WO2024097620A1 - COMPOSITIONS ET MÉTHODES IMPLIQUANT L'INTÉGRINE α3β1 - Google Patents

COMPOSITIONS ET MÉTHODES IMPLIQUANT L'INTÉGRINE α3β1 Download PDF

Info

Publication number
WO2024097620A1
WO2024097620A1 PCT/US2023/078109 US2023078109W WO2024097620A1 WO 2024097620 A1 WO2024097620 A1 WO 2024097620A1 US 2023078109 W US2023078109 W US 2023078109W WO 2024097620 A1 WO2024097620 A1 WO 2024097620A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
sequence
antibody
cdr
isolated antibody
Prior art date
Application number
PCT/US2023/078109
Other languages
English (en)
Inventor
Santiago Balza PINEDA
Darlah LOPEZ-RODRIGUEZ
Carl J. ABRAHAMS
Antonio J. Barbosa
Vineet Gupta
Original Assignee
149 Bio, Llc
Rush University Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 149 Bio, Llc, Rush University Medical Center filed Critical 149 Bio, Llc
Publication of WO2024097620A1 publication Critical patent/WO2024097620A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], 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 [IGs], 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • 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
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Integrin ⁇ 3 ⁇ 1 is a key integrin on the surface of cells, including podocytes, which are cells in Bowman’s capsule in the kidneys that wrap around capillaries of the glomerulus. Integrin ⁇ 3 ⁇ 1 is essential for podocyte attachment to the outside of blood vessels to form a healthy glomerulus in the kidney.
  • the disclosure features an isolated antibody that binds to integrin ⁇ 3 ⁇ 1 or a portion thereof, comprising: (1) a heavy chain complementarity-determining region 1 (CDR H1) comprising the sequence of X1X2SGX3TFX4X5YX6X7X8 (SEQ ID NO:38), wherein X1 is A or K; X2 is A or T; X3 is F, G, or F; X4 is S or T; X5 is S or N; X6 is G, S, or A; X7 is M or I; and X8 is H, N, or S; (2) a CDR H2 comprising a sequence having up to two amino acid substitutions relative to the sequence of GISGSADTTY (SEQ ID NO:6), SISSSSSYIY (SEQ ID NO:9)
  • the CDR H1 comprises a sequence of any one of AASGFTFSSYGMH (SEQ ID NO:1), KASGYTFTSYGIS (SEQ ID NO:2), KTSGFTFTNYGIS (SEQ ID NO:3), AASGFTFSSYSMN (SEQ ID NO:4), and KASGGTFSSYAIN (SEQ ID NO:5);
  • the CDR H2 comprises a sequence of any one of GISGSADTTY (SEQ ID NO:6), WISAYNGNTN (SEQ ID NO:7), WISANNGNSN (SEQ ID NO:8), SISSSSSYIY (SEQ ID NO:9), and GIIPIFGTAN (SEQ ID NO:10);
  • the CDR H3 comprises a sequence of any one of VRDDIQLRD (SEQ ID NO:11), ARDYSGSWYPSNGPALDY (SEQ ID NO:12), AREFPGWYFDY (SEQ ID NO:13), AREY
  • the CDR H1 comprises the sequence of SEQ ID NO:1; the CDR H2 comprises the sequence of SEQ ID NO:6; and the CDR H3 comprises the sequence of SEQ ID NO:11. [0006] In some embodiments, the CDR H1 comprises the sequence of SEQ ID NO:2; the CDR H2 comprises the sequence of SEQ ID NO:7; and the CDR H3 comprises the sequence of SEQ ID NO:12. [0007] In some embodiments, the CDR H1 comprises the sequence of SEQ ID NO:3; the CDR H2 comprises the sequence of SEQ ID NO:8; and the CDR H3 comprises the sequence of SEQ ID NO:13.
  • the CDR H1 comprises the sequence of SEQ ID NO:4; the CDR H2 comprises the sequence of SEQ ID NO:9; and the CDR H3 comprises the sequence of SEQ ID NO:14.
  • the CDR H1 comprises the sequence of SEQ ID NO:5; the CDR H2 comprises the sequence of SEQ ID NO:10; and the CDR H3 comprises the sequence of SEQ ID NO:15.
  • the CDR L1 comprises the sequence of SEQ ID NO:21; the CDR L2 comprises the sequence of SEQ ID NO:25; and the CDR L3 comprises the sequence of SEQ ID NO:28.
  • the CDR L1 comprises the sequence of SEQ ID NO:22; the CDR L2 comprises the sequence of SEQ ID NO:25; and the CDR L3 comprises the sequence of SEQ ID NO:29.
  • the CDR L1 comprises the sequence of SEQ ID NO:21; the CDR L2 comprises the sequence of SEQ ID NO:25; and the CDR L3 comprises the sequence of SEQ ID NO:30.
  • the CDR L1 comprises the sequence of SEQ ID NO:23; the CDR L2 comprises the sequence of SEQ ID NO:26; and the CDR L3 comprises the sequence of SEQ ID NO:31.
  • the CDR L1 comprises the sequence of SEQ ID NO:24; the CDR L2 comprises the sequence of SEQ ID NO:27; and the CDR L3 comprises the sequence of SEQ ID NO:32.
  • the antibody comprises a heavy chain variable region having at least 90% identity to a sequence of any one of SEQ ID NOS:16-20. In some embodiments, the antibody comprises a light chain variable region having at least 90% identity to a sequence of any one of SEQ ID NOS:33-37.
  • the antibody comprises a HCDR1 having the sequence of SEQ ID NO:1, a HCDR2 having the sequence of SEQ ID NO:6, a HCDR3 having the sequence of SEQ ID NO:11, a LCDR1 having the sequence of SEQ ID NO:21, a LCDR2 having the sequence of SEQ ID NO:25, and a LCDR3 having the sequence of SEQ ID NO:28.
  • the antibody comprises a heavy chain variable region having at least 90% identity to the sequence of SEQ ID NO:16.
  • the antibody comprises a light chain variable region having at least 90% identity to the sequence of SEQ ID NO:33.
  • the antibody comprises a HCDR1 having the sequence of SEQ ID NO:2, a HCDR2 having the sequence of SEQ ID NO:7, a HCDR3 having the sequence of SEQ ID NO:12, a LCDR1 having the sequence of SEQ ID NO:22, a LCDR2 having the sequence of SEQ ID NO:25, and a LCDR3 having the sequence of SEQ ID NO:29.
  • the antibody comprises a heavy chain variable region having at least 90% identity to the sequence of SEQ ID NO:17.
  • the antibody comprises a light chain variable region having at least 90% identity to the sequence of SEQ ID NO:34.
  • the antibody comprises a HCDR1 having the sequence of SEQ ID NO:3, a HCDR2 having the sequence of SEQ ID NO:8, a HCDR3 having the sequence of SEQ ID NO:13, a LCDR1 having the sequence of SEQ ID NO:21, a LCDR2 having the sequence of SEQ ID NO:25, and a LCDR3 having the sequence of SEQ ID NO:30.
  • the antibody comprises a heavy chain variable region having at least 90% identity to the sequence of SEQ ID NO:18.
  • the antibody comprises a light chain variable region having at least 90% identity to the sequence of SEQ ID NO:35.
  • the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO:9, a HCDR3 having the sequence of SEQ ID NO:14, a LCDR1 having the sequence of SEQ ID NO:23, a LCDR2 having the sequence of SEQ ID NO:26, and a LCDR3 having the sequence of SEQ ID NO:31.
  • the antibody comprises a heavy chain variable region having at least 90% identity to the sequence of SEQ ID NO:19.
  • the antibody comprises a light chain variable region having at least 90% identity to the sequence of SEQ ID NO:36.
  • the antibody comprises a HCDR1 having the sequence of SEQ ID NO:5, a HCDR2 having the sequence of SEQ ID NO:10, a HCDR3 having the sequence of SEQ ID NO:15, a LCDR1 having the sequence of SEQ ID NO:24, a LCDR2 having the sequence of SEQ ID NO:27, and a LCDR3 having the sequence of SEQ ID NO:32.
  • the antibody comprises a heavy chain variable region having at least 90% identity to the sequence of SEQ ID NO:20.
  • the antibody comprises a light chain variable region having at least 90% identity to the sequence of SEQ ID NO:37.
  • the antibody comprises an Fc polypeptide having at least 90% identity to a sequence of SEQ ID NO:43.
  • the antibody binds to a cell expressing integrin ⁇ 3 ⁇ 1 or a portion thereof.
  • the cell is a podocyte, a T cell, a cancer cell, or a neutrophil.
  • the antibody binds to the ⁇ 3 portion of the integrin ⁇ 3 ⁇ 1. In some embodiments, the antibody binds to a sequence within a thigh-genu region in the ⁇ 3 portion.
  • the antibody binds to the sequence of SEQ ID NO:44 or a sequence within the sequence of SEQ ID NO:44. In some embodiments, the antibody binds to a specific conformation of ⁇ 3. In some embodiments, the antibody binds ⁇ 3 and stabilizes it in a specific conformation. [0024] In certain embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a full-length antibody, a Fab, a Fab’, a F(ab’)2, an Fv, or a single chain Fv (scFv) antibody. In some embodiments, the antibody is a bispecific antibody.
  • the disclosure also provides an isolated nucleic acid encoding the isolated antibody described herein.
  • the disclosure provides an expression vector comprising the nucleic acid that encodes the isolated antibody described herein.
  • the disclosure provides an isolated host cell comprising the vector described above.
  • the disclosure provides a pharmaceutical composition comprising the isolated antibody described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides a method for treating a disease or condition associated with a loss of podocytes in a subject in need thereof, comprising administering to the subject an isolated antibody described herein.
  • the disease or condition is a kidney disease, an autoimmune disease, a cancer, or an inflammation.
  • the disease or condition is a transplant procedure.
  • the kidney disease is a glomerular disease, such as a nephritic disease, a nephrotic disease, Alport’s syndrome, or Focal Segmental Glomerulosclerosis (FSGS).
  • FSGS Focal Segmental Glomerulosclerosis
  • the disclosure features a method for identifying antibodies that bind to an integrin ⁇ 3 ⁇ 1 or a portion thereof, comprising: 1) removing antibodies that bind against the ⁇ 1 chain of the integrin ⁇ 3 ⁇ 1 in the presence or absence of a ligand mimetic peptide and/or an antibody; 2) from the remaining antibodies from step 1), selecting for antibodies that bind to the integrin ⁇ 3 ⁇ 1 in the presence or absence of a ⁇ 1 agonist antibody; 3) counter selecting for antibodies that bind to the integrin ⁇ 3 ⁇ 1 against immobilized ⁇ 1 agonist antibodies or a ligand mimetic peptide alone; and 4) repeating steps 1), 2), and 3) above to enrich for antibodies that are integrin ⁇ 3 allosteric agonists in the presence of cell surface-expressed integrin ⁇ 3 ⁇ 1.
  • the ligand mimetic peptide is LXY2.
  • step 1) and/or 3) is performed using ⁇ 1 containing integrin dimers that are not ⁇ 3 ⁇ 1, such as ⁇ 4 ⁇ 1 and ⁇ 5 ⁇ 1.
  • step 1) and/or 2) is performed using human K562 cells that primarily express human ⁇ 5 ⁇ 1 integrin and not over-express ⁇ 3 ⁇ 1.
  • step 2) and/or 3) is performed using human K562 cells that over-express ⁇ 3 ⁇ 1.
  • step 1) and/or 2) and/or 3) are performed in the presence of agents that block the ligand binding site or domain of the integrin, such as antibodies and ligands.
  • agents that block the ligand binding site or domain of the integrin such as antibodies and ligands.
  • integrin ⁇ 3 ⁇ 1 is stabilized in a specific conformation by pre- complexing it with activating or inhibitory agents, such as activating antibody 9EG7 or TS2/16.
  • integrin ⁇ 3 ⁇ 1 is stabilized in a specific conformation by pre- complexing with agents that selectively bind the beta-chain of the integrin dimer.
  • 1A-1D Binding characterization of integrin agonist antibodies by Direct Integrin ELISA.
  • Bovine Serum Albumin (BSA) Bovine Serum Albumin
  • recombinant human integrin ⁇ 3 ⁇ 1 ECD recombinant human integrin ⁇ 4 ⁇ 1 ECD
  • recombinant mouse integrin ⁇ 3 ⁇ 1 ECD were coated on the plate and incubated with human anti- ⁇ 3 Abs or isotype (n of 8 per coated protein, n of 4 for BSA).
  • FIG. 2 Epitope mapping of integrin agonist antibodies by Direct Integrin ELISA. Recombinant integrin ⁇ 3 ⁇ 1 domains or Bovine Serum Albumin (BSA) were coated on the plate and incubated with human anti-a3 Abs (red) or isotype (blue) (n of 4).
  • BSA Bovine Serum Albumin
  • FIGS. 3A-3D Increased ligand binding by mouse integrin ⁇ 3 ⁇ 1 expressing cells in the presence of agonist antibodies.
  • ⁇ 3 ⁇ 1-expressing K562 cells are incubated with ⁇ 3 ⁇ 1 ligand mimetic LXY2-biotin conjugate and either an integrin agonist antibody or isotype antibody control. Cells are then stained by Streptavidin-fluorophore conjugate and cells are measured in the flow cytometer.
  • FIGS. 4A-4E Reduced cell migration in the presence of integrin agonist antibodies by Wound Healing Assay. ⁇ 3 ⁇ 1-expressing SK-OV-3 cells are plated on ligand-coated wells and allowed to adhere for 16 hours at 37 °C. (A) Scratch wound created on the cell layer using a sterile pipette tip before adding treatment. (B) Isotype antibody control and (C) blocking anti- ⁇ 3 antibody do not reduce cell migration, allowing cells to close the wound.
  • FIG.5 Schematic illustration of domain swapped mammalian expression constructs.
  • FIGS. 6A-6C Staining of podocytes with antibodies shows that novel anti-integrin ⁇ 3 antibodies stain podocyte expressed integrin ⁇ 3 ⁇ 1.
