in morphology or function in the endothelial cells, or a change in the interactions between endothelial cells or astrocytes or between endothelial cells and astrocytes that form the BBB.
[0138] [00138] Therapeutically effective doses of the compositions of the present invention, for the decrease in BBB permeability vary depending on many different factors, including means of administration, target site, patient's physiological state, whether the patient is human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. In certain embodiments the patient is a human being, but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
[0139] [00139] The amount of at least one anti-SEMA4D binding molecule, anti-Plexin B1 binding molecule, or combination thereof, for example, antibody or binding fragment, variant, or derivative thereof, to be administered is easily determined by a person of ordinary skill in the art without undue experimentation given the disclosure of the present invention. Factors influencing the mode of administration and the respective amount of at least one antiSEMA4D binding molecule, anti-Plexin B1 binding molecule, or combination thereof, for example, antibody, antigen binding fragment, variant or derivative thereof include, but are not limited to, they are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual experimentation therapy. Similarly, the amount of anti-SEMA4D binding molecule, anti-Plexin B1 binding molecule, or combination thereof, for example, antibody, or fragment, variant, or derivative thereof, to be administered will be dependent on the mode of administration and whether the subject will endure a single dose or multiple doses of this agent.
[0140] [00140] The invention also takes into account the use of an anti-SEMA4D binding molecule, an anti-Plexin B1 binding molecule, or combination thereof, for example, the invention's antibody, or antigen-binding fragment, variant, or derived from these, in the manufacture of a drug to treat a subject to treat a neuroinflammatory disorder, in which the drug is used in a subject who has been pre-treated with at least one other therapy. By "pre-treated" or "pre-treatment" is meant that the subject received one or more other therapies (for example, he was treated with at least one other neuroinflammatory therapy) before receiving the drug comprising the anti-SEMA4D binding molecule , an anti-Plexin B1 binding molecule, or combination thereof, for example, antibody or antigen binding fragment, variant, or derivative thereof. “Pre-treated” or “pre-treatment” includes subjects who have been treated with at least one other therapy within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within 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 within 1 day prior to the start of treatment with the drug comprising the anti-SEMA4D binding molecule, for example, the monoclonal antibody VX15 / 2503 disclosed herein, or antigen binding fragment, variant, or derivative thereof. It is not necessary for the subject to be a responder to pretreatment with previous therapy or therapies. Thus, the subject receiving the drug comprising the anti-SEMA4D binding molecule, an anti-Plexin B1 binding molecule, or a combination thereof, for example, an antibody or antigen binding fragment, variant, or derivative thereof may have responded , or may have failed to respond, either to pretreatment with the previous therapy, or to one or more of the previous therapies where the pretreatment comprised multiple therapies.
[0141] [00141] The practice of the present invention will use, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained completely 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. Pat. U.S. No. 4,683,195; 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 Mice 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.).
[0142] [00142] General principles of antibody engineering are presented in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed .; Oxford Univ. Press). General principles of protein engineering are presented in Rickwood et al., Eds. (1995) Protein Engineering, A Practical Method (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles of antibodies and antibody-hapten binding are presented 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 immunology known in the art and not specifically described are generally followed as 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).
[0143] [00143] Standard reference papers presenting general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); 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., (Elsevere, Amsterdam); Goldsby et al., Eds. (2000) Kuby Immunnology (4th ed .; H. Freemand &Co.); 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 Verlan); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).
