WO2023025194A1 - Fap/cd40结合分子及其医药用途 - Google Patents

Fap/cd40结合分子及其医药用途 Download PDF

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WO2023025194A1
WO2023025194A1 PCT/CN2022/114522 CN2022114522W WO2023025194A1 WO 2023025194 A1 WO2023025194 A1 WO 2023025194A1 CN 2022114522 W CN2022114522 W CN 2022114522W WO 2023025194 A1 WO2023025194 A1 WO 2023025194A1
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amino acid
seq
fap
binding
antibody
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English (en)
French (fr)
Inventor
林�源
陈思萌
吴婷婷
胡榕婷
廖成
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Priority to US18/683,212 priority Critical patent/US20250051467A1/en
Priority to MX2024002261A priority patent/MX2024002261A/es
Priority to CA3228641A priority patent/CA3228641A1/en
Priority to CN202280047669.9A priority patent/CN117616049A/zh
Priority to JP2024512158A priority patent/JP2024531462A/ja
Priority to EP22860540.8A priority patent/EP4393950A4/en
Priority to KR1020247008866A priority patent/KR20240049318A/ko
Priority to AU2022332499A priority patent/AU2022332499A1/en
Publication of WO2023025194A1 publication Critical patent/WO2023025194A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07K2317/75—Agonist effect on antigen
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    • 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

Definitions

  • the present disclosure relates to the field of biomedicine, especially the field of treating or intervening diseases related to CD40/CD40L signaling pathway.
  • the present disclosure relates to FAP/CD40 binding molecules, pharmaceutical compositions thereof, and methods and related pharmaceutical uses for treating diseases, especially tumors or cancers.
  • CD40 (TNFRSF5) is a transmembrane phosphorylated glycoprotein that belongs to the tumor necrosis factor receptor superfamily (TNFRS) member.
  • CD40 is expressed in a variety of cell types, including B cells, follicular dendritic cells (DC), epithelial cells, monocytes, macrophages, smooth muscle cells, and tumor cells. Its ligand CD40L is mainly expressed in activated T cells, activated B cells, platelets and smooth muscle cells. CD40L binding multimerizes CD40, generating downstream activation, growth and differentiation signals.
  • DC follicular dendritic cells
  • CD40L is mainly expressed in activated T cells, activated B cells, platelets and smooth muscle cells. CD40L binding multimerizes CD40, generating downstream activation, growth and differentiation signals.
  • CD40 signal transduction activates a variety of downstream signaling pathways, such as NF- ⁇ B, MAPK and STAT3 (Pype S, et al.J Biol Chem.2000Jun.16; 275(24):1858693), these pathways through the regulation of activator protein c- Jun, ATF2 and Rel transcription factors to regulate gene expression.
  • Binding of CD40 to CD40L induces resting B cell proliferation, immunoglobulin switching, antibody secretion, and plays an important role in tissue germinal center development and B cell survival, all of which are essential for humoral immune responses (Kehry M R. J Immunol 1996 ; 156:2345-2348).
  • CD40L binds to CD40 on DCs to induce DC maturation, which is manifested by the upregulation of the expression of co-stimulatory factor B7 family (CD80, CD86) and the secretion of pro-inflammatory cytokines such as interleukin 12 (IL-12).
  • CD40 and CD40L provides co-stimulatory signals for T cell activation and promotes the presentation of antigens by DC cells to T cells.
  • CD40 agonists CP-870 and 893 from Pfizer and CD40L-Fc fusion protein MEDI5083 from Medimmune a new fusion protein from AstraZeneca that can activate the CD40 signaling pathway, consisting of three fusion proteins of CD40L and IgG4-Fc in series
  • TAA tumor antigen
  • Fibroblast activation protein FAP fibroblast activation protein
  • FAP fibroblast activation protein
  • ⁇ is a tumor-associated antigen. FAP is less expressed in normal tissues of healthy adults, and is selectively expressed in 93% of tumor tissues, of which 30% are highly expressed. Examples include colon, pancreas, breast, stomach, prostate, bladder, and oral squamous cell carcinomas.
  • the present disclosure provides an anti-FAP antibody, an anti-CD40 antibody, and a bispecific antibody of the two.
  • the bispecific antibody can mediate tumor-specific CD40 activation through FAP, and has a stimulating effect on APC (such as dendritic cell DC). It has a mature and activating effect, can remove liver toxicity, peripheral blood toxicity and other peripheral toxicity, has excellent drug delivery window and druggability, and provides a solution for clinical application.
  • the present disclosure provides CD40-binding molecules, FAP-binding molecules, FAP/CD40-binding molecules (for example, anti-FAP/CD40 bispecific antibodies) with novel structures, encoding nucleic acids, vectors, host cells, pharmaceutical compositions, and uses thereof. Methods for treating, alleviating or preventing cell proliferative diseases (such as tumors or cancers) and related pharmaceutical uses.
  • the present disclosure provides a CD40 binding molecule comprising at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2, CDR3, wherein:
  • CDR1 in the immunoglobulin single variable domain contains or is the amino acid sequence shown in SEQ ID NO:39, and/or CDR2 contains or is the amino acid sequence shown in SEQ ID NO:40, and/or CDR3 comprises or is the amino acid sequence shown in SEQ ID NO:41;
  • the amino acid sequence in the immunoglobulin single variable domain comprises or is as shown in any of SEQ ID NO:22, 23, 32 to 38, or any of SEQ ID NO: 22, 23, 32 to 38 have at least 90%, at least 95%, at least 98%, at least 99% sequence identity; and/or
  • CDR1, CDR2 and CDR3 in the immunoglobulin single variable domain are as shown in CDR1, CDR2 and CDR3 in SEQ ID NO:22, or as shown in CDR1, CDR2 and CDR3 in SEQ ID NO:23 or as shown in CDR1, CDR2 and CDR3 among SEQ ID NO:32, or as shown in CDR1, CDR2 and CDR3 among SEQ ID NO:33, or as shown in CDR1, CDR2 and CDR3 among SEQ ID NO:34 or as shown in CDR1, CDR2 and CDR3 among SEQ ID NO:35, or as shown in CDR1, CDR2 and CDR3 among SEQ ID NO:36, or as shown in CDR1, CDR2 and CDR3 among SEQ ID NO:37 Shown in CDR3, or as shown in CDR1, CDR2 and CDR3 in SEQ ID NO:38; Said CDR is defined according to Kabat, IMGT, Chothia, AbM or Contact numbering system; In some specific embodiments,
  • a CD40 binding molecule comprising any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the aforementioned immunoglobulin single variable domains , said immunoglobulin single variable domains may be identical or different.
  • the aforementioned immunoglobulin single variable domain is a Nanobody or VHH.
  • the aforementioned CD40-binding molecule further includes a human immunoglobulin Fc region.
  • the Fc region is the Fc region of human IgG1, IgG2, IgG4.
  • the Fc region of the human IgG1 has a mutation that can increase the affinity with Fc ⁇ RIIb and weaken the ADCC effect, such as S267E/L328F.
  • Exemplary mutations that attenuate the ADCC effect are L234A/L235A on IgG1, V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P/F234A/L235A on IgG4, IgG1, N297A on IgG2, IgG3 or IgG4, V234A/G237A on IgG2, K214T/E233P/L234V/L235A/G236 deletion/A327G/P331A/D365E/L358M on IgG1, H268Q/V309L/A310S/P3 on IgG2 S267E/L328F on IgG1, L234F/L235E/D265A on IgG1, L234A/L235A/G237A/P238S/H268A/A
  • Hybrid IgG2/4 Fc domains may also be used, for example an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.
  • Exemplary mutations that increase affinity for FcyRIIb also include E233D/G237D/P238D/H268D/P271G/A330R on IgG1.
  • the aforementioned Fc region can make the anti-CD40 antibody or antigen-binding fragment thereof form a dimer or multimer molecule.
  • the immunoglobulin single variable domain in the aforementioned CD40-binding molecule is connected to the Fc region directly or through a linker.
  • the linker may be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure.
  • the linker is a flexible linker, such as G 4 S, GS, GAP, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 5 , ASGS, and the like.
  • a CD40 binding molecule comprising an amino acid sequence as set forth in, or at least 80% identical to, any one of SEQ ID NOs: 57-63.
  • the aforementioned CD40-binding molecule is directly or indirectly linked to at least one antibody that binds to another antigen, for example, to form a heterodimer.
  • the aforementioned CD40-binding molecule has at least one activity selected from the following:
  • APCs e.g. dendritic cells DC
  • the aforementioned CD40-binding molecule inhibits the binding of CD40 to CD40L, or competes with CD40L for binding to CD40.
  • the aforementioned CD40-binding molecule is capable of inhibiting tumor growth and/or metastasis by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% %.
  • the aforementioned CD40-binding molecule reduces the binding of CD40L to CD40 by at least 45%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or at least 95%.
  • the aforementioned CD40-binding molecule binds human CD40 with a KD of 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M or lower.
  • the aforementioned CD40-binding molecule is an anti-CD40 antibody or an antigen-binding fragment thereof.
  • the aforementioned CD40-binding molecule is an agonist anti-CD40 antibody or an antigen-binding fragment thereof.
  • the aforementioned anti-CD40 antibody is a camelid antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the aforementioned anti-CD40 antibody is a Nanobody or VHH.
  • the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof include, but are not limited to: linear antibodies, single-chain antibodies, nanobodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabody , triabody and tetrabody, tandem di-scFv, tandem tri-scFv).
  • the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof encompass variants, e.g., comprising one or more amino acid substitutions, e.g., conservative amino acid substitutions, compared to any of SEQ ID NOs: 22, 23, 32-38 for example, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.
  • the present disclosure provides an anti-CD40 antibody or an antigen-binding fragment thereof obtained by affinity maturation of SEQ ID NO: 22, 23, wherein the affinity-matured anti-CD40 antibody can have one or more CDRs having one or more changes that result in increased affinity for CD40 compared to the parental anti-CD40 antibody.
  • an anti-CD40 antibody or antigen-binding fragment thereof that binds or competes for binding to the same epitope as the aforementioned anti-CD40 antibody or antigen-binding fragment thereof.
  • an anti-CD40 antibody or antigen-binding fragment thereof that blocks the binding of the aforementioned anti-CD40 antibody or antigen-binding fragment thereof to CD40 (eg, human CD40) is provided.
  • an anti-CD40 antibody or antigen-binding fragment thereof whose binding to CD40 (eg, human CD40) is blocked by the aforementioned anti-CD40 antibody or antigen-binding fragment thereof.
  • conjugates comprising the aforementioned anti-CD40 antibodies of the present disclosure or antigen-binding fragments thereof.
  • At least 90% (sequence) identity encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or 100% (sequence) identity;
  • at least 80% (sequence) identity encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86 %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% (sequence) identity.
  • the present disclosure provides a FAP binding molecule comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, HCDR3, the light chain variable region comprising LCDR1, LCDR2, LCDR3, where:
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:9, and/or the amino acid sequence of the HCDR2 is shown in SEQ ID NO:10, and/or the amino acid sequence of the HCDR3 is shown in SEQ ID NO:18 and/or the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:12, and/or the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:13, and/or the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:13 ID NO:19 shown;
  • the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown in SEQ ID NO: 9, 10, and 18; and/or the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively shown in SEQ ID NO: 12, 13, and 19 shown;
  • amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:9, 10, and 15 respectively, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 12, 13, and 14, respectively;
  • amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown in SEQ ID NO:9, 10, and 15; the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively shown in SEQ ID NO:12, 13, and 16; or
  • amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:9, 10, and 11 respectively; the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:12, 13, and 17, respectively;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1 or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2 or at least 90% identical thereto the identity of the amino acid sequence;
  • the heavy chain variable region comprises an amino acid sequence as set forth in or at least 90% identical to SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:4 or at least 90% identical thereto amino acid sequence;
  • the heavy chain variable region comprises an amino acid sequence as set forth in or at least 90% identical to SEQ ID NO:5, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:6 or at least 90% identical thereto the amino acid sequence of; or
  • the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 7 or has at least 90% identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8 or has at least 90% identity thereto amino acid sequence;
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 1, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 2;
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 4;
  • the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 in SEQ ID NO: 5, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 in SEQ ID NO: 6; or
  • the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 7, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 8, wherein the CDR is based on Kabat, IMGT , Chothia, AbM, or Contact numbering systems, such as those defined for the Kabat numbering system.
  • a FAP-binding molecule (eg, an anti-FAP antibody or an antigen-binding fragment thereof) is provided, comprising any one or a combination of the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the aforementioned FAP-binding molecule further comprises a human immunoglobulin Fc region.
  • the Fc region is the Fc region of human IgG1, IgG2, IgG4.
  • the Fc of the human IgG1 has a mutation that removes or reduces the Fc effector function, such as N297A or D265A/N297A on the IgG1.
  • exemplary mutations that abolish or reduce Fc effector function include L234A/L235A, L234A/L235A/P329G, L234E, L234F, L234E/L235F, L234E/L235F/P329G on IgG1, V234A/G237A/P238S/H268A on IgG2 /V309L/A330S/P331S, F234A/L235A on IgG4, S228P/F234A/L235A on IgG4, N297A on IgG2 or IgG4, V234A/G237A on IgG2, K214T/E233P/L234V/L2365A/G on IgG1 deletion /A327G/P331A/D365E/L358M, H268Q/V309L/A330S/P331S on IgG2, S267E/L328F on IgG
  • a FAP-binding molecule (e.g., an anti-FAP antibody or antigen-binding fragment thereof) comprising a heavy chain (HC) and a light chain (LC) is provided, wherein:
  • the heavy chain is an amino acid sequence set forth in or at least 80% identical to SEQ ID NO: 49, and the light chain is an amino acid sequence set forth in SEQ ID NO: 50 or at least 80% identical thereto;
  • the heavy chain is an amino acid sequence set forth in or at least 80% identical to SEQ ID NO:51, and the light chain is an amino acid sequence set forth in SEQ ID NO:52 or at least 80% identical thereto;
  • the heavy chain is an amino acid sequence set forth in or at least 80% identical to SEQ ID NO: 53, and the light chain is an amino acid sequence set forth in or at least 80% identical to SEQ ID NO: 54; or
  • the heavy chain is an amino acid sequence shown in or at least 80% identical to SEQ ID NO:55, and the light chain is an amino acid sequence shown in or at least 80% identical to SEQ ID NO:56.
  • the heavy chain variable region of the aforementioned FAP binding molecule has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes; There are 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes.
  • the amino acid changes are conservative substitutions, substitutions or modifications, and/or deletions and additions that do not affect functions.
  • the aforementioned FAP-binding molecule has at least one activity selected from the following:
  • APCs e.g., dendritic cells DC
  • the aforementioned FAP-binding molecule binds FAP (e.g., human FAP) or a fragment thereof with a KD of 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M or lower .
  • the aforementioned FAP-binding molecule is an anti-FAP antibody or an antigen-binding fragment thereof; in some specific embodiments, it is a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragments of the aforementioned anti-FAP antibodies include, but are not limited to: Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single-chain antibody, scFv, sdAb, sdFv, Nanobody, Peptibodies peptibodies, domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), eg as scFv, Fv, Fab or Fab' fragments.
  • an anti-FAP antibody or antigen-binding fragment thereof that binds or competes for binding to the same epitope of FAP (eg, human FAP) as the aforementioned anti-FAP antibody or antigen-binding fragment thereof.
  • an anti-FAP antibody or antigen-binding fragment thereof that blocks the binding of the aforementioned anti-FAP antibody or antigen-binding fragment thereof to FAP (eg, human FAP) is provided.
  • an anti-FAP antibody or antigen-binding fragment thereof whose binding to FAP (eg, human FAP) is blocked by the aforementioned anti-FAP antibody or antigen-binding fragment thereof.
  • conjugates comprising the aforementioned anti-FAP antibodies or antigen-binding fragments thereof are provided.
  • the present disclosure provides a FAP/CD40 binding molecule comprising a first antigen-binding domain specifically binding to FAP and a second antigen-binding domain specifically binding to CD40, the second antigen-binding domain specifically binding to CD40 Comprising (at least one) immunoglobulin single variable domain.
  • the FAP/CD40 binding molecule comprises an immunoglobulin single variable domain that specifically binds CD40. In other embodiments, the FAP/CD40 binding molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more immunoglobulin single variable domains that specifically bind CD40, The immunoglobulin single variable domains may be identical or different.
  • the FAP/CD40 binding molecule comprises at least one (eg, 2, 3, 4) antigen binding domains that specifically bind FAP.
  • the immunoglobulin single variable domain specifically binding to CD40 comprises three complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: Shown in SEQ ID NO:39,40,41.
  • the amino acid sequences of the CDR1, CDR2, and CDR3 are shown in SEQ ID NO:24, 25, and 26, respectively; or the amino acid sequences of the CDR1, CDR2, and CDR3 are shown in SEQ ID NO:27, and 28, respectively. , 29 shown.
  • a FAP/CD40 binding molecule comprising a first antigen-binding domain that specifically binds FAP and a second antigen-binding domain that specifically binds CD40, the first antigen that specifically binds FAP
  • the binding domain comprises a heavy chain variable region comprising HCDR1, HCDR2, HCDR3 and a light chain variable region comprising LCDR1, LCDR2, LCDR3, wherein:
  • amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:9, 10, and 18, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:12, 13, and 19, respectively.
  • amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:9, 10, and 11, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:12, 13, and 14, respectively;
  • amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:9, 10, and 15, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:12, 13, and 14, respectively;
  • amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively shown in SEQ ID NO:9, 10, and 15; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively shown in SEQ ID NO:12, 13, and 16; or
  • amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:9, 10, and 11, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:12, 13, and 17, respectively.
  • the heavy chain variable region of the first antigen-binding domain that specifically binds FAP comprises SEQ ID NO: 1 or has at least 80% identity therewith
  • the amino acid sequence of the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 80% identity thereto;
  • the heavy chain variable region comprises an amino acid sequence as set forth in or at least 80% identical to SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:4 or at least 80% identical thereto amino acid sequence;
  • the heavy chain variable region comprises an amino acid sequence as set forth in or at least 80% identical to SEQ ID NO:5, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:6 or at least 80% identical thereto the amino acid sequence of; or
  • the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 7 or has at least 80% identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8 or has at least 80% identity thereto amino acid sequence.
  • the first antigen-binding domain that specifically binds FAP comprises a heavy chain (HC) and a light chain (LC);
  • the heavy chain is of the IgG1 or IgG4 isotype and the light chain is of the Kappa isotype;
  • the heavy chain is an amino acid sequence shown in or at least 80% identical to SEQ ID NO: 49, and the light chain is an amino acid sequence shown in SEQ ID NO: 50 or at least 80% identical thereto sequence;
  • the heavy chain is an amino acid sequence shown in or at least 80% identical to SEQ ID NO:51, and the light chain is an amino acid sequence shown in SEQ ID NO:52 or at least 80% identical thereto;
  • the heavy chain is an amino acid sequence set forth in or at least 80% identical to SEQ ID NO: 53, and the light chain is an amino acid sequence set forth in or at least 80% identical to SEQ ID NO: 54; or
  • the heavy chain is an amino acid sequence shown in or at least 80% identical to SEQ ID NO:55, and the light chain is an amino acid sequence shown in or at least 80% identical to SEQ ID NO:56.
  • the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region and a light chain variable region, wherein:
  • the immunoglobulin single variable domain that specifically binds the second antigen-binding domain of CD40 is located at the N-terminal of the heavy chain variable region of the first antigen-binding domain that specifically binds FAP;
  • the immunoglobulin single variable domain that specifically binds the second antigen-binding domain of CD40 is located at the C-terminus of the heavy chain variable region of the first antigen-binding domain that specifically binds FAP;
  • the immunoglobulin single variable domain that specifically binds the second antigen-binding domain of CD40 is located at the N-terminal of the light chain variable region of the first antigen-binding domain that specifically binds FAP;
  • the immunoglobulin single variable domain that specifically binds the second antigen-binding domain of CD40 is located at the C-terminal of the light chain variable region of the first antigen-binding domain that specifically binds FAP.
  • the aforementioned immunoglobulin single variable domain that specifically binds to the second antigen-binding domain of CD40 is connected directly or via a linker to the first antigen-binding domain that specifically binds to FAP; for example, the The linker has an amino acid sequence shown as (G 4 S) x , wherein x is independently selected from an integer of 1-20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); for example, the linker is the amino acid sequence shown by (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 .
  • the second antigen-binding domain that specifically binds CD40 is multivalent (for example, one aforementioned FAP/CD40 binding molecule contains 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10 second antigen-binding domains that specifically bind CD40).
  • the second antigen-binding domain that specifically binds CD40 is bivalent, tetravalent or hexavalent.
  • the second antigen-binding domain that specifically binds CD40 comprises 2, 3, 4, 5 or 6 of said immunoglobulin single variable domains.
  • a FAP/CD40 binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises any one of SEQ ID NO: 42, 44-48 or An amino acid sequence having at least 80% identity thereto, the second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 43 or having at least 80% identity thereto.
  • a FAP/CD40 binding molecule comprises two first polypeptide chains and two second polypeptide chains; in some specific embodiments, the two first polypeptide chains are the same, so The two second polypeptide chains are identical.
  • the aforementioned FAP/CD40 binding molecule is capable of inhibiting tumor growth (e.g., increased volume, increased tumor weight) and/or metastasis (e.g., multi-organ or multi-tissue metastasis, distant metastasis) by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%.
  • tumor growth e.g., increased volume, increased tumor weight
  • metastasis e.g., multi-organ or multi-tissue metastasis, distant metastasis
  • the aforementioned FAP/CD40 binding molecule is an anti-FAP/CD40 bispecific antibody or an antigen-binding fragment thereof, which includes but is not limited to: Fab, Fv, sFv, Fab', F(ab') 2.
  • the anti-FAP/CD40 bispecific antibody contains the immunoglobulin single variable domain in the second antigen-binding domain that specifically binds CD40 provided by the foregoing disclosure, and the specific binding domain provided by the disclosure above.
  • the first antigen-binding domain that specifically binds FAP is a first antibody comprising a heavy chain (HC) and a light chain (LC); and
  • the second antigen-binding domain that specifically binds CD40 is a second antibody, which is a VHH and has CDR1, CDR2, and CDR3 in the CD40-binding molecules provided in the present disclosure.
  • the VHH as the second antibody is located at the N-terminal and/or C-terminal of the heavy chain or light chain of the first antibody.