  • Kidney sections from C57B/L6 wildtype mouse were immunofluorescently stained with various antibodies (5 ⁇ g/mL) and imaged using confocal microscopy. Representative images showing staining with either Ab74_A100 (A), 9EG7 (B), or human anti-mouse IgG1 isotype control antibody (C).
  • the antibodies can be useful for treating diseases and/or conditions associated with a loss of podocytes, e.g., a kidney disease such as nephritic disease, a nephrotic disease, Alport’s syndrome, or Focal Segmental Glomerulosclerosis (FSGS).
  • a kidney disease such as nephritic disease, a nephrotic disease, Alport’s syndrome, or Focal Segmental Glomerulosclerosis (FSGS).
  • FSGS Focal Segmental Glomerulosclerosis
  • antibody includes antibody fragments that retain binding specificity. For example, there are a number of well characterized antibody fragments.
  • pepsin digests an antibody C-terminal to the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond.
  • the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments).
  • an antibody as described herein can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antibody is IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, or IgE.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD).
  • variable light chain VL
  • variable heavy chain VH
  • substitution variants have at least one amino acid residue removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but framework alterations are also contemplated. Examples of conservative substitutions are described above.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a ⁇ -sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties: (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile; (2) Polar without charge: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His. Non-conservative substitutions are made by exchanging a member of one of these classes for another class.
  • substitution is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine.
  • alanine or serine such as, without limitation, alanine or serine.
  • substitution can be made in a CDR or framework region of a variable domain or in the constant region of an antibody.
  • the cysteine is canonical (e.g., involved in di- sulfide bond formation).
  • Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking.
  • Antibodies include VH-VL dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light region are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • VH-VL dimers including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light region are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • the single chain Fv antibody is a covalently linked VH-VL which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker (e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the VH and VL are connected to each as a single polypeptide chain, the V H and V L domains associate non-covalently. Alternatively, the antibody can be another fragment. Other fragments can also be generated, e.g., using recombinant techniques, as soluble proteins or as fragments obtained from display methods. Antibodies can also include diantibodies and miniantibodies.
  • Antibodies of the disclosure also include heavy chain dimers, such as antibodies from camelids.
  • an antibody is dimeric.
  • the antibody may be in a monomeric form that has an active isotype.
  • the antibody is in a multivalent form, e.g., a trivalent or tetravalent form.
  • the terms “variable region” and “variable domain” refer to the portions of the light and heavy chains of an antibody that include amino acid sequences of complementary determining regions (CDRs, e.g., HCDR1, HCDR2, HCR3, LCDR1, LCDR2, and LCDR3) and framework regions (FRs).
  • CDRs complementary determining regions
  • the variable region for the heavy and light chains is commonly designated VH and VL, respectively.
  • variable region is included on Fab, F(ab’) 2 , Fv and scFv antibody fragments described herein, and involved in specific antigen recognition.
  • CDR complementarity-determining region
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
  • the amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, North method (see, e.g., North et al., J Mol Biol.
  • antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci.
  • allosteric agonist refers to a molecule (e.g., an antibody) that binds to its target (e.g., integrin ⁇ 3 ⁇ 1 or a portion thereof, a sequence within ⁇ 3 portion of the integrin ⁇ 3 ⁇ 1, a sequence of SEQ ID NO:44 or a portion thereof), at a site or area that is not the target’s active site, to enhance, activate, or increase the target’s response to the binding of its natural ligand.
  • target e.g., integrin ⁇ 3 ⁇ 1 or a portion thereof, a sequence within ⁇ 3 portion of the integrin ⁇ 3 ⁇ 1, a sequence of SEQ ID NO:44 or a portion thereof
  • chimeric antibody refers to an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region, or portion thereof, having a different or altered antigen specificity; or with corresponding sequences from another species or from another antibody class or subclass.
  • humanized antibody refers to an immunoglobulin molecule in CDRs from a donor antibody are grafted onto human framework sequences. Humanized antibodies may also comprise residues of donor origin in the framework sequences. The humanized antibody can also comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • Humanization can be performed using methods known in the art (e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Patent No. 4,816,567), including techniques such as “superhumanizing” antibodies (Tan et al., J. Immunol. 169: 1119, 2002) and "resurfacing” (e.g., Staelens et al., Mol. Immunol.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • antigen a molecule, compound, or complex that is recognized by an antibody, i.e., can be specifically bound by the antibody.
  • the term can refer to any molecule that can be specifically recognized by an antibody, e.g., a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.).
  • a polypeptide polynucleotide
  • carbohydrate e.g., lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.).
  • Antibodies bind to an “epitope” on an antigen.
  • the epitope is the localized site on the antigen that is recognized and bound by the antibody.
  • Epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids.
  • the epitope includes non-protein components, e.g., from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three- dimensional moiety.
  • the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope).
  • a discontinuous epitope e.g., a discontinuous epitope.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, Glenn E. Morris, Ed (1996).
  • the terms “specific for,” “specifically binds,” and like terms refer to a molecule (e.g., antibody or antibody fragment) that binds to a target with at least 2-fold greater affinity than non-target compounds, e.g., at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater affinity.
  • a target e.g., antibody or antibody fragment
  • an antibody that specifically binds a target will typically bind the target with at least a 2-fold greater affinity than a non-target.
  • Specificity can be determined using standard methods, e.g., solid-phase ELISA immunoassays (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • an antibody target e.g., antigen, analyte, immune complex
  • an antibody that binds a given antibody target typically binds to at least 2/3 of the antibody targets in a solution (e.g., at least any of 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%).
  • a solution e.g., at least any of 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.
  • a “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample.
  • a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control).
  • a control can also represent an average value or a range gathered from a number of tests or results.
  • controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of benefit and/or side effects). Controls can be designed for in vitro applications.
  • Controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site ncbi.nlm.nih.gov/BLAST/ or the like).
  • sequences are then said to be “substantially identical.” As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 or more amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • default program parameters can be used, or alternative parameters can be designated.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art.
  • An algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Antibodies that bind integrin ⁇ 3 ⁇ 1 or a portion thereof Antibodies (including antibody fragments) that specifically bind to integrin ⁇ 3 ⁇ 1 or a portion thereof (e.g., a sequence within a thigh-genu region of integrin ⁇ 3 ⁇ 1) are provided herein. Integrin ⁇ 3 ⁇ 1 is an integrin heterodimer of ⁇ 3 and ⁇ 1 portions.
  • Integrin ⁇ 3 ⁇ 1 is highly expressed on the surface of kidney podocyte cells and is essential for podocyte attachment to the outside of blood vessels to form a healthy glomerulus in the kidney. This integrin is also expressed on other cells, such as T-cells (Park et al., Integrin ⁇ 3 promotes TH17 cell polarization and extravasation during autoimmune neuroinflammation, Science Immunology, Vol 8 (88), 2023), cancer cells (Ke et al., Novel monoclonal antibody against integrin ⁇ 3 shows therapeutic potential for ovarian cancer, Cancer Sci., 111 (10), p3478, 2020) and neutrophils (Lerman et al., Sepsis lethality via exacerbated tissue infiltration and TLR-induced cytokine production by neutrophils is integrin ⁇ 3 ⁇ 1-dependent, Blood, 2014 Dec 4;124(24):3515-23) and keratinocytes (Has et al, Integrin a3 mutations with kidney, lung, and skin disease
  • the antibodies described herein act as allosteric agonist antibodies to integrin ⁇ 3 ⁇ 1 and can enhance integrin-dependent ligand binding and cell adhesion, thus, preventing podocyte cell loss in the urine and protecting from loss in kidney function.
  • the antibodies can also reduce T-cell transmigration and infiltration to reduce autoimmune diseases, cancer cell migration to reduce tumor growth and metastases, and pro-inflammatory neutrophil activation and tissue recruitment.
  • the anti- ⁇ 3 ⁇ 1 antibody is isolated (e.g., separated from a component of its natural environment (e.g., an animal, a biological sample)).
  • the anti- ⁇ 3 ⁇ 1 antibody is a humanized antibody, or an antigen binding fragment thereof. In some embodiments, the anti- ⁇ 3 ⁇ 1 antibody is a derivative of a humanized antibody that binds ⁇ 3 ⁇ 1 or a portion thereof. In some embodiments, the anti- ⁇ 3 ⁇ 1 antibody binds ⁇ 3 ⁇ 1 under laboratory conditions (e.g., binds ⁇ 3 ⁇ 1 in vitro, binds ⁇ 3 ⁇ 1 in a flow cytometry assay, binds ⁇ 3 ⁇ 1 in an ELISA).
  • the anti- ⁇ 3 ⁇ 1 antibody binds ⁇ 3 ⁇ 1 under physiological conditions (e.g., binds ⁇ 3 ⁇ 1 in a cell (e.g., a podocyte) in a subject).
  • the ⁇ 3 portion of the heterodimer integrin ⁇ 3 ⁇ 1 has a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of: MGPGPSRAPRAPRLMLCALALMVAAGGCVVSAFNLDTRFLVVKEAGNPGSLFGYS VALHRQTERQQRYLLLAGAPRELAVPDGYTNRTGAVYLCPLTAHKDDCERMNITV KNDPGHHIIEDMWLGVTVASQGPAGRVLVCAHRYTQVLWSGSEDQRRMVGKCYV RGNDLELDSSDDWQTYHNEMCNSNTDYLETGMCQLGTSGGFTQN
  • the ⁇ 1 portion of the heterodimer integrin ⁇ 3 ⁇ 1 has a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of: MNLQPIFWIGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEG MPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQI QPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLM NEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKG EVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAG
  • the antibody binds to the ⁇ 3 portion (e.g., SEQ ID NO:45) of the integrin ⁇ 3 ⁇ 1. In some embodiments, the antibody binds to a sequence within a thigh- genu region in the ⁇ 3 portion. In particular embodiments, the antibody binds to the sequence of SEQ ID NO:44 or a portion within the sequence of SEQ ID NO:44.
  • the anti- ⁇ 3 ⁇ 1 antibodies provided herein comprise at least one immunoglobulin heavy chain variable region and at least one immunoglobulin light chain variable region.
  • an anti- ⁇ 3 ⁇ 1 antibody described herein comprises two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions.
  • each immunoglobulin heavy chain variable region of the anti- ⁇ 3 ⁇ 1 antibody comprises first, second, and third heavy chain complementarity determining regions (CDRs; HCDR1, HCDR2, and HCDR3)
  • each immunoglobulin light chain variable region of the anti- ⁇ 3 ⁇ 1 antibody comprises first, second, and third light chain CDRs (LCDR1, LCDR2, and LCDR3).
  • the antibodies are antibody fragments such as Fab, F(ab’)2, Fv or scFv.
  • the antibody fragments can be generated using any means known in the art including, chemical digestion (e.g., papain or pepsin) and recombinant methods.
