FAP / 4-1BB / CD40 binding molecules and their pharmaceutical uses

Abstract

The present invention relates to FAP / 4-1BB / CD40 binding molecules and pharmaceutical uses thereof, particularly FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules, polynucleotides encoding the binding molecules, vectors, pharmaceutical compositions, and methods for treating cancer and related pharmaceutical uses thereof.

Description

[Technical Field]

[0001] This disclosure claims priority to Chinese Patent Application No. 202310186509.9, filed on March 1, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the biomedical field, and in particular to FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules, polynucleotides encoding the binding molecules, vectors, pharmaceutical compositions, and methods for treating cancer and related pharmaceutical uses thereof. [Background technology]

[0003] CD40 (TNFRSF5) is a transmembrane phosphoglycoprotein belonging to the tumor necrosis factor receptor superfamily (TNFRS). It is primarily expressed on the surface of DCs, macrophages, and B cells, but also on non-immune cells such as platelets and endothelial cells. Signaling through the receptor CD40 is optimal when its ligand CD40L is restricted to a trimeric configuration, thereby enhancing CD40 aggregation and subsequent recruitment and interaction with TNF receptor-associated factors. CD40L binding to CD40 on DCs induces DC maturation, which is manifested by upregulation of the expression of the B7 family of costimulatory factors (CD80, CD86) and increased secretion of proinflammatory cytokines such as interleukin-12 (IL-12). The interaction of CD40 with CD40L provides costimulatory signals for T cell activation and promotes DCs to present antigens to T cells.

[0004] 4-1BB (CD137, TNFRSF9) is a transmembrane protein that also belongs to the tumor necrosis factor receptor superfamily. It is primarily expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells. 4-1BB is expressed on the cell surface in a monomeric or dimeric form and, after binding to its ligand (4-1BBL), signals through trimerization. On T cells, 4-1BB is not constitutively expressed but is induced upon T cell receptor (TCR) activation. Stimulation from its natural ligand, 4-1BBL, or antibody agonists can further upregulate 4-1BB expression. The 4-1BB intracellular domain is thought to function by recruiting TRAF1 and TRAF2, or may be an adaptor protein for TRAF3-mediated K63 polyubiquitination. 4-1BBL trimerization leads to 4-1BB receptor aggregation and activation of TRAF-mediated NF-κB and MAPK intracellular signaling pathways, resulting in T cell proliferation, maturation, prolonged survival, and cytokine production.

[0005] Fibroblast activation protein (FAP) is a tumor-associated antigen. FAP is expressed at relatively low levels in normal tissues of healthy adults, but is selectively expressed in 93% of tumor tissues, of which 30% are highly expressed, including colon cancer, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, bladder cancer, and oral squamous cell carcinoma. Currently, no multispecific antibodies against CD40, 4-1BB, or FAP are commercially available.

[0006] Therefore, there is a strong need in the art for the development of a multispecific antibody that can mediate tumor-specific activation of CD40 and 4-1BB through FAP, and that combines good tumor therapeutic efficacy with low toxicity. The present disclosure provides a novel anti-FAP / 4-1BB / CD40 trispecific antibody that can mediate tumor-specific activation of CD40 and 4-1BB through FAP, has maturation-promoting and activation effects on APCs (e.g., dendritic cells (DC)) and T cells, can eliminate liver toxicity, peripheral blood toxicity, and other peripheral toxicities, has an excellent administration window, has potential for drug discovery, and provides a solution for clinical use. Summary of the Invention

[0007] The present disclosure provides FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules and nucleic acids encoding them, vectors, host cells, pharmaceutical compositions, and methods for treating and alleviating cell proliferative disorders (e.g., tumors or cancers) and related pharmaceutical uses thereof.

[0008] FAP binding molecule The present disclosure provides a FAP-binding molecule comprising a first antigen-binding domain that specifically binds to a FAP. In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 1, and / or the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 2.

[0009] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0010] In some embodiments, the FAP binding molecule comprises a VH and / or VL as shown below, wherein the VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 3, 4, and 5, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 6, 7, and 8, respectively.

[0011] In some embodiments, the FAP-binding molecule comprises a VH and a VL, wherein any HCDR contained in the VH has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any one of the HCDRs, and / or any LCDR contained in the VL has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any one of the LCDRs.

[0012] In some specific embodiments, the amino acid mutations are conservative replacements, substitutions or modifications, and / or deletions, additions that do not affect function.

[0013] In some embodiments, the disclosure provides FAP binding molecules comprising any one or any multiple (eg, 2, 3, 4, 5, or 6) combination of the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3.

[0014] In some embodiments, the FAP binding molecule comprises a VH and a VL, wherein: The VH comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity thereto; and / or The VL comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity thereto.

[0015] In some embodiments, the present disclosure provides FAP binding molecules comprising any one or any two combinations of the above VH, VL.

[0016] In some embodiments, the FAP-binding molecule further comprises a light chain constant region and / or a heavy chain constant region.

[0017] In some embodiments, the light chain constant region is derived from a κ light chain, a λ light chain, or a variant of any one of the above. For example, it is derived from a human κ light chain, a human λ light chain, or a variant of any one of the above. In some specific embodiments, the light chain constant region is derived from a human κ light chain or a variant thereof.

[0018] In some embodiments, the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any one of the above, for example, human IgG1, human IgG2, human IgG3, human IgG4, or a variant of any one of the above.