  • the anti-FAP/CD40 bispecific antibody comprises 1 primary antibody and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (such as 2, 4, 6 A) the second antibody of VHH;
  • the first antibody includes two HCs and two LCs, the VH of one HC and the VL of one LC of the first antibody form an antigen-binding site, and the VH of the other HC and the VL of another LC The VL forms the antigen binding site.
  • the first antibody of the anti-FAP/CD40 bispecific antibody or its antigen-binding fragment may be linked with a second antibody with 1, 2, 3, 4, 5, 6, 7, or 8 VHHs, said The second antibody of VHH can be the same or different, and both can be linked to the N-terminal of the heavy chain of the first antibody, or both can be linked to the C-terminal of the heavy chain of the first antibody, or both can be linked to the N-terminal of the light chain of the first antibody. terminal, or all connected to the C-terminal of the light chain of the first antibody, or any combination of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain.
  • the anti-FAP/CD40 bispecific antibody comprises a first polypeptide chain and a second polypeptide chain:
  • the first polypeptide chain from the N-terminal to the C-terminal is: [heavy chain of the first antibody]-joint 1-[second antibody]; the second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from N-terminus to C-terminus is: [the heavy chain of the first antibody]-joint 1-[second antibody] 1 -joint 2-[second antibody] 2 ; the second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from N-terminal to C-terminal is: [heavy chain of the first antibody] - linker 1 - [second antibody] 1 - linker 2 - [second antibody] 2 - linker 3 - [second antibody] Second antibody] 3 ;
  • the second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from the N-terminal to the C-terminal is: [second antibody]-joint 1-[heavy chain of the first antibody]; the second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from N-terminus to C-terminus is: [second antibody] 2 -linker 2-[second antibody] 1 -linker 1-[heavy chain of first antibody]; second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from N-terminal to C-terminal is: [Second Antibody] 3 -Linker 3-[Second Antibody] 2 -Linker 2-[Second Antibody] 1 -Linker 1-[First Antibody heavy chain]; the second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from N-terminus to C-terminus is: [second antibody] 1 -joint 1-[heavy chain of first antibody]-joint 2-[second antibody] 2 ; second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from N-terminal to C-terminal is: [Second Antibody] 1 - Linker 1 - [Second Antibody] 2 - Linker 2 - [Heavy Chain of First Antibody] - Linker 3 - [Second Antibody] Second antibody] 3 ;
  • the second polypeptide chain is the light chain of the first antibody;
  • the first polypeptide chain from N-terminal to C-terminal is: [second antibody] 1 -linker 1-[heavy chain of first antibody]-linker 2-[second antibody] 2 -linker 3-[second antibody Second antibody] 3 ;
  • the second polypeptide chain is the light chain of the first antibody;
  • [Second Antibody] 1 , [Second Antibody] 2 , and [Second Antibody] 3 may be the same or different.
  • the second antibody of the VHH in the aforementioned anti-FAP/CD40 bispecific antibody is linked to the first antibody directly or through a linker.
  • the linker is selected from the amino acid sequence shown as (G m S n ) x or (GGNGT) x or (YGNGT) x , wherein m and n are each independently selected from an integer of 1-8 (for example , 1, 2, 3, 4, 5, 6, 7 or 8), x is independently selected from an integer of 1-20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker is an amino acid sequence represented by G 4 S, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 5 , (G 4 S) 6 .
  • Linker 1, Linker 2, and Linker 3 can be the same or different.
  • the heavy chain of the first antibody of the anti-FAP/CD40 bispecific antibody comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) and Light chain constant region (CL).
  • the primary antibody can be a full length antibody.
  • the heavy chain of the first antibody of the anti-FAP/CD40 bispecific antibody is an IgG isotype, such as IgG1, IgG2, IgG3 or IgG4, such as an IgG1 isotype; and/or, the first antibody The light chain of is of the Kappa isotype.
  • the two HCs of the anti-FAP/CD40 bispecific antibody comprise the same CDRs and/or the two LCs comprise the same CDRs.
  • the two HCs of the first antibody comprise the same VH and/or the two LCs comprise the same VL.
  • the two HCs of the first antibody have the same amino acid sequence and/or the two LCs have the same amino acid sequence.
  • the second antibody of the two VHHs of the anti-FAP/CD40 bispecific antibody have identical or non-identical amino acid sequences.
  • two of said VHH's secondary antibodies have the same amino acid sequence.
  • the anti-FAP/CD40 bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain: a) each of the first polypeptide chains independently comprises VHH The heavy chain (HC) of the second antibody and the first antibody; and b) the second polypeptide chain each independently comprises the light chain (LC) of the first antibody; wherein, VHH is connected to the N-terminus of the HC of the second antibody through a linker and/or connected to the C-terminal;
  • the first polypeptide chains each independently comprise the heavy chain (HC) of the first antibody; and ii) the second polypeptide chains each independently comprise the VHH of the second antibody and the light chain (LC) of the first antibody ; Wherein, the VHH is connected to the N-terminal and/or C-terminal of the LC of the first antibody directly or through a linker.
  • the anti-FAP/CD40 bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.
  • antibodies that compete for binding to the same epitope as an anti-FAP/CD40 bispecific antibody of the disclosure are provided.
  • mutations are introduced in the Fc region of a FAP/CD40 binding molecule or anti-FAP/CD40 bispecific antibody of the present disclosure.
  • the mutation is, for example, a mutation that removes or reduces the Fc effector function of IgG, including but not limited to: N297A or D265A/N297A of IgG1, or L234A/L235A, L234A/L235A/P329G, L234E, L234F, L234E on IgG1 /L235F, L234E/L235F/P329G, V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P/F234A/L235A on IgG4, IgG2 or IgG234A on Ng97A V234A/G237A on IgG1
  • the aforementioned FAP/CD40 binding molecule or anti-FAP/CD40 bispecific antibody provided in the present disclosure has one or more of the following characteristics:
  • APCs such as dendritic cells
  • APCs such as dendritic cells
  • the anti-CD40 antibody, anti-FAP antibody, and FAP/CD40 binding molecule of the present disclosure are all resistant to heat treatment or have high stability. For example, no obvious aggregation or degradation was seen after treatment at 40°C for 30 days, and it was at least stable at 60°C.
  • the present disclosure provides polynucleotides encoding CD40-binding molecules, FAP-binding molecules, FAP/CD40-binding molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof of the present disclosure.
  • a nucleic acid of the present disclosure may be RNA, DNA or cDNA.
  • nucleic acids of the present disclosure are substantially isolated nucleic acids.
  • a nucleic acid of the disclosure can also be in the form of, can be present in, and/or can be part of a vector, such as a plasmid, cosmid, YAC, or viral vector.
  • the vector may especially be an expression vector, ie a vector providing for in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system) expression of the CD40 binding molecule.
  • the expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). The selection of said elements and their sequences for expression in a particular host is within the general knowledge of those skilled in the art.
  • Regulatory elements and other elements useful or necessary for the expression of CD40 binding molecules, FAP binding molecules, FAP/CD40 binding molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof of the present disclosure are, for example, promoters, enhancers, terminators, Son, integrator, selectable marker, leader sequence, reporter gene.
  • Nucleic acids of the disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA techniques) based on the amino acid sequence information of the polypeptides of the disclosure, and/or can be isolated from suitable natural sources.
  • the present disclosure provides expressing or capable of expressing one or more CD40 binding molecules, FAP binding molecules, FAP/CD40 binding molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments of the present disclosure, and/or containing the present disclosure Recombinant host cells for nucleic acids or vectors.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains
  • Fungal cells include, for example, cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of Saccharomyces (Hansenula).
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Hansen A cell of a species of Saccharomyces Hansenula
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure can also use amphibian cells, insect cells, plant cells, and any other cell used in the art to express heterologous proteins.
  • the cells of the present disclosure do not develop into finished plants or individual animals.
  • the present disclosure provides methods for preparing CD40-binding molecules, FAP-binding molecules, FAP/CD40-binding molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof of the present disclosure, the methods generally comprising the following steps:
  • a host cell of the present disclosure under conditions that allow expression of a CD40-binding molecule of the present disclosure, a FAP-binding molecule, a FAP/CD40-binding molecule, an anti-FAP/CD40 bispecific antibody or an antigen-binding fragment thereof; and
  • a CD40-binding molecule, FAP-binding molecule, FAP/CD40-binding molecule, anti-FAP/CD40 bispecific antibody or antigen-binding fragment thereof of the present disclosure can be expressed intracellularly (e.g., in the cytoplasm, in the periplasm, or produced in inclusion bodies), then isolated from the host cell and optionally further purified; or it may be produced extracellularly (for example in the medium in which the host cell is grown), then isolated from the medium and optionally further purified.
  • Recombinant immunoglobulin expression vectors can stably transfect cells.
  • Mammalian-like expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid that secretes the antibody can be purified and collected using conventional techniques.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange.
  • the obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
  • CD40-binding molecules, FAP-binding molecules, FAP/CD40-binding molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof of the present disclosure may also be obtained by other protein-producing methods known in the art, such as chemical synthesis , including solid-phase or liquid-phase synthesis.
  • compositions comprising a prophylactically or therapeutically effective amount of a CD40-binding molecule, a FAP-binding molecule, a FAP/CD40-binding molecule, an anti-FAP/CD40 bispecific antibody or Its antigen-binding fragment, or encoding polynucleotide, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of CD40-binding molecules, FAP/CD40-binding molecules or anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof.
  • the amount of the CD40 binding molecule, FAP/CD40 binding molecule or anti-FAP/CD40 bispecific antibody contained in the unit dose of the pharmaceutical composition is 0.1-2000 mg; in some specific embodiments, it is 1-1000 mg.
  • the present disclosure provides a kit comprising a CD40-binding molecule, a FAP-binding molecule, a FAP/CD40-binding molecule, an anti-FAP/CD40 bispecific antibody or an antigen-binding fragment thereof, and/or an encoding polynucleotide of the present disclosure.
  • a diagnostic reagent comprising the above-mentioned polynucleotide is also provided, as well as related diagnostic uses.
  • the present disclosure provides CD40-binding molecules, FAP-binding molecules, FAP/CD40-binding molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof, encoding polynucleotides or pharmaceutical compositions of the present disclosure in the prevention and/or treatment of diseases
  • the disease may or may not be associated with the CD40 signaling pathway.
  • the present disclosure provides a method for preventing and/or treating a disease associated with CD40, the method comprising administering to a subject a preventive and/or therapeutically effective amount of a CD40-binding molecule of the present disclosure, or comprising the present disclosure.
  • Pharmaceutical compositions of CD40 binding molecules are disclosed. And, the use of the CD40-binding molecule of the present disclosure in the prevention and/or treatment of CD40- or CD40L-related diseases, CD40- or CD40L-related disorders is also provided.
  • CD40-binding molecules, FAP-binding molecules, FAP/CD40-binding molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof, encoding polynucleotides or pharmaceutical compositions of the present disclosure can be used alone, or combined with other anti-cancer or tumor Therapeutic approaches are used in combination (eg, with other immunogenic agents, standard cancer therapies, or other antibody molecules) to inhibit the growth of cancer or tumors.
  • the present disclosure provides a method for preventing and/or treating cancer or tumors, comprising administering a preventive and/or therapeutically effective amount of the disclosed CD40 binding molecules, FAP binding molecules, FAP/ The CD40 binding molecule, anti-FAP/CD40 bispecific antibody or antigen-binding fragment thereof, encoding polynucleotide, pharmaceutical composition inhibits tumor cell growth in a patient or subject.
  • the cancer is preferably but not limited to a cancer that responds to immunotherapy.
  • non-limiting examples of cancer or tumor include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (such as metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma cancer, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor and osteosarcoma.
  • melanoma such as metastatic malignant melanoma
  • cancers examples include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer Cancer, Vaginal Cancer, Vaginal Cancer, Hodgkin's Disease, Non-Hodgkin's Lymphoma, Esophageal Cancer, Small Intestine Cancer, Endocrine System Cancer, Thyroid Cancer, Parathyroid Cancer, Adrenal Cancer, Soft Tissue Sarcoma, Urethral Cancer, Penile Cancer , chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, Renal pelvis carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brains
  • CNS central nervous system
  • the present disclosure also provides a method for preventing and/or treating infectious diseases in a subject or a patient, comprising administering to the subject or patient the CD40 binding molecule, FAP binding molecule, FAP/CD40 binding molecule of the present disclosure.
  • Molecules, anti-FAP/CD40 bispecific antibodies or antigen-binding fragments thereof, encoding polynucleotides or pharmaceutical compositions enable the prevention and/or treatment of infectious diseases in the subject.
  • CD40 binding molecules can be used alone or in combination with vaccines to stimulate immune responses to pathogens, toxins and self-antigens.
  • pathogens for which this method of treatment is particularly applicable include pathogens for which there is currently no effective vaccine, or for which conventional vaccines are not fully effective. These include but are not limited to HIV, hepatitis viruses (A, B, C), influenza virus, herpes virus, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.
  • the present disclosure provides compositions for detecting CD40 or FAP comprising a CD40-binding molecule or a FAP-binding molecule according to the present disclosure.
  • the present disclosure also provides methods, systems or devices for detecting CD40 or FAP in vivo or in vitro, comprising treating a sample with a CD40-binding molecule or a FAP-binding molecule of the present disclosure.
  • an in vitro detection method, system or device may, for example, include:
  • a change eg, a statistically significant change in complex formation in a sample or subject, as compared to a control sample or subject, is indicative of the presence of CD40 or FAP in the sample.
  • the in vivo detection method, system or device may comprise:
  • Detection can include determining where or when a complex is formed.
  • the CD40-binding molecule or the FAP-binding molecule is labeled with a detectable substance, and detection is achieved by detecting the label.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • Complex formation between a CD40-binding molecule and CD40, or a FAP-binding molecule and FAP can be detected by measuring or visualizing antibody binding or not binding to CD40 or FAP.
  • Conventional detection assays may be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry.
  • a CD40-binding molecule or a FAP-binding molecule of the disclosure can be labeled with a fluorophore chromophore.
  • kits comprising a CD40-binding molecule or a FAP-binding molecule, and may further comprise instructions for diagnostic use.
  • the kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent.
  • the CD40-binding molecule or the FAP-binding molecule can be formulated as a pharmaceutical composition.
  • CD40 and CD40 antigen refer to an approximately 48kD glycoprotein expressed on the surface of normal and neoplastic B cells that acts as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al., 1987, J. Immunol. 138 :788-785).
  • a cell endogenously expressing CD40 is any cell characterized by surface expression of CD40, including but not limited to normal and neoplastic B cells, interdigitated cells, basal epithelial cells, cancer cells, macrophages, endothelial cells, follicular trees adenocytes, tonsil cells, and plasma cells of bone marrow origin.
  • CD40 in the present disclosure refers to any native CD40 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise indicated.
  • a cDNA molecule encoding CD40 has been isolated from a library prepared from the Burkitt's lymphoma cell line Raji (Stamenkovic et al., 1989, EMBO J. 8:1403). Sequence information can be found in Table 8 of this disclosure.
  • "CD40" of the present disclosure encompasses full-length unprocessed CD40 as well as any form of CD40 that results from processing in a cell, as well as naturally occurring variants of CD40, such as splice variants or allelic variants.
  • a CD40-binding molecule of the present disclosure is capable of specifically binding human, mouse and/or cynomolgus CD40.
  • FAP Fibroblast Activation Protein
  • FAP Antigen also known as prolyl endopeptidase FAP or Seprase (EC 3.4.21)
  • FAP Fibroblast Activation Protein
  • FAP Antigen also known as prolyl endopeptidase FAP or Seprase (EC 3.4.21)
  • mammals such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys) and rodents (eg, mice and rats), unless otherwise indicated.
  • FAP of the present disclosure encompasses full-length unprocessed FAP as well as any form of FAP derived from processing in the cell, and also naturally occurring variants of FAP, such as splice variants or allelic variants.
  • a FAP binding molecule of the present disclosure is capable of specifically binding human, mouse and/or cynomolgus FAP.
  • the amino acid sequence of human FAP is in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO:2), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2, GeneBank Accession NO .AAC51668 is displayed.
  • the extracellular domain (ECD) of human FAP extends from amino acid positions 26 to 760. Amino acid sequences such as human FAP ECD with His tag are shown in Table 2 of the present disclosure.
  • mouse FAP The amino acid sequence of mouse FAP is shown in UniProt Accession No. P97321 (version 126, SEQ ID NO: 143), or NCBI RefSeq NP_032012.1.
  • the extracellular domain (ECD) of mouse FAP extends from amino acid positions 26 to 761.
  • a FAP-binding molecule of the disclosure binds the extracellular domain of FAP.
  • Antibody is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are mu, delta, and gamma chains, respectively , ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes 3 hypervariable regions (CDRs) and 4 framework regions (FRs) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) are composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • Bispecific antibody encompasses antibodies (including antibodies or antigen-binding fragments thereof such as single chain antibodies) that specifically bind to two different antigens or at least two different epitopes of the same antigen.
  • Typical bispecific antibody structure models such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one /DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent- Bispecific antibodies such as DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(
  • deterministic delineation of the CDRs and identification of the residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • Various analytical methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformation definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see eg Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural ring regions. (See eg Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge base and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, "AbM” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Initio Protein Structure Prediction Using a Combined Hierarchical Approach” described).
  • a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The correspondence between various numbering systems is well known to those skilled in the art, and is exemplary, as shown in Table 1 below.
  • the CDR amino acid residues of the VL and VH regions of the disclosed antibodies conform to the known Kabat numbering system in number and position.
  • a “domain” of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for a single functional property of a protein, and in many cases can be added, removed or transferred to other proteins without loss of the rest of the protein and/or the function of the domain.
  • Immunoglobulin domain refers to a globular region of an antibody chain (eg, a chain of a conventional tetrapeptide chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by their maintenance of the immunoglobulin fold characteristic of antibody molecules, consisting of a 2-layer sandwich of approximately seven antiparallel beta-sheet strands, optionally stabilized by conserved disulfide bonds, arranged in two beta-sheets .
  • Immunoglobulin variable domain refers to the term “framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” or “FR3” essentially defined in the art and hereinafter as “FR1” or “FR1”, respectively. , and four “framework regions” of “framework region 4" or “FR4", wherein the framework regions are respectively referred to in the art and hereinafter as “complementarity determining region 1" or "CDR1", The three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains are endowed with antigen-specificity by having an antigen-binding site.
  • Immunoglobulin variable domain refers to the term “framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” or “FR3” essentially defined in the art and hereinafter as “FR1” or “FR1”, respectively. , and four “framework regions” of “framework region 4" or “FR4", wherein the framework regions are respectively referred to in the art and hereinafter as “complementarity determining region 1" or "CDR1", The three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains are endowed with antigen-specificity by having an antigen-binding site.
  • Antibody framework (FR) refers to the portion of a variable domain that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain.
  • Immunoglobulin single variable domain is commonly used to refer to a variable domain that can be used without interaction with other variable domains (e.g. without the need for between the VH and VL domains of a conventional four-chain monoclonal antibody).
  • an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that forms a functional antigen binding site.
  • immunoglobulin single variable domains include Nanobodies (including VHH, humanized VHH and/or camelized VH, such as camelized human VH), IgNAR, domains, as VH domains or derived from VH Domain (single domain) antibodies such as dAbs TM and (single domain) antibodies that are or are derived from VL domains such as dAbs TM .
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains, such as VH or VHH domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments, VHH antibodies, nanobodies, are termed “heavy chain antibodies” (i.e. "antibodies lacking light chains”) ) antigen-binding variable domains of immunoglobulins (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)).
  • VHH domain is used to distinguish the variable domain from the variable domain of the heavy chain (which is referred to in this disclosure as the "VH domain”) and the variable domain of the light chain present in antibodies of conventional tetrapeptide chain structure.
  • variable domains (which are referred to as “VL domains” in this disclosure).
  • the VHH domain specifically binds the epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain) .
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain includes, but is not limited to, natural antibodies produced by camelids, humanized antibodies produced by camelids, or obtained by screening with phage display technology.
  • the total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this disclosure.
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e. one according to Kabat numbering). or positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering).
  • Kabat numbering i.e. one according to Kabat numbering
  • numbers may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering.
  • numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • Other numbering systems or coding conventions include Chothia, IMGT, AbM.
  • Humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to an antibody produced by grafting a non-human CDR sequence into a human antibody variable region framework. It can overcome the strong immune response induced by chimeric antibodies due to carrying a large number of non-human protein components. In order to avoid a decrease in activity while reducing immunogenicity, minimal reverse mutations can be performed on the variable region of the fully human antibody to maintain activity.
  • Examples of "humanization” include that the VHH domain derived from Camelidae can be replaced by the amino acid sequence of the original VHH sequence with one or more amino acid residues present in the corresponding position in the VH domain of an antibody with a conventional tetrapeptide chain structure.
  • Humanization by one or more amino acid residues in VHH (also referred to as “sequence optimization” in this disclosure. Mutations are other modifications to the sequence such as removal of potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences.
  • minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity.
  • Fully human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). "Fully human antibody” does not include “humanized antibody”.
  • the CD40 binding molecules, FAP binding molecules of the present disclosure will be present at preferably 10 ⁇ 7 to 10 ⁇ 10 moles per liter (M), more preferably 10 ⁇ 8 to 10 ⁇ 10 moles as measured in a Biacore or KinExA or Fortibio assay. /L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and/or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, More preferably an association constant (KA) of at least 10-10 M binds the desired antigen (ie CD40 or FAP). Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays as described in this disclosure (for example radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay).
  • SPR surface plasmon resonance
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competitive assay sandwich competitive assay
  • “competition” in the context of antigen binding proteins that compete for the same epitope (such as neutralizing antigen binding proteins or neutralizing antibodies), it means competition between antigen binding proteins, which is determined by the following assay:
  • the detected antigen binding protein eg, antibody or immunologically functional fragment thereof
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619
  • solid-phase Phase direct labeling assay solid phase direct labeling sandwich assay
  • Such assays involve the use of purified antigen (on a solid surface or cell surface) capable of binding to an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antigen binding protein to be tested.
  • the antigen binding protein to be tested is present in excess.
  • Antigen binding proteins identified by competition assays include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and, epitopes that bind in sufficient proximity to the reference antigen binding protein.
  • a competing antigen binding protein when present in excess it will inhibit (e.g. reduce) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% - specific binding of 75% or 75% or more of the reference antigen binding protein to the common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • Antibodies can be competitively screened for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for antigen binding. A high-throughput method to obtain antibodies that bind the same epitope based on their cross-competition is described in International Patent Publication WO 03/48731. Thus, antibodies that compete for binding to the same epitope on CD40 or FAP as the antibody molecules of the disclosure can be obtained using routine techniques known to those skilled in the art.