  • the antibodies can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, and HeLa cells lines and myeloma cell lines.
  • antibodies of the disclosure can comprise sequences of a heavy chain complementary determining region 1 (HCDR1), an HCDR2, an HCDR3, a light chain complementary determining region 1 (LCDR1), a LCDR2, a LCDR3, a heavy chain variable region (VH), and/or a light chain variable region (VL) as described in Table 1.
  • HCDR1 heavy chain complementary determining region 1
  • LCDR2 light chain complementary determining region 1
  • LCDR2 a LCDR3, a heavy chain variable region (VH), and/or a light chain variable region (VL) as described in Table 1.
  • an antibody of the disclosure comprises: an HCDR1 having a sequence of any one of SEQ ID NOS:1-5 or a variant thereof that has a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS:1-5.
  • an antibody of the disclosure comprises: an HCDR2 having a sequence of any one of SEQ ID NOS:6-10 or a variant thereof that has a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS:6-10.
  • an antibody of the disclosure comprises: an HCDR3 having a sequence of any one of SEQ ID NOS:11-15 or a variant thereof that has a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS:11-15.
  • an antibody of the disclosure comprises: a LCDR1 having a sequence of any one of SEQ ID NOS:21-24 or a variant thereof that has a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS:21-24.
  • an antibody of the disclosure comprises: a LCDR2 having a sequence of any one of SEQ ID NOS:25-27 or a variant thereof that has a sequence having one substitution relative to a sequence of any one of SEQ ID NOS:25-27.
  • an antibody of the disclosure comprises: a LCDR3 having a sequence of any one of SEQ ID NOS:28-32 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:28-32.
  • an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:1 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:1, an HCDR2 having the sequence of SEQ ID NO:6 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:6, and an HCDR3 having the sequence of SEQ ID NO:11 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:11.
  • an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:2 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:2, an HCDR2 having the sequence of SEQ ID NO:7 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:7, and an HCDR3 having the sequence of SEQ ID NO:12 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:12.
  • an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:3 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO:8 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:8, and an HCDR3 having the sequence of SEQ ID NO:13 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:13.
  • an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:4 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:4, an HCDR2 having the sequence of SEQ ID NO:9 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:9, and an HCDR3 having the sequence of SEQ ID NO:14 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:14.
  • an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:5 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:5, an HCDR2 having the sequence of SEQ ID NO:10 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:10, and an HCDR3 having the sequence of SEQ ID NO:15 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:15.
  • An antibody of the disclosure can comprise a heavy chain variable region (VH) having an HCDR1, an HCDR2, and an HCDR3 as described herein.
  • VH heavy chain variable region
  • an antibody of the disclosure can comprise a heavy chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS:16-20.
  • an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:1, 6, and 11, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:16.
  • an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:2, 7, and 12, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:17.
  • an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 8, and 13, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:18.
  • an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:4, 9, and 14, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:19.
  • an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:5, 10, and 15, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:20.
  • an antibody of the disclosure can comprise an LCDR1 having the sequence of SEQ ID NO:21 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:21, an LCDR2 having the sequence of SEQ ID NO:25 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:25, and an LCDR3 having the sequence of SEQ ID NO:28 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:28.
  • an antibody of the disclosure can comprise an LCDR1 having the sequence of SEQ ID NO:22 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:22, an LCDR2 having the sequence of SEQ ID NO:25 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:25, and an LCDR3 having the sequence of SEQ ID NO:29 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:29.
  • an antibody of the disclosure can comprise an LCDR1 having the sequence of SEQ ID NO:21 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:21, an LCDR2 having the sequence of SEQ ID NO:25 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:25, and an LCDR3 having the sequence of SEQ ID NO:30 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:30.
  • an antibody of the disclosure can comprise an LCDR1 having the sequence of SEQ ID NO:23 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:23, an LCDR2 having the sequence of SEQ ID NO:26 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:26, and an LCDR3 having the sequence of SEQ ID NO:31 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:31.
  • an antibody of the disclosure can comprise an LCDR1 having the sequence of SEQ ID NO:24 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:24, an LCDR2 having the sequence of SEQ ID NO:27 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:27, and an LCDR3 having the sequence of SEQ ID NO:32 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:32.
  • An antibody of the disclosure can comprise a light chain variable region (VL) having a LCDR1, a LCDR2, and a LCDR3 as described herein.
  • VL light chain variable region
  • an antibody of the disclosure can comprise a light chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS:33-37.
  • an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:21, 25, and 28, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:33.
  • an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:22, 25, and 29, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:34.
  • an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:21, 25, and 30, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:35.
  • an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:23, 26, and 31, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:36.
  • an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:24, 27, and 32, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:37.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:1 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:1; (2) an HCDR2 having the sequence of SEQ ID NO:6 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:6; (3) an HCDR3 having the sequence of SEQ ID NO:11 or a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:11; (4) a LCDR1 having the sequence of SEQ ID NO:21 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:21; (5) a LCDR2 having the sequence of SEQ ID NO:25 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:25; and (6) a LCDR3 having the sequence of SEQ ID NO:28 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:28.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:1; (2) an HCDR2 having the sequence of SEQ ID NO:6; (3) an HCDR3 having the sequence of SEQ ID NO:11; (4) a LCDR1 having the sequence of SEQ ID NO:21; (5) a LCDR2 having the sequence of SEQ ID NO:25; and (6) a LCDR3 having the sequence of SEQ ID NO:28.
  • the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:1, 6, and 11, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:16, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:21, 25, and 28, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:33.
  • the antibody comprises a heavy chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:47: EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWLSGISGSAD TTYYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCVRDDIQLRDWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
  • the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:16 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:47; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:33 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48.
  • a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:16 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:2 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:2; (2) an HCDR2 having the sequence of SEQ ID NO:7 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO:12 or a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:12; (4) a LCDR1 having the sequence of SEQ ID NO:22 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:22; (5) a LCDR2 having the sequence of SEQ ID NO:25 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:25; and (6) a LCDR3 having the sequence of SEQ ID NO:29 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:29.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:2; (2) an HCDR2 having the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO:12; (4) a LCDR1 having the sequence of SEQ ID NO:22; (5) a LCDR2 having the sequence of SEQ ID NO:25; and (6) a LCDR3 having the sequence of SEQ ID NO:29.
  • the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:2, 7, and 12, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:17, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:22, 25, and 29, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:34.
  • the antibody comprises a heavy chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYN GNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYSGSWYPSNGP ALDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
  • the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:17 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:34 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:50.
  • a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:17 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:50.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO:8 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:8; (3) an HCDR3 having the sequence of SEQ ID NO:13 or a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:13; (4) a LCDR1 having the sequence of SEQ ID NO:21 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:21; (5) a LCDR2 having the sequence of SEQ ID NO:25 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:25; and (6) a LCDR3 having the sequence of SEQ ID NO:30 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:30.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO:8; (3) an HCDR3 having the sequence of SEQ ID NO:13; (4) a LCDR1 having the sequence of SEQ ID NO:21; (5) a LCDR2 having the sequence of SEQ ID NO:25; and (6) a LCDR3 having the sequence of SEQ ID NO:30.
  • the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 8, and 13, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:18, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:21, 25, and 30, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:35.
  • the antibody comprises a heavy chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:51: EVQLVQSGAEVKKPGASVKVSCKTSGFTFTNYGISWVRQAPGQGLEWMGWISANN GNSNYAQDHQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREFPGWYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
  • the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:18 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:51; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:35 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52.
  • a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:18 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:4 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:4; (2) an HCDR2 having the sequence of SEQ ID NO:9 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:9; (3) an HCDR3 having the sequence of SEQ ID NO:14 or a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:14; (4) a LCDR1 having the sequence of SEQ ID NO:23 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:26 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:26; and (6) a LCDR3 having the sequence of SEQ ID NO:31 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:31.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:4; (2) an HCDR2 having the sequence of SEQ ID NO:9; (3) an HCDR3 having the sequence of SEQ ID NO:14; (4) a LCDR1 having the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:26; and (6) a LCDR3 having the sequence of SEQ ID NO:31.