[0144] [00144] All references cited above, as well as all references cited here, are incorporated here as a reference in their entirety.
[0145] [00145] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES
[0146] [00146] The following examples demonstrate the effectiveness of the antiSEMA4D antibody (VX15 / 2503) in reducing or preventing BBB rupture, that is, a decrease in BBB permeability, in a DIV-BBB model in vitro as well as in a model of EAE in vivo. An in vivo Alzheimer's disease model experiment is also released here. A detailed description of the DIV-BBB model in vitro can be found in, for example, Cucullo et al., Brain Research. 951 243 - 254 (2002); and Cucullo et al., Journal of Cerebral Blood Flow & Metabolism. 1 - 11 (2010). Models of EAE and Alzheimer's disease in vivo are disclosed, for example, in Miller et al., Curr Protoc Immunol. CHAPTER: Unit-15.1, 2007; Colton et al., J Alzheimers Dis 15: 571 - 587, 2008 and Wilcock et al., J. Neuroscience, 29: 7957 - 7965, 2009, respectively. Example 1: Testing the ability of an anti-SEMA4D binding molecule, for example, an antibody or antigen-binding fragment, variant, or derivative thereof, for example, VX15 / 2503, to restore the integrity of the BBB following disruption SEMA4D-induced reaction of BBB in an in vitro DIV-BBB model
[0147] [00147] * Experimental Project. An in vitro dynamic BBB model (“DIVBBB”) was performed to study the effect of recombinant human SEMA4D (huSEMA4D-his) and VX15 / 2503 (described in detail in US 2010/0285036 A1, incorporated here as a reference in its entirety) BBB's integrity. Two DIV-BBB cartridges were tested on the model. The basic experimental design is shown in FIG. 1. Increasing concentrations of recombinant SEMA4D (rSEMA4D) were added to the lumen at 12 hour intervals, considering equilibrium (approximately 12 hours / concentration). rSEMA4D was initially added to the lumen at a concentration of 0.05 µg / ml at time 0. The concentration of rSEMA4D increased by 10 times in each interval, for example, 0.5 µg / ml in 12 hours, 5.0 µg / ml ml in 24 hours, and 50.0 µg / ml in 36 hours. TEER measurements were taken between each interval as a reflection of changes in BBB permeability at varying concentrations of rSEMA4D. Following the addition of the final dose of rSEMA4D at 50.0 µg / ml in 36 hours, VX15 / 2503 was added to the lumen at a concentration of 250 µg / ml in 48 hours. Within 72 hours, 24 hours after the addition of VX15 / 2503, the BBB permeability was measured again.
[0148] [00148] Measurement of Transendothelial Electrical Resistance (TEER) was used to monitor the integrity of the BBB in real time. As mentioned above, the TEER system uses electronic multiplexing to measure multiple cartridges in rapid succession and assesses the integrity and viability of tissue culture bilayers quickly and safely (Cucullo et al., 2002; Santaguida et al, 2006). In this dynamic in vitro model, the cartridges, or hollow tubes, were configured to represent the blood-brain barrier with the inside of the cartridge representing the blood side of the blood-brain barrier and the outside of the cartridge representing the brain side of the blood-brain barrier. The inside of the cartridge was lined with adult human brain microvascular endothelial cells and the outside was lined with adult human astrocytes. As a blood-brain barrier modifying agent, such as SEMA4D, was introduced into the cartridge lumen, the electrical current between the inside and outside of the tube was monitored using TEER. In operation, the TEER system applies an excitation voltage (0.06 V) through the excitation electrodes inserted in each cartridge in the luminous and extraluminal compartments. A microcontroller computes the resistivity and capacitance (per cm2) of the barrier based on physical parameters. Capacitance values are calculated by comparing the voltage and current waveforms. The peak-to-peak delay of the two waveforms is proportional to the capacitance value, which is expressed as arc voltage. TEER was measured from the initial configuration throughout the course of each experiment.
[0149] [00149] Increase induced by rSEMA4D in BBB permeability. Following the formation of the BBB, the effect of rSEMA4D on the integrity of the BBB was measured by adding increasing concentrations of recombinant SEMA4D (rSEMA4D) in the lumen of the two cartridges. rSEMA4D was initially added to the lumen at a concentration of 0.05 µg / ml at time 0. The concentration of rSEMA4D was increased 10 times in each 12-hour interval, for example, 0.5 µg / ml in 12 hours, 5 µg / ml in 24 hours and 50.0 µg / ml in 36 hours. TEER measurements were taken between and during each interval as a reflection of changes in BBB permeability at varying concentrations of rSEMA4D. BBB permeability generally remained relatively stable at 0.05 µg / ml rSEMA4D. Starting at 0.5 µg / ml, increasing concentrations of rSEMA4D (ie 0.5 µg / ml, 5 µg / ml and 50 µg / ml) resulted in decreased TEER measurement reflecting the increased permeability of the endothelial cell layer. These results are shown in FIG. two.