  • CD40 binding molecule means any protein capable of specifically binding CD40 or any molecule comprising said protein.
  • a CD40 binding molecule may comprise an antibody against CD40 as defined in the present disclosure or a conjugate thereof.
  • CD40 binding molecules also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • a "CD40-binding molecule” of the present disclosure may comprise at least one immunoglobulin single variable domain (eg, VHH) that binds CD40.
  • a "CD40-binding molecule” may comprise 2, 3, 4 or more immunoglobulin single variable domains (eg, VHH) that bind CD40.
  • the CD40-binding molecules of the present disclosure may also comprise linkers and/or moieties with effector functions, such as half-life extending moieties (such as serum albumin-binding immunoglobulin single variable domains). variable domain), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region.
  • linkers and/or moieties with effector functions such as half-life extending moieties (such as serum albumin-binding immunoglobulin single variable domains). variable domain
  • a fusion partner such as serum albumin
  • a conjugated polymer such as PEG
  • a "CD40-binding molecule" of the present disclosure also encompasses bi/multispecific antibodies comprising immunoglobulins that bind different antigens (e.g., a first antibody that binds a first antigen (e.g., CD40) and a second antibody that binds
  • the second antibody to an antigen optionally includes a third antibody that binds to a third antigen, and further optionally includes a fourth antibody that binds to a fourth antigen.
  • FAP binding molecule means any protein capable of specifically binding FAP or any molecule comprising said protein.
  • a FAP binding molecule may comprise an antibody as defined in the present disclosure against FAP or a conjugate thereof.
  • FAP/CD40 binding molecule means any protein capable of specifically binding CD40 and FAP or any molecule comprising said protein.
  • a FAP/CD40 binding molecule may comprise an antibody as defined in the present disclosure against CD40 and FAP or a conjugate thereof.
  • Binding to CD40 or “binding to CD40” refers to being capable of interacting with CD40 or its epitope, which may be of human origin.
  • Binding to FAP or “binding to FAP” refers to being able to interact with FAP or its epitope, which may be of human origin.
  • the "antigen-combining site” of the present disclosure refers to a discontinuous three-dimensional site on the antigen recognized by the antibody of the present disclosure.
  • Antigen refers to a molecule used to immunize an immunocompetent vertebrate, to generate antibodies recognizing the antigen, or to screen expression libraries (such as, inter alia, phage, yeast or ribosome display libraries).
  • antigen is defined more broadly to include target molecules specifically recognized by antibodies, and to include portions or mimetics of molecules used in immunization procedures for antibody generation or library screening for antibody selection.
  • monomers and multimers e.g., dimers, trimers, etc.
  • truncated and other variants of human CD40 are referred to as antigen.
  • Epitope refers to the site on an antigen to which an immunoglobulin or antibody binds. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids that are juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents.
  • An epitope typically comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods of determining the spatial conformation of an epitope include techniques in the art and techniques described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • Specific binding and “selective binding” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody when human CD40 or its epitope is used as the analyte and the antibody is used as the ligand, when the surface plasmon resonance (SPR) technique is used in the instrument, the antibody is at an equilibrium below about 10 ⁇ 7 M or even less
  • the dissociation constant (K D ) for binding to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is nonspecific to that of the predetermined antigen (or its epitope) or closely related antigens Antigen (such as BSA, etc.) at least twice the binding affinity.
  • An "antibody that recognizes an antigen” may be used interchangeably with a “specifically binding antibody” in this disclosure.
  • Binding affinity or “affinity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or portion thereof and an antigen).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the Surface Plasmon Resonance (SPR) method (Biacore).
  • SPR Surface Plasmon Resonance
  • the rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362).
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432).
  • Other standard assays to assess the binding ability of antibodies against a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure.
  • Binding kinetics and binding affinity of antibodies can also be assessed by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the Biacore TM system or KinExA. Binding affinities associated with different molecular interactions can be compared by comparing the KD values of the individual antibody/antigen complexes, eg, a comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the KD value of an interaction of interest (e.g., a specific interaction between an antibody and an antigen) with that of an unintended interaction (e.g., a control antibody known not to bind CD40). KD value was evaluated.
  • SPR surface plasmon resonance
  • a “conservative substitution” refers to a substitution for another amino acid residue that has similar properties to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine , valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains.
  • “Homology”, “identity” or “sequence identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position of.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100%. For example, two sequences are 60% homologous if there are 6 matches or homology at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when two sequences are aligned to yield the greatest percent homology.
  • a “host cell” includes any cell or cell culture that can be or has been a recipient of a vector for the incorporation of a polynucleotide insert.
  • a host cell includes the progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation.
  • Host cells include cells transfected and/or transformed in vivo with polynucleotides of the present disclosure.
  • Cell “cell line,” and “cell culture” are used interchangeably, and all such designations include progeny. It should also be understood that all progeny may not be precisely identical in DNA content due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cells are included.
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. “Inhibition of growth” (eg, in relation to cells) is intended to include any measurable decrease in cell growth.
  • Antagonist activity refers to the function of a substance as an agonist. Binding of an agonist to a cellular receptor elicits a response or activity similar or identical to that elicited by the receptor's natural ligand.
  • a CD40 agonist or CD40 agonistic antibody can induce any or all of the following responses: cell proliferation and/or differentiation; upregulation of adhesion between cells by molecules such as ICAM-1, E-selectin, VCAM, etc.; secretion of Pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, IL-12, TNF, etc.; transduce signals through the CD40 receptor, such as TRAF (eg, TRAF2 and/or TRAF3), MAP Kinases, such as NIK (NF- ⁇ B-inducing kinase), 1- ⁇ B kinase (IKK ⁇ / ⁇ ), transcription factors NF- ⁇ B, Ras and MEK/ERK pathway, PI3K/Akt pathway, P38MAPK pathway, etc.; through XIAP, Mcl-1 , BCLx and other molecules transduce anti-apoptotic signals; B and/or T cell memory generation; B cell antibody production; B cell isotype switching; up-regulation of class II M
  • CD40-associated disease indicates a condition in which CD40-expressing cells are modified or eliminated.
  • These cells include CD40-expressing cells that exhibit abnormal proliferation or that are associated with cancerous or malignant growth. More specific examples of cancers exhibiting aberrant expression of the CD40 antigen include B-lymphoblastoid, Burkitt's lymphoma, multiple myeloma, T-cell lymphoma, Kaposi's sarcoma, osteosarcoma, epidermal and endothelial tumors, Cancers of the pancreas, lung, breast, ovary, colon, prostate, head and neck, skin (melanoma), bladder and kidney.
  • Such disorders include, but are not limited to, leukemias, lymphomas (including B-cell lymphomas and non-Hodgkin's lymphomas), multiple myeloma, Waldenstrom's macroglobulinemia; solid tumors, including sarcomas such as Osteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinoma (including ovarian adenocarcinoma), Kaposi's sarcoma/Kaposi's tumor, and squamous cell carcinoma.
  • sarcomas such as Osteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinoma (including ovarian adenocarcinoma), Kaposi's sarcoma/Kaposi's tumor, and squamous cell carcinoma.
  • Proliferative disease refers to a disorder associated with some degree of abnormal cell proliferation.
  • the proliferative disorder is cancer.
  • Preventing cancer means delaying, inhibiting or preventing the onset of cancer in a subject in which the onset of cancer or tumorigenesis has not been proven, but has been determined, for example, by genetic screening or other methods, identified susceptibility to cancer. This also includes treating a subject with a precancerous condition to halt progression of the precancerous condition to a malignancy or to cause regression thereof.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the interaction of an exogenous drug, therapeutic, diagnostic, or composition with an animal. , humans, subjects, cells, tissues, organs or biological fluids, such as therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contacting the reagents with the cells, and contacting the reagents with a fluid, wherein the fluid contacts the cells.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition or by another cell. When applied to human, veterinary or research subjects, it refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, for example, comprising any antibody of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, the subject has suffered from, is suspected of having, or tends to suffer from There is one or more proliferative diseases or symptoms thereof for which the therapeutic agent is known to have a therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated in an amount effective to alleviate one or more symptoms of a disease, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent.
  • the amount of a therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on factors such as the disease state, age, and weight of the subject, and the extent to which the drug produces the desired therapeutic effect in the subject. ability. Whether disease symptoms have been alleviated can be assessed by any of the clinical tests commonly used by a physician or other professional health care practitioner to assess the severity or progression of such symptoms.
  • any statistical test method known in the art such as Student's t-test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should reduce the target disease symptoms in a statistically significant number of subjects.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dose of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • a subject of the present disclosure can be an animal or a human subject.
  • Subject and “patient” in the present disclosure refer to mammals, especially primates, especially humans.
  • Figure 1 shows the binding of anti-FAP antibodies Ab9, Ab10, Ab14, and Ab15 to human FAP on the cell surface, using 28H1 as a positive control, and IgG1 isotype (as a negative control.
  • Figure 2 shows the binding of anti-FAP antibodies Ab9, Ab10, Ab14, Ab15 to mouse FAP on the cell surface, using 28H1 as a positive control and IgG1 isotype as a negative control.
  • Figure 3 shows the binding of anti-CD40 antibodies A12 and A297 to human CD40 on the surface of Raji cells, using 9E5-SELFNS as a positive control and IgG1 as a negative control.
  • Figure 4 shows the binding of anti-CD40 antibodies A12 and A297 to human CD40 on the surface of HEK293 cells, using 9E5-SELFNS as a positive control and IgG1 as a negative control, and the ordinate is the percentage of agonistic activity relative to 200nM 9E5-SELFNS.
  • Figure 5A shows the affinity flow cytometric detection results of humanized anti-CD40 antibodies A12_V1, A12_V2, A12_V3 and HEK293 cells overexpressing human CD40
  • Figure 5B shows the humanized anti-CD40 antibodies A297_V1, A297_V2, A297_V3, A297_V4 and HEK293 cells overexpressing human CD40
  • 9E5-SELFNS was used as a positive control
  • hIgG was used as a negative control.
  • Figure 6A shows the activation of HEK-Blue TM CD40L cells mediated by humanized anti-CD40 antibodies A12_V1, A12_V2, and A12_V3 through Fc ⁇ RIIb
  • Figure 6B shows the activation of HEK-Blue TM CD40L cells mediated by humanized anti-CD40 antibodies A297_V1, A297_V2, A297_V3, and A297_V4 through Fc ⁇ RIIb.
  • 9E5-SELFNS was used as a positive control
  • hIgG was used as a negative control.
  • Figure 7 is a schematic diagram of the structure of an anti-FAP/CD40 bispecific antibody.
  • Figure 8A shows the affinity flow between anti-FAP/CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, Ab10-A297V3-4 and CHOK1/FAP stable transfectants that highly express human FAP antigen
  • Figure 8B and Figure 8C are the affinity flow cytometric detection results of the aforementioned anti-FAP/CD40 bispecific antibody and CHOK1/FAP stably transformed strains highly expressing mouse FAP antigen and cynomolgus monkey FAP antigen.
  • Figure 9A is the affinity flow of anti-FAP/CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, Ab10-A297V3-4 and HEK-BlueTM CD40L stable transfection strains highly expressing human CD40 antigen
  • Figure 9B shows the affinity flow cytometric detection results of the aforementioned anti-FAP/CD40 bispecific antibody and HEK293 cells highly expressing cynomolgus CD40 antigen.
  • Figure 10 shows the flow cytometric detection results of anti-FAP/CD40 bispecific antibodies Ab10-A297V3-2, Ab10-A297V3-4 and CD40 affinity on human immature DC.
  • Figure 11 shows the concentration gradient anti-FAP/CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, Ab10-A297V3-4 promote the cross-linking of cells in the absence of CHOK1/FAP stabilizing For DC maturation, LPC, hIgG1, and vehicle were used as controls.
  • Figure 12 shows that anti-FAP/CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, Ab10-A297V3-4 with concentration gradients promote DC under the condition of cross-linking of CHOK1/FAP stably transfected cells
  • LPC, hIgG1, and vehicle were used as controls.
  • Figure 13A is the tumor growth curve of mice injected with a single dose of antibody Ab10-A297V3-2 or Ab10-A297V3-4 into mFAP-MC38 tumor-bearing hCD40 humanized mice;
  • Figure 13B is the activation of corresponding peripheral blood B cells in mice;
  • Figure 13C is the corresponding body weight change curve of the mouse;
  • Figure 13D is the corresponding change of the platelet count of the mouse;
  • Figure 13E is the effect of the corresponding liver function of the mouse.
  • Figure 14A is the mouse tumor growth curve after injecting multiple doses of antibody Ab10-A297V3-2 or Ab10-A297V3-4 into mFAP-MC38 tumor-bearing hCD40 humanized mice;
  • Figure 14B is the activation of the corresponding peripheral blood B cells in the mice;
  • Figure 14C is the corresponding body weight change curve of the mice;
  • Figure 14D is the corresponding change of platelet count in the mice;
  • Figure 14E is the influence of the corresponding liver function in the mice.
  • Figure 15A is the tumor growth curve of mFAP-MC38 tumor-bearing hCD40 humanized mice injected with multiple doses of antibody Ab10-A12V2-2;
  • Figure 15B is the corresponding body weight change curve of the mice;
  • Figure 15C is the corresponding liver function curve of the mice Effect;
  • Figure 15D shows the corresponding changes in platelet count in mice.
  • Figure 16A is the tumor growth curve of mFAP-MC38 tumor-bearing hCD40 humanized mice injected with a single dose of Ab10-A12V2-4;
  • Figure 16B is the corresponding body weight change curve of the mice;
  • Figure 16C is the corresponding liver function of the mice ;
  • Figure 16D is the effect of corresponding platelet count in mice.
  • Fig. 17 is the pharmacokinetic comparison results of bispecific antibodies Ab10-A12V2-2 and Ab10-A297V3-2.
  • the P values are all calculated relative to the control hIgG1.
  • Student't-test where, ns means no significant difference, * is p ⁇ 0.05, ** is p ⁇ 0.01, *** is p ⁇ 0.001, **** is p ⁇ 0.0001 .
  • Human fibroblast activation protein (FAP; GeneBank accession number AAC51668) is a serine oligopeptidase with a molecular weight of about 170 kDa, which is a homodimer.
  • Table 2 shows the recombinant FAP protein sequence, source and use used in this disclosure.
  • antigen-specific phages were captured using magnetic beads with avidin on the surface, and the phages were enriched with a magnetic frame. Antigen-specific phage were eluted with a glycine solution at pH 2.2.
  • 284 clones were randomly selected, and the positive clones were screened by phage ELISA, including the following specific steps: counting plate growth colonies, a total of 284, inoculated into 96-well plates, each well containing 400 ⁇ L culture solution (2YT+Amp+0.2 % glucose), shake at 250rpm at 37°C for 6h. Coat the plate with the antigen h-FAP-His, 100ng/100 ⁇ L/well, overnight at 4°C.
  • h-FAP antigen-specific full-length antibody cell display library including: transforming with full-length antibody cell display gene library vector DNA to construct full-length antibody CHO cell display library. Cell banks were pressure screened with hygromycin. Obtain a stable transformed cell bank, double-stain the cell bank with PE-labeled mouse anti-human ⁇ (or ⁇ ) light chain antibody and FITC-labeled h-FAP antigen, FACS sort PE and FITC double positive cells, one cell per well, ⁇ Two 96-well plates for light chain library sorting, one 96-well plate for lambda light chain library sorting, hygromycin pressurized culture.
  • Cloning antibody genes including: analyzing the affinity of positive clones through FACS detection, and deciding to carry out PCR amplification of antibody genes on 45 clones.
  • the positive clones were collected by centrifugation, the supernatant was discarded, the genomic DNA was extracted, VH and LC were amplified by PCR, the amplified fragments were separated by electrophoresis, and sequenced and analyzed to determine 6 unique VH and 6 unique ⁇ , combined to obtain 12 unique sequences Cloning, the sequences of VH and VL of 4 clones are shown in Table 3.
  • the present disclosure also provides the full-length heavy chain and full-length light chain sequences of Ab9, Ab10, Ab14, Ab15.
  • the above-mentioned anti-FAP antibody has the following general structure:
  • HCDR1 are all SEQ ID NO:9;
  • HCDR2 are all SEQ ID NO:10;
  • HCDR3 are all DX 1 SLTARPYYFYGFDV (SEQ ID NO: 18), wherein X 1 is E or A;
  • LCDR1 is SEQ ID NO: 12;
  • LCDR2 is SEQ ID NO: 13;
  • LCDR3 are all QQANSFPX 2 X 3 (SEQ ID NO: 19), wherein, X 2 is P or L, and X 3 is A or T.
  • soluble antibody expression vector including: digesting and purifying positive VH fragments and LC fragments with unique sequences respectively. Insert the VH fragment into the soluble heavy chain expression vector (IgG1), insert the LC fragment into the soluble light chain expression vector, send colony sequencing to determine and extract DNA.
  • VH fragment into the soluble heavy chain expression vector (IgG1)
  • LC fragment into the soluble light chain expression vector
  • the His-tagged FAP recombinant protein was directly coated, and after adding the antibody, the activity of the antibody binding to the antigen was detected by adding a secondary antibody (HRP-coupled anti-human IgG antibody) and HRP substrate TMB.
  • a secondary antibody HRP-coupled anti-human IgG antibody
  • HRP substrate TMB HRP substrate TMB
  • the CM5 sensor chip was selected, and the mobile phase was HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20).
  • the anti-human IgG (Fc) antibody was formulated into a 30 ⁇ g/mL solution with 10 mM sodium acetate buffer (pH 5.0), and immobilized by amino coupling.
  • Each antibody to be tested was prepared with HBS-EP+ buffer solution, and captured by the anti-human IgG (Fc) antibody on the chip channel.
  • the diluted antibody flowed through the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min, combined for 1 min, and dissociated for 15 min.
  • Table 6 show that compared with the control anti-FAP antibody 28H1 (sequence 219 and sequence 233 of the patent US9266938B2), the affinity of Ab9, Ab10, Ab14, Ab15 to the antigen FAP is 3-6 times higher, and the highest affinity of Ab15 K D reaches 2.15x10 -10 .
  • other anti-FAP antibodies obtained through simultaneous screening in the present disclosure such as Ab2, Ab4, and Ab5 (sequence not shown), have lower binding affinity to antigen than 28H1.
  • the heavy chain light chain variable region sequence of the control antibody 28H1 is as follows:
  • the binding properties of anti-FAP antibody on cells were detected by FACS experiment. Including: constructing a CHO cell line overexpressing human FAP, and plating (1E5/well). Add the antibody to be tested, 100 ⁇ L/well, the highest concentration is 100 nM, 5-fold dilution, a total of 8 concentrations, and incubate at 4 °C for 1 h. Anti-hIgG Alexa Fluor-647 was added as a secondary antibody, incubated at 4°C for 30 min at a ratio of 1:500, and detected by FACS. Antibodies that bind FAP can label cells. The relationship between the number of labeled cells overexpressing human FAP as a percentage of all cells and the concentration of the antibody is shown in Figure 1. The EC50 values for the binding of human FAP to the antibody are shown in Table 7; The relationship between the number of cells as a percentage of all cells and the antibody concentration is shown in Figure 2.
  • the alpacas were immunized with h-CD40-his once every two weeks for a total of four times.
  • For the first immunization mix 0.5 mg of antigen with 1 mL of Freund's complete adjuvant (CFA) and inject subcutaneously, and mix 0.25 mg of antigen with 1 mL of Freund's incomplete adjuvant (IFA) for subcutaneous injection in the next three immunizations.
  • Blank serum was collected before immunization, and 50 mL of peripheral blood was collected one week after the third immunization and one week after the fourth immunization to separate lymphocytes and detect the serum titer.
  • the serum titer of the alpaca was tested after four times of immunization. After the titer is qualified, isolate PBMC, extract total RNA, check the purity, reverse transcribe into DNA, and after two rounds of nested PCR, the purified vector is digested and ligated with the VHH target fragment. Electroporation, clones were selected, and phage library A library and B library were obtained.
  • the h-CD40 antigen was used as the target molecule, and the Gly-HCL acid elution method was used for screening to elute the specific phage.
  • 384 clones were randomly selected from the first and second rounds of titer determination plates, positive clones were screened by phage ELISA, and the optical density at 450nm was detected. According to the sequencing results, sequence alignment and phylogenetic tree analysis were performed on the sequences, and 16 sequences were screened out.
  • the amino acid sequences of two anti-CD40 antibodies with better functions are shown in Table 9 and Table 10.
  • the underline is the CDR under the Kabat numbering system.
  • VHHs were respectively connected to human IgG1-Fc containing the N297A mutation, and the sequence of the connected VHH-Fc fusion protein is shown in Table 11. Human IgG1-Fc is underlined and N297A mutation is bolded.
  • 9E5-SELFNS was used as a CD40 agonist positive control (see sequence 58 and sequence 59 of WO2020108611A1).
  • Plasmids were constructed, cells were transiently transfected, antibodies were expressed, and purified. After detection, the target antibody was obtained.
  • the binding properties of anti-CD40 antibody on cells were detected by FACS experiment. Including: obtaining Raji single cells, plating. Add the antibody to be tested, 100 ⁇ L/well, the highest concentration point is 100 nM, dilute 5 times to obtain a total of 8 gradient points, and incubate at 4 °C for 1 h. Anti-hIgG Alexa Fluor-647 was added as a secondary antibody, and incubated at 4°C for 30 min at a ratio of 1:500. FACS detection. A CD40 agonist was used as a positive control, and the EC 50 of each antibody is shown in Figure 3 and Table 12.
  • the EC 50 of its affinity for CD40 should not be too high, otherwise the bispecific antibody will preferentially bind to CD40, and it is more desirable that the bispecific antibody Preferentially combined with FAP, and then play a role. Therefore, A12 and A297 have the advantage of being more consistent with the above characteristics, which is also reflected in Table 13.
  • the activation effect of anti-CD40 antibody on CD40 reporter gene cells was detected, and the agonist activity of CD40 antibody was evaluated according to EC 50 .
  • preparation of HEK-Blue TM CD40L cells purchased from Invivogen Cat#hkb-cd40, which are stably transfected with human CD40 gene and NF- ⁇ B-mediated SEAP genome, and can be detected by SEAP substrate QUANTI-Blue in the supernatant Secreted SEAP content to characterize the activation level of CD40 signaling pathway
  • the medium was DMEM containing 10% FBS, 100 ⁇ g/mL Normocin, 100 ⁇ g/mL Zeocin and 30 pg/mL Blasticidin.
  • A12 and A297 were humanized.
