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WO2025166228A1 - Anti-il12p35 antibodies and uses thereof - Google Patents

Anti-il12p35 antibodies and uses thereof

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Publication number
WO2025166228A1
WO2025166228A1 PCT/US2025/014119 US2025014119W WO2025166228A1 WO 2025166228 A1 WO2025166228 A1 WO 2025166228A1 US 2025014119 W US2025014119 W US 2025014119W WO 2025166228 A1 WO2025166228 A1 WO 2025166228A1
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WO
WIPO (PCT)
Prior art keywords
seq
amino acid
nos
set forth
cdrs
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
PCT/US2025/014119
Other languages
French (fr)
Inventor
Nan Bing
Xiaodong Jiang
Dong Zhang
Guangan HU
Quanju ZHAO
Zhengwang SUN
Tianfei HOU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
D2m Biotherapeutics Ltd
D2m Biotherapeutics Inc
Original Assignee
D2m Biotherapeutics Ltd
D2m Biotherapeutics Inc
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Filing date
Publication date
Application filed by D2m Biotherapeutics Ltd, D2m Biotherapeutics Inc filed Critical D2m Biotherapeutics Ltd
Publication of WO2025166228A1 publication Critical patent/WO2025166228A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • This disclosure relates to antibodies that can bind to IL12p35 (interleukin 12 subunit alpha), and the uses thereof.
  • autoimmune diseases primarily involve the use of immunosuppressive medications and biologics/small molecule drugs targeting inflammatory cytokines, immune cells, and intracellular kinases.
  • An effective therapeutic strategy focuses on the IL12/IL23 pathway, resulting in multiple drug approvals for various autoimmune diseases.
  • evidence shows that IL12/IL23 pathway-specific genes are effective targets for a set of autoimmune diseases, but not for other autoimmune diseases.
  • This disclosure relates to anti-IL12p35 antibodies, antigen-binding fragment thereof, and the uses thereof.
  • the disclosure also demonstrates that developing IL12p35/ IL12R02 antagonist would be used for treating a cluster of autoimmune diseases, e.g., systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to interleukin 12 subunit alpha (IL12p35), comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1 , 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL
  • the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, 49, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 50, 51, 52, respectively; (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 53, 54, 55, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 56, 57, 58, respectively; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, 61, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 62, 63, 64, respectively; (4) the selected VH CDR
  • the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 185, 186, 187, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 188, 189, 190, respectively; (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 191, 192, 193, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 194, 195, 196, respectively; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 197, 198, 199, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 200, 201, 202,
  • the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, 157, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, 160, respectively, according to Kabat definition; (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 179, 180, 181, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 182, 183, 184, respectively, according to Kabat definition; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 293, 294, 295, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to IL12p35 comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence
  • VH heavy chain variable region
  • VL light chain variable region
  • the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 1, and the selected VL sequence is SEQ ID NO: 2; (2) the selected VH sequence is SEQ ID NO: 3, and the selected VL sequence is
  • the selected VH sequence is SEQ ID NO: 7; (4) the selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8; (5) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10; (6) the selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (7) the selected VH sequence is SEQ ID NO: 13, and the selected VL sequence is SEQ ID NO: 14; (8) the selected VH sequence is SEQ ID NO: 15, and the selected VL sequence is SEQ ID NO: 16; (9) the selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18; (10) the selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; (11) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (12) the selected VH sequence is SEQ ID NO: 23, and the selected
  • the VH and/or VL comprise one or more back-to-germline (B2G) mutations.
  • the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is 38.
  • the selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46.
  • the selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336.
  • the selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338.
  • the antibody or antigen-binding fragment thereof can block the binding between interleukin 12 (e.g., human interleukin 12 (IL 12)) and interleukin 12 receptor, beta 2 subunit (e.g., human interleukin 12 receptor, beta 2 subunit (IL12R02)).
  • interleukin 12 e.g., human interleukin 12 (IL 12)
  • interleukin 12 receptor e.g., interleukin 12 receptor, beta 2 subunit (IL12R02)
  • the antibody or antigen-binding fragment thereof can block IL12-induced intracellular signaling (e.g., JAK-STAT signaling), optionally the IL12 is human or monkey IL 12.
  • the antibody or antigen-binding fragment thereof can inhibit IL 12- induced IFN-y production in human PBMCs.
  • the antibody or antigenbinding fragment thereof can prevent IFN-y production by CD4+ T cells cocultured with allogenic dendritic cells.
  • the antibody or antigen-binding fragment specifically binds to human IL12p35 and/or monkey IL12p35.
  • the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment is a F(ab’)2 fragment, a single-chain variable fragment (scFV) or a multi-specific antibody (e.g., a bispecific antibody).
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to IL12p35 comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, a first immunoglobulin light chain, and a second immunoglobulin light chain, in some embodiments, the first immunoglobulin heavy chain and the first immunoglobulin light chain associates with each other, forming a first antigen-binding site that binds to IL12p35, in some embodiments, the second immunoglobulin heavy chain and the second immunoglobulin light chain associates with each other, forming a first antigen-binding site that binds to IL12p35, in some embodiments: (1) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 325, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 326; (2) the first and second immunoglobulin heavy chains comprise an amino acid sequence
  • the first and second immunoglobulin heavy chains are identical, in some embodiments, the first and second immunoglobulin light chains are identical.
  • the first and second immunoglobulin heavy chains comprise an Fc region (e.g., an IgGl Fc region).
  • the Fc region comprises YTE mutations (M252Y/S254T/T256E according to EU numbering).
  • the disclosure is related to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1 , VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, in some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are identical to VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the antibody or antigen-binding fragment thereof described herein.
  • VH heavy chain variable region
  • VL light chain variable region
  • the disclosure is related to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.
  • the disclosure is related to an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent.
  • the therapeutic agent is a cytotoxic or cytostatic agent.
  • the disclosure is related to a method of inhibiting immune response in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, to the subject.
  • the subject has an immune disorder (e.g., an autoimmune disease or an inflammatory disease).
  • the disclosure is related to a method of treating a subject having an autoimmune disease, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, to the subject.
  • the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
  • the subject does not have psoriasis (PsO), Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel diseases (IBD), or ankylosing spondylitis (AS).
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.
  • the disclosure is related to a method of reducing IL12-induced intracellular signaling in a cell, the method comprising contacting the cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, to the subject.
  • the disclosure is related to a method of identifying a subject as having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject, using the antibody or antigen-binding fragment thereof described herein.
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the disclosure is related to a method of determining the risk of a subject having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject, using the antibody or antigen-binding fragment thereof described herein.
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the disclosure is related to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: (1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1 , 2, and 3 comprising VH CDR 1, 2, 3 set forth in FIG. 26 or FIG. 27, and in some embodiments, the VH, when paired with a corresponding light chain variable region (VL) binds to IL12p35; or (2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1 , 2, and 3 comprising VL CDR 1, 2, 3 set forth in FIG. 26 or FIG. 27, when paired with a corresponding VH binds to IL12p35.
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the polypeptide comprises: (1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1 , 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively (or SEQ ID NOs: 185, 186, and 187, respectively), and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 2 binds to IL12p35; (2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively (or SEQ ID NOs: 188, 189, and 190, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 bind
  • the VH when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 10 binds to IL12p35; (10) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 74, 75, and 76, respectively (or SEQ ID NOs:
  • the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 binds to IL12p35; (11) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 77, 78, and 79, respectively (or SEQ ID NOs: 215, 216, and 217, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 12 binds to IL12p35; (12) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 80, 81, and 82, respectively (or SEQ ID NOs: 218, 219, and 220, respectively), and in some embodiments, the VL
  • the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 23 binds to IL12p35; (25) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs:
  • the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 26 binds to IL12p35;
  • the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 25 binds to IL12p35;
  • an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 125, 126, and 127, respectively or SEQ ID NOs: 263,
  • the VL when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 35 binds to IL12p35; (37) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs:
  • the VH when paired with a VL specifically binds to human IL12p35; or the VL when paired with a VH specifically binds to human IL12p35.
  • the immunoglobulin heavy chain or the fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof
  • the immunoglobulin light chain or the fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.
  • the nucleic acid encodes a F(ab’)2 fragment, a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody).
  • the nucleic acid is cDNA.
  • the disclosure is related to a vector comprising one or more of the nucleic acids described herein. In one aspect, the disclosure is related to a vector comprising two of the nucleic acids described herein, in some embodiments, the vector encodes the VL region and the VH region that together bind to IL12p35. In one aspect, the disclosure is related to a pair of vectors, in some embodiments, each vector comprises one of the nucleic acids described herein, in some embodiments, together the pair of vectors encodes the VL region and the VH region that together bind to IL12p35.
  • the disclosure is related to a cell comprising the vector or the pair of vectors described herein.
  • the cell is a CHO cell.
  • the disclosure is related to a cell comprising one or more of the nucleic acids described herein.
  • the disclosure is related to a cell comprising two of the nucleic acids described herein. In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to IL12p35.
  • the disclosure is related to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and (b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.
  • the disclosure is related to a method of treating a subject having an autoimmune disease, the method comprising administering a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof that binds to IL12p35 and/or an antibody or antigen-binding fragment thereof that binds to IL12R01, to the subject.
  • the antibody or antigen-binding fragment thereof that binds to IL12p35 does not bind to IL12p40.
  • the antibody or antigen-binding fragment thereof that binds to IL12R02 does not bind to IL12R01.
  • the antibody or antigenbinding fragment thereof does not interfere IL23 pathway and/or IL35 pathway.
  • the subject is a human subject.
  • the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the antibody or antigen-binding fragment thereof is a human or humanized antibody or antigen-binding fragment thereof.
  • the subject is a non-human mammal, e.g., a monkey, a dog, or a mouse.
  • the mammal has a similar disease or disorder as systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the subject is a dog.
  • the antibody or antigenbinding fragment thereof is a canine or caninized antibody or antigen-binding fragment thereof.
  • antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope.
  • CDR complementary determining region
  • Nonlimiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bi-specific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
  • the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain).
  • variable domains include, e.g., Fab, Fab’, F(ab’)2, and Fv fragments.
  • human antibody refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human.
  • a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells).
  • a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line).
  • a human antibody is produced in a bacterial or yeast cell.
  • a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus).
  • a transgenic non-human animal e.g., a bovine
  • human immunoglobulin locus e.g., heavy or light chain human immunoglobulin locus
  • chimeric antibody refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody).
  • a non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
  • humanized antibody refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin.
  • humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody), e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity.
  • the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues.
  • humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance.
  • the humanized antibody contains at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin.
  • CDRs hypervariable loops
  • the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically, that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
  • single-chain antibody refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen.
  • immunoglobulin variable domains e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain
  • single-chain antibodies are described herein.
  • multimeric antibody refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains.
  • the multimeric antibody is able to crosslink one target molecule (e.g., IL12p35) to at least one second target molecule (e.g., IL12p35) on the surface of a mammalian cell.
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided.
  • Veterinary and non-veterinary applications are contemplated by the present invention.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old).
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • swine e.g., pig, miniature pig
  • equine canine
  • feline bovine
  • other domestic, farm, and zoo animals equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
  • the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., IL12p35) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general.
  • a target-specific antibody an antibody that specifically binds to a IL12p35 molecule may be referred to as a IL12p35-specific antibody or an anti-IL12p35 antibody.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
  • nucleic acid molecule As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
  • FIG. 1 is a table showing the preliminary binding affinities of anti-IL12p35 antibodies against human IL12p70 measured by the Gator® biolayer interferometry platform.
  • Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
  • FIG. 2A is a table showing the binding affinities of selected anti-IL12p35 antibodies and their YTE variants to human, rhesus, canine or mouse IL12p70 measured by the Gator® biolayer interferometry platform.
  • 27H28L is an engineered antibody composed of the heavy chain of D2M008-A1A8 and the light chain of D2M008-A1A9.
  • YTE mutations of anti-IL12p35 antibodies 27H28L and D2M008-4A4 were introduced via amino acid substitutions (M252Y/S254T/T256E) in the Fc region, generating “27H28L YTE” and “D2M-4A4 YTE,” respectively.
  • 27H28L-hblb-YTE and 4A4-hblb2-YTE have back-to-germline mutations in the frameworks in variable regions of “27H28L YTE” and “D2M-4A4 YTE,” respectively.
  • O.R. means out of range.
  • N.D. means not performed.
  • N.B. means no binding was detected.
  • FIG. 2B shows the binding kinetics and fitting curves of 27H28L-hblb-YTE and 4A4- hblb2-YTE to human IL12p70 measured by the BiaCoreTM SPR platform.
  • FIG. 2C is a table showing the method information and parameters acquired by the BiaCoreTM 8K SRP platform for 27H28L-hblb-YTE and 4A4-hblb2-YTE.
  • FIG. 3A shows the binding affinities of 27H28L and its YTE variant (“27H28L YTE”) to a recombinant FcRn protein at pH 6.0, pH 6.5, or pH 7.0, measured by the Gator® biolayer interferometry platform.
  • FIG. 3B shows the binding affinities of D2M008-4A4 and its YTE variant (“D2M-4A4 YTE”) to a recombinant FcRn protein at pH 6.0, pH 6.5, or pH 7.0, measured by the Gator® biolayer interferometry platform.
  • FIG. 4 A shows the inhibition rate of different anti-IL12p35 antibodies blocking human IL12p70 binding to its receptor IL12R 2. All antibodies at a fixed concentration were preincubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA.
  • FIG. 4B shows the inhibition rate of selected anti-IL12p35 antibodies blocking human IL12p70 binding to its receptor IL12R 2.
  • Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA.
  • FIG. 4C shows blocking of human IL12p70 binding to its receptor IL12R 2 by 27H28L and additional anti-IL12p35 antibodies.
  • Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2.
  • the ICso values of 27H28L and the selected anti-IL12p35 antibodies were calculated.
  • FIG. 4D shows the inhibition rate of anti-IL12p35 antibodies 27H28L, D2M008-4A4 and their YTE variants blocking human IL12p70 binding to its receptor IL12R 2.
  • Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA.
  • FIG. 4E shows the inhibition rate of anti-IL12p35 antibodies 27H28L-YTE and D2M008-4A4 YTE and their variants with back-to-germline mutations blocking human IL12p70 binding to its receptor IL12R 2.
  • Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA.
  • FIG. 5 A shows the inhibition rate of different anti-IL12p35 antibodies blocking human IL12p70-induced signaling in HEK-BlueTM IL12 reporter cells. All antibodies at a fixed concentration were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring and calculating based on the reporter gene signal at OD620.
  • FIG. 5B shows the inhibition rate of selected anti-IL12p35 antibodies blocking human IL12p70-induced signaling in HEK-BlueTM IL12 reporter cells.
  • Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
  • Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was calculated based on the reporter gene signal at OD620.
  • FIG. 5C shows the inhibition rate of selected anti-IL12p35 antibodies (D2M008-A1A8, D2M008-A1A9, and 27H28L), anti-IL12p40 (Ustekinumab, anti-hP40), and a combination of D2M008-A1 A8 and anti-hP40 blocking human IL12p70-induced signaling in HEK-BlueTM IL12 reporter cells.
  • Serially diluted antibodies were pre- incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was calculated based on the reporter gene signal at OD620.
  • the IC50 values of the antibodies and the combination of D2M008-A1A8 and anti-hP40 were calculated.
  • FIG. 5D shows the inhibition rate of selected anti-IL12p35 antibodies and 27H28L blocking human IL12p70-induced signaling in HEK-BlueTM IL12 reporter cells.
  • Anti-human IL12p40 (Ustekinumab, anti-hP40) was used as a reference antibody.
  • Serially diluted antibodies were pre- incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was calculated based on the reporter gene signal at OD620. The IC50 values of the antibodies were calculated.
  • FIG. 5E shows the inhibition level of anti-IL12p35 antibodies 27H28L, D2M008-4A4, and their YTE variants blocking human IL12p70-induced signaling in HEK-BlueTM IL12 reporter cells.
  • Serially diluted antibodies were pre- incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
  • FIG. 5F shows the inhibition level of anti-IL12p35 antibodies 27H28L-YTE and D2M008-4A4 YTE and their variants with back-to-germline mutations blocking human IL12p70-induced signaling in HEK-BlueTM IL12 reporter cells.
  • Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
  • FIG. 6 shows the inhibition level of anti-IL12p35 antibody D2M008-4A4 and 27H28L blocking rhesus IL12-induced signaling in HEK-BlueTM IL12 reporter cells.
  • Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
  • Serially diluted antibodies were preincubated with rhesus IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
  • FIG. 7A-7C show the level of IFN-y production by exogenous IL 12- stimulated human PBMCs in vitro.
  • PBMCs from three healthy donors (#20, #22 and #23) were used.
  • Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
  • Serially diluted anti-IL12p35 antibodies were pre- incubated with the exogenous IL12p70 at room temperature for 30 minutes before incubating with PBMCs. IFN-y production was measured by a sandwich ELISA.
  • FIG. 8A-8B show the level of IFN-y production by human CD4+ T cells co-cultured with allogeneic dendritic cells in vitro.
  • the data in FIG. 8A was obtained using CD4+ T cell from Donor #4, and the data in FIG. 8B was obtained using CD4+ T cell from Donor #8.
  • Antihuman IL12p40 (anti-hP40) was used as a reference antibody.
  • Serially diluted antibodies were individually mixed with monocyte-derived dendritic cells (DC) from donor #18 before coculture with human CD4+ T cells.
  • the ratio of dendritic cells and CD4+ T cells was 1: 10. IFN-y production was measured by a sandwich ELISA.
  • FIGS. 9A-9B show the inhibition level of anti-IL12p35 antibody 27H28L (FIG. 9A), D2M007-4A4 (FIG. 9B), and their YTE variants neutralizing IL23 -induced transducing signaling in HEK-BlueTM IL23 reporter cells.
  • Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
  • Serially diluted antibodies were pre-incubated with human IL23 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
  • FIG. 10A shows the melting curves of full IgG and the F(ab’)2 fragment of anti-IL12p35 antibodies 27H28L and D2M008-4A4.
  • Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
  • the melting curves were measured by the Protein Thermal ShiftTM Dye Kit using a qPCR thermocycler. Fluorescence intensity was subtracted by the lowest intensity (bottom) before the peak and normalized to the amplitude from bottom to peak.
  • FIG. 10B shows the T m of full IgG and the F(ab’)2 fragment of anti-IL12p35 antibodies 27H28L and D2M008-4A4.
  • Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
  • FIG. 11A shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70 binding to its receptor IL12R 2, after being exposed in stress conditions.
  • the antibodies were stressed in a pH 5.5, pH 7.4, or pH 8.5 buffer, respectively, at 40°C for two weeks. Their blocking kinetics was measured by ELISA.
  • FIG. 11B shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70-induced transducing signaling in HEK-BlueTM IL12 reporter cells, after being exposed in stress conditions.
  • the antibodies were stressed in a pH 5.5, pH 7.4, or pH 8.5 buffer, respectively, at 40°C for two weeks. Their blocking kinetics was evaluated by measuring the reporter gene signal at OD620.
  • FIG. 12A shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70 binding to its receptor IL12R 2, after being exposed to human plasma or PBS.
  • the antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for two weeks. Their blocking kinetics was measured by ELISA.
  • Plasma 20 and plasma 21 indicate human plasma samples from two different donors.
  • FIG. 12B shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70-induced transducing signaling in HEK-BlueTM IL12 reporter cells, after being exposed to human plasma or PBS.
  • the antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for two weeks. Their blocking kinetics was evaluated by measuring the reporter gene signal at OD620.
  • Plasma 20 and plasma 21 indicate human plasma samples from two different donors.
  • FIG. 12C shows the functional stability of anti-IL12p35 antibodies 27H28L-hblb-YTE in blocking IL12p70 binding to its receptor IL12R 2, after being exposed to human plasma for up to 3 weeks.
  • the antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for 0, 1, 2, or 3 weeks. Their blocking kinetics was measured by ELISA.
  • Plasma 42, 45, 53 and 54 indicate human plasma samples from four different donors.
  • FIG. 12D shows the functional stability of anti-IL12p35 antibodies 4A4-hblb2-YTE in blocking IL12p70 binding to its receptor IL12R 2, after being exposed to human plasma for up to 3 weeks.
  • the antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for 0, 1, 2, or 3 weeks. Their blocking kinetics was measured by ELISA.
  • Plasma 42, 45, 53 and 54 indicate human plasma samples from four different donors.
  • FIG. 13B shows the competition in binding to IL12 between anti-IL12p35 antibody 27H28L and D2M008-4A4.
  • the anti-human Fc probes were used to immobilize one antibody (D2M008-4A4 and 27H28L, respectively), which was then incubated with human IL12p70, followed by an additional incubation with the second antibody (27H28L and D2M008-4A4, respectively).
  • the binding ability was measured and analyzed using the Gator® biolayer interferometry platform.
  • FIG. 13C shows the competition in binding to IL12 between anti-IL12p35 antibody 27H28L and D2M008-4A4.
  • the anti-His probes were used to immobilize human IL12p70, and then incubated with one antibody (D2M008-4A4 and 27H28L, respectively), followed by an additional incubation with the second antibody (27H28L or D2M008-4A4, respectively).
  • the binding ability was measured and analyzed using the Gator® biolayer interferometry platform.
  • FIG. 14 shows IL12 related cytokines, receptors and signaling pathways.
  • the schematic diagrams are adapted from Choi, J., et al. "IL-35 and autoimmunity: a comprehensive perspective.” Clinical Reviews in Allergy & Immunology 49 (2015): 327-332.
  • FIGS. 15A-15C show regional plot of genetic marker association in IL12A locus with IL12A expression (IL12A eQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL12A gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of -logio(p value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x l O' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL12 eQTL variant (rs4680586).
  • FIGS. 16A-16C show regional plot of genetic marker association in IL12R02 locus with IL12RB2 expression (IL12RB2 eQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL12R02 gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x 10' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL12RP2 eQTL variant (rsl7129778).
  • FIGS. 17A-17C show regional plot of genetic marker association in IL12B locus with IL12B serum protein levels (IL12B pQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL12B gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x 10' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL12B pQTL variant (rs6556416).
  • FIGS. 18A-18C show regional plot of genetic marker association in IL23R locus with IL23R serum protein levels (IL23R pQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL23R gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x 10' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL23R pQTL variant (rsl 1581607).
  • FIGS. 19A-19C show regional plot of genetic marker association in EBB locus with EBB serum protein levels (EBB pQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of EBB gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x 10' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top EBI3 pQTL variant (rs60160662).
  • FIGS. 20A-20C show regional plot of genetic marker association in IL6ST locus with IL6ST serum protein levels (IL6ST pQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL6ST gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x 10' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top EBI3 pQTL variant (rsl 1574765).
  • FIGS. 21A-21C show regional plot of genetic marker association in STAT4 locus with STAT4 gene expression levels (STAT4 eQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of STAT4 gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p Value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x 10' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top STAT4 eQTL variant (rsl6833249).
  • FIGS. 22A-22C show regional plot of genetic marker association in STAT3 locus with STAT3 gene expression levels (STAT3 eQTL) and 9 inflammatory diseases.
  • Each dot in the plot is a genetic marker.
  • the X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of STAT3 gene with a spanning genomics length of 500 KB.
  • the Y-axis represents the significant p value of each genetic marker in the metrics of - loglO(p Value).
  • the horizontal straight line represents the GWAS significant threshold p value of 5 x 10' 8 .
  • the circle size of each genetic marker is categorized by correlation of the genetic marker with the top STAT3 pQTL variant (rsl053004).
  • FIG. 23 shows scatter plots comparing the genetic effects on IL12A expression vs. genetic effects on SSc, PBC, SjS, and SLE disease risk. Genetic variants significantly associated with IL12A’s expression were selected first. Variants overlapping with SSc, PBC, SjS, or SLE disease GWAS data were finally picked for each disease. Each dot in the plot is a finally picked genetic variant.
  • X-axis represents the effect of such genetic variant on IL12A’s gene expression.
  • Y-axis represents the effect of such genetic variant’s same allele on disease risk.
  • the straight line represents a fit regression line across all the selected genetic variants. An upward line indicated that genetically increased IL12A gene expression leads to an increased disease risk.
  • the R- squared and p-value explain the strength of the relationship between genetically determined IL12A gene expression and genetically determined disease risk.
  • FIG. 24 shows scatter plots comparing the genetic effects on IL12R02 expression vs. genetic effects on SSc, PBC, SjS, and SLE disease risk. Genetic variants significantly associated with IL12Rp2’s expression were selected first. Variants overlapping with SSc, PBC, SjS, or SLE disease GWAS data were finally picked for each disease. Each dot in the plot is a finally picked genetic variant.
  • X-axis represents the effect of such genetic variant on IL12Rp2’s gene expression.
  • Y-axis represents the effect of such genetic variant’s same allele on disease risk.
  • the straight line represents a fit regression line across all the selected genetic variants. An upward line indicated that genetically increased IL12RP2 gene expression leads to an increased disease risk.
  • the R-squared and p-value explain the strength of the relationship between genetically determined IL12RP2 gene expression and genetically determined disease risk.
  • FIG. 25 shows the heavy chain variable region (VH) and light chain variable region (VL) sequences of anti-IL12p35 antibodies.
  • FIG. 26 shows VH and VL CDR sequences of anti-IL12p35 antibodies according to Kabat definition.
  • FIG. 27 shows VH and VL CDR sequences of anti-IL12p35 antibodies according to IMGT definition.
  • FIG. 28 shows the heavy chain (HC) and light chain (LC) sequences of antibodies discussed in the disclosure.
  • FIG. 29 shows the serum concentration of anti-IL12p35 antibodies 4A4-hblb2-YTE and 27H28L-hblb-YTE post a single i.v. dose (1 mg/kg) in human FcRn transgenic mice.
  • FIG. 30 shows pharmacokinetic parameters of anti-IL12p35 antibodies 4A4-hblb2-YTE and 27H28L-hblb-YTE after 1 mg/kg i.v. single dose in human FcRn transgenic mice.
  • FIG. 31A shows the experimental scheme of using a DNFB-induced chronic skin inflammation model to determine the in vivo effects of anti-IL12p35 antibodies on inflammation diseases.
  • FIG. 3 IB shows the average ear thickness of mice treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an anti-mouse IL12p40 antibody (“anti-mP40”) in a DNFB-induced chronic skin inflammation model.
  • FIGS. 32A shows the experimental scheme of using STZ-induced Sjogren's syndrome model to determine the in vivo effects of anti-IL12p35 antibodies on Sjogren's syndrome.
  • FIG. 32B shows the excretion of saliva relative to the body weight of mice treated with an anti-mouse IL12p35 antibody (“anti-P35”) or an isotype antibody control (“Isotype”) in a STZ-induced Sjogren's syndrome model.
  • anti-P35 anti-mouse IL12p35 antibody
  • Isotype isotype antibody control
  • FIG. 33A shows the average body weight of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an isotype antibody control (“Isotype”) in a IMQ-induced SLE model.
  • anti-mP35 anti-mouse IL12p35 antibody
  • Isotype isotype antibody control
  • FIG. 33B shows the ratio of urine albumin and creatinine of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an isotype antibody control (“Isotype”) in a IMQ-induced SLE (systemic lupus erythematosus) model.
  • anti-mP35 anti-mouse IL12p35 antibody
  • Isotype isotype antibody control
  • FIG. 33C shows representative mouse kidney histology images of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-P35”) or an isotype antibody control (“Isotype”) in a IMQ-induced SLE model.
  • anti-P35 anti-mouse IL12p35 antibody
  • Isotype isotype antibody control
  • FIG. 34A shows the ratio of urine albumin and creatinine (“Cr”) of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an isotype antibody control (“Isotype”) in a spontaneous SLE (systemic lupus erythematosus) model.
  • anti-mP35 anti-mouse IL12p35 antibody
  • Isotype isotype antibody control
  • FIG. 34B shows representative mouse kidney histology images of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (panels e-h) or an isotype antibody control (panels a-d) in a spontaneous SLE model.
  • Scale panels a and e: 3 mm; panels b and f: 100 pm; panels c and g: 200 pm; and panels d and h: 50 pm.
  • FIG. 34C shows detailed scores and a summary of the kidney pathology scores evaluated by independent histopathologists.
  • IL12 is composed of two subunits: p35 (encoded by IL12A, and the UniProt ID as P29459) and p40 (encoded by IL12B, and the UniProt ID as P29460) with a receptor consisting of IL12R01 (binding to p40, encoded by IL12RB1 and IL12R02 (binding to p35, encoded by IL12RB2).
  • IL23 shares p40 and its receptor IL12R01 with IL12, while having unique subunits: pl9 (encoded by I 23A) and its receptor IL23R (encoded by IL23R).
  • IL-35 shares p35 and its receptor IL12R02 with IL 12, with unique subunits: EBB and its receptor GP130 (encoded by IL6ST).
  • Schematic structures of IL12, IL23, IL35, their respective receptors and downstream signaling pathways are shown in FIG. 14.
  • IL23 promotes the differentiation of naive T helper cells into Thl7 phenotype, leading to the secretion of inflammatory cytokines such as IL17 and IL22.
  • IL12 induces Thl polarization and the production of critical cytokines like interferon-y (IFN- y) and tumor necrosis factor.
  • IFN- y interferon-y
  • IL12 is a pro-inflammatory heterodimeric cytokine primarily produced by dendritic cells, monocytes, and macrophages
  • IL35 is an inflammation inhibitory heterodimeric cytokine mainly produced by regulatory T cells and regulatory B cells.
  • PsO psoriasis
  • PsA psoriatic arthritis
  • CD Crohn’s disease
  • UC ulcerative colitis
  • Ustekinumab (Stelara) targeting p40 was approved for CD, UC, PsO and PsA; Risankizumab (Skyrizi) targeting pl 9 was approved for PsO, PsA, and CD; Guselkumab (Tremfya) targeting pl9 was approved for PsO; Tildrakizumab (Ilumya) targeting pl9 was approved for PsO; and Mirikizumab (Omvoh) targeting pl 9 was approved for UC.
  • PBC primary biliary cholangitis
  • SLE systemic erythematosus lupus indications
  • mice deficient in IL12p35 showed no protection or exacerbated disease in some preclinical models, where genetic or antibody-mediated inhibition of IL12p40 or IL12R.pi ameliorated (Cua, D. J., et al. "Interleukin-23 rather than interleukin- 12 is the critical cytokine for autoimmune inflammation of the brain.” Nature 421.6924 (2003): 744-748.; Teng, M.WL, et al.
  • IL-12 and IL-23 cytokines from discovery to targeted therapies for immune- mediated inflammatory diseases.” Nature Medicine 21.7 (2015): 719-729; and Yen, D., et al. "IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL- 17 and IL-6.” The Journal of Clinical Investigation 116.5 (2006): 1310-1316).
  • a common (mis)-concept was formed that anti-IL12-p35 should be less effective comparing to anti-IL12-p40, and if anti- p40 did not work in an autoimmune indication, anti-p35 would not work either.
  • anti-IL12p35 there is no publicly revealed ongoing activities in developing anti-IL12p35 or its receptor IL12RP2 therapies for autoimmune diseases either clinically or pre-clinically.
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the present disclosure provides examples of antibodies, antigen-binding fragment thereof, that bind to IL12p35 (interleukin 12 subunit alpha), which can be used for treating systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • IL12 consists of two subunits connected by disulfide bonds.
  • the smaller p35 monomer 35 kDa a-chain, IL12A, IL12p35, or IL12A-p35
  • the gene for the larger p40 monomer 40 kDa P-chain, IL12B, IL12p40, or IL12B-p40
  • Co-expression results in the formation of the biologically active p70 heterodimer.
  • monomers combine with different partners to create various cytokines.
  • IL35 may also pair with Epstein-Barr virus induced gene 3 (EBB) to yield IL35
  • EBB Epstein-Barr virus induced gene 3
  • the p40 subunit in combination with the pl9 monomer leads to the formation of IL-23.
  • the fourth member of the family is IL-27, which is composed of EBI3 and the p28 subunit.
  • p40 homodimers Due to the lower expression of the IL12 a-chain compared to the P-chain, only free P- chains, p40 homodimers or the heterodimer are secreted. Structurally, the p40 subunit shares some features with the IL6 receptor, whereas the p35 subunit is similar to the granulocyte colony-stimulating factor (G-CSE) and IL-6. It has been demonstrated in mice that p40 homodimers regulate the activity of IL12 by counteracting IL12-induced signaling via competition with IL12p70 for binding to the receptor. Eurther functions of p40 homodimers have been described, e.g., roles in the migration of dendritic cells (DCs), allograft rejection or chemotactic activity with regard to macrophages.
  • DCs dendritic cells
  • DCs dendritic cells
  • chemotactic activity with regard to macrophages.
  • IL12-receptor pi (IL12RP1) is encoded on chromosome 19 and has a molecular weight of 100 kDa. It is a transmembrane protein with the extracellular domain consisting of 516 amino acids that is responsible for the interaction with IL12p40. Consistently, it is also part of the receptor for IL23, where it pairs with IL23R.
  • the gene for IL12RP2 is located on chromosome 1 and is translated to a 130 kDa transmembrane protein, with 595 amino acids forming the extracellular domain.
  • IL12RP2 Signal transduction into the cell derives from IL12RP2, which interacts with IL12p35 and is, thus, in combination with glycoprotein 130 (gpl30), also part of the IL35 receptor.
  • the IL27 receptor as the fourth receptor of the family is composed of gpl30 together with the interleukin 27 receptor subunit alpha (WSX1). Since NK cells and T cells are the main targets of IL12, the expression of IL12R is predominantly confined to these cell types. In particular, antigen contact of naive T cells induces upregulation of IL12RP2, which is subsequently maintained by interferon gamma (IFN-y) signaling, but may be counteracted by IL-4.
  • IFN-y interferon gamma
  • T cells to different effector T (Teff) cell lineages such as cells with a T helper type 1 (TH1), but not a TH2 phenotype and, consistently, only the former cells express IL12R02.
  • Teff effector T
  • IL12 is primarily produced by professional antigen-presenting cells (APCs) such as B cells and DCs as well as phagocytes including monocytes, macrophages and granulocytes. While the production of IL12p35 is predominantly regulated at the translational level, transcriptional regulation is responsible for the amount of IL12p40 expressed.
  • the initial signal triggering IL 12 expression is the exposure of the above mentioned cells to bacteria, viruses, fungi or parasites.
  • Pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) or CpG DNA expressed or contained in such commensals or pathogens are recognized by pattern recognition receptors (PRRs) of the toll like receptor (TLR) family. This leads to the activation of several transcription factors regulating IL12 production, most importantly NF-KB and interferon regulatory elements (IRFs).
  • TLRs are linked to the expression of IL12p40, while expression of p35 is induced by only a limited subset of these receptors, including TLR3, 4 and 8.
  • activation of TLRs also leads to the secretion of IFN-0 and IFN-y, whose signaling, in turn, induces activation of IRF-1, IRF-7 and IRF-8. All three IRFs induce p35 and IRF-7 and IRF-8 also induce p40.
  • IL12 expression is regulated via interaction of APCs with T-cells through CD40 and its ligand CD40L.