[0150] [00150] Antibody-induced decrease in BBB permeability treated with rSEMA4D. To measure the effect of an anti-SEMA4D antibody on the BBB following exposure to the rising dose of rSEMA4D, VX15 / 2503 was added at a concentration of 250 µg / ml in 48 hours. TEER measurements were taken in 72 hours. Treatment with VX15 / 2503 resulted in an overall decrease in BBB permeability (or increase in resistivity) in both cartridges. This decrease in permeability reflects the restoration of BBB. The results are shown in FIG. two. Example 2: Testing the ability of an anti-SEMA4D binding molecule, for example, an antibody or antigen-binding fragment, variant, or derivative thereof, for example, VX15 / 2503, to restore the integrity of the BBB following disruption SEMA4D-induced reaction of BBB in an in vitro DIV-BBB model
[0151] [00151] Experimental Project. A second experiment using the DIV-BBB in vitro model was performed to study the effect of SEMA4D and VX15 / 2503 on the integrity of the BBB. The basic experimental design was similar to that shown in example 1, and FIG. 1, above. For two weeks, DIV-BBB cartridges underwent BBB formation in endothelial and astrocytic cell compartments. The formation of the BBB as reflected in TEER is shown in FIGS. 3 and 4.
[0152] [00152] Increase induced by rSEMA4D in BBB permeability. Following the formation of the BBB, the effect of rSEMA4D on the integrity of the BBB was measured by adding increasing concentrations of recombinant SEMA4D (rSEMA4D) in the lumen of the first cartridge in a set of three cartridges at 12 hour intervals, considering balance ( approximately 12 hours / concentration). rSEMA4D was initially added to the lumen at a concentration of 0.5 µg / ml at time 0. The concentration of rSEMA4D increased by 10 times in each interval, for example, 5 µg / ml in 12 hours and 50.0 µg / ml in 24 hours. TEER measurements were taken between each interval as a reflection of changes in BBB permeability at varying concentrations of rSEMA4D. Generally, increasing concentrations of rSEMA4D resulted in decreased TEER measurement reflecting the increased BBB permeability. These results are shown in FIG. 3.
[0153] [00153] To test the integrity of the BBB in the presence of an antigen that does not target the endothelial cell layer, a similarly prepared recombinant protein control (CTRL, protein C35) was added in equimolar concentrations at the same 12-hour intervals (ie , 0.25 µg / ml at time 0, 2.5 µg / ml at 12 hours, and 25.0 µg / ml at 24 hours) to the two additional control cartridges. Unlike the effect of rSEMA4D, the CTRL protein did not induce a significant change in TEER reflecting no significant change in BBB permeability. If, however, 50.0 µg / ml of rSEMA4D were added 12 hours after adding the highest concentration of CTRL protein, a rapid decrease in TEER similar to that seen with rising doses of rSEMA4D was induced. The results are shown in FIG. 4.
[0154] [00154] Antibody-induced decrease in BBB permeability treated with rSEMA4D. Following the addition of the final dose of rSEMA4D at 50.0 µg / ml in 24 hours, the effect of VX15 / 2503 on TEER and the BBB permeability was measured. In FIG. 3, the VX15 / 2503 antibody was added at a concentration of 250 µg / ml in 36 hours to two of the three cartridges that received rising doses of rSEMA4D while the same concentration of an isotype control antibody was added to the only remaining cartridge that received ascending doses of rSEMA4D. TEER measurements were taken at several subsequent time points. Treatment with VX15 / 2503 resulted in an increase in TEER back to peak levels at the beginning of the experiment, reflecting an overall decrease in BBB permeability (ie, BBB restoration). In the only cartridge that received isotype control antibody, TEER levels remained at the relatively low levels induced by treatment with rSEMA4D, indicating no significant decrease in BBB permeability. Similar results are shown in FIG. 4. In FIG. 4, the VX15 / 2503 antibody was added at a concentration of 250 µg / ml in 48 hours to the two cartridges that received recombinant C35 protein from initial control followed by 50 µg / ml of rSEMA4D for 12 hours. Treatment with VX15 / 2503 resulted in an increase in TEER back to peak levels at the beginning of the experiment, reflecting an overall decrease in BBB permeability (ie, BBB restoration). Example 3: Testing the ability of an anti-Plexin-B1 binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof, to restore BBB integrity following BBB SEMA4D-induced disruption in an in vitro DIV-BBB model
