Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2839—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2839—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
  • C07K16/2842—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
  • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM

Definitions

• a5b1 integrin binding agents include antibodies (e.g., monospecific or multispecific, including bispecific) that bind to a5b1 integrin, including antibodies that bind to human a5b1 integrin.
• An epitope may be part of a larger a5 integrin antigen, which may be part of a larger a5 integrin polypeptide fragment, which, in turn, may be part of a larger a5 integrin polypeptide.
• An a5 integrin may exist in a native or denatured form.
• An a5 integrin polypeptide described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
• An a5 integrin polypeptide may comprise a polypeptide having the same amino acid sequence as a corresponding a5 integrin polypeptide derived from nature.
• Fibronectin is known in the art to interact (e.g., via RGD) with integrins and is a ligand for a5b1 integrin, a8b1 integrin and anb3 integrin.
• identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
• the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage to or conjugation with (directly or indirectly) a moiety such as a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage to or conjugation with (directly or indirectly) a moiety such as a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification
• an “antigen” is a moiety or molecule that contains an epitope to which a binding agent (e.g., an antibody) can bind.
• a binding agent e.g., an antibody
• an antigen can be bound by an antibody.
• the antigen, to which a binding agent (e.g., an antibody) described herein binds is a5b1 integrin (e.g., human a5b1 integrin), or a fragment thereof.
• the terms “specifically binds,” “specifically recognizes,” “immunospecifically binds,” “selectively binds,” “immunospecifically recognizes” and “immunospecific” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope) as such binding is understood by one skilled in the art.
• “specifically binds” means, for instance that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins.
• Biacore surface plasmon resonance assays by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM- 3000 BIAcore, Inc., Piscataway, NJ).
• SPR surface plasmon resonance
• a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and/or changed ⁇ e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human.
• Native sequence human Fc regions include a native sequence human lgG1 Fc region (non-A and A allotypes); native sequence human lgG2 Fc region; native sequence human lgG3 Fc region; and native sequence human lgG4 Fc region as well as naturally occurring variants thereof. Exemplary lgG1 and lgG4 Fc sequences are shown in FIG. 3.
• the “heavy chain” can refer to any distinct types, e.g., for example, alpha (a), delta (d), epsilon (e), gamma (y) and mu (m), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., lgG1, lgG2, lgG3 and lgG4.
• Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., lgG1, lgG2, lgG3, lgG4, lgA1 or lgA2), or any subclass (e.g., lgG2a or lgG2b) of immunoglobulin molecule.
• antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human lgG1 , lgG2, lgG3 or lgG4) or subclass thereof.
• Specific binding indicates that the antibody or fragment thereof binds to a5b1 integrin with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme).
• the antibody or fragment thereof may bind a5b1 integrin substantially exclusively (e.g., is able to distinguish a5b1 integrin from other known polypeptides, for example, by virtue of measurable differences in binding affinity).
• An a5b1 integrin- mediated disease, disorder, or condition includes a cancer, an angiogenesis- mediated disease (e.g., a disease with abnormal angiogenesis), and an inflammatory disease (e.g., a neuroinflammatory disease, including MS and ALS).
• an a5b1 integrin-mediated disease includes a disease, disorder or condition that is a cancer that is characterized by or associated with tumor cells that express or overexpress an a5b1 integrin.
• an a5b1 integrin- mediated disease includes a disease, disorder or condition that is characterized by or associated with abnormally increased angiogenic activity of cells (e.g., tumor
• a therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule/agent are outweighed by the therapeutically beneficial effects.
• the term “therapeutically effective amount” refers to an amount of an antibody or other agent ( e.g or drug) effective to “treat” a disease, disorder, or condition, in a subject or mammal.
• a “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of a disease, disorder or condition, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, or condition or associated symptom(s).
• the full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
• a therapeutically or prophylactically effective amount may be administered in one or more administrations.
• carrier can also refer to a diluent, adjuvant (e.g ., Freund’s adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered.
• adjuvant e.g ., Freund’s adjuvant (complete or incomplete)
• excipient or vehicle with which the therapeutic is administered.
• Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.
• VL sequence that is SEQ ID NO:92 or a humanized variant thereof.
• the antibody designated C-14D12 comprises CDR sequences according to Kabat and/or Chothia, AbM, Contact, or IMGT as shown in Table 6 and in some embodiments can comprise a VH sequence that is SEQ ID NO: 109 or a humanized variant thereof and a VL sequence that is SEQ ID NO: 110 or a humanized variant thereof.
• a5b1 integrin binding agents e.g., antibodies, such as bispecific antibodies
• a human a5b1 integrin binding agent described herein comprises one or more CDRs, including six CDRs, for example, VH CDR1 ,
• VL CDR1 VL CDR2
• VL CDR3 Kabat and/or Chothia, AbM, Contact, or IMGT listed in Table 1.
• a5b1 integrin binding agents ⁇ e.g., antibodies, such as bispecific antibodies), including human a5b1 integrin binding agents, described herein comprise one or more CDRs, including three CDRs, for example, VL CDR1, VL CDR2, and/or VL CDR3 according to Kabat and/or Chothia, AbM, Contact, or IMGT listed in Table 5.
• an a5b1 integrin binding agent e.g., an antibody, such as a bispecific antibody
• a VH CDR2 having the amino acid sequence of any one of SEQ ID NOS:2, 8, 14, 19, 24, 28, 54, 60, 66, 71, 76, 79, 82, 84, 87, and 90.
• an a5b1 integrin binding agent e.g., an antibody, such as a bispecific antibody
• a heavy chain variable (VH) region comprising: (1 ) a VH CDR1 having an amino acid sequence of selected from the group consisting of: (i) SEQ ID NO:1 , 27, 53, or 93, (ii) SEQ ID NO:7, 31 ,
• VH CDR2 having an amino acid sequence of selected from the group consisting of: (i) SEQ ID NO:2, 28, 54, or 79, (ii) SEQ ID NO:8, 60, or 82, (iii) SEQ ID NO:14, 66, or 84 (iv) SEQ ID NO:19, 71 , or 87, and (v) SEQ ID NO:24, 76; or 90, and (3) a VH CDR3 having an amino acid sequence of selected from the group consisting of: (i) SEQ ID NO:3, 29, 55, 80, or 94, (ii) SEQ ID NO:9, 32, 61 , 83, or 98, (iii) SEQ ID NO:15, 36, 67, 85
• VH CDR2 having an amino acid sequence of selected from the group consisting of: (i) SEQ ID NO:2, 28, 54, or 79, (ii) SEQ ID NO:8, 60, or 82, (iii) SEQ ID NO:14, 66, or 84 (iv) SEQ ID NO: 19, 71 , or 87, and (v) SEQ ID NO:24, 76; or 90, and (3) a VH CDR3 having an amino acid sequence of selected from the group consisting of: (i) SEQ ID NO:2, 28, 54, or 79, (ii) SEQ ID NO:8, 60, or 82, (iii) SEQ ID NO:14, 66, or 84 (iv) SEQ ID NO: 19, 71 , or 87, and (v) SEQ ID NO:24, 76; or 90, and (3) a VH CDR3 having an amino acid sequence of selected from the group consisting of: (i) SEQ ID NO:2, 28, 54, or 79, (ii
• an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 7, 12, 13, and 18;
• an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:34; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:28; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:29; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:30; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.
• VH heavy chain variable
• VL light chain variable
• an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:53; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:76; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:55; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:56; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:57; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:58.