  • CD40 signaling through H-Ras and K-Ras enhances p38 mitogen- activated protein kinase (MAPK)-mediated pro-inflammatory IL12 production.
  • MPK mitogen- activated protein kinase
  • An important positive feedback loop increasing IL12 secretion is so-called IFN-y priming. IFN-y release downstream of IL12 further boosts IL12 production via induction of IL12p35 by IRF-1 and of p40 by ICSBP.
  • IL12R01 subsequently recruits the JAK family member tyrosine kinase 2 (TYK2), whereas IL12R02 associates with JAK2, resulting in phosphorylation of JAK2.
  • TYK2 JAK family member tyrosine kinase 2
  • IL12R02 associates with JAK2, resulting in phosphorylation of JAK2. This activates the kinase activity of JAK2, which now, vice versa, phosphorylates a tyrosine residue of the associated receptor subunit.
  • STAT molecules contain SRC homology domains (SH2), which, in a next step, bind to phospho-IL12Rp2 exposing the STATs to JAK and leading to their phosphorylation. Association of these activated transcription factors to homo- or heterodimers enables subsequent nuclear translocation. By binding to specific DNA sequences, they promote or repress gene transcription. STAT4 is the most important downstream target of IL12, while effects on STAT1, STAT3 and STAT5 molecules play minor roles. Moreover, IL12R signaling activates mitogen-activated protein kinase kinase 3/6 (MKK) and p38 MAPK, which support the secretion of IFN-y in activated T cells and TH1 cells. Importantly, this pathway is mediated by a STAT4-independent mechanism and correlates with increased STAT2
  • IL12 A main effect of IL12 is the induction of IFN-y production, by which the cytokine is importantly implicated in adaptive as well as innate immune processes. Additionally, it has been shown that IL12 also primed CD4+ and CD8+ T cells to produce IL10, when present early during clonal expansion. This might result in the development of IL10 secreting Type 1 regulatory (Tri) cells in response to IL12 and IL27, which is consistent with the observation of IL12-dependent Tri cell development in visceral leishmaniasis patients.
  • Tri Type 1 regulatory
  • TH2 differentiation to the contrary, is counteracted by IL12, since GATA binding protein 3 (GAT A3), which is indispensable for TH2 polarization, is repressed in CD4+ and CD 8+ T cell populations upon treatment with IL12 or in vivo expansion in the presence of IL12-producing DCs.
  • GATA binding protein 3 GAT A3
  • IBDs Inflammatory Bowel Diseases
  • SLE systemic lupus erythematosus
  • PBC primary biliary cholangitis
  • SjS Sjogren's syndrome
  • IL 12, IL 12 receptor, and their functions are described e.g., in Ullrich, K. A-M., et al. "Immunology of IL- 12: An update on functional activities and implications for disease.” EXCLI Journal 19 (2020): 1563; Wojno, E.D., et al. "The immunobiology of the interleukin- 12 family: room for discovery.” Immunity 50.4 (2019): 851-870; and Sun, L., et al. "Interleukin 12 (IL-12) family cytokines: Role in immune pathogenesis and treatment of CNS autoimmune disease.” Cytokine 75.2 (2015): 249-255; each of which is incorporated herein by reference in its entirety. Anti-IL12p35 Antibodies and Antigen-Binding Fragments
  • the disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to IL12p35.
  • the antibodies and antigen-binding fragments thereof that specifically bind to IL12p35 described herein are fully human antibodies or antigen-binding fragments thereof.
  • the antibodies and antigen-binding fragments described herein are capable of binding to IL12p35 (e.g., human IL12p35), blocking the binding of IL12 and its receptor IL12R02, and blocking the IL12-induced intracellular signaling pathways.
  • anti-IL12p35 antibodies D2M008-A1C6, D2M008-A1A12, D2M008- A1E5, D2M008-A1A2, D2M008-A1E7, D2M008-A1G9, D2M008-A1A8, D2M008-A1A9, D2M008-A1B4, D2M008-A1E10, D2M008-B2C5, D2M008-B2B10, D2M008-B2B1, D2M008- B2B12, D2M008-B2C3, D2M008-B2B7, D2M008-3A4, D2M008-3A8, D2M008-4A4, D2M008-4A5, D2M008-3A11, D2M008-3A2, D2M008-27H28L 4A4-hblb2, 27H28
  • the CDR sequences for “D2M008-A1C6”, and “D2M008-A1C6” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 47, 48, and 49, and CDRs of the light chain variable domain, SEQ ID NOs: 50, 51, and 52, as defined by Kabat numbering.
  • the CDRs can also be defined by IMGT numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 185, 186, 187
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 188, 189, and 190.
  • the CDR sequences for “D2M008-A1 A12”, and “D2M008-A1A12” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 53, 54, and 55, and CDRs of the light chain variable domain, SEQ ID NOs: 56, 57, and 58, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 191, 192, and 193
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 194, 195, and 196.
  • the CDR sequences for “D2M008-A1E5”, and “D2M008-A1E5” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 59, 60, and 61, and CDRs of the light chain variable domain, SEQ ID NOs: 62, 63, and 64, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 197, 198, and 199
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 200, 201, and 202.
  • the CDR sequences for “D2M008-A1A2”, and “D2M008-A1 A2” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 65, 66, and 67, and CDRs of the light chain variable domain, SEQ ID NOs: 68, 69, and 70, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 203, 204, and 205
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 206, 207, and 208.
  • the CDR sequences for “D2M008-A1E7”, and “D2M008-A1E7” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 71, 72, and 73, and CDRs of the light chain variable domain, SEQ ID NOs: 74, 75, and 76, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 209, 210, and 211
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 212, 213, and 214.
  • the CDR sequences for “D2M008-A1G9”, and “D2M008-A1G9” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 77, 78, and 79, and CDRs of the light chain variable domain, SEQ ID NOs: 80, 81, and 82, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 215, 216, and 217
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 218, 219, and 220.
  • the CDR sequences for “D2M008-A1A8”, and “D2M008-A1 A8” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 83, 84, and 85, and CDRs of the light chain variable domain, SEQ ID NOs: 86, 87, and 88, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 221, 222, and 223, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 224, 225, and 226.
  • the CDR sequences for “D2M008-A1A9”, and “D2M008-A1 A9” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 89, 90, and 91, and CDRs of the light chain variable domain, SEQ ID NOs: 92, 93, and 94, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 227, 228, and 229
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 230, 231, and 232.
  • the CDR sequences for “D2M008-A1B4”, and “D2M008-A1B4” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 95, 96, and 97, and CDRs of the light chain variable domain, SEQ ID NOs: 98, 99, and 100, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 233, 234, and 235
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 236, 237, and 238.
  • the CDR sequences for “D2M008-A1E10”, and “D2M008-A1E10” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 101, 102, and 103, and CDRs of the light chain variable domain, SEQ ID NOs: 104, 105, and 106, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 239, 240, and 241
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 242, 243, and 244.
  • the CDR sequences for “D2M008-B2C5”, and “D2M008-B2C5” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 107, 108, and 109, and CDRs of the light chain variable domain, SEQ ID NOs: 110, 111, and 112, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 245, 246, and 247
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 248, 249, and 250.
  • the CDR sequences for “D2M008-B2B10”, and “D2M008-B2B 10” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 113, 114, and 115, and CDRs of the light chain variable domain, SEQ ID NOs: 116, 117, and 118, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 251, 252, and 253
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 254, 255, and 256.
  • the CDR sequences for “D2M008-B2B1”, and “D2M008-B2B1” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 119, 120, and 121, and CDRs of the light chain variable domain, SEQ ID NOs: 122, 123, and 124, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 257, 258, and 259
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 260, 261, and 262.
  • the CDR sequences for “D2M008-B2B12”, and “D2M008-B2B12” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 125, 126, and 127, and CDRs of the light chain variable domain, SEQ ID NOs: 128, 129, and 130, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 263, 264, and 265, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 266, 267, and 268.
  • the CDR sequences for “D2M008-B2C3”, and “D2M008-B2C3” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 131, 132, and 133, and CDRs of the light chain variable domain, SEQ ID NOs: 134, 135, and 136, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 269, 270, and 271
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 272, 273, and 274.
  • the CDR sequences for “D2M008-B2B7”, and “D2M008-B2B7” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 137, 138, and 139, and CDRs of the light chain variable domain, SEQ ID NOs: 140, 141, and 142, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 275, 276, and 277
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 278, 279, and 280.
  • the CDR sequences for “D2M008-3A4”, and “D2M008-3A4” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 143, 144, and 145, and CDRs of the light chain variable domain, SEQ ID NOs: 146, 147, and 148, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 281, 282, and 283, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 284, 285, and 286.
  • the CDR sequences for “D2M008-3A8”, and “D2M008-3A8” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 149, 150, and 151, and CDRs of the light chain variable domain, SEQ ID NOs: 152, 153, and 154, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 287, 288, and 289
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 290, 291, and 292.
  • the CDR sequences for “D2M008-4A4”, and “D2M008-4A4” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 155, 156, and 157, and CDRs of the light chain variable domain, SEQ ID NOs: 158, 159, and 160, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 293, 294, and 295
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 296, 297, and 298.
  • the CDR sequences for “4A4-hblb2”, and “4A4-hblb2” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 155, 156, and 157, and CDRs of the light chain variable domain, SEQ ID NOs: 158, 159, and 160, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 293, 294, and 295
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 296, 297, and 298.
  • the CDR sequences for “D2M008-4A5”, and “D2M008-4A5” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 161, 162, and 163, and CDRs of the light chain variable domain, SEQ ID NOs: 164, 165, and 166, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 299, 300, and 301
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 302, 303, and 304.
  • the CDR sequences for “D2M008-3A11”, and “D2M008-3A11” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 167, 168, and 169, and CDRs of the light chain variable domain, SEQ ID NOs: 170, 171, and 172, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 305, 306, and 307
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 308, 309, and 310.
  • the CDR sequences for “D2M008-3A2”, and “D2M008-3A2” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 173, 174, and 175, and CDRs of the light chain variable domain, SEQ ID NOs: 176, 177, and 178, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 311, 312, and 313, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 314, 315, and 316.
  • the CDR sequences for “D2M008-27H28L”, and “D2M008-27H28L” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 179, 180, and 181, and CDRs of the light chain variable domain, SEQ ID NOs: 182, 183, and 184, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 317, 318, and 319
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 320, 321, and 322.
  • the CDR sequences for “27H28L-hblb”, and “27H28L-hblb” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 179, 180, and 181, and CDRs of the light chain variable domain, SEQ ID NOs: 182, 183, and 184, as defined by Kabat numbering.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 317, 318, and 319
  • CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 320, 321, and 322.
  • the disclosure provides an antibody or antigen-binding fragment thereof that binds to IL12p35 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least amino acid sequence that
  • the VH and VL of D2M008-A1C6 are set forth in SEQ ID NOs: 1 and 2.
  • the VH and VL of D2M008-A1A12 are set forth in SEQ ID NOs: 3 and 4.
  • the VH and VL of D2M008-A1E5 are set forth in SEQ ID NOs: 5 and 6.
  • the VH and VL of D2M008-A1 A2 are set forth in SEQ ID NOs: 7 and 8.
  • the VH and VL of D2M008-A1E7 are set forth in SEQ ID NOs: 9 and 10.
  • the VH and VL of D2M008-A1G9 are set forth in SEQ ID NOs: 11 and 12.
  • the VH and VL of D2M008-A1A8 are set forth in SEQ ID NOs: 13 and 14.
  • the VH and VL of D2M008-A1A9 are set forth in SEQ ID NOs: 15 and 16.
  • the VH and VL of D2M008-A1B4 are set forth in SEQ ID NOs: 17 and 18.
  • the VH and VL of D2M008-A1E10 are set forth in SEQ ID NOs: 19 and 20.
  • the VH and VL of D2M008-B2C5 are set forth in SEQ ID NOs: 21 and 22.
  • the VH and VL of D2M008-B2B10 are set forth in SEQ ID NOs: 23 and 24.
  • the VH and VL of D2M008-B2B1 are set forth in SEQ ID NOs: 25 and 26.
  • the VH and VL of D2M008-B2B12 are set forth in SEQ ID NOs: 27 and 28.
  • the VH and VL of D2M008-B2C3 are set forth in SEQ ID NOs: 29 and 30.
  • VH and VL of D2M008-B2B7 are set forth in SEQ ID NOs: 31 and 32.
  • VH and VL of D2M008-3A4 are set forth in SEQ ID NOs: 33 and 34.
  • the VH and VL of D2M008-B2B7 are set forth in SEQ ID NOs: 31 and 32.
  • the VH and VL of D2M008-3A4 are set forth in SEQ ID NOs: 33 and 34.
  • the VH and VL of D2M008-3A4 are set forth in SEQ ID NOs: 33 and 34.
  • D2M008-3A8 are set forth in SEQ ID NOs: 35 and 36.
  • D2M008-4A4 are set forth in SEQ ID NOs: 37 and 38.
  • D2M008-4A5 are set forth in SEQ ID NOs: 39 and 40.
  • D2M008-3 Al 1 are set forth in SEQ ID NOs: 41 and 42.
  • the VH and VL of D2M008-3A2 are set forth in SEQ ID NOs: 43 and 44.
  • the VH and VL of D2M008-27H28L are set forth in SEQ ID NOs: 45 and 46.
  • the VH and VL of 4A4- hblb2 are set forth in SEQ ID NOs: 335 and 336.
  • the VH and VL of 27H28L-hblb are set forth in SEQ ID NOs: 337 and 338.
  • These antibodies can be human or humanized antibodies.
  • any of these heavy chain variable region sequences can be paired with any of these light chain variable region sequences (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 336, and 338).
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from FIGS. 26-27; and/or one, two, or three light chain variable region CDRs selected from FIGS. 26-27.
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs and/or one, two, or three light chain variable region CDRs as shown in FIG. 26, under Kabat numbering scheme.
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs and/or one, two, or three light chain variable region CDRs as shown in FIG. 27, under IMGT numbering scheme.
  • the antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of any one of the VH CDR1 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions; any one of the VH CDR2 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions; any one of the VH CDR3 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of any one of the VL CDR1 shown in FIGS.
  • the insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • the disclosure also provides antibodies or antigen-binding fragments thereof that bind to IL12p35.
  • the antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence.
  • the selected VH sequence is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 335, or 337.
  • the selected VL sequence is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 336, or 338.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain.
  • the immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs (under Kabat, or IMGT numbering) as shown in FIGS. 26-27.
  • the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to IL12p35.
  • the anti-IL12p35 antibodies and antigen-binding fragments thereof can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (e.g., bi-specific) antibodies or antibody fragments.
  • Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof.
  • the antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.
  • the antibody or antigen-binding fragment thereof is an IgG antibody or antigenbinding fragment thereof.
  • Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody (e.g., a full IgG antibody).
  • a fragment of an antibody that binds to IL12p35 will retain an ability to bind to IL12p35.
  • An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer.
  • the six CDRs or a subset thereof confer antigen binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • the anti-IL12p35 antibodies and antigen-binding fragments thereof described herein have an Fc region (e.g., a human IgGl Fc region).
  • the Fc region described herein includes one or more of YTE mutations (M252Y/S254T/T256E according to EU numbering).
  • the YTE mutations are located at the CH2-CH3 interface in the Fc domain, which have been shown to increase the binding affinity of the antibody Fc at pH 6.0 to the MHC Class I neonatal FcR (FcRn), located primarily in the acidic endosomes of endothelial and haematopoietic cells, thereby permitting more efficient recycling of administered IgGl antibody and longer retention in the plasma.
  • the increased FcRn binding at pH 6.0 by a YTE triple- mutant antibody is mediated by the creation of one additional salt bridge between Glu 256 (E) of Fc-YTE and Gin 2(Q) of the b2-microglobulin chain of FcRn compared to the original IgGl Fc structure.
  • YTE mutations can result in higher FcRn binding, and has been shown to be well tolerated and extended the half-life of antibodies in human. Details of the YTE mutations can be found, e.g., in Oganesyan, V, et al. "Structural insights into neonatal Fc receptor-based recycling mechanisms.” Journal of Biological Chemistry 289.11 (2014): 7812-7824; and Wang, X. et al., "IgGFc engineering to modulate antibody effector functions.” Protein & Cell 9.1 (2016): 63-73; each of which is incorporated herein by reference in its entirety.
  • Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • the Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain.
  • F(ab')2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
  • Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
  • Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgGl molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
  • purified antibody preparations e.g., purified IgGl molecules
  • antibody homodimers may be formed through chemical linkage techniques known in the art.
  • heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane- 1 -carboxylate) and SATA (N- succinimidyl S-acethylthio-acetate) can be used to form antibody multimers.
  • SMCC succinimidyl 4-(maleimidomethyl)cyclohexane- 1 -carboxylate
  • SATA N- succinimidyl S-acethylthio-acetate
  • An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Set. U.S.A. 94: 7509-7514, 1997).
  • Antibody homodimers can be converted to F(ab’)2 homodimers through digestion with pepsin.
  • the multi-specific antibody is a bi-specific antibody.
  • Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the interface can contain at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
  • Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin.
  • Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigenbinding fragment thereof in a subject or in solution).
  • stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin).
  • the conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).
  • the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent.
  • the antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent.
  • the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).
  • cytotoxic or cytostatic agent e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenopos
  • the antibodies or antigen-binding fragments thereof described herein include one or more back-to-germline (B2G) mutations, e.g., through amino acid substitutions, deletions, and/or insertions in framework regions (FRs) of any of the VHs and VLs described herein.
  • B2G mutations may reduce potential immunogenicity to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% as compared to that of an antibody or antigen-binding fragment thereof without the one or more B2G mutations.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 B2G mutations can be introduced to any of the VHs and VLs described herein.
  • one or more B2G mutations are made before VH CDR1 , between VH CDR1 and VH CDR2, between VH CDR2 and VH CDR3, and/or after VH CDR3, in any of the VHs described herein.
  • one or more B2G mutations are made before VL CDR1, between VL CDR1 and VL CDR2, between VL CDR2 and VL CDR3, and/or after VL CDR3, in any of the VLs described herein.
  • the one or more B2G mutations are in the framework regions (FRs) of the VH, e.g., in FR1, FR2, FR3, and/or FR4 of the VH. In some embodiments, the one or more B2G mutations are in the framework regions (FRs) of the VL, e.g., in FR1, FR2, FR3, and/or FR4 of the VL. In some embodiments, the one or more B2G mutations are not within CDRs of the VH, e.g., in CDR1, CDR2, and/or CDR3 of the VH. In some embodiments, the one or more B2G mutations are not within CDRs of the VL, e.g., in CDR1, CDR2, and/or CDR3 of the VL.
  • sequences of the VH e.g., any of the VHs described herein
  • the VL e.g., any of the VLs described herein
  • sequences of the VH and/or the VL can be aligned with the closest germline sequences, and the B2G mutations within the framework regions (e.g., FRs) can be identified.
  • the B2G mutations in the VH are also referred to as heavy chain B2G mutations (“hb”).
  • the B2G mutations in the VL are also referred to as light chain B2G mutations (“lb”).
  • more than one sets of B2G mutations may be identified, e.g., when different germline sequences are used for alignment.
  • the first set of light chain B2G mutations may be referred to as “lb” and the second set of light chain B2G mutations may be referred to as “lb2. ”
  • an antibody or antigen-binding fragment thereof e.g., any of the antibodies or antigen-binding fragments thereof described herein
  • the antibody or antigen-binding fragment thereof may be referred to as having “hblb” or “hblb2” mutations, e.g., 27H28L-hblb- YTE and 4A4-hblb2-YTE.
  • the B2G mutations described herein may be used to increase the sequence pool of the antibodies or antigen-binding fragments thereof described herein so as to identifying variants having a reduced immunogenicity. Details of B2G mutations can be found, e.g., in Rossotti, M.A., et al. "Immunogenicity and humanization of single - domain antibodies.” The FEBS Journal 289.14 (2022): 4304-4327; and Clavero- Alvarez, A., et al. "Humanization of antibodies using a statistical inference approach.” Scientific Reports 8.1 (2016): 14820; each of which is incorporated herein by reference in its entirety.
  • antibodies also called immunoglobulins
  • a non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains (e.g., a full IgG antibody described herein).
  • the heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc.
  • the light chain can be a kappa light chain or a lambda light chain.
  • An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain.
  • the heavy chains which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions), bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody.
  • the light chains which each contain one variable domain (or variable region, VL) and one constant domain (or constant region), each bind to one heavy chain via disulfide binding.
  • the variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound.
  • variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR). These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principle antigen binding surface of the antibody.
  • CDRs complementary determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
  • the CDRs are important for recognizing an epitope of an antigen.
  • an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody.
  • the minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three- dimensional configuration based on the antigen’s secondary and tertiary structure.
  • the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA).
  • the IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains.
  • the sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions.” Frontiers in Immunology 5 (2014); Irani, et al.
  • the antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid).
  • Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide.
  • the term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F(ab')2, and variants of these fragments.
  • an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi- specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain.
  • Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
  • the antigen binding fragment can form a part of a chimeric antigen receptor (CAR).
  • the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane- and endodomain.
  • the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS).
  • the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency.
  • the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.
  • F(ab) and F(ab')2 fragments are smaller antibody fragments that retain full-length antibodies' antigen-binding specificity.
  • the F(ab) fragment is generated by the enzymatic digestion of the Ig molecule with the proteolytic enzyme papain. Papain cleaves the antibody molecule below the hinge region, resulting in two separate Fab fragments and a smaller Fc fragment.
  • the F(ab')2 fragment is generated by the enzymatic digestion of the Ig molecule with the proteolytic enzyme pepsin.
  • F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa.
  • Divalent antibody fragments e.g., F(ab')2 fragments
  • F(ab')2 fragments are smaller than full IgG antibodies and enable a better penetration into tissue thus facilitating better antigen recognition in immunohistochemistry.
  • the use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A/G.
  • F(ab’)2 fragments and its production methods can be found, e.g., in Rosenstein, S., et al. "Production of F (ab’) 2 from Monoclonal and Polyclonal Antibodies.” Current Protocols in Molecular Biology 131.1 (2020): el l9, which is incorporated herein by reference in its entirety.
  • the antibodies or antigen-binding fragments thereof described herein can block the binding of IL12 (e.g., IL12p70) to its receptor IL12RP2, thereby inhibiting IL12p35/IL12Rp2- specific downstream pathways that are involved in a series of autoimmune diseases (e.g., SSc, PBC, SLE, and SjS), according to the human genetics analysis described in Example 12.
  • IL12p35 is not shared by IL23
  • the antibodies or antigen-binding fragments thereof described herein can specifically neutralize IL12 without interfering the IL23 pathway.
  • Some tested antibodies also exhibited excellent thermostability, endured different pH stress conditions, and exhibited serum stability.
  • the binning competition assay results also indicate that some antibodies can target different epitopes on IL12p35.
  • the antibodies or antigen-binding fragments thereof described herein can reduce the binding of IL12 (e.g., IL12p70) to its receptor IL12RP2 to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl).
  • a reference antibody e.g., human IgGl
  • ICso of the binding curves can be determined.
  • the IC50 of the antibodies or antigen-binding fragments thereof described herein is less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to that of a reference antibody (e.g., human IgGl).
  • YTE mutations and/or the B2G mutations (e.g., any of the YTE and/or B2G mutations described herein) of the antibodies or antigen-binding fragments thereof described herein do not have a significant impact on the binding between IL12 (e.g., IL12p70) and IL12RP2.
  • the binding between IL12 (e.g., IL12p70) and IL12RP2 is determined by ELISA.
  • the antibodies or antigen-binding fragments thereof described herein can reduce human IL12-induced intracellular signaling in a cell (e.g., a human reporter cell) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl) or an anti-IL12p40 antibody (e.g., anti-hP40).
  • the IL12-induced intracellular signaling is JAK-STAT signaling.
  • IC50 of the signaling curves can be determined.
  • the IC50 of the antibodies or antigen-binding fragments thereof described herein is less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to that of a reference antibody (e.g., human IgGl) or an anti-IL12p40 antibody (e.g., anti-hP40).
  • a reference antibody e.g., human IgGl
  • an anti-IL12p40 antibody e.g., anti-hP40
  • YTE mutations and/or the B2G mutations (e.g., any of the YTE and/or B2G mutations described herein) of the antibodies or antigen-binding fragments thereof described herein do not have a significant impact on the inhibition of the IL12-induced intracellular signaling.
  • the inhibition of the IL12-induced intracellular signaling is evaluated using a report cell system.
  • the antibodies or antigen-binding fragments thereof described herein can reduce monkey IL12-induced intracellular signaling in a cell (e.g., a human reporter cell) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl).
  • the antibodies or antigen-binding fragments thereof described herein can reduce monkey IL 12- induced intracellular signaling in a cell (e.g., a human reporter cell) to a level that is comparable to an anti-IL12p40 antibody (e.g., anti-hP40).
  • the antibodies or antigenbinding fragments thereof described herein do not reduce monkey IL12-induced intracellular signaling in a cell (e.g., a human reporter cell) as compared to a reference antibody (e.g., human IgGl).
  • the antibodies or antigen-binding fragments thereof described herein can reduce IL12-induced IFN-y production or secretion by a human cell (e.g., human PBMC) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl) or an anti-IL12p40 antibody (e.g., anti-hP40).
  • the IL12 is an exogenous IL12 (e.g., a recombinant human IL12).
  • the antibodies or antigen-binding fragments thereof described herein can reduce IL12-induced IFN-y production or secretion by T cells (e.g., CD4+ T cells) cocultured with antigen-presenting cells (e.g., allogeneic dendritic cells) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl).
  • the IL12 is an exogenous IL 12 (e.g., a recombinant human IL 12).
  • the antibodies or antigen-binding fragments thereof described herein can reduce IL12-induced IFN-y production or secretion by T cells (e.g., CD4+ T cells) cocultured with antigen-presenting cells (e.g., allogeneic dendritic cells) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to an anti- IL12p40 antibody (e.g., anti-hP40).
  • T cells e.g., CD4+ T cells
  • antigen-presenting cells e.g., allogeneic dendritic cells
  • the antibodies, antigen-binding fragments thereof, or YTE and/or B2G variants thereof described herein do not interfere IL23 binding to its receptors and/or IL23- induced signaling pathways.
  • the antibody specifically binds to IL12p35 (e.g., human IL12p35, monkey IL12p35, dog IL12p35, mouse IL12p35, and/or chimeric IL12p35), or IL12p70 (e.g., human IL12p70, monkey IL12p70, dog IL12p70, mouse IL12p70, and/or chimeric IL12p70) with a dissociation rate (koff or kd) of less than 0.1 s’ 1 , less than 0.01 s’ 1 , less than 0.001 s’ 1 , less than 0.0001 s’ 1 , or less than 0.0001 s’ 1 .
  • IL12p35 e.g., human IL12p35, monkey IL12p35, dog IL12p35, mouse IL12p35, and/or chimeric IL12p35
  • IL12p70 e.g., human IL12p70, monkey
  • the dissociation rate (koff) is greater than 0.01 s’ 1 , greater than 0.001 s’ 1 , greater than 0.0001 s’ 1 , greater than 0.00001 s’ 1 , or greater than 0.000001 s’ 1 .
  • kinetic association rates is greater than 1 x 10 2 /Ms, greater than 1 x 10 3 /Ms, greater than 1 x 10 4 /Ms, greater than 1 x 10 5 /Ms, or greater than 1 x 10 6 /MS. In some embodiments, kinetic association rates (kon) is less than 1 x 10 5 /Ms, less than 1 x 10 6 /MS, or less than 1 x 10 7 /Ms.
  • KD is less than 1 x 1 O' 6 M, less than 1 x 1 O' 7 M, less than 1 x 1 O' 8 M, less than 1 x ICT 9 M, less than 1 x IO' 10 M, less than 1 x 10' 11 M, or less than 1 x 10' 12 M.
  • the KD is less than 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM 10 pM, 5 pM, or 1 pM.
  • KD is greater than 1 x 1 O' 7 M, greater than 1 x 10' 8 M, greater than 1 x 10' 9 M, greater than 1 x 10' 10 M, greater than 1 x 10' 11 M, or greater than 1 x 10' 12 M.
  • General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR).
  • the measurement is conducted using Gator® Prime BLI system or Carterra® SPR imaging system.
  • the antibody binds to human IL12p35, monkey IL12p35 (e.g., rhesus or cynomolgus IL12p35), dog IL12p35 (e.g., canine IL12p35), mouse IL12p35, and/or chimeric IL12p35.
  • the antibody binds to human IL12p70, monkey IL12p70 (e.g., rhesus or cynomolgus IL12p70), dog IL12p70 (e.g., canine IL12p70), mouse IL12p70, and/or chimeric IL12p70.
  • the human IL12p70 described herein is formed by human IL12p35 and human IL12p40.
  • the monkey IL12p70 described herein is formed by monkey IL12p35 and monkey IL12p40.
  • the dog IL12p70 described herein is formed by dog IL12p35 and dog IL12p40.
  • the mouse IL12p70 described herein is formed by mouse IL12p35 and mouse IL12p40.
  • the antibody does not bind to human IL12p40, monkey IL12p40 (e.g., rhesus or cynomolgus IL12p40), dog IL12p40 (e.g., canine IL12p40), mouse IL12p40, or chimeric IL12p40.
  • YTE mutations of the antibodies or antigen-binding fragments thereof described herein can improve FcRn binding affinity at acidic pHs (e.g., about pH 6.0, pH 6.5, or pH 7.0) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold as compared to that of the parental antibodies (e.g., the antibodies or antigen-binding fragments thereof described herein without YTE mutations).
  • acidic pHs e.g., about pH 6.0, pH 6.5, or pH 7.0
  • the YTE mutations can increase the half-life of the antibodies or antigen-binding fragments thereof described herein in vivo (e.g., when administered in a subject) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to that of the parent antibodies (e.g., the antibodies or antigenbinding fragments thereof described herein without YTE mutations).
  • thermostabilities are determined.
  • the antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67,
  • Tm DI first denaturation temperature
  • Tm D2 second denaturation temperature
  • the antibodies or antigen binding fragments as described herein has a Tm DI (e.g., for full IgG antibodies) greater than 60, 61, 62, 63, 64, 65, 66, 67, 68,
  • the antibodies or antigen binding fragments as described herein has a Tm D2 (e.g., for F(ab’)2 fragments) greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • Tm D2 e.g., for F(ab’)2 fragments
  • Tm, Tm DI, Tm D2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • the full IgG antibodies described herein (e.g., D2M008-4A4 or 27H28L) has a Tm that is greater than 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.1, 68.2, 68.3, 68.4,
  • the F(ab’)2 fragments of the antibodies described herein has a Tm that is greater than 71,
  • the antibodies or antigen-binding fragments thereof described herein after being stored in stress conditions, can maintain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity as compared to an unstressed control sample.
  • the antibodies or antigen-binding fragments thereof described herein are incubated in a water bath (at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, or 45°C) for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days.
  • a water bath at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, or 45°C
  • the antibodies or antigen-binding fragments thereof described herein are in a buffer at an acidic pH (e.g., about pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0) or a basic pH (e.g., about pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9 or pH 9.0).
  • an acidic pH e.g., about pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0
  • a basic pH e.g., about pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9 or pH 9.0.
  • the unstressed control sample described herein is in a buffer at a neutral pH (e.g., about pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, or pH 7.9).
  • a neutral pH e.g., about pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, or pH 7.9
  • the activity of the antibodies or antigen-binding fragments thereof described herein, or the unstressed control sample is determined by measuring the blocking of the binding between human IL12p70 to its receptor IL12R02 (e.g., as described in Example 3 by ELISA), and/or measuring the inhibition of IL12-induced intracellular signaling transduction (e.g., as described in Example 4 by cell-based assays).
  • the antibodies or antigen-binding fragments thereof described herein after being mixed with human serum (e.g., fresh human serum collected from healthy donors) and stored in a water bath (at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, or 45°C) for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days, can maintain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity as compared to a control sample.
  • human serum e.g., fresh human serum collected from healthy donors
  • a water bath at about 30°C, 31°C, 32°C, 33°C,
  • control sample contains the same antibodies or antigen-binding fragments thereof described herein that were mixed with PBS.
  • the antibodies or antigen-binding fragments thereof described herein after being mixed with human serum (e.g., fresh human serum collected from healthy donors) and stored in a water bath (at about 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C) for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or longer, can maintain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity as compared to a sample that has not been stored at the same condition.
  • the activity of the antibodies or antigen-binding fragments thereof described herein, or the control sample is determined by measuring the blocking of the binding between human IL12p70 to its receptor IL12R02 (e.g., as described in Example 3 by ELISA), and/or measuring the inhibition of IL12- induced intracellular signaling transduction (e.g., as described in Example 4 by cell-based assays).
  • the antibodies or antigen-binding fragments thereof described herein can bind to different epitopes on IL12p35. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to the same epitope on IL12p35.
  • the anti-IL12p35 antibodies described herein can decrease immune response in patients having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS) after treatment with a therapeutically effective amount of the anti-IL12p35 antibodies by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to no treatment with the anti-IL12p35 antibodies or treatment with a reference antibody (e.g., Ustekinumab).
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the anti-IL12p35 antibodies described herein are more effective (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds, or 10 folds more effective) than a reference antibody (e.g., Ustekinumab).
  • a reference antibody e.g., Ustekinumab
  • the heavy chain and light chain sequences of Ustekinumab can be found in FIG. 28.
  • the antibodies or antigen-binding fragments thereof described herein may have a good in vivo stability and/or pharmacokinetic properties.
  • the terminal elimination half-life (T1/2) of the antibody or antigen-binding fragment thereof can be at least 100 hours, at least 150 hours, at least 200 hours, at least 250 hours, at least 300 hours, at least 350 hours, at least 375 hours, at least 400 hours, at least 425 hours, at least 450 hours, at least 475 hours, or at least 500 hours.
  • the T1/2 is determined in a subject expressing human FcRn (e.g., a human FcRn transgenic mouse).
  • the antibodies or antigen-binding fragments thereof described herein include one or more YTE mutations and/or B2G mutations.
  • the antibodies or antigen-binding fragments thereof described herein can reduce the average ear thickness of mice in a DNFB-induced chronic contact hypersensitivity mouse model.
  • the average ear thickness can be reduced to less than 1.0 mm, less than 0.9 mm, less than 0.8 mm, less than 0.6 mm, less than 0.5 mm, less than 0.4 mm, or less than 0.3 mm.
  • the average ear thickness can be reduced to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% as compared to that when the mice are treated with PBS or an anti-IL12p40 antibody.
  • the ear thickness is measured after about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days post initial DNFB sensitization.
  • the changes described herein are significant, e.g., with a p value that is less than 0.01 or less than 0.005.
  • the antibodies or antigen-binding fragments thereof described herein can alleviate the decrease of saliva excretion of mice in a STZ-induced Sjogren’s Syndrome mouse model.
  • the decrease of saliva excretion level relative to the body weight can be higher than 4 mg/g, higher than 3 mg/g, higher than 2 mg/g, or higher than 1 mg/g.
  • the saliva excretion level relative to the body weight can be improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to that when the mice are treated with an isotype control antibody.