[0019] In some embodiments, the FAP binding molecule comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1), and the Fab light chain comprises a light chain variable region (VL) and a light chain constant region (CL).

[0020] In some specific embodiments, the FAP binding molecule comprises a Fab heavy chain and a Fab light chain, of which: The Fab heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity thereto, and / or the Fab light chain comprises the amino acid sequence set forth in SEQ ID NO: 67, or an amino acid sequence having at least 80% sequence identity thereto.

[0021] In the context of the present disclosure, "at least 80% sequence identity" covers 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, and "at least 90% sequence identity" covers 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.

[0022] In some embodiments, the FAP-binding molecule further comprises a human immunoglobulin Fc region. In some specific embodiments, the Fc region is a human IgG1, IgG2, or IgG4 Fc region.

[0023] In some embodiments, the FAP-binding molecule further comprises a human immunoglobulin Fc region, e.g., the Fc region is a human IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region has a mutation, exemplary mutations being L234A / L235A, L234A / L235A / P329G, L234E, L234F, L234E / L235F, L234E / L235F / P329G in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in IgG2 or IgG4, V23 4A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M in IgG1, H268Q / V309L / A330S / P331S in IgG2, S267E / L328F in IgG1, L234F / L235E / D265A in IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1, and S228P / F234A / L235A / G237A / P238S in IgG4. A hybrid IgG2 / 4 Fc region may also be used, for example, an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4. In some embodiments, mutations are introduced into the Fc region to reduce or eliminate effector functions of the Fc region (e.g., to reduce or eliminate ADCC effect).

[0024] In some specific embodiments, the FAP binding molecule comprises a human IgG1 Fc region, wherein the human IgG1 Fc region has at least one of the following mutations: 234A, 235A, 220A, 297A, or 297Q, 267E, 328F.

[0025] Illustratively, the Fc region of human IgG1 comprises 234A / 235A, 234A / 235A / 297A, 220A / 234A / 235A, 220A / 267E / 328F, 220A / 234A / 235A / 297A, 267E / 328F or 234A / 235A / 297A mutations. In some specific embodiments, the human IgG1 Fc region comprises L234A / L235A, L234A / L235A / N297A, C220A / L234A / L235A, C220A / 267E / 328F, C220A / L234A / L235A / N297A, S267E / L328F or L234A / L235A / N297A mutations.

[0026] In some specific embodiments, the FAP-binding molecule comprises a human IgG4 Fc region, and the human IgG4 Fc region has at least one of the following mutations: 228P, 234A, 235A, and 447A. Illustratively, the human IgG4 Fc region comprises the S228P mutation.

[0027] In the present disclosure, in the context of mutations contained in the Fc region, " / " indicates "and", for example, "L234A / L235A" indicates "L234A and L235A", i.e., the Fc contains the L234A and L235A mutations, and the amino acid positions of the mutations are numbered according to the EU numbering system.

[0028] In some embodiments, the FAP-binding molecule comprises a heavy chain (HC) and a light chain (LC), of which: the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity thereto; and / or The light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80% sequence identity thereto.

[0029] In some embodiments, the present disclosure provides FAP binding molecules comprising any one or any two combinations of the heavy chains (HC) and light chains (LC) described above.

[0030] In some embodiments, the FAP-binding molecule has a -7 K D and the FAP-binding molecule binds to human FAP or an epitope thereof at a concentration of 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower K D The FAP (e.g., human FAP) or a fragment thereof is bound to the FAP (e.g., human FAP) or a fragment thereof.

[0031] In some embodiments, the FAP-binding molecule is an anti-FAP antibody or antigen-binding fragment thereof; illustratively, the anti-FAP antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0032] In some embodiments, the FAP-binding molecule is a human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

[0033] In some specific embodiments, the antigen-binding fragment of the anti-FAP antibody includes, but is not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv), for example, the anti-FAP antigen-binding fragment is an scFv, Fv, Fab, or Fab' fragment.

[0034] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof is provided that binds to or competes with the same epitope of a FAP (eg, human FAP) as the anti-FAP antibody or antigen-binding fragment thereof.

[0035] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof is provided that blocks binding of the anti-FAP antibody or antigen-binding fragment thereof to a FAP (eg, a human FAP).

[0036] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof is provided whose binding to a FAP (eg, a human FAP) is blocked by the anti-FAP antibody or antigen-binding fragment thereof.

[0037] CD40 binding molecule The present disclosure provides CD40 binding molecules comprising a second antigen-binding domain that specifically binds to CD40. In some embodiments, the second antigen-binding domain comprises at least one immunoglobulin single variable domain that specifically binds to CD40.

[0038] In some embodiments, the immunoglobulin single variable domain that specifically binds to CD40 is 1) CDR1, CDR2, and CDR3 in the amino acid sequence shown in SEQ ID NO: 11, 15, or 32; 2) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, and / or CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and / or CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14; 3) CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 12, 13 and 14, respectively; and CDR1, CDR2 and CDR3 as shown in any one of:

[0039] The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the CDR1, CDR2, and CDR3 are defined according to the Kabat numbering system.

[0040] In some embodiments, the present disclosure provides an immunoglobulin single variable domain that specifically binds to CD40, wherein the CDR1 comprises 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any one of the CDR1s described above, and / or the CDR2 comprises 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any one of the CDR2s described above, and / or the CDR3 comprises 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any one of the CDR3s described above.

[0041] In some specific embodiments, the amino acid mutations in CDR1, CDR2 and / or CDR3 are conservative substitutions.