[0155] [00155] Another study was conducted to measure the effects of the antiPlexin-B1 antibody (MAB37491 Human Plexin-B1 MAb (Clone 559830), R&D Systems) on the integrity of the BBB. This antibody blocks the binding of SEMA4D to the Plexin-B1 receptor. The results of this study are shown in FIG. 5. As shown in FIG. 5, human endothelial cells and astrocytes in four DIV-BBB cartridges underwent BBB formation similar to the experiments described above. After BBB formation, rSEMA4D was added at a concentration of 50.0 µg / ml, inducing an increase in BBB permeability (ie, destruction of BBB). Following the addition of rSEMA4D, the anti-Plexin-B1 antibody was added at a concentration of 125 µg / ml in 6 hours to two of the four cartridges, the antibody VX15 / 2503 was added at a concentration of 250 µg / ml to one of the four cartridges, and the isotype control antibody was added at a concentration of 250 µg / ml to the remaining cartridge. TEER measurements were taken at several subsequent time points. Treatment with VX15 / 2503 or anti-Plexin-B1 antibody resulted in an increase in TEER levels with both agents. Treatment with VX15 / 2503 resulted in a slightly greater increase in TEER than treatment with anti-Plexin-B1 antibody at the last point in time. The effect of the two antibodies is indistinguishable at all other points in time. The increase in TEER reflects an overall decrease in BBB permeability (i.e., BBB restoration) in the presence of VX15 / 2503 or anti-Plexin-B1 antibody. In the only cartridge that received isotype control antibody, TEER levels remained at the relatively low levels induced by treatment with rSEMA4D, indicating no significant decrease in BBB permeability. It should be assessed that treatment can also be conducted using a combination of VX15 / 2503 and anti-Plexin-B1. Example 4: Testing the ability of an anti-SEMA4D binding molecule, for example, an antibody or antigen-binding fragment, variant, or derivative thereof, for example, VX15 / 2503, to restore the integrity of the BBB following disruption of BBB induced by activated PBMC and flow cessation in an in vitro DIVBBB model
[0156] [00156] Experimental Project. Another experiment using the DIV-BBB model in vitro was carried out to study the effect of VX15 / 2503 on restoring BBB integrity following BBB rupture induced by activated peripheral blood mononuclear cells (PBMC) and flow cessation. For two weeks, two DIV-BBB cartridges underwent BBB formation in endothelial and astrocytic cell compartments.
[0157] [00157] Increase induced by activated PBMC in BBB permeability. Following the formation of the BBB, the effect of activated PBMC on the integrity of the BBB was measured. PBMC were activated with PMA / ionomycin for 2 hours and then added at a concentration of 106 / ml in the lumen of the two cartridges. TEER measurements were taken before and after the addition of activated PBMCs as a reflection of changes in BBB permeability. Generally, adding activated PBMC to the cartridges at 106 / ml resulted in decreased TEER measurement reflecting increased BBB permeability. These results are shown in FIG. 6.
[0158] [00158] In approximately 2 to 4 hours after the addition of activated PBMCs to the cartridges, the flow cessation was performed for 1 hour. TEER measurements were taken before and after flow cessation as a reflection of changes in BBB permeability. Generally, the flow cessation resulted in an additional decrease in the TEER measurement reflecting the increased BBB permeability. These results are also shown in FIG. 6.
[0159] [00159] Antibody-induced decrease in BBB permeability exposed to activated PBMCs. Following exposure to activated PBMC and flow cessation, the effect of VX15 / 2503 on TEER and BBB permeability was measured. The VX15 / 2503 antibody was added at a concentration of 250 µg / ml to one of the two cartridges that received activated PBMC while the same concentration of an isotype control antibody (Isotype Control Ig, 2269) was added to the remaining cartridge. TEER measurements were taken at several subsequent time points. As shown in FIG. 6, treatment with VX15 / 2503 resulted in an increase in TEER back to peak levels at the beginning of the experiment, reflecting an overall decrease in BBB permeability (ie, BBB restoration). In the cartridge that received isotype control antibody, TEER levels remained at the relatively low levels induced by treatment with activated PBMC and flow cessation, indicating no significant decrease in BBB permeability. Example 5: Testing the ability of an anti-SEMA4D binding molecule, for example, an antibody or antigen-binding fragment, variant, or derivative thereof, for example, VX15 / 2503, to protect the integrity of the BBB in a model of EAE in vivo