  • the saliva excretion level is determined using methods described herein, after about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days post initial STZ immunization.
  • the changes described herein are significant, e.g., with a p value that is less than 0.06, less than 0.05, less than 0.04, or less than 0.03.
  • the antibodies or antigen-binding fragments thereof described herein can increase the body weight of mice in a IMQ-induced SLE model.
  • the body weight drop after initial IMQ treatment is less than 10 g, less than 9 g, less than 8 g, less than 7 g, less than 6 g, less than 5 g, less than 4 g, less than 3 g, less than 2 g, or less than 1 g.
  • the body weight can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%, as compared to that when the mice are treated with an isotype control antibody.
  • the body weight is measured after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks post initial IMQ treatment.
  • the antibodies or antigen-binding fragments thereof described herein can increase the urine albumin level of mice in a IMQ-induced SLE model.
  • the ratio of urine albumin/creatinine levels can be above 100 pg/mg, above 1000 pg/mg, above 10000 pg/mg, or above 100000 pg/mg.
  • the ratio of urine albumin/creatinine levels can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, as compared to that when the mice are treated with an isotype control antibody.
  • the ratios described herein is determined after about 1, 2, 3, 4, 5, 6, 7, or 8 weeks post initial IMQ treatment.
  • the antibodies or antigen-binding fragments thereof described herein can increase the urine albumin level of mice in a spontaneous SLE model.
  • the ratio of urine albumin/creatinine levels can be above 1 mg/mg, above 10 mg/mg, above 20 mg/mg, above 30 mg/mg, above 40 mg/mg, above 50 mg/mg, above 60 mg/mg, above 70 mg/mg, above 80 mg/mg, above 90 mg/mg, or above 100 mg/mg.
  • the ratio of urine albumin/creatinine levels can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, as compared to that when the mice are treated with an isotype control antibody.
  • the ratios described herein is determined after about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks from the first day of the experiment using the spontaneous SLE model.
  • the antibodies or antigen-binding fragments thereof described herein can significantly reduce kidney pathologic symptoms associated with SLE, as compared that when the mice are treated with an isotype control antibody. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can slow down disease progression in the spontaneous SLE model. In some embodiments, the antibodies or antigenbinding fragments thereof described herein can increase the activity score by at least 50%, at least 75%, or at least 100% as compared to that when the mice are treated with an isotype control antibody in the spontaneous SLE model.
  • the antibodies or antigenbinding fragments thereof described herein can decrease the chronicity score to less than 50%, less than 40%, less than 30%, or less than 20% as compared to that when the mice are treated with an isotype control antibody in the spontaneous SLE model. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can decrease the independent lesion score to less than 80%, less than 60%, less than 40%, or less than 20% as compared to that when the mice are treated with an isotype control antibody in the spontaneous SLE model.
  • An isolated fragment of human IL12p35 can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
  • Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein.
  • the antigenic peptide or protein is injected with at least one adjuvant.
  • the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times).
  • the full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens.
  • the antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of IL12p35 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
  • the full length sequence of human IL12p35 is known in the art.
  • an Fc-tagged or His- tagged human IL12p35 protein is used as the immunogen.
  • An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus).
  • a suitable subject e.g., human or transgenic animal expressing at least one human immunoglobulin locus.
  • An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., the recombinant chimeric IL12 described herein, or a fragment of human IL12p35).
  • the preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject (e.g., a RenMabTM mouse) with the recombinant chimeric IL12 described herein as an immunogen.
  • a suitable subject e.g., a RenMabTM mouse
  • a IL12p35 polypeptide or an antigenic peptide thereof e.g., part of IL12p35
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using an immobilized human IL12p35 or a fragment thereof.
  • ELISA enzyme-linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol.
  • Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
  • Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis.
  • Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigenbinding domain.
  • some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., IL12p35.
  • any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target.
  • the amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.
  • Antibodies disclosed herein can be derived from any species of animal, including mammals.
  • Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies.
  • a mouse e.g., a RenMabTM mouse with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used to generate antibodies.
  • the heavy chain immunoglobulin locus is a region on the chromosome that contains genes for the heavy chains of antibodies.
  • the locus can include e.g., human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes.
  • the kappa chain immunoglobulin locus is a region on the chromosome that contains genes that encode the light chains of antibodies (kappa chain).
  • the kappa chain immunoglobulin locus can include e.g., human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes.
  • human IGKV variable
  • human IGKJ joining
  • mouse light chain constant domain genes e.g., RenMabTM mice.
  • a recombinant chimeric IL12 comprising mouse IL12p40 and human IL12p35 is used as the immunogen to immunize a mouse (e.g., a RenMabTM mouse).
  • a mouse e.g., a RenMabTM mouse
  • the obtained antibodies can specifically target human IL12p35, but not mouse IL12p40.
  • Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • a humanized antibody typically has a human framework (FR) grafted with non-human CDRs.
  • FR human framework
  • a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.
  • VH and VL domains are very important for reducing immunogenicity.
  • the sequence of the V domain of a mouse antibody is screened against the entire library of known human-domain sequences.
  • the human sequence which is closest to that of the mouse is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol. , 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).
  • humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • amino acid sequence variants of the human, humanized, or chimeric anti- IL12p35 antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with a sequence present in the light or heavy chain of the original antibody.
  • the antibodies generated by the mice have a full human VH, a full human VL, and mouse constant regions.
  • the human VH and human VL is linked to a human IgG constant regions (e.g., IgGl, IgG2, IgG3, and IgG4).
  • Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric anti-IL12p35 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo.
  • Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional crosslinkers as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993).
  • an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).
  • a covalent modification can be made to the anti-IL12p35 antibody or antigen-binding fragment thereof.
  • These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage.
  • Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide)
  • a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly- A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly- A tail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with recombinant virus).
  • vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus
  • viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Set.
  • the DNA insert comprising an antibody-encoding or polypeptide- encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan.
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors can include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
  • coli Streptomyces, and Salmonella typhimurium cells
  • fungal cells such as yeast cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells
  • plant cells Appropriate culture mediums and conditions for the host cells described herein are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • the antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.
  • the disclosure provides methods for treating, preventing, or reducing the risk of developing disorders associated with an abnormal or unwanted immune response, e.g., an autoimmune disorder, e.g., by inhibiting IL12p35/IL12Rp2-specific downstream pathways.
  • the methods described herein can inhibit the IL 12 signaling pathway without interfering the IL23 signaling pathway.
  • the methods described herein can neutralize IL12 but reserve IL23 in vivo.
  • SSc Systemic sclerosis
  • PBC Primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • SSc Systemic sclerosis
  • SSc-ILD SSc-associated interstitial lung disease
  • PBC Primary biliary cholangitis
  • SLE Systemic lupus erythematosus
  • Sjogren's syndrome a prevalent chronic autoimmune rheumatic disease, features lymphocytic infiltration of exocrine glands and various extra-glandular organs. Treatment mainly focuses on symptom management and complication prevention, lacking a cure (Zhan, Q., et al. "Pathogenesis and treatment of Sjogren’s syndrome: Review and update.” Frontiers in Immunology 14 (2023): 1127417).
  • the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein can block the binding of IL12 to its receptor IL12RP2, thereby inhibiting IL12- induced intracellular pathways (e.g., IL12p35/ IL12Rp2-specific pathways).
  • these anti-IL12p35 antibodies or antigenbinding fragments thereof can be used for treating a cluster of immune disorders (e.g., SSc, PBC, SLE, and SjS).
  • the subject described herein has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic lupus erythematosus
  • SjS Sjogren's syndrome
  • the subject is a human subject.
  • the subject is a non-human mammal, e.g., a monkey, a dog, a mouse, or any commonly used model animals known in the art.
  • the subject is a dog having a similar disease or disorder as SSc, PBC, SLE, and/or SjS.
  • the dog has an immune disorder (e.g., an autoimmune disease), e.g., Hypothyroidism, Lupus, Immune-Mediated Polyarthritis (IMP A), Inflammatory Bowel Disease (IBD), Immune-Mediated Hemolytic Anemia (IMHA), Immune-Mediated Thrombocytopenia (IMT), Diabetes, Myasthenia Gravis, Rheumatoid Arthritis, Addison’s Disease (Hypoadrenocorticism), Bullous Autoimmune Skin Diseases, and Periodontal disease. Details can be found, e.g., in Pedersen, N. C. "A review of immunologic diseases of the dog.” Veterinary Immunology and Immunopathology 69.2-4 (1999): 251-342, which is incorporated herein by reference in its entirety.
  • an immune disorder e.g., an autoimmune disease
  • the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein cannot block the binding of IL12p40 to its receptor IL12RP1.
  • these anti- IL12p35 antibodies or antigen-binding fragments thereof do not interfere IL23 binding to its receptors or transducing signals (e.g., IL12p40/IL12Rpi-specific pathways).
  • the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein may not be suitable for treating a cluster of immune disorders (e.g., PsO, CD, UC, IBD, and AS).
  • the subject described herein does not have psoriasis (PsO), Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel diseases (IBD), or ankylosing spondylitis (AS).
  • the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein can inhibit the IL 12 signaling pathway without interfering the IL23 signaling pathway.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., any of the autoimmune diseases described herein.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.
  • Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal.
  • the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein
  • at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).
  • at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition).
  • At least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
  • the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein).
  • the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigenbinding fragments described herein) in the subject.
  • the subject can be administered the at least one antibody, antigenbinding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigenbinding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years).
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer).
  • a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).
  • compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination.
  • the pharmaceutical compositions may be formulated in any manner known in the art.
  • compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).
  • the compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol),
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin).
  • an agent that delays absorption e.g., aluminum monostearate and gelatin.
  • controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).
  • biodegradable, biocompatible polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.
  • compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).
  • parenteral e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage.
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
  • Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
  • antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys).
  • One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50.
  • Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects).
  • Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
  • a therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer), or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured), decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human, a dog, a monkey, or a mouse).
  • a subject e.g., a human, a dog, a monkey, or a mouse.
  • any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human, a dog, a monkey, or a mouse). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases).
  • Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject’s weight (e.g., about 1 pg/kg to about 500 mg/kg; about 100 pg/kg to about 500 mg/kg; about 100 pg/kg to about 50 mg/kg; about 10 pg/kg to about 5 mg/kg; about 10 pg/kg to about 0.5 mg/kg; or about 1 pg/kg to about 50 pg/kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art.
  • relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained.
  • the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half- life of the antibody or antibody fragment in vivo.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.
  • Example 1 Generation of anti-IL12p35 antibodies This example describes how anti-IL12p35 antibodies were generated.
  • a panel of antibodies that selectively bind to human and cynomolgus (cyno) monkey IL12p35 antigens were generated in RenMabTM mice (Biocytogen) by immunizing with recombinant chimeric IL 12.
  • Recombinant chimeric IL 12 is a heterodimer composed of mouse IL12p40 and human IL12p35, which was designed to promote immunogenicity towards human IL12p35 subunit of IL12p70.
  • a total of seven mice were initially immunized subcutaneously through neck and hock injections with a recombinant chimeric IL 12 emulsion prepared in complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • all mice were boosted with the recombinant chimeric IL12 emulsion prepared in incomplete Freund's adjuvant (CFA) three times.
  • Five days after the last boost four mice with a higher serum titer were scarified and proceeded to Beacon binder selection. The rest three mice were further boosted three times.
  • Four days after the last boost two mice with a higher serum titer were scarified and proceeded to Beacon binder selection.
  • Lymphoid organs including lymph nodes, bone marrow, and spleen were harvested from scarified mice. The harvested organs were homogenized to break down tissue structures. Single-cell suspensions were obtained by passing the homogenized material through a 40 pm cell strainer. Cell numbers and viability were assessed. Plasma B cells were enriched using the mouse CD 138+ Plasma Cell Isolation Kit (StemCell Technologies, Cat#: 18957).
  • Beacon screening Plasma B cells were subjected to Beacon screening assays using Beacon® Optofluidic System (Model: 110-08023) and OptoSelect® UK Chips (Berkeley Lights, Cat#: 500-12012).
  • the Beacon bloom screening assays employed streptavidin beads (Berkeley Lights, Cat#: 520-00053) complexed with biotinylated chimeric IL12 or biotinylated chimeric IL 12 to capture secreted binder antibodies and simultaneously quantify bound antibodies with a fluorescently labeled anti-mouse Fc.
  • a total of 128 cells were successfully unloaded from blooming Beacon pins and were subjected to RNA isolation, followed by RT-PCR to amplify nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL).
  • PCR amplicons were sent to Genewiz/Azenta for sequencing. The sequences were analyzed with the IMGT program. The unique clones were converted into full human IgGl format and synthesized for further characterization.
  • Certain clones were engineered to improve pharmacokinetics by introducing an FcRn affinity-enhancing Fc mutations through amino acid substitutions (e.g., M252Y/S254T/T256E, or YTE mutations) and to reduce potential immunogenicity by introducing back-to-germline mutations through amino acid substitutions in frameworks in variable regions.
  • amino acid substitutions e.g., M252Y/S254T/T256E, or YTE mutations
  • Example 2 Anti-IL12p35 antibodies bound to human and monkey IL12p70 with high affinities
  • the binding kinetics of the antibodies identified in Example 1 were determined.
  • the antibodies that were successfully produced were measured using the Gator® Prime BLI (Biolayer Interferometry) System (Gator Bio, USA) and selected variants were measured using the BiaCoreTM SPR platform.
  • the anti-IL12p35 antibodies were loaded onto HFC probes (Anti-hlgGFc, Gator Bio, Cat#: 160003).
  • the probes were incubated with corresponding monomeric antigens human IL12p70-His (Sino Bio, Cat#: CT011-H08H), rhesus IL12-H1S (Sino Bio, Cat#: CT045-C08H), canine IL12p70 (Kingfisher Biotech, Cat#: RP2228D- 005), or mouse IL12p70 (BioLegend, Cat#: 577002) for 3-4 minutes at 25°C in 250 pl K buffer (Gator Bio, Cat#: 120011). Dissociation was monitored for approximately 10 minutes. The probes were regenerated between binding cycles with Regeneration Buffer (Gator Bio, Cat#: 120012).
  • Binding kinetics was analyzed using software supplied by the manufacturer. In particular, the following setting parameters were used: all tested antibodies: 4 pg/ml; canine IL12p70: 2 pg/ml; and mouse IL12p70: 1 pg/ml.
  • BiaCoreTM 8K SPR platform To more accurately measure the binding kinetics, selected antibodies were analyzed using the BiaCoreTM 8K SPR platform. Specifically, the anti-IL12p35 antibodies were loaded on to Protein A chip (Cytiva, Cat#:29127556). Subsequently, the chips were incubated with serially diluted monomeric antigens human IL12p70-His (Sino Bio, Cat#: CT011-H08H) to allow association for 90 seconds and dissociation for 210 seconds, and the kinetics was monitored. The chips were regenerated between cycles with a Glycine hydrochloride buffer at pH 1.5. Binding kinetics was analyzed using BiaCoreTM Insight Evaluation Software supplied by the manufacturer.
  • the anti-IL12p35 antibodies were loaded with HFC (Anti-hlgGFc, Gator Bio, Cat#: 160003), the probes were incubated with a recombinant FcRn protein (Sino Bio, Cat#: CT009- H08H) in K buffer prepared in-house at pH 6.0, pH 6.5, or pH 7.0, and the dissociation steps were performed in the corresponding K buffers.
  • the preliminary binding affinities of antibodies to human IL12p70 were measured by the Gator® Prime System, and results are shown in FIG. 1.
  • their binding affinities were too high (e.g., with estimated KD values less than 1 x 10' 11 M or less than 1 x 10' 12 M), which were out of range (“O.R.”) of the detecting capability by the Gator BLI technology, their KD values were not calculable.
  • D2M008-A1A8, D2M008-A1 A9 and D2M008-4A4 exhibited high binding affinities to human and rhesus IL12p70.
  • Both D2M008-A1 A8 and D2M008-A1 A9 showed an approximately 100- fold difference in binding affinity for human and monkey IL12.
  • Antibody D2M008-4A4 demonstrated very high binding affinities to human and rhesus IL12p70. Because the IL12 protein sequences of cynomolgus monkey and rhesus monkey are identical, the high binding affinity to rhesus IL12p70 suggested antibodies that can also bind to cynomolgus monkey IL12p70, indicating that cynomolgus monkeys (as a relevant species) can be used for future nonclinical studies. As shown in FIGS. 2B-2C, YTE mutations and back-to- germline mutations showed no impact on binding affinities of 27H28L and D2M008-4A4.
  • 27H28L variant 27H28L-hblb-YTE showed a very high affinity to canine IL- 12, which was comparable to its affinity to human IL- 12, as both were out of range on Gator.
  • YTE variants exhibited improved FcRn binding affinity at acidic pHs compared to the parental antibodies, indicating that YTE variants theoretically can improve pharmacokinetics in human compared to parental antibodies.
  • anti-IL12p35 antibodies to block the interaction of human IL12p70 with a recombinant IL12RP2 (interleukin 12 receptor, beta 2 subunit) protein was determined by an ELISA-based assay. Specifically, 1 pg/ml biotinylated human IL12p70 (Sino Bio, Cat#: CT011- H08H-B) was pre- incubated with a fixed concentration of (or serially diluted) anti-IL12p35 or control antibody anti-IL12p40 (Ustekinumab, Biointron, Cat#: B779149; or “anti-hP40”) at room temperature (RT) for 30 minutes, and then added to a 96- well plate coated with 1 pg/mL IL12RP2 (Biointron, Cat#: B21709001), followed by an incubation at RT for 2 hours with gentle shaking.
  • IL12RP2 interleukin 12 receptor, beta 2 subunit
  • Bound IL12p70 was detected using HRP-conjugated streptavidin (BioLegend, Cat#: 405210) and TMB substrate (Surmodics, Cat#: TMBS-1000-01). OD450 was measured using a VarioskanTM Lux plate reader (Thermo Scientific).
  • the 27 binders identified in Examples 2 and listed in FIG. 1 demonstrated differences in the ability to block the interaction between human IL12p70 and recombinant IL12RP2.
  • Eight selected anti-IL12p35 antibodies were further analyzed, which blocked the interaction between human IL12p70 and recombinant IL12RP2 in a dose-dependent manner (FIG. 4B) Titrated 27H28L and several additional anti-IL12p35 antibodies were tested.
  • 27H28L, D2M008-4A4 and D2M008-4A5 exhibited dose-dependent blocking of the interaction between human IL12p70 and recombinant IL12RP2 (FIG. 4C).
  • YTE mutations showed no significant impact on the blocking function of 27H28L and D2M008-4A4 (FIG. 4D).
  • back-to-germline mutations showed no impact on the blocking function (FIG. 4E)
  • anti-IL12p35 antibodies to inhibit IL12p70-induced signaling was evaluated in HEK-BlueTM IL12 reporter cells (InvivoGen, Cat#: hkb-il 12). Specifically, 20 ng/ml recombinant human IL12 (BioLegend, Cat#: 573004) or rhesus IL12 (Sino Bio, Cat#: CT045- C08H) was pre-incubated with different concentrations of IgGl, anti-hP40, or anti-IL12p35 antibodies at room temperature for 30 minutes, and then added to a flat-bottom 96- well plate seeded with 5 10 4 HEK-BlueTM IL12 reporter cells, followed by an incubation at 37°C for 24 hours.
  • the 27 binders identified in Examples 2 and listed in FIG. 1 demonstrated differences in the ability to inhibit IL12p70-induced intracellular signaling in HEK-BlueTM IL12 reporter cells.
  • D2M008-A1 A8 and D2M008-A1 A9 showed the best blocking ability among these IL12 binders.
  • D2M008-A1B4 and D2M008-A1E10 also exhibited blocking capabilities. These four selected anti-IL12p35 antibodies were further analyzed.
  • D2M008-A1 A8 and D2M008-A1 A9 potently inhibited IL12p70-induced intracellular signaling in HEK-BlueTM IL 12 reporter cells in a dose-dependent manner.
  • engineered variant 27H28L which composed of heavy chain of D2M008-A1A8 and light chain of D2M008-A1A9, showed a significantly stronger potency than its parental antibodies D2M008-A1 A8 and D2M008-A1A9 in a dose-dependent manner.
  • 27H28L was also more potent than anti-IL12p40 (Ustekinumab) and was equally active as the combination of anti-IL12p40 (Ustekinumab) and D2M008-A1 A8.
  • D2M008-A1 A8 and D2M008-A1 A9 were slightly better than anti- IL12p40 (Ustekinumab), or at least comparable in inhibiting IL12p70 activity. These results indicate that the engineered variant of anti-IL12p35, 27H28L, can block the interaction of IL12p70 with its receptors via a mode of action that is distinct from D2M008-A1 A8 and D2M008-A1 A9, which is superior to the mode of action of anti-IL12p40 (Ustekinumab).
  • IL 12 The principal function of IL 12 is the activation of T cells and NK cells, leading to an increased production of INF -y, proliferation, and cytotoxic potential.
  • the ability of the identified anti-IL12p35 antibodies to inhibit human IFN-y production in response to exogenous IL12p70 was evaluated using human PBMCs.
  • 10 ng/ml recombinant human IL12 (BioLegend, Cat#: 573004) was pre-incubated with different concentrations of IgGl, anti- IL12p40, or anti-IL12p35 antibodies for 30 minutes at room temperature, and then added to a U- bottom 96- well plate seeded with 1-2 x 10 5 human PBMCs per well, followed by an incubation at 37°C for 24 hours. Supernatant was collected from each well and human IFN-y was measured using the MAXTM Deluxe Set Human IFN-y ELISA kit (BioLegend, Cat#: 430116).
  • anti-IL12p35 antibodies inhibited exogenous IL12-induced IFN-y production in human PBMCs in a dose-dependent manner.
  • D2M008-4A4 and 27H28L were more potent than anti- IL12p40 (Ustekinumab) in inhibiting IL12-induced IFN-y production in human PBMCs.
  • IL12p70 The ability to inhibit endogenous IL12p70 activity was evaluated in an MLR assay using primary human CD4+ T cells and allogenic dendritic cells. Specifically, human dendritic cells were generated from monocytes through incubation with 50 ng/ml of human GM-CSF (BioLegend, Cat#: 766106) and 20 ng/ml of human IL-4 (BioLegend, Cat#: 766206) for 6 days, followed by maturation with 1 mg/ml of LPS (Sigma, Cat#: L2630-10MG) for 1 day.
  • human dendritic cells were generated from monocytes through incubation with 50 ng/ml of human GM-CSF (BioLegend, Cat#: 766106) and 20 ng/ml of human IL-4 (BioLegend, Cat#: 766206) for 6 days, followed by maturation with 1 mg/ml of LPS (Sigma, Cat#: L2630-10MG) for 1 day.
  • 1 x 10 4 dendritic cells in 50 ml of complete 1640 RPMI culture medium were seeded to each well of a flat-bottom 96- well plate, and then 100 ml of different concentrations of IgGl, anti-IL12p40, 27H28L, or D2M008-4A4 (starting at 60 pg/ml, then 3-fold dilutions) in complete 1640 RPMI culture medium were added to the indicated well.
  • 50 pl of 1 x 10 5 allogeneic CD4+ T cells in complete 1640 RPMI culture medium was added to the indicated wells. The cells were mixed and incubated at 37°C for 24 hours. Supernatant from each well was collected and human IFN-y was measured using MAXTM Deluxe Set Human IFN-y ELISA kit (BioLegend, Cat#: 430116).
  • anti-IL12p35 antibodies engineered variant 27H28L and D2M008-4A4
  • anti-IL12p40 Ustekinumab
  • D2M008-4A4 and 27H28L, especially 27H28L were more effective than anti- IL12p40 (Ustekinumab) in inhibiting IFN-y production at low concentrations.
  • anti-IL12p40 Ustekinumab
  • anti-IL12p40 Ustekinumab
  • IL12p35/IL12Rp2 a more efficient strategy for suppressing IL12’s biological activity.
  • This enhanced potency may be attributed to the distinct roles of IL12R 1 and IL12R 2 in the heterodimeric IL12 receptor complex.
  • inhibiting the IL12p35/IL12R 2 interaction offers an efficient strategy to specifically suppress IL12’s biological activity without interfering IL23, whereas blocking the IL12p40/IL12Rpi interaction cannot achieve the same specificity.
  • HEK-BlueTM IL23 reporter cells InvivoGen, Cat#: hkb- il23. Specifically, 10 ng/ml recombinant human IL23 (BioLegend, Cat#: 574104) was preincubated with different concentrations of IgGl, anti-hP40, or anti-IL12p35 antibodies for 30 minutes at room temperature, and then added to a flat-bottom 96-well plate seeded with 5 10 4 per well of HEK-BlueTM IL23 reporter cells, followed by an incubation at 37°C for 24 hours.
  • anti-IL12p40 (Ustekinumab) efficiently neutralized IL23
  • 27H28L, D2M008-4A4 nor their YTE variants inhibited IL23 biological activity.
  • anti-IL12p35 antibodies can specifically neutralize IL 12, unlike anti- IL12p40 which can neutralize both IL 12 and IL23.
  • Example 8 Anti-IL12p35 antibodies exhibited excellent thermostability
  • the melting points of full IgG and F(ab’)2 of humanized anti-IL12p35 antibodies were measured using the Protein Thermal ShiftTM Dye Kit (ThermoFisher, Cat#: 4461146) and a qPCR machine.
  • F(ab’)2 17.25 pL (1 mg/mL) full IgG antibody was digested with 0.25 pL IdeZ Protease (IgG- specific) (NEB, Cat#: P0770S) together with 2 pL 10x GlycoBuffer 2 (NEB, Cat#: P0770S) in a 37°C water bath for 2 hours.
  • anti-IL12p35 antibodies 27H28L and D2M008-4A4 both demonstrated excellent thermostability and showed a T m of 73.99 °C and 79.53 °C, respectively, in their F(ab’)2 format.
  • Example 9 Anti-IL12p35 antibodies exhibited stability under different pH stress conditions
  • antibodies include deamidation and isomerization. Asparagine deamidation and aspartic acid isomerization may be induced in vitro at high and low pH conditions, respectively.
  • 100 pL antibody (1 mg/mL) was exchanged with a pH 5.5 buffer (50 mM Sodium Acetate) or a pH 8.5 buffer (20 mM Tris and 10 mM EDTA) using ZebraTM Spin Desalting columns (40K MWCO, ThermoFisher, Cat#: 87767), respectively. The antibodies were then incubated in a 40°C water bath for 2 weeks.
  • anti-IL12p35 antibodies under different stress conditions was measured using the methods described in Examples 3-4, which detected the ability of anti-IL12p35 antibodies to block the binding of human IL12p70 to its receptor IL12RP2 and inhibit IL12-induced signaling transduction, respectively.
  • the control sample in a pH 7.4 buffer was not subjected to the 40°C water bath for 2 weeks.
  • anti-IL12p35 antibodies 50 pg anti-IL12p35 (about 1 mg/mL) was mixed with an equal volume of fresh human serum collected from healthy volunteers and incubated in a 37°C water bath for 2 weeks. Anti-IL12p35 antibodies exposed to fresh plasma serum were then compared with their untreated stock samples (in PBS without incubation at 37°C water bath for 2 weeks) using ELISA- and cell-based assays described in Examples 3 and 4, measuring their ability to block IL12 binding to IL12RP2 and transducing intracellular signals, respectively.
  • anti-IL12p35 antibodies incubated with human serum for up to 2 weeks exhibited similar activities compared to anti-IL12p35 antibodies incubated with PBS, indicating their serum stability.
  • the stability of 27H28L-hblb-YTE and 4A4-hblb2-YTE were also determined by mixing 50 pg anti-IL12p35 (about 1 mg/mL) with an equal volume of fresh human serum collected from healthy volunteers and incubated in a 37°C water bath for 1, 2, or 3 weeks. Afterwards, 1 pg/ml biotinylated human IL12p70 (Sino Bio, Cat#: CT011-H08H-B) was preincubated with serially diluted anti-IL12p35 antibodies at room temperature (RT) for 30 minutes. Next, 100 pl of the pre-incubated solution was added to the indicated well and incubated at RT for 2 hours.
  • RT room temperature
  • the binning competition assays were carried out using the Gator® Prime BLI (Biolayer Interferometry) System (Gator Bio, USA), for anti-IL12p35 antibodies selected from FIG. 1, in a matrix format. Specifically, individual anti-IL12p35 antibodies were immobilized to anti-human Fc probes, respectively (Anti-hlgG Fc, Gator Bio, Cat#: 160003). Subsequently, the probes were incubated with the monomeric antigen human IL12p70-His (Sino Bio, Cat#: CT011-H08H) in K buffer (Gator Bio, Cat#: 120011) at 25°C for 3-4 minutes. The probes were then incubated with individual anti-IL12p35 antibodies in K buffer for 3-4 minutes at 25°C. The binding kinetics was analyzed using software provided by the manufacturer.
  • Gator® Prime BLI Biolayer Interferometry
  • anti-IL12p35 antibodies 27H28L and D2M008-4A4 were individually immobilized on probes by binding to anti-human Fc probes (Anti-hlgGFc, Gator Bio, Cat#: 160003). Subsequently, the probes were incubated with the monomeric antigen human IL12p70- His (Sino Bio, Cat#: CT011-H08H) in K buffer (Gator Bio, Cat#: 120011) at 25°C for 3-4 minutes. The probes were then incubated with 27H28L or D2M008-4A4 in K buffer for 3-4 minutes at 25°C.
  • human IL12p70-His was immobilized by binding to anti -His probes (Gator Bio, Cat#: 160009). The probes were then incubated with 27H28L and D2M008-4A4, respectively, for 3-4 minutes, followed by an additional incubation with 27H28L or D2M008-4A4 for 3-4 minutes. The binding kinetics was analyzed using software provided by the manufacturer.
  • all anti-IL12p35 could be further placed into three smaller bins: the first bin including D2M008-4A4, A1E7, D2M008-A1C6, D2M008-B2B1 and D2M008-B2C5; the second bin including D2M008-A1A8, D2M008-A1A9, D2M008-B2B7, D2M008-A1E5, D2M008-B2C3, D2M008-B2B10, D2M008-A1A2, D2M008-A1G9 and D2M008-B2B12, and the third bin including D2M008-A1B4 and D2M008-A1E10.
  • D2M008- A1 A8 and D2M008-A1 A9 were derived from the same IGHV and IGKV alleles.
  • D2M008- A1B4 and D2M008-A1E10 were derived from the same IGHV and IGKV alleles.
  • Human immune system is complex. Different autoimmune diseases share similar or distinct mechanisms. Animal models contribute to our understanding of some of the autoimmune diseases; however, they have limitations and cannot fully replicate the human immune system complexity. Many of the human autoimmune diseases are lacking suitable animal models. In this situation, relying on human data, especially human genetics associations on immune diseases may bring more relevant insights.
  • GWAS genome- wide association studies
  • eQTL expression quantitative loci
  • pQTL protein quantitative loci
  • IL12A p35
  • IL12B IL12B
  • IL23R are specifically causal to immune diseases of psoriasis (PsO), Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel diseases (IBD), and ankylosing spondylitis (AS) but not to other autoimmune diseases.
  • PsO psoriasis
  • CD Crohn’s disease
  • UC ulcerative colitis
  • IBD inflammatory bowel diseases
  • AS ankylosing spondylitis
  • IL12A (p35) and IL12R02 are specifically causal to autoimmune diseases of systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic erythematosus lupus (SLE), and Sjogren's syndrome (SjS), with no evidence of associations to PsO, CD, UC, IBD, and AS.
  • SSc systemic sclerosis
  • PBC primary biliary cholangitis
  • SLE systemic erythematosus lupus
  • SjS Sjogren's syndrome
  • the genetics determinant on the molecular phenotypes of IL12/IL23/IL35 pathway ligands and receptors, such as the GWAS of their gene expression (eQTL) in blood cells and GWAS of their protein levels (pQTL) in serum were collected from different resources.
  • the data resources are as below: Table 2.
  • Colocalization is a statistical method of determining whether two distinct traits (such as the expression level of a gene and a disease risk) share a common genetic cause. The process typically involves a Bayesian statistical method to estimate the probabilities of shared and distinct causal variants in overlapping genomic regions. To further evaluate the causal relationship between the selected genes and diseases of interest, colocalization analyses were conducted using the coloc R package v5.2.2 described at chrlswallace.github.io/coloc/articles/a01_intro.html.
  • a beta value represents the effect size of a genetic variant (usually as a Single Nucleotide Polymorphism, SNP) on an outcome, such as gene expression level, protein level, or disease risk.
  • SNP Single Nucleotide Polymorphism
  • the X-axis represents the genetic effect of a Quantitative Trait Locus (QTL) for a gene or protein, while the Y-axis shows the genetic effect for a disease outcome.
  • QTL Quantitative Trait Locus
  • Each dot on the plot corresponds to a genetic variant. Only genetic variants that are common to both the gene/protein and the disease outcome are applicable for inclusion in a beta-beta plot. In our analysis, we restricted genetic variants that were significant associated with gene/protein levels (e.g. eQTL/pQTL) to reduce the noise of the data representation.
  • IL12A IL12p35
  • its receptor IL12R02 are associated with SSc, PBC, SLE, and SjS, but not with PsO, CD, UC, IBD, or AS
  • IL12A eQTL analysis revealed SNP rs4680536, located near the IL12A gene, as the top variant associated with IL12A gene expression levels (plots of the 4th row).
  • the genetic markers in this region also showed significant associations with SSc, SLE, and PBC (with p value ⁇ 5 x 10' 8 ), where the top associated markers were highly correlated with IL12A eQTL top variant (rs4680536).
  • IL12RP2 eQTL analysis revealed SNP rsl7129778, located near the IL12RP2 gene, associated with IL12RP2 gene expression levels (plots of the 4th row).
  • the genetic markers in this region also showed significant associations with SSc, PBC, and SLE (with p value ⁇ 5 x 10' 8 ), where the top associated markers were highly correlated with IL12RB2 eQTL top variant (rsl 7129778).
  • the pattern of significance of association between genotype and IL12RP2 gene expression levels matched closely with that of association with SSc, PBC, SLE, and SjS risk.
  • IL12Rp2 gene expression levels and the SSc, PBC, SLE risk share a common causal genomic variant.
  • different sets of genetic variants not correlated with IL12Rp2’s eQTLs in this genetic region were significantly (with p value ⁇ 5 x 10' 8 ) associated with PsO, CD, UC, IBD, or AS.
  • IL23R is a nearby gene upstream of IL12RP2.
  • the set of variants associated with PsO, CD, UC, IBD, or AS were actually correlated with those affecting IL23R’s protein expression (pQTLs).
  • IL12B and IL23R are associated with PsO, CD, UC, IBD, and AS, but not with SSc, PBC, SLE, or SjS
  • IL12B pQTL analysis revealed SNP rs6556416, located near the IL12B gene, associated with IL12B serum protein levels (plots of the 4th row).
  • the genetic markers in this region also showed significant associations with PsO, CD, UC, IBD, and AS (with p value ⁇ 5 x 10' 8 ), where the top associated markers were highly correlated with IL12B pQTL top variant (rs6556416).
  • the pattern of significance of association between genotype and IL12B protein levels matched closely with that of association with PsO, CD, UC, IBD, and AS risk.