[0042] In some embodiments, the present disclosure provides immunoglobulin single variable domains comprising any one or any multiple (eg, 2 or 3) combination of the above CDR1, CDR2, CDR3.

[0043] In some embodiments, the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, reduced antibody aggregation, reduced antibody isomerization, reduced pre-anti-drug antibody (pre-ADA) binding, and / or reduced anti-drug antibody (ADA) binding.

[0044] In some specific embodiments, the immunoglobulin single variable domain has been modified by humanization. Illustratively, the framework region template of the human germline gene used for the humanization modification is derived from IGHV3-48*03.

[0045] In some specific embodiments, the immunoglobulin single variable domain is modified by reducing and / or eliminating binding to pre-anti-drug antibodies (pre-ADA) and / or by reducing binding to anti-drug antibodies (ADA). Illustratively, to eliminate and / or reduce binding of the 4-1BB binding molecule to pre-ADA, the C-terminal sequence of the immunoglobulin single variable domain is modified to "TVS AA " (SEQ ID NO: 69). In some specific embodiments, the C-terminal sequence of the immunoglobulin single variable domain is mutated to "TVS S " (SEQ ID NO: 68) to "TVS AA " (SEQ ID NO: 69).

[0046] In some embodiments, the immunoglobulin single variable domain in the second antigen-binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11, 15 or 32, or an amino acid sequence having at least 80% sequence identity thereto.

[0047] In some embodiments, the CD40 binding molecule comprises or is an antibody or antigen-binding fragment thereof that specifically binds to CD40 or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, or a multispecific antibody (such as a bispecific antibody, a diabody, a triabody, a tetrabody, a tandem di-scFv, or a tandem tri-scFv).

[0048] In some embodiments, the immunoglobulin single variable domain in the CD40 binding molecule is a VHH.

[0049] In some embodiments, the present disclosure provides CD40 binding molecules comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the above-described immunoglobulin single variable domains, which may be the same or different, and between any two of the immunoglobulin single variable domains may be directly linked or may be linked via a linker.

[0050] In some embodiments, an anti-CD40 antibody or antigen-binding fragment thereof that binds to the same epitope as, or competes with, the anti-CD40 antibody or antigen-binding fragment thereof is provided.

[0051] In some embodiments, an anti-CD40 antibody or antigen-binding fragment thereof is provided that blocks binding of the anti-CD40 antibody or antigen-binding fragment thereof to CD40 (eg, human CD40).

[0052] In some embodiments, an anti-CD40 antibody or antigen-binding fragment thereof is provided whose binding to CD40 (eg, human CD40) is blocked by the anti-CD40 antibody or antigen-binding fragment thereof.

[0053] In some embodiments, the CD40 binding molecule further comprises a human immunoglobulin Fc region, e.g., the Fc region is a human IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region may comprise a mutation.

[0054] Exemplary human IgG1 Fc regions have at least one of the following mutations: 234A, 235A, 220A, 297A, or 297Q, 267E, 328F. In some specific embodiments, the human IgG1 Fc region comprises 234A / 235A, 234A / 235A / N297A, C220A / 234A / 235A, C220A / 267E / L328F, C220A / L234A / L235A / N297A, S267E / L328F, or L234A / L235A / N297A mutations.

[0055] Exemplarily, the Fc region of human IgG4 has at least one of the following mutations: 228P, 234A, 235A, 447A. In some specific embodiments, the Fc region of human IgG4 comprises the S228P mutation.

[0056] In some embodiments, the immunoglobulin single variable domain in the CD40 binding molecule is linked to the Fc region directly or via a linker. The linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and lacking secondary or higher structure. For example, the linker may be a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, etc.

[0057] 4-1BB binding molecule The present disclosure provides a 4-1BB binding molecule comprising a third antigen-binding domain that specifically binds to 4-1BB. In some embodiments, the third antigen-binding domain comprises at least one immunoglobulin single variable domain that specifically binds to 4-1BB.

[0058] In some embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 28, 23 to 26, 18 and 33.

[0059] The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0060] In some embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 19, 30 and 31, wherein the amino acid sequence set forth in SEQ ID NO: 30 is DINSGGX1STFYX2DSVKG, wherein X1 is E or Q, and wherein X2 is E or A, and the amino acid sequence set forth in SEQ ID NO: 31 is HPLTX3TIATMNDYDY, wherein X3 is F or Y.

[0061] In some embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises: CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20 or 29, and CDR3 shown in SEQ ID NO: 21 or 27, Includes:

[0062] In some embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises: a-1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 29 and 27, respectively; a-2) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 20 and 27, respectively; a-3) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 20 and 21, respectively; and CDR1, CDR2 and CDR3 as shown in any one of:

[0063] In some embodiments, the CDR1 comprised in the immunoglobulin single variable domain provided by the present disclosure has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any one of the CDR1s above, and / or the CDR2 contained in said immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any one of said CDR2s, and / or The CDR3 contained in said immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any one of said CDR3s.

[0064] In some specific embodiments, the amino acid mutations in CDR1, CDR2 and / or CDR3 are conservative substitutions.

[0065] In some embodiments, the present disclosure provides immunoglobulin single variable domains comprising any one or any multiple (eg, 2 or 3) combination of the above CDR1, CDR2, CDR3.