  • the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:4, 9, and 14, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:19, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:23, 26, and 31, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:36.
  • the antibody comprises a heavy chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAREYYDFWSGYPSGYAF DIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVSV
  • the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:19 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:36 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:54.
  • a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:19 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:54.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:5 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:5; (2) an HCDR2 having the sequence of SEQ ID NO:10 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:10; (3) an HCDR3 having the sequence of SEQ ID NO:15 or a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:15; (4) a LCDR1 having the sequence of SEQ ID NO:24 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:24; (5) a LCDR2 having the sequence of SEQ ID NO:27 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:27; and (6) a LCDR3 having the sequence of SEQ ID NO:32 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:32.
  • an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:5; (2) an HCDR2 having the sequence of SEQ ID NO:10; (3) an HCDR3 having the sequence of SEQ ID NO:15; (4) a LCDR1 having the sequence of SEQ ID NO:24; (5) a LCDR2 having the sequence of SEQ ID NO:27; and (6) a LCDR3 having the sequence of SEQ ID NO:32.
  • the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:5, 10, and 15, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:20, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:24, 27, and 32, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:37.
  • the antibody comprises a heavy chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGT ANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGVPSGSGYYLGLDYW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTV
  • the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:20 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:37 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:56.
  • a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:20 and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:56.
  • An anti- ⁇ 3 ⁇ 1 antibody provided herein can comprise a fragment crystallizable region (Fc region), also referred to as an Fc polypeptide herein.
  • An Fc polypeptide is part of each of the two heavy chains in the antibody and can interact with certain cell surface receptors and certain components of the complement system.
  • An Fc polypeptide typically includes the CH2 domain and the CH3 domain, which are immunoglobulin constant region domain polypeptides.
  • the Fc polypeptide in an antibody described herein can be a wild-type Fc polypeptide, e.g., a human IgG1 Fc polypeptide.
  • an antibody described herein can comprise a wild-type Fc polypeptide having the sequence of SEQ ID NO:43: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
  • an antibody described herein can comprise a variant of the wild-type Fc polypeptide that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity to the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO:43) and at least one amino acid substitution relative to the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO:43).
  • an Fc polypeptide includes one or more modifications (e.g., one or more amino acid substitutions, insertions, or deletions relative to a comparable wild- type Fc region).
  • Antibodies comprising modified Fc polypeptides typically have altered phenotypes relative to antibodies comprising wild-type Fc polypeptides.
  • antibodies comprising modified Fc polypeptides can have altered serum half-life, altered stability, altered susceptibility to cellular enzymes, and/or altered effector function (e.g., as assayed in an NK-dependent or macrophage-dependent assay).
  • an Fc polypeptide in an antibody described herein can include amino acid substitutions that modulate effector function.
  • an Fc polypeptide in an antibody described herein can include amino acid substitutions that reduce or eliminate effector function.
  • Illustrative Fc polypeptide amino acid substitutions that reduce effector function include, but are not limited to, substitutions in a CH2 domain, e.g., at positions 4 and 5 (position numbering relative to the sequence of SEQ ID NO:43) (see, e.g., Lund et al., J Immunol. 147(8):2657-62, 1991).
  • one or both Fc polypeptides in an antibody described herein can comprise L4A and L5A substitutions.
  • Additional Fc polypeptide amino acid substitutions that modulate an effector function include, e.g., substitution at position 99 (position numbering relative to the sequence of SEQ ID NO:43).
  • one or both Fc polypeptides in an antibody described herein can comprise P99G substitution.
  • one or both Fc polypeptides in an antibody described herein can have L4A, L5A, and P99G substitutions.
  • an Fc polypeptide includes one or more modifications that alter (relative to a wild-type Fc polypeptide) the Ratio of Affinities of the modified Fc polypeptide to an activating Fc ⁇ R (such as Fc ⁇ RIIA or Fc ⁇ RIIIA) relative to an inhibiting Fc ⁇ R (such as Fc ⁇ RIIB): .
  • an anti- ⁇ 3 ⁇ 1 antibody herein may have particular use in providing a therapeutic or prophylactic treatment of a disease, disorder, or infection, or the amelioration of a symptom thereof, where an enhanced efficacy of effector cell function (e.g., ADCC) mediated by Fc ⁇ R is desired, e.g., cancer or infectious disease.
  • ADCC effector cell function
  • an anti- ⁇ 3 ⁇ 1 antibody herein may have particular use in providing a therapeutic or prophylactic treatment of a disease or disorder, or the amelioration of a symptom thereof, where a decreased efficacy of effector cell function mediated by Fc ⁇ R is desired, e.g., autoimmune or inflammatory disorders.
  • Table 2 lists examples of single, double, triple, quadruple, and quintuple amino acid substitutions in an Fc polypeptide that provide a Ratio of Affinities greater than 1 or less than 1 (see e.g., PCT Publication Nos.
  • Antibodies that competitively bind with an anti- ⁇ 3 ⁇ 1 antibody are also provided herein.
  • anti- ⁇ 3 ⁇ 1 antibodies that competitively bind, or are capable of competitively binding e.g., competitor antibodies
  • an antibody i.e., competitor antibody
  • an antibody i.e., competitor antibody
  • an antibody may be considered to compete for binding to ⁇ 3 ⁇ 1 when the competitor binds to the exact same region of ⁇ 3 ⁇ 1 as an anti- ⁇ 3 ⁇ 1 antibody described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)).
  • an antibody i.e., competitor antibody
  • an antibody may be considered capable of competing for binding to ⁇ 3 ⁇ 1 when the competitor binds to the same general region of ⁇ 3 ⁇ 1 as an anti- ⁇ 3 ⁇ 1 antibody described herein (i.e., a sequence within a thigh-genu region of integrin ⁇ 3 ⁇ 1) under suitable assay conditions.
  • an antibody i.e., competitor agent
  • an antibody may be considered capable of competing for binding to ⁇ 3 ⁇ 1 when the competitor binds to the exact same region of ⁇ 3 ⁇ 1 as an anti- ⁇ 3 ⁇ 1 antibody described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)) under suitable assay conditions.
  • an antibody i.e., competitor antibody
  • an antibody may be considered to compete for binding to ⁇ 3 ⁇ 1 when the competitor blocks the binding of one or more anti- ⁇ 3 ⁇ 1 antibodies described herein to ⁇ 3 ⁇ 1, for example, under suitable assay conditions.
  • Whether a competitor blocks the binding of one or more anti- ⁇ 3 ⁇ 1 antibodies described herein to ⁇ 3 ⁇ 1 may be determined using a suitable competition assay or blocking assay, such as, for example, a blocking assay as described in herein.
  • a competitor antibody may block binding of one or more anti- ⁇ 3 ⁇ 1 antibodies described herein to ⁇ 3 ⁇ 1 in a competition or blocking assay by 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%), and conversely, one or more anti- ⁇ 3 ⁇ 1 antibodies described herein may block binding of the competitor antibody to ⁇ 3 ⁇ 1 in a competition or blocking assay by about 50% or more (e.g., e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%).
  • an antibody i.e., competitor antibody
  • an antibody may be considered to compete for binding to ⁇ 3 ⁇ 1 when the competitor binds to ⁇ 3 ⁇ 1 with a similar affinity as one or more anti- ⁇ 3 ⁇ 1 antibodies described herein, for example, under suitable assay conditions.
  • an antibody i.e., competitor antibody
  • an antibody is considered to compete for binding to ⁇ 3 ⁇ 1 when the competitor binds to ⁇ 3 ⁇ 1 with an affinity that is at least about 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the affinity of one or more anti- ⁇ 3 ⁇ 1 antibodies described herein.
  • anti- ⁇ 3 ⁇ 1 antibodies that bind to, or are capable of binding to, the same epitope as one or more anti- ⁇ 3 ⁇ 1 antibodies described herein.
  • anti- ⁇ 3 ⁇ 1 antibodies that compete with one or more anti- ⁇ 3 ⁇ 1 antibodies described herein for binding to the same epitope (e.g., same peptide (linear epitope) or same surface amino acids (conformational epitope)) on ⁇ 3 ⁇ 1.
  • epitope competitors Such antibodies that bind the same epitope may be referred to as epitope competitors.
  • Polyclonal and monoclonal antibodies may be raised in animals (vertebrate or invertebrates, including mammals, birds and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a relevant antigen and an adjuvant.
  • animals vertebrate or invertebrates, including mammals, birds and fish, including cartilaginous fish
  • sc subcutaneous
  • ip intraperitoneal
  • a protein or other carrier that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor
  • Non-protein carriers e.g., colloidal gold
  • Animals can be immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with one-fifth to one- tenth of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • Monoclonal antibodies may be made using a hybridoma, e.g., the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by other methods such as recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).
  • a mouse or other appropriate host animal such as a hamster or macaque monkey
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as SP-2 or X63- Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation, by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA), or by flow cytometric analysis of cells expressing the membrane antigen.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • cDNA may be prepared from mRNA and the cDNA then subjected to DNA sequencing.
  • the hybridoma cells serve as a preferred source of such genomic DNA or RNA for cDNA preparation.
  • the DNA may be placed into expression vectors, which are well known in the art, and which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclo
  • the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts.
  • a humanized antibody i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., PNAS USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec.
  • polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments.
  • Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells.
  • the CDRs for producing the immunoglobulins of the present disclosure can be similarly derived from monoclonal antibodies capable of specifically binding to ⁇ 3 ⁇ 1.
  • Amino acid sequence variants of the anti- ⁇ 3 ⁇ 1 antibody can be prepared by introducing appropriate nucleotide changes into the anti- ⁇ 3 ⁇ 1 antibody DNA, or by peptide synthesis.
  • Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti- ⁇ 3 ⁇ 1 antibodies for the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processes of the humanized or variant anti- ⁇ 3 ⁇ 1 antibody, such as changing the number or position of glycosylation sites.
  • alanine scanning mutagenesis One method for identification of certain residues or regions of the anti- ⁇ 3 ⁇ 1 antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis,” as described by, e.g., Cunningham and Wells, Science, 244:1081-1085 (1989).