  • the VHH variable region sequences were compared with the antibody germline database to obtain human germline templates with high homology.
  • the preferred human germline templates of antibodies A12 and A297 of the present disclosure are both IGHV3-48*03.
  • the CDR was transplanted into human FR, and the key amino acids that affect the structure and function of the antibody were back mutated to restore the binding force and activity.
  • Each humanized sequence is shown in Table 14.
  • the anti-CD40 nanobody of the present disclosure has the following general structure:
  • CDR1 is X 4 YGMK (SEQ ID NO: 39), wherein X 4 is N or R;
  • CDR2 is TIX 5 SX 6 GX 7 X 8 TYYADSVKG (SEQ ID NO: 40), wherein X 5 is N or L, X 6 is Q or H, X 7 is G or D, and X 8 is S or T;
  • CDR3 is X 9 D X 10 SDYSX 11 (SEQ ID NO: 41), wherein X 9 is V or I, X 10 is W or Y, and X 11 is P or S.
  • VHHs were ligated into human IgG1-Fc containing S267E/L328F mutation (according to EU numbering).
  • the sequence of the connected VHH-Fc fusion protein is shown in Table 15. Human IgG1-Fc is underlined, and S267E/L328F mutation is bolded.
  • VHH-Fcs were transiently transfected into cells and expressed, and the antibodies were purified. After detection, the target antibody protein is obtained.
  • CD40 plasmid CD40cDNA ORF Clone, Human, C-DYKDDDDK Tag; Sino Biological; HG10774-CF
  • PBS+2% FBS flow staining solution
  • a goat anti-human Fc antibody labeled with fluorophore Alexa Fluor 647 was added as a secondary antibody, stained in ice bath for 1 h, washed twice with PBS, and then detected with FACS for fluorescent signals on the cell surface.
  • the results are shown in Figures 5A and 5B, and the EC50 values are shown in Table 16.
  • the humanized antibodies of the present disclosure have higher affinity to CD40 high-expression cell lines.
  • Antibody EC50 (nM) Emax MFI A12 1.09 17256 A12_V1 2.13 16608 A12_V2 0.42 16609 A12_V3 1.74 16909 A297 1.12 16728 A297_V1 1.11 7300 A297_V2 1.07 12764 A297_V3 1.25 12827
  • the in vitro agonistic activity of CD40 antibody was evaluated by detecting the activation effect of CD40 antibody on HEK-Blue TM CD40L cells cross-linked by Fc ⁇ RIIb.
  • the S267E/L328F mutation in the Fc of the humanized CD40 antibody enhances the affinity of IgG1Fc to Fc ⁇ RIIb, thus also enhances the cross-linking of Fc ⁇ RIIb to the antibody.
  • Activation of HEK-Blue TM CD40L cells by FcyRIIb-mediated CD40 antibody mimics the agonist activity of CD40 antibody when fully cross-linked.
  • HEK293 cells were transiently transfected with Fc ⁇ RIIb plasmid (CD32B/Fcgr2b cDNA ORF Clone, Human, N-His tag; Sino Biological; HG10259-NH) to obtain HEK293 cells highly expressing Fc ⁇ RIIb.
  • the antibody to be tested was incubated overnight at 37°C.
  • CD40 antibody The activation effect of CD40 antibody on HEK-Blue TM CD40L cells mediated by Fc ⁇ RIIb is shown in Figure 6A and 6B, and the EC50 value and Emax value (relative fluorescence intensity) are shown in Table 17.
  • A12V2 and A297V3 were selected; according to the screening results of anti-FAP antibodies, Ab10 was selected to construct anti-FAP/CD40 bispecific antibodies.
  • the bispecific antibody uses Ab10 as the IgG backbone, and one or two or three anti-CD40 nanobodies are connected in series at the C-terminals of the two heavy chains of Ab10.
  • the linker between CD40 nanobodies and the C-terminus of Ab10 heavy chain adopts "GGGGSGGGGS".
  • Each bispecific antibody contains 2-valent, 4-valent and 6-valent CD40 nanobodies, respectively, as shown in FIG. 7 .
  • Ab10 represents the anti-FAP antibody used
  • A12V2 represents the humanized anti-CD40 antibody used
  • the last 2 represents the valency of CD40 is 2. All other antibodies adopt this naming convention.
  • the method in 2.5 of Example 2 was used for transient transfection, expression and purification of antibodies. After detection, the target bispecific antibody molecule is obtained.
  • the amino acid sequence of each bispecific antibody molecule is as follows.
  • the light chains of Ab10-A12V2-4, Ab10-A12V2-6, Ab10-A297V3-2, Ab10-A297V3-4, and Ab10-A297V3-6 are all shown in SEQ ID NO:43.
  • CM5 sensor chip was selected, and the mobile phase was HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20).
  • HBS-EP+ buffer solution 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20.
  • Anti-human IgG (Fc) antibodies were immobilized by amino coupling. Each antibody to be tested was prepared as a ligand with HBS-EP+ buffer solution, and captured by the anti-human IgG (Fc) antibody on the chip channel.
  • Antigenic proteins of different species were used as analytes. The results are shown in Table 18 and Table 19, which show that the preparation of bispecific antibodies does not affect the binding of antibodies to FAP, and the bispecific antibodies have lower affinity to CD40, and will preferentially bind to FAP to play a role.
  • FACS was used to detect the binding of anti-FAP/CD40 bispecific antibody to CHOK1 cell lines stably transfected with high expression of human FAP, cynomolgus monkey FAP and mouse FAP. It shows that the bispecific antibody can bind to the FAP antigen on the cell surface of human, mouse, and cynomolgus monkey, and the binding ability is similar to that of the monoclonal antibody, see Figures 8A-8C, and the EC 50 values are shown in Table 20.
  • Table 20 EC 50 values of anti-FAP/CD40 bispecific antibodies binding to FAP antigen on the surface of human and mouse cells
  • FACS was used to detect the binding of bispecific antibodies to CD40 on the cell surface, including HEK-BlueTM CD40L cells highly expressing human CD40 (Invivogen, Cat#hkb-cd40), and transiently transfected cynomolgus monkey CD40 plasmid (Sino Biological, cat#CG90970- UT) HEK293 cells that highly express cynomolgus monkey CD40.
  • the results showed that the bispecific antibody could bind to CD40 on the cell membrane surface of human and cynomolgus monkeys. See Figure 9A, 9B, Figure 11 and Table 21 for details.
  • Table 21 EC 50 values of bispecific antibodies binding to human cell surface CD40 antigen
  • the antibody to be tested was added in a gradient dilution, 1 ⁇ g/mL LPS was used as a positive control, and 100 nM hIgG1 and no antibody group were used as a negative control. After culturing for 48 hours, the expression of dendritic cell surface molecule CD83 was detected by FACS.
  • B-hCD40 humanized mice were from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. (species Mus musc ⁇ Lus, strain C57BL/6, female). Transfect mouse full-length FAP plasmid in mouse intestinal cancer MC38 cells to construct a stable transfection strain, inoculate cells 5 ⁇ 10 5 /0.1 mL/only in the right axillary skin of mice, when the average tumor volume grows to 80-100mm 3 At the same time, mice with appropriate individual tumor volumes were selected and randomly divided into 6 mice in each group. Mice were given intraperitoneal injection twice a week. Peripheral blood was drawn 48 hours after the first administration to detect the number of B cells and the expression of CD86 on the cell surface.
  • ALT Alanine Aminotransferase
  • AST Aspartate aminotransferase
  • the antitumor activity of Ab10-A297V3-2 or Ab10-A297V3-4 was compared with the CD40 agonist mAb 9E5-mIgG1 at a single dose.
  • 9E5-mIgG1 and 9E5-SELFNS have the same variable region, but the heavy and light chain constant regions adopt the constant region of mouse mIgG1 heavy chain and the constant region of mouse kappa chain. Since the activity of CD40 agonist monoclonal antibody depends on the cross-linking of Fc ⁇ RIIb to its Fc, 9E5-mIgG1 using the heavy chain constant region of mouse IgG1 can better display CD40 agonist activity in mice. In the drug efficacy experiment in mice, 9E5-mIgG1 is a better control antibody than 9E5-SELFNS.
  • the anti-tumor activity of Ab10-A297V3-4 is stronger than that of CD40 monoclonal antibody 9E5-mIgG1 at the same molar dose in terms of TGI (Tumor growth inhibition, tumor growth inhibition) and CR (Complete Response, complete response).
  • TGI Tumor growth inhibition, tumor growth inhibition
  • CR Complete Response, complete response
  • the antitumor activity of Ab10-A297V3-2 at twice the molar concentration was still weaker than that of Ab10-A297V3-4, and comparable to that of 9E5-mIgG1.
  • peripheral CD40 The activation of peripheral CD40 by bispecific antibody molecules was detected by detecting the activation of peripheral blood B lymphocytes in mice, and the results were consistent with those of in vitro experiments.
  • Ab10-A297V3-4 can partially activate peripheral B lymphocytes due to its partially FAP-independent CD40 activation activity, while Ab10-A297V3-2 has no significant peripheral B lymphocytes detected because the activation of CD40 is completely dependent on FAP.
  • Activation of cells One-Way ANOVA.
  • Table 22 The information of the tested antibodies is shown in Table 22, and the in vivo drug efficacy results are shown in Figure 13A (P ⁇ 0.0001 for each group of antibodies relative to the hIgG1 control group) and Figure 13B.
  • 9E5-mIgG1 can cause a certain degree of thrombocytopenia, which is similar to liver toxicity; Ab10-A297V3-2 did not cause thrombocytopenia; Ab10-A297V3-4 It can induce the same degree of platelet drop as 9E5-mIgG1, see Fig. 13D. See Table 23 for details of the in vivo dosage and tumor growth inhibition rate of Ab10-A297V3-2/4 in mice.
  • TGI (tumor volume of the blank group on the day-group tumor volume on the day of administration)/(tumor volume of the blank group on the day)*100%
  • Ab10-A297V3-4 was compared with another quadrivalent CD40 bispecific antibody without FAP binding function (isotype-A297V3-4) antitumor activity.
  • Isoform-A297V3-4 is identical in construction to Ab10-A297V3-4, except that the anti-FAP portion of Ab10 is replaced by another isotype antibody that does not bind any murine protein.
  • Ab10-A12V2-2 and Ab10-A297V3-2 have similar pharmacological characteristics in mice (both doses are P ⁇ 0.0001 compared with the hIgG1 control group), and the antitumor The activity is better than 9E5-mIgG1. Elevating the dose can not further improve its anti-tumor activity, but there is no increase in ALT/AST and decrease in platelets, showing a wider therapeutic window and lower side effects.
  • this example explores the efficacy and toxicity of a single dose of Ab10-A12V2-4 in mice.
  • Ab10-A12V2-4 group has P ⁇ 0.0001, which is better than 9E5-mIgG1 in terms of anti-tumor activity, and there is no increase in ALT/AST and decrease in platelets, see Table 27 and Figures 16A-16D.
  • the disclosure compares the pharmacokinetic characteristics of anti-FAP/CD40 bispecific antibodies from three different directions in mFAP-MC38 tumor-bearing hCD40 humanized mice. Antibodies of different concentrations were given by intravenous injection, and then blood was collected at different time points, and the blood drug concentration/time curve of each antibody molecule was analyzed by detecting the content of human Fc in the mouse blood.
  • HRP-labeled goat anti-human IgG-Fc secondary antibody 1% BSA PBS dilution, 1:20000

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Abstract

FAP/CD40结合分子及其医药用途；具体地，提供FAP结合分子、CD40结合分子及FAP/CD40结合分子，以及其治疗、预防疾病(例如肿瘤或癌症)的方法和医药用途。

Description

FAP/CD40结合分子及其医药用途
本公开要求2021年08月24日提交的中国专利申请(申请号CN202110973560.5)的优先权。
技术领域
本公开涉及生物医药领域，特别是治疗或干预与CD40/CD40L信号通路相关疾病的领域。具体而言，本公开涉及FAP/CD40结合分子、其药物组合物，及用于治疗疾病，特别是肿瘤或癌症的方法和相关制药用途。
背景技术
CD40(TNFRSF5)是一种跨膜磷酸化糖蛋白，属于肿瘤坏死因子受体超家族(TNFRS)成员。CD40在多种细胞类型中表达，包括B细胞、滤泡树突细胞(DC)、上皮细胞、单核细胞、巨噬细胞、平滑肌细胞和肿瘤细胞。其配体CD40L主要表达在激活的T细胞、激活的B细胞、血小板和平滑肌细胞等。CD40L结合使CD40多聚化，产生下游激活、生长和分化信号。CD40信号转导激活多种下游信号通路，如NF-κB、MAPK和STAT3(Pype S,et al.J Biol Chem.2000Jun.16；275(24):1858693)，这些途径通过调节激活蛋白c-Jun、ATF2和Rel转录因子来调节基因表达。CD40与CD40L结合诱导静息B细胞增殖，免疫球蛋白转换，抗体分泌，并在组织生发中心发育和B细胞存活发挥重要作用，所有这些对于体液免疫反应是必不可少的(Kehry M R.J Immunol 1996；156:2345-2348)。CD40L与DC上的CD40结合，诱导DC成熟，表现为共刺激因子B7家族(CD80，CD86)的表达上调和促炎细胞因子如白细胞介素12(IL-12)的分泌增加。CD40和CD40L相互作用为T细胞激活提供共刺激信号，并促进DC细胞递呈抗原给T细胞。
随着以CD40、CTLA-4和PD-L1为靶点的免疫检查点抑制(ICI)疗法在肿瘤治疗中取得的临床胜利，免疫治疗已成为第三代肿瘤治疗中最令人期待的研究领域。其中通过激活CD40从而增加DC细胞对T细胞的抗原递呈及激活为机制的抗肿瘤免疫疗法已展示出临床有效性。但是，CD40存在系统性激活，这会导致一些靶点特异的毒性,如细胞因子风暴、肝毒性、血液毒性以及外周CD40激活导致的静脉血栓。这些毒性限制了CD40激动型抗体的治疗窗口。Pfizer公司的CD40激动剂CP-870、893和Medimmune公司的CD40L-Fc融合蛋白MEDI5083(AstraZeneca公司的可激活CD40信号通路的新型融合蛋白，由3个串联的CD40L与IgG4-Fc的融合蛋白)先后终止了临床研究。因此。在第二代CD40激动剂抗体的研发中，研究者期望通过肿瘤抗原(Tumor-associated antigen，TAA)介导CD40激活，使得CD40在肿瘤内部特异性激活，同时降低外周CD40的激活，从而提高CD40激动型抗体的治疗窗。
成纤维细胞活化蛋白FAP(fibroblast activation protein)α是肿瘤相关抗原，FAP在健康成年人的正常组织中表达量较低，选择性地表达在93％的肿瘤组织中，其中30％为高表达，例如结肠癌、胰腺癌、乳腺癌、胃癌、前列腺癌、膀胱癌和口腔鳞癌。本公开提供了抗FAP抗体、抗CD40抗体及两者的双特异性抗体，所述双特异性抗体能通过FAP介导针对肿瘤特异性的CD40激活，对APC(例如树突细胞DC)具有促成熟、激活作用，能去除肝毒性、外周血液毒性及其它外周毒性，具有优良的给药窗口和成药性，为临床应用提供了解决方案。
发明内容
本公开提供了具有新结构的CD40结合分子、FAP结合分子、FAP/CD40结合分子(例如，抗FAP/CD40双特异性抗体)，其编码核酸、载体、宿主细胞、药物组合物，及其用于治疗、缓解或预防细胞增殖性疾病(例如肿瘤或癌症)的方法和相关制药用途。
CD40结合分子
本公开提供了一种CD40结合分子，其包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2、CDR3，其中：