• VH heavy chain variable
• VL light chain variable
• the CDRs of an a5b1 integrin binding agent can be determined according to the Chothia system, which will be referred to herein as the “Chothia
• the carboxy terminus of a VH and/or VL CDR1 , CDR2, and/or CDR3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs (according to Kabat and/or Chothia, AbM, Contact, or IMGT) described by SEQ ID NOS: 1-24, 27-41, 44-50, 53-76, 79-90 and 93-108, so long as binding to a5b1 integrin ⁇ e.g., human a5b1 integrin) is maintained ⁇ e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
• the carboxy terminus of a VH and/or VL CDR1 , CDR2, and/or CDR3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs (according to Kabat and/or Chothia, AbM, Contact, or IMGT) described by SEQ ID NOS: 1-24, 27-41, 44-50, 53-76, 79-90 and 93-108, so long as binding to a5b1 integrin ⁇ e.g., human a5b1 integrin) is maintained ⁇ e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
• the a5b1 integrin binding agents ⁇ e.g., antibodies), including human a5b1 integrin binding agents, presented herein that bind to a5b1 integrin, comprise conservative sequence modifications ⁇ e.g., modifications of one or more amino acids in one or more CDRs as described above).
• conservative sequence modifications include conservative amino acid substitutions that include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• Such diagnosis and/or detection can be accomplished, for example, by coupling an a5b1 integrin binding agent ⁇ e.g., an antibody) to detectable substances ⁇ e.g., a labeled agent, including a labeled antibody) including, for example: enzymes, including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, including, but not limited to, streptavidin/biotin or avidin/biotin; fluorescent materials, including, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, including, but not limited to, luminol; bioluminescent
• fusion proteins comprising an antigen-binding fragment of an a5b1 integrin binding agent (e.g., an antibody), including a human a5b1 integrin binding agent, described herein (e.g., comprising CDR1, CDR2, and/or CDR3 of VH and/or VL) and a heterologous protein, polypeptide, or peptide.
• an a5b1 integrin binding agent e.g., an antibody
• a human a5b1 integrin binding agent described herein (e.g., comprising CDR1, CDR2, and/or CDR3 of VH and/or VL) and a heterologous protein, polypeptide, or peptide.
• a5b1 integrin binding agents including human a5b1 integrin binding agents, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination.
• An a5b1 integrin binding agent e.g ., an antibody
• a human a5b1 integrin binding agents described herein may also be attached to solid supports, which are useful for immunoassays or purification of the target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
• selenocysteine is cotranslationally inserted into an antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids.
• an a5b1 integrin binding agent e.g., an antibody
• a human a5b1 integrin binding agent described herein is conjugated to a cytotoxic agent.
• an a5b1 integrin binding agent e.g., an antibody
• an a5b1 integrin binding agent e.g., an antibody
• a human a5b1 integrin binding agent disclosed herein can be optionally conjugated with one or more cytotoxic agent(s) disclosed herein or known in the art in order to generate an ADC.
• a multispecific (e.g ., bispecific) antibody disclosed herein comprises an a5b1 integrin binding domain that comprises the CDRs (according to Kabat and/or Chothia, AbM, Contact, or IMGT) of the VH and/or VL amino acid sequences of Table 2.
• a multispecific ⁇ e.g., bispecific antibody comprising a binding domain which binds to a5b1 integrin that comprises VH and VL CDRs ⁇ e.g., VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDR3 according to Kabat and/or Chothia, AbM, Contact, or IMGT) as set forth in Table 1.
• VL CDR3 according to Kabat and/or Chothia, AbM, Contact, or IMGT
• a multispecific (e.g ., bispecific) antibody comprising a binding domain which binds to a5b1 integrin that comprises VH and VL CDRs (e.g., VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDR3 according to Kabat and/or Chothia, AbM, Contact, or IMGT) as set forth in Table 4.
• VH and VL CDRs e.g., VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDR3 according to Kabat and/or Chothia, AbM, Contact, or IMGT
• a multispecific (e.g., bispecific) antibody comprising a binding domain which binds to a5b1 integrin that comprises VH and VL CDRs (e.g., VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDR3 according to Kabat and/or Chothia, AbM, Contact, or IMGT) as set forth in Table 6.
• VH and VL CDRs e.g., VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDR3 according to Kabat and/or Chothia, AbM, Contact, or IMGT
• a hybridoma may produce a monoclonal antibody or antibody fragment that binds a5b1 integrin.