[0160] [00160] Anti-SEMA4D binding molecules, for example, antibodies or antigen-binding fragments, variants or derivatives thereof, for example, VX15 / 2503, were tested in the experimental autoimmune encephalomyelitis (EAE) model in vivo.
[0161] [00161] In an in vivo EAE model, BBB rupture was investigated by examining changes in brain permeability as reflected in the penetration of blood fibrinogen into the brain parenchyma and by examining proteins from the endothelial occlusion junction, including Claudina -5. In this model, EAE was induced in mice by immunization with PLP peptide (139 to 151). Certainly, those skilled in the art will assess that other EAE-inducing proteins can also be used (for example, a myelin antigen, for example myelin-oligodendrocyte glycoprotein peptide 35 to 55) and that, for greater efficiency, these proteins or induction peptides can vary from one species to another and from one strain of mice to another, Steinman, L. Neuron 24: 511-514 (1999). Sections of tissue from the central nervous system (CNS) of animals at different stages of the disease were then subjected to immunostaining for proteins (fibrinogen and claudin-5, which serve as markers for BBB disruption).
[0162] [00162] Experimental Project. In an EAE model in vivo, EAE was induced in 12-week-old SJL / J mice (10 mice per group) by immunization with PLP peptide (139 to 151) in CFA (Freund's complete adjuvant). The mice were then treated once a week from 7 days post-induction with 600 µg of anti-SEMA4D antibody (antibody VX15 / 2503) or control IgG. Neurological signs were first observed at 11 d post-induction (dpi). At 13 days post-induction, during the acute phase of the disease, 4 mice per group were sacrificed and samples of the lumbar spinal cord were prepared for histopathological analysis. To detect BBB rupture in the samples, these samples were subjected to immunostaining for fibrinogen and claudin-5. The procedure for immunostaining is as follows: Sections were rinsed twice in PBS, then incubated in PBS with 0.1% glycine for 10 min, blocked in PBS with 0.3% Triton X-100 and 10% goat serum. % for 1 h, and incubated with primary Abs in blocking buffer overnight at 4 ° C. For claudin-5 (CLN-5), before blocking, the sections were soaked in EDTA, pH 8, 100 ° C. The primary antibodies used were anti-CLN-5 (1:50), and anti-fibrinogen (1: 1,000). After washing three times in 0.3% PBS, Triton X-100 the sections were then incubated in relevant species-specific secondary antibodies conjugated to AlexaFluor 488 and / or AlexaFluor 594 (1/100; Molecular Probes) in blocking buffer for 1 h at 25 ° C, washed again three times, and counter-treated with 4,6-diamidino-2-phenylindole (DAPI). All samples were examined and photographed using a Zeiss LSM 510 META confocal laser scanning system attached to an Axiovert 200 inverted fluorescence microscope.
[0163] [00163] The clinical disease in the mice was classified as follows: 0 = no symptoms; 1 = soft tail; 2 = weakness in the hind leg; 4 = weakness in the front and rear paw; 5 = death. Neurological signs were first seen at 11 days post-induction. In mice treated with the VX15 / 2503 antibody, the clinical disease reached an average severity rating of 0.75, indicative of mild tail weakness, while the clinical disease in mice in the control group reached an average severity rating of 2, 25, indicative of partial paralysis.
[0164] [00164] The results of immunostaining in 13 days post-induction are shown in FIGS. 7A to 7C. Fibrinogen does not normally penetrate the blood-brain (BBB) brain barrier. In EAE, with the BBB compromised, the color of green fibrinogen was detected in the brain substance (left panel). In addition, the expression of claudin-5 (CLN-5, red), a component of the occlusion junctions that make up the BBB, has been reduced. Mice in the control group showed reduced expression of claudin-5 and increased levels of extravascular fibrinogen loss, which correlated with a disruption in the BBB. In mice treated with VX15 / 2503 antibody, on the other hand, the expression of claudin-5 was maintained and the loss of fibrinogen was significantly reduced. These results demonstrated the protective effect of the VX15 / 2503 antibody against BBB disruption in these treated mice, and specifically demonstrated how the anti-SEMA4D antibody prevents the rupture of the BBB, prevents extravascular loss of fibrinogen (left panel 7A and quantification in 7B) , and preserves claudin-5 as detected by the color red (right panel 7A and quantification in 7C). Example 6: Effect of SEMA4D on Occlusion Junction Proteins in brain endothelial cell cultures