1)所述免疫球蛋白单一可变结构域中的CDR1包含或为SEQ ID NO:39所示的氨基酸序列，和/或CDR2包含或为SEQ ID NO:40所示的氨基酸序列，和/或CDR3包含或为SEQ ID NO:41所示的氨基酸序列；
2)所述免疫球蛋白单一可变结构域中的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:39、40、41所示；
3)所述免疫球蛋白单一可变结构域中的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:24、25、26所示，或如SEQ ID NO:27、28、29所示；
4)所述免疫球蛋白单一可变结构域中的氨基酸序列包含或如SEQ ID NO:22、23、32至38任一所示，或与SEQ ID NO:22、23、32至38任一具有至少90％、至少95％、至少98％、至少99％的序列同一性；和/或
5)所述免疫球蛋白单一可变结构域中的CDR1、CDR2和CDR3如SEQ ID NO:22中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:23中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:32中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:33中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:34中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:35中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:36中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:37中的CDR1、CDR2和CDR3所示，或如SEQ ID NO:38中的CDR1、CDR2和CDR3所示；所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；一些具体实施方案中，所述CDR是根据Kabat编号系统定义的。
一些实施方案中，提供CD40结合分子，其包含前述任意一个或多个(例如，1、2、3、4、5、6、7、8、9、10个)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域可以是相同的或不同的。
一些实施方案中，前述免疫球蛋白单一可变结构域为纳米抗体或VHH。
一些实施方案中，前述CD40结合分子中还包含人免疫球蛋白Fc区。一些具体实施方案中，所述Fc区是人IgG1、IgG2、IgG4的Fc区。一些具体实施方案中，所述人IgG1的Fc区具有能够增加与FcγRIIb亲和力、减弱ADCC效应的突变，例如S267E/L328F。减弱ADCC效应的示例性突变是IgG1上的L234A/L235A，IgG2上的V234A/G237A/P238S/H268A/V309L/A330S/P331S，IgG4上的F234A/L235A，IgG4上的S228P/F234A/L235A，IgG1、IgG2、IgG3或IgG4上的N297A，IgG2上的V234A/G237A，IgG1上的K214T/E233P/L234V/L235A/G236缺失/A327G/P331A/D365E/L358M，IgG2上的H268Q/V309L/A330S/P331S，IgG1上的S267E/L328F，IgG1上的L234F/L235E/D265A，IgG1上的L234A/L235A/G237A/P238S/H268A/A330S/P331S，IgG4上的S228P/F234A/L235A/G237A/P238S，以及IgG4上的S228P/F234A/L235A/G236缺失/G237A/P238S。还可使用杂合IgG2/4Fc域，例如具有来自IgG2的残基117-260和来自IgG4的残基261-447的Fc。增加与FcγRIIb亲和力的示例性突变还包括IgG1上的E233D/G237D/P238D/H268D/P271G/A330R。
一些实施方案中，前述Fc区可以使所述抗CD40抗体或其抗原结合片段形成二聚体或多聚体分子。
一些实施方案中，前述CD40结合分子中的免疫球蛋白单一可变结构域与Fc区直接或通过接头相连接。所述接头可以是长度为1-20个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如G 4S、GS、GAP、(G 4S) 2、(G 4S) 3、(G 4S) 4、(G 4S) 5、ASGS等。
一些实施方案中，提供CD40结合分子，其包含如SEQ ID NO：57至63中任一所示或与之具有至少80％同一性的氨基酸序列。
一些实施方案中，前述CD40结合分子与至少一个结合其它抗原的抗体直接或间接相连接，例如，形成异二聚体。
一些实施方案中，前述CD40结合分子，具有选自以下至少一项的活性：
(a)以≤10 -7M K D值与人CD40或其表位结合；
(b)增加或促进APC(例如树突细胞DC)的活化和/或增殖；和
(c)抑制肿瘤生长和/或转移。
一些实施方案中，前述CD40结合分子抑制CD40与CD40L的结合，或与CD40L竞争性结合CD40。
一些实施方案中，前述CD40结合分子能够抑制肿瘤生长和/或转移至少约10％，例如至少约20％、约30％、约40％、约50％、约60％、约70％、约80％。
一些实施方案中，前述CD40结合分子使CD40L与CD40的结合降低至少45％、至少50％、至少60％、至少75％、至少80％、至少90％或至少95％。
一些实施方案中，前述CD40结合分子以10 -7M、10 -8M、10 -9M、10 -10M、10 -11M或更低的K D结合人CD40。
一些实施方案中，前述CD40结合分子为抗CD40抗体或其抗原结合片段。
一些实施方案中，前述CD40结合分子为激动剂型抗CD40抗体或其抗原结合片段。
一些实施方案中，前述抗CD40抗体为骆驼抗体、嵌合抗体、人源化抗体、全人抗体。一些实施方案中，前述抗CD40抗体为纳米抗体或VHH。
一些实施方案中，前述抗CD40抗体或其抗原结合片段包括但不限于：线性抗体、单链抗体、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)。
一些实施方案中，前述抗CD40抗体或其抗原结合片段涵盖变体，例如与SEQ ID NO:22、23、32至38中任一相比，包含一或多个氨基酸取代，例如，保守氨基酸取代；例如，所述变体包含1、2、3、4、5、6、7、8、9或10个保守氨基酸取代。
一些实施方案中，本公开提供抗CD40抗体或其抗原结合片段，其是SEQ ID NO:22、23经过亲合力成熟获得的，所述经亲合力成熟的抗CD40抗体可以在一个或多个CDR中具有一个或多个变化，所述变化导致对CD40的亲合力相比于亲本抗CD40抗体有所增加。
一些实施方案中，提供抗CD40抗体或其抗原结合片段，其与前述抗CD40抗体或其抗原结合片段结合或竞争结合相同的表位。
一些实施方案中，提供抗CD40抗体或其抗原结合片段，其阻断前述抗CD40抗体或其抗原结合片段与CD40(例如人CD40)的结合。
一些实施方案中，提供抗CD40抗体或其抗原结合片段，其与CD40(例如人CD40)的结合被前述抗CD40抗体或其抗原结合片段阻断。
一些实施方案中，提供缀合物，包含前述本公开的抗CD40抗体或其抗原结合片段。
本公开中，“至少90％(序列)同一性”涵盖至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性；“至少80％(序列)同一性”涵盖至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性。
FAP结合分子
本公开提供了一种FAP结合分子，其包含重链可变区和轻链可变区，所述重 链可变区包含HCDR1、HCDR2、HCDR3，所述轻链可变区包含LCDR1、LCDR2、LCDR3，其中：
1)所述HCDR1的氨基酸序列如SEQ ID NO:9所示，和/或所述HCDR2的氨基酸序列如SEQ ID NO:10所示，和/或所述HCDR3的氨基酸序列如SEQ ID NO:18所示；和/或所述LCDR1的氨基酸序列如SEQ ID NO:12所示，和/或所述LCDR2的氨基酸序列如SEQ ID NO:13所示，和/或所述LCDR3的氨基酸序列如SEQ ID NO:19所示；
2)所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、18所示；和/或所述LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、19所示；
3)所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、11所示，所述LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、14所示；
所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、15所示，所述LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、14所示；
所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、15所示；所述LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、16所示；或
所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、11所示；所述LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、17所示；
4)重链可变区包含如SEQ ID NO：1所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：2所示或与之具有至少90％同一性的氨基酸序列；
重链可变区包含如SEQ ID NO：3所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：4所示或与之具有至少90％同一性的氨基酸序列；
重链可变区包含如SEQ ID NO：5所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：6所示或与之具有至少90％同一性的氨基酸序列；或
重链可变区包含如SEQ ID NO：7所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：8所示或与之具有至少90％同一性的氨基酸序列；
5)重链可变区中包含SEQ ID NO：1中的HCDR1、HCDR2、HCDR3，轻链可变区中包含SEQ ID NO：2中的LCDR1、LCDR2、LCDR3；
重链可变区中包含SEQ ID NO：3中的HCDR1、HCDR2、HCDR3，轻链可变区中包含SEQ ID NO：4中的LCDR1、LCDR2、LCDR3；
重链可变区中包含SEQ ID NO：5中的HCDR1、HCDR2、HCDR3，轻链可变区中包含SEQ ID NO：6中的LCDR1、LCDR2、LCDR3；或
重链可变区中包含SEQ ID NO：7中的HCDR1、HCDR2、HCDR3，轻链可变区中包含SEQ ID NO：8中的LCDR1、LCDR2、LCDR3，其中，所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，例如为Kabat编号系统定义的。
一些实施方案中，提供FAP结合分子(例如，抗FAP抗体或其抗原结合片段)，包含前述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3中的任一个或几个的组合。
一些实施方案中，前述FAP结合分子还包含人免疫球蛋白Fc区。一些具体实施方案中，所述Fc区是人IgG1、IgG2、IgG4的Fc区。一些实施方案中，所述人IgG1的Fc具有去除或降低Fc效应器功能的突变，例如IgG1上具有N297A或D265A/N297A。其它去除或降低Fc效应器功能的示例性突变包括IgG1上的L234A/L235A、L234A/L235A/P329G、L234E、L234F、L234E/L235F、L234E/L235F/P329G，IgG2上的V234A/G237A/P238S/H268A/V309L/A330S/P331S，IgG4上的F234A/L235A，IgG4上的S228P/F234A/L235A，IgG2或IgG4上的N297A，IgG2上的V234A/G237A，IgG1上的K214T/E233P/L234V/L235A/G236缺失/A327G/P331A/D365E/L358M，IgG2上的H268Q/V309L/A330S/P331S，IgG1上的S267E/L328F，IgG1上的L234F/L235E/D265A，IgG1上的L234A/L235A/G237A/P238S/H268A/A330S/P331S，IgG4上的S228P/F234A/L235A/G237A/P238S。
一些实施方案中，提供FAP结合分子(例如，抗FAP抗体或其抗原结合片段)，其包含重链(HC)和轻链(LC)，其中：
重链为SEQ ID NO：49所示或与之具有至少80％同一性的氨基酸序列，轻链为SEQ ID NO：50所示或与之具有至少80％同一性的氨基酸序列；
重链为SEQ ID NO：51所示或与之具有至少80％同一性的氨基酸序列，轻链为SEQ ID NO：52所示或与之具有至少80％同一性的氨基酸序列；
重链为SEQ ID NO：53所示或与之具有至少80％同一性的氨基酸序列，轻链为SEQ ID NO：54所示或与之具有至少80％同一性的氨基酸序列；或
重链为SEQ ID NO：55所示或与之具有至少80％同一性的氨基酸序列，轻链为SEQ ID NO：56所示或与之具有至少80％同一性的氨基酸序列。
一些实施方案中，前述FAP结合分子的重链可变区有0至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸变化；轻链可变区有0至10个(1、2、3、4、5、6、 7、8、9、10个)氨基酸变化。在一些具体实施方案中，所述氨基酸变化为保守的替换、取代或修饰，和/或不影响功能的缺失、添加。
一些实施方案中，前述FAP结合分子，具有选自以下至少一项的活性：
(a)以≤10 -7的K D值与人FAP或其表位结合；
(b)增加或促进APC(例如树突细胞DC)的活化和/或增殖；
(c)抑制肿瘤生长和/或转移。
一些实施方案中，前述FAP结合分子以10 -7M、10 -8M、10 -9M、10 -10M、10 -11M或更低的K D结合FAP(例如人FAP)或其片段。
一些实施方案中，前述FAP结合分子为抗FAP抗体或其抗原结合片段；一些具体实施方案中，为嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。
一些实施方案中，前述抗FAP抗体的抗原结合片段包括但不限于：Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，例如为scFv、Fv、Fab或Fab’片段。
一些实施方案中，提供抗FAP抗体或其抗原结合片段，其与前述抗FAP抗体或其抗原结合片段结合或竞争结合相同的FAP(例如人FAP)表位。
一些实施方案中，提供抗FAP抗体或其抗原结合片段，其阻断前述抗FAP抗体或其抗原结合片段与FAP(例如人FAP)的结合。
一些实施方案中，提供抗FAP抗体或其抗原结合片段，其与FAP(例如人FAP)的结合被前述抗FAP抗体或其抗原结合片段阻断。
一些实施方案中，提供缀合物，其包含前述抗FAP抗体或其抗原结合片段。
FAP/CD40结合分子
本公开提供一种FAP/CD40结合分子，其包含特异性结合FAP的第一抗原结合结构域和特异性结合CD40的第二抗原结合结构域，所述特异性结合CD40的第二抗原结合结构域包含(至少一个)免疫球蛋白单一可变结构域。
一些实施方案中，所述FAP/CD40结合分子包含一个特异性结合CD40的免疫球蛋白单一可变结构域。在另一些实施方案中，所述FAP/CD40结合分子包含2、3、4、5、6、7、8、9、10或更多个特异性结合CD40的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域可以是相同的或不同的。
一些实施方案中，所述FAP/CD40结合分子包含至少一个(例如2、3、4个)特异性结合FAP的抗原结合结构域。
一些实施方案中，前述FAP/CD40结合分子中，特异性结合CD40的免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，所述CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:39、40、41所示。一些具体实施方案中，所述CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:24、25、26所示；或所述CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:27、28、29所示。
一些实施方案中，提供一种FAP/CD40结合分子，其包含特异性结合FAP的第一抗原结合结构域和特异性结合CD40的第二抗原结合结构域，所述特异性结合FAP的第一抗原结合结构域包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2、HCDR3，所述轻链可变区包含LCDR1、LCDR2、LCDR3，其中：
HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、18所示；LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、19所示。
一些具体实施方案中，前述特异性结合FAP的第一抗原结合结构域中：
HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、11所示；LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、14所示；
HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、15所示；LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、14所示；
HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、15所示；LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、16所示；或
HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:9、10、11所示；LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:12、13、17所示。
一些实施方案中，前述FAP/CD40结合分子中，所述特异性结合FAP的第一抗原结合结构域的重链可变区包含如SEQ ID NO：1所示或与之具有至少80％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：2所示或与之具有至少80％同一性的氨基酸序列；
重链可变区包含如SEQ ID NO：3所示或与之具有至少80％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：4所示或与之具有至少80％同一性的氨基酸序列；
重链可变区包含如SEQ ID NO：5所示或与之具有至少80％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：6所示或与之具有至少80％同一性的氨基酸序列；或
重链可变区包含如SEQ ID NO：7所示或与之具有至少80％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：8所示或与之具有至少80％同一性的氨基酸序列。
一些实施方案中，前述FAP/CD40结合分子中，所述特异性结合FAP的第一抗原结合结构域包含重链(HC)和轻链(LC)；
例如，重链为IgG1或IgG4同种型，轻链为Kappa同种型；
一些具体实施方案中，重链为SEQ ID NO：49所示或与之具有至少80％同一性的氨基酸序列，轻链为SEQ ID NO：50所示或与之具有至少80％同一性的氨基酸序列；
重链为SEQ ID NO：51所示或与之具有至少80％同一性的氨基酸序列，轻链 为SEQ ID NO：52所示或与之具有至少80％同一性的氨基酸序列；
重链为SEQ ID NO：53所示或与之具有至少80％同一性的氨基酸序列，轻链为SEQ ID NO：54所示或与之具有至少80％同一性的氨基酸序列；或
重链为SEQ ID NO：55所示或与之具有至少80％同一性的氨基酸序列，轻链为SEQ ID NO：56所示或与之具有至少80％同一性的氨基酸序列。
一些实施方案中，前述FAP/CD40结合分子中，所述特异性结合FAP的第一抗原结合结构域包含重链可变区和轻链可变区，其中：
所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的重链可变区的N端；
所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的重链可变区的C端；
所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的轻链可变区的N端；和/或
所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的轻链可变区的C端。
一些实施方案中，前述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域与特异性结合FAP的第一抗原结合结构域直接或通过连接子相连接；例如，所述连接子为具有如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10)；例如，所述连接子为(G 4S) 2、(G 4S) 3、(G 4S) 4所示的氨基酸序列。
一些实施方案中，前述FAP/CD40结合分子中，特异性结合CD40的第二抗原结合结构域为多价(例如，一个前述FAP/CD40结合分子中含有1、2、3、4、5、6、7、8、9、10个特异性结合CD40的第二抗原结合结构域)。一些具体实施方案中，特异性结合CD40的第二抗原结合结构域为二价、四价或六价。一些具体实施方案中，特异性结合CD40的第二抗原结合结构域包含2、3、4、5或6个所述免疫球蛋白单一可变结构域。
一些实施方案中，提供FAP/CD40结合分子，其包含第一多肽链和第二多肽链，其中，第一多肽链包含如SEQ ID NO：42、44-48中任一所示或与之具有至少80％同一性的氨基酸序列，第二多肽链包含如SEQ ID NO：43所示或与之具有至少80％同一性的氨基酸序列。一些具体实施方案中，一个FAP/CD40结合分子中包含两条第一多肽链和两条第二多肽链；一些具体实施方案中，所述两条第一多肽链是相同的，所述两条第二多肽链是相同的。
一些实施方案中，前述FAP/CD40结合分子能够抑制肿瘤生长(例如体积增大、瘤重增加)和/或转移(例如多器官或多组织转移、远端转移)至少约10％，例如至少约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％。
一些实施方案中，前述FAP/CD40结合分子为抗FAP/CD40双特异性抗体或其 抗原结合片段，所述抗原结合片段包括但不限于：Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，例如为scFv、Fv、Fab或Fab’片段。
一些实施方案中，抗FAP/CD40双特异性抗体含有前述本公开提供的特异性结合CD40的第二抗原结合结构域中的免疫球蛋白单一可变结构域，和前述本公开提供的特异性结合FAP的第一抗原结合结构域中的重链可变区(VH)和轻链可变区(VL)。
一些实施方案中，抗FAP/CD40双特异性抗体中：
特异性结合FAP的第一抗原结合结构域为第一抗体，其包括重链(HC)和轻链(LC)；和
特异性结合CD40的第二抗原结合结构域为第二抗体，其是VHH，具有前述本公开提供的CD40结合分子中的CDR1、CDR2、CDR3。
一些具体实施方案中，所述VHH作为第二抗体位于第一抗体的重链或轻链的N端和/或C端。
一些具体实施方案中，抗FAP/CD40双特异性抗体包含1个第一抗体和1、2、3、4、5、6、7、8、9、10个(例如2个、4个、6个)VHH的第二抗体；所述第一抗体包括两条HC和两条LC，第一抗体的一条HC的VH与一条LC的VL形成抗原结合部位，另一条HC的VH与另一条LC的VL形成抗原结合部位。
一些具体实施方案中，抗FAP/CD40双特异性抗体或其抗原结合片段的第一抗体可以连接有1、2、3、4、5、6、7、8个VHH的第二抗体，所述VHH的第二抗体可以是相同的或不同的，可以均连接在第一抗体的重链N端，或均连接在第一抗体的重链C端，或均连接在第一抗体的轻链N端，或均连接在第一抗体的轻链C端，或重链N端、重链C端、轻链N端、轻链C端的任意组合。一些具体实施方案中，抗FAP/CD40双特异性抗体包含第一多肽链和第二多肽链：
(i)第一多肽链从N端至C端为：[第一抗体的重链]-接头1-[第二抗体]；第二多肽链为第一抗体的轻链；
(ii)第一多肽链从N端至C端为：[第一抗体的重链]-接头1-[第二抗体] 1-接头2-[第二抗体] 2；第二多肽链为第一抗体的轻链；
(iii)第一多肽链从N端至C端为：[第一抗体的重链]-接头1-[第二抗体] 1-接头2-[第二抗体] 2-接头3-[第二抗体] 3；第二多肽链为第一抗体的轻链；
(iv)第一多肽链从N端至C端为：[第二抗体]-接头1-[第一抗体的重链]；第二多肽链为第一抗体的轻链；
(v)第一多肽链从N端至C端为：[第二抗体] 2-接头2-[第二抗体] 1-接头1-[第一抗体的重链]；第二多肽链为第一抗体的轻链；
(vi)第一多肽链从N端至C端为：[第二抗体] 3-接头3-[第二抗体] 2-接头2-[第 二抗体] 1-接头1-[第一抗体的重链]；第二多肽链为第一抗体的轻链；
(vii)第一多肽链从N端至C端为：[第二抗体] 1-接头1-[第一抗体的重链]-接头2-[第二抗体] 2；第二多肽链为第一抗体的轻链；
(viii)第一多肽链从N端至C端为：[第二抗体] 1-接头1-[第二抗体] 2-接头2-[第一抗体的重链]-接头3-[第二抗体] 3；第二多肽链为第一抗体的轻链；
(ix)第一多肽链从N端至C端为：[第二抗体] 1-接头1-[第一抗体的重链]-接头2-[第二抗体] 2-接头3-[第二抗体] 3；第二多肽链为第一抗体的轻链；
其中，[第二抗体] 1、[第二抗体] 2、[第二抗体] 3可以相同或不同。
一些实施方案中，前述抗FAP/CD40双特异性抗体中的VHH的第二抗体直接或通过连接子与第一抗体连接。所述连接子(接头)选自：如(G mS n) x或(GGNGT) x或(YGNGT) x所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，x独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。例如，连接子为G 4S、(G 4S) 2、(G 4S) 3、(G 4S) 4、(G 4S) 5、(G 4S) 6所示的氨基酸序列。一些实施方案中，接头1、接头2、接头3可以相同或不同。
一些实施方案中，抗FAP/CD40双特异性抗体的第一抗体的重链包含重链可变区(VH)和重链恒定区(CH)，轻链包含轻链可变区(VL)和轻链恒定区(CL)。第一抗体可以为全长抗体。
一些实施方案中，抗FAP/CD40双特异性抗体第一抗体的重链为IgG同种型，例如IgG1、IgG2、IgG3或IgG4，例如为IgG1同种型；和/或，所述第一抗体的轻链为Kappa同种型。
一些实施方案中，抗FAP/CD40双特异性抗体的两条HC包含相同的CDR和/或两条LC包含相同的CDR。一些具体实施方案中，第一抗体的两条HC包含相同的VH和/或两条LC包含相同的VL。一些具体实施方案中，第一抗体的两条HC具有相同的氨基酸序列和/或两条LC具有相同的氨基酸序列。
一些实施方案中，抗FAP/CD40双特异性抗体两个VHH的第二抗体具有相同或不相同的氨基酸序列。例如，两个所述VHH的第二抗体具有相同的氨基酸序列。
一些实施方案中，抗FAP/CD40双特异性抗体包含两条第一多肽链和两条第二多肽链，其中对于每条多肽链：a)第一多肽链各自独立地包含VHH的第二抗体和第一抗体的重链(HC)；和b)第二多肽链各自独立地包含第一抗体的轻链(LC)；其中，VHH通过接头与第二抗体的HC的N端和/或C端相连；
或者，i)第一多肽链各自独立地包含第一抗体的重链(HC)；和ii)第二多肽链各自独立地包含VHH的第二抗体和第一抗体的轻链(LC)；其中，VHH直接或通过连接子与第一抗体的LC的N端和/或C端相连。
一些具体实施方案中，抗FAP/CD40双特异性抗体包含两条相同的第一多肽链和两条相同的第二多肽链。一些实施方案中，提供与本公开的抗FAP/CD40双特异 性抗体竞争性结合相同表位的抗体。