  • a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably Ala or poly- Ala) to affect the interaction of the amino acids with ⁇ 3 ⁇ 1 antigen (e.g., a sequence within a thigh-genu region of integrin ⁇ 3 ⁇ 1).
  • amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an N-terminal methionyl residue or the antibody fused to an epitope tag.
  • Other insertional variants include the fusion of an enzyme or a polypeptide that increases the serum half-life of the antibody to the N- or C-terminus of the antibody.
  • Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred, but more substantial changes may be introduced and the products may be screened.
  • substitutions are listed below: Ala (A): Val; Leu; Ile; Val Arg (R): Lys; Gln; Asn; Lys Asn (N): Gln; His; Asp, Lys; Gln; Arg Asp (D): Glu; Asn Cys (C): Ser; Ala Gln (Q): Asn; Glu Glu (E): Asp; Gln Gly (G): Ala His (H): Asn; Gln; Lys; Arg Ile (I): Leu; Val; Met; Ala; Leu; Phe; Norleucine Leu (L): Norleucine; Ile; Val; Ile; Met; Ala; Phe Lys (K): Arg; Gln; Asn Met (M): Leu; Phe; Ile Phe (F): Leu; Val; Ile; Ala; Tyr Pro (P): Ala Ser (S): Thr Thr (T): Ser Tr
  • Naturally occurring residues are divided into groups based on common side- chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: Asn, Gln, His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe [0130] Non-conservative substitutions will entail exchanging a member of one of the above classes for another class. [0131] Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.
  • the antibody variants thus generated can be displayed in the monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle.
  • the phage- displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • alanine- scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one of more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • Glycosylation of antibodies is typically either N-linked and/or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the most common recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O- linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used.
  • Addition of glycosylation sites to the antibody can be accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate), or a cytotoxic drug.
  • Conjugates can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyldithi
  • Anti- ⁇ 3 ⁇ 1 antibodies may be formulated as immunoliposomes.
  • Liposomes containing an antibody are prepared by methods know in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of an antibody provided herein can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem.257:286- 288 (1982) via a disulfide interchange reaction. Another active ingredient is optionally contained within the liposome.
  • Enzymes or other polypeptides can be covalently bound to an anti- ⁇ 3 ⁇ 1 antibody by techniques well known in the art such as the use of the heterobifunctional cross-linking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody provided herein linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984)).
  • the antibody fragment in order to increase its serum half-life.
  • This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis; see, e.g., WO96/32478 published Oct.17, 1996).
  • a modification can optionally be introduced into the antibodies (e.g., within the polypeptide chain or at either the N- or C-terminal), e.g., to extend in vivo half-life, such as PEGylation or incorporation of long-chain polyethylene glycol polymers (PEG).
  • PEG polyethylene glycol polymers
  • Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the polypeptides, for example, to prevent rapid filtration into the urine.
  • a lysine residue in the sequence is conjugated to PEG directly or through a linker.
  • linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain.
  • An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue is then site- specifically attached to a PEG chain containing, for example, a maleimide function.
  • Methods for incorporating PEG or long chain polymers of PEG are known in the art (described, for example, in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et al., Adv. Drug Deliv. Rev.55: 217-50 (2003); Roberts, M. J., et al., Adv. Drug Deliv.
  • Covalent modifications of an anti- ⁇ 3 ⁇ 1 antibody are also included within the scope of this technology. For example, modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of an anti- ⁇ 3 ⁇ 1 antibody. Other types of covalent modifications of an antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • Example covalent modifications of polypeptides are described in U.S. Pat. No. 5,534,615, specifically incorporated herein by reference.
  • a preferred type of covalent modification of the antibody comprises linking the antibody to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in, e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • Nucleic acids, vectors, host cells, and recombinant methods [0140] The disclosure also provides isolated nucleic acids encoding an anti- ⁇ 3 ⁇ 1 antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody.
  • a nucleic acid herein may include one or more subsequences, each referred to as a polynucleotide.
  • nucleic acids e.g., isolated nucleic acids
  • a nucleic acid encodes an immunoglobulin heavy chain variable domain of an anti- ⁇ 3 ⁇ 1 antibody provided herein.
  • a nucleic acid encodes an immunoglobulin light chain variable domain of an anti- ⁇ 3 ⁇ 1 antibody provided herein.
  • a nucleic acid encodes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of an anti- ⁇ 3 ⁇ 1 antibody provided herein.
  • a nucleic acid comprises a nucleotide sequence that encodes an amino acid sequence of any one of SEQ ID NOS: 1-37.
  • DNA encoding an anti- ⁇ 3 ⁇ 1 antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, and origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Suitable host cells for cloning or expressing DNA in vectors herein can be prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
  • E. coli cloning host is E.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti- ⁇ 3 ⁇ 1 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • K. lactis K. fragilis
  • K. bulgaricus ATCC 16,045)
  • K. wickeramii ATCC 24,178
  • K. waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K.
  • Suitable host cells for the expression of anti- ⁇ 3 ⁇ 1 antibodies can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells.
  • baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk moth) have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present technology, particularly for transfection of Spodoptera frugiperda cells.
  • Suitable host cells for the expression of anti- ⁇ 3 ⁇ 1 antibodies also may include vertebrate cells (e.g., mammalian cells). Vertebrate cells may be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.
  • Host cells may be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Host cells used to produce antibodies provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of the media described in Ham et al., Meth. Enz.58:44 (1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., EMBO J. 5:15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • Bakerbond ABX.TM. resin J. T. Baker, Phillipsburg, N.J. is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between, e.g., about 2.5-4.5, and may be performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • elution buffer at a pH between, e.g., about 2.5-4.5
  • salt concentrations e.g., from about 0-0.25M salt.
  • Anti- ⁇ 3 ⁇ 1 antibodies may be formulated in a pharmaceutical composition that is useful for a variety of purposes, including the treatment of diseases or disorders.
  • Pharmaceutical compositions comprising one or more anti- ⁇ 3 ⁇ 1 antibodies may be administered using a pharmaceutical device to a patient in need thereof, and according to one embodiment of the technology, kits are provided that include such devices. Such devices and kits may be designed for routine administration, including self-administration, of the pharmaceutical compositions herein.
  • Therapeutic formulations of an antibody may be prepared for storage by mixing the agent or antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues ) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • Formulations for in vivo administration generally are sterile. This may be accomplished for instance by filtration through sterile filtration membranes, for example.
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent/antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule.
  • sustained-release matrices examples include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • polyesters for example, poly(2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)
  • polylactides U.S. Pat. No.3,773,919
  • copolymers of L-glutamic acid and gamma ethyl-L-glutamate non-
  • stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • anti- ⁇ 3 ⁇ 1 antibodies provided herein are administered to a mammal, e.g., a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra- cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • a mammal e.g., a human
  • a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra- cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the appropriate dosage of agent or antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventative or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody may be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily or weekly dosage might range from about 1 ⁇ g/kg to about 20 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods using the antibody to determine ⁇ 3 ⁇ 1 levels in bodily fluids or tissues may be used in order to optimize patient exposure to the therapeutic antibody.
  • a composition comprising an anti- ⁇ 3 ⁇ 1 antibody herein can be administered as a monotherapy, and in some embodiments, the composition comprising the anti- ⁇ 3 ⁇ 1 antibody can be administered as part of a combination therapy.
  • the effectiveness of the antibody in preventing or treating diseases may be improved by administering the antibody serially or in combination with another drug that is effective for those purposes, such as a chemotherapeutic drug for treatment of cancer or a microbial infection.
  • the anti- ⁇ 3 ⁇ 1 antibody may serve to enhance or sensitize cells to chemotherapeutic treatment, thus permitting efficacy at lower doses and with lower toxicity.
  • Certain combination therapies include, in addition to administration of the composition comprising an antibody that reduces the number of ⁇ 3 ⁇ 1-expressing cells, delivering a second therapeutic regimen selected from the group consisting of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing.
  • a second therapeutic regimen selected from the group consisting of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing.
  • Such other agents may be present in the composition being administered or may be administered separately.
  • the anti- ⁇ 3 ⁇ 1 antibody may be suitably administered serially or in combination with the other agent or modality, e.g., chemotherapeutic drug or radiation for treatment of cancer, infection, and the like, or an immunosuppressive drug.
  • integrin ⁇ 3 ⁇ 1 is a key integrin on the surface of podocytes, which are cells in Bowman’s capsule in the kidneys that wrap around capillaries of the glomerulus. Integrin ⁇ 3 ⁇ 1 is essential for podocyte attachment to the outside of blood vessels to form a healthy glomerulus in the kidney.
  • the antibodies described herein can act as allosteric agonist antibodies to integrin ⁇ 3 ⁇ 1 and can enhance integrin-dependent ligand binding and cell adhesion, thus, preventing cell loss in the urine and protecting from loss in kidney function.
  • diseases and/or conditions associated with a loss of podocytes in a subject in need thereof by administering to the subject an anti- ⁇ 3 ⁇ 1 antibody described herein that binds to integrin ⁇ 3 ⁇ 1 or a portion thereof (e.g., a sequence within a thigh- genu region of integrin ⁇ 3 ⁇ 1).
  • diseases and/or conditions that are associated with a loss of podocytes can be diseases and/or conditions that are caused by a loss of podocytes (i.e., a loss in cell number and/or a loss in cell function).
  • diseases and/or conditions that are associated with a loss of podocytes can be diseases and/or conditions that affect the kidney, and thus, the loss of podocytes (i.e., a loss in cell number and/or a loss in cell function) can be a result or manifestation of the kidney disease and/or condition.