  • IL23R pQTL analysis revealed SNP rsl 1581607, located near the IL23R gene, associated with IL23R serum protein levels (plots of the 4th row).
  • the genetic markers in this region also showed significant associations with PsO, CD, UC, IBD, and AS (with p value ⁇ 5 x 1 O' 8 ), where the top associated markers were highly correlated with IL23R pQTL top variant (rsl 1581607).
  • the pattern of significance of association between genotype and IL23R protein levels matched closely with that of association with PsO, CD, UC, IBD, and AS risk.
  • no genetic variants in this IL23R genetic region were significantly (with p value ⁇ 5 x 1 O' 8 ) associated with SjS either in the data shown here or in other published GWAS studies.
  • different sets of genetic variants not correlated with IL23R’s pQTLs in this genetic region were significantly (with p value ⁇ 5 x 1 O' 8 ) associated with SSc, PBC, and SLE.
  • IL12RP2 is a nearby gene downstream of IL23R. As shown earlier in FIGS. 16A-16C, the set of variants associated with SSc, PBC, and SLE were actually correlated with those affecting IL12Rp2’s gene expression (eQTLs).
  • EBI3 and IL6ST are not associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS.
  • EBB pQTL analysis revealed SNP rs60160662, located near the EBB gene, associated with EBB serum protein levels (plots of the 4th row).
  • SNP rs60160662 located near the EBB gene, associated with EBB serum protein levels (plots of the 4th row).
  • no genetic variants in this EBB genetic region were significantly (with p value ⁇ 5 x 1 O' 8 ) associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS, either in the data shown here or in other published GWAS studies.
  • EBB’s protein level is unlikely a causing effect to these diseases.
  • IL6ST pQTL analysis revealed SNP rsl 1574765, located near the IL6ST gene, associated with IL6ST serum protein levels (plots of the 4th row).
  • SNP rsl 1574765 located near the IL6ST gene, associated with IL6ST serum protein levels (plots of the 4th row).
  • no genetic variants in this IL6ST genetic region were significantly (with p value ⁇ 5 x 1 O' 8 ) associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS, either in the data shown here or in other published GWAS studies.
  • STAT4 is associated with SSc, PBC, SLE, SjS, IBD, CD, and UC; whereas STAT3 is associated with CD, UC, and IBD
  • STAT4 eQTL analysis revealed SNP rsl 6833249, located near the STAT4 gene, associated with STAT4 gene expression levels (plots of the 4th row).
  • the genetic markers in this region also showed significant associations with SSc, PBC, SLE, SjS, IBD, CD, and UC (with p value ⁇ 5 x 1 O' 8 ), where the significant associated markers were correlated with STAT4 eQTL top variant (rsl6833249).
  • the pattern of significance of association between genotype and STAT4 gene expression levels matched closely with that of association with SSc, PBC, SLE, SjS, IBD, CD, UC risk.
  • STAT3 eQTL analysis revealed SNP rsl 053004, located near the STAT3 gene, associated with STAT3 gene expression levels (plots of the 4th row).
  • the genetic markers in this region also showed significant associations with IBD, CD, UC (with p value ⁇ 5 x 1 O' 8 ), where the significant associated markers were correlated with STAT3 eQTL top variant (rsl 053004).
  • the pattern of significance of association between genotype and STAT3 gene expression levels matched closely with that of association with IBD, CD, UC risk. The results indicate that STAT3’s gene expression levels and the IBD, CD, UC risk share a common causal genomic variant.
  • a positive regression line fits the scatter plots comparing the genetic effects on IL12RP2 expression vs. genetic effects on SSc, PBC, SjS, and SLE disease risk (FIG. 24). This indicates the increased IL12RP2 expression through genetic perturbation positively associated with the increased disease risk of SSc, PBC, SLE, and SjS.
  • IL12A-p35/IL12Rp2 were specifically associated with immune diseases of SSc, PBC, SLE, and SjS. IL12B, although together with IL12A forms IL12, showed a differential immune disease association pattern.
  • IL12B was associated with PsO, CD, UC, IBD and AS, but not with SSc, PBC, SLE, and SjS.
  • IL12B-p40 is also a component of IL23.
  • the disease association pattern for IL12B was the same as that of IL23R.
  • IL12B or IL23A- pl9 have been successful in IBD and PsO, but failed in PBC and SLE, which is consistent to the genetic association pattern for IL12B and IL23R. These genetic association profiles reflected that pharmacologically targeting IL12B would have effects similarly to targeting the IL23 pathway, but could be different from targeting IL 12 A.
  • STAT4 is the major downstream signaling molecule of the IL12 cytokine pathway. STAT4 was associated with all diseases SSc, PBC, SLE, and SjS that IL12A- p35/IL12Rp2 were associated with. STAT3 is one downstream signaling molecule of the IL23 cytokine pathway, and STAT3 was only associated with IBD, UC, CD diseases. The differential immune disease associations from STAT4 and STAT3 also supported the different indications of targeting IL 12 vs. IL23.
  • IL12A-p35/IL12Rp2 is also a component of IL35 cytokine and receptor.
  • IL12A and IL12B are also a component of IL35 cytokine and receptor.
  • IL12A and IL12B are also a component of IL35 cytokine and receptor.
  • IL12A and IL12B are differential genetic association could be explained by the differential effect of IL35 and IL12 cytokines.
  • genetic variants significantly affect the protein levels of EBI3/IL6ST, the other components of IL35 and its receptor, they showed no association to any of the above evaluated autoimmune diseases.
  • IL12A-p35/IL12Rp2 affect the immune diseases through the immune regulatory effect of IL35.
  • hFcRN transgenic C57BL/6 mice (Shanghai Model Organisms Center, Inc.) were intravenously (/. v.) dosed with anti-IL12p35 antibodies 4A4-hblb2-YTE or 27H28L-hblb-YTE at 1 mg/kg.
  • the pharmacokinetic (PK) blood samples were collected 2 hours (“hr”), 6 hours, 24 hours, 48 hours, 72 hours, 7 days (“D”), 10 days, 14 days, 17 days, 21 days, 24 days, and 28 days post dosing.
  • Serum concentration of anti-IL12p35 antibodies was analyzed by ELISA.
  • human IL-12 was used to capture the anti-IL12p35 antibodies in serum and an anti-human Fc antibody was used to detect the human IL-12-bound anti-IL12p35 antibodies.
  • the serum concentrations 4A4-hblb2-YTE and 27H28L-hblb-YTE at corresponding time points are shown in FIG. 29.
  • the pharmacokinetic parameters of 27H28L-hblb-YTE and 4A4- hblb2-YTE are shown in FIG. 30.
  • the anti-IL12p35 antibodies with TYE and back-to-germline mutations demonstrated a good in vivo stability and exhibited excellent PK properties in human FcRn transgenic mice.
  • 4A4-hblb2-YTE and 27H28L-hblb- YTE exhibited a T1/2 of 375 hours and 500 hours, respectively, after the single dose at 1 mg/kg in human FcRn transgenic mice.
  • DNFB 1,4-dinitrobenzene
  • mice were randomly placed into three groups (8 mice per group), and intraperitoneally (zip.) dosed with 100 pL PBS (control), 10 mg/kg of a rat anti-mouse IL12p35 antibody (anti-mP35) (BioXcell, Cat#: BE0371), or 10 mg/kg of a rat anti-mouse IL12p40 antibody (anti-mP40) (BioXcell, Cat#: BE0051) on Day 0, Day 4, Day 7 and Day 11, respectively.
  • Ear thickness was determined using a digital portable thickness gage from Day 5 to Day 12 before the DNFB application. Each ear was measured three times to determine the average thickness of each data point.
  • FIG. 31A The experimental scheme is shown in FIG. 31A.
  • FIG. 31B the average ear thickness increased in all DNFB-induced skin inflammation groups.
  • Anti-mouse IL12p35 antibody treatment significantly reduced the ear thickness compared to that of the control group (treated with PBS), whereas anti-mouse IL12p40 antibody treatment did not significantly affect the ear thickness as compared to the control group.
  • Sjogren’s Syndrome is an autoimmune disease mediated by lymphocytic infiltration into exocrine glands, resulting in progressive lacrimal and salivary destruction and dysfunctional glandular secretion.
  • an anti-mouse IL12p35 antibody was used to treat NOD mice (NOD/MrkTac, Taconic US). In NOD/ShiLtJ mice, type 1 diabetes accelerates the progression of the Sjogren’s syndrome.
  • mice 8-week-old NOD/ShiLtJ mice were fasted for 24 hours and then intraperitoneally (zip.) dosed with 3.6 mg streptozotocin (STZ; dissolved in 0.1 M sodium citrate, pH 4.5).
  • STZ streptozotocin
  • mice were randomly placed into two groups and treated with 10 mg/kg of a rat IgG2a isotype antibody (BioXcell, Cat#: BE0089) or a rat anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371), respectively on Day 0, Day 3, Day 6 and Day 9.
  • mice were anesthetized using an isoflurane machine.
  • a pre- weighted absorbent material was inserted into the mouth of mice. Each mouse received 100 pg pilocarpine intraperitoneally (i.p.). 12 minutes later, the absorbent material was removed and weighted to determine saliva release.
  • FIG. 32A The experimental scheme is shown in FIG. 32A.
  • FIG. 32B the release of saliva decreased significantly in response to the pilocarpine stimulation during the development of Sjogren’s syndrome induced by STZ in NOD/ShiLtJ mice.
  • the anti-mouse IL12-p35 antibody treatment significantly alleviated the decrease of saliva excretion in the mice with Sjogren’s syndrome.
  • Anti-IL12p35 antibodies significantly inhibited weight loss, and reduced urine albumin levels and kidney pathology in a IMQ-induced SLE model
  • SLE Systemic lupus erythematosus
  • NZBWF1/J mice serve as a classic model for SLE, as they spontaneously develop immune responses similar to human SLE.
  • TLR toll-like receptor 7
  • TLR7 agonist of imiquimod IMQ
  • mice On Day 0, all mice topically received 2.5 mg of 2.5% IMQ cream per 25 g mouse weight on the right inner ear. IMQ was applied twice per week for a total of 5 doses.
  • the IMQ cream was prepared by mixing 100 mg ( ⁇ 100 pL) Vanish cream with 5 mg IMQ with extra 100 pL pure H2O.
  • mice On Day 0/Week 0, mice were randomly placed into two groups according to body weight, treated with 10 mg/kg of a rat IgG2a isotype antibody (BioXcell, Cat#: BE0089; as the control group) or an anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371, as the treatment group), respectively. All mice were dosed twice per week until the experimental endpoint.
  • the plate was then washed and 100 pL diluted urine samples and standards (Sigma, Cat#: 126674-25MG) were added into wells and incubated for 2 hours at room temperature. After the incubation, the plate was washed and 100 pL anti-mouse albumin Polyclonal Antibody-HRP (1:25000 dilution with 1% BSA, Bethyl Laboratories, Cat#: A90-134P) was added and incubated for 1 hour at room temperature. The plate was then washed and developed with TMB to determine the concentrations of albumin based on the standard.
  • the urine creatinine concentrations were measured using a creatinine assay kit (Bioassay Systems, Cat#: DICT500) according to the manufacture’s instructions. In Week 5, the mice were euthanized. Their kidneys were collected. The left ones were frozen while the right ones were fixed with 10% formalin and sent to iHisto (Salem, MA) for histology analysis.
  • the anti- IL12p35 antibody treatment group displayed mostly intact glomerular structures, with only a few instances of mild glomerulonephritis (as indicated by a black arrow); and no fibrosis or interstitial infiltration was observed.
  • topical application of IMQ accelerated the development of SLE in young NZBWF1/J mice, enabling a rapid evaluation of the effects of anti-IL12p35 antibody treatment on SLE.
  • the anti-IL12p35 antibody treatment significantly inhibited weight loss, reduced urine albumin levels, and markedly reduced kidney pathology associated with SLE.
  • Example 17 Anti-IL12p35 antibodies significantly reduced kidney pathology in a spontaneous SLE model
  • NZBWF1/J mice Jackson Laboratory, Cat#100008
  • NZBWF1/J female mice were received at 18-week- old and acclimated for one week. All mice were randomly placed into two groups in Week 0 according to body weight, treated with 15 mg/kg of a rat IgG2a isotype antibody (BioXcell, Cat#: BE0089; as the control group) or an anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371; as the treatment group) intraperitoneally (z. >.) every week until the experimental endpoint.
  • a rat IgG2a isotype antibody BioXcell, Cat#: BE0089; as the control group
  • an anti-mouse IL12p35 antibody BioXcell, Cat#: BE0371; as the treatment group
  • anti-IL12p35 antibody treatment significantly reduced SLE kidney symptoms.
  • the urine albumin level showed a significant decrease 14 weeks post treatment when the NZBWF1/J mice spontaneously developed SLE.
  • the onset time of SLE symptoms varied significantly among the mice.
  • some mice in the control group did not exhibit an increase in the albumin/creatinine ratio throughout the experiment, whereas some mice in both treated and control groups had very early disease onset.
  • the development of anti-drug antibodies led to poor pharmacokinetic (PK) exposure of the anti-IL12p35 antibody, compromising its therapeutic efficacy. This was particularly evident in mice with an elevated albumin/creatinine ratio, which also correlated with poor PK.
  • kidney histology analysis demonstrated that kidney tissues primarily exhibited end-stage lesions in the isotype control group (panels a-d; #42): 1) 5 out of 7 samples showed glomerular sclerosis (as indicated by black arrows), and some accompanied by fibrous crescent formation (as indicated by white arrows); 2) tubular degeneration or atrophy was observed (as indicated by black triangles), along with protein casts in the tubules (as indicated by a black diamond); and 3) interstitial inflammation of varying degrees was observed, with some samples showing interstitial fibrosis.
  • the WTS whole-tissue score
  • AS activity score
  • CS chronicity score
  • anti-IL12p35 antibody treatment reduced urine albumin levels, markedly reduced kidney pathology associated with SLE, and slowed down disease progression in the spontaneous SLE model.

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Abstract

This disclosure relates to anti-IL12p35 (interleukin 12 subunit alpha) antibodies, antigen-binding fragments, and the uses thereof. In some embodiments, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein can be used for treating systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).

Description

ANTI-IL12P35 ANTIBODIES AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
This disclosure claims priority to and benefit of U.S. Provisional Patent Application Serial No. 63/627,553, filed on January 31, 2024, which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
This disclosure relates to antibodies that can bind to IL12p35 (interleukin 12 subunit alpha), and the uses thereof.
BACKGROUND
Autoimmune diseases arise when the immune system malfunctions, mistakenly attacking healthy cells. There are over 80 recognized autoimmune conditions, affecting approximately one in ten individuals (Conrad, N., et al. "Incidence, prevalence, and co-occurrence of autoimmune disorders over time and by age, sex, and socioeconomic status: a population-based cohort study of 22 million individuals in the UK." The Lancet 401.10391 (2023): 1878-1890). In the United States alone, over 24 million people are suffering with one or more of these diseases.
Current therapeutic options for autoimmune diseases primarily involve the use of immunosuppressive medications and biologics/small molecule drugs targeting inflammatory cytokines, immune cells, and intracellular kinases. An effective therapeutic strategy focuses on the IL12/IL23 pathway, resulting in multiple drug approvals for various autoimmune diseases. However, evidence shows that IL12/IL23 pathway-specific genes are effective targets for a set of autoimmune diseases, but not for other autoimmune diseases. Thus, there is a need to develop more therapies for treating autoimmune diseases with different pathological mechanisms.
SUMMARY
This disclosure relates to anti-IL12p35 antibodies, antigen-binding fragment thereof, and the uses thereof. The disclosure also demonstrates that developing IL12p35/ IL12R02 antagonist would be used for treating a cluster of autoimmune diseases, e.g., systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to interleukin 12 subunit alpha (IL12p35), comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1 , 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, in some embodiments, the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are selected from VH CDRS 1, 2, 3 and VL CDRS 1, 2, 3 listed in PIG. 26 or FIG. 27.
In some embodiments, the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, 49, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 50, 51, 52, respectively; (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 53, 54, 55, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 56, 57, 58, respectively; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, 61, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 62, 63, 64, respectively; (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 65, 66, 67, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 68, 69, 70, respectively; (5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 71, 72, 73, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 74, 75, 76, respectively; (6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 77, 78, 79, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 80, 81, 82, respectively; (7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 83, 84, 85, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 86, 87, 88, respectively; (8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 89, 90, 91, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 92, 93, 94, respectively; (9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 95, 96, 97, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 98, 99, 100, respectively; (10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 101, 102, 103, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 104, 105, 106, respectively; (11) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 107, 108, 109, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 110, 111, 112, respectively; (12) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 113, 114, 115, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 116, 117, 118, respectively; (13) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 119, 120, 121, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 122, 123, 124, respectively; (14) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 125, 126, 127, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 128, 129, 130, respectively; (15) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 131, 132, 133, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 134, 135, 136, respectively; (16) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 137, 138, 139, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 140, 141, 142, respectively; (17) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 143, 144, 145, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 146, 147, 148, respectively; (18) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 149, 150, 151, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 152, 153, 154, respectively; (19) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, 157, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, 160, respectively; (20) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 161, 162, 163, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 164, 165, 166, respectively; (21) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 167, 168, 169, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 170, 171, 172, respectively; (22) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 173, 174, 175, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 176, 177, 178, respectively; and (23) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 179, 180, 181, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 182, 183, 184, respectively. In some embodiments, CDR is determined by Kabat definition.
In some embodiments, the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 185, 186, 187, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 188, 189, 190, respectively; (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 191, 192, 193, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 194, 195, 196, respectively; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 197, 198, 199, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 200, 201, 202, respectively; (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 203, 204, 205, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 206, 207, 208, respectively; (5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 209, 210, 211, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 212, 213, 214, respectively; (6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 215, 216, 217, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 218, 219, 220, respectively; (7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 221, 222, 223, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 224, 225, 226, respectively; (8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 227, 228, 229, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 230, 231, 232, respectively; (9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 233, 234, 235, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 236, 237, 238, respectively; (10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 239, 240, 241, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 242, 243, 244, respectively; (11) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 245, 246, 247, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 248, 249, 250, respectively; (12) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 251, 252, 253, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 254, 255, 256, respectively; (13) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 257, 258, 259, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 260, 261, 262, respectively; (14) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 263, 264, 265, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 266, 267, 268, respectively; (15) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 269, 270, 271, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 272, 273, 274, respectively; (16) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 275, 276, 277, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 278, 279, 280, respectively; (17) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 281, 282, 283, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 284, 285, 286, respectively; (18) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 287, 288, 289, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 290, 291, 292, respectively; (19) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 293, 294, 295, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 296, 297, 298, respectively; (20) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 299, 300, 301, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 302, 303, 304, respectively; (21) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 305, 306, 307, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 308, 309, 310, respectively; (22) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 311, 312, 313, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 314, 315, 316, respectively; and (23) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 317, 318, 319, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 320, 321, 322, respectively. In some embodiments, CDR is determined by IMGT definition.
In some embodiments, the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, 157, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, 160, respectively, according to Kabat definition; (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 179, 180, 181, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 182, 183, 184, respectively, according to Kabat definition; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 293, 294, 295, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 296, 297, 298, respectively, according to IMGT definition; and (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 317, 318, 319, respectively, and the selected VL CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 320, 321, 322, respectively, according to Kabat definition.
In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to IL12p35 comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 1, and the selected VL sequence is SEQ ID NO: 2; (2) the selected VH sequence is SEQ ID NO: 3, and the selected VL sequence is
SEQ ID NO: 4; (3) the selected VH sequence is SEQ ID NO: 5, and the selected VL sequence is
SEQ ID NO: 6; (4) the selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8; (5) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10; (6) the selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (7) the selected VH sequence is SEQ ID NO: 13, and the selected VL sequence is SEQ ID NO: 14; (8) the selected VH sequence is SEQ ID NO: 15, and the selected VL sequence is SEQ ID NO: 16; (9) the selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18; (10) the selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; (11) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (12) the selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 24; (13) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 26; (14) the selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 28; (15) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30; (16) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32; (17) the selected VH sequence is SEQ ID NO: 33, and the selected VL sequence is SEQ ID NO: 34; (18) the selected VH sequence is SEQ ID NO: 35, and the selected VL sequence is SEQ ID NO: 36; (19) the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38; (20) the selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40; (21) the selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42; (22) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44; (23) the selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; (24) the selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336; and (25) the selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338. In some embodiments, the VH and/or VL comprise one or more back-to-germline (B2G) mutations. In some embodiments, the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is 38. In some embodiments, the selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46. In some embodiments, the selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336. In some embodiments, the selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338.
In some embodiments, the antibody or antigen-binding fragment thereof can block the binding between interleukin 12 (e.g., human interleukin 12 (IL 12)) and interleukin 12 receptor, beta 2 subunit (e.g., human interleukin 12 receptor, beta 2 subunit (IL12R02)). In some embodiments, the antibody or antigen-binding fragment thereof can block IL12-induced intracellular signaling (e.g., JAK-STAT signaling), optionally the IL12 is human or monkey IL 12. In some embodiments, the antibody or antigen-binding fragment thereof can inhibit IL 12- induced IFN-y production in human PBMCs. In some embodiments, the antibody or antigenbinding fragment thereof can prevent IFN-y production by CD4+ T cells cocultured with allogenic dendritic cells. In some embodiments, the antibody or antigen-binding fragment specifically binds to human IL12p35 and/or monkey IL12p35. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a F(ab’)2 fragment, a single-chain variable fragment (scFV) or a multi-specific antibody (e.g., a bispecific antibody).
In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to IL12p35 comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, a first immunoglobulin light chain, and a second immunoglobulin light chain, in some embodiments, the first immunoglobulin heavy chain and the first immunoglobulin light chain associates with each other, forming a first antigen-binding site that binds to IL12p35, in some embodiments, the second immunoglobulin heavy chain and the second immunoglobulin light chain associates with each other, forming a first antigen-binding site that binds to IL12p35, in some embodiments: (1) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 325, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 326; (2) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 327, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 326; (3) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 328, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 329; (4) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 330, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 329; (5) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 331, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 332; and (6) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 333, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 334. In some embodiments, the first and second immunoglobulin heavy chains are identical, in some embodiments, the first and second immunoglobulin light chains are identical. In some embodiments, the first and second immunoglobulin heavy chains comprise an Fc region (e.g., an IgGl Fc region). In some embodiments, the Fc region comprises YTE mutations (M252Y/S254T/T256E according to EU numbering).
In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1 , VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, in some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are identical to VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the antibody or antigen-binding fragment thereof described herein.
In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.
In one aspect, the disclosure is related to an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.
In one aspect, the disclosure is related to a method of inhibiting immune response in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, to the subject. In some embodiments, the subject has an immune disorder (e.g., an autoimmune disease or an inflammatory disease).
In one aspect, the disclosure is related to a method of treating a subject having an autoimmune disease, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, to the subject. In some embodiments, the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS). In some embodiments, the subject does not have psoriasis (PsO), Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel diseases (IBD), or ankylosing spondylitis (AS).
In one aspect, the disclosure is related to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.
In one aspect, the disclosure is related to a method of reducing IL12-induced intracellular signaling in a cell, the method comprising contacting the cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof or the antibody-drug conjugate described herein, to the subject.
In one aspect, the disclosure is related to a method of identifying a subject as having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject, using the antibody or antigen-binding fragment thereof described herein.
In one aspect, the disclosure is related to a method of determining the risk of a subject having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject, using the antibody or antigen-binding fragment thereof described herein.
In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: (1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1 , 2, and 3 comprising VH CDR 1, 2, 3 set forth in FIG. 26 or FIG. 27, and in some embodiments, the VH, when paired with a corresponding light chain variable region (VL) binds to IL12p35; or (2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1 , 2, and 3 comprising VL CDR 1, 2, 3 set forth in FIG. 26 or FIG. 27, when paired with a corresponding VH binds to IL12p35.
In some embodiments, the polypeptide comprises: (1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1 , 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively (or SEQ ID NOs: 185, 186, and 187, respectively), and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 2 binds to IL12p35; (2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively (or SEQ ID NOs: 188, 189, and 190, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 binds to IL12p35; (3) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 53, 54, and 55, respectively (or SEQ ID NOs: 191, 192, and 193, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 4 binds to IL12p35; (4) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 57, and 58, respectively (or SEQ ID NOs: 194, 195, and 196, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 binds to IL12p35; (5) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively (or SEQ ID NOs: 197, 198, and 199, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 6 binds to IL12p35; (6) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively (or SEQ ID NOs: 200, 201, and 202, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 5 binds to IL12p35; (7) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively (or SEQ ID NOs: 203, 204, and 205, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 8 binds to IL12p35; (8) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 68, 69, and 70, respectively (or SEQ ID NOs: 206, 207, and 208, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 binds to IL12p35; (9) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1 , 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 71, 72, and 73, respectively (or SEQ ID NOs: 209, 210, and
211, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 10 binds to IL12p35; (10) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 74, 75, and 76, respectively (or SEQ ID NOs:
212, 213, and 214, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 binds to IL12p35; (11) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 77, 78, and 79, respectively (or SEQ ID NOs: 215, 216, and 217, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 12 binds to IL12p35; (12) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 80, 81, and 82, respectively (or SEQ ID NOs: 218, 219, and 220, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 11 binds to IL12p35; (13) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 83, 84, and 85, respectively (or SEQ ID NOs: 221, 222, and 223, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 14 binds to IL12p35; (14) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 86, 87, and 88, respectively (or SEQ ID NOs: 224, 225, and 226, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 13 binds to IL12p35; (15) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 89, 90, and 91, respectively (or SEQ ID NOs: 227, 228, and 229, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 binds to IL12p35; (16) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 92, 93, and 94, respectively (or SEQ ID NOs: 230, 231, and 232, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 binds to IL12p35; (17) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 95, 96, and 97, respectively (or SEQ ID NOs: 233, 234, and 235, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 18 binds to IL12p35; (18) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 98, 99, and 100, respectively (or SEQ ID NOs: 236, 237, and 238, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 binds to IL12p35; (19) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 101, 102, and 103, respectively (or SEQ ID NOs: 239, 240, and 241, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 20 binds to IL12p35; (20) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively (or SEQ ID NOs: 242, 243, and 244, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 19 binds to IL12p35; (21) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 107, 108, and 109, respectively (or SEQ ID NOs: 245, 246, and 247, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 22 binds to IL12p35; (22) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively (or SEQ ID NOs: 248, 249, and 250, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 21 binds to IL12p35; (23) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 113, 114, and 115, respectively (or SEQ ID NOs: 251, 252, and 253, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 24 binds to IL12p35; (24) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 116, 117, and
118, respectively (or SEQ ID NOs: 254, 255, and 256, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 23 binds to IL12p35; (25) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs:
119, 120, 121, respectively (or SEQ ID NOs: 257, 258, and 259, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 26 binds to IL12p35; (26) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 122, 123, and 124, respectively (or SEQ ID NOs: 260, 261, and 262, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 25 binds to IL12p35; (27) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 125, 126, and 127, respectively (or SEQ ID NOs: 263, 264, and 265, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 28 binds to IL12p35; (28) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 128, 129, and 130, respectively (or SEQ ID NOs: 266, 267, and 268, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 27 binds to IL12p35; (29) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 131, 132, and 133, respectively (or SEQ ID NOs: 269, 270, and 271, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 30 binds to IL12p35; (30) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 134, 135, and 136, respectively (or SEQ ID NOs: 272, 273, and 274, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 29 binds to IL12p35; (31) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 137, 138, and 139, respectively (or SEQ ID NOs: 275, 276, and 277, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 32 binds to IL12p35; (32) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 140, 141, and 142, respectively (or SEQ ID NOs: 278, 279, and 280, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 31 binds to IL12p35; (33) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 143, 144, and 145, respectively (or SEQ ID NOs: 281, 282, and 283, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 34 binds to IL12p35; (34) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 146, 147, and 148, respectively (or SEQ ID NOs: 284, 285, and 286, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 33 binds to IL12p35; (35) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 149, 150, and 151, respectively (or SEQ ID NOs: 287, 288, and 289, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 36 binds to IL12p35; (36) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 152, 153, and
154, respectively (or SEQ ID NOs: 290, 291, and 292, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 35 binds to IL12p35; (37) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs:
155, 156, and 157, respectively (or SEQ ID NOs: 293, 294, and 295, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 38 or 336 binds to IL12p35; (38) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 158, 159, and 160, respectively (or SEQ ID NOs: 296, 297, and 298, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 37 or 335 binds to IL12p35; (39) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 161, 162, and 163, respectively (or SEQ ID NOs: 299, 300, and 301, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 40 binds to IL12p35; (40) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 164, 165, and 166, respectively (or SEQ ID NOs: 302, 303, and 304, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 39 binds to IL12p35; (41) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 167, 168, and 169, respectively (or SEQ ID NOs: 305, 306, and 307, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 42 binds to IL12p35; (42) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively (or SEQ ID NOs: 308, 309, and 310, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 41 binds to IL12p35; (43) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 173, 174, and 175, respectively (or SEQ ID NOs: 311, 312, and 313, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 44 binds to IL12p35; (44) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1 , 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 176, 177, and 178, respectively (or SEQ ID NOs: 314, 315, and 316, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 43 binds to IL12p35; (45) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 179, 180, and 181, respectively (or SEQ ID NOs: 317, 318, and 319, respectively), and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 46 or 338 binds to IL12p35; or (46) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 182, 183, and 184, respectively (or SEQ ID NOs: 320, 321, and 322, respectively), and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 45 or 337 binds to IL12p35.
In some embodiments, the VH when paired with a VL specifically binds to human IL12p35; or the VL when paired with a VH specifically binds to human IL12p35. In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof. In some embodiments, the nucleic acid encodes a F(ab’)2 fragment, a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody). In some embodiments, the nucleic acid is cDNA.
In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids described herein. In one aspect, the disclosure is related to a vector comprising two of the nucleic acids described herein, in some embodiments, the vector encodes the VL region and the VH region that together bind to IL12p35. In one aspect, the disclosure is related to a pair of vectors, in some embodiments, each vector comprises one of the nucleic acids described herein, in some embodiments, together the pair of vectors encodes the VL region and the VH region that together bind to IL12p35.
In one aspect, the disclosure is related to a cell comprising the vector or the pair of vectors described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure is related to a cell comprising one or more of the nucleic acids described herein. In one aspect, the disclosure is related to a cell comprising two of the nucleic acids described herein. In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to IL12p35.
In one aspect, the disclosure is related to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and (b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.
In one aspect, the disclosure is related to a method of treating a subject having an autoimmune disease, the method comprising administering a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof that binds to IL12p35 and/or an antibody or antigen-binding fragment thereof that binds to IL12R01, to the subject. In some embodiments, the antibody or antigen-binding fragment thereof that binds to IL12p35 does not bind to IL12p40. In some embodiments, the antibody or antigen-binding fragment thereof that binds to IL12R02 does not bind to IL12R01. In some embodiments, the antibody or antigenbinding fragment thereof does not interfere IL23 pathway and/or IL35 pathway. In some embodiments, the subject is a human subject. In some embodiments, the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS). In some embodiments, the antibody or antigen-binding fragment thereof is a human or humanized antibody or antigen-binding fragment thereof. In some embodiments, the subject is a non-human mammal, e.g., a monkey, a dog, or a mouse. In some embodiments, the mammal has a similar disease or disorder as systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS). In some embodiments, the subject is a dog. In some embodiments, the antibody or antigenbinding fragment thereof is a canine or caninized antibody or antigen-binding fragment thereof.
As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Nonlimiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bi-specific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain). Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F(ab’)2, and Fv fragments.
As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus).
As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody). A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody), e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, the humanized antibody contains at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically, that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.
As used herein, the term “multimeric antibody” refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is able to crosslink one target molecule (e.g., IL12p35) to at least one second target molecule (e.g., IL12p35) on the surface of a mammalian cell.
As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non- veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., IL12p35) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a IL12p35 molecule may be referred to as a IL12p35-specific antibody or an anti-IL12p35 antibody.
As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1 is a table showing the preliminary binding affinities of anti-IL12p35 antibodies against human IL12p70 measured by the Gator® biolayer interferometry platform. Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
FIG. 2A is a table showing the binding affinities of selected anti-IL12p35 antibodies and their YTE variants to human, rhesus, canine or mouse IL12p70 measured by the Gator® biolayer interferometry platform. 27H28L is an engineered antibody composed of the heavy chain of D2M008-A1A8 and the light chain of D2M008-A1A9. YTE mutations of anti-IL12p35 antibodies 27H28L and D2M008-4A4 were introduced via amino acid substitutions (M252Y/S254T/T256E) in the Fc region, generating “27H28L YTE” and “D2M-4A4 YTE,” respectively. 27H28L-hblb-YTE and 4A4-hblb2-YTE have back-to-germline mutations in the frameworks in variable regions of “27H28L YTE” and “D2M-4A4 YTE,” respectively. O.R. means out of range. N.D. means not performed. N.B. means no binding was detected.
FIG. 2B shows the binding kinetics and fitting curves of 27H28L-hblb-YTE and 4A4- hblb2-YTE to human IL12p70 measured by the BiaCore™ SPR platform.
FIG. 2C is a table showing the method information and parameters acquired by the BiaCore™ 8K SRP platform for 27H28L-hblb-YTE and 4A4-hblb2-YTE.
FIG. 3A shows the binding affinities of 27H28L and its YTE variant (“27H28L YTE”) to a recombinant FcRn protein at pH 6.0, pH 6.5, or pH 7.0, measured by the Gator® biolayer interferometry platform.
FIG. 3B shows the binding affinities of D2M008-4A4 and its YTE variant (“D2M-4A4 YTE”) to a recombinant FcRn protein at pH 6.0, pH 6.5, or pH 7.0, measured by the Gator® biolayer interferometry platform.
FIG. 4 A shows the inhibition rate of different anti-IL12p35 antibodies blocking human IL12p70 binding to its receptor IL12R 2. All antibodies at a fixed concentration were preincubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA.
FIG. 4B shows the inhibition rate of selected anti-IL12p35 antibodies blocking human IL12p70 binding to its receptor IL12R 2. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA.
FIG. 4C shows blocking of human IL12p70 binding to its receptor IL12R 2 by 27H28L and additional anti-IL12p35 antibodies. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The ICso values of 27H28L and the selected anti-IL12p35 antibodies were calculated.
FIG. 4D shows the inhibition rate of anti-IL12p35 antibodies 27H28L, D2M008-4A4 and their YTE variants blocking human IL12p70 binding to its receptor IL12R 2. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA. FIG. 4E shows the inhibition rate of anti-IL12p35 antibodies 27H28L-YTE and D2M008-4A4 YTE and their variants with back-to-germline mutations blocking human IL12p70 binding to its receptor IL12R 2. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before being incubated with IL12R 2. The level of inhibition was calculated based on OD450 measured by sandwich ELISA.
FIG. 5 A shows the inhibition rate of different anti-IL12p35 antibodies blocking human IL12p70-induced signaling in HEK-Blue™ IL12 reporter cells. All antibodies at a fixed concentration were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring and calculating based on the reporter gene signal at OD620.