一些实施方案中，在本公开的FAP/CD40结合分子或抗FAP/CD40双特异性抗体的Fc区引入突变。所述突变例如是使得IgG的Fc效应器功能去除或降低的突变，包括但不限于：IgG1的N297A或D265A/N297A，或IgG1上的L234A/L235A、L234A/L235A/P329G、L234E、L234F、L234E/L235F、L234E/L235F/P329G，IgG2上的V234A/G237A/P238S/H268A/V309L/A330S/P331S，IgG4上的F234A/L235A，IgG4上的S228P/F234A/L235A，IgG2或IgG4上的N297A，IgG2上的V234A/G237A，IgG1上的K214T/E233P/L234V/L235A/G236缺失/A327G/P331A/D365E/L358M，IgG2上的H268Q/V309L/A330S/P331S，IgG1上的S267E/L328F，IgG1上的L234F/L235E/D265A，IgG1上的L234A/L235A/G237A/P238S/H268A/A330S/P331S，IgG4上的S228P/F234A/L235A/G237A/P238S。
在本文中Fc区所包含的突变的上下文中，“/”表示“和”，例如，“D265A/N297A”表示“D265A和N297A”，即，Fc中包含D265A和N297A突变；突变的氨基酸位置是根据EU编号系统编号的。
一些实施方案中，前述本公开提供的FAP/CD40结合分子或抗FAP/CD40双特异性抗体具有如下的一项或多项特征：
(a)以≤10 -7的K D值与人FAP或其表位结合；
(b)以≤10 -7的K D值与人CD40或其表位结合；
(c)诱导CD40表达性抗原呈递细胞(APC)的免疫刺激；
(d)增加APC(例如树突细胞)的活化，和/或促进APC(例如树突细胞)的增殖；
(e)刺激肿瘤特异性T细胞应答；
(f)引起或促进肿瘤细胞的凋亡；和/或
(g)抑制肿瘤生长和/或转移。
此外，本公开的抗CD40抗体、抗FAP抗体、FAP/CD40结合分子对热处理均具有抗性或具有较高的稳定性。例如，在40℃下处理30天未见明显聚集或降解，至少在60℃下稳定。
多核苷酸
本公开提供编码本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段的多核苷酸。本公开的核酸可为RNA、DNA或cDNA。一些实施方案中，本公开的核酸是基本上分离的核酸。
本公开的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提供CD40结合分子体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主 中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段的表达有用或必需的调控元件及其它元件例如为启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的核酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
宿主细胞
本公开提供表达或能够表达一种或多种本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段，和/或含有本公开的核酸或载体的重组宿主细胞。
一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其它细胞。
本公开的细胞不能发育成完成的植株或动物个体。
生产或制备方法
本公开提供制备本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段的方法，所述方法通常包含以下步骤：
-在允许表达本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段的条件下培养本公开的宿主细胞；及
-从培养物回收由所述宿主细胞表达的目的蛋白；及
-任选的，包括进一步纯化和/或修饰本公开的目的蛋白。
本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40 双特异性抗体或其抗原结合片段可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的结合分子或抗体等得目的蛋白分离及纯化技术为本领域技术人员所公知。生产和纯化抗体的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。本公开工程化的抗体也可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
然而，本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
组合物
本公开提供组合物(例如药物组合物)，其含有预防或治疗有效量的如上所述的本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段、或编码多核苷酸，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
一些具体实施方案中，所述药物组合物单位剂量中可含有0.01至99重量％的CD40结合分子、FAP/CD40结合分子或抗FAP/CD40双特异性抗体或其抗原结合片段。另一些具体实施方案中，药物组合物单位剂量中含CD40结合分子、FAP/CD40结合分子或抗FAP/CD40双特异性抗体的量为0.1-2000mg；一些具体实施方案中为1-1000mg。
试剂盒
本公开提供试剂盒，包含本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段、和/或编码多核苷酸。一些实施方案中，还提供包含上述多核苷酸的诊断试剂，以及提供相关诊断用途。
治疗疾病的方法和制药用途
本公开提供了本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段、编码多核苷酸或药物组合物在预防 和/或治疗疾病中用途和方法，所述疾病可以是与CD40信号通路相关或不相关的。一些实施方案中，本公开提供一种预防和/或治疗与CD40相关的疾病的方法，所述方法包括向受试者施用预防和/或治疗有效量的本公开的CD40结合分子，或包含本公开CD40结合分子的药物组合物。以及，还提供在制备本公开的CD40结合分子在预防和/或治疗与CD40或CD40L相关疾病、CD40或CD40L相关障碍的药物中的用途。
本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段、编码多核苷酸或药物组合物可以单独使用，或者与其它抗癌症或肿瘤治疗手段联合使用(例如与其它免疫原性剂、标准癌症疗法或其它抗体分子联合使用)，以抑制癌症或肿瘤的生长。
一些实施方案中，本公开提供一种预防和/或治疗癌症或肿瘤的方法，包括给患者或受试者施用预防和/或治疗有效量的本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段、编码多核苷酸、药物组合物，抑制患者或受试者中的肿瘤细胞生长。一些具体实施方案中，所述癌症优选但不限于对免疫治疗有应答的癌症。
以上方法或用途中，癌症或肿瘤的非限制性的例子包括肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤(例如转移的恶性黑色素瘤)、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本公开的方法治疗的其它癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病，包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。一些实施方案中，上述肿瘤或癌症是转移性的和/或晚期的。
此外，本公开还提供一种预防和/或治疗受试者或患者中的感染性疾病的方法，包括给该受试者或患者施用本公开的CD40结合分子、FAP结合分子、FAP/CD40结合分子、抗FAP/CD40双特异性抗体或其抗原结合片段、编码多核苷酸或药物组合物，使得所述对象的感染性疾病得到预防和/或治疗。类似于对于如上所述的癌症或肿瘤的应用，CD40结合分子可以单独使用，或者与疫苗组合使用来刺激对病原体、毒素和自身抗原的免疫应答。特别可以应用该治疗方法的病原体的示例包括当前没有有效疫苗的病原体，或常规疫苗不完全有效的病原体。其中包括但不 限于HIV、肝炎病毒(甲、乙、丙)、流感病毒、疱疹病毒、贾第虫、疟疾、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。
检测
本公开提供检测CD40或FAP的组合物，所述组合物包含根据本公开的CD40结合分子或FAP结合分子。本公开还提供用于体内或体外检测CD40或FAP的方法、系统或装置，其包括用本公开的CD40结合分子或FAP结合分子处理样品。
一些实施方案中，体外检测方法、系统或装置可能例如包括：
(1)使样品与结合CD40或FAP的抗体接触；
(2)检测在结合CD40或FAP的抗体和样品之间形成的复合物；和/或
(3)使参比样品(例如，对照样品)与抗体接触；和
(4)通过与参比样品比较，确定复合物形成的程度。如与对照样品或受试者中相比，样品或受试者中复合物形成的变化(例如，统计学上的显著变化)表示样品中存在CD40或FAP。
另一些实施方案中，体内检测方法、系统或装置可以包括：
(1)向受试者施用结合CD40结合分子或FAP结合分子；和
(2)检测CD40结合分子或FAP结合分子和受试者之间复合物的形成。
检测可以包括确定形成复合物的位置或时间。用可检测物质对CD40结合分子或FAP结合分子标记，通过对所述标记检测以实现检测。合适的可检测物质包括多种酶、辅基、荧光物质、发光物质和放射性物质。可以通过测量与CD40或FAP结合或不结合的抗体或使其可视化，检测CD40结合分子和和CD40，或FAP结合分子和FAP之间的复合物形成。可以使用常规检测测定法，例如，酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)或组织免疫组织化学。出于检测目的，本公开的CD40结合分子或FAP结合分子可以用荧光团发色团标记。
一些实施方案中，还提供试剂盒，所述试剂盒包含CD40结合分子或FAP结合分子，还可以包含诊断使用说明。试剂盒还可以含有至少一种额外的试剂，如标记物或额外的诊断剂。对于体内使用，CD40结合分子或FAP结合分子可以配制为药物组合物。
定义
为了更容易理解本公开，以下具体定义了某些技术和科学。除非在本文中另有明确定义，本文使用的所有其它技术和科学都具有本公开所属领域的一般技术人员通常理解的含义。
除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968) 中所述。
“CD40”和“CD40抗原”是指在正常和赘生性B细胞表面上表达的约48kD糖蛋白，其充当参与细胞增殖和分化的信号的受体(Ledbetter等人,1987,J.Immunol.138:788-785)。内源性表达CD40的细胞是特征在于CD40的表面表达的任何细胞，包括但不限于正常和赘生性B细胞、交错突细胞、基底上皮细胞、癌细胞、巨噬细胞、内皮细胞、滤泡树突细胞、扁桃体细胞和骨髓来源的浆细胞。本公开“CD40”指来自任何脊椎动物来源,包括哺乳动物例如灵长类(例如人)和啮齿类(例如小鼠和大鼠)的任何天然CD40,除非另有说明。已从由伯基特氏淋巴瘤细胞系Raji制备的文库中分离出编码CD40的cDNA分子(Stamenkovic等人,1989,EMBO J.8:1403)。序列信息可参见本公开表8。本公开“CD40”涵盖全长未加工的CD40以及源自细胞中的加工的任何形式的CD40，还涵盖CD40的天然发生变体，例如剪接变体或等位变体。在一个实施方案中，本公开的CD40结合分子能够特异性结合人，小鼠和/或食蟹猴CD40。
“成纤维细胞激活蛋白(FAP)”和“FAP抗原”，也称作脯氨酰内肽酶FAP或分离酶(Seprase)(EC 3.4.21)，指来自任何脊椎动物来源的任何天然FAP，包括哺乳动物，例如灵长类(例如人)，非人灵长类(例如食蟹猴)和啮齿类(例如小鼠和大鼠)，除非另外指明。本公开“FAP”涵盖全长未加工的FAP以及源自细胞中的加工的任何形式的FAP，还涵盖FAP的天然发生变体，例如剪接变体或等位变体。在一个实施方案中，本公开的FAP结合分子能够特异性结合人，小鼠和/或食蟹猴FAP。人FAP的氨基酸序列在UniProt(www.uniprot.org)登录号Q12884(版本149，SEQ ID NO:2)，或NCBI(www.ncbi.nlm.nih.gov/)RefSeq NP_004451.2、GeneBank Accession NO.AAC51668中显示。人FAP的细胞外结构域(ECD)自氨基酸位置26延伸至760。带His标签的人FAP ECD等氨基酸序列在本公开表2中显示。小鼠FAP的氨基酸序列在UniProt登录号P97321(版本126，SEQ ID NO:143)，或NCBI RefSeq NP_032012.1中显示。小鼠FAP的细胞外结构域(ECD)自氨基酸位置26延伸至761。一些实施方案中，本公开的FAP结合分子结合FAP的细胞外结构域。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠 近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(CDR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
“双特异性抗体”涵盖对两个不同抗原或同一抗原的至少两个不同抗原表位特异性结合的抗体(包括抗体或其抗原结合片段，如单链抗体)。典型的双特异性抗体结构模型如KiH、CrossMAb、Triomab quadroma、FcΔAdp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2:1TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab)2-CrossMAb、IgG-(scFv)2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab)4-CrossMAb等双特异性抗体(参见Aran F.Labrijn等，Nature Reviews Drug Discovery volume 18，pages585–608(2019)；Chen S1等，J Immunol Res.2019Feb 11；2019:4516041)。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM编号系统、IMGT编号系统、接触定义、构象定义。
Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置。(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号系统使用知识数据库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与 Kabat CDR的至少一部分重叠，尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。各种编号系统之间的对应关系是本领域技术人员熟知的，示例性的，如下表1中所示。
表1.CDR编号系统之间的关系
CDR IMGT Kabat AbM Chothia Contact
HCDR1 27-38 31-35 26-35 26-32 30-35
HCDR2 56-65 50-65 50-58 52-56 47-58
HCDR3 105-117 95-102 95-102 95-102 93-101
LCDR1 27-38 24-34 24-34 24-34 30-36
LCDR2 56-65 50-56 50-56 50-56 46-55
LCDR3 105-117 89-97 89-97 89-97 89-96
本公开的抗体的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号系统。
多肽或蛋白的“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个功能性质，且在许多情况下可添加、移除或转移至其它蛋白而不损失蛋白的其余部分和/或结构域的功能。
“免疫球蛋白结构域”是指抗体链(例如常规四肽链结构抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征，其由排列在两个β折叠中任选由保守二硫键稳定的约7个反平行β折叠股的2层夹层组成。
“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
“抗体框架(FR)”，是指可变结构域的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。
“免疫球蛋白单一可变结构域”通常用于指可以在不与其他可变结构域相互作用的情况下(例如在没有如常规四链单克隆抗体的VH和VL结构域之间所需要的VH/VL相互作用的情况下)，形成功能性抗原结合位点的免疫球蛋白可变结构域(其可以是重链或轻链结构域，包括VH、VHH或VL结构域)。“免疫球蛋白单一可变结构域”的实例包括纳米抗体(包括VHH、人源化VHH和/或骆驼化VH，例如骆驼化人VH)、IgNAR、结构域、作为VH结构域或衍生自VH结构域的(单结构域)抗体(诸如dAbs TM)和作为VL结构域或衍生自VL结构域的(单结构域)抗体(诸如dAbs TM)。基于和/或衍生自重链可变结构域(诸如VH或VHH结构域)的免疫球蛋白单一可变结构域通常是优选的。免疫球蛋白单一可变结构域的一个具体实例为如下文定义的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、V HH结构域、VHH抗体片段、VHH抗体、纳米抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature363，446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规四肽链结构抗体中的重链可变结构域(其在本公开中称为“VH结构域”)以及轻链可变结构域(其在本公开中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规四肽链结构抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。术语“重链单域抗体”、“VHH结构域”、“VHH”、“V HH结构域”、“VHH抗体片段”、“VHH抗体”、
Figure PCTCN2022114522-appb-000001
以及““
Figure PCTCN2022114522-appb-000002
结构域””(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。“VHH结构域”包括但不限于经骆驼科动物产生的天然抗体，也可以是骆驼科动物产生的抗体后再经人源化的，也可以是经噬菌体体展示技术筛选获得的。VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本公开所述的目的。获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.，Journal of Immunological Methods，240(2000)185-195；Li et al.，J Biol Chem.，287(2012)13713-13721；Deffar et al.，African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有 多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。其它的编号系统或编码规则包括Chothia、IMGT、AbM。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的全人抗体可变区可进行最少反向突变，以保持活性。“人源化”的例子包括可将源自骆驼科的VHH结构域通过以人常规四肽链结构抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而“人源化”(本公开中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其它修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列。此外，为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
“全人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的全人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。“全人抗体”不包括“人源化抗体”。
通常，本公开的CD40结合分子、FAP结合分子将以如于Biacore或KinExA或Fortibio测定中测量的优选10 -7至10 -10摩尔/升(M)、更优选10 -8至10 -10摩尔/升、甚至更优选10 -9至10 -10或更低的解离常数(K D)，和/或以至少10 -7M、优选至少10 -8M、更优选至少10 -9M，更优选至少10 -10M的缔合常数(KA)结合所要结合的抗原(即CD40或FAP)。任何大于10 -4M的K D值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本公开所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定)。
当“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如CD40或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane， 1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化抗原(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与充分接近参考抗原结合蛋白结合的表位所邻近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利公开WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合CD40或FAP上的相同表位的抗体。
“CD40结合分子”意指任何能够特异性结合CD40的蛋白或包含所述蛋白的任何分子。CD40结合分子可以包括针对CD40的如本公开定义的抗体或其缀合物。CD40结合分子还涵盖免疫球蛋白超家族抗体(IgSF)或CDR移植分子。本公开的“CD40结合分子”可以包含至少一个结合CD40的免疫球蛋白单一可变结构域(如VHH)。在一些实施方案中，“CD40结合分子”可以包含2、3、4或更多个结合CD40的免疫球蛋白单一可变结构域(如VHH)。本公开的CD40结合分子除结合包含CD40的免疫球蛋白单一可变结构域外，也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中，本公开的“CD40结合分子”还涵盖双/多特异性抗体，其含有结合不同抗原的免疫球蛋白(如结合第一抗原(如CD40)的第一抗体和结合第二抗原(如FAP)的第二抗体，可选的，包括结合第三抗原的第三抗体，进一步可选的，包括结合第四抗原的第四抗体。
“FAP结合分子”意指任何能够特异性结合FAP的蛋白或包含所述蛋白的任何分子。FAP结合分子可以包括针对FAP的如本公开定义的抗体或其缀合物。
“FAP/CD40结合分子”意指任何能够特异性结合CD40和FAP的蛋白或包含 所述蛋白的任何分子。FAP/CD40结合分子可以包括针对CD40和FAP的如本公开定义的抗体或其缀合物。
“与CD40结合”或“结合CD40”，指能与CD40或其表位相互作用，所述CD40或其表位可以是人源的。“与FAP结合”或“结合FAP”，指能与FAP或其表位相互作用，所述FAP或其表位可以是人源的。本公开的“抗原结合位点”指抗原上不连续的，由本公开抗体识别的三维空间位点。
“抗原”指用于免疫接种免疫活性的脊椎动物的分子，以产生识别抗原的抗体，或筛选表达文库(例如尤其是噬菌体、酵母或核糖体展示文库)。在本公开中，抗原被更广义地定义，包括由抗体特异性识别的靶分子，以及包括用于产生抗体的免疫接种过程或用于选择抗体的文库筛选中使用的分子的一部分或模拟物。例如，对于本公开的与人CD40结合的抗体，人CD40的单体和多聚体(例如二聚体、三聚体等)，以及人CD40的截短变体和其它变体均被称为抗原。
“表位”是指抗原上与免疫球蛋白或抗体结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本公开所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。例如，当使用人CD40或其表位作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲和力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。“识别抗原的抗体”在本公开中可以与“特异性结合的抗体”互换使用。
“结合亲和力”或“亲和力”在本公开中用作两个分子(例如抗体或其部分与抗原)之间的非共价相互作用的强度量度。两个分子之间的结合亲和力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。K D通过方程K D＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且可通过方法例如Caceci等人(1984，Byte 9：340-362)中所述的那些甚至对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定K D。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如 ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本公开其它地方例举的其它测定。抗体的结合动力学和结合亲和力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用Biacore TM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的K D值来比较与不同分子相互作用相关的结合亲和力，例如，不同抗体对于给定抗原的结合亲和力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的K D值与非目的相互作用(例如已知不结合CD40的对照抗体)的K D值进行评价。
“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
“同源性”、“同一性”或“序列同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“宿主细胞”包括各个细胞或细胞培养物，其可为或已是用于掺入多核苷酸插入片段的载体的受体。宿主细胞包括单个宿主细胞的子代，并且由于天然、偶然或有意的突变，子代可不一定与原始亲本细胞完全相同(在形态学或基因组DNA互补体中)。宿主细胞包括用本公开的多核苷酸在体内转染和/或转化的细胞。“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
“激动活性”、“激动剂活性”或“激动性”指作为激动剂的物质功能。激动剂与细胞受体的结合引起了与该受体的天然配体所引起的相类似或相同的反应或活性。例如，CD40激动剂或CD40激动型抗体可诱导任何或全部的下列应答：细胞增殖和/或分化；通过例如ICAM-1、E-选凝素、VCAM等分子上调细胞之间的 粘附；分泌促炎性细胞因子，例如IL-1、IL-6、IL-8、IL-12、TNF等；经CD40受体通过以下途径转导信号，例如TRAF(例如，TRAF2和/或TRAF3)、MAP激酶，如NIK(NF-κB诱导激酶)、1-κB激酶(IKKα/β)、转录因子NF-κB、Ras和MEK/ERK途径、PI3K/Akt途径、P38MAPK途径等；通过XIAP、Mcl-1、BCLx等分子转导抗-凋亡信号；B和/或T细胞记忆的产生；B细胞抗体产生；B细胞同种型转换；上调II类MHC和CD80/86的细胞表面表达等。
“CD40相关疾病”或“CD40相关病症”指示表达CD40的细胞的被修饰或消除的病症。这些细胞包括表现出异常增殖的表达CD40的细胞或与癌性或恶性生长相关联的表达CD40的细胞。表现出CD40抗原的异常表达的癌症的更具体例子包括B淋巴母细胞、伯基特氏淋巴瘤、多发性骨髓瘤、T细胞淋巴瘤、卡波西氏肉瘤、骨肉瘤、表皮和内皮肿瘤、胰腺癌、肺癌、乳腺癌、卵巢癌、结肠癌、前列腺癌、头颈癌、皮肤癌(黑色素瘤)、膀胱癌和肾癌。此类障碍包括但不限于白血病、淋巴瘤(包括B细胞淋巴瘤和非霍奇金淋巴瘤)、多发性骨髓瘤、瓦尔登斯特罗姆巨球蛋白血症；实体瘤，包括肉瘤，例如骨肉瘤、尤因氏肉瘤、恶性黑色素瘤、腺癌(包括卵巢腺癌)、卡波西氏肉瘤/卡波西氏肿瘤和鳞状细胞癌。
“增殖性疾病”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，增殖性病症指癌症。