  • the subject has a kidney disease that is associated with a loss of podocytes (i.e., a loss in cell number and/or a loss in cell function).
  • a kidney disease can be a glomerular disease, such as a nephritic disease, a nephrotic disease, Alport’s syndrome, or Focal Segmental Glomerulosclerosis (FSGS).
  • the subject is undergoing, has undergone, or about to undergo a transplant procedure.
  • the transplant procedure is kidney transplant.
  • the antibody is administered after the transplant procedure.
  • the antibody is administered after the kidney transplant to protect, maintain, and/or improve kidney function and health.
  • the disease or condition that is associated with a loss of podocytes in the subject is an autoimmune disease.
  • the autoimmune disease affects kidney function and/or kidney health.
  • the autoimmune disease is lupus nephritis.
  • the autoimmune disease is Goodpasture syndrome.
  • the autoimmune disease is anti-glomerular basement membrane (anti-GBM).
  • the autoimmune disease is ANCA-associated vasculitis and glomerulonephritis.
  • the disease or condition that is associated with a loss of podocytes is cancer, in particular, cancers that affect kidney function and/or health.
  • the cancer is kidney cancer.
  • the cancer is renal cell carcinoma, urothelial carcinoma, kidney sarcoma, Wilms tumor, or lymphoma.
  • the disease or condition that is associated with a loss of podocytes is an inflammation, in particular, inflammations that affect kidney function and/or health. In particular, the inflammation is glomerulonephritis.
  • the inflammation is membranoproliferative glomerulonephritis (MPGN), interstitial nephritis, IgA nephropathy (Berger's disease), pyelonephritis, lupus nephritis, or Wegener's granulomatosis.
  • MPGN membranoproliferative glomerulonephritis
  • IgA nephropathy Boger's disease
  • pyelonephritis pyelonephritis
  • lupus nephritis or Wegener's granulomatosis.
  • the disclosure also features a method for identifying antibodies that bind to an integrin ⁇ 3 ⁇ 1 or a portion thereof, comprising: 1) removing antibodies that bind against the ⁇ 1 chain of the integrin ⁇ 3 ⁇ 1 in the presence or absence of a ligand mimetic peptide and/or an antibody; 2) from the remaining antibodies from step 1), selecting for antibodies that bind to the integrin ⁇ 3 ⁇ 1 in the presence or absence of a ⁇ 1 agonist antibody; 3) counter selecting for antibodies that bind to the integrin ⁇ 3 ⁇ 1 against immobilized ⁇ 1 agonist antibodies or a ligand mimetic peptide alone; and 4) repeating steps 1), 2), and 3) above to enrich for antibodies that are integrin ⁇ 3 allosteric agonists in the presence of cell surface-expressed integrin ⁇ 3 ⁇ 1.
  • the ligand mimetic peptide is LXY2.
  • step 1) and/or 3) is performed using human K562 cells that primarily express human ⁇ 5 ⁇ 1 integrin and not over-express ⁇ 3 ⁇ 1.
  • step 2) and/or 3) is performed using human K562 cells that over-express ⁇ 3 ⁇ 1.
  • step 1) and/or 2) and/or 3) are performed in the presence of agents that block the ligand binding site or domain of the integrin, such as antibodies and ligands.
  • integrin ⁇ 3 ⁇ 1 is stabilized in a specific conformation by pre- complexing it with activating or inhibitory agents, such as activating antibody 9EG7 or TS2/16. In some other embodiments, integrin ⁇ 3 ⁇ 1 is stabilized in a specific conformation by pre- complexing with agents that selectively bind the beta-chain of the integrin dimer. [0170] We clear for ⁇ 1 binders by clearing against "other" ⁇ 1 integrins - either as recombinant proteins or using cell lines such as K562 that express ⁇ 5 ⁇ 1 etc.
  • the strategy uses ligand blockers (such as integrin ⁇ 3 ⁇ 1 complexed with ligand mimetic peptide LXY2 or ligand laminin, in the absence or presence of ⁇ 1 activating antibody, to block the highly antigenic ligand binding pocket and the MIDAS site) to deliberately exclude binders to the ligand binding pocket.
  • ligand blockers such as integrin ⁇ 3 ⁇ 1 complexed with ligand mimetic peptide LXY2 or ligand laminin, in the absence or presence of ⁇ 1 activating antibody, to block the highly antigenic ligand binding pocket and the MIDAS site
  • Selection 1 utilized recombinant integrin
  • Selection 2 utilized cell surface expressed integrin.
  • a round of screening used three steps: pre-depletion, selection and counter selection.
  • the pre-depletion step was used to remove binders against the ⁇ 1 chain of the ⁇ 3 ⁇ 1 dimer by using a negative selection, where phage binders are selected away from the screening pool, against immobilized recombinant human integrin ⁇ 4 ⁇ 1. Additionally, a commercial ⁇ 1 agonist antibody (TS2/16) and ligand mimetic peptide (LXY2) were included with the immobilized integrin ⁇ 4 ⁇ 1 in some of the steps, to further remove any phage binders to those agents.
  • TS2/16 ⁇ 1 agonist antibody
  • LXY2 ligand mimetic peptide
  • the non-binding phages were used in the next step, selection step, for positive selection, where the phages were incubated with immobilized recombinant human or mouse integrin ⁇ 3 ⁇ 1 in the presence or absence of ⁇ 1 agonist antibodies (antibody clone TS2/16 for human ⁇ 3 ⁇ 1 and antibody clone 9EG7 for mouse ⁇ 3 ⁇ 1) and ligand mimetic peptide LXY2.
  • the non binding phages were removed and discarded. Bound phages were eluted and used in the final step, where any binders to the ⁇ 1 agonist antibodies and LXY2 were removed using a counter selection against immobilized ⁇ 1 agonist antibodies and LXY2 alone.
  • a round of screening is structured as follows: 1) a pre-depletion step against K562 cells (primarily expressing human ⁇ 5 ⁇ 1 integrin and not over-expressing ⁇ 3 ⁇ 1), in the presence or absence of ⁇ 1 agonist antibodies and ligand mimetic peptide LXY2; 2) a positive selection against human K562 cells expressing integrin ⁇ 3 ⁇ 1, in the presence or absence of ⁇ 1 agonist antibody TS2/16, and a counter selection against immobilized ⁇ 1 agonist antibodies and LXY2 alone. Cell line generation is described in the methods. [0175] The enriched phage pool from Selection 2 was optionally further amplified and plated using standard methods.
  • the light human Kappa variable regions and the Lambda variable region DNA sequences of each scFv were appended to full-length human light constant IGKC1 and IGLC1 DNA respectively, retaining their heavy/light pairing from the scFv level.
  • the heavy and light DNA constructs were cloned into separate cloning vectors then shuttled into mammalian expression vectors. [0178] Each of the five paired constructs were transfected and expressed in 10 mL of mammalian cells, then the antibodies were isolated using Protein A purification. Antibody samples were run on reducing and non-reducing SDS-PAGE as well as SEC-HPLC for quality control.
  • Antibody samples were found at the expected molecular weights on the SDS-PAGE gels and SEC-HPLC peaks determined the samples are reasonably pure.
  • BSA recombinant human integrin ⁇ 3 ⁇ 1 ECD
  • recombinant human integrin ⁇ 4 ⁇ 1 ECD recombinant mouse integrin ⁇ 3 ⁇ 1 ECD
  • recombinant mouse integrin ⁇ 3 ⁇ 1 ECD were coated overnight on the plate then incubated with either each of the five Ab74 individually or an isotype human IgG1 antibody negative control or a commercial anti-human ⁇ 3 antibody positive control. Binding was detected by incubating with anti-human IgG1 antibody Horse Radish Peroxidase (HRP) conjugate, then treating with fluorometric substrate, developing the reaction, and reading mean fluorescence intensity using a plate reader.
  • HRP Horse Radish Peroxidase
  • ELISA results show that the five Ab74 preferentially bind human ⁇ 3 ⁇ 1 ECD over every other antigen coated on the plate (FIGS.1A-1D).
  • the isotype negative control and anti- ⁇ 3 positive control yield expected negative and positive results, confirming the low background of the assay and the positive signal against human ⁇ 3 respectively.
  • Two of the five Ab74 antibodies demonstrate low binding towards mouse ⁇ 3, and all five Ab74 show only background signal against coated BSA and human ⁇ 4 ⁇ 1 ECD, confirming by ELISA that Ab74 bind human ⁇ 3 ECD and not human ⁇ 1 ECD.
  • individual human ⁇ 3 domains were recombinantly expressed and purified in mammalian cells.
  • ⁇ 3 ⁇ 1 domain mapping performed by Binding ELISA and Flow Cytometry using recombinant protein domains and domain-swapped integrin-expressing cell lines, respectively.
  • human ⁇ 3-expressing K562 were treated with the anti- ⁇ 3 Ab74 or negative control isotype antibody or positive control commercial ⁇ 1 agonist antibody TS2/16 in the presence of biotinylated ligand mimetic peptide LXY2 in a low- affinity Ca2+/Mg2+ buffer.
  • Binding of LXY2 is detected by treating the cells with Streptavidin fluorophore conjugate and reading the cells in a flow cytometer. Negative and positive controls correctly show low-to-none and high ligand binding respectively in the low-affinity buffer. Results in Table 4 show Ab74 increases LXY2 binding in the low-affinity buffer compared to isotype antibody. Table 4: Increased ligand binding by human integrin ⁇ 3 ⁇ 1 expressing cells in the presence of agonist antibodies . ⁇ 3 ⁇ 1-expressing K562 cells are incubated with ⁇ 3 ⁇ 1 ligand mimetic LXY2-biotin conjugate and either an integrin agonist antibody or isotype control.
  • FIGS.4A-4E demonstrate inhibition of wound closure in wells treated with all Ab74 antibodies or positive control anti- ⁇ 1 agonist antibody when compared to isotype treated negative control or blocking anti- ⁇ 3 which yielded wound closure.
  • Example 8 Integrin agonist antibodies target the thigh-genu domain [0187]
  • ⁇ 3 integrin domains were individually ‘exchanged’ with their homologous counterpart in the similar protein human integrin ⁇ 7 (as described in the methods), which also contains the Thigh-Genu, Calf 1, and Calf 2 domains.
  • integrin DNA constructs were created where the Thigh-genu, Calf 1, and Calf 2 domains on integrin ⁇ 7 replace the equivalent domains on the integrin ⁇ 3 inside the mammalian expression plasmid. These three constructs were individually transfected into HEK-293 cells using Lipofectamine and allowed to expand for 48 hours under culture conditions. These cells were then treated with Ab74 or negative control isotype antibody or positive control commercial ⁇ 3 antibody P1B5, before staining with a fluorophore conjugated anti-human IgG1 for detection in a flow cytometer.
  • Results in Table 5 show Ab74 detection in cells transfected with either the full-length integrin ⁇ 3 DNA and construct with integrin ⁇ 3 containing calf-2 of integrin ⁇ 7 and construct with integrin ⁇ 3 containing calf-1 of integrin ⁇ 7 whereas little to no binding occurs in the integrin ⁇ 3 construct replaced with Thigh-genu domain of integrin ⁇ 7 suggesting that the five Ab74 antibodies recognize an epitope in the Thigh-Genu region of ⁇ 3.
  • a DNA construct was created where the respective domain is swapped out for its homologous integrin ⁇ 7 counterpart.
  • HEK293 cells were cultured in serum-free CD medium (Sino Biological Cat # SMM 293-TI) until they reached optimum cell density.
  • the expression vector was added to the cells with the presence of TF1 transfection reagent and serum-free feeder solution (Sino Biological Cat # M293-SUPI-100) was added to the culture at days 1, 3, and 5 upon transfection. Cells were harvested at culture day 7 and proceeded to protein purification.
  • Protein purification from HEK293 cells [0192] Cells were removed via centrifugation and the culture supernatant was collected for protein purification. [0193] Affinity purification: the column was equilibrated with loading buffer and the culture supernatant was loaded onto the column. The column was re-equilibrated, and the target protein was eluted by a gradient of buffer contain imidazole (Ni-affinity), or Glycine and NaCl (Protein A affinity or FLAG affinity). The protein is subjected to further buffer exchange to remove excessive imidazole or other salts. The protein solution was concentrated and protein concentration as well as purity was assayed by corresponding methods.
  • N-term FLAG Tag MDYKDDDDK (SEQ ID NO:58) [0198] C-term 6X His Tag HHHHHH (SEQ ID NO:59) [0199] (Gly4Ser)3 linker GGGGSGGGGSGGGGS (SEQ ID NO:60) [0200] PreScission protease (3C) LEVLFQGP (SEQ ID NO:61) cleavage site (LEVLFQ/GP (SEQ ID NO:61), (where “/” indicates the cleavage site)) Domain swapped mammalian expression constructs [0201] To test antibodies for domain specificity, individual domains from ITGA3 are replaced with the homologous respective domains from ITGA7 in the DNA sequence (FIG.5).