• An a5b1 integrin binding agent (e.g., antibody) can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length a5b1 integrin binding agent (e.g., an antibody) clone using VH and/or VL sequences ⁇ e.g., the Fv sequences), or various CDR sequences from VFI and VL sequences, from the phage clone of interest and suitable constant region ⁇ e.g., Fc) sequences described in Kabat etai, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.
• human antibodies that bind a5b1 integrin may be obtained from transgenic animals that have been engineered to produce specific human antibodies in response to antigenic challenge.
• International Patent Publication No. WO 98/24893 discloses transgenic animals having a human Ig locus, wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci.
• Transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin encoding loci are substituted or inactivated also have been described.
• the present disclosure provides humanized antibodies that bind a5b1 integrin, including human a5b1 integrin.
• a humanized antibody can have one or more amino acid residues introduced into it from a source that is non human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
• FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
• the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
• the a5b1 integrin binding agent provided herein comprises a VH comprising a VH CDR1 , VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 144; and a VL comprising a VL CDR1 , VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO:145.
• the CDRs are according to Kabat numbering.
• the CDRs are according to AbM numbering.
• the CDRs are according to Chothia numbering.
• the CDRs are according to Contact numbering.
• the CDRs are according to IMGT.
• the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.
• the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:136 and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:137, and the binding of the antibody or fragment thereof to a5b1 integrin (e.g ., human a5b1 integrin) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
• a5b1 integrin e.g ., human a5b1 integrin
• a5b1 integrin e.g ., human a5b1 integrin
• a5b1 integrin e.g ., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
• the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence of SEQ ID NO: 144 and a VL comprising an amino acid sequence of SEQ ID NO: 145.
• the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence of SEQ ID NO: 146 and a VL comprising an amino acid sequence of SEQ ID NO: 147.
• an isolated cell e.g., a hybridoma, a transformed or transfected cell
• an a5b1 integrin binding agent e.g., antibody or antibody fragment
• a cell e.g., an isolated cell
• Exemplary expression control sequences include promoter/enhancer sequences, including, for example, cytomegalovirus promoter/enhancer (Lehner et al., J. Clin. Microbiol., 29: 2494-2502, 1991; Boshart et al., Cell, 41: 521-530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Then, 4: 151, 1993); Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); simian virus 40 promoter; DRA (downregulated in adenoma; Alrefai et al., Am. J. Physiol.
• polypeptides e.g., a5b1 integrin binding agents such as antibodies), including human a5b1 integrin binding agents
• polypeptides may be glycosylated or non-glycosylated and/or have been covalently modified to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
• Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.
• host cells are useful for amplifying polynucleotides and also for expressing polypeptides encoded by the polynucleotides.
• a process for the production of an a5b1 integrin binding agent may comprise culturing a host cell and isolating the a5b1 integrin binding agent.
• described herein is a method for alleviating one or more symptoms associated with an angiogenesis-mediated disease, disorder, or condition in a subject comprising administering to the subject an a5b1 integrin binding agent (e.g., an antibody) described herein or fragment thereof or a pharmaceutical composition comprising the binding agent (e.g., antibody) described herein.
• an a5b1 integrin binding agent e.g., an antibody
• a pharmaceutical composition comprising the binding agent (e.g., antibody) described herein.
• an a5b1 integrin binding agent e.g., an antibody
• an a5b1 integrin binding agent e.g., an antibody
• a pharmaceutical composition comprising an a5b1 integrin binding agent (e.g., an antibody) such as a human a5b1 integrin binding agent is, in one aspect, placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions.
• a5b1 integrin binding agent e.g., an antibody
• such instructions include a tangible expression describing the reagent concentration, as well as, in some embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.
• the immunotherapeutic agent is an agent that inhibits suppressor cells or suppressor cell activity. In some embodiments, the immunotherapeutic agent is an agent or therapy that inhibits Treg activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of inhibitory immune checkpoint receptors.
• an a5b1 integrin binding agent e.g ., an antibody
• CTLA-4 inhibitors e.g., an anti- CTLA-4 antibody, for example, ipilimumab (Yervoy), or with antibodies to cytokines, or with bispecific antibodies that bind to PD-L1 and CTLA-4 or PD-1 and CTLA-4, or with other anti-cancer agents.