FIG. 5B shows the inhibition rate of selected anti-IL12p35 antibodies blocking human IL12p70-induced signaling in HEK-Blue™ IL12 reporter cells. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was calculated based on the reporter gene signal at OD620.
FIG. 5C shows the inhibition rate of selected anti-IL12p35 antibodies (D2M008-A1A8, D2M008-A1A9, and 27H28L), anti-IL12p40 (Ustekinumab, anti-hP40), and a combination of D2M008-A1 A8 and anti-hP40 blocking human IL12p70-induced signaling in HEK-Blue™ IL12 reporter cells. Serially diluted antibodies were pre- incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was calculated based on the reporter gene signal at OD620. The IC50 values of the antibodies and the combination of D2M008-A1A8 and anti-hP40 were calculated.
FIG. 5D shows the inhibition rate of selected anti-IL12p35 antibodies and 27H28L blocking human IL12p70-induced signaling in HEK-Blue™ IL12 reporter cells. Anti-human IL12p40 (Ustekinumab, anti-hP40) was used as a reference antibody. Serially diluted antibodies were pre- incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was calculated based on the reporter gene signal at OD620. The IC50 values of the antibodies were calculated.
FIG. 5E shows the inhibition level of anti-IL12p35 antibodies 27H28L, D2M008-4A4, and their YTE variants blocking human IL12p70-induced signaling in HEK-Blue™ IL12 reporter cells. Serially diluted antibodies were pre- incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
FIG. 5F shows the inhibition level of anti-IL12p35 antibodies 27H28L-YTE and D2M008-4A4 YTE and their variants with back-to-germline mutations blocking human IL12p70-induced signaling in HEK-Blue™ IL12 reporter cells. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
FIG. 6 shows the inhibition level of anti-IL12p35 antibody D2M008-4A4 and 27H28L blocking rhesus IL12-induced signaling in HEK-Blue™ IL12 reporter cells. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were preincubated with rhesus IL12p70 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
FIG. 7A-7C show the level of IFN-y production by exogenous IL 12- stimulated human PBMCs in vitro. PBMCs from three healthy donors (#20, #22 and #23) were used. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted anti-IL12p35 antibodies were pre- incubated with the exogenous IL12p70 at room temperature for 30 minutes before incubating with PBMCs. IFN-y production was measured by a sandwich ELISA.
FIG. 8A-8B show the level of IFN-y production by human CD4+ T cells co-cultured with allogeneic dendritic cells in vitro. The data in FIG. 8A was obtained using CD4+ T cell from Donor #4, and the data in FIG. 8B was obtained using CD4+ T cell from Donor #8. Antihuman IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were individually mixed with monocyte-derived dendritic cells (DC) from donor #18 before coculture with human CD4+ T cells. The ratio of dendritic cells and CD4+ T cells was 1: 10. IFN-y production was measured by a sandwich ELISA.
FIGS. 9A-9B show the inhibition level of anti-IL12p35 antibody 27H28L (FIG. 9A), D2M007-4A4 (FIG. 9B), and their YTE variants neutralizing IL23 -induced transducing signaling in HEK-Blue™ IL23 reporter cells. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were pre-incubated with human IL23 at room temperature for 30 minutes before incubating with the reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.
FIG. 10A shows the melting curves of full IgG and the F(ab’)2 fragment of anti-IL12p35 antibodies 27H28L and D2M008-4A4. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. The melting curves were measured by the Protein Thermal Shift™ Dye Kit using a qPCR thermocycler. Fluorescence intensity was subtracted by the lowest intensity (bottom) before the peak and normalized to the amplitude from bottom to peak.
FIG. 10B shows the Tm of full IgG and the F(ab’)2 fragment of anti-IL12p35 antibodies 27H28L and D2M008-4A4. Anti-human IL12p40 (anti-hP40) was used as a reference antibody.
FIG. 11A shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70 binding to its receptor IL12R 2, after being exposed in stress conditions. The antibodies were stressed in a pH 5.5, pH 7.4, or pH 8.5 buffer, respectively, at 40°C for two weeks. Their blocking kinetics was measured by ELISA.
FIG. 11B shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70-induced transducing signaling in HEK-Blue™ IL12 reporter cells, after being exposed in stress conditions. The antibodies were stressed in a pH 5.5, pH 7.4, or pH 8.5 buffer, respectively, at 40°C for two weeks. Their blocking kinetics was evaluated by measuring the reporter gene signal at OD620.
FIG. 12A shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70 binding to its receptor IL12R 2, after being exposed to human plasma or PBS. The antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for two weeks. Their blocking kinetics was measured by ELISA. Plasma 20 and plasma 21 indicate human plasma samples from two different donors.
FIG. 12B shows the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70-induced transducing signaling in HEK-Blue™ IL12 reporter cells, after being exposed to human plasma or PBS. The antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for two weeks. Their blocking kinetics was evaluated by measuring the reporter gene signal at OD620. Plasma 20 and plasma 21 indicate human plasma samples from two different donors.
FIG. 12C shows the functional stability of anti-IL12p35 antibodies 27H28L-hblb-YTE in blocking IL12p70 binding to its receptor IL12R 2, after being exposed to human plasma for up to 3 weeks. The antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for 0, 1, 2, or 3 weeks. Their blocking kinetics was measured by ELISA. Plasma 42, 45, 53 and 54 indicate human plasma samples from four different donors.
FIG. 12D shows the functional stability of anti-IL12p35 antibodies 4A4-hblb2-YTE in blocking IL12p70 binding to its receptor IL12R 2, after being exposed to human plasma for up to 3 weeks. The antibodies were stressed in fresh human plasma from different donors or PBS at 37°C for 0, 1, 2, or 3 weeks. Their blocking kinetics was measured by ELISA. Plasma 42, 45, 53 and 54 indicate human plasma samples from four different donors.
FIG. 13A shows the results of binning competition assays performed for selected anti- IL12p35 antibodies. White indicates no competition, and black indicates competition between the antibody pairs.
FIG. 13B shows the competition in binding to IL12 between anti-IL12p35 antibody 27H28L and D2M008-4A4. The anti-human Fc probes were used to immobilize one antibody (D2M008-4A4 and 27H28L, respectively), which was then incubated with human IL12p70, followed by an additional incubation with the second antibody (27H28L and D2M008-4A4, respectively). The binding ability was measured and analyzed using the Gator® biolayer interferometry platform.
FIG. 13C shows the competition in binding to IL12 between anti-IL12p35 antibody 27H28L and D2M008-4A4. The anti-His probes were used to immobilize human IL12p70, and then incubated with one antibody (D2M008-4A4 and 27H28L, respectively), followed by an additional incubation with the second antibody (27H28L or D2M008-4A4, respectively). The binding ability was measured and analyzed using the Gator® biolayer interferometry platform.
FIG. 14 shows IL12 related cytokines, receptors and signaling pathways. The schematic diagrams are adapted from Choi, J., et al. "IL-35 and autoimmunity: a comprehensive perspective." Clinical Reviews in Allergy & Immunology 49 (2015): 327-332.
FIGS. 15A-15C show regional plot of genetic marker association in IL12A locus with IL12A expression (IL12A eQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL12A gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of -logio(p value). The horizontal straight line represents the GWAS significant threshold p value of 5 x l O' 8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL12 eQTL variant (rs4680586).
FIGS. 16A-16C show regional plot of genetic marker association in IL12R02 locus with IL12RB2 expression (IL12RB2 eQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL12R02 gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value). The horizontal straight line represents the GWAS significant threshold p value of 5 x 10'8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL12RP2 eQTL variant (rsl7129778).
FIGS. 17A-17C show regional plot of genetic marker association in IL12B locus with IL12B serum protein levels (IL12B pQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL12B gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value). The horizontal straight line represents the GWAS significant threshold p value of 5 x 10'8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL12B pQTL variant (rs6556416).
FIGS. 18A-18C show regional plot of genetic marker association in IL23R locus with IL23R serum protein levels (IL23R pQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL23R gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value). The horizontal straight line represents the GWAS significant threshold p value of 5 x 10'8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top IL23R pQTL variant (rsl 1581607).
FIGS. 19A-19C show regional plot of genetic marker association in EBB locus with EBB serum protein levels (EBB pQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of EBB gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value). The horizontal straight line represents the GWAS significant threshold p value of 5 x 10'8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top EBI3 pQTL variant (rs60160662).
FIGS. 20A-20C show regional plot of genetic marker association in IL6ST locus with IL6ST serum protein levels (IL6ST pQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of IL6ST gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p value). The horizontal straight line represents the GWAS significant threshold p value of 5 x 10'8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top EBI3 pQTL variant (rsl 1574765).
FIGS. 21A-21C show regional plot of genetic marker association in STAT4 locus with STAT4 gene expression levels (STAT4 eQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of STAT4 gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of - logio(p Value). The horizontal straight line represents the GWAS significant threshold p value of 5 x 10'8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top STAT4 eQTL variant (rsl6833249).
FIGS. 22A-22C show regional plot of genetic marker association in STAT3 locus with STAT3 gene expression levels (STAT3 eQTL) and 9 inflammatory diseases. Each dot in the plot is a genetic marker. The X-axis represents genomic location of the genetic marker. This genetic region is centered on the genomic location of STAT3 gene with a spanning genomics length of 500 KB. The Y-axis represents the significant p value of each genetic marker in the metrics of - loglO(p Value). The horizontal straight line represents the GWAS significant threshold p value of 5 x 10'8. The circle size of each genetic marker is categorized by correlation of the genetic marker with the top STAT3 pQTL variant (rsl053004).
FIG. 23 shows scatter plots comparing the genetic effects on IL12A expression vs. genetic effects on SSc, PBC, SjS, and SLE disease risk. Genetic variants significantly associated with IL12A’s expression were selected first. Variants overlapping with SSc, PBC, SjS, or SLE disease GWAS data were finally picked for each disease. Each dot in the plot is a finally picked genetic variant. X-axis represents the effect of such genetic variant on IL12A’s gene expression. Y-axis represents the effect of such genetic variant’s same allele on disease risk. The straight line represents a fit regression line across all the selected genetic variants. An upward line indicated that genetically increased IL12A gene expression leads to an increased disease risk. The R- squared and p-value explain the strength of the relationship between genetically determined IL12A gene expression and genetically determined disease risk.
FIG. 24 shows scatter plots comparing the genetic effects on IL12R02 expression vs. genetic effects on SSc, PBC, SjS, and SLE disease risk. Genetic variants significantly associated with IL12Rp2’s expression were selected first. Variants overlapping with SSc, PBC, SjS, or SLE disease GWAS data were finally picked for each disease. Each dot in the plot is a finally picked genetic variant. X-axis represents the effect of such genetic variant on IL12Rp2’s gene expression. Y-axis represents the effect of such genetic variant’s same allele on disease risk. The straight line represents a fit regression line across all the selected genetic variants. An upward line indicated that genetically increased IL12RP2 gene expression leads to an increased disease risk. The R-squared and p-value explain the strength of the relationship between genetically determined IL12RP2 gene expression and genetically determined disease risk.
FIG. 25 shows the heavy chain variable region (VH) and light chain variable region (VL) sequences of anti-IL12p35 antibodies.
FIG. 26 shows VH and VL CDR sequences of anti-IL12p35 antibodies according to Kabat definition.
FIG. 27 shows VH and VL CDR sequences of anti-IL12p35 antibodies according to IMGT definition.
FIG. 28 shows the heavy chain (HC) and light chain (LC) sequences of antibodies discussed in the disclosure.
FIG. 29 shows the serum concentration of anti-IL12p35 antibodies 4A4-hblb2-YTE and 27H28L-hblb-YTE post a single i.v. dose (1 mg/kg) in human FcRn transgenic mice.
FIG. 30 shows pharmacokinetic parameters of anti-IL12p35 antibodies 4A4-hblb2-YTE and 27H28L-hblb-YTE after 1 mg/kg i.v. single dose in human FcRn transgenic mice.
FIG. 31A shows the experimental scheme of using a DNFB-induced chronic skin inflammation model to determine the in vivo effects of anti-IL12p35 antibodies on inflammation diseases. FIG. 3 IB shows the average ear thickness of mice treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an anti-mouse IL12p40 antibody (“anti-mP40”) in a DNFB-induced chronic skin inflammation model.
FIGS. 32A shows the experimental scheme of using STZ-induced Sjogren's syndrome model to determine the in vivo effects of anti-IL12p35 antibodies on Sjogren's syndrome.
FIG. 32B shows the excretion of saliva relative to the body weight of mice treated with an anti-mouse IL12p35 antibody (“anti-P35”) or an isotype antibody control (“Isotype”) in a STZ-induced Sjogren's syndrome model.
FIG. 33A shows the average body weight of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an isotype antibody control (“Isotype”) in a IMQ-induced SLE model.
FIG. 33B shows the ratio of urine albumin and creatinine of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an isotype antibody control (“Isotype”) in a IMQ-induced SLE (systemic lupus erythematosus) model.
FIG. 33C shows representative mouse kidney histology images of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-P35”) or an isotype antibody control (“Isotype”) in a IMQ-induced SLE model.
FIG. 34A shows the ratio of urine albumin and creatinine (“Cr”) of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (“anti-mP35”) or an isotype antibody control (“Isotype”) in a spontaneous SLE (systemic lupus erythematosus) model.
FIG. 34B shows representative mouse kidney histology images of NZBWF1/J mice that were treated with an anti-mouse IL12p35 antibody (panels e-h) or an isotype antibody control (panels a-d) in a spontaneous SLE model. Scale: panels a and e: 3 mm; panels b and f: 100 pm; panels c and g: 200 pm; and panels d and h: 50 pm.
FIG. 34C shows detailed scores and a summary of the kidney pathology scores evaluated by independent histopathologists.
DETAILED DESCRIPTION
IL12 is composed of two subunits: p35 (encoded by IL12A, and the UniProt ID as P29459) and p40 (encoded by IL12B, and the UniProt ID as P29460) with a receptor consisting of IL12R01 (binding to p40, encoded by IL12RB1 and IL12R02 (binding to p35, encoded by IL12RB2).
Two other IL12 related cytokines are IL23 and IL35. IL23 shares p40 and its receptor IL12R01 with IL12, while having unique subunits: pl9 (encoded by I 23A) and its receptor IL23R (encoded by IL23R). IL-35 shares p35 and its receptor IL12R02 with IL 12, with unique subunits: EBB and its receptor GP130 (encoded by IL6ST). Schematic structures of IL12, IL23, IL35, their respective receptors and downstream signaling pathways are shown in FIG. 14.
IL23 promotes the differentiation of naive T helper cells into Thl7 phenotype, leading to the secretion of inflammatory cytokines such as IL17 and IL22. In contrast, IL12 induces Thl polarization and the production of critical cytokines like interferon-y (IFN- y) and tumor necrosis factor. Despite sharing the IL12A subunit, IL12 is a pro-inflammatory heterodimeric cytokine primarily produced by dendritic cells, monocytes, and macrophages, while IL35 is an inflammation inhibitory heterodimeric cytokine mainly produced by regulatory T cells and regulatory B cells.
Within IL 12 family cytokines, multiple selective p40 or pl 9 targeting drugs have been approved for multiple autoimmune diseases including psoriasis (PsO), psoriatic arthritis (PsA), Crohn’s disease (CD), and ulcerative colitis (UC), which is listed below. For example, Ustekinumab (Stelara) targeting p40 was approved for CD, UC, PsO and PsA; Risankizumab (Skyrizi) targeting pl 9 was approved for PsO, PsA, and CD; Guselkumab (Tremfya) targeting pl9 was approved for PsO; Tildrakizumab (Ilumya) targeting pl9 was approved for PsO; and Mirikizumab (Omvoh) targeting pl 9 was approved for UC.
Meanwhile, targeting p40 failed to demonstrate efficacy in primary biliary cholangitis (PBC) (Hirschfield, G.M., et al. "Ustekinumab for patients with primary biliary cholangitis who have an inadequate response to ursodeoxycholic acid: a proof-of-concept study." Hepatology 64.1 (2016): 189-199) and systemic erythematosus lupus indications (SLE) (van Vollenhoven, R.F., et al. "Phase 3, multicentre, randomised, placebo-controlled study evaluating the efficacy and safety of ustekinumab in patients with systemic lupus erythematosus." Annals of the Rheumatic Diseases 81.11 (2022): 1556-1563) in clinical trials. In addition, case reports exist where patient taking anti-pl 9 antibody though with effective control of PsO can develop new- onset of SLE (Barber, C. et al. "New-onset systemic lupus erythematous in a patient receiving risankizumab for psoriasis." JAAD Case Reports 25 (2022): 104-106) or PBC (Tomse, P. et al. "Primary biliary cholangitis-cause or association with psoriasis: a case report." Acta Dermatovenerologica Alpina, Pannonica, etAdriatica 32.1 (2023): 23-26). This evidence highlighted that IL12p40 or IL23 pathway specific genes are effective targets for a set of autoimmune diseases of PsO, PsA, CD, and UC, but not for other autoimmune diseases including SLE and PBC.
Though extensive clinic successes and failures demonstrated in p40 and pl 9 targeting drugs, the role of targeting IL12p35 in autoimmune diseases is mostly neglected. This is likely driven by the observations that mice deficient in IL12p35 showed no protection or exacerbated disease in some preclinical models, where genetic or antibody-mediated inhibition of IL12p40 or IL12R.pi ameliorated (Cua, D. J., et al. "Interleukin-23 rather than interleukin- 12 is the critical cytokine for autoimmune inflammation of the brain." Nature 421.6924 (2003): 744-748.; Teng, M.WL, et al. "IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune- mediated inflammatory diseases." Nature Medicine 21.7 (2015): 719-729; and Yen, D., et al. "IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL- 17 and IL-6." The Journal of Clinical Investigation 116.5 (2006): 1310-1316). Thus, a common (mis)-concept was formed that anti-IL12-p35 should be less effective comparing to anti-IL12-p40, and if anti- p40 did not work in an autoimmune indication, anti-p35 would not work either. To our knowledge, there is no publicly revealed ongoing activities in developing anti-IL12p35 or its receptor IL12RP2 therapies for autoimmune diseases either clinically or pre-clinically.
Pollowed by human genetics analysis in the present disclosure, we challenged this (mis)- concept and provide evidences that targeting IL12p35 and IL12RP2 would be effective for specific autoimmune diseases of systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
The present disclosure provides examples of antibodies, antigen-binding fragment thereof, that bind to IL12p35 (interleukin 12 subunit alpha), which can be used for treating systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
IL12 and IL12 receptor
IL12 consists of two subunits connected by disulfide bonds. The smaller p35 monomer (35 kDa a-chain, IL12A, IL12p35, or IL12A-p35) is encoded on chromosome 3, while the gene for the larger p40 monomer (40 kDa P-chain, IL12B, IL12p40, or IL12B-p40) is located on chromosome 5. Co-expression results in the formation of the biologically active p70 heterodimer. Within the “IL12 family” of cytokines, monomers combine with different partners to create various cytokines. While p35 may also pair with Epstein-Barr virus induced gene 3 (EBB) to yield IL35, the p40 subunit in combination with the pl9 monomer leads to the formation of IL-23. The fourth member of the family is IL-27, which is composed of EBI3 and the p28 subunit.
Due to the lower expression of the IL12 a-chain compared to the P-chain, only free P- chains, p40 homodimers or the heterodimer are secreted. Structurally, the p40 subunit shares some features with the IL6 receptor, whereas the p35 subunit is similar to the granulocyte colony-stimulating factor (G-CSE) and IL-6. It has been demonstrated in mice that p40 homodimers regulate the activity of IL12 by counteracting IL12-induced signaling via competition with IL12p70 for binding to the receptor. Eurther functions of p40 homodimers have been described, e.g., roles in the migration of dendritic cells (DCs), allograft rejection or chemotactic activity with regard to macrophages.
Reflecting the heterodimeric structure of the cytokines of the IL 12 family, the corresponding receptors also consist of two subunits. IL12-receptor pi (IL12RP1) is encoded on chromosome 19 and has a molecular weight of 100 kDa. It is a transmembrane protein with the extracellular domain consisting of 516 amino acids that is responsible for the interaction with IL12p40. Consistently, it is also part of the receptor for IL23, where it pairs with IL23R. The gene for IL12RP2 is located on chromosome 1 and is translated to a 130 kDa transmembrane protein, with 595 amino acids forming the extracellular domain. Signal transduction into the cell derives from IL12RP2, which interacts with IL12p35 and is, thus, in combination with glycoprotein 130 (gpl30), also part of the IL35 receptor. The IL27 receptor as the fourth receptor of the family is composed of gpl30 together with the interleukin 27 receptor subunit alpha (WSX1). Since NK cells and T cells are the main targets of IL12, the expression of IL12R is predominantly confined to these cell types. In particular, antigen contact of naive T cells induces upregulation of IL12RP2, which is subsequently maintained by interferon gamma (IFN-y) signaling, but may be counteracted by IL-4. This hints at a vital role for the commitment of T cells to different effector T (Teff) cell lineages such as cells with a T helper type 1 (TH1), but not a TH2 phenotype and, consistently, only the former cells express IL12R02.
IL12 is primarily produced by professional antigen-presenting cells (APCs) such as B cells and DCs as well as phagocytes including monocytes, macrophages and granulocytes. While the production of IL12p35 is predominantly regulated at the translational level, transcriptional regulation is responsible for the amount of IL12p40 expressed. The initial signal triggering IL 12 expression is the exposure of the above mentioned cells to bacteria, viruses, fungi or parasites. Pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) or CpG DNA expressed or contained in such commensals or pathogens are recognized by pattern recognition receptors (PRRs) of the toll like receptor (TLR) family. This leads to the activation of several transcription factors regulating IL12 production, most importantly NF-KB and interferon regulatory elements (IRFs).
Due to the different chromosomal locations and as mentioned above, there are important differences in the regulation of p40 and p35 production. The synthesis of the p40 chain greatly exceeds the production of the p35 chain, suggesting that the synthesis of the p35 chain is the rate-limiting step of IL 12 secretion. Moreover, most of the TLRs are linked to the expression of IL12p40, while expression of p35 is induced by only a limited subset of these receptors, including TLR3, 4 and 8. In addition to direct regulation of IL 12 production, activation of TLRs also leads to the secretion of IFN-0 and IFN-y, whose signaling, in turn, induces activation of IRF-1, IRF-7 and IRF-8. All three IRFs induce p35 and IRF-7 and IRF-8 also induce p40.
In addition, IL12 expression is regulated via interaction of APCs with T-cells through CD40 and its ligand CD40L. CD40 signaling through H-Ras and K-Ras enhances p38 mitogen- activated protein kinase (MAPK)-mediated pro-inflammatory IL12 production. An important positive feedback loop increasing IL12 secretion is so-called IFN-y priming. IFN-y release downstream of IL12 further boosts IL12 production via induction of IL12p35 by IRF-1 and of p40 by ICSBP.
Binding of the two IL12 subunits to the two chains of the IL12 receptor, IL12R01 and IL12R02, activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway of signal transduction. Specifically, IL12R01 subsequently recruits the JAK family member tyrosine kinase 2 (TYK2), whereas IL12R02 associates with JAK2, resulting in phosphorylation of JAK2. This activates the kinase activity of JAK2, which now, vice versa, phosphorylates a tyrosine residue of the associated receptor subunit. STAT molecules contain SRC homology domains (SH2), which, in a next step, bind to phospho-IL12Rp2 exposing the STATs to JAK and leading to their phosphorylation. Association of these activated transcription factors to homo- or heterodimers enables subsequent nuclear translocation. By binding to specific DNA sequences, they promote or repress gene transcription. STAT4 is the most important downstream target of IL12, while effects on STAT1, STAT3 and STAT5 molecules play minor roles. Moreover, IL12R signaling activates mitogen-activated protein kinase kinase 3/6 (MKK) and p38 MAPK, which support the secretion of IFN-y in activated T cells and TH1 cells. Importantly, this pathway is mediated by a STAT4-independent mechanism and correlates with increased STAT2
A main effect of IL12 is the induction of IFN-y production, by which the cytokine is importantly implicated in adaptive as well as innate immune processes. Additionally, it has been shown that IL12 also primed CD4+ and CD8+ T cells to produce IL10, when present early during clonal expansion. This might result in the development of IL10 secreting Type 1 regulatory (Tri) cells in response to IL12 and IL27, which is consistent with the observation of IL12-dependent Tri cell development in visceral leishmaniasis patients. TH2 differentiation, to the contrary, is counteracted by IL12, since GATA binding protein 3 (GAT A3), which is indispensable for TH2 polarization, is repressed in CD4+ and CD 8+ T cell populations upon treatment with IL12 or in vivo expansion in the presence of IL12-producing DCs.
Consistent with its central role in orchestrating immune responses, various studies in animal models and humans suggested that IL 12 contributes to the pathogenesis of several immune-mediated inflammatory diseases, e.g., Inflammatory Bowel Diseases (IBDs), multiple sclerosis, cancer, psoriasis, diabetes mellitus, systemic lupus erythematosus (SLE), primary biliary cholangitis (PBC), Sjogren's syndrome (SjS), and rheumatoid arthritis.
The function of IL 12, IL 12 receptor, and their functions are described e.g., in Ullrich, K. A-M., et al. "Immunology of IL- 12: An update on functional activities and implications for disease." EXCLI Journal 19 (2020): 1563; Wojno, E.D., et al. "The immunobiology of the interleukin- 12 family: room for discovery." Immunity 50.4 (2019): 851-870; and Sun, L., et al. "Interleukin 12 (IL-12) family cytokines: Role in immune pathogenesis and treatment of CNS autoimmune disease." Cytokine 75.2 (2015): 249-255; each of which is incorporated herein by reference in its entirety. Anti-IL12p35 Antibodies and Antigen-Binding Fragments
The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to IL12p35. In some embodiments, the antibodies and antigen-binding fragments thereof that specifically bind to IL12p35 described herein are fully human antibodies or antigen-binding fragments thereof. The antibodies and antigen-binding fragments described herein are capable of binding to IL12p35 (e.g., human IL12p35), blocking the binding of IL12 and its receptor IL12R02, and blocking the IL12-induced intracellular signaling pathways. The disclosure provides e.g., anti-IL12p35 antibodies D2M008-A1C6, D2M008-A1A12, D2M008- A1E5, D2M008-A1A2, D2M008-A1E7, D2M008-A1G9, D2M008-A1A8, D2M008-A1A9, D2M008-A1B4, D2M008-A1E10, D2M008-B2C5, D2M008-B2B10, D2M008-B2B1, D2M008- B2B12, D2M008-B2C3, D2M008-B2B7, D2M008-3A4, D2M008-3A8, D2M008-4A4, D2M008-4A5, D2M008-3A11, D2M008-3A2, D2M008-27H28L 4A4-hblb2, 27H28L-hblb, chimeric antibodies thereof, and humanized antibodies thereof. The CDR sequences, VH, and VL of these antibodies or antibodies derived therefrom are shown in FIGS. 25-27.
For example, the CDR sequences for “D2M008-A1C6”, and “D2M008-A1C6” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 47, 48, and 49, and CDRs of the light chain variable domain, SEQ ID NOs: 50, 51, and 52, as defined by Kabat numbering. The CDRs can also be defined by IMGT numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 185, 186, 187, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 188, 189, and 190.
For example, the CDR sequences for “D2M008-A1 A12”, and “D2M008-A1A12” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 53, 54, and 55, and CDRs of the light chain variable domain, SEQ ID NOs: 56, 57, and 58, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 191, 192, and 193, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 194, 195, and 196.
For example, the CDR sequences for “D2M008-A1E5”, and “D2M008-A1E5” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 59, 60, and 61, and CDRs of the light chain variable domain, SEQ ID NOs: 62, 63, and 64, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 197, 198, and 199, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 200, 201, and 202.
For example, the CDR sequences for “D2M008-A1A2”, and “D2M008-A1 A2” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 65, 66, and 67, and CDRs of the light chain variable domain, SEQ ID NOs: 68, 69, and 70, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 203, 204, and 205, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 206, 207, and 208.
For example, the CDR sequences for “D2M008-A1E7”, and “D2M008-A1E7” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 71, 72, and 73, and CDRs of the light chain variable domain, SEQ ID NOs: 74, 75, and 76, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 209, 210, and 211, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 212, 213, and 214.
For example, the CDR sequences for “D2M008-A1G9”, and “D2M008-A1G9” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 77, 78, and 79, and CDRs of the light chain variable domain, SEQ ID NOs: 80, 81, and 82, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 215, 216, and 217, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 218, 219, and 220.
For example, the CDR sequences for “D2M008-A1A8”, and “D2M008-A1 A8” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 83, 84, and 85, and CDRs of the light chain variable domain, SEQ ID NOs: 86, 87, and 88, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 221, 222, and 223, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 224, 225, and 226.
For example, the CDR sequences for “D2M008-A1A9”, and “D2M008-A1 A9” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 89, 90, and 91, and CDRs of the light chain variable domain, SEQ ID NOs: 92, 93, and 94, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 227, 228, and 229, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 230, 231, and 232.
For example, the CDR sequences for “D2M008-A1B4”, and “D2M008-A1B4” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 95, 96, and 97, and CDRs of the light chain variable domain, SEQ ID NOs: 98, 99, and 100, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 233, 234, and 235, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 236, 237, and 238.
For example, the CDR sequences for “D2M008-A1E10”, and “D2M008-A1E10” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 101, 102, and 103, and CDRs of the light chain variable domain, SEQ ID NOs: 104, 105, and 106, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 239, 240, and 241, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 242, 243, and 244.
For example, the CDR sequences for “D2M008-B2C5”, and “D2M008-B2C5” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 107, 108, and 109, and CDRs of the light chain variable domain, SEQ ID NOs: 110, 111, and 112, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 245, 246, and 247, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 248, 249, and 250.
For example, the CDR sequences for “D2M008-B2B10”, and “D2M008-B2B 10” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 113, 114, and 115, and CDRs of the light chain variable domain, SEQ ID NOs: 116, 117, and 118, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 251, 252, and 253, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 254, 255, and 256.
For example, the CDR sequences for “D2M008-B2B1”, and “D2M008-B2B1” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 119, 120, and 121, and CDRs of the light chain variable domain, SEQ ID NOs: 122, 123, and 124, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 257, 258, and 259, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 260, 261, and 262.
For example, the CDR sequences for “D2M008-B2B12”, and “D2M008-B2B12” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 125, 126, and 127, and CDRs of the light chain variable domain, SEQ ID NOs: 128, 129, and 130, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 263, 264, and 265, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 266, 267, and 268.
For example, the CDR sequences for “D2M008-B2C3”, and “D2M008-B2C3” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 131, 132, and 133, and CDRs of the light chain variable domain, SEQ ID NOs: 134, 135, and 136, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 269, 270, and 271, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 272, 273, and 274.
For example, the CDR sequences for “D2M008-B2B7”, and “D2M008-B2B7” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 137, 138, and 139, and CDRs of the light chain variable domain, SEQ ID NOs: 140, 141, and 142, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 275, 276, and 277, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 278, 279, and 280.
For example, the CDR sequences for “D2M008-3A4”, and “D2M008-3A4” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 143, 144, and 145, and CDRs of the light chain variable domain, SEQ ID NOs: 146, 147, and 148, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 281, 282, and 283, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 284, 285, and 286.
For example, the CDR sequences for “D2M008-3A8”, and “D2M008-3A8” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 149, 150, and 151, and CDRs of the light chain variable domain, SEQ ID NOs: 152, 153, and 154, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 287, 288, and 289, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 290, 291, and 292.
For example, the CDR sequences for “D2M008-4A4”, and “D2M008-4A4” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 155, 156, and 157, and CDRs of the light chain variable domain, SEQ ID NOs: 158, 159, and 160, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 293, 294, and 295, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 296, 297, and 298.
For example, the CDR sequences for “4A4-hblb2”, and “4A4-hblb2” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 155, 156, and 157, and CDRs of the light chain variable domain, SEQ ID NOs: 158, 159, and 160, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 293, 294, and 295, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 296, 297, and 298.
For example, the CDR sequences for “D2M008-4A5”, and “D2M008-4A5” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 161, 162, and 163, and CDRs of the light chain variable domain, SEQ ID NOs: 164, 165, and 166, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 299, 300, and 301, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 302, 303, and 304.
For example, the CDR sequences for “D2M008-3A11”, and “D2M008-3A11” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 167, 168, and 169, and CDRs of the light chain variable domain, SEQ ID NOs: 170, 171, and 172, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 305, 306, and 307, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 308, 309, and 310.
For example, the CDR sequences for “D2M008-3A2”, and “D2M008-3A2” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 173, 174, and 175, and CDRs of the light chain variable domain, SEQ ID NOs: 176, 177, and 178, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 311, 312, and 313, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 314, 315, and 316.
For example, the CDR sequences for “D2M008-27H28L”, and “D2M008-27H28L” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 179, 180, and 181, and CDRs of the light chain variable domain, SEQ ID NOs: 182, 183, and 184, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 317, 318, and 319, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 320, 321, and 322.
For example, the CDR sequences for “27H28L-hblb”, and “27H28L-hblb” derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 179, 180, and 181, and CDRs of the light chain variable domain, SEQ ID NOs: 182, 183, and 184, as defined by Kabat numbering. Under the IMGT definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 317, 318, and 319, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 320, 321, and 322.
Thus, in one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to IL12p35 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are selected from VH CDRS 1, 2, 3 and VL CDRS 1, 2, 3 listed in FIGS. 26-27. The amino acid sequence for heavy chain variable region and light variable region of anti-IL12p35 antibodies are also provided. These VH and VL sequences are shown in FIG. 25.
For example, the VH and VL of D2M008-A1C6 are set forth in SEQ ID NOs: 1 and 2. For example, the VH and VL of D2M008-A1A12 are set forth in SEQ ID NOs: 3 and 4. For example, the VH and VL of D2M008-A1E5 are set forth in SEQ ID NOs: 5 and 6. For example, the VH and VL of D2M008-A1 A2 are set forth in SEQ ID NOs: 7 and 8. For example, the VH and VL of D2M008-A1E7 are set forth in SEQ ID NOs: 9 and 10. For example, the VH and VL of D2M008-A1G9 are set forth in SEQ ID NOs: 11 and 12. For example, the VH and VL of D2M008-A1A8 are set forth in SEQ ID NOs: 13 and 14. For example, the VH and VL of D2M008-A1A9 are set forth in SEQ ID NOs: 15 and 16. For example, the VH and VL of D2M008-A1B4 are set forth in SEQ ID NOs: 17 and 18. For example, the VH and VL of D2M008-A1E10 are set forth in SEQ ID NOs: 19 and 20. For example, the VH and VL of D2M008-B2C5 are set forth in SEQ ID NOs: 21 and 22. For example, the VH and VL of D2M008-B2B10 are set forth in SEQ ID NOs: 23 and 24. For example, the VH and VL of D2M008-B2B1 are set forth in SEQ ID NOs: 25 and 26. For example, the VH and VL of D2M008-B2B12 are set forth in SEQ ID NOs: 27 and 28. For example, the VH and VL of D2M008-B2C3 are set forth in SEQ ID NOs: 29 and 30. For example, the VH and VL of D2M008-B2B7 are set forth in SEQ ID NOs: 31 and 32. For example, the VH and VL of D2M008-3A4 are set forth in SEQ ID NOs: 33 and 34. For example, the VH and VL of
D2M008-3A8 are set forth in SEQ ID NOs: 35 and 36. For example, the VH and VL of
D2M008-4A4 are set forth in SEQ ID NOs: 37 and 38. For example, the VH and VL of
D2M008-4A5 are set forth in SEQ ID NOs: 39 and 40. For example, the VH and VL of
D2M008-3 Al 1 are set forth in SEQ ID NOs: 41 and 42. For example, the VH and VL of D2M008-3A2 are set forth in SEQ ID NOs: 43 and 44. For example, the VH and VL of D2M008-27H28L are set forth in SEQ ID NOs: 45 and 46. For example, the VH and VL of 4A4- hblb2 are set forth in SEQ ID NOs: 335 and 336. For example, the VH and VL of 27H28L-hblb are set forth in SEQ ID NOs: 337 and 338. These antibodies can be human or humanized antibodies.