  • SEQ ID NO:62 Full-length human ITGA3 with swapped ITGA7 Thigh-Genu: MGPGPSRAPRAPRLMLCALALMVAAGGCVVSAFNLDTRFLVVKEAGNPGSLFGYS VALHRQTERQQRYLLLAGAPRELAVPDGYTNRTGAVYLCPLTAHKDDCERMNITV KNDPGHHIIEDMWLGVTVASQGPAGRVLVCAHRYTQVLWSGSEDQRRMVGKCYV RGNDLELDSSDDWQTYHNEMCNSNTDYLETGMCQLGTSGGFTQNTVYFGAPGAYN WKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQVGSFILHPKNITIVTGAPRHR HMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDGWQDLLVGAP
  • K562 mouse ⁇ 3 ⁇ 1 and K562 cynomolgus monkey ⁇ 3 ⁇ 1 K562 cells from ATCC were transfected via electroporation with linearized expression plasmid containing either mouse or cynomolgus monkey integrin ⁇ 3 containing a C-terminus FLAG tag and kept under 0.8 mg/mL of puromycin selection for 2 weeks.
  • Target integrin-expressing K562 cells expressing human integrin ⁇ 3 ⁇ 1 were washed with TBS and 50,000 cells / well were transferred to ligand-coated wells of high binding clear 384-well microplates (Corning Incorporated, One Riverfront Plaza, NY, USA) in total 90 ⁇ L of assay buffer (HEPES 20mM/ 2mg/mL Glucose/ 140mM NaCl containing 1 mM each of Ca2+ and Mg2+ denoted HEPES-CaMg).
  • assay buffer HEPES 20mM/ 2mg/mL Glucose/ 140mM NaCl containing 1 mM each of Ca2+ and Mg2+ denoted HEPES-CaMg.
  • HEPES buffer containing 10 mM EDTA denoted HEPES-EDTA
  • TBS-Mn positive control assay buffer
  • Target integrin-expressing K562 cells expressing human integrin ⁇ 3 ⁇ 1 were washed with TBS and 50,000 cells / well were transferred to ligand-coated wells of high binding clear 384-well microplates (Corning Incorporated, One Riverfront Plaza, NY, USA) in total 90 ⁇ L of assay buffer (HEPES 20mM/ 2mg/mL Glucose/ 140mM NaCl containing 1 mM each of Ca2+ and Mg2+ denoted HEPES-CaMg). Plates were incubated at 37°C in the presence of antibodies for 30 minutes.
  • assay buffer HEPES 20mM/ 2mg/mL Glucose/ 140mM NaCl containing 1 mM each of Ca2+ and Mg2+ denoted HEPES-CaMg
  • TBS-EDTA negative control assay buffer
  • TBS-Mn positive control assay buffer
  • TBS-Mn positive control assay buffer
  • Direct integrin ELISA High binding black 384-well microplates (Corning Incorporated, One Riverfront Plaza, NY, USA) were coated overnight with 30 ⁇ L TBS containing 3 ⁇ g/mL recombinant integrin at 4°C.
  • the plates were flicked to remove any liquid and blocked by adding 90 ⁇ L of TBS containing 5% Bovine Serum Albumin (w/v), 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA) and incubating for 1 hour. After incubation, the plate was washed three times with 100 ⁇ L TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 ⁇ L assay buffer (TBS containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68, denoted TBS-T) were added to each well.
  • TBS-T Triton X-100
  • Pluronic F68 denoted TBS-T
  • the plate was washed three times with 100 ⁇ L in an automated plate washer.30 ⁇ L of substrate buffer is added (TBS containing 100 ⁇ M Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) and incubated for 30 minutes at room temperature. The plate was analyzed in a fluorescent microplate reader (Agilent Technologies, Santa Clara, CA, USA) at 563/587 nm. Integrin sandwich ELISA [0210] High binding black 384-well microplates (Corning Incorporated, One Riverfront Plaza, NY, USA) were coated overnight with 30 ⁇ L TBS containing 4 ⁇ g/mL anti-integrin antibody at 4°C.
  • TBS-T Assay buffer (TBS containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA), denoted TBS-T) was prepared ahead of the following steps. The plate was flicked to remove any liquid and blocked by adding 90 ⁇ L of TBS-T containing 5% Bovine Serum Albumin (w/v) (Sigma-Aldrich, St. Louis, MO, USA) and incubating for 1 hour.
  • TBS-T Triton X-100
  • Pluronic F68 Sigma-Aldrich, St. Louis, MO, USA
  • the plate was washed three times with 100 ⁇ L TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA).30 ⁇ L assay buffer (TBS-T containing 4 ⁇ g/mL tagged recombinant integrin) was added to each well. The plate was centrifuged at 1000 g for 1 minute and incubated at room temperature for 1 hour. The plate was washed three times with 100 ⁇ L TBS using the automated washer.30 ⁇ L of staining buffer (TBS-T containing a 1:2000 dilution of anti-tag antibody HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated for 30 minutes at room temperature.
  • TBS-T containing 4 ⁇ g/mL tagged recombinant integrin
  • the plate was washed three times with 100 ⁇ L in an automated plate washer.30 ⁇ L of substrate buffer (TBS containing 100 ⁇ M Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and developed for 30 minutes at room temperature. The plate was analyzed in a fluorescent microplate reader (Agilent Technologies, Santa Clara, CA, USA) at 563/587 nm. Recombinant integrin functional assay (SoLISA), integrin detection [0211] High binding black 384-well microplates (Corning Incorporated, One Riverfront Plaza, NY, USA) were coated overnight with 30 ⁇ L TBS containing 8 ⁇ g/mL ligand at 4°C.
  • substrate buffer TBS containing 100 ⁇ M Amplex Red and 4 mM hydrogen peroxide
  • SoLISA Recombinant integrin functional assay
  • integrin detection [0211] High binding black 384-well microplates (Corning Incorporated,
  • Assay buffer TBS containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA), denoted TBS-T
  • TBS-T Assay buffer
  • the plate was flicked to remove any liquid and blocked by adding 90 ⁇ L of TBS-T containing 5% Bovine Serum Albumin (w/v) (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 hour.
  • the plate was washed three times with 100 ⁇ L TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA).30 ⁇ L assay buffer (TBS-T containing 4 ⁇ g/mL tagged recombinant integrin and one of 1 mM Ca 2+ /1 mM Mg2+, 1 mM Mn2+/200 ⁇ M Ca2+, or 10 mM EDTA) was added to each well. 1 ⁇ L of agonist antibody (or isotype control) stock solution at an appropriate concentration was added to each well. The plate was centrifuged at 1000 g for 1 minute and incubated at room temperature for 3 hours.
  • assay buffer TBS-T containing 4 ⁇ g/mL tagged recombinant integrin and one of 1 mM Ca 2+ /1 mM Mg2+, 1 mM Mn2+/200 ⁇ M Ca2+, or 10 mM EDTA
  • the plate was washed three times with 100 ⁇ L TBS using the automated washer.
  • 30 ⁇ L of staining buffer (TBS-T containing a 1:2000 dilution of anti-tag antibody HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated for 30 minutes at room temperature.
  • the plate was washed three times with 100 ⁇ L in an automated plate washer.
  • 30 ⁇ L of substrate buffer (TBS containing 100 ⁇ M Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and developed for 30 minutes at room temperature.
  • the plate was analyzed in a fluorescent microplate reader (Agilent Technologies, Santa Clara, CA, USA) at 563/587 nm.
  • Recombinant integrin functional assay (SoLISA), ligand detection [0212] High binding black 384-well microplates (Corning Incorporated, One Riverfront Plaza, NY, USA) were coated overnight with 30 ⁇ L TBS containing 4 ⁇ g/mL anti-tag antibody at 4 °C.
  • Assay buffer TBS containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA), denoted TBS-T) were prepared ahead of the following steps.
  • the plate was flicked to remove any liquid and blocked by adding 90 ⁇ L of TBS-T containing 5% Bovine Serum Albumin (w/v) then incubated for 1 hour. After incubation, the plate was washed three times with 100 ⁇ L TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 ⁇ L capture buffer (TBS-T containing 4 ⁇ g/mL tagged recombinant integrin) was added to each well.
  • TBS-T containing 4 ⁇ g/mL tagged recombinant integrin
  • assay buffer TBS-T containing 8 ⁇ g/mL tagged recombinant ligand and one of 1 mM Ca2+ /1mM Mg2+, 1 mM Mn 2+ /200 ⁇ M Ca 2+ , or 10 mM EDTA
  • 1 ⁇ L of agonist antibody (or isotype control) stock solution at an appropriate concentration was added to each well.
  • the plate was centrifuged at 1000 g for 1 minute and incubated at room temperature for 3 hours. The plate was washed three times with 100 ⁇ L TBS using the automated washer.
  • Recombinant integrin functional assay (SoLISA), antibody detection [0213]
  • High binding black 384-well microplates (Corning Incorporated, One Riverfront Plaza, NY, USA) were coated overnight with 30 ⁇ L TBS containing 8 ug/mL ligand at 4°C.
  • Assay buffer (TBS containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA), denoted TBS-T) is prepared ahead of the following steps.
  • the plate was flicked to remove any liquid and blocked by adding 90 ⁇ L of TBS-T containing 5% Bovine Serum Albumin (w/v) (Sigma-Aldrich, St. Louis, MO, USA) then incubated for 1 hour. After incubation, the plate was washed three times with 100 ⁇ L TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA).30 ⁇ L assay buffer (TBS-T containing 4 ⁇ g/mL tagged recombinant integrin and one of 1 mM Ca2+ /1mM Mg 2+ 1 mM Mn 2+ /200 ⁇ M Ca 2+ , or 10 mM EDTA) was added to each well.
  • TBS-T containing 4 ⁇ g/mL tagged recombinant integrin and one of 1 mM Ca2+ /1mM Mg 2+ 1 mM Mn 2+ /200 ⁇ M Ca 2+ , or 10 mM
  • Laminin-511 E8 Fragment Fc (Acro Biosystems, Newark, DE, USA) were conjugated with anti-human IgG Alexa Fluor 647 conjugate (Jackson Immuno Research Labs, West Grove, PA, USA) in a 1:1.5 molar ratio (5 ⁇ g Laminin-511 E8 Fc per test group) incubated for 30 minutes at RT in the dark. The cells were centrifuged and resuspended in FACS buffer (PBS containing 2% Fetal Bovine Serum) (Summerlin Scientific, Hampton, NH, USA) at 10 million cells/mL.
  • FACS buffer PBS containing 2% Fetal Bovine Serum
  • Human Fc block were added to cells (BD Biosciences, Franklin Lakes, NJ, USA) to a final 25 ⁇ g/mL and incubated for 15 minutes on ice. After incubation, add FACS buffer to dilute the concentration to 1 million cells/mL. Once cells are transferred to a V-bottom 96-well plate [Cat. No. 290-8116-01V], 40 ⁇ L of assay buffer containing one of 1 mM Ca 2+ /1mM Mg2+ or 1 mM Mn2+/200 ⁇ M Ca2+ or 10mM EDTA was added to each respective well followed by agonist antibody or isotype and incubated for 5 minutes at room temperature.
  • FACS buffer PBS containing 2% Fetal Bovine Serum
  • FACS buffer PBS containing 2% Fetal Bovine Serum
  • Human Fc block were added to cells (BD Biosciences, Franklin Lakes, NJ, USA) to a final 25 ⁇ g/mL and incubated for 15 minutes on ice. After incubation, add FACS buffer to dilute the concentration to 1 million cells/mL. Once cells are transferred to a V-bottom 96-well plate [Cat. No.
  • Biotinylated ligand mimic was detected by fluorescently tagged streptavidin and incubated for 30 minutes in the dark at room temperature. Plate was washed with 200 ⁇ L assay buffer and pellet was resuspended in 100 ⁇ L freshly prepared fixation buffer (PBS containing 4% paraformaldehyde) [Cat No. AA47377-9M] and incubated on ice for 10 minutes. Finally, cells were resuspended PBS and cells were analyzed on a CytoFLEX Flow Cytometer (Beckman Coulter, Pasadena, CA, USA).
  • a vertical wound/scratch in the middle of the well was created using a sterile p200 tip.
  • Agonist or isotype antibody treatment were prepared in warm complete media, and 100uL of treatment were added per well.
  • Ca 2+ Mg 2+ or Mn 2+ were added to a final concentration of 0.5mM in 10 ⁇ L per well.
  • the wound healing process was observed at 24 hours for wound closure, when complete media treated only is near closing, the media was aspirated, and cell layer was washed with 200 ⁇ L of PBS and fixed with 4% of PFA at 4 °C for 10 minutes. 200 ⁇ L of 0.5% Crystal violet were added to each well and stained for 30 minutes at RT.
  • Transient transfection of 293HEK with integrin alpha subunit chimeric DNA constructs [0217] 293HEK cells were plated in a 6-well plate at 500,000 cells per well. The day of the transfection, complete media was aspirated, cell layer was washed with 2mL of PBS then 800 ⁇ L of Opti-MEM was gently added to cells. Transfection agent was prepared with 2.5 ⁇ g of DNA and 3 ⁇ L of lipofectamine 2000 in 250 ⁇ L of Opti-MEM then incubated for 5 minutes at room temperature. Solution was dispensed dropwise into wells, incubated overnight followed by complete media change. Cells were analyzed for Ab74 binding by flow cytometry and detected by anti-human IgG1 antibody conjugated to a fluorophore.
  • Mouse recombinant integrin ⁇ 3 ⁇ 1 Mouse ⁇ 3 ECD sequence; Protein sequence: 1036aa MGWSCIILFLVATATGVHSFNLDTRFLVVKEAVNPGSLFGYSVALHRQTERQQRYLL LAGAPRDLAVGDDYTNRTGAVYLCPLTAHKDDCERMDISEKSDPDHHIIEDMWLGV TVASQGPAGRVLVCAHRYTKVLWSGLEDQRRMVGKCYVRGNDLQLDPGDDWQTY HNEMCNSNTDYLQTGMCQLGTSGGFTQNTVYFGAPGAYNWKGNSYMIQRKDWDL SEYSYRGSEEQGNLYIGYTVQVGNAILHPTDIITVVTGAPRHQHMGAVFLLKQESGG DLQRKQVLKGTQVGAYFGSAIALADLNNDGWQDLLVGAPYYFERKEEVGGAVYVF MNQAGASFPDQPSLLLHGPSRSAFGISIASIGDINQDGFQDIAVG