[0165] [00165] Experimental Project. The expression of the Claudina-5 key endothelial occlusion junction protein following treatment of CNS-derived endothelial cells with soluble recombinant SEMA4D was investigated. In this model, endothelial cultures of the primary mouse central nervous system (CNS) were isolated and plated on a plate coated with a matrix of 6 reservoirs (MBCEC isolated from 10 brains were resuspended in 3 ml of primary endothelial cell culture medium and plated at 250 ul per reservoir). Cultures were used on day 7 after isolation. Cultures were treated with 1 ng / ml, 10 ng / ml or 100 ng / ml recombinant mouse SEMA4D or 100 ng / ml mouse VEGF-A (positive control) for 24 hours. The endothelial cultures of the animals were then subjected to SDS-electrophoresis in polyacrylamide gel (SDSPAGE) and immunoblotting to control the claudin-5 occlusion junction and actin protein load. Data were scanned and subjected to densitometry using the ImageJ (NIH) software.
[0166] [00166] The results of the immunoblotting are shown in FIG. 8. As provided in FIG, 8, endothelial cell cultures treated with 100 ng / ml of recombinant SEMA4D showed a significant reduction in Claudine-5 protein expression. Endothelial cell cultures treated with 100 ng / ml of VEGF-A were tested as a positive control for Claudina-5 infraregulation. This demonstrates the important role of SEMA4D in regulating the expression of a key BBB occlusion junction protein. Example 7: Testing the ability of an anti-SEMA4D or anti-Plexin B1 binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof to decrease BBB permeability in an Alzheimer's Disease model (AD) in vivo
[0167] [00167] Anti-SEMA4D or anti-Plexin B1 binding molecules, for example, antibodies or antigen binding fragments, variants or derivatives thereof, for example, MAb 67 (described in detail in US 2010/0285036 A1, incorporated herein as reference in its entirety), are tested in several model systems of neuroinflammatory disorders, including, but not limited to, a transgenic mouse model with experimental Alzheimer's disease (AD) in vivo APPSwDI / NOSC - / -. These mice were generated by crossing the APP-Swedish-Dutch-Iowa mutant mouse with nitric oxide synthase 2 knock-out mouse (Colton et al., J Alzheimers Dis, 15: 571 - 587, 2008; Van Nostrand et al. , Stroke 41: S135 - S138, 2010). APPSwDI / NOSC - / - mice develop age-related neurovascular amyloidosis with interrupted BBB function, intraparenchymal amyloid plaques, mouse tau hyperphosphorylation, neuroinflammation, neuronal cell death, and cognitive deficits. Wilcock et al. showed that the treatment of APPSwDI / NOSC - / - mice with active immunotherapy directed with amyloid-beta leads to a marked reduction in amyloid deposition, but with an increased incidence of microhemorrhages (Wilcock et al., J Neurosci. 29: 7957 - 7965, 2009).
[0168] [00168] In an in vivo AD model, the progression of AD is investigated by examining immunohistochemical signatures of amyloid deposition, tau hyperphosphorylation, and loss of BBB (fibrinogen), as well as assessing cognitive abilities in behavioral paradigms with based on spatial memory. In this model, the transgenic mice are administered with MAb 67 or Control Ig (Mab 2B8) intravenously at a concentration of 30 mg / kg from the age of 26 to 38 weeks for a total of 13 doses.
[0169] [00169] Mice are initially subjected to comparative behavioral testing at the age of 10 to 12 weeks, for example, Open Field, RAWn and Barnes Maze tests, and mice meeting the activity and learning / memory criteria are included in the follow-up. Behavioral deficits are again measured at the age of 38, 39 and 40 weeks and body weight is recorded. Mice that do not meet the criteria for the study protocol will be sacrificed. At the equivalence point at 41 weeks of age, the animals will be euthanized and the brains will be processed for biochemical and immunohistological analyzes regarding levels and deposits of soluble and insoluble beta amyloid. The serum is collected pre-dosing, during dosing and at the equivalence point for PK at the age of 10, 25 and 41 weeks. Sections of tissue from the central nervous system (CNS) of animals at different stages of the disease can be subjected to immunostaining for fibrinogen, so that they can be used as markers for BBB disruption.
[0170] [00170] Many modifications and other embodiments of the inventions presented here will come to the mind of a person skilled in the technique to which these inventions belong, having the benefit of the teachings presented in the preceding descriptions and in the associated drawings. Therefore, it should be understood that inventions should not be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims and list of embodiments disclosed herein. Although specific terms are used here, they are used in a generic and descriptive sense only and not for purposes of limitation.