• Example 1 Antibody Generation and Initial Screening for a5 Integrin Binding
• NZBW and CD-1 mice four of each, were injected with 100pg purified recombinant human a5b1 integrin heterodimer (rh-adb ⁇ ; Aero Biosystems, Newark, DE; cat. no. IT1-H52W5).
• rh-adb ⁇ purified recombinant human a5b1 integrin heterodimer
• hybridoma supernatants were incubated with the activated K562 cells for 20 minutes, washed, then incubated with a fluorescent conjugated detecting antibody for 20 minutes, washed, resuspended in 7- Aminoactinomycin D, and Mean Fluorescence Intensity (MFI) measured on a Guava
• lmmulon4 HBX ELISA 96-well plates were coated with FN by incubation overnight at 4°C with 2.5pg/mL human FN (R&D Systems, Minneapolis, MN 55413, cat. no. 1918-FN) in 1xPBS (0.01 M phosphate buffer and 0.154M NaCI, pH 7.4). Plates were then washed 3 times with Wash Buffer (1x Tris Buffered Saline containing 0.05% Tween20), blocked with 2% BSA in 1xTBS for 2 hours at room temperature (RT).
• Wash Buffer (1x Tris Buffered Saline containing 0.05% Tween20
• the chip was then blocked with 5% (w/v) non-fat milk in water overnight, and washed with 10x PBST for 10 min, 1x PBST for 10 min, and deionized water twice for 10 min before being dried under a stream of nitrogen prior to use.
• SPR measurements were performed using PlexArray FIT (Plexera Bioscience, Seattle, WA), a high-throughput surface plasmon resonance imaging (SPRi) platform. Collimated light (660 nm) passes through the coupling prism,
• ligand site competitive antagonists such as cRGD are not effective at dissociation of ligand-integrin complexes as are allosteric antagonist antibodies suggesting that an allosteric mechanism is involved in the dissociation activity seen with the antibodies tested here.
• Results are shown in FIG. 9.
• the highest concentration of antibodies tested at 3pg/ml_ inhibited adhesion ranging from 78% to 86% for the antibodies that are strong blockers of FN binding, A-15B08, A-15B08-T62A and C- 14D12, and 25% for antibody A2-7A05 that was shown to be a partial blocker of FN binding (Table 15).
• the data from this cellular adhesion blocking assay demonstrated the inhibitory effects of the present antibodies on the binding of a5b1 with Fn and on cellular adhesion.
• VH heavy chain variable
• VL light chain variable
• VH heavy chain variable
• VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 7, 12, 13, and 18;
• VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 8, 14, 19, and 24;
• VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:3, 9, 15, and 20;
• VL light chain variable
• VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:6, 17, and 23.
• VH CDR1 having the amino acid sequence of SEQ ID NO:1 ;
• VH CDR2 having the amino acid sequence of SEQ ID NO:2;
• VL light chain variable
• VL CDR1 having the amino acid sequence of SEQ ID NO:4;
• VL CDR3 having the amino acid sequence of SEQ ID NO:6.
• VH heavy chain variable
• VL light chain variable
• VL CDR3 having the amino acid sequence of SEQ ID NO:6.
• VH heavy chain variable
• VL CDR3 having the amino acid sequence of SEQ ID NO:6.
• VH heavy chain variable
• VH CDR1 having the amino acid sequence of SEQ ID NO:13;
• VH heavy chain variable
• VL CDR1 having the amino acid sequence of SEQ ID NO:21 ;
• VL light chain variable
• VL CDR3 having the amino acid sequence of SEQ ID NO:6.
• VH heavy chain variable
• VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS:27, 31 , 34, 35, and 38;
• VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:29, 32, 36, and 39;
• VL light chain variable
• VH CDR1 having the amino acid sequence of SEQ ID NO:27;
• VL light chain variable
• VL CDR2 having the amino acid sequence of SEQ ID NO: 11 ;
• VH heavy chain variable

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