[0066] In some embodiments, the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, reduced antibody aggregation, reduced antibody isomerization, reduced pre-anti-drug antibody (pre-ADA) binding, and / or reduced anti-drug antibody (ADA) binding.

[0067] In some specific embodiments, the immunoglobulin single variable domain has been modified by humanization. Illustratively, the framework region templates of human germline genes used for the humanization are derived from IGHV3-64*04, IGHV3-23*03, and / or IGHV3-74*01. For example, FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03, and FR3 is derived from IGHV3-74*01.

[0068] In some specific embodiments, the immunoglobulin single variable domain has been modified by the removal and / or reduction of a T-cell epitope (TCE). Reduction and / or removal of TCE sites in one or more of CDR1, CDR3, and CDR3 can reduce the immunogenicity of the 4-1BB binding molecule. Illustratively, an immunoglobulin single variable domain modified by the removal of a T-cell epitope contains the amino acid mutation 99Y, numbered according to the Kabat numbering convention.

[0069] In some specific embodiments, the immunoglobulin single variable domain is modified by reducing and / or eliminating binding to pre-anti-drug antibodies (pre-ADA) and / or by reducing binding to anti-drug antibodies (ADA). Illustratively, to eliminate and / or reduce binding of the 4-1BB binding molecule to pre-ADA, the C-terminal sequence of the immunoglobulin single variable domain is modified to "TVS AA " (SEQ ID NO: 69). In some specific embodiments, the C-terminal sequence of the immunoglobulin single variable domain is mutated to "TVS S " (SEQ ID NO: 68) to "TVS AA " (SEQ ID NO: 69).

[0070] In some specific embodiments, the immunoglobulin single variable domain has been modified by eliminating and / or reducing antibody aggregation. Illustratively, to reduce the risk of antibody aggregation, the C-terminal sequence of the immunoglobulin single variable domain is modified to "T Q In some specific embodiments, the C-terminal sequence of the immunoglobulin single variable domain is mutated to "T VTVSS" (SEQ ID NO: 71). L VTVSS" (sequence number 70) to "T Q VTVSS" (SEQ ID NO: 71).

[0071] In some embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18, 23 to 26, 28, and 33, or an amino acid sequence having at least 80% sequence identity thereto.

[0072] In some embodiments, the 4-1BB-binding molecule comprises or is an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, or a multispecific antibody (bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv).

[0073] In some embodiments, the immunoglobulin single variable domain in the 4-1BB binding molecule is a VHH.

[0074] In some embodiments, the present disclosure provides 4-1BB binding molecules comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the above-mentioned immunoglobulin single variable domains, which may be the same or different, and between any two of the immunoglobulin single variable domains may be directly linked or may be linked via a linker.

[0075] In some embodiments, the 4-1BB binding molecules of the present disclosure include variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 18, 23-26, 28, and 33, and the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function, and the amino acid mutations may occur in the CDR and / or FR regions.

[0076] In some embodiments, an anti-4-1BB antibody or antigen-binding fragment thereof is provided that binds to the same epitope as, or competes with, an immunoglobulin single variable domain in a 4-1BB binding molecule of the present disclosure.

[0077] In some embodiments, an anti-4-1BB antibody or antigen-binding fragment thereof is provided that blocks binding of an immunoglobulin single variable domain in a 4-1BB-binding molecule of the present disclosure to 4-1BB (e.g., human 4-1BB). In some specific embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof can also activate T cells and / or promote T cell proliferation.

[0078] In some embodiments, there is provided an anti-4-1BB antibody or antigen-binding fragment thereof whose binding to 4-1BB (eg, human 4-1BB) is blocked by an immunoglobulin single variable domain in a 4-1BB binding molecule of the present disclosure described above.

[0079] In some embodiments, the 4-1BB binding molecule further comprises a human immunoglobulin Fc region, for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region may have a mutation.

[0080] Exemplary human IgG1 Fc regions have at least one of the following mutations: 234A, 235A, 220A, 297A, or 297Q, 267E, 328F. In some specific embodiments, the human IgG1 Fc region comprises 234A / 235A, 234A / 235A / N297A, C220A / 234A / 235A, C220A / 267E / L328F, C220A / L234A / L235A / N297A, S267E / L328F, or L234A / L235A / N297A mutations.

[0081] Exemplarily, the Fc region of human IgG4 has at least one of the following mutations: 228P, 234A, 235A, 447A. In some specific embodiments, the Fc region of human IgG4 comprises the S228P mutation.

[0082] In some embodiments, the immunoglobulin single variable domain in the 4-1BB binding molecule is linked to the Fc region directly or via a linker. The linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and lacking secondary or higher structure. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, etc.

[0083] In some embodiments, the 4-1BB binding molecule further comprises ii) a first antigen-binding domain that specifically binds to a FAP, wherein the first antigen-binding domain is the first antigen-binding domain in any FAP-binding molecule provided by the present disclosure.

[0084] In some embodiments, the 4-1BB binding molecule further comprises iii) a second antigen-binding domain that specifically binds to CD40, wherein the second antigen-binding domain is the second antigen-binding domain in any of the CD40-binding molecules provided by the present disclosure.

[0085] In some embodiments, the 4-1BB binding molecule comprises a third antigen-binding domain that specifically binds 4-1BB, a second antigen-binding domain that specifically binds CD40, and an Fc region.