In some embodiments, any of these heavy chain variable region sequences (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 335, and 337) can be paired with any of these light chain variable region sequences (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 336, and 338).
Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from FIGS. 26-27; and/or one, two, or three light chain variable region CDRs selected from FIGS. 26-27. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs and/or one, two, or three light chain variable region CDRs as shown in FIG. 26, under Kabat numbering scheme. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs and/or one, two, or three light chain variable region CDRs as shown in FIG. 27, under IMGT numbering scheme.
In some embodiments, the antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of any one of the VH CDR1 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions; any one of the VH CDR2 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions; any one of the VH CDR3 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions. In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of any one of the VL CDR1 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions; any one of the VL CDR2 shown in FIGS. 26- 27 with zero, one or two amino acid insertions, deletions, or substitutions; any one of the VL CDR3 shown in FIGS. 26-27 with zero, one or two amino acid insertions, deletions, or substitutions.
The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
The disclosure also provides antibodies or antigen-binding fragments thereof that bind to IL12p35. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 335, or 337. In some embodiments, the selected VL sequence is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 336, or 338.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs (under Kabat, or IMGT numbering) as shown in FIGS. 26-27. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to IL12p35.
The anti-IL12p35 antibodies and antigen-binding fragments thereof can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigenbinding fragment thereof.
Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody (e.g., a full IgG antibody). Thus, a fragment of an antibody that binds to IL12p35 will retain an ability to bind to IL12p35. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
In some embodiments, the anti-IL12p35 antibodies and antigen-binding fragments thereof described herein have an Fc region (e.g., a human IgGl Fc region). In some embodiments, the Fc region described herein includes one or more of YTE mutations (M252Y/S254T/T256E according to EU numbering). The YTE mutations are located at the CH2-CH3 interface in the Fc domain, which have been shown to increase the binding affinity of the antibody Fc at pH 6.0 to the MHC Class I neonatal FcR (FcRn), located primarily in the acidic endosomes of endothelial and haematopoietic cells, thereby permitting more efficient recycling of administered IgGl antibody and longer retention in the plasma. The increased FcRn binding at pH 6.0 by a YTE triple- mutant antibody is mediated by the creation of one additional salt bridge between Glu 256 (E) of Fc-YTE and Gin 2(Q) of the b2-microglobulin chain of FcRn compared to the original IgGl Fc structure. Thus, YTE mutations can result in higher FcRn binding, and has been shown to be well tolerated and extended the half-life of antibodies in human. Details of the YTE mutations can be found, e.g., in Oganesyan, V, et al. "Structural insights into neonatal Fc receptor-based recycling mechanisms." Journal of Biological Chemistry 289.11 (2014): 7812-7824; and Wang, X. et al., "IgGFc engineering to modulate antibody effector functions." Protein & Cell 9.1 (2018): 63-73; each of which is incorporated herein by reference in its entirety. Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgGl molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane- 1 -carboxylate) and SATA (N- succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Set. U.S.A. 94: 7509-7514, 1997). Antibody homodimers can be converted to F(ab’)2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J Immunol. 25:396-404, 2002).
In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigenbinding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).
In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).
In some embodiments, the antibodies or antigen-binding fragments thereof described herein include one or more back-to-germline (B2G) mutations, e.g., through amino acid substitutions, deletions, and/or insertions in framework regions (FRs) of any of the VHs and VLs described herein. Such one or more B2G mutations may reduce potential immunogenicity to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% as compared to that of an antibody or antigen-binding fragment thereof without the one or more B2G mutations. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 B2G mutations can be introduced to any of the VHs and VLs described herein. In some embodiments, one or more B2G mutations are made before VH CDR1 , between VH CDR1 and VH CDR2, between VH CDR2 and VH CDR3, and/or after VH CDR3, in any of the VHs described herein. In some embodiments, one or more B2G mutations are made before VL CDR1, between VL CDR1 and VL CDR2, between VL CDR2 and VL CDR3, and/or after VL CDR3, in any of the VLs described herein. In some embodiments, the one or more B2G mutations are in the framework regions (FRs) of the VH, e.g., in FR1, FR2, FR3, and/or FR4 of the VH. In some embodiments, the one or more B2G mutations are in the framework regions (FRs) of the VL, e.g., in FR1, FR2, FR3, and/or FR4 of the VL. In some embodiments, the one or more B2G mutations are not within CDRs of the VH, e.g., in CDR1, CDR2, and/or CDR3 of the VH. In some embodiments, the one or more B2G mutations are not within CDRs of the VL, e.g., in CDR1, CDR2, and/or CDR3 of the VL.
Methods of designing B2G mutations are generally known in the art. For example, sequences of the VH (e.g., any of the VHs described herein) and/or the VL (e.g., any of the VLs described herein) can be aligned with the closest germline sequences, and the B2G mutations within the framework regions (e.g., FRs) can be identified. The B2G mutations in the VH are also referred to as heavy chain B2G mutations (“hb”). The B2G mutations in the VL are also referred to as light chain B2G mutations (“lb”). In some cases, more than one sets of B2G mutations may be identified, e.g., when different germline sequences are used for alignment. As a result, the first set of light chain B2G mutations may be referred to as “lb” and the second set of light chain B2G mutations may be referred to as “lb2. ” When an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments thereof described herein) includes B2G mutations in both VH and VL sequences, the antibody or antigen-binding fragment thereof may be referred to as having “hblb” or “hblb2” mutations, e.g., 27H28L-hblb- YTE and 4A4-hblb2-YTE.
The B2G mutations described herein may be used to increase the sequence pool of the antibodies or antigen-binding fragments thereof described herein so as to identifying variants having a reduced immunogenicity. Details of B2G mutations can be found, e.g., in Rossotti, M.A., et al. "Immunogenicity and humanization of single - domain antibodies." The FEBS Journal 289.14 (2022): 4304-4327; and Clavero- Alvarez, A., et al. "Humanization of antibodies using a statistical inference approach." Scientific Reports 8.1 (2018): 14820; each of which is incorporated herein by reference in its entirety.
Antibodies and Antigen Binding Fragments
The present disclosure provides anti-IL12p35 antibodies and antigen-binding fragments thereof. In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains (e.g., a full IgG antibody described herein). The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions), bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region), each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR). These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular Immunology 45.14 (2008): 3832-3839; Wu, T.T., et al. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(l-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan 1998); Chothia et al., Nature 342(6252): 877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety. Other definitions are also known in the art, including e.g., IMGT, Aho (Honneger’s Numbering Scheme) and North. In some embodiments, a combination of CDR definitions is used.
The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three- dimensional configuration based on the antigen’s secondary and tertiary structure.
In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in Immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular Immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.
The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi- specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane- and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein. F(ab) and F(ab')2 fragments are smaller antibody fragments that retain full-length antibodies' antigen-binding specificity. The F(ab) fragment is generated by the enzymatic digestion of the Ig molecule with the proteolytic enzyme papain. Papain cleaves the antibody molecule below the hinge region, resulting in two separate Fab fragments and a smaller Fc fragment. The F(ab')2 fragment is generated by the enzymatic digestion of the Ig molecule with the proteolytic enzyme pepsin. Pepsin cleaves the antibody molecule below the hinge region, but leaves a small portion of the hinge region intact, resulting in two separate Fab fragments covalently linked by disulfide bonds. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa. Divalent antibody fragments (e.g., F(ab')2 fragments) are smaller than full IgG antibodies and enable a better penetration into tissue thus facilitating better antigen recognition in immunohistochemistry. The use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A/G. Details of F(ab’)2 fragments and its production methods can be found, e.g., in Rosenstein, S., et al. "Production of F (ab’) 2 from Monoclonal and Polyclonal Antibodies." Current Protocols in Molecular Biology 131.1 (2020): el l9, which is incorporated herein by reference in its entirety.
Antibody Characteristics
The antibodies or antigen-binding fragments thereof described herein can block the binding of IL12 (e.g., IL12p70) to its receptor IL12RP2, thereby inhibiting IL12p35/IL12Rp2- specific downstream pathways that are involved in a series of autoimmune diseases (e.g., SSc, PBC, SLE, and SjS), according to the human genetics analysis described in Example 12. Because IL12p35 is not shared by IL23, the antibodies or antigen-binding fragments thereof described herein can specifically neutralize IL12 without interfering the IL23 pathway. Some tested antibodies also exhibited excellent thermostability, endured different pH stress conditions, and exhibited serum stability. The binning competition assay results also indicate that some antibodies can target different epitopes on IL12p35.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce the binding of IL12 (e.g., IL12p70) to its receptor IL12RP2 to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl). In some cases, ICso of the binding curves can be determined. In some cases, the IC50 of the antibodies or antigen-binding fragments thereof described herein is less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to that of a reference antibody (e.g., human IgGl). In some embodiments, YTE mutations and/or the B2G mutations (e.g., any of the YTE and/or B2G mutations described herein) of the antibodies or antigen-binding fragments thereof described herein do not have a significant impact on the binding between IL12 (e.g., IL12p70) and IL12RP2. In some embodiments, the binding between IL12 (e.g., IL12p70) and IL12RP2 is determined by ELISA.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce human IL12-induced intracellular signaling in a cell (e.g., a human reporter cell) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl) or an anti-IL12p40 antibody (e.g., anti-hP40). In some embodiments, the IL12-induced intracellular signaling is JAK-STAT signaling. In some cases, IC50 of the signaling curves can be determined. In some cases, the IC50 of the antibodies or antigen-binding fragments thereof described herein is less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to that of a reference antibody (e.g., human IgGl) or an anti-IL12p40 antibody (e.g., anti-hP40). In some embodiments, YTE mutations and/or the B2G mutations (e.g., any of the YTE and/or B2G mutations described herein) of the antibodies or antigen-binding fragments thereof described herein do not have a significant impact on the inhibition of the IL12-induced intracellular signaling. In some embodiments, the inhibition of the IL12-induced intracellular signaling is evaluated using a report cell system.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce monkey IL12-induced intracellular signaling in a cell (e.g., a human reporter cell) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl). In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce monkey IL 12- induced intracellular signaling in a cell (e.g., a human reporter cell) to a level that is comparable to an anti-IL12p40 antibody (e.g., anti-hP40). In some embodiments, the antibodies or antigenbinding fragments thereof described herein do not reduce monkey IL12-induced intracellular signaling in a cell (e.g., a human reporter cell) as compared to a reference antibody (e.g., human IgGl). In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce IL12-induced IFN-y production or secretion by a human cell (e.g., human PBMC) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl) or an anti-IL12p40 antibody (e.g., anti-hP40). In some embodiments, the IL12 is an exogenous IL12 (e.g., a recombinant human IL12).
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce IL12-induced IFN-y production or secretion by T cells (e.g., CD4+ T cells) cocultured with antigen-presenting cells (e.g., allogeneic dendritic cells) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference antibody (e.g., human IgGl). In some embodiments, the IL12 is an exogenous IL 12 (e.g., a recombinant human IL 12). In some embodiments, particularly at low concentrations (e.g., less than 0.1 pg/mL, less than 0.05 pg/mL, less than 0.01 pg/mL, less than 0.005 pg/mL, or less than 0.001 pg/mL), the antibodies or antigen-binding fragments thereof described herein can reduce IL12-induced IFN-y production or secretion by T cells (e.g., CD4+ T cells) cocultured with antigen-presenting cells (e.g., allogeneic dendritic cells) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to an anti- IL12p40 antibody (e.g., anti-hP40).
In some embodiments, the antibodies, antigen-binding fragments thereof, or YTE and/or B2G variants thereof described herein do not interfere IL23 binding to its receptors and/or IL23- induced signaling pathways.
In some implementations, the antibody (or antigen-binding fragments thereof) specifically binds to IL12p35 (e.g., human IL12p35, monkey IL12p35, dog IL12p35, mouse IL12p35, and/or chimeric IL12p35), or IL12p70 (e.g., human IL12p70, monkey IL12p70, dog IL12p70, mouse IL12p70, and/or chimeric IL12p70) with a dissociation rate (koff or kd) of less than 0.1 s’1, less than 0.01 s’1, less than 0.001 s’1, less than 0.0001 s’1, or less than 0.0001 s’1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s’1, greater than 0.001 s’1, greater than 0.0001 s’1, greater than 0.00001 s’1, or greater than 0.000001 s’1.
In some embodiments, kinetic association rates (kon or ka) is greater than 1 x 102/Ms, greater than 1 x 103/Ms, greater than 1 x 104/Ms, greater than 1 x 105/Ms, or greater than 1 x 106/MS. In some embodiments, kinetic association rates (kon) is less than 1 x 105/Ms, less than 1 x 106/MS, or less than 1 x 107/Ms.
Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon). In some embodiments, KD is less than 1 x 1 O'6 M, less than 1 x 1 O'7 M, less than 1 x 1 O'8 M, less than 1 x ICT9 M, less than 1 x IO'10 M, less than 1 x 10'11 M, or less than 1 x 10'12 M. In some embodiments, the KD is less than 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM 10 pM, 5 pM, or 1 pM. In some embodiments, KD is greater than 1 x 1 O'7 M, greater than 1 x 10'8 M, greater than 1 x 10'9 M, greater than 1 x 10'10 M, greater than 1 x 10'11 M, or greater than 1 x 10'12 M.
General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the measurement is conducted using Gator® Prime BLI system or Carterra® SPR imaging system. In some embodiments, the antibody binds to human IL12p35, monkey IL12p35 (e.g., rhesus or cynomolgus IL12p35), dog IL12p35 (e.g., canine IL12p35), mouse IL12p35, and/or chimeric IL12p35. In some embodiments, the antibody binds to human IL12p70, monkey IL12p70 (e.g., rhesus or cynomolgus IL12p70), dog IL12p70 (e.g., canine IL12p70), mouse IL12p70, and/or chimeric IL12p70. In some embodiments, the human IL12p70 described herein is formed by human IL12p35 and human IL12p40. In some embodiments, the monkey IL12p70 described herein is formed by monkey IL12p35 and monkey IL12p40. In some embodiments, the dog IL12p70 described herein is formed by dog IL12p35 and dog IL12p40. In some embodiments, the mouse IL12p70 described herein is formed by mouse IL12p35 and mouse IL12p40. In some embodiments, the antibody does not bind to human IL12p40, monkey IL12p40 (e.g., rhesus or cynomolgus IL12p40), dog IL12p40 (e.g., canine IL12p40), mouse IL12p40, or chimeric IL12p40.
In some embodiments, YTE mutations of the antibodies or antigen-binding fragments thereof described herein can improve FcRn binding affinity at acidic pHs (e.g., about pH 6.0, pH 6.5, or pH 7.0) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold as compared to that of the parental antibodies (e.g., the antibodies or antigen-binding fragments thereof described herein without YTE mutations). As a result, the YTE mutations can increase the half-life of the antibodies or antigen-binding fragments thereof described herein in vivo (e.g., when administered in a subject) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to that of the parent antibodies (e.g., the antibodies or antigenbinding fragments thereof described herein without YTE mutations).
In some embodiments, thermostabilities are determined. The antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. As IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, with a first denaturation temperature, Tm DI, and a second denaturation temperature Tm D2. The presence of these two peaks often indicates the denaturation of the Fc domains (Tm DI) and Fab domains (Tm D2), respectively. When there are two peaks, Tm usually refers to Tm D2.
Thus, in some embodiments, the antibodies or antigen binding fragments as described herein has a Tm DI (e.g., for full IgG antibodies) greater than 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D2 (e.g., for F(ab’)2 fragments) greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, Tm, Tm DI, Tm D2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
In some embodiments, the full IgG antibodies described herein (e.g., D2M008-4A4 or 27H28L) has a Tm that is greater than 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.1, 68.2, 68.3, 68.4,
68.5, 68.6, 68.7, 68.8, 68.9, 69, 69.5, or 70 °C. In some embodiments, the F(ab’)2 fragments of the antibodies described herein (e.g., D2M008-4A4 or 27H28L) has a Tm that is greater than 71,
71.5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81,
81.5, 82, 82.5, 83, 83.5, 84, 84.5, or 85 °C.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein, after being stored in stress conditions, can maintain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity as compared to an unstressed control sample. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are incubated in a water bath (at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, or 45°C) for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are in a buffer at an acidic pH (e.g., about pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0) or a basic pH (e.g., about pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9 or pH 9.0). In some embodiments, the unstressed control sample described herein is in a buffer at a neutral pH (e.g., about pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, or pH 7.9). In some embodiments, the activity of the antibodies or antigen-binding fragments thereof described herein, or the unstressed control sample, is determined by measuring the blocking of the binding between human IL12p70 to its receptor IL12R02 (e.g., as described in Example 3 by ELISA), and/or measuring the inhibition of IL12-induced intracellular signaling transduction (e.g., as described in Example 4 by cell-based assays).
In some embodiments, the antibodies or antigen-binding fragments thereof described herein, after being mixed with human serum (e.g., fresh human serum collected from healthy donors) and stored in a water bath (at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, or 45°C) for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days, can maintain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity as compared to a control sample. In some embodiments, the control sample contains the same antibodies or antigen-binding fragments thereof described herein that were mixed with PBS. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, after being mixed with human serum (e.g., fresh human serum collected from healthy donors) and stored in a water bath (at about 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C) for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or longer, can maintain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity as compared to a sample that has not been stored at the same condition. In some embodiments, In some embodiments, the activity of the antibodies or antigen-binding fragments thereof described herein, or the control sample, is determined by measuring the blocking of the binding between human IL12p70 to its receptor IL12R02 (e.g., as described in Example 3 by ELISA), and/or measuring the inhibition of IL12- induced intracellular signaling transduction (e.g., as described in Example 4 by cell-based assays).
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to different epitopes on IL12p35. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to the same epitope on IL12p35.
In some embodiments, the anti-IL12p35 antibodies described herein can decrease immune response in patients having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS) after treatment with a therapeutically effective amount of the anti-IL12p35 antibodies by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to no treatment with the anti-IL12p35 antibodies or treatment with a reference antibody (e.g., Ustekinumab).
In some embodiments, the anti-IL12p35 antibodies described herein are more effective (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds, or 10 folds more effective) than a reference antibody (e.g., Ustekinumab). The heavy chain and light chain sequences of Ustekinumab can be found in FIG. 28.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein may have a good in vivo stability and/or pharmacokinetic properties. For example, after administration at about 0.1-10 mg/kg (e.g., about 1 mg/kg), the terminal elimination half-life (T1/2) of the antibody or antigen-binding fragment thereof can be at least 100 hours, at least 150 hours, at least 200 hours, at least 250 hours, at least 300 hours, at least 350 hours, at least 375 hours, at least 400 hours, at least 425 hours, at least 450 hours, at least 475 hours, or at least 500 hours. In some embodiments, the T1/2 is determined in a subject expressing human FcRn (e.g., a human FcRn transgenic mouse). In some embodiments, the antibodies or antigen-binding fragments thereof described herein include one or more YTE mutations and/or B2G mutations.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce the average ear thickness of mice in a DNFB-induced chronic contact hypersensitivity mouse model. For example, the average ear thickness can be reduced to less than 1.0 mm, less than 0.9 mm, less than 0.8 mm, less than 0.6 mm, less than 0.5 mm, less than 0.4 mm, or less than 0.3 mm. For example, the average ear thickness can be reduced to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% as compared to that when the mice are treated with PBS or an anti-IL12p40 antibody. In some embodiments, the ear thickness is measured after about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days post initial DNFB sensitization. In some embodiments, the changes described herein are significant, e.g., with a p value that is less than 0.01 or less than 0.005.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can alleviate the decrease of saliva excretion of mice in a STZ-induced Sjogren’s Syndrome mouse model. For example, the decrease of saliva excretion level relative to the body weight can be higher than 4 mg/g, higher than 3 mg/g, higher than 2 mg/g, or higher than 1 mg/g. For example, the saliva excretion level relative to the body weight can be improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to that when the mice are treated with an isotype control antibody. In some embodiments, the saliva excretion level is determined using methods described herein, after about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days post initial STZ immunization. In some embodiments, the changes described herein are significant, e.g., with a p value that is less than 0.06, less than 0.05, less than 0.04, or less than 0.03.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the body weight of mice in a IMQ-induced SLE model. For example, the body weight drop after initial IMQ treatment is less than 10 g, less than 9 g, less than 8 g, less than 7 g, less than 6 g, less than 5 g, less than 4 g, less than 3 g, less than 2 g, or less than 1 g. For example, the body weight can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%, as compared to that when the mice are treated with an isotype control antibody. In some embodiments, the body weight is measured after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks post initial IMQ treatment.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the urine albumin level of mice in a IMQ-induced SLE model. For example, the ratio of urine albumin/creatinine levels can be above 100 pg/mg, above 1000 pg/mg, above 10000 pg/mg, or above 100000 pg/mg. In some embodiments, the ratio of urine albumin/creatinine levels can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, as compared to that when the mice are treated with an isotype control antibody. In some embodiments, the ratios described herein is determined after about 1, 2, 3, 4, 5, 6, 7, or 8 weeks post initial IMQ treatment.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the urine albumin level of mice in a spontaneous SLE model. For example, the ratio of urine albumin/creatinine levels can be above 1 mg/mg, above 10 mg/mg, above 20 mg/mg, above 30 mg/mg, above 40 mg/mg, above 50 mg/mg, above 60 mg/mg, above 70 mg/mg, above 80 mg/mg, above 90 mg/mg, or above 100 mg/mg. In some embodiments, the ratio of urine albumin/creatinine levels can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, as compared to that when the mice are treated with an isotype control antibody. In some embodiments, the ratios described herein is determined after about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks from the first day of the experiment using the spontaneous SLE model.
In some embodiments, the antibodies or antigen-binding fragments thereof described herein can significantly reduce kidney pathologic symptoms associated with SLE, as compared that when the mice are treated with an isotype control antibody. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can slow down disease progression in the spontaneous SLE model. In some embodiments, the antibodies or antigenbinding fragments thereof described herein can increase the activity score by at least 50%, at least 75%, or at least 100% as compared to that when the mice are treated with an isotype control antibody in the spontaneous SLE model. In some embodiments, the antibodies or antigenbinding fragments thereof described herein can decrease the chronicity score to less than 50%, less than 40%, less than 30%, or less than 20% as compared to that when the mice are treated with an isotype control antibody in the spontaneous SLE model. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can decrease the independent lesion score to less than 80%, less than 60%, less than 40%, or less than 20% as compared to that when the mice are treated with an isotype control antibody in the spontaneous SLE model.
Methods of Making Anti-IL12p35 Antibodies An isolated fragment of human IL12p35 can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times).
The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of IL12p35 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein. As described above, the full length sequence of human IL12p35 is known in the art. In some embodiments, an Fc-tagged or His- tagged human IL12p35 protein is used as the immunogen.
An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., the recombinant chimeric IL12 described herein, or a fragment of human IL12p35). The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject (e.g., a RenMab™ mouse) with the recombinant chimeric IL12 described herein as an immunogen. Alternative, a IL12p35 polypeptide or an antigenic peptide thereof (e.g., part of IL12p35) can be used as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using an immobilized human IL12p35 or a fragment thereof. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigenbinding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., IL12p35. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.
Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies.
In some embodiments, a mouse (e.g., a RenMab™ mouse) with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used to generate antibodies. The heavy chain immunoglobulin locus is a region on the chromosome that contains genes for the heavy chains of antibodies. The locus can include e.g., human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes. The kappa chain immunoglobulin locus is a region on the chromosome that contains genes that encode the light chains of antibodies (kappa chain). The kappa chain immunoglobulin locus can include e.g., human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. A detailed description regarding RenMab™ mice can be found in WO/2020/169022, which is incorporated herein by reference in its entirety.
In some embodiments, a recombinant chimeric IL12 comprising mouse IL12p40 and human IL12p35 is used as the immunogen to immunize a mouse (e.g., a RenMab™ mouse). As a result, the obtained antibodies can specifically target human IL12p35, but not mouse IL12p40.
Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in 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); each of which is incorporated by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.
The choice of human VH and VL domains to be used in making the humanized antibodies is very important for reducing immunogenicity. According to the so-called “best-fit” method, the sequence of the V domain of a mouse antibody is screened against the entire library of known human-domain sequences. The human sequence which is closest to that of the mouse is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol. , 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).
It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
Ordinarily, amino acid sequence variants of the human, humanized, or chimeric anti- IL12p35 antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with a sequence present in the light or heavy chain of the original antibody.
The antibodies generated by the mice have a full human VH, a full human VL, and mouse constant regions. In some embodiments, the human VH and human VL is linked to a human IgG constant regions (e.g., IgGl, IgG2, IgG3, and IgG4).
Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric anti-IL12p35 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
Additional modifications to the anti-IL12p35 antibodies or antigen-binding fragments can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo. Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional crosslinkers as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993).
Alternatively, an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).
In some embodiments, a covalent modification can be made to the anti-IL12p35 antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
Recombinant Vectors
The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.
As used herein, a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly- A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non- pathogenic (defective), replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Set. 569:86-103; Flexner et al., 1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner et al., Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91 :215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498- 11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.
For expression, the DNA insert comprising an antibody-encoding or polypeptide- encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Non-limiting bacterial promoters suitable for use include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grant etal., Methods Enzymol., 153: 516-544 (1997).
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.
Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
Methods of Treatment
The antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.
In one aspect, the disclosure provides methods for treating, preventing, or reducing the risk of developing disorders associated with an abnormal or unwanted immune response, e.g., an autoimmune disorder, e.g., by inhibiting IL12p35/IL12Rp2-specific downstream pathways. In some embodiments, the methods described herein can inhibit the IL 12 signaling pathway without interfering the IL23 signaling pathway. In some embodiments, the methods described herein can neutralize IL12 but reserve IL23 in vivo.
Systemic sclerosis (SSc),, Primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS) represent a set of inflammatory diseases commonly associated with each other, which implies potential commonly shared pathological mechanisms (Conrad, N., et al. "Incidence, prevalence, and co-occurrence of autoimmune disorders over time and by age, sex, and socioeconomic status: a population-based cohort study of 22 million individuals in the UK." The Lancet 401.10391 (2023): 1878-1890; and Selmi, C. et al. "Chronic autoimmune epithelitis in Sjogren’s syndrome and primary biliary cholangitis: a comprehensive review." Rheumatology and Therapy 4 (2017): 263-279). Most of the widely used immunological therapeutics such as anti-TNF, JAK inhibitors, and anti-p40 are not effective for them, highlighting significant unmet needs in developing novel therapies for this cluster of autoimmune disorders.
Systemic sclerosis (SSc), or scleroderma, presents a complex autoimmune profile marked by vascular damage and fibrosis across various organs. It bears the highest case-specific mortality among systemic autoimmune diseases (Scherlinger, M., et al. "Worldwide trends in allcause mortality of auto-immune systemic diseases between 2001 and 2014." Autoimmunity reviews 19.6 (2020): 102531). Treatment options for fibrotic lesions, skin sclerosis, and SSc- associated interstitial lung disease (SSc-ILD) have been constrained. Primary biliary cholangitis (PBC), a chronic autoimmune cholestatic liver disease, showcases progressive intrahepatic cholestasis and eventual end-stage liver disease necessitating transplantation (Onofrio, F.Q., et al. "A practical review of primary biliary cholangitis for the gastroenterologist." Gastroenterology & Hepatology 15.3 (2019): 145). Systemic lupus erythematosus (SLE) is a challenging autoimmune disease with diverse clinical manifestations and an unpredictable disease course. Despite advances in pathophysiology understanding and some newly developed treatments, SLE patients face significant mortality and a risk of progressive organ damage (Piga, M., et al. "The main challenges in systemic lupus erythematosus: where do we stand?." Journal of Clinical Medicine 10.2 (2021): 243). Sjogren's syndrome (SjS), a prevalent chronic autoimmune rheumatic disease, features lymphocytic infiltration of exocrine glands and various extra-glandular organs. Treatment mainly focuses on symptom management and complication prevention, lacking a cure (Zhan, Q., et al. "Pathogenesis and treatment of Sjogren’s syndrome: Review and update." Frontiers in Immunology 14 (2023): 1127417).
In some embodiments, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein can block the binding of IL12 to its receptor IL12RP2, thereby inhibiting IL12- induced intracellular pathways (e.g., IL12p35/ IL12Rp2-specific pathways). According to the human genetics analysis discussed in Example 12, these anti-IL12p35 antibodies or antigenbinding fragments thereof can be used for treating a cluster of immune disorders (e.g., SSc, PBC, SLE, and SjS). In some embodiments, the subject described herein has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS). In some embodiments, the subject is a human subject. In some embodiments, the subject is a non-human mammal, e.g., a monkey, a dog, a mouse, or any commonly used model animals known in the art. In some embodiments, the subject is a dog having a similar disease or disorder as SSc, PBC, SLE, and/or SjS. In some embodiments, the dog has an immune disorder (e.g., an autoimmune disease), e.g., Hypothyroidism, Lupus, Immune-Mediated Polyarthritis (IMP A), Inflammatory Bowel Disease (IBD), Immune-Mediated Hemolytic Anemia (IMHA), Immune-Mediated Thrombocytopenia (IMT), Diabetes, Myasthenia Gravis, Rheumatoid Arthritis, Addison’s Disease (Hypoadrenocorticism), Bullous Autoimmune Skin Diseases, and Periodontal disease. Details can be found, e.g., in Pedersen, N. C. "A review of immunologic diseases of the dog." Veterinary Immunology and Immunopathology 69.2-4 (1999): 251-342, which is incorporated herein by reference in its entirety.
In some embodiments, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein cannot block the binding of IL12p40 to its receptor IL12RP1. Thus, these anti- IL12p35 antibodies or antigen-binding fragments thereof do not interfere IL23 binding to its receptors or transducing signals (e.g., IL12p40/IL12Rpi-specific pathways). According to the human genetics analysis discussed in Example 12, because IL12p40 and IL23R are associated with PsO, CD, UC, IBD, and AS, but not with SSc, PBC, SLE, or SjS, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein may not be suitable for treating a cluster of immune disorders (e.g., PsO, CD, UC, IBD, and AS). In some embodiments, the subject described herein does not have psoriasis (PsO), Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel diseases (IBD), or ankylosing spondylitis (AS).
In some embodiments, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein can inhibit the IL 12 signaling pathway without interfering the IL23 signaling pathway.
As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., any of the autoimmune diseases described herein. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.
Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal. Guidance in selecting appropriate doses for antibody or antigen binding fragment can be found in the literature on therapeutic uses of antibodies and antigen binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.
In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigenbinding fragments described herein) in the subject.
In some embodiments, the subject can be administered the at least one antibody, antigenbinding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigenbinding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer). As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).
Pharmaceutical Compositions and Routes of Administration
Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.
Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).
Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).
Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human, a dog, a monkey, or a mouse). A therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer), or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured), decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human, a dog, a monkey, or a mouse). The effectiveness and dosing of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human, a dog, a monkey, or a mouse). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases).
Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject’s weight (e.g., about 1 pg/kg to about 500 mg/kg; about 100 pg/kg to about 500 mg/kg; about 100 pg/kg to about 50 mg/kg; about 10 pg/kg to about 5 mg/kg; about 10 pg/kg to about 0.5 mg/kg; or about 1 pg/kg to about 50 pg/kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half- life of the antibody or antibody fragment in vivo.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1: Generation of anti-IL12p35 antibodies This example describes how anti-IL12p35 antibodies were generated. A panel of antibodies that selectively bind to human and cynomolgus (cyno) monkey IL12p35 antigens were generated in RenMab™ mice (Biocytogen) by immunizing with recombinant chimeric IL 12.
Immunization: Recombinant chimeric IL 12 is a heterodimer composed of mouse IL12p40 and human IL12p35, which was designed to promote immunogenicity towards human IL12p35 subunit of IL12p70. A total of seven mice were initially immunized subcutaneously through neck and hock injections with a recombinant chimeric IL 12 emulsion prepared in complete Freund's adjuvant (CFA). Subsequently, all mice were boosted with the recombinant chimeric IL12 emulsion prepared in incomplete Freund's adjuvant (CFA) three times. Five days after the last boost, four mice with a higher serum titer were scarified and proceeded to Beacon binder selection. The rest three mice were further boosted three times. Four days after the last boost, two mice with a higher serum titer were scarified and proceeded to Beacon binder selection.
Preparation of B cells: Lymphoid organs including lymph nodes, bone marrow, and spleen were harvested from scarified mice. The harvested organs were homogenized to break down tissue structures. Single-cell suspensions were obtained by passing the homogenized material through a 40 pm cell strainer. Cell numbers and viability were assessed. Plasma B cells were enriched using the mouse CD 138+ Plasma Cell Isolation Kit (StemCell Technologies, Cat#: 18957).
Beacon screening: Plasma B cells were subjected to Beacon screening assays using Beacon® Optofluidic System (Model: 110-08023) and OptoSelect® UK Chips (Berkeley Lights, Cat#: 500-12012). The Beacon bloom screening assays employed streptavidin beads (Berkeley Lights, Cat#: 520-00053) complexed with biotinylated chimeric IL12 or biotinylated chimeric IL 12 to capture secreted binder antibodies and simultaneously quantify bound antibodies with a fluorescently labeled anti-mouse Fc. A total of 128 cells were successfully unloaded from blooming Beacon pins and were subjected to RNA isolation, followed by RT-PCR to amplify nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL). PCR amplicons were sent to Genewiz/Azenta for sequencing. The sequences were analyzed with the IMGT program. The unique clones were converted into full human IgGl format and synthesized for further characterization. Certain clones were engineered to improve pharmacokinetics by introducing an FcRn affinity-enhancing Fc mutations through amino acid substitutions (e.g., M252Y/S254T/T256E, or YTE mutations) and to reduce potential immunogenicity by introducing back-to-germline mutations through amino acid substitutions in frameworks in variable regions.