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions et des méthodes comprenant des anticorps qui se lient à l'intégrine α3β1.
PCT/US2023/078109 2022-10-31 2023-10-27 COMPOSITIONS ET MÉTHODES IMPLIQUANT L'INTÉGRINE α3β1 WO2024097620A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263420964P 2022-10-31 2022-10-31
US63/420,964 2022-10-31

Publications (1)

Publication Number Publication Date
WO2024097620A1 true WO2024097620A1 (fr) 2024-05-10

Family

ID=90931444

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/078109 WO2024097620A1 (fr) 2022-10-31 2023-10-27 COMPOSITIONS ET MÉTHODES IMPLIQUANT L'INTÉGRINE α3β1

Country Status (1)

Country Link
WO (1) WO2024097620A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200392228A1 (en) * 2019-05-20 2020-12-17 Pandion Therapeutics, Inc. MAdCAM TARGETED IMMUNOTOLERANCE
WO2020257296A1 (fr) * 2019-06-21 2020-12-24 Board Of Regents, The University Of Texas System Ciblage de l'intégrine alpha3bêta1 pour le traitement du cancer et d'autres maladies
WO2022173949A1 (fr) * 2021-02-10 2022-08-18 WUGEN, Inc. Polypeptides et leur utilisation dans le traitement d'une maladie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200392228A1 (en) * 2019-05-20 2020-12-17 Pandion Therapeutics, Inc. MAdCAM TARGETED IMMUNOTOLERANCE
WO2020257296A1 (fr) * 2019-06-21 2020-12-24 Board Of Regents, The University Of Texas System Ciblage de l'intégrine alpha3bêta1 pour le traitement du cancer et d'autres maladies
WO2022173949A1 (fr) * 2021-02-10 2022-08-18 WUGEN, Inc. Polypeptides et leur utilisation dans le traitement d'une maladie

Similar Documents

Publication Publication Date Title
CA2597717C (fr) Anticorps contre cxcr4 et leurs procedes d'utilisation
CN117098561A (zh) Ccr8抗体及其应用
KR101442266B1 (ko) 항-알파2 인테그린 항체 및 이의 용도
KR102047248B1 (ko) 침식성 세포의 선택적 제거
KR101695056B1 (ko) 카드헤린-11의 ec1 도메인을 표적으로 하는 인간화 항체 및 관련 조성물 및 방법
NZ719092A (en) Tumor necrosis factor-like ligand 1a specific antibodies and compositions and uses thereof
KR102523150B1 (ko) 항-테나신 c 항체 및 이의 용도
JP2015145397A (ja) 抗α２インテグリン抗体を使用する処置
US9587024B2 (en) Monoclonal antibodies for enhancing or inhibiting insulin-like growth factor 1 (IGF-1)
US20100056760A1 (en) Sialoadhesin factor-2 antibodies
CN112996815A (zh) 人pd-l1抗体
KR20170016501A (ko) 콜로니 자극 인자 1 수용체 (ｃｓｆ1ｒ)에 결합하는 항체를 이용하여 병태를 치료하는 방법
RU2741802C2 (ru) АНТИТЕЛО К Myl9
CN115315442A (zh) Sars-cov-2抗体及其应用
EP4448581A2 (fr) Anticorps se liant à cd28 et leurs fragments de liaison à l'antigène
WO2024097620A1 (fr) COMPOSITIONS ET MÉTHODES IMPLIQUANT L'INTÉGRINE α3β1
KR20190049866A (ko) 신규한 항-ｐｃｓｋ９ 항체
KR20230124001A (ko) Cd47을 표적으로 하는 항체 및 이의 응용
AU2014230640B2 (en) Monoclonal antibody inhibiting enzymatic activity of endothelial lipase
EP3774916A2 (fr) Agents anti-tétraspanine 33 et compositions et procédés pour la fabrication et l'utilisation de ces agents
WO2022221314A1 (fr) Compositions et méthodes impliquant le domaine de liaison du récepteur 2 du coronavirus du syndrome respiratoire aigu sévère
CN107840893B (zh) 新型抗-pcsk9抗体
CN113166264B (zh) 一种分离的抗原结合蛋白及其用途
AU2022346049A1 (en) Novel wnt agonist antibodies and therapeutic uses thereof
WO2023133470A2 (fr) Anticorps se liant à tmprss2 et leurs fragments de liaison à l'antigène

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23886850

Country of ref document: EP

Kind code of ref document: A1