[0086] In some specific embodiments, the third antigen-binding domain that specifically binds to 4-1BB is located at the N-terminus of the Fc region, and the second antigen-binding domain that specifically binds to CD40 is located at the C-terminus of the Fc region; alternatively, the third antigen-binding domain that specifically binds to 4-1BB is located at the C-terminus of the Fc region, and the second antigen-binding domain that specifically binds to CD40 is located at the N-terminus of the Fc region.

[0087] In some specific embodiments, the 4-1BB binding molecule comprises a polypeptide set forth in the amino acid sequence set forth in SEQ ID NO: 44, or an amino acid sequence having at least 90% sequence identity thereto.

[0088] FAP / 4-1BB / CD40 binding molecule The present disclosure provides a FAP / 4-1BB / CD40 binding molecule that comprises a first antigen-binding domain that specifically binds to a FAP, a second antigen-binding domain that specifically binds to CD40, and a third antigen-binding domain that specifically binds to 4-1BB, and that can specifically bind to a FAP, 4-1BB, and CD40 simultaneously or separately.

[0089] In some embodiments, the third antigen-binding domain is a third antigen-binding domain that specifically binds to 4-1BB in any of the 4-1BB-binding molecules provided by the present disclosure above.

[0090] Illustratively, in some specific embodiments, the third antigen-binding domain comprises at least one immunoglobulin single variable domain that specifically binds to 4-1BB, and the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 28, 23 to 26, 18, and 33.

[0091] The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the CDR1, CDR2, and CDR3 are defined according to the Kabat numbering system.

[0092] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in said third antigen-binding domain comprises the CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 19, 30 and 31, respectively.

[0093] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises: CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20 or 29, and CDR3 shown in SEQ ID NO: 21 or 27, Includes:

[0094] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises: a-1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 29 and 27, respectively; a-2) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 20 and 27, respectively; a-3) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 20 and 21, respectively; and CDR1, CDR2 and CDR3 as shown in any one of:

[0095] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the third antigen-binding domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, reduced antibody aggregation, reduced antibody isomerization, reduced pre-anti-drug antibody (pre-ADA) binding, and / or reduced anti-drug antibody (ADA) binding.

[0096] Exemplarily, the immunoglobulin single variable domain in the third antigen-binding domain is modified by humanization, and the templates of the framework regions of the human germline genes used for said humanization modification are derived from IGHV3-64*04, IGHV3-23*03, and / or IGHV3-74*01. For example, FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03, and FR3 is derived from IGHV3-74*01.

[0097] Illustratively, the immunoglobulin single variable domain in the third antigen-binding domain has been modified by the removal and / or reduction of a T-cell epitope (TCE). For example, the immunoglobulin single variable domain modified by the removal of a T-cell epitope comprises the amino acid mutation 99Y, numbered according to the Kabat numbering convention.

[0098] Illustratively, the immunoglobulin single variable domain in the third antigen-binding domain is modified by reducing and / or eliminating binding to pre-anti-drug antibodies (pre-ADA) and / or by reducing binding to anti-drug antibodies (ADA). For example, the C-terminal sequence of the immunoglobulin single variable domain may be modified to have a "TVS" sequence. S " (SEQ ID NO: 68) to "TVS AA " (SEQ ID NO: 69).

[0099] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the third antigen-binding domain is modified to eliminate and / or reduce antibody aggregation. For example, the C-terminal sequence of the immunoglobulin single variable domain is changed from "TLVTVSS" (SEQ ID NO: 70) to "T Q VTVSS" (SEQ ID NO: 71).

[0100] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the third antigen-binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18, 23-26, 28 and 33, or having at least 80% sequence identity thereto.

[0101] Illustratively, in some specific embodiments, the third antigen-binding domain comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the immunoglobulin single variable domains, wherein any two of the immunoglobulin single variable domains may be the same or different, and wherein any two of the immunoglobulin single variable domains may be directly linked or may be linked via a linker.

[0102] In some embodiments, the second antigen-binding domain is a second antigen-binding domain that specifically binds to CD40 in any of the CD40-binding molecules provided by the present disclosure above.

[0103] Illustratively, in some specific embodiments, the second antigen-binding domain comprises at least one immunoglobulin single variable domain that specifically binds to CD40, wherein the immunoglobulin single variable domain is 1) CDR1, CDR2 and CDR3 in the amino acid sequences shown in SEQ ID NO: 11, 15 or 32, respectively; 2) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, and / or CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and / or CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14; 3) CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 12, 13 and 14, respectively; and CDR1, CDR2 and CDR3 as shown in any one of:

[0104] The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the CDR1, CDR2, and CDR3 are defined according to the Kabat numbering system.

[0105] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the second antigen-binding domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, reduced antibody aggregation, reduced antibody isomerization, reduced pre-anti-drug antibody (pre-ADA) binding, and / or reduced anti-drug antibody (ADA) binding.

[0106] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the second antigen-binding domain has been modified by humanization, and the template framework region of the human germline gene used for the humanization modification is derived from IGHV3-48*03.

[0107] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the second antigen-binding domain has been modified by reducing and / or eliminating pre-anti-drug antibody (pre-ADA) binding and / or by reducing anti-drug antibody (ADA) binding. For example, the C-terminal sequence of the immunoglobulin single variable domain may be modified to have a "TVS" sequence. S " (SEQ ID NO: 68) to "TVS AA " (SEQ ID NO: 69).

[0108] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the second antigen-binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 11, 15 or 32, or having at least 80% sequence identity thereto.