权利要求:
Claims (8)
[0001]
Use of an effective amount of an isolated antibody or antigen-binding fragment that specifically binds to semaphorin-4D (SEMA4D), CHARACTERIZED by the fact that it is for the manufacture of a drug to decrease blood brain barrier permeability in an individual with increased blood-brain barrier permeability and a neuroinflammatory disorder, in which the antibody or antigen-binding fragment thereof inhibits the interaction of SEMA4D with the SEMA4D receptor, Plexin-B1, and in which the antibody or antigen-binding fragment of it even comprises a variable heavy chain (VH) comprising VHCDRs 1 to 3 comprising SEQ ID NOs 6, 7 and 8, respectively, and a variable light chain (VL) comprising VLCDRs 1 to 3 comprising SEQ ID NOs 14, 15 and 16, respectively.
[0002]
Use, according to claim 1, CHARACTERIZED by the fact that the individual is a mammal.
[0003]
Use, according to claim 2, CHARACTERIZED by the fact that the mammal is a human.
[0004]
Use according to claim 1, CHARACTERIZED by the fact that the antibody or antigen-binding fragment thereof competitively inhibits a reference monoclonal antibody comprising: 1) the amino acid sequence of the variable heavy chain (VH) of SEQ ID NO : 9 and the amino acid sequence of the variable light chain (VL) of SEQ ID NO: 17, or 2) the amino acid sequence of the VH of SEQ ID NO: 10 and the amino acid sequence of the VL of SEQ ID NO: 18.
[0005]
Use according to claim 1, CHARACTERIZED by the fact that the antibody or antigen-binding fragment thereof specifically binds to the same SEMA4D epitope as a reference monoclonal antibody comprising: 1) the amino acid sequence of the variable heavy chain (VH) of SEQ ID NO: 9 and the variable light chain (VL) amino acid sequence of SEQ ID NO: 17, or 2) the VH amino acid sequence of SEQ ID NO: 10 and the VL amino acid sequence of SEQ ID NO: 18.
[0006]
Use according to any one of claims 1 to 4, CHARACTERIZED by the fact that the antibody or antigen-binding fragment thereof competitively inhibits a reference monoclonal antibody selected from the group consisting of VX15 / 2503 or 67 of se call SEMA4D specifically.
[0007]
Use according to any one of claims 1 to 6, CHARACTERIZED by the fact that the antibody or antigen-binding fragment thereof specifically binds to the same SEMA4D epitope as a reference monoclonal antibody selected from the group consisting of VX15 / 2503 or 67.
[0008]
Use according to any one of claims 1 to 7, CHARACTERIZED by the fact that the neuroinflammatory disorder is selected from the group consisting of Multiple Sclerosis, Amyotrophic Lateral Sclerosis, epilepsy, Alzheimer's Disease, Parkinson's Disease, meningitis, edema cerebral and brain trauma.