Example 2: Anti-IL12p35 antibodies bound to human and monkey IL12p70 with high affinities
The binding kinetics of the antibodies identified in Example 1 were determined. The antibodies that were successfully produced were measured using the Gator® Prime BLI (Biolayer Interferometry) System (Gator Bio, USA) and selected variants were measured using the BiaCore™ SPR platform. Specifically, the anti-IL12p35 antibodies were loaded onto HFC probes (Anti-hlgGFc, Gator Bio, Cat#: 160003). Subsequently, the probes were incubated with corresponding monomeric antigens human IL12p70-His (Sino Bio, Cat#: CT011-H08H), rhesus IL12-H1S (Sino Bio, Cat#: CT045-C08H), canine IL12p70 (Kingfisher Biotech, Cat#: RP2228D- 005), or mouse IL12p70 (BioLegend, Cat#: 577002) for 3-4 minutes at 25°C in 250 pl K buffer (Gator Bio, Cat#: 120011). Dissociation was monitored for approximately 10 minutes. The probes were regenerated between binding cycles with Regeneration Buffer (Gator Bio, Cat#: 120012). Binding kinetics was analyzed using software supplied by the manufacturer. In particular, the following setting parameters were used: all tested antibodies: 4 pg/ml; canine IL12p70: 2 pg/ml; and mouse IL12p70: 1 pg/ml.
To more accurately measure the binding kinetics, selected antibodies were analyzed using the BiaCore™ 8K SPR platform. Specifically, the anti-IL12p35 antibodies were loaded on to Protein A chip (Cytiva, Cat#:29127556). Subsequently, the chips were incubated with serially diluted monomeric antigens human IL12p70-His (Sino Bio, Cat#: CT011-H08H) to allow association for 90 seconds and dissociation for 210 seconds, and the kinetics was monitored. The chips were regenerated between cycles with a Glycine hydrochloride buffer at pH 1.5. Binding kinetics was analyzed using BiaCore™ Insight Evaluation Software supplied by the manufacturer.
To compare the binding kinetics of YTE variants with the corresponding parental antibodies, the anti-IL12p35 antibodies were loaded with HFC (Anti-hlgGFc, Gator Bio, Cat#: 160003), the probes were incubated with a recombinant FcRn protein (Sino Bio, Cat#: CT009- H08H) in K buffer prepared in-house at pH 6.0, pH 6.5, or pH 7.0, and the dissociation steps were performed in the corresponding K buffers.
The preliminary binding affinities of antibodies to human IL12p70 were measured by the Gator® Prime System, and results are shown in FIG. 1. For some antibodies, because their binding affinities were too high (e.g., with estimated KD values less than 1 x 10'11 M or less than 1 x 10'12 M), which were out of range (“O.R.”) of the detecting capability by the Gator BLI technology, their KD values were not calculable.
As shown in FIG. 2A, further analysis revealed that some of the antibodies, e.g., D2M008-A1A8, D2M008-A1 A9 and D2M008-4A4, exhibited high binding affinities to human and rhesus IL12p70. Both D2M008-A1 A8 and D2M008-A1 A9 showed an approximately 100- fold difference in binding affinity for human and monkey IL12. A recombinant antibody composed of the heavy chain of D2M008-A1 A8 and the light chain of D2M008-A1A9, named 27H28L, exhibited a similar magnitude of difference when binding to human and rhesus IL12p70. Antibody D2M008-4A4 demonstrated very high binding affinities to human and rhesus IL12p70. Because the IL12 protein sequences of cynomolgus monkey and rhesus monkey are identical, the high binding affinity to rhesus IL12p70 suggested antibodies that can also bind to cynomolgus monkey IL12p70, indicating that cynomolgus monkeys (as a relevant species) can be used for future nonclinical studies. As shown in FIGS. 2B-2C, YTE mutations and back-to- germline mutations showed no impact on binding affinities of 27H28L and D2M008-4A4. Notably, 27H28L variant 27H28L-hblb-YTE showed a very high affinity to canine IL- 12, which was comparable to its affinity to human IL- 12, as both were out of range on Gator. As shown in FIGS. 3A-3B, YTE variants exhibited improved FcRn binding affinity at acidic pHs compared to the parental antibodies, indicating that YTE variants theoretically can improve pharmacokinetics in human compared to parental antibodies.
Example 3: Anti-IL12p35 antibodies blocked the binding of IL12 to its receptor IL12RP2
The ability of anti-IL12p35 antibodies to block the interaction of human IL12p70 with a recombinant IL12RP2 (interleukin 12 receptor, beta 2 subunit) protein was determined by an ELISA-based assay. Specifically, 1 pg/ml biotinylated human IL12p70 (Sino Bio, Cat#: CT011- H08H-B) was pre- incubated with a fixed concentration of (or serially diluted) anti-IL12p35 or control antibody anti-IL12p40 (Ustekinumab, Biointron, Cat#: B779149; or “anti-hP40”) at room temperature (RT) for 30 minutes, and then added to a 96- well plate coated with 1 pg/mL IL12RP2 (Biointron, Cat#: B21709001), followed by an incubation at RT for 2 hours with gentle shaking. Bound IL12p70 was detected using HRP-conjugated streptavidin (BioLegend, Cat#: 405210) and TMB substrate (Surmodics, Cat#: TMBS-1000-01). OD450 was measured using a Varioskan™ Lux plate reader (Thermo Scientific).
As shown in FIG. 4A, the 27 binders identified in Examples 2 and listed in FIG. 1 demonstrated differences in the ability to block the interaction between human IL12p70 and recombinant IL12RP2. Eight selected anti-IL12p35 antibodies were further analyzed, which blocked the interaction between human IL12p70 and recombinant IL12RP2 in a dose-dependent manner (FIG. 4B) Titrated 27H28L and several additional anti-IL12p35 antibodies were tested. In particular, 27H28L, D2M008-4A4 and D2M008-4A5 exhibited dose-dependent blocking of the interaction between human IL12p70 and recombinant IL12RP2 (FIG. 4C). In addition, YTE mutations showed no significant impact on the blocking function of 27H28L and D2M008-4A4 (FIG. 4D). Furthermore, back-to-germline mutations showed no impact on the blocking function (FIG. 4E)
Example 4: Anti-IL12p35 antibodies blocked IL12-induced intracellular signaling in IL12 reporter cells
The ability of anti-IL12p35 antibodies to inhibit IL12p70-induced signaling was evaluated in HEK-Blue™ IL12 reporter cells (InvivoGen, Cat#: hkb-il 12). Specifically, 20 ng/ml recombinant human IL12 (BioLegend, Cat#: 573004) or rhesus IL12 (Sino Bio, Cat#: CT045- C08H) was pre-incubated with different concentrations of IgGl, anti-hP40, or anti-IL12p35 antibodies at room temperature for 30 minutes, and then added to a flat-bottom 96- well plate seeded with 5 104 HEK-Blue™ IL12 reporter cells, followed by an incubation at 37°C for 24 hours. 20 pl supernatant from each well was collected and mixed with 180 pl QUANTI-Blue™ Solution (Invivogen, Cat#: rep-qbs) in a new flat-bottom 96-well plate, and incubated at 37°C for 50 minutes. OD620 was measured using a Varioskan™ Lux plate reader (Thermo Scientific, SN#3020-80467).
As shown in FIG. 5A, the 27 binders identified in Examples 2 and listed in FIG. 1 demonstrated differences in the ability to inhibit IL12p70-induced intracellular signaling in HEK-Blue™ IL12 reporter cells. D2M008-A1 A8 and D2M008-A1 A9 showed the best blocking ability among these IL12 binders. D2M008-A1B4 and D2M008-A1E10 also exhibited blocking capabilities. These four selected anti-IL12p35 antibodies were further analyzed. As shown in FIG. 5B, D2M008-A1 A8 and D2M008-A1 A9 potently inhibited IL12p70-induced intracellular signaling in HEK-Blue™ IL 12 reporter cells in a dose-dependent manner.
As shown in FIG. 5C, engineered variant 27H28L, which composed of heavy chain of D2M008-A1A8 and light chain of D2M008-A1A9, showed a significantly stronger potency than its parental antibodies D2M008-A1 A8 and D2M008-A1A9 in a dose-dependent manner. Interestingly, 27H28L was also more potent than anti-IL12p40 (Ustekinumab) and was equally active as the combination of anti-IL12p40 (Ustekinumab) and D2M008-A1 A8. Thus, as shown in FIG. 5B and FIG. 5C, D2M008-A1 A8 and D2M008-A1 A9 were slightly better than anti- IL12p40 (Ustekinumab), or at least comparable in inhibiting IL12p70 activity. These results indicate that the engineered variant of anti-IL12p35, 27H28L, can block the interaction of IL12p70 with its receptors via a mode of action that is distinct from D2M008-A1 A8 and D2M008-A1 A9, which is superior to the mode of action of anti-IL12p40 (Ustekinumab).
Although the ability of D2M008-4A4 to inhibit IL12 activity was not as potent as 27H28L, it was significantly better than anti-IL12p40 (Ustekinumab) in a dose-dependent manner (FIG. 5D). YTE and back-to-germline mutations showed no significant impact on the ability of 27H28L and D2M008-4A4 to inhibit IL12 activity (FIGS. 5E-5F). While both 27H28L and D2M008-4A4 inhibited human IL12p70 more potently than anti-IL12p40 (Ustekinumab), only D2M008-4A4 inhibited rhesus IL 12p70- induced signaling in HEK-Blue™ IL12 reporter cells (similar to Ustekinumab), but 27H28L did not (FIG. 6).
Example 5: Anti-IL12p35 antibodies inhibited IL12-induced IFN-y production in human PBMCs
The principal function of IL 12 is the activation of T cells and NK cells, leading to an increased production of INF -y, proliferation, and cytotoxic potential. The ability of the identified anti-IL12p35 antibodies to inhibit human IFN-y production in response to exogenous IL12p70 was evaluated using human PBMCs. Specifically, 10 ng/ml recombinant human IL12 (BioLegend, Cat#: 573004) was pre-incubated with different concentrations of IgGl, anti- IL12p40, or anti-IL12p35 antibodies for 30 minutes at room temperature, and then added to a U- bottom 96- well plate seeded with 1-2 x 105 human PBMCs per well, followed by an incubation at 37°C for 24 hours. Supernatant was collected from each well and human IFN-y was measured using the MAX™ Deluxe Set Human IFN-y ELISA kit (BioLegend, Cat#: 430116).
As shown in FIGS. 7A-7C, anti-IL12p35 antibodies inhibited exogenous IL12-induced IFN-y production in human PBMCs in a dose-dependent manner. Significantly, in line with the results observed in IL12 reporter cells, D2M008-4A4 and 27H28L were more potent than anti- IL12p40 (Ustekinumab) in inhibiting IL12-induced IFN-y production in human PBMCs.
Example 6: Anti-IL12p35 antibodies prevented IFN-y production by CD4+ T cells cocultured with allogeneic dendritic cells
The ability to inhibit endogenous IL12p70 activity was evaluated in an MLR assay using primary human CD4+ T cells and allogenic dendritic cells. Specifically, human dendritic cells were generated from monocytes through incubation with 50 ng/ml of human GM-CSF (BioLegend, Cat#: 766106) and 20 ng/ml of human IL-4 (BioLegend, Cat#: 766206) for 6 days, followed by maturation with 1 mg/ml of LPS (Sigma, Cat#: L2630-10MG) for 1 day. 1 x 104 dendritic cells in 50 ml of complete 1640 RPMI culture medium were seeded to each well of a flat-bottom 96- well plate, and then 100 ml of different concentrations of IgGl, anti-IL12p40, 27H28L, or D2M008-4A4 (starting at 60 pg/ml, then 3-fold dilutions) in complete 1640 RPMI culture medium were added to the indicated well. Next, 50 pl of 1 x 105 allogeneic CD4+ T cells in complete 1640 RPMI culture medium was added to the indicated wells. The cells were mixed and incubated at 37°C for 24 hours. Supernatant from each well was collected and human IFN-y was measured using MAX™ Deluxe Set Human IFN-y ELISA kit (BioLegend, Cat#: 430116).
As shown in FIGS. 8A-8B, in the MLR assay using allogeneic dendritic cells to stimulate primary CD4 T cells, anti-IL12p35 antibodies (engineered variant 27H28L and D2M008-4A4) and anti-IL12p40 (Ustekinumab) inhibited IFN-y production in a dose-dependent manner. Interestingly, D2M008-4A4 and 27H28L, especially 27H28L, were more effective than anti- IL12p40 (Ustekinumab) in inhibiting IFN-y production at low concentrations. However, at high concentrations, anti-IL12p40 (Ustekinumab) exhibited a greater efficacy, suggesting that anti- IL12p40 not only prevents IL12-induced IFN-y production but also inhibits IFN-y production via other mechanisms (e.g., by other factors) through IL12p40. Taken together with the observations related to exogenous IL12, the results indicate that compared to blocking the IL12p40/IL12Rpi interaction, specifically inhibiting the IL12p35/IL12Rp2 interaction can be a more efficient strategy for suppressing IL12’s biological activity. This enhanced potency may be attributed to the distinct roles of IL12R 1 and IL12R 2 in the heterodimeric IL12 receptor complex. Moreover, inhibiting the IL12p35/IL12R 2 interaction offers an efficient strategy to specifically suppress IL12’s biological activity without interfering IL23, whereas blocking the IL12p40/IL12Rpi interaction cannot achieve the same specificity.
Example 7: Anti-IL12p35 antibodies did not interfere IL23 binding to its receptors and transducing signals
The impact of anti-IL12p35 antibodies on IL23 -activated downstream JAK-STAT signaling pathways was examined in HEK-Blue™ IL23 reporter cells (InvivoGen, Cat#: hkb- il23). Specifically, 10 ng/ml recombinant human IL23 (BioLegend, Cat#: 574104) was preincubated with different concentrations of IgGl, anti-hP40, or anti-IL12p35 antibodies for 30 minutes at room temperature, and then added to a flat-bottom 96-well plate seeded with 5 104 per well of HEK-Blue™ IL23 reporter cells, followed by an incubation at 37°C for 24 hours. 20 pl supernatant from each well was collected and mixed with 180 pl QUANTI-Blue™ Solution (Invivogen, Cat#: rep-qbs) in a new flat-bottom 96-well plate, followed by an incubation at 37°C for 30-60 minutes. OD620 was measured using a Varioskan™ Lux plate reader (Thermo Scientific).
As shown in FIGS. 9A-9B, whereas anti-IL12p40 (Ustekinumab) efficiently neutralized IL23, neither 27H28L, D2M008-4A4, nor their YTE variants inhibited IL23 biological activity. The results confirmed that anti-IL12p35 antibodies can specifically neutralize IL 12, unlike anti- IL12p40 which can neutralize both IL 12 and IL23.
Example 8: Anti-IL12p35 antibodies exhibited excellent thermostability
The melting points of full IgG and F(ab’)2 of humanized anti-IL12p35 antibodies were measured using the Protein Thermal Shift™ Dye Kit (ThermoFisher, Cat#: 4461146) and a qPCR machine. To prepare the F(ab’)2, 17.25 pL (1 mg/mL) full IgG antibody was digested with 0.25 pL IdeZ Protease (IgG- specific) (NEB, Cat#: P0770S) together with 2 pL 10x GlycoBuffer 2 (NEB, Cat#: P0770S) in a 37°C water bath for 2 hours. 10 pL Captures elect™ IgG-Fc Magnetic Agarose Beads (ThermoFisher, Cat#: 2882852005) were washed with PBS and added to the digestion reaction. The mixture was incubated for 15 minutes at room temperature. The beads were separated by centrifuging for 2 minutes at 300g to deplete the undigested antibody and Fc fragments. The supernatant with F(ab’)2 or full IgG antibodies (12.5 pL aliquots) was added to the wells of 0.2 ml Non-skirted Low profile 96-well PCR plate (Thermo Scientific, Cat#: AB-0700/W). 5 pL buffer and 2.5 pL diluted dye (8 ) from the Protein Thermal Shift™ Dye Kit (ThermoFisher, Cat#: 4461146) were added to each of the wells and mixed well. The plate was heated from 25°C to 99°C at a rate of 0.05 °C/second and fluorescence was detected at each temperature using QuantStudio™ 6 software (ThermoFisher, Cat#: A43180). The fluorescence intensity was subtracted by the lowest intensity (bottom) before the peak and normalized to the amplitude of peak to bottom. The Tm for each antibody was calculated based on the temperature of 50% of amplitude from bottom to peak, using an in-house Perl script.
As shown in FIGS. 10A-10B, anti-IL12p35 antibodies 27H28L and D2M008-4A4 both demonstrated excellent thermostability and showed a Tm of 73.99 °C and 79.53 °C, respectively, in their F(ab’)2 format.
Example 9: Anti-IL12p35 antibodies exhibited stability under different pH stress conditions
Common chemical modifications of antibodies include deamidation and isomerization. Asparagine deamidation and aspartic acid isomerization may be induced in vitro at high and low pH conditions, respectively. To investigate the stability of anti-IL12p35 antibodies under stress conditions, 100 pL antibody (1 mg/mL) was exchanged with a pH 5.5 buffer (50 mM Sodium Acetate) or a pH 8.5 buffer (20 mM Tris and 10 mM EDTA) using Zebra™ Spin Desalting columns (40K MWCO, ThermoFisher, Cat#: 87767), respectively. The antibodies were then incubated in a 40°C water bath for 2 weeks. The function of anti-IL12p35 antibodies under different stress conditions was measured using the methods described in Examples 3-4, which detected the ability of anti-IL12p35 antibodies to block the binding of human IL12p70 to its receptor IL12RP2 and inhibit IL12-induced signaling transduction, respectively. The control sample (in a pH 7.4 buffer) was not subjected to the 40°C water bath for 2 weeks.
As shown in FIGS. 11A-11B, stressed anti-IL12p35 antibodies, 27H28L and D2M008- 4A4, exhibited similar blocking abilities compared to antibodies incubated at a normal pH, indicating that the anti-IL12p35 antibodies were stable under high or low pH stressed conditions. Example 10: Anti-IL12p35 antibodies exhibited serum stability
To further investigate the stability of anti-IL12p35 antibodies, 50 pg anti-IL12p35 (about 1 mg/mL) was mixed with an equal volume of fresh human serum collected from healthy volunteers and incubated in a 37°C water bath for 2 weeks. Anti-IL12p35 antibodies exposed to fresh plasma serum were then compared with their untreated stock samples (in PBS without incubation at 37°C water bath for 2 weeks) using ELISA- and cell-based assays described in Examples 3 and 4, measuring their ability to block IL12 binding to IL12RP2 and transducing intracellular signals, respectively.
As shown in FIGS. 12A-12B, anti-IL12p35 antibodies incubated with human serum for up to 2 weeks exhibited similar activities compared to anti-IL12p35 antibodies incubated with PBS, indicating their serum stability.
Similarly, the stability of 27H28L-hblb-YTE and 4A4-hblb2-YTE were also determined by mixing 50 pg anti-IL12p35 (about 1 mg/mL) with an equal volume of fresh human serum collected from healthy volunteers and incubated in a 37°C water bath for 1, 2, or 3 weeks. Afterwards, 1 pg/ml biotinylated human IL12p70 (Sino Bio, Cat#: CT011-H08H-B) was preincubated with serially diluted anti-IL12p35 antibodies at room temperature (RT) for 30 minutes. Next, 100 pl of the pre-incubated solution was added to the indicated well and incubated at RT for 2 hours. Next, 100 pl HRP- conjugated streptavidin was added to each well and incubated for 30 minutes. Next, 100 pl of HPR substrate was added to each and incubated at RT for 3 minutes. Next, 100 pl stop solution was added to each well. OD450 was then measured using a Varioskan™ Lux plate reader (Thermo Scientific).
As shown in FIGS. 12C-12D, 27H28L-hblb-YTE and 4A4-hblb2-YTE incubated with human serum for up to 3 weeks exhibited similar activities compared to the control sample (“0 week”), indicating their serum stability.
Example 11: Anti-IL12p35 antibodies 27H28L and D2M008-4A4 did not compete with each other in binding to IL12
The binning competition assays were carried out using the Gator® Prime BLI (Biolayer Interferometry) System (Gator Bio, USA), for anti-IL12p35 antibodies selected from FIG. 1, in a matrix format. Specifically, individual anti-IL12p35 antibodies were immobilized to anti-human Fc probes, respectively (Anti-hlgG Fc, Gator Bio, Cat#: 160003). Subsequently, the probes were incubated with the monomeric antigen human IL12p70-His (Sino Bio, Cat#: CT011-H08H) in K buffer (Gator Bio, Cat#: 120011) at 25°C for 3-4 minutes. The probes were then incubated with individual anti-IL12p35 antibodies in K buffer for 3-4 minutes at 25°C. The binding kinetics was analyzed using software provided by the manufacturer.
Whether anti-IL12p35 antibodies 27H28L and D2M008-4A4 compete or not was further verified. Specifically, anti-IL12p35 antibodies 27H28L and D2M008-4A4 were individually immobilized on probes by binding to anti-human Fc probes (Anti-hlgGFc, Gator Bio, Cat#: 160003). Subsequently, the probes were incubated with the monomeric antigen human IL12p70- His (Sino Bio, Cat#: CT011-H08H) in K buffer (Gator Bio, Cat#: 120011) at 25°C for 3-4 minutes. The probes were then incubated with 27H28L or D2M008-4A4 in K buffer for 3-4 minutes at 25°C. In a separate experiment, human IL12p70-His was immobilized by binding to anti -His probes (Gator Bio, Cat#: 160009). The probes were then incubated with 27H28L and D2M008-4A4, respectively, for 3-4 minutes, followed by an additional incubation with 27H28L or D2M008-4A4 for 3-4 minutes. The binding kinetics was analyzed using software provided by the manufacturer.
As shown in FIG. 13A, there were two big bins, one composed of antibodies competed with almost every anti-IL12p35 antibodies except the antibodies D2M008-4A4, D2M008-A1E7, D2M008-A1C6, D2M008-B2B1 and D2M008-B2C5; the other one composed of antibodies competed with almost every anti-IL12p35 antibodies except the antibodies D2M008-A1B4 and D2M008-A1E10. Both of the two big bins overlapped with D2M008-A1A8 and D2M008-A1A9, D2M008-B2B7, D2M008-A1E5, D2M008-B2C3, D2M008-B2B10, D2M008-A1A2, D2M008- A1G9 and D2M008-B2B12. Therefore, all anti-IL12p35 could be further placed into three smaller bins: the first bin including D2M008-4A4, A1E7, D2M008-A1C6, D2M008-B2B1 and D2M008-B2C5; the second bin including D2M008-A1A8, D2M008-A1A9, D2M008-B2B7, D2M008-A1E5, D2M008-B2C3, D2M008-B2B10, D2M008-A1A2, D2M008-A1G9 and D2M008-B2B12, and the third bin including D2M008-A1B4 and D2M008-A1E10. D2M008- A1 A8 and D2M008-A1 A9 were derived from the same IGHV and IGKV alleles. D2M008- A1B4 and D2M008-A1E10 were derived from the same IGHV and IGKV alleles.
As shown in FIGS. 13B-13C, the results from two separate experiments obtained by either initially immobilizing an anti-IL12 antibody with an anti-human Fc probe first, adding the antigen IL12 next, and then allowing the other antibody binding to IL12 (FIG. 13B); or initially immobilizing the antigen IL12 with an anti -His probe first, and allowing 27H28L and D2M008- 4A4 sequentially binding to IL12 next (FIG. 13C), revealed that 27H28L and D2M008-4A4 exhibited no competition for binding to IL 12, and there was competition observed between 27H28L or D2M008-4A4 with anti-hP40. In summary, 27H28L and D2M008-4A4 represent two top performers within their corresponding functional series with entirely distinct binding epitopes on IL 12, according to the results described above.
Example 12. Human genetics analysis
Human immune system is complex. Different autoimmune diseases share similar or distinct mechanisms. Animal models contribute to our understanding of some of the autoimmune diseases; however, they have limitations and cannot fully replicate the human immune system complexity. Many of the human autoimmune diseases are lacking suitable animal models. In this situation, relying on human data, especially human genetics associations on immune diseases may bring more relevant insights.
Here, we implemented a thorough human genetics analysis of IL12/IL23/IL35 pathway components, via integrating data of genome- wide association studies (GWAS) of a broad set of autoimmune diseases, GWAS of gene expression (eQTL: expression quantitative loci) and GWAS of protein expressions (pQTL: protein quantitative loci), using the methodology of colocalization test and comparison of the genetic effect directions in eQTL/pQTL vs. disease risks.
Our broad and deep genetics analysis demonstrated that IL12A (p35) and IL12B (p40), though are heterodimers of the same IL 12 cytokine, are genetically differentially associated with different human diseases. IL12B (p40) and IL23R are specifically causal to immune diseases of psoriasis (PsO), Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel diseases (IBD), and ankylosing spondylitis (AS) but not to other autoimmune diseases. On the other hand, IL12A (p35) and IL12R02 are specifically causal to autoimmune diseases of systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic erythematosus lupus (SLE), and Sjogren's syndrome (SjS), with no evidence of associations to PsO, CD, UC, IBD, and AS.
Based on our deep genetics analysis across a broad set of autoimmune diseases, it was concluded that targeting IL12p35/ IL12R02 would be specifically beneficial to patients with SSc, PBC, SLE, and SjS. Following this insight, we discovered and developed specific anti-IL12Ap35 antibodies for the treatment of this cluster of immune diseases of SSc, PBC, SLE, and SjS, as discussed in previous Examples.
Example 12.1: Methods (1) Data collection
To make full use of immune disease and human genetics results, detailed associations from each genetic variant are needed. The full disease GW AS data were collected from GWAS catalog:
Table 1.
The genetics determinant on the molecular phenotypes of IL12/IL23/IL35 pathway ligands and receptors, such as the GWAS of their gene expression (eQTL) in blood cells and GWAS of their protein levels (pQTL) in serum were collected from different resources. The data resources are as below: Table 2.
*eQTL: expression quantitative trait loci; #pQTL: protein quantitative trait loci
(2) Data cleaning and harmonization
To ensure all GWAS datasets were coded in a consistent manner of genomic locations, variant name, and effect allele types, data preprocessing was a necessary step. The preprocessing of datasets was conducted in the following manner:
First, datasets aligned to the human genome build hg38 were converted to hg37 using the UCSC Genome Browser's LiftOver tool: (genome.ucsc.edu/cgi- bin/hgLiftOver?hgsid=1770184142_UzmzHb9sIFwTgvO2zwt0sLLOTsM7).
In cases where datasets only included effect alleles, the corresponding other alleles were sourced from dbSNP. Furthermore, for datasets lacking effect allele frequency (EAF) data, we utilized the minor allele frequency (MAF) details from dbSNP to infer the necessary EAF information. At last, the datasets were harmonized using the IEU GWAS catalog harmonization pipeline (https : // www. ebi . ac. uk/g was/ docs/ methods/ summary-statistics) .
(3) Genetic association regional plot
To effectively visualize the association results centered on a specific gene region, regional plots were generated using LocusZoom vl.4, which can be accessed at github.com/statgen/locuszoom-standalone. For these plots, set build = “hgl9”, pop = “EUR”, and source = “1000G_Nov2014”.
(4) Disease loci, gene expression eQTL and protein expression pQTL colocalization Colocalization is a statistical method of determining whether two distinct traits (such as the expression level of a gene and a disease risk) share a common genetic cause. The process typically involves a Bayesian statistical method to estimate the probabilities of shared and distinct causal variants in overlapping genomic regions. To further evaluate the causal relationship between the selected genes and diseases of interest, colocalization analyses were conducted using the coloc R package v5.2.2 described at chrlswallace.github.io/coloc/articles/a01_intro.html.
We first calculated the center of the gene by averaging the start and stop positions, then took all SNPs that fall within 500 kb flanking the gene center for colocalization analysis of the gene. SNPs with MAF = 0 were removed. Assuming each trait had at most one causal variant in the region of consideration, the function “coloc.abf(..)” was used to perform the colocalization analysis.
(5) Comparing the genetics effect on gene/protein expression vs. disease risk
To explore the impact of gene expression or protein concentration change on disease outcomes, beta-beta plots were created. A beta value represents the effect size of a genetic variant (usually as a Single Nucleotide Polymorphism, SNP) on an outcome, such as gene expression level, protein level, or disease risk. In a beta-beta scatter plot, the X-axis represents the genetic effect of a Quantitative Trait Locus (QTL) for a gene or protein, while the Y-axis shows the genetic effect for a disease outcome. Each dot on the plot corresponds to a genetic variant. Only genetic variants that are common to both the gene/protein and the disease outcome are applicable for inclusion in a beta-beta plot. In our analysis, we restricted genetic variants that were significant associated with gene/protein levels (e.g. eQTL/pQTL) to reduce the noise of the data representation.
Example 12.2: Results
(1) IL12A (IL12p35) and its receptor IL12R02 are associated with SSc, PBC, SLE, and SjS, but not with PsO, CD, UC, IBD, or AS
As shown in FIGS. 15A-15C, IL12A eQTL analysis revealed SNP rs4680536, located near the IL12A gene, as the top variant associated with IL12A gene expression levels (plots of the 4th row). The genetic markers in this region also showed significant associations with SSc, SLE, and PBC (with p value < 5 x 10'8), where the top associated markers were highly correlated with IL12A eQTL top variant (rs4680536). Although in this dataset, no genetic variants reached GWAS significance for SjS, rs485497 (p value =1.17 x 1 O'10) in IL12A region was reported to be associated with SjS in a more recent publication (Lessard, Christopher J., et al. "Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjogren's syndrome." Nature Genetics 45.11 (2013): 1284-1292). In addition, rs4680536 and rs485497 were correlated (R2= 0.27 in 1000 genome European population). The pattern of significance of association between genotype and IL12A gene expression levels matched closely with that of association with SSc, PBC, SLE, and SjS risk. The posterior probabilities that IL12’s eQTLs colocalize with genetic markers associated with SSc, PBC, SLE and SjS were 0.948, 0.975, 0.985, and 0.744, respectively. The results strongly indicate that IL12A gene expression levels and the SSc, PBC, SLE and SjS risk share a common causal genomic variant. To the contrary, no genetic variant in this IL12A region was significantly (with p value < 5 x 10'8) associated with PsO, CD, UC, IBD, or AS, either in the data shown here or in other published GWAS studies.
As shown in FIGS. 16A-16C, IL12RP2 eQTL analysis revealed SNP rsl7129778, located near the IL12RP2 gene, associated with IL12RP2 gene expression levels (plots of the 4th row). The genetic markers in this region also showed significant associations with SSc, PBC, and SLE (with p value < 5 x 10'8), where the top associated markers were highly correlated with IL12RB2 eQTL top variant (rsl 7129778). The pattern of significance of association between genotype and IL12RP2 gene expression levels matched closely with that of association with SSc, PBC, SLE, and SjS risk. The results indicate that IL12Rp2’s gene expression levels and the SSc, PBC, SLE risk share a common causal genomic variant. To the contrary, different sets of genetic variants not correlated with IL12Rp2’s eQTLs in this genetic region were significantly (with p value < 5 x 10'8) associated with PsO, CD, UC, IBD, or AS. IL23R is a nearby gene upstream of IL12RP2. As shown later in FIGS. 18A-18C, the set of variants associated with PsO, CD, UC, IBD, or AS were actually correlated with those affecting IL23R’s protein expression (pQTLs).
(2) IL12B (IL12p40) and IL23R are associated with PsO, CD, UC, IBD, and AS, but not with SSc, PBC, SLE, or SjS
As shown in FIGS. 17A-17C, IL12B pQTL analysis revealed SNP rs6556416, located near the IL12B gene, associated with IL12B serum protein levels (plots of the 4th row). The genetic markers in this region also showed significant associations with PsO, CD, UC, IBD, and AS (with p value < 5 x 10'8), where the top associated markers were highly correlated with IL12B pQTL top variant (rs6556416). The pattern of significance of association between genotype and IL12B protein levels matched closely with that of association with PsO, CD, UC, IBD, and AS risk. The results indicate that IL12B’s protein levels and the PsO, CD, UC, IBD, and AS risk share a common causal genomic variant. To the contrary, no genetic variants in this IL12B genetic region were significantly (with p value < 5 x 1 O'8) associated with SSc, PBC, SLE, or SjS either in the data shown here or in other published GWAS studies.
As shown in FIG. 18A-18C, IL23R pQTL analysis revealed SNP rsl 1581607, located near the IL23R gene, associated with IL23R serum protein levels (plots of the 4th row). The genetic markers in this region also showed significant associations with PsO, CD, UC, IBD, and AS (with p value < 5 x 1 O'8), where the top associated markers were highly correlated with IL23R pQTL top variant (rsl 1581607). The pattern of significance of association between genotype and IL23R protein levels matched closely with that of association with PsO, CD, UC, IBD, and AS risk. The results indicate that IL23R’s protein levels and the PsO, CD, UC, IBD, and AS risk share a common causal genomic variant. To the contrary, no genetic variants in this IL23R genetic region were significantly (with p value < 5 x 1 O'8) associated with SjS either in the data shown here or in other published GWAS studies. In addition, different sets of genetic variants not correlated with IL23R’s pQTLs in this genetic region were significantly (with p value < 5 x 1 O'8) associated with SSc, PBC, and SLE. IL12RP2 is a nearby gene downstream of IL23R. As shown earlier in FIGS. 16A-16C, the set of variants associated with SSc, PBC, and SLE were actually correlated with those affecting IL12Rp2’s gene expression (eQTLs).
(3) EBI3 and IL6ST are not associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS.
As shown in FIGS. 19A-19C, EBB pQTL analysis revealed SNP rs60160662, located near the EBB gene, associated with EBB serum protein levels (plots of the 4th row). However, no genetic variants in this EBB genetic region were significantly (with p value < 5 x 1 O'8) associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS, either in the data shown here or in other published GWAS studies. As the functional genetic variants regulating EBB’s protein level exist in this region but have no impact on the disease risks, EBB’s protein level is unlikely a causing effect to these diseases.
As shown in FIGS. 20A-20C, IL6ST pQTL analysis revealed SNP rsl 1574765, located near the IL6ST gene, associated with IL6ST serum protein levels (plots of the 4th row). However, no genetic variants in this IL6ST genetic region were significantly (with p value < 5 x 1 O'8) associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS, either in the data shown here or in other published GWAS studies. As the functional genetic variants regulating IL6ST’s protein level exist in this region but have no impact on the disease risks, IL6ST’s protein level is unlikely a causing effect to these diseases.
(4) STAT4 is associated with SSc, PBC, SLE, SjS, IBD, CD, and UC; whereas STAT3 is associated with CD, UC, and IBD
As shown in FIGS. 21A-21C, STAT4 eQTL analysis revealed SNP rsl 6833249, located near the STAT4 gene, associated with STAT4 gene expression levels (plots of the 4th row). The genetic markers in this region also showed significant associations with SSc, PBC, SLE, SjS, IBD, CD, and UC (with p value < 5 x 1 O'8), where the significant associated markers were correlated with STAT4 eQTL top variant (rsl6833249). The pattern of significance of association between genotype and STAT4 gene expression levels matched closely with that of association with SSc, PBC, SLE, SjS, IBD, CD, UC risk. The results indicate that STAT4’s gene expression levels and the SSc, PBC, SLE, SjS, IBD, CD, UC risk share a common causal genomic variant. To the contrary, no genetic variants in this STAT4 genetic region were significantly (with p value < 5 x 1 O'8) associated with PsO or AS, either in the data shown here or in other published GWAS studies.