“癌症”、“癌性”、“增殖性病症”和“肿瘤”在本公开中提到时并不互相排斥。
“预防癌症”是指在受试者中延迟、抑制或防止癌症发作，所述受试者中癌症发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定了癌症易感性。该还包括治疗具有癌变前病症的受试者以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触，例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本公开的任一种抗体或其药物组合物作为治疗剂，所述受试者已经患有、疑似患有、倾向于患有一种或多种增殖性疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床能测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化， 例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。本公开的受试者可以是动物或人类受试者。
本公开的“受试者”、“患者”意指哺乳动物，尤其灵长类动物，尤其是人。
附图说明
图1为抗FAP抗体Ab9、Ab10、Ab14、Ab15与细胞表面人FAP的结合，使用28H1作为阳性对照，IgG1同种型(作为阴性对照。
图2为抗FAP抗体Ab9、Ab10、Ab14、Ab15与细胞表面小鼠FAP的结合，使用28H1作为阳性对照，IgG1同种型作为阴性对照。
图3为抗CD40抗体A12、A297与Raji细胞表面人CD40的结合，使用9E5-SELFNS作为阳性对照，IgG1作为阴性对照。
图4为抗CD40抗体A12、A297与HEK293细胞表面人CD40的结合，使用9E5-SELFNS作为阳性对照，IgG1作为阴性对照，纵坐标为相对于200nM9E5-SELFNS的激动活性百分比。
图5A为人源化抗CD40抗体A12_V1、A12_V2、A12_V3与过表达人CD40的HEK293细胞的亲和力流式检测结果，图5B为人源化抗CD40抗体A297_V1、A297_V2、A297_V3、A297_V4与过表达人CD40的HEK293细胞的亲和力流式检测结果，均使用9E5-SELFNS作为阳性对照，hIgG作为阴性对照。
图6A为人源化抗CD40抗体A12_V1、A12_V2、A12_V3通过FcγRIIb介导的HEK-Blue TMCD40L细胞的活化作用，图6B为人源化抗CD40抗体A297_V1、A297_V2、A297_V3、A297_V4通过FcγRIIb介导的HEK-Blue TMCD40L细胞的活化作用，均使用9E5-SELFNS作为阳性对照，hIgG作为阴性对照。
图7为抗FAP/CD40双特异性抗体结构示意图。
图8A为抗FAP/CD40双特异性抗体Ab10-A12V2-2、Ab10-A12V2-4、Ab10-A297V3-2、Ab10-A297V3-4与高表达人FAP抗原的CHOK1/FAP稳转株的亲和力流式检测结果；图8B、图8C分别为前述抗FAP/CD40双特异性抗体与高 表达鼠FAP抗原、食蟹猴FAP抗原的CHOK1/FAP稳转株的亲和力流式检测结果。
图9A为抗FAP/CD40双特异性抗体Ab10-A12V2-2、Ab10-A12V2-4、Ab10-A297V3-2、Ab10-A297V3-4与高表达人CD40抗原的HEK-BlueTMCD40L稳转株的亲和力流式检测结果；图9B为前述抗FAP/CD40双特异性抗体与高表达食蟹猴CD40抗原的HEK293细胞的亲和力流式检测结果。
图10为抗FAP/CD40双特异性抗体Ab10-A297V3-2、Ab10-A297V3-4与人未成熟DC上CD40亲和力流式检测结果。
图11为浓度梯度的抗FAP/CD40双特异性抗体Ab10-A12V2-2、Ab10-A12V2-4、Ab10-A297V3-2、Ab10-A297V3-4在没有CHOK1/FAP稳转细胞交联情况下促DC成熟作用，使用LPC、hIgG1、载剂作为对照。
图12为浓度梯度的抗FAP/CD40双特异性抗体Ab10-A12V2-2、Ab10-A12V2-4、Ab10-A297V3-2、Ab10-A297V3-4在CHOK1/FAP稳转细胞交联情况下促DC成熟作用，使用LPC、hIgG1、载剂作为对照。
图13A为向mFAP-MC38荷瘤hCD40人源化小鼠注射单剂量抗体Ab10-A297V3-2或Ab10-A297V3-4后小鼠肿瘤生长曲线；图13B为小鼠相应外周血B细胞的激活；图13C为小鼠相应体重变化曲线；图13D为小鼠相应血小板计数的变化；图13E为小鼠相应肝功能的影响。
图14A为向mFAP-MC38荷瘤hCD40人源化小鼠注射多剂量抗体Ab10-A297V3-2或Ab10-A297V3-4后小鼠肿瘤生长曲线；图14B为小鼠相应外周血B细胞的激活；图14C为小鼠相应体重变化曲线；图14D为小鼠相应血小板计数的变化；图14E为小鼠相应肝功能的影响。
图15A为向mFAP-MC38荷瘤hCD40人源化小鼠注射多剂量抗体Ab10-A12V2-2后小鼠肿瘤生长曲线；图15B为小鼠相应体重变化曲线；图15C为小鼠相应肝功能的影响；图15D为小鼠相应血小板计数变化。
图16A为向mFAP-MC38荷瘤hCD40人源化小鼠注射单剂量Ab10-A12V2-4后小鼠肿瘤生长曲线；图16B为小鼠相应体重变化曲线；图16C为小鼠相应肝功能的影响；图16D为小鼠相应血小板计数的影响。
图17为双特异性抗体Ab10-A12V2-2和Ab10-A297V3-2的药代动力学比较结果。
其中，图13A至图17中，除图中另有标识外，P值均是相对于对照hIgG1计算。使用统计学方法Student’t-test，其中，ns是指不具有显著性差异，*为p<0.05，**为p<0.01，***为p<0.001，****为p<0.0001。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗FAP单克隆抗体的筛选和制备
1.1筛选抗原、检测抗原的序列和制备
人成纤维细胞激活蛋白(FAP；GeneBank登录号AAC51668)是一种分子量约为170kDa的丝氨酸寡肽酶，是同源二聚体。本公开使用的重组FAP蛋白序列、来源和用途如表2。
表2.重组蛋白氨基酸序列来源
Figure PCTCN2022114522-appb-000003
1.2从人天然Fab噬菌体展示库中筛选h-FAP特异性结合抗体片段
以h-FAP-biotin抗原为靶分子，采用表面带有亲和素的磁珠捕获抗原特异性噬菌体，并用磁力架进行噬菌体的富集。用pH2.2的甘氨酸溶液洗脱抗原特异性噬菌体。
经二轮筛选，随机挑选284个克隆，采用噬菌体ELISA筛选阳性克隆，包括如下具体步骤：计数铺皿生长菌落，共计284个，接种96孔板，每孔含有400μL培养液(2YT+Amp+0.2％葡萄糖)，37℃下250rpm振摇培养6h。用抗原h-FAP-His包板，100ng/100μL/孔，4℃过夜。第二天，洗涤封闭96孔板后，每孔加100μL过夜培养的菌液，37℃下孵育1h，洗涤，加二抗(HRP标记的抗人IgG-Fab的抗体)，37℃下孵育40min，洗涤，加显色液，避光保存30min，酶标仪读取OD600数值。测序经两次筛选得到的共计122个阳性克隆，得到74个独特的VH(构成了VH富集库)、48个独特的κ轻链(构成了KLC富集库)和10个独特的λ轻链(构成了LLC富集库)。
1.3构建CHO细胞表面全长抗体展示库
1)构建全长抗体CHO细胞展示基因库，包括：用三套引物分别PCR扩增VH富集库、KLC富集库和LLC富集库。将三个富集库经酶切后插入相应的组件载体，构建相应的组件库。酶切KLC、LLC、VH组件库，电泳分析纯化酶切后的KLC片段库、LLC片段库和VH片段库。酶切全长抗体细胞展示载体，纯化载体片段。将酶切纯化的载体片段、KLC片段库、LLC片段库、VH片段库混合并连接。纯化连接产物，电转进入大肠杆菌，铺皿，37℃培养过夜，计数菌落。经检测，库容达到3.6×10E6，是理论多样性(74×58＝4292)的800倍以上。收集全部菌落，提取载体DNA，得到全长抗体细胞展示基因库。
2)构建h-FAP抗原特异性全长抗体细胞展示库，包括：用全长抗体细胞展示基因库载体DNA转化，构建全长抗体CHO细胞展示库。用潮霉素加压筛选细胞库。得到稳定转化细胞库，用PE标记的鼠抗人κ(或者λ)轻链抗体和FITC标记的h-FAP抗原双染细胞库，FACS分选PE和FITC双阳性细胞，每孔一个细胞，κ轻链库分选2块96孔板，λ轻链库分选1块96孔板，潮霉素加压培养。
3)FACS分析鉴定展示h-FAP抗原特异性抗体单细胞克隆，包括：潮霉素加压培养14天，得到稳定转化κ链单细胞克隆153个，λ链单细胞克隆50个。用0.5mM EDTA-PBS缓冲液消化细胞，用PE标记的鼠抗人κ(或者λ)轻链抗体和FITC标记的h-FAP抗原双染单细胞克隆，经FACS分析，得到PE和FITC荧光双阳性单细胞克隆138个。
4)克隆抗体基因，包括：通过FACS检测分析阳性克隆的亲和力，决定对45个克隆进行抗体基因PCR扩增。离心收集阳性克隆，弃上清，提取细胞基因组DNA，PCR扩增VH和LC，电泳分离扩增片段，测序分析，确定6个独特VH和6个独特κ，组合可得到12个有独特序列的克隆，其中的4个克隆的VH和VL的序列参见表3。
表3.h-FAP抗体VH及VL(KLC的κ)氨基酸序列
Figure PCTCN2022114522-appb-000004
Figure PCTCN2022114522-appb-000005
注：下划线为Kabat编号规则的CDR。
本公开还提供Ab9、Ab10、Ab14、Ab15的重链全长和轻链全长序列。
>Ab9重链全长
Figure PCTCN2022114522-appb-000006
>Ab9轻链全长
Figure PCTCN2022114522-appb-000007
>Ab10重链全长
Figure PCTCN2022114522-appb-000008
>Ab10轻链全长
Figure PCTCN2022114522-appb-000009
>Ab14重链全长
Figure PCTCN2022114522-appb-000010
Figure PCTCN2022114522-appb-000011
>Ab14轻链全长
Figure PCTCN2022114522-appb-000012
>Ab15重链全长
Figure PCTCN2022114522-appb-000013
>Ab15轻链全长
Figure PCTCN2022114522-appb-000014
表4.抗FAP抗体的CDR
Figure PCTCN2022114522-appb-000015
Figure PCTCN2022114522-appb-000016
也即，上述抗FAP抗体有如下通式结构：
HCDR1均为SEQ ID NO:9；
HCDR2均为SEQ ID NO:10；
HCDR3均为DX 1SLTARPYYFYGFDV(SEQ ID NO:18)，其中，X 1为E或A；
LCDR1均为SEQ ID NO:12；
LCDR2均为SEQ ID NO:13；
LCDR3均为QQANSFPX 2X 3(SEQ ID NO:19)，其中，X 2为P或L，X 3为A或T。
1.4构建CHO细胞表面全长抗体展示库
1)构建可溶性抗体表达载体，包括：分别酶切纯化具有独特序列阳性VH片段和LC片段。将VH片段插入可溶性重链表达载体(IgG1)，将LC片段插入可溶性轻链表达载体，送菌落测序确定并提取DNA。
2)表达纯化可溶性抗体，包括：悬浮培养扩增Expi293细胞。按上述确定的轻重链配对，轻链表达载体18μg，重链表达载体12μg的比例混合。将载体DNA与PEI以重量比1：2.5的比例混合，转化Expi293细胞，第六天收集培养液上清，Protein-A法纯化抗体。SDS-PAGE变性凝胶电泳分析，抗体纯度均达到90％以上，-80℃保存。
1.5 ELISA结合测试
直接包被带His标签的FAP重组蛋白，加入抗体后，通过加入二抗(HRP偶联的抗人IgG的抗体)和HRP底物TMB检测抗体与抗原结合的活性。包括：用人FAP-His蛋白包被96孔酶标板，按0.5μg/mL浓度每孔100μL，4℃孵育过夜。充分洗涤，加入200μL/孔封闭液室温孵育2h。充分洗涤，每孔加入100μL用稀释液稀释好的抗FAP待测抗体。室温孵育1h，充分洗涤，每孔加入100μL用稀释液按1：20000倍稀释的HRP标记的羊抗人IgG二抗。室温孵育1h，充分洗涤，每孔加入100μL TMB，避光反应15min。加入50μL/孔的0.16M硫酸。Thermo MultiSkanFc酶标仪读取450nm OD值，计算抗FAP抗体的结合EC 50值，结果见表5。
表5.抗FAP抗体与人FAP抗原结合的亲和力EC 50值
编号 EC 50(mg/mL)
Ab9 0.1014
Ab10 0.0920
Ab14 0.4168
Ab15 0.3206
1.6表面等离子共振技术(surface plasmon resonance,SPR)结合测试
选用CM5传感器芯片，流动相采用HBS-EP+缓冲溶液(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)。抗人IgG(Fc)抗体用10mM醋酸钠缓冲液(pH 5.0)配制成30μg/mL溶液，进行氨基偶联固定。用HBS-EP+缓冲溶液配制各待测抗体，通过芯片通道上的抗人IgG(Fc)抗体进行捕获。用HBS-EP+缓冲溶液配制h-FAP-His作为分析物，2倍梯度稀释。将稀释好的抗体在30μL/min的流速下流过实验通道和参比通道，结合1min，解离15min。用10mM Glycine pH1.5作为再生缓冲液，10μL/min的流速下运行30秒。分析数据，表6结果显示，与对照抗FAP抗体28H1(专利US9266938B2的序列219和序列233)相比较，Ab9、Ab10、Ab14、Ab15与抗原FAP的亲和力要高3-6倍，最高的Ab15亲和力K D达到2.15x10 -10。而本公开同步筛选获得的其它抗FAP抗体，例如Ab2、Ab4、Ab5(序列未出示)与抗原结合亲和力则低于28H1。
表6.抗FAP抗体的SPR亲和力Ka、Kd及K D值
Figure PCTCN2022114522-appb-000017
其中，对照抗体28H1的重链轻链可变区序列如下：
>28H1的VH
Figure PCTCN2022114522-appb-000018
>28H1的VL
Figure PCTCN2022114522-appb-000019
1.7 FACS结合测试
用FACS实验检测抗FAP抗体在细胞上的结合特性。包括：构建过表达人FAP 的CHO细胞系，铺板(1E5/孔)。加入待测抗体，100μL/孔，最高浓度为100nM，5倍稀释，共8个浓度，4℃孵育1h。加入抗hIgG Alexa Fluor-647作为二抗，按1:500比例4℃孵育30min，利用FACS进行检测。能结合FAP的抗体可以标记细胞。被标记的过表达人FAP的细胞的数量占所有细胞的百分比与抗体浓度之间的关系如图1所示，人FAP与抗体的结合EC 50值参见表7；被标记的过表达小鼠FAP的细胞的数量占所有细胞百分比与抗体浓度之间的关系如图2所示。
表7.抗FAP抗体与人FAP抗原结合的亲和力EC 50值
抗体编号 EC 50(μg/mL)
28H1 0.0007839
Ab9 0.0003684
Ab10 0.0004320
Ab14 0.0004738
Ab15 0.0004858
同种型(NC) -
实施例2.抗CD40纳米抗体(VHH)的筛选和制备
2.1免疫抗原，筛选抗原的序列和制备
选择带His标签的人CD40重组蛋白(h-CD40-His)，C端生物素标签的人CD40重组蛋白(h-CD40-biotin)，C端带His标签的猴CD40重组蛋白(cyno-CD40-His)。序列和来源见表8。所述蛋白试剂可用于下述各实施例实验中。
表8.重组蛋白氨基酸序列及来源
Figure PCTCN2022114522-appb-000020
2.2羊驼免疫流程及效价检测
用h-CD40-his免疫羊驼，每两周免疫一次，共免疫四次。首次免疫时，将0.5mg抗原与弗氏完全佐剂(CFA)1mL混匀皮下注射，后三次免疫将0.25mg抗原与弗氏不完全佐剂(IFA)1mL混匀皮下注射。免疫前采集空白血清，第3次免疫后一周和第4次免疫后一周分别采50mL外周血分离淋巴细胞，检测血清效价。
2.3效价检测及噬菌体文库构建
羊驼经四次免疫后检测血清效价。效价合格后，分离PBMC，提取总RNA，检测纯度，反转录为DNA，两轮巢式PCR后将纯化载体与VHH目的片段酶切连接。电转，挑选克隆，获得噬菌体文库A库和B库。
2.4噬菌体文库亲和淘选及ELISA鉴定
以h-CD40抗原为靶分子，采用Gly-HCL酸洗脱方法进行筛选，洗脱特异噬菌体。经二轮筛选，从第一、二轮滴度测定平板随机挑选384个克隆，采用噬菌 体ELISA筛选阳性克隆，检测450nm下光密度。根据测序结果，对序列进行序列比对和进化树分析，筛选出16条序列，其中两个功能较好的抗CD40抗体的氨基酸序列见表9和表10。
表9.抗CD40单域抗体序列
Figure PCTCN2022114522-appb-000021
注:下划线为Kabat编号规则下的CDR。
表10.抗CD40抗体的CDR
Figure PCTCN2022114522-appb-000022
2.5抗CD40单域抗体-Fc融合蛋白的表达纯化
将以上2条VHH分别与含有N297A突变的人IgG1-Fc进行连接，连接后的VHH-Fc融合蛋白序列见表11。下划线为人IgG1-Fc，加粗为N297A突变。
采用9E5-SELFNS作为CD40激动剂阳性对照(参见WO2020108611A1的序列58和序列59)。
表11.抗CD40单域抗体与人IgG1-Fc的融合蛋白序列
Figure PCTCN2022114522-appb-000023
Figure PCTCN2022114522-appb-000024
构建质粒，瞬时转染细胞，表达抗体，并纯化。经检测，获得目的抗体。
2.6 FACS结合测试
用FACS实验检测抗CD40抗体在细胞上的结合特性。包括：获得Raji单细胞，铺板。加入待测抗体，100μL/孔，最高浓度点为100nM，5倍稀释，获得共8个梯度点，4℃孵育1h。加入抗hIgG Alexa Fluor-647作为二抗，按1:500比例4℃孵育30min。FACS检测。使用CD40激动剂为阳性对照，各抗体EC 50见图3及表12。
表12.抗CD40抗体与人CD40抗原结合的亲和力EC 50值
编号 EC 50(nM)
A12 0.3657
A297 0.5293
9E5-SELFNS 0.1961
对于用做FAP/CD40双特异性抗体中的CD40激动性抗体而言，其对CD40的亲和力EC 50不宜太高，否则会导致双特异性抗体优先结合CD40，更期望的是，双特异性抗体优先结合FAP，进而发挥作用。因此，A12和A297具有更符合以上特性的优势，这在表13中也有所体现。
2.7抗CD40纳米抗体的活性检测
检测抗CD40抗体对CD40报告基因细胞的活化作用，根据EC 50评价CD40抗体的激动剂活性。包括：制备HEK-Blue TMCD40L细胞(购自Invivogen Cat#hkb-cd40，该细胞稳定转染了人CD40基因和NF-κB介导的SEAP基因组，可以通过SEAP底物QUANTI-Blue检测上清中分泌的SEAP含量来表征CD40信号通路的活化水平)，培养基为含10％FBS、100μg/mL Normocin、100μg/mL Zeocin和30pg/mL Blasticidin的DMEM。将细胞以5E4/孔铺入96孔板，培养基为含10％FBS和100μg/mL Normocin的DMEM，培养过夜，细胞贴壁后，每孔加入100μL梯度稀释的待测抗体，37℃过夜培养。将细胞离心，转移细胞上清到一个新96孔板中，加入180μL QUANTI-Blue底物溶液，避光孵育15分钟，用Envision酶标仪测定在620nm处的吸光度，计算EC 50值。使用CD40激动剂(9E5-SELFNS)为阳性对照，相对激动活性百分比(与200nM 9E5-SELFNS相比)与抗体浓度之间的关系EC 50见图4和表13。
表13.抗CD40抗体的激动活性EC 50值及其与阳性对照的相对激活活性百分比％
编号 EC 50(nM) 最大响应(相对200nM 9E5-SELFNS的百分比)
A12 0.231 51.76
A297 0.556 84.15
9E5-SELFNS 0.466 102.55
IgG1 不激活 不激活
2.8抗CD40纳米抗体的人源化
根据以上结果，将A12、A297进行人源化。在所获得的纳米抗体A12和A297的VHH典型结构的基础上，将VHH可变区序列与抗体种系数据库比较，获得同源性高的人种系模板。本公开抗体A12和A297优选人种系模板均为IGHV3-48*03。再把CDR移植到人FR中，对影响抗体结构和功能的关键氨基酸进行回复突变，以恢复结合力和活性。各人源化序列如表14所示。
表14.抗CD40人源化抗体序列
Figure PCTCN2022114522-appb-000025
也即，本公开的抗CD40纳米抗体有如下通式结构：
CDR1为X 4YGMK(SEQ ID NO:39)，其中，X 4为N或R；
CDR2为TIX 5SX 6GX 7X 8TYYADSVKG(SEQ ID NO:40)，其中，X 5为N或L，X 6为Q或H，X 7为G或D，X 8为S或T；
CDR3为X 9D X 10SDYSX 11(SEQ ID NO:41)，其中，X 9为V或I，X 10为W或Y，X 11为P或S。
将以上7条VHH分别连入含有S267E/L328F突变(根据EU编号)的人IgG1-Fc。连接后的VHH-Fc融合蛋白序列见表15。下划线为人IgG1-Fc，加粗为S267E/L328F突变。
表15.人源化抗CD40单域抗体与人IgG1-Fc(S267E/L328F)的融合蛋白序列
Figure PCTCN2022114522-appb-000026
Figure PCTCN2022114522-appb-000027
将以上7条VHH-Fc瞬时转染细胞并表达，纯化抗体。经检测，获得目标抗体蛋白。
2.9人源化CD40抗体对CD40高表达细胞株的亲和力
采用FACS检测人源化抗体与高表达人抗原蛋白CD40的细胞的结合能力。用CD40质粒(CD40cDNA ORF Clone,Human,C-DYKDDDDK
Figure PCTCN2022114522-appb-000028
Tag；Sino Biological；HG10774-CF)瞬时转染HEK293细胞，获得高表达CD40的HEK293细胞，用流式染色液(PBS+2％FBS)重悬，加入梯度稀释后的不同浓度的待检测抗体。冰上孵育1h后，PBS洗涤，400g离心5min。加入带有荧光基团Alexa Fluor 647标记的羊抗人Fc抗体作为二抗，冰浴染色1h，PBS清洗两遍后用FACS检测细胞表面的荧光信号。结果如图5A和5B所示，EC 50值见表16。本公开人源化抗体对CD40高表达细胞株均有较高的亲和力。
表16.各人源化抗CD40抗体与人CD40高表达细胞株的亲和力EC 50值
抗体 EC 50(nM) Emax MFI
A12 1.09 17256
A12_V1 2.13 16608
A12_V2 0.42 16609
A12_V3 1.74 16909
A297 1.12 16728
A297_V1 1.11 7300
A297_V2 1.07 12764
A297_V3 1.25 12827
A297_V4 2.70 8255
9E5-SELFNS 1.12 12120
2.10人源化CD40抗体对HEK-Blue TM CD40L细胞的活化作用
通过检测CD40抗体在被FcγRIIb交联的情况下对HEK-Blue TMCD40L细胞的活化作用来评价CD40抗体的体外激动活性。人源化CD40抗体的Fc中带有的S267E/L328F突变，增强了IgG1Fc与FcγRIIb的亲合力，因此也增强了FcγRIIb对抗体的交联。通过FcγRIIb介导的CD40抗体对HEK-Blue TM CD40L细胞的活化作用模拟了CD40抗体在被完全交联的情况下的激动剂活性。用FcγRIIb质粒(CD32B/Fcgr2b cDNA ORF Clone,Human,N-His tag；Sino Biological；HG10259-NH)瞬时转染HEK293细胞，获得高表达FcγRIIb的HEK293细胞。将HEK-Blue TMCD40L细胞以5E4/孔铺入96孔细胞培养板(培养基为DMEM，10％FBS，100μg/mL Normocin)，加入5E4/孔表达FcγRIIb的HEK293细胞,每孔加入100μL梯度稀释的待测抗体，37℃过夜培养。将细胞离心，转移20μL细胞上清到一个新96孔板中，加入180μL QUANTI-Blue底物溶液，避光孵育30min，用Envision酶标仪测定在620nm处的吸光度，计算EC 50值以及Emax值(相对无抗体组荧光强度)，以EC 50值来评价CD40抗体的体外细胞激动剂活性。
CD40抗体通过FcγRIIb介导的HEK-Blue TMCD40L细胞的活化作用如图6A、6B所示,EC 50值及Emax值(相对荧光强度)见表17。
结果显示，在上述报告基因细胞体系下，人源化CD40抗体在被FcγRIIb交联的情况下，人源化CD40抗体的激动剂活性均较强。
表17.各人源化抗CD40抗体对HEK-Blue TMCD40L细胞的活化作用
Figure PCTCN2022114522-appb-000029
实施例3.抗FAP/CD40双特异性抗体的设计和制备
3.1抗FAP/CD40双特异性抗体的设计及表达纯化
根据人源化后的抗CD40抗体筛选结果，选择A12V2和A297V3；根据抗FAP 抗体的筛选结果，选择Ab10，进行抗FAP/CD40双特异性抗体的构建。双特异性抗体采用Ab10为IgG骨架，在Ab10的两条重链的C端各串联1个或2个或3个抗CD40纳米抗体。CD40纳米抗体之间以及与Ab10重链C端的连接子采用“GGGGSGGGGS”。每个双特异性抗体分别含有2价、4价和6价的CD40纳米抗体，如图7所示。对于抗体名称，例如，对于Ab10-A12V2-2，Ab10代表采用的抗FAP抗体，A12V2代表采用的人源化抗CD40抗体，最后的2代表CD40的价数为2价。其它抗体均采用该命名规则。瞬时转染与表达、纯化抗体采用实施例2的2.5中方法。经检测，获得目的双特异性抗体分子。各双特异性抗体分子氨基酸序列如下。
>Ab10-A12V2-2的重链
Figure PCTCN2022114522-appb-000030
>Ab10-A12V2-2的轻链
Figure PCTCN2022114522-appb-000031
>Ab10-A12V2-4的重链
Figure PCTCN2022114522-appb-000032
Figure PCTCN2022114522-appb-000033
>Ab10-A12V2-6的重链
Figure PCTCN2022114522-appb-000034
>Ab10-A297V3-2的重链
Figure PCTCN2022114522-appb-000035
>Ab10-A297V3-4的重链
Figure PCTCN2022114522-appb-000036
Figure PCTCN2022114522-appb-000037
>Ab10-A297V3-6的重链
Figure PCTCN2022114522-appb-000038
Ab10-A12V2-4、Ab10-A12V2-6、Ab10-A297V3-2、Ab10-A297V3-4、Ab10-A297V3-6的轻链均为SEQ ID NO：43所示。
注：重链的下划线为CH1和Fc区，轻链的下划线为CL区，斜体为连接子，加粗为LALA(L234A和L235A突变)。
3.2抗FAP/CD40双特异性抗体的抗原结合亲和力检测
采用SPR(GE Healthcare；Biacore 8K)检测抗FAP/CD40双特异性抗体与其抗原人FAP蛋白(Acro公司，FAP-H5244)、人CD40蛋白(Acro公司，CD0-H5228)之间的亲和力。选用CM5传感器芯片，流动相采用HBS-EP+缓冲溶液(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)。将抗人IgG(Fc)抗体通过氨基偶联固定。用HBS-EP+缓冲溶液分别配制各待测抗体作为配体，通过芯片通道上的抗人IgG(Fc)抗体进行捕获。不同种属的抗原蛋白作为分析物。结果见表18、表19，显示制备成双特异性抗体后，不影响抗体与FAP的结合，且双特异性抗体对CD40的亲和力更低，将优先结合FAP，进而发挥作用。
表18.抗FAP/CD40双特异性抗体与人FAP的亲和力
抗体 Ka(1/Ms) Kd(1/s) KD(M)
Ab10 2.17E+05 7.39E-05 3.40E-10
Ab10-A12V2-4 1.75E+05 5.14E-05 2.94E-10
Ab10-A297V3-2 2.03E+05 8.22E-05 4.05E-10
Ab10-A297V3-4 1.84E+05 6.96E-05 3.78E-10
表19.抗FAP/CD40双特异性抗体与人CD40的亲和力
抗体 Ka(1/Ms) Kd(1/s) KD(M)
9E5-SELFNS 6.04E+06 6.70E-03 1.11E-09
Ab10-A12V2-4 6.90E+04 2.00E-03 2.90E-08
Ab10-A297V3-2 2.09E+05 2.94E-02 1.41E-07
Ab10-A297V3-4 5.24E+05 1.37E-01 2.62E-07
3.3抗FAP/CD40双特异性抗体对高表达FAP细胞株的亲和力检测