类似技术:

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公开号 | 公开日 | 专利标题

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BR112014008885B1|2021-02-17|use of an isolated antibody or antigen-binding fragment that specifically binds to semaphorin-4d to decrease blood-brain barrier | permeability in an individual with increased bbb permeability and a neuroinflammatory disorder

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AU2019257379B2|2021-04-01|Use of semaphorin-4D binding molecules for treating neurodegenerative disorders

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AU2013259192B2|2018-02-22|Use of semaphorin-4D binding molecules to promote neurogenesis following stroke

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NZ623856B2|2016-08-02|Use of semaphorin-4d binding molecules for modulation of blood brain barrier permeability

同族专利:

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公开号 | 公开日

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EP2766093B1|2018-02-21|

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EA029023B1|2018-01-31|

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IL232072A|2021-01-31|

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WO2013055922A1|2013-04-18|

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AU2012322721A1|2013-05-16|

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AU2012322721B2|2016-05-05|

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KR101999872B1|2019-07-12|

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CN110105452A|2019-08-09|

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IL232072D0|2014-05-28|

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JP2017193569A|2017-10-26|

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ES2669209T3|2018-05-24|

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JP2014530239A|2014-11-17|

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MX2014004457A|2014-06-05|

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DK2766093T3|2018-06-06|

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US20130095118A1|2013-04-18|

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PT2766093T|2018-05-18|

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CN104168956A|2014-11-26|

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EA201490768A1|2014-09-30|

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US20190358321A1|2019-11-28|

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SG11201401458UA|2014-07-30|

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EP2766093A4|2015-03-25|

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CA2851805C|2021-12-28|

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NZ623856A|2016-04-29|

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CA2851805A1|2013-04-18|

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BR112014008885A2|2017-04-18|

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MX353550B|2018-01-18|

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KR20140076616A|2014-06-20|

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EP2766093A1|2014-08-20|

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JP6395606B2|2018-09-26|

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法律状态:
2017-04-25| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|

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2017-06-13| B08G| Application fees: restoration [chapter 8.7 patent gazette]|

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2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-21| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-08-18| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-01-26| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |

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