As shown in FIGS. 22A-22C, STAT3 eQTL analysis revealed SNP rsl 053004, located near the STAT3 gene, associated with STAT3 gene expression levels (plots of the 4th row). The genetic markers in this region also showed significant associations with IBD, CD, UC (with p value < 5 x 1 O'8), where the significant associated markers were correlated with STAT3 eQTL top variant (rsl 053004). The pattern of significance of association between genotype and STAT3 gene expression levels matched closely with that of association with IBD, CD, UC risk. The results indicate that STAT3’s gene expression levels and the IBD, CD, UC risk share a common causal genomic variant. To the contrary, no genetic variants in this STAT3 genetic region were significantly (with p value < 5 x 10'8) associated with SSc, PBC, SLE, SjS, PsO or AS, either in the data shown here or in other published GWAS studies.
(5) Genetically determined higher IL12A and IL12RP2 expression is associated with higher SSc, PBC, SLE, SjS disease risk Based on the genetic variants that are significantly (p value < 5 x 10'8) associated with IL12A’s expression, a positive regression line fits the scatter plots comparing the genetic effects on IL12A expression vs. genetic effects on SSc, PBC, SjS, and SLE disease risk (FIG. 23). This indicates the increased IL12A expression through genetic perturbation positively associated with the increased disease risk of SSc, PBC, SLE, and SjS.
Based on the genetic variants that are significantly (p value < 5 x 1 O'8) associated with IL12Rp2’s expression, a positive regression line fits the scatter plots comparing the genetic effects on IL12RP2 expression vs. genetic effects on SSc, PBC, SjS, and SLE disease risk (FIG. 24). This indicates the increased IL12RP2 expression through genetic perturbation positively associated with the increased disease risk of SSc, PBC, SLE, and SjS.
Example 12.3: Conclusion
IL12A-p35/IL12Rp2 were specifically associated with immune diseases of SSc, PBC, SLE, and SjS. IL12B, although together with IL12A forms IL12, showed a differential immune disease association pattern. IL12B was associated with PsO, CD, UC, IBD and AS, but not with SSc, PBC, SLE, and SjS. IL12B-p40 is also a component of IL23. The disease association pattern for IL12B was the same as that of IL23R. Pharmacologically targeting IL12B or IL23A- pl9 have been successful in IBD and PsO, but failed in PBC and SLE, which is consistent to the genetic association pattern for IL12B and IL23R. These genetic association profiles reflected that pharmacologically targeting IL12B would have effects similarly to targeting the IL23 pathway, but could be different from targeting IL 12 A.
In addition, STAT4 is the major downstream signaling molecule of the IL12 cytokine pathway. STAT4 was associated with all diseases SSc, PBC, SLE, and SjS that IL12A- p35/IL12Rp2 were associated with. STAT3 is one downstream signaling molecule of the IL23 cytokine pathway, and STAT3 was only associated with IBD, UC, CD diseases. The differential immune disease associations from STAT4 and STAT3 also supported the different indications of targeting IL 12 vs. IL23.
At the same time, IL12A-p35/IL12Rp2 is also a component of IL35 cytokine and receptor. One explanation for IL12A and IL12B’s differential genetic association could be explained by the differential effect of IL35 and IL12 cytokines. However, although genetic variants significantly affect the protein levels of EBI3/IL6ST, the other components of IL35 and its receptor, they showed no association to any of the above evaluated autoimmune diseases. Thus, it is unlikely that IL12A-p35/IL12Rp2 affect the immune diseases through the immune regulatory effect of IL35.
Furthermore, a positive correlation between genetic effects on IL12A and IL12Rp2’s expression and the SSc, PBC, SLE, and Sj S ’ disease risk was identified. As higher expression of IL12A/IL12RP2 was associated with a higher disease risk, we propose that developing IL12A- p35/ IL12RP2 antagonist would be specifically beneficial to patients with SSc, PBC, SLE, and/or SjS.
Example 13. 27H28L-hblb-YTE and 4A4-hblb2-YTE exhibited excellent pharmacokinetic properties in human FcRn transgenic mice
To investigate pharmacokinetic properties of anti-IL12p35 antibodies in human, hFcRN transgenic C57BL/6 mice (Shanghai Model Organisms Center, Inc.) were intravenously (/. v.) dosed with anti-IL12p35 antibodies 4A4-hblb2-YTE or 27H28L-hblb-YTE at 1 mg/kg. The pharmacokinetic (PK) blood samples were collected 2 hours (“hr”), 6 hours, 24 hours, 48 hours, 72 hours, 7 days (“D”), 10 days, 14 days, 17 days, 21 days, 24 days, and 28 days post dosing. Serum concentration of anti-IL12p35 antibodies was analyzed by ELISA. For example, human IL-12 was used to capture the anti-IL12p35 antibodies in serum and an anti-human Fc antibody was used to detect the human IL-12-bound anti-IL12p35 antibodies.
The serum concentrations 4A4-hblb2-YTE and 27H28L-hblb-YTE at corresponding time points are shown in FIG. 29. The pharmacokinetic parameters of 27H28L-hblb-YTE and 4A4- hblb2-YTE are shown in FIG. 30. As shown in FIG. 30, the anti-IL12p35 antibodies with TYE and back-to-germline mutations demonstrated a good in vivo stability and exhibited excellent PK properties in human FcRn transgenic mice. Specifically, 4A4-hblb2-YTE and 27H28L-hblb- YTE exhibited a T1/2 of 375 hours and 500 hours, respectively, after the single dose at 1 mg/kg in human FcRn transgenic mice.
Example 14. Anti-IL12p35 antibodies but not anti-IL12p40 antibodies reduced DNFB- induced skin inflammation
To investigate the in vivo effects of anti-IL12p35 antibodies on inflammation diseases, a 1 -fluoro, 2,4-dinitrobenzene (DNFB)-induced chronic contact hypersensitivity mouse model was established and treated with an anti-mouse IL12p35 surrogate antibody. Specifically, twenty-four 8-week-old CB57BL/6 mice were sensitized with 50 pL of 0.5% DNFB (%v/v) on the shaved dorsal right skin on Day 0. DNFB was freshly dissolved in acetone and olive oil (4: 1, v/v) prior to the application. Then all animals received 10 pL of 0.3% DNFB (%v/v) on the dorsum of both ears on Days 5-7. Subsequently, 10 pL of 0.1% DNFB was applied to the dorsum of both ears on Day 8, Day 9, Day 10, Day 11 and Day 12. On Day 0, mice were randomly placed into three groups (8 mice per group), and intraperitoneally (zip.) dosed with 100 pL PBS (control), 10 mg/kg of a rat anti-mouse IL12p35 antibody (anti-mP35) (BioXcell, Cat#: BE0371), or 10 mg/kg of a rat anti-mouse IL12p40 antibody (anti-mP40) (BioXcell, Cat#: BE0051) on Day 0, Day 4, Day 7 and Day 11, respectively. Ear thickness was determined using a digital portable thickness gage from Day 5 to Day 12 before the DNFB application. Each ear was measured three times to determine the average thickness of each data point.
The experimental scheme is shown in FIG. 31A. As shown in FIG. 31B, the average ear thickness increased in all DNFB-induced skin inflammation groups. Anti-mouse IL12p35 antibody treatment significantly reduced the ear thickness compared to that of the control group (treated with PBS), whereas anti-mouse IL12p40 antibody treatment did not significantly affect the ear thickness as compared to the control group.
Example 15. Anti-IL12p35 antibodies alleviated Sjogren's syndrome in a mouse model
Sjogren’s Syndrome (SjS) is an autoimmune disease mediated by lymphocytic infiltration into exocrine glands, resulting in progressive lacrimal and salivary destruction and dysfunctional glandular secretion. To investigate the in vivo effects of anti-IL12-p35 antibodies on Sjogren's syndrome, an anti-mouse IL12p35 antibody was used to treat NOD mice (NOD/MrkTac, Taconic US). In NOD/ShiLtJ mice, type 1 diabetes accelerates the progression of the Sjogren’s syndrome. Briefly, 8-week-old NOD/ShiLtJ mice were fasted for 24 hours and then intraperitoneally (zip.) dosed with 3.6 mg streptozotocin (STZ; dissolved in 0.1 M sodium citrate, pH 4.5). On Day 0, mice were randomly placed into two groups and treated with 10 mg/kg of a rat IgG2a isotype antibody (BioXcell, Cat#: BE0089) or a rat anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371), respectively on Day 0, Day 3, Day 6 and Day 9. On Day 0, Day 3, Day 7, and Day 10, mice were anesthetized using an isoflurane machine. A pre- weighted absorbent material was inserted into the mouth of mice. Each mouse received 100 pg pilocarpine intraperitoneally (i.p.). 12 minutes later, the absorbent material was removed and weighted to determine saliva release.
The experimental scheme is shown in FIG. 32A. As shown in FIG. 32B, the release of saliva decreased significantly in response to the pilocarpine stimulation during the development of Sjogren’s syndrome induced by STZ in NOD/ShiLtJ mice. The anti-mouse IL12-p35 antibody treatment significantly alleviated the decrease of saliva excretion in the mice with Sjogren’s syndrome.
Example 16. Anti-IL12p35 antibodies significantly inhibited weight loss, and reduced urine albumin levels and kidney pathology in a IMQ-induced SLE model
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease. NZBWF1/J mice serve as a classic model for SLE, as they spontaneously develop immune responses similar to human SLE. Studies have shown that mutations and activation of toll-like receptor (TLR) 7 are implicated in the onset of various autoimmune diseases, including SLE. To investigate the therapeutic effect of anti-IL12p35 antibodies on mouse SLE, the model of NZBWF1/J (Jackson Laboratory, Cat#100008) mice induced by a TLR7 agonist of imiquimod (IMQ) was employed. Specifically, NZBWF1/J female mice were received at 18- week-old and acclimated for one week. On Day 0, all mice topically received 2.5 mg of 2.5% IMQ cream per 25 g mouse weight on the right inner ear. IMQ was applied twice per week for a total of 5 doses. The IMQ cream was prepared by mixing 100 mg (~ 100 pL) Vanish cream with 5 mg IMQ with extra 100 pL pure H2O. On Day 0/Week 0, mice were randomly placed into two groups according to body weight, treated with 10 mg/kg of a rat IgG2a isotype antibody (BioXcell, Cat#: BE0089; as the control group) or an anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371, as the treatment group), respectively. All mice were dosed twice per week until the experimental endpoint.
In Week 0, 1, 2, 3, 4 and 5, urine samples were collected to measure urine albumin and creatinine levels using an in-house developed ELISA method and a commercial creatinine assay kit, respectively. The ratio of albumin/creatinine was calculated by dividing the albumin concentration with the creatinine concentration. To measure the urine albumin concentrations, 100 pL anti-mouse albumin (2 pg/mL, Exalpha, Cat#: 3 RAM/Alb/7S) were coated in a 96- well assay plate overnight. The plate was then washed and blocked with 300 pL of a blocking buffer (containing 1% BSA) for 1 hour at room temperature. The plate was then washed and 100 pL diluted urine samples and standards (Sigma, Cat#: 126674-25MG) were added into wells and incubated for 2 hours at room temperature. After the incubation, the plate was washed and 100 pL anti-mouse albumin Polyclonal Antibody-HRP (1:25000 dilution with 1% BSA, Bethyl Laboratories, Cat#: A90-134P) was added and incubated for 1 hour at room temperature. The plate was then washed and developed with TMB to determine the concentrations of albumin based on the standard. The urine creatinine concentrations were measured using a creatinine assay kit (Bioassay Systems, Cat#: DICT500) according to the manufacture’s instructions. In Week 5, the mice were euthanized. Their kidneys were collected. The left ones were frozen while the right ones were fixed with 10% formalin and sent to iHisto (Salem, MA) for histology analysis.
As shown in FIGS. 33A-33C, anti-IL12p35 antibody treatment significantly inhibited weight loss and reduced urine albumin levels. The most notable difference in urine albumin levels between the treatment and control groups occurred at the Week 3 time point. After three weeks, likely due to a decline in the effects of IMQ, SLE symptoms began to diminish in some mice, resulting in a decrease in the albumin/creatinine ratio for such mice. As shown in FIG. 33C with representative mouse kidney histology images from each group (both had a high level of urine albumin), there was evidence of multifocal glomerulonephritis and multifocal glomerular atrophy (as indicated by black arrows), with partial replacement by fibrous connective tissue (as indicated by a black diamond), multifocal renal tubular distention and hyaline casts (as indicated by a hollow star), significant interstitial infiltration (as indicated by a black triangle) by a mixed cell population in the isotype treatment group. In contrast, the anti- IL12p35 antibody treatment group displayed mostly intact glomerular structures, with only a few instances of mild glomerulonephritis (as indicated by a black arrow); and no fibrosis or interstitial infiltration was observed. These results indicated that topical application of IMQ accelerated the development of SLE in young NZBWF1/J mice, enabling a rapid evaluation of the effects of anti-IL12p35 antibody treatment on SLE. The anti-IL12p35 antibody treatment significantly inhibited weight loss, reduced urine albumin levels, and markedly reduced kidney pathology associated with SLE. Example 17. Anti-IL12p35 antibodies significantly reduced kidney pathology in a spontaneous SLE model
To further investigate the therapeutic effect of anti-mouse IL12-p35 antibody on mouse SLE, a spontaneously developed SLE model using NZBWF1/J mice (Jackson Laboratory, Cat#100008) was established. Specifically, NZBWF1/J female mice were received at 18-week- old and acclimated for one week. All mice were randomly placed into two groups in Week 0 according to body weight, treated with 15 mg/kg of a rat IgG2a isotype antibody (BioXcell, Cat#: BE0089; as the control group) or an anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371; as the treatment group) intraperitoneally (z. >.) every week until the experimental endpoint. In Week 0, 2, 4, 6, 8, 10, 12, 14 and 15, urine samples were collected to measure albumin and creatinine levels using an in-house developed ELISA method and a creatinine assay kit, respectively, as described in Example 16. The ratio of albumin/creatinine was calculated by dividing the albumin concentration with the creatinine concentration. On the second day of Week 15, the mice were euthanized. Their kidneys were collected. The left ones were frozen while the right ones were fixed with 10% formalin and sent for histology in iHisto (Salem, MA). The pathological analysis based on histology was performed in-house and by independent histopathologists from Alliance Bio (Hangzhou, China).
As shown in FIG. 34A, anti-IL12p35 antibody treatment significantly reduced SLE kidney symptoms. For example, the urine albumin level showed a significant decrease 14 weeks post treatment when the NZBWF1/J mice spontaneously developed SLE. It is important to note that the onset time of SLE symptoms varied significantly among the mice. Notably, some mice in the control group did not exhibit an increase in the albumin/creatinine ratio throughout the experiment, whereas some mice in both treated and control groups had very early disease onset. Additionally, the development of anti-drug antibodies (mouse anti-rat) led to poor pharmacokinetic (PK) exposure of the anti-IL12p35 antibody, compromising its therapeutic efficacy. This was particularly evident in mice with an elevated albumin/creatinine ratio, which also correlated with poor PK.
As shown in the images in FIG. 34B, kidney histology analysis demonstrated that kidney tissues primarily exhibited end-stage lesions in the isotype control group (panels a-d; #42): 1) 5 out of 7 samples showed glomerular sclerosis (as indicated by black arrows), and some accompanied by fibrous crescent formation (as indicated by white arrows); 2) tubular degeneration or atrophy was observed (as indicated by black triangles), along with protein casts in the tubules (as indicated by a black diamond); and 3) interstitial inflammation of varying degrees was observed, with some samples showing interstitial fibrosis. However, in the antimouse IL12p35 antibody treatment group (panels e-h; #51), pathological changes in kidney tissues mainly reflected early-stage damages: 1) glomerular mesangial proliferation accompanied by inflammatory cell infiltration was common; 2) 3 out of 7 samples displayed mild to moderate- to-severe glomerular sclerosis, but no fibrous crescent formation was observed; 3) tubular degeneration ranged from moderate to moderate-to-severe, with 3 out of 7 samples showing protein casts; and 4) inflammatory cell infiltration in the interstitial area was noted, with one sample showing mild interstitial fibrosis. Overall, in the treatment group, the WTS (whole-tissue score) was significantly lower compared to the isotype control group, while the AS (activity score) increased, and the CS (chronicity score) decreased. Detailed scores and a summary of the pathology scores evaluated by independent histopathologists are shown in FIG. 34C.
In summary, despite the development of anti-drug antibody compromised its therapeutic efficacy, anti-IL12p35 antibody treatment reduced urine albumin levels, markedly reduced kidney pathology associated with SLE, and slowed down disease progression in the spontaneous SLE model.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. An antibody or antigen-binding fragment thereof that binds to interleukin 12 subunit alpha
(IL12p35), comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are selected from VH CDRS 1, 2, 3 and VL CDRS 1, 2, 3 listed in FIG. 26 or FIG. 27.
2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, 49, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 50, 51, 52, respectively;
(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 53, 54, 55, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 56, 57, 58, respectively;
(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, 61, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 62, 63, 64, respectively; (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 65, 66, 67, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 68, 69, 70, respectively;
(5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 71, 72, 73, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 74, 75, 76, respectively;
(6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 77, 78, 79, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 80, 81, 82, respectively;
(7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 83, 84, 85, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 86, 87, 88, respectively;
(8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 89, 90, 91, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 92, 93, 94, respectively;
(9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 95, 96, 97, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 98, 99, 100, respectively;
(10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 101, 102, 103, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 104, 105, 106, respectively;
(11) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 107, 108, 109, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 110, 111, 112, respectively;
(12) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 113, 114, 115, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 116, 117, 118, respectively;
(13) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 119, 120, 121, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 122, 123, 124, respectively; (14) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 125, 126, 127, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 128, 129, 130, respectively;
(15) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 131, 132, 133, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 134, 135, 136, respectively;
(16) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 137, 138, 139, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 140, 141, 142, respectively;
(17) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 143, 144, 145, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 146, 147, 148, respectively;
(18) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 149, 150, 151, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 152, 153, 154, respectively;
(19) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, 157, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, 160, respectively;
(20) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 161, 162, 163, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 164, 165, 166, respectively;
(21) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 167, 168, 169, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 170, 171, 172, respectively;
(22) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 173, 174, 175, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 176, 177, 178, respectively; and
(23) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 179, 180, 181, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 182, 183, 184, respectively.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein CDR is determined by Kabat definition.
4. The antibody or antigen-binding fragment thereof of claim 1 , wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 185, 186, 187, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 188, 189, 190, respectively;
(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 191, 192, 193, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 194, 195, 196, respectively;
(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 197, 198, 199, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 200, 201, 202, respectively;
(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 203, 204, 205, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 206, 207, 208, respectively;
(5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 209, 210, 211, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 212, 213, 214, respectively;
(6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 215, 216, 217, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 218, 219, 220, respectively;
(7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 221, 222, 223, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 224, 225, 226, respectively;
(8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 227, 228, 229, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 230, 231, 232, respectively; (9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 233, 234, 235, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 236, 237, 238, respectively;
(10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 239, 240, 241, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 242, 243, 244, respectively;
(11) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 245, 246, 247, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 248, 249, 250, respectively;
(12) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 251, 252, 253, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 254, 255, 256, respectively;
(13) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 257, 258, 259, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 260, 261, 262, respectively;
(14) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 263, 264, 265, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 266, 267, 268, respectively;
(15) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 269, 270, 271, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 272, 273, 274, respectively;
(16) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 275, 276, 277, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 278, 279, 280, respectively;
(17) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 281, 282, 283, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 284, 285, 286, respectively;
(18) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 287, 288, 289, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 290, 291, 292, respectively; (19) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 293,
294, 295, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 296, 297, 298, respectively;
(20) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 299, 300, 301, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 302, 303, 304, respectively;
(21) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 305, 306, 307, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 308, 309, 310, respectively;
(22) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 311, 312, 313, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 314, 315, 316, respectively; and
(23) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 317, 318, 319, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 320, 321, 322, respectively.
5. The antibody or antigen-binding fragment thereof of claim 1 or 4, wherein CDR is determined by IMGT definition.
6. The antibody or antigen-binding fragment thereof of claim 1 , wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, 157, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, 160, respectively, according to Kabat definition;
(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 179, 180, 181, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 182, 183, 184, respectively, according to Kabat definition;
(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 293, 294,
295, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 296, 297, 298, respectively, according to IMGT definition; and (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 317, 318, 319, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 320, 321, 322, respectively, according to Kabat definition.
7. An antibody or antigen-binding fragment thereof that binds to IL12p35 comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:
(1) the selected VH sequence is SEQ ID NO: 1, and the selected VL sequence is SEQ ID NO: 2;
(2) the selected VH sequence is SEQ ID NO: 3, and the selected VL sequence is SEQ ID NO: 4;
(3) the selected VH sequence is SEQ ID NO: 5, and the selected VL sequence is SEQ ID NO: 6;
(4) the selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8;
(5) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10;
(6) the selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12;
(7) the selected VH sequence is SEQ ID NO: 13, and the selected VL sequence is SEQ ID NO: 14;
(8) the selected VH sequence is SEQ ID NO: 15, and the selected VL sequence is SEQ ID NO: 16;
(9) the selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18;
(10) the selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20;
(11) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (12) the selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 24;
(13) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 26;
(14) the selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 28;
(15) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30;
(16) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32;
(17) the selected VH sequence is SEQ ID NO: 33, and the selected VL sequence is SEQ ID NO: 34;
(18) the selected VH sequence is SEQ ID NO: 35, and the selected VL sequence is SEQ ID NO: 36;
(19) the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38;
(20) the selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40;
(21) the selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42;
(22) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44;
(23) the selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46;
(24) the selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336; and
(25) the selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338, optionally wherein the VH and/or VL comprise one or more back-to-germline (B2G) mutations.
8. The antibody or antigen-binding fragment thereof of claim 7, wherein the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is 38; or wherein the selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336.
9. The antibody or antigen-binding fragment thereof of claim 7, wherein the selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; or wherein the selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338.
10. The antibody or antigen-binding fragment thereof of any one of claims 1 -9, wherein the antibody or antigen-binding fragment thereof can block the binding between interleukin 12 (e.g., human interleukin 12 (IL12)) and interleukin 12 receptor, beta 2 subunit (e.g., human interleukin 12 receptor, beta 2 subunit (IL12RP2)).
11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof can block IL12-induced intracellular signaling (e.g., JAK-STAT signaling), optionally the IL12 is human or monkey IL12.
12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof can inhibit IL12-induced IFN-y production in human PBMCs.
13. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof can prevent IFN-y production by CD4+ T cells cocultured with allogenic dendritic cells.
14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen-binding fragment specifically binds to human IL12p35 and/or monkey IL12p35.
15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.
16. The antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the antibody or antigen-binding fragment is a F(ab’)2 fragment, a single-chain variable fragment (scFV) or a multi-specific antibody (e.g., a bispecific antibody).
17. An antibody or antigen-binding fragment thereof that binds to IL12p35 comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, a first immunoglobulin light chain, and a second immunoglobulin light chain, wherein the first immunoglobulin heavy chain and the first immunoglobulin light chain associates with each other, forming a first antigen-binding site that binds to IL12p35, wherein the second immunoglobulin heavy chain and the second immunoglobulin light chain associates with each other, forming a first antigen-binding site that binds to IL12p35, wherein:
(1) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 325, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 326;
(2) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 327, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 326;
(3) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 328, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 329;
(4) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 330, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 329; (5) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 331, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 332; and
(6) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 333, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 324.
18. The antibody or antigen-binding fragment thereof of claim 17, wherein the first and second immunoglobulin heavy chains are identical, wherein the first and second immunoglobulin light chains are identical.
19. The antibody or antigen-binding fragment thereof of claim 17 or 18, wherein the first and second immunoglobulin heavy chains comprise an Fc region (e.g., an IgGl Fc region).
20. The antibody or antigen-binding fragment thereof of claim 19, wherein the Fc region comprises YTE mutations (M252Y/S254T/T256E according to EU numbering).
21. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are identical to VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the antibody or antigen-binding fragment thereof of any one of claims 1-20.
22. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-21.
23. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22 covalently bound to a therapeutic agent.
24. The antibody drug conjugate of claim 23, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
25. A method of inhibiting immune response in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigenbinding fragment thereof of any one of claims 1-22, or the antibody-drug conjugate of claims 23 or 24, to the subject.
26. The method of claim 25, wherein the subject has an immune disorder (e.g., an autoimmune disease or an inflammatory disease).
27. A method of treating a subject having an autoimmune disease, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22, or the antibody-drug conjugate of claims 23 or 24, to the subject.
28. The method of claim 27, wherein the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
29. The method of claim 27 or 28, wherein the subject does not have psoriasis (PsO), Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel diseases (IBD), or ankylosing spondylitis (AS).
30. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22, or the antibody-drug conjugate of claims 23 or 24, and a pharmaceutically acceptable carrier.
31. A method of reducing IL12-induced intracellular signaling in a cell, the method comprising
I l l contacting the cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22, or the antibody-drug conjugate of claims 23 or 24, to the subject.
32. A method of identifying a subject as having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject, using the antibody or antigen-binding fragment thereof of any one of claims 1-22.
33. A method of determining the risk of a subject having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject, using the antibody or antigen-binding fragment thereof of any one of claims 1-22.
34. A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising VH CDR 1, 2, 3 set forth in FIG. 26 or FIG. 27, and wherein the VH, when paired with a corresponding light chain variable region (VL) binds to IL12p35; or
(2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1 , 2, and 3 comprising VL CDR 1, 2, 3 set forth in FIG. 26 or FIG. 27, when paired with a corresponding VH binds to IL12p35.
35. The nucleic acid of claim 31, wherein the polypeptide comprises:
(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively (or SEQ ID NOs: 185, 186, and 187, respectively), and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 2 binds to IL12p35;
(2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively (or SEQ ID NOs: 188, 189, and 190, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 binds to IL12p35;
(3) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 53, 54, and 55, respectively (or SEQ ID NOs: 191, 192, and 193, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 4 binds to IL12p35;
(4) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 57, and 58, respectively (or SEQ ID NOs: 194, 195, and 196, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 3 binds to IL12p35;
(5) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively (or SEQ ID NOs: 197, 198, and 199, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 6 binds to IL12p35;
(6) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively (or SEQ ID NOs: 200, 201, and 202, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 5 binds to IL12p35;
(7) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively (or SEQ ID NOs: 203, 204, and 205, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO:
8 binds to IL12p35;
(8) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 68, 69, and 70, respectively (or SEQ ID NOs: 206, 207, and 208, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 binds to IL12p35;
(9) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 71, 72, and 73, respectively (or SEQ ID NOs: 209, 210, and 211, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO:
10 binds to IL12p35;
(10) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 74, 75, and 76, respectively (or SEQ ID NOs: 212, 213, and 214, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:
9 binds to IL12p35;
(11) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 77, 78, and 79, respectively (or SEQ ID NOs: 215, 216, and 217, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 12 binds to IL12p35;
(12) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 80, 81, and 82, respectively (or SEQ ID NOs: 218, 219, and 220, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:
11 binds to IL12p35;
(13) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 83, 84, and 85, respectively (or SEQ ID NOs: 221, 222, and 223, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 14 binds to IL12p35;
(14) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 86, 87, and 88, respectively (or SEQ ID NOs: 224, 225, and 226, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 13 binds to IL12p35;
(15) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 89, 90, and 91, respectively (or SEQ ID NOs: 227, 228, and 229, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 binds to IL12p35;
(16) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 92, 93, and 94, respectively (or SEQ ID NOs: 230, 231, and 232, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 binds to IL12p35;
(17) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 95, 96, and 97, respectively (or SEQ ID NOs: 233, 234, and 235, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 18 binds to IL12p35;
(18) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 98, 99, and 100, respectively (or SEQ ID NOs: 236, 237, and 238, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 binds to IL12p35;
(19) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 101, 102, and 103, respectively (or SEQ ID NOs: 239, 240, and 241, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 20 binds to IL12p35;
(20) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively (or SEQ ID NOs: 242, 243, and 244, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 19 binds to IL12p35;
(21) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 107, 108, and 109, respectively (or SEQ ID NOs: 245, 246, and 247, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 22 binds to IL12p35;
(22) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively (or SEQ ID NOs: 248, 249, and 250, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 21 binds to IL12p35;
(23) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 113, 114, and 115, respectively (or SEQ ID NOs: 251, 252, and 253, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 24 binds to IL12p35;
(24) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 116, 117, and 118, respectively (or SEQ ID NOs: 254, 255, and 256, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 23 binds to IL12p35;
(25) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 119, 120, 121, respectively (or SEQ ID NOs: 257, 258, and 259, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 26 binds to IL12p35;
(26) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 122, 123, and 124, respectively (or SEQ ID NOs: 260, 261, and 262, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 25 binds to IL12p35;
(27) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 125, 126, and 127, respectively (or SEQ ID NOs: 263, 264, and 265, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 28 binds to IL12p35;
(28) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 128, 129, and 130, respectively (or SEQ ID NOs: 266, 267, and 268, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 27 binds to IL12p35;
(29) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 131, 132, and 133, respectively (or SEQ ID NOs: 269, 270, and 271, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 30 binds to IL12p35;
(30) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 134, 135, and 136, respectively (or SEQ ID NOs: 272, 273, and 274, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 29 binds to IL12p35;
(31) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 137, 138, and 139, respectively (or SEQ ID NOs: 275, 276, and 277, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 32 binds to IL12p35;
(32) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 140, 141, and 142, respectively (or SEQ ID NOs: 278, 279, and 280, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 31 binds to IL12p35;
(33) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 143, 144, and 145, respectively (or SEQ ID NOs: 281, 282, and 283, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 34 binds to IL12p35;
(34) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 146, 147, and 148, respectively (or SEQ ID NOs: 284, 285, and 286, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 33 binds to IL12p35;
(35) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 149, 150, and 151, respectively (or SEQ ID NOs: 287, 288, and 289, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 36 binds to IL12p35;
(36) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 152, 153, and 154, respectively (or SEQ ID NOs: 290, 291, and 292, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 35 binds to IL12p35;
(37) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 155, 156, and 157, respectively (or SEQ ID NOs: 293, 294, and 295, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 38 or 336 binds to IL12p35;
(38) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 158, 159, and 160, respectively (or SEQ ID NOs: 296, 297, and 298, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 37 or 335 binds to IL12p35;
(39) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 161, 162, and 163, respectively (or SEQ ID NOs: 299, 300, and 301, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 40 binds to IL12p35;
(40) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 164, 165, and 166, respectively (or SEQ ID NOs: 302, 303, and 304, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 39 binds to IL12p35;
(41) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 167, 168, and 169, respectively (or SEQ ID NOs: 305, 306, and 307, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 42 binds to IL12p35;
(42) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively (or SEQ ID NOs: 308, 309, and 310, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 41 binds to IL12p35;
(43) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 173, 174, and 175, respectively (or SEQ ID NOs: 311, 312, and 313, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 44 binds to IL12p35;
(44) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 176, 177, and 178, respectively (or SEQ ID NOs: 314, 315, and 316, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 43 binds to IL12p35;
(45) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 179, 180, and 181, respectively (or SEQ ID NOs: 317, 318, and 319, respectively), and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 46 or 338 binds to IL12p35; or
(46) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 182, 183, and 184, respectively (or SEQ ID NOs: 320, 321, and 322, respectively), and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 45 or 337 binds to IL12p35.
36. The nucleic acid of claim 34 or 35, wherein the VH when paired with a VL specifically binds to human IL12p35; or the VL when paired with a VH specifically binds to human IL12p35.
37. The nucleic acid of any one of claims 34-36, wherein the immunoglobulin heavy chain or the fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.
38. The nucleic acid of any one of claims 34-37, wherein the nucleic acid encodes a F(ab’)2 fragment, a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody).
39. The nucleic acid of any one of claims 34-38, wherein the nucleic acid is cDNA.
40. A vector comprising one or more of the nucleic acids of any one of claims 34-39.
41. A vector comprising two of the nucleic acids of any one of claims 34-39, wherein the vector encodes the VL region and the VH region that together bind to IL12p35.
42. A pair of vectors, wherein each vector comprises one of the nucleic acids of any one of claims 34-39, wherein together the pair of vectors encodes the VL region and the VH region that together bind to IL12p35.
43. A cell comprising the vector of claim 40 or 41, or the pair of vectors of claim 42.
44. The cell of claim 43, wherein the cell is a CHO cell.
45. A cell comprising one or more of the nucleic acids of any one of claims 34-39.
46. A cell comprising two of the nucleic acids of any one of claims 34-39.
47. The cell of claim 46, wherein the two nucleic acids together encode the VL region and the VH region that together bind to IL12p35.
48. A method of producing an antibody or an antigen-binding fragment thereof, the method comprising
(a) culturing the cell of any one of claims 43-47 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and
(b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.
49. A method of treating a subject having an autoimmune disease, the method comprising administering a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof that binds to IL12p35 and/or an antibody or antigenbinding fragment thereof that binds to IL12R.pi, to the subject.
50. The method of claim 49, wherein the antibody or antigen-binding fragment thereof that binds to IL12p35 does not bind to IL12p40.
51. The method of claim 49 or 50, wherein the antibody or antigen-binding fragment thereof that binds to IL12R02 does not bind to IL12RP1.
52. The method of any one of claims 49-51, wherein the antibody or antigen-binding fragment thereof does not interfere IL23 pathway and/or IL35 pathway.
53. The method of any one of claims 49-52, wherein the subject is a human subject.
54. The method of claim 53, wherein the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
55. The method of any one of claims 49-54, wherein the antibody or antigen-binding fragment thereof is a human or humanized antibody or antigen-binding fragment thereof.
56. The method of any one of claims 49-52, wherein the subject is a non-human mammal, e.g., a monkey, a dog, or a mouse, optionally the mammal has a similar disease or disorder as systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and/or Sjogren's syndrome (SjS).
57. The method of claim 56, wherein the subject is a dog, optionally the antibody or antigenbinding fragment thereof is a canine or caninized antibody or antigen-binding fragment thereof.
PCT/US2025/014119 2024-01-31 2025-01-31 Anti-il12p35 antibodies and uses thereof Pending WO2025166228A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060286112A1 (en) * 2005-05-16 2006-12-21 Sirid-Aimee Kellermann Human monoclonal antibodies that bind to very late antigen-1 for the treatment of inflammation and other disorders
US20150203580A1 (en) * 2014-01-23 2015-07-23 Regeneron Pharmaceuticals, Inc. Human Antibodies to PD-L1
US20160264661A1 (en) * 2008-08-14 2016-09-15 Teva Pharmaceuticals Australia Pty Ltd Anti-il-12/il-23 antibodies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060286112A1 (en) * 2005-05-16 2006-12-21 Sirid-Aimee Kellermann Human monoclonal antibodies that bind to very late antigen-1 for the treatment of inflammation and other disorders
US20160264661A1 (en) * 2008-08-14 2016-09-15 Teva Pharmaceuticals Australia Pty Ltd Anti-il-12/il-23 antibodies
US20150203580A1 (en) * 2014-01-23 2015-07-23 Regeneron Pharmaceuticals, Inc. Human Antibodies to PD-L1

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