[0109] Illustratively, in some specific embodiments, the second antigen-binding domain comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the immunoglobulin single variable domains, wherein any two of the immunoglobulin single variable domains may be the same or different, and wherein any two of the immunoglobulin single variable domains may be directly linked or may be linked via a linker.

[0110] In some embodiments, the first antigen-binding domain is a first antigen-binding domain that specifically binds to a FAP in any of the FAP-binding molecules provided by this disclosure above.

[0111] Illustratively, in some specific embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence set forth in SEQ ID NO: 1, and / or the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence set forth in SEQ ID NO: 2.

[0112] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0113] Illustratively, in some specific embodiments, the first antigen-binding domain comprises a VH and / or VL as shown below, wherein the VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 3, 4, and 5, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 6, 7, and 8, respectively.

[0114] In some exemplary specific embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity thereto, and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 80% sequence identity thereto.

[0115] Illustratively, in some specific embodiments, the first antigen-binding domain further comprises a light chain constant region and / or a heavy chain constant region. Illustratively, the light chain constant region is derived from a human κ light chain, a human λ light chain, or a variant of any one of the above. In some specific embodiments, the light chain constant region is derived from a human κ light chain or a variant thereof. Illustratively, the heavy chain constant region is derived from a human IgG1, human IgG2, human IgG3, human IgG4, or a variant of any one of the above.

[0116] Illustratively, in some specific embodiments, the first antigen-binding domain comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1), and the Fab light chain comprises a light chain variable region (VL) and a light chain constant region (CL).

[0117] Illustratively, in some specific embodiments, the Fab heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity thereto, and / or the Fab light chain comprises the amino acid sequence set forth in SEQ ID NO: 67, or an amino acid sequence having at least 80% sequence identity thereto.

[0118] In some embodiments, the FAP / 4-1BB / CD40 binding molecule further comprises an Fc region. In some specific embodiments, the Fc region is a human IgG1, IgG2, or IgG4 Fc region.

[0119] In some embodiments, the Fc region comprises amino acid mutations, exemplary mutations are L234A / L235A, L234A / L235A / P329G, L234E, L234F, L234E / L235F, L234E / L235F / P329G in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in IgG2 or IgG4, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, 34A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M in IgG1, H268Q / V309L / A330S / P331S in IgG2, S267E / L328F in IgG1, L234F / L235E / D265A in IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1, and S228P / F234A / L235A / G237A / P238S in IgG4. A hybrid IgG2 / 4 Fc region may also be used, for example an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0120] In some specific embodiments, the FAP / 4-1BB / CD40 binding molecule comprises a human IgG1 Fc region, wherein the human IgG1 Fc region has at least one of the following mutations: 234A, 235A, 220A, 297A, or 297Q, 267E, 328F.

[0121] Illustratively, the Fc region of human IgG1 comprises 234A / 235A, 234A / 235A / 297A, 220A / 234A / 235A, 220A / 267E / 328F, 220A / 234A / 235A / 297A, 267E / 328F or 234A / 235A / 297A mutations. In some specific embodiments, the human IgG1 Fc region comprises L234A / L235A, L234A / L235A / N297A, C220A / L234A / L235A, C220A / 267E / 328F, C220A / L234A / L235A / N297A, S267E / L328F or L234A / L235A / N297A mutations.

[0122] In some specific embodiments, the FAP / 4-1BB / CD40 binding molecule comprises a human IgG4 Fc region, wherein the human IgG4 Fc region has at least one of the following mutations: 228P, 234A, 235A, 447A. Illustratively, the human IgG4 Fc region comprises the S228P mutation.

[0123] In some embodiments, mutations are introduced into the Fc region to reduce or eliminate effector functions of the Fc region (e.g., to reduce or eliminate ADCC effect).

[0124] In some embodiments, mutations can be introduced into the Fc region that cause the two subunits (Fc1 and Fc2) of the Fc region to pair and form a dimer. For example, within the CH3 / CH3 interface, one or more amino acid residues in the CH3 domain of Fc1 are mutated with amino acid residues having one or more larger side chain volumes, thereby creating a protrusion (or knob) on the surface of the CH3 domain of Fc1. Alternatively, one or more, preferably two or three, amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having smaller side chain volumes, thereby creating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the input residues having larger side chain volumes are phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the input residue with the smaller side chain volume is serine (S), alanine (A), valine (V), or threonine (T). Illustratively, the knob mutation comprises a T366W substitution, and the hole mutation is selected from at least one, at least two, or three of the following mutations: T366S, L368A, and Y407V.

[0125] In some embodiments, the Fc region is capable of increasing the in vivo half-life of the binding molecule upon formation of a dimeric molecule.

[0126] In some embodiments, in the FAP / 4-1BB / CD40 binding molecule, the first antigen-binding domain comprises a Fab heavy chain and a Fab light chain, the Fab heavy chain is located at the N-terminus of the Fc region, the third antigen-binding domain is located at the C-terminus of the Fc region, and the second antigen-binding domain is located at the C-terminus of the third antigen-binding domain; the Fab heavy chain is located at the N-terminus of the Fc region, the third antigen-binding domain is located at the C-terminus of the Fc region, and the second antigen-binding domain is located at the C-terminus of the Fab light chain; the Fab heavy chain is located at the N-terminus of the Fc region, the second antigen-binding domain is located at the C-terminus of the Fc region, and the third antigen-binding domain is located at the C-terminus of the second antigen-binding domain; Or, The Fab heavy chain is located at the N-terminus of the Fc region, the second antigen-binding domain is located at the C-terminus of the Fc region, and the third antigen-binding domain is located at the C-terminus of the Fab light chain.