本实验通过FACS检测抗FAP/CD40双特异性抗体对高表达人FAP、食蟹猴FAP和小鼠FAP的CHOK1细胞稳转株的结合情况。显示双特异性抗体能结合人、小鼠、食蟹猴的细胞表面FAP抗原，且结合能力与单抗相类似，参见图8A-8C，EC 50值见表20。
表20.抗FAP/CD40双特异性抗体与人、鼠细胞表面FAP抗原结合的EC 50值
Figure PCTCN2022114522-appb-000039
3.4抗FAP/CD40双特异性抗体对高表达CD40细胞株的亲和力检测
利用FACS检测双特异性抗体对于细胞表面CD40的结合情况，包括高表达人CD40的HEK-BlueTMCD40L细胞(Invivogen,Cat#hkb-cd40)，瞬转食蟹猴CD40质粒(Sino Biological,cat#CG90970-UT)使其高表达食蟹猴CD40的HEK293细胞。结果显示，双特异性抗体能够结合人和食蟹猴的细胞膜表面CD40。详见图9A、9B，图11和表21。
表21.双特异性抗体与人细胞表面CD40抗原结合的EC 50值
Figure PCTCN2022114522-appb-000040
3.5人源化抗FAP/CD40双特异性抗体FAP依赖性促DC成熟活性检测
为了验证抗FAP/CD40双特异性抗体对树突状细胞成熟的影响，利用CD14阳性磁珠从新鲜PBMC中分离单核细胞，用含50ng/mL的GM-CSF和50ng/mL的hIL-4的1640培养基培养5天。每2-3天半数换液，第5天将诱导成的树突状细胞以1E5/孔的密度加入96孔板中，再分别加入处于对期生长的CHOK1和过表达人FAP的CHOK1细胞。同时加入梯度稀释好的待测抗体，1μg/mL LPS作为阳性对照，100nM hIgG1和无抗体组作为阴性对照。培养48h后，用FACS检测树突状细胞表面分子CD83的表达。
如图11(无CHOK1/FAP)和图12(有CHOK1/FAP)，结果显示，2价分子Ab10-A12V2-2和Ab10-A297V3-2在没有CHOK1/FAP情况下不能活化DC，其CD40激动剂活性完全依赖于FAP介导的交联(crosslink)。而4价分子Ab10-A12V2-4和Ab10-A297V3-4在没有CHOK1/FAP细胞存在情况下能进够诱导DC细胞的成熟，在有CHOK1/FAP存在的情况下，DC细胞的激活能进一步增强。因此，无论对于2价分子和4价分子而言，树突细胞表面表达的FAP均能提供双特异性抗体的激活窗口。
实施例4.抗FAP/CD40双特异性抗体Ab10-A297V3-2/4的小鼠体内药效
B-hCD40人源化小鼠来自百奥赛图江苏基因生物技术有限公司(种属Mus musc μLus，品系C57BL/6，雌性)。在小鼠肠癌MC38细胞中转染小鼠全长FAP质粒，构建稳转株，接种细胞5×10 5/0.1mL/只于小鼠右腋皮下，当平均肿瘤体积长至80-100mm 3时，挑选个体肿瘤体积合适的小鼠，随机分组，每组6只。小鼠腹腔注射给药，每周两次，首次给药48h后抽取外周血检测B细胞数目及细胞表面CD86表达，分组后第8天检测血液中的谷丙转氨酶ALT(Alanine Aminotransferase)和谷草转氨酶AST(Aspartate aminotransferase)浓度，给药4次后停止给药，直至hIgG1对照组有小鼠肿瘤体积超过2000mm 3给药最后一次，最后一次给药24h后检测血常规。
4.1 Ab10-A297V3-2或Ab10-A297V3-4单剂量小鼠体内药效及毒性
在单一剂量的情况下比较了Ab10-A297V3-2或Ab10-A297V3-4与CD40激动剂单抗9E5-mIgG1的抗肿瘤活性。9E5-mIgG1与9E5-SELFNS具有相同的可变区，只是重、轻链恒定区采用小鼠mIgG1重链的恒定区以及小鼠kappa链的恒定区。由于CD40激动剂单抗的活性依赖于FcγRIIb对其Fc的交联，因此采用了小鼠IgG1重链恒定区的9E5-mIgG1可以更好地在小鼠体内展示CD40的激动剂活性。在小鼠体内药效实验中，9E5-mIgG1是比9E5-SELFNS更好的对照抗体。
Ab10-A297V3-4抗肿瘤活性无论是从TGI(Tumor growth inhibition，肿瘤生长抑制)还是CR(Complete Response，完全响应)的角度上，均强于同摩尔剂量的CD40单抗9E5-mIgG1，序列如下：
>9E5-mIgG1重链
Figure PCTCN2022114522-appb-000041
>9E5-mIgG1轻链
Figure PCTCN2022114522-appb-000042
两倍摩尔浓度下的Ab10-A297V3-2的抗肿瘤活性依然弱于Ab10-A297V3-4，与9E5-mIgG1相当。
通过检测小鼠外周血B淋巴细胞的激活来检测双特异性抗体分子对于外周CD40的激活，结果与体外实验一致。Ab10-A297V3-4由于具有部分不依赖于FAP的CD40激活活性，所以能部分激活外周B淋巴细胞，而Ab10-A297V3-2由于对CD40的激活完全依赖于FAP，所以未检测到显著的外周B细胞的激活(One-Way ANOVA)。所测抗体信息如表22，体内药效结果如图13A(各组抗体相对hIgG1对照组均P<0.0001)和图13B。
表22.所测试抗体信息
药物 浓度 分子量(kDa) 给药剂量
hIgG1 14.17mg/mL 150 3mg/kg
Ab10-A297V3-2 2.95mg/mL 174 6.96mg/kg
Ab10-A297V3-4 3mg/mL 200.03 4mg/kg
9E5-mIgG1 4.4mg/mL 150 3mg/kg
从小鼠毒性上来看，Ab10-A297V3-2和Ab10-A297V3-4都未产生小鼠体重上的变化，参见图13C。
从血常规的检测来看，与同摩尔剂量hIgG1对照组相比，9E5-mIgG1能引发一定程度的血小板下降，与肝毒性相类似；Ab10-A297V3-2没有引发血小板下降；Ab10-A297V3-4能引发与9E5-mIgG1同等程度的血小板下降，参见图13D。Ab10-A297V3-2/4的小鼠体内给药剂量和肿瘤生长抑制率统计详见表23。
其中，TGI的计算公式为：
TGI＝(空白组当天肿瘤体积-给药当天组肿瘤体积)/(空白组当天肿瘤体 积)*100％
表23.Ab10-A297V3-2或4的小鼠体内给药剂量和肿瘤生长抑制率
抗体编号 给药量(mpk) TGI
hIgG1 3 -
Ab10-A297V3-2 6.96(二倍摩尔剂量) 54.6％
Ab10-A297V3-4 4(等摩尔剂量) 96.4％
9E5-mIgG1 3 59.2％
从ALT和AST的检测中可以看出，9E5-mIgG1通过CD32B(即FcγRIIB)介导的交联，在给药后第8天显示出一定的肝毒性。Ab10-A297V3-4由于具有一定的本底激活，所以也具有一定的肝毒性，但是由于其激活不依赖于CD32B，所以肝毒性低于9E5-mIgG1；Ab10-A297V3-2由于依赖FAP才能激活CD40，因此完全没有肝毒性，参见图13E。
4.2 Ab10-A297V3-2或Ab10-A297V3-4多剂量小鼠体内药效和毒性
为了摸索Ab10-A297V3-2或4的治疗窗，进一步提高了Ab10-A297V3-2的剂量，并降低了Ab10-A297V3-4分子的剂量，详见表24。当继续升高2价Ab10-A297V3-2分子的给药剂量时，其抗肿瘤活性并能没有得到进一步的提高，也没有观测到外周B淋巴细胞的激活。降低Ab10-A297V3-4的给药剂量至1.3mg/kg(即9E5-mIgG1的1/3摩尔剂量)时，其仍有92.6％的TGI，远远好于9E5-mIgG1。
为了在体内实验中评估4价CD40双特异性抗体中FAP抗体的作用，比较了Ab10-A297V3-4和另一个没有FAP结合功能的4价CD40双特异性抗体(同种型-A297V3-4)的抗肿瘤活性。同种型-A297V3-4与Ab10-A297V3-4的构造完全一致，除了将抗FAP部分的Ab10替换为另一个不结合任何鼠源蛋白的同种型抗体。
图14A-14E的结果显示，4价CD40双特异性抗体即使不结合FAP就有较好的抗肿瘤活性，而结合FAP则抗肿瘤活性更强。从毒性上来看，Ab10-A297V3-2提高剂量后仍未见体重、ALT/AST以及血小板数目的异常，而对于Ab10-A297V3-4，在降低剂量后，之前在高剂量下观测到的ALT/AST以及血小板数目上的异常消失了，而仍保留了很强的抗肿瘤效果(P<0.0001)，说明对比CD40单抗9E5-mIgG1，无论是Ab10-A297V3-2和Ab10-A297V3-4都具有更宽的治疗窗。抗体信息见表24，Ab10-A297V3-2或Ab10-A297V3-4多剂量体内药效统计数据见表25。
表24.所测试抗体信息
Figure PCTCN2022114522-appb-000043
Figure PCTCN2022114522-appb-000044
表25.Ab10-A297V3-2/4多剂量体内药效统计
药物 给药量(mg/kg) TGI CR比率(<100mm 3)
hIgG1 3 - 0％
Ab10-A297V3-2 6.96(二倍摩尔剂量) 32.8％ 0％
Ab10-A297V3-2 13.92(四倍摩尔剂量) 46.9％ 0％
Ab10-A297V3-4 4(等摩尔剂量) 101.6％ 100％
Ab10-A297V3-4 1.3(1/3摩尔剂量) 92.6％ 33％
同种型-A297V3-4 1.3(1/3摩尔剂量) 79.3％ 0％
9E5-mIgG1 3 59.2％ 0％
4.3 Ab10-A12V2-2多剂量小鼠体内药效和毒性
参见图15A-15D和表26，Ab10-A12V2-2与Ab10-A297V3-2具有相似的小鼠体内药理学特征(两个剂量与hIgG1对照组相比，均为P<0.0001)，从抗肿瘤活性上优于9E5-mIgG1。升高剂量不能进一步提高其抗肿瘤活性，但是没有ALT/AST的升高及血小板的下降，显示其更宽的治疗窗和更低的副作用。
表26.所测试抗体信息及TGI
Figure PCTCN2022114522-appb-000045
4.4 Ab10-A12V2-4单剂量小鼠体内药效和毒性
与前类似地，本实施例探索Ab10-A12V2-4单剂量小鼠药效及毒性。Ab10-A12V2-4与hIgG1对照组相比，Ab10-A12V2-4组P<0.0001，从抗肿瘤活性上优于9E5-mIgG1，且没有ALT/AST的升高及血小板的下降，参见表27和图16A-16D。
表27.所测试抗体信息及TGI
药物 浓度 分子量(kDa) 给药量 TGI
hIgG1 14.17mg/ 150 3mg/kg -
  mL      
Ab10-A12V2-4 3mg/mL 200.03 4mg/kg 88.3％
9E5-mIgG1 4.4mg/mL 150 3mg/kg 59.2％
4.5抗FAP/CD40双特异性抗体药代动力学检测
本公开在mFAP-MC38荷瘤hCD40人源化小鼠中，从三个不同方向比较了抗FAP/CD40双特异性抗体的药代动力学特征。分别通过静脉注射的方式给予不同浓度的抗体，然后在不同时间点取血，通过检测小鼠血液中人Fc的含量来分析每个抗体分子的血药浓度/时间曲线。
用PBS稀释抗人IgG-Fc抗体至1.5μg/mL,用此抗体包板，每孔100μL,4℃过夜。去除溶液，每孔加入200μL PBST洗三次，弃去PBST后每孔加100μL 1％BSA，37℃封闭2h。去除溶液，每孔加入200μL PBST洗三次后每孔加入50μL稀释后的抗体或者血清样品(均用1％BSA PBS稀释，血清分别10倍、100倍、1000倍、10000倍稀释)，37℃孵育1h。去除溶液，每孔加入200μL PBST洗三次后每孔加入50μL HRP标记的羊抗人IgG-Fc二抗(1％BSA PBS稀释，1:20000)，37℃孵育20min。去除溶液，每孔加入200μL PBST洗五次后每孔加入50μL TMB显色，观察到抗体有梯度颜色反应后，每孔加入50μL ELISA终止液。Envision上机读值OD450，取血清OD450读值在抗体标准曲线线性范围内的数值换算浓度后作图分析PK曲线。
结果见图17，比较了Ab10-A297V3-2和Ab10-A12V2-2分子在13.92mg/kg给药剂量下的药代动力学特征。结果发现两个分子的药代动力学特征类似，因此，本公开双特异性抗体的药代动力学特征不因CD40抗体的序列不同而变化。

Claims (29)

  1. FAP/CD40结合分子，其包含特异性结合FAP的第一抗原结合结构域和特异性结合CD40的第二抗原结合结构域，所述特异性结合FAP的第一抗原结合结构域包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2、HCDR3，所述轻链可变区包含LCDR1、LCDR2、LCDR3，其中：
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、18所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、19所示的氨基酸序列；
    优选地，
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、15所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、14所示的氨基酸序列；
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、11所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、14所示的氨基酸序列；
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、15所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、16所示的氨基酸序列；或
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、11所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、17所示的氨基酸序列。
  2. 如权利要求1所述的FAP/CD40结合分子，其中所述特异性结合CD40的第二抗原结合结构域包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:39、40、41所示的氨基酸序列；
    优选地，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:24、25、26所示的氨基酸序列；或所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:27、28、29所示的氨基酸序列。
  3. 如权利要求1或2所述的FAP/CD40结合分子，其中所述特异性结合FAP的第一抗原结合结构域中：
    所述重链可变区包含如SEQ ID NO：3所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：4所示或与之具有至少90％同一性的氨基酸序列；
    所述重链可变区包含如SEQ ID NO：1所示或与之具有至少90％同一性的氨 基酸序列，所述轻链可变区包含如SEQ ID NO：2所示或与之具有至少90％同一性的氨基酸序列；
    所述重链可变区包含如SEQ ID NO：5所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：6所示或与之具有至少90％同一性的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO：7所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：8所示或与之具有至少90％同一性的氨基酸序列。
  4. 如权利要求2或3所述的FAP/CD40结合分子，其中所述特异性结合CD40的第二抗原结合结构域中的免疫球蛋白单一可变结构域包含如SEQ ID NO：22、23、32至38任一所示或与SEQ ID NO：22、23、32至38任一具有至少90％序列同一性的氨基酸序列。
  5. 如权利要求2至4任一项所述的FAP/CD40结合分子，其中，特异性结合CD40的第二抗原结合结构域包含2、3、4、5或6个所述免疫球蛋白单一可变结构域。
  6. 如权利要求2至5中任一项所述的FAP/CD40结合分子，其中所述特异性结合FAP的第一抗原结合结构域包含重链可变区和轻链可变区，其中，
    所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的重链可变区的N端；
    所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的重链可变区的C端；
    所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的轻链可变区的N端；和/或
    所述特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合FAP的第一抗原结合结构域的轻链可变区的C端。
  7. 如权利要求2至6中任一项所述的FAP/CD40结合分子，其中，特异性结合CD40的第二抗原结合结构域的免疫球蛋白单一可变结构域与特异性结合FAP的第一抗原结合结构域直接或通过连接子相连接；
    优选地，所述连接子为具有如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20的整数；
    更优选地，所述连接子为(G 4S) 2、(G 4S) 3或(G 4S) 4所示的氨基酸序列。
  8. 如权利要求1至7中任一项所述的FAP/CD40结合分子，其还包含人免疫 球蛋白Fc区；
    优选地，所述Fc区是人IgG1或IgG4的Fc区；
    更优选地，所述人IgG1具有去除或降低Fc效应器功能的突变；
    最优选地，所述人IgG1具有选自N297A、D265A/N297A、L234A/L235A、L234A/L235A/P329G、L234E、L234F、L234E/L235F和L234E/L235F/P329G的突变。
  9. 如权利要求1至8中任一项所述的FAP/CD40结合分子，所述特异性结合FAP的第一抗原结合结构域包含重链和轻链：
    优选地，所述重链为IgG1或IgG4同种型，所述轻链为Kappa同种型；
    更优选地，所述重链为SEQ ID NO：51所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：52所示或与之具有至少90％同一性的氨基酸序列；
    所述重链为SEQ ID NO：49所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：50所示或与之具有至少90％同一性的氨基酸序列；
    所述重链为SEQ ID NO：53所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：54所示或与之具有至少90％同一性的氨基酸序列；或
    所述重链为SEQ ID NO：55所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：56所示或与之具有至少90％同一性的氨基酸序列。
  10. 如权利要求1至9中任一项所述的FAP/CD40结合分子，其包含第一多肽链和第二多肽链，其中：
    所述第一多肽链包含如SEQ ID NO：42、44-48中任一所示或与之具有至少90％同一性的氨基酸序列，第二多肽链包含如SEQ ID NO：43所示或与之具有至少90％同一性的氨基酸序列；
    优选地，所述FAP/CD40结合分子中含有两条相同的第一多肽链和两条相同的第二多肽链。
  11. FAP结合分子，包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2、HCDR3，轻链可变区包含LCDR1、LCDR2、LCDR3，其中：
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、18所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、19所示的氨基酸序列；
    优选地，
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、15所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、14所 示的氨基酸序列；
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、11所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、14所示的氨基酸序列；
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、15所示的氨基酸序列；所述LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、16所示的氨基酸序列；或
    所述HCDR1、HCDR2、HCDR3分别包含如SEQ ID NO:9、10、11所示的氨基酸序列；LCDR1、LCDR2、LCDR3分别包含如SEQ ID NO:12、13、17所示的氨基酸序列。
  12. 如权利要求11所述的FAP结合分子，其中，
    所述重链可变区包含如SEQ ID NO：3所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：4所示或与之具有至少90％同一性的氨基酸序列；
    所述重链可变区包含如SEQ ID NO：1所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：2所示或与之具有至少90％同一性的氨基酸序列；
    所述重链可变区包含如SEQ ID NO：5所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：6所示或与之具有至少90％同一性的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO：7所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：8所示或与之具有至少90％同一性的氨基酸序列。
  13. 如权利要求11或12所述的FAP结合分子，其还包含人免疫球蛋白Fc区；
    优选地，所述Fc区是人IgG1或IgG4的Fc区；
    更优选地，所述人IgG1具有去除或降低Fc效应器功能的突变；
    最优选地，所述人IgG1具有选自N297A、D265A/N297A、L234A/L235A、L234A/L235A/P329G、L234E、L234F、L234E/L235F和L234E/L235F/P329G的突变。
  14. 如权利要求11至13中任一项所述的FAP结合分子，其包含重链和轻链，其中：
    所述重链为SEQ ID NO：51所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：52所示或与之具有至少90％同一性的氨基酸序列；
    所述重链为SEQ ID NO：49所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：50所示或与之具有至少90％同一性的氨基酸序列；
    所述重链为SEQ ID NO：53所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：54所示或与之具有至少90％同一性的氨基酸序列；或
    所述重链为SEQ ID NO：55所示或与之具有至少90％同一性的氨基酸序列，所述轻链为SEQ ID NO：56所示或与之具有至少90％同一性的氨基酸序列。
  15. 如权利要求11至14中任一项所述的FAP结合分子，其为抗FAP抗体或其抗原结合片段；
    优选地，所述抗FAP抗体或其抗原结合片段选自：双或多特异性抗体、scFv、scFv二聚体、dsFv、(dsFv)2、dsFv-dsFv'、Fv片段、Fab、Fab'或F(ab')2；
    优选地，所述抗FAP抗体或其抗原结合片段为嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。
  16. CD40结合分子，其包含结合CD40的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2、CDR3，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:39、40、41所示的氨基酸序列；
    优选地，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:24、25、26所示的氨基酸序列；或，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:27、28、29所示的氨基酸序列。
  17. 如权利要求16所述的CD40结合分子，其包含SEQ ID NO:22、23、32至38中任一所示或与之具有至少90％同一性的氨基酸序列。
  18. 如权利要求16或17所述的CD40结合分子，其还包含人免疫球蛋白Fc区；
    优选地，所述Fc区是人IgG1或IgG4的Fc区；
    更优选地，所述人IgG1的Fc区具有能够增加与FcγRIIb亲和力和/或减弱ADCC效应的突变；
    最优选地，所述人IgG1的Fc区具有选自S267E/L328F、L234A/L235A、N297A、L234F、L235E、L234F/L235E/D265A、L234A/L235A/G237A/P238S/H268A/A330S/P331S、和E233D/G237D/P238D/H268D/P271G/A330R中的突变。
  19. 如权利要求16至18中任一项所述的CD40结合分子，其包含如SEQ ID NO：57至63中任一所示或与之具有至少90％同一性的氨基酸序列。
  20. 如权利要求16至19任一项所述的CD40结合分子，其为抗CD40抗体或其抗原结合片段；
    优选地，所述抗CD40抗体或其抗原结合片段选自线性抗体、单链抗体、纳米抗体、肽抗体、结构域抗体和多特异性抗体；
    优选地，所述抗CD40抗体或其抗原结合片段为骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。
  21. FAP/CD40结合分子，其包含特异性结合FAP的第一抗原结合结构域和特异性结合CD40的第二抗原结合结构域，所述特异性结合CD40的第二抗原结合结构域包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:39、40、41所示的氨基酸序列；
    优选地，所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:24、25、26所示的氨基酸序列；或所述CDR1、CDR2、CDR3分别包含如SEQ ID NO:27、28、29所示的氨基酸序列。
  22. 如权利要求21所述的FAP/CD40结合分子，其中所述特异性结合CD40的第二抗原结合结构域中的免疫球蛋白单一可变结构域包含如SEQ ID NO：22、23、32至38任一所示或与SEQ ID NO：22、23、32至38任一具有至少90％序列同一性的氨基酸序列。
  23. 如权利要求1至10和21至22中任一项所述的FAP/CD40结合分子，其为抗FAP/CD40双特异性抗体。
  24. 多核苷酸，其编码权利要求1至10和21至23中任一项所述的FAP/CD40结合分子、权利要求11至15中任一项所述的FAP结合分子、或权利要求16至20中任一项所述的CD40结合分子。
  25. 载体，其含有权利要求24所述的多核苷酸。
  26. 宿主细胞，其含有或表达权利要求24所述的多核苷酸或权利要求25所述的载体。
  27. 药物组合物，其含有权利要求1至10和21至23中任一项所述的FAP/CD40结合分子、权利要求11至15中任一项所述的FAP结合分子、或权利要求16至20中任一项所述的CD40结合分子，以及至少一种可药用的赋形剂、稀释剂或载体。
  28. 权利要求1至10和21至23中任一项所述的FAP/CD40结合分子、权利要求11至15中任一项所述的FAP结合分子、权利要求16至20中任一项所述的CD40结合分子、权利要求24所述的多核苷酸、或权利要求27所述的药物组合物在制备治疗或缓解疾病或病症的药物中的用途；
    所述疾病或病症优选为肿瘤或癌症；
    更优选为肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症。
  29. 治疗或缓解肿瘤或癌症的方法，所述方法包括：
    向有需要的受试者施用治疗有效量的权利要求1至10和21至23中任一项所述的FAP/CD40结合分子、权利要求11至15中任一项所述的FAP结合分子、权利要求16至20中任一项所述的CD40结合分子、权利要求24所述的多核苷酸、或权利要求27所述的药物组合物；
    所述肿瘤或癌症优选为肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症。
PCT/CN2022/114522 2021-08-24 2022-08-24 Fap/cd40结合分子及其医药用途 Ceased WO2023025194A1 (zh)

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