[0127] In some embodiments, the third antigen-binding domain that specifically binds to 4-1BB and the second antigen-binding domain that specifically binds to CD40 may be linked directly or via a linker.

[0128] In some embodiments, the third antigen-binding domain that specifically binds to 4-1BB may be linked directly to the Fc region or may be linked via a linker.

[0129] In some embodiments, the second antigen-binding domain that specifically binds to CD40 may be linked directly to the Fc region or may be linked via a linker.

[0130] In some embodiments, the third antigen-binding domain that specifically binds to 4-1BB and the Fab light chain having the first antigen-binding domain that specifically binds to FAP may be linked directly or via a linker.

[0131] In some embodiments, the second antigen-binding domain that specifically binds to CD40 and the Fab light chain having the first antigen-binding structure that specifically binds to a FAP may be linked directly or via a linker.

[0132] In some embodiments, the third antigen-binding domain that specifically binds to 4-1BB comprises two, three, four, five, six, seven, eight, etc. immunoglobulin single variable domains, of which any two immunoglobulin single variable domains may be directly linked or linked via a linker.

[0133] In some embodiments, the second antigen-binding domain that specifically binds to CD40 comprises two, three, four, five, six, seven, eight, etc. immunoglobulin single variable domains, wherein any two of the immunoglobulin single variable domains may be directly linked or may be linked via a linker.

[0134] Illustratively, the linker is m S n ) h or (GGNGT) h (SEQ ID NO: 72) or (YGNGT) h (SEQ ID NO: 73) or (EPKSS) h (SEQ ID NO: 74), in which m and n are each independently selected from integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0135] In some embodiments, the linker is (G x S) y a linker, wherein x is selected from an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5) and y is selected from an integer of 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6); in some specific embodiments, the linker is selected from G4S (SEQ ID NO: 75), GS, GAP, (G4S)2 (SEQ ID NO: 76), (G4S)3 (SEQ ID NO: 77), (G4S)4 (SEQ ID NO: 78), (G4S)5 (SEQ ID NO: 79), ASGS (SEQ ID NO: 80), for example, (G4S)2.

[0136] In some embodiments, when the number of linkers contained in a FAP / 4-1BB / CD40 binding molecule is two or more, any two linkers may be the same or different.

[0137] In some specific embodiments, the FAP / 4-1BB / CD40 binding molecule comprises an anti-FAP antibody comprising two heavy chains (HC) and two light chains (LC), wherein the VH of one HC and the VL of one LC form an antigen-binding site, and the VH of the other HC and the VL of the other LC form an antigen-binding site.

[0138] In some specific embodiments, the FAP / 4-1BB / CD40 binding molecule comprises at least one VHH (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) that specifically binds to CD40 and at least one VHH (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) that specifically binds to 4-1BB.

[0139] In some specific embodiments, any one of the VHHs that specifically bind to CD40 can be linked to the C-terminus of the heavy chain of an anti-FAP antibody and / or the C-terminus of the light chain of an anti-FAP antibody. The C-terminus of the VHH that specifically binds to CD40 can continue to link one or more VHHs that specifically bind to CD40 and / or one or more VHHs that specifically bind to 4-1BB.

[0140] In some specific embodiments, any one of the VHHs that specifically bind to 4-1BB can be linked to the C-terminus of the anti-FAP antibody heavy chain and / or the C-terminus of the anti-FAP antibody light chain. The C-terminus of the VHH that specifically binds to 4-1BB can continue to link one or more VHHs that specifically bind to 4-1BB and / or one or more VHHs that specifically bind to CD40.

[0141] In some embodiments, in the FAP / 4-1BB / CD40 binding molecule, the valency ratio among the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is (1-2):(1-5):(1-5), and in some embodiments, the valency ratio among the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is 1:(1-4):(1-4). In some specific embodiments, the valency ratio is 1:2:2, 1:2:4, 1:1:1, or 1:1:2.

[0142] In this disclosure, "valency" is described as the number of antigen-binding moieties present in an antigen-binding molecule (e.g., antigen-binding domain). Thus, a single binding molecule can bind to more than one binding site on a target molecule.

[0143] In some embodiments, the FAP / 4-1BB / CD40 binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein, from N-terminus to C-terminus, the FAP / 4-1BB / CD40 binding molecule comprises: (I) a first polypeptide chain: [a Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[a third antigen-binding domain that specifically binds to 4-1BB]-[linker]b-[a second antigen-binding domain that specifically binds to CD40]; and second polypeptide chain: [a Fab light chain of the first antigen-binding domain that specifically binds to the FAP], (II) a first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[a third antigen-binding domain that specifically binds to 4-1BB], and second polypeptide chain: [Fab light chain of a first antigen-binding domain that specifically binds to FAP]-[linker]b-[second antigen-binding domain that specifically binds to CD40]; (III) first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[third antigen-binding domain that specifically binds to 4-1BB]-[linker]b-[third antigen-binding domain that specifically binds to 4-1BB], and second polypeptide chain: [Fab light chain of a first antigen-binding domain that specifically binds to FAP]-[linker]c-[second antigen-binding domain that specifically binds to CD40]; (IV) a first polypeptide chain: [a Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[a second antigen-binding domain that specifically binds to CD40]-[linker]b-[a third antigen-binding domain that specifically binds to 4-1BB]; and second polypeptide chain: [a Fab light chain of the first antigen-binding domain that specifically binds to the FAP], (V) a first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[a second antigen-binding domain that specifically binds to CD40], and second polypeptide chain: [Fab light chain of the first antigen-binding domain that specifically binds to FAP]-[linker]b-[third antigen-binding domain that specifically binds to 4-1BB]; any group or combination of: wherein - represents a peptide bond, any two linkers may be the same or different, and a, b, and c are each independently selected from 0 or 1.

[0144] Illustratively, the linker is m S n ) h or (GGNGT) h (SEQ ID NO: 72) or (YGNGT) h (SEQ ID NO: 73) or (EPKSS) h(SEQ ID NO: 74), in which m and n are each independently selected from integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0145] In some embodiments, the linker is (G x S) y a linker, wherein x is selected from an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5) and y is selected from an integer of 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6); in some specific embodiments, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, and ASGS, for example, (G4S)2.

[0146] In some embodiments, the FAP / 4-1BB / CD40 binding molecule comprises two or more first antigen-binding domains that specifically bind to a FAP, wherein any two first antigen-binding domains that specifically bind to a FAP may be the same or different and are selected from any first antigen-binding domain provided by the present disclosure; In some embodiments, the FAP / 4-1BB / CD40 binding molecule comprises two or more second antigen-binding domains that specifically bind to CD40, wherein any two second antigen-binding domains that specifically bind to CD40 may be the same or different and are selected from any second antigen-binding domain provided by the present disclosure; In some embodiments, the FAP / 4-1BB / CD40 binding molecule comprises two or more third antigen-binding domains that specifically bind to 4-1BB, and any two third antigen-binding domains that specifically bind to 4-1BB may be the same or different and are selected from any third antigen-binding domain provided by the present disclosure.

[0147] In some embodiments, the FAP / 4-1BB / CD40 binding molecule comprises a first polypeptide chain and a second polypeptide chain shown in any one of the following: 1) the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 49 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence having 80% sequence identity thereto.

[0148] 2) the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 35 or an amino acid sequence having 80% sequence identity thereto.

[0149] 3) the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 36 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 37 or an amino acid sequence having 80% sequence identity thereto.

[0150] 4) The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 38 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 39 or an amino acid sequence having 80% sequence identity thereto.

[0151] 5) The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 40 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having 80% sequence identity thereto.

[0152] 6) The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 42 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence having 80% sequence identity thereto.

[0153] 7) The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 46 or an amino acid sequence having 80% sequence identity thereto.

[0154] 8) The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 47 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having 80% sequence identity thereto.

[0155] In some embodiments, the FAP / 4-1BB / CD40 binding molecule comprises two first polypeptide chains and two second polypeptide chains, hi some embodiments, the FAP / 4-1BB / CD40 binding molecule has two identical first polypeptide chains and two identical second polypeptide chains.

[0156] In some embodiments, the FAP / 4-1BB / CD40 binding molecule has at least one of the following properties:

[0157] a) specifically binding to human FAP or an epitope thereof; b) specifically binds to human 4-1BB or an epitope thereof; c) specifically binds to human CD40 or an epitope thereof; d) weakly or not activating the 4-1BB signaling pathway when not cross-linked by FAP and CD40; e) relatively strong or strong activation of the 4-1BB signaling pathway after cross-linking by FAP and / or CD40, e.g., EC 50 ≦1 nM, and illustratively, 50 ≤0.9 nM, EC 50 ≤0.8nM, EC 50 ≤0.7 nM, EC 50 ≤0.6 nM, EC 50 ≤0.5nM, EC 50 ≤0.4nM, EC 50 ≤0.3 nM, EC 50 ≤0.2nM, EC 50 ≤0.1 nM, EC 50 ≤0.09nM, EC 50 ≤0.08nM, EC 50 ≤0.07nM, EC 50 ≤0.06nM, EC 50 ≤0.05nM, EC 50 ≤0.04nM, EC 50 ≤0.03nM, EC 50 ≦0.02 nM or less, f) weakly or not activating the CD40 signaling pathway when not cross-linked by FAP and 4-1BB; g) relatively strong or strong activation of the CD40 signaling pathway after cross-linking by FAP and / or 4-1BB, e.g., EC 50 ≦1 nM, and illustratively, 50 ≤0.9 nM, EC 50 ≤0.8nM, EC 50 ≤0.7 nM, EC 50 ≤0.6 nM, EC 50 ≤0.5nM, EC 50 ≤0.4nM, EC 50 ≤0.3 nM, EC 50 ≤0.2nM, EC 50 ≤0.1 nM, EC 50 ≤0.09nM, EC 50 ≤0.08nM, EC 50 ≤0.07nM, EC 50 ≤0.06nM, EC 50 ≤0.05nM, EC 50 ≤0.04nM, EC50 ≤0.03nM, EC 50 ≦0.02 nM or less, h) activating T cells and / or promoting T cell proliferation; I) promoting the activation of APCs (e.g., dendritic cells DC) and / or proliferation of APCs (e.g., dendritic cells DC); j) low immunogenicity and / or low levels of binding to pre-drug antibodies (pre-ADA); k) Inhibiting tumor growth.

[0158] In some embodiments, the FAP / 4-1BB / CD40 binding molecules of the present disclosure can inhibit tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0159] In some embodiments, the FAP / 4-1BB / CD40 binding molecule is an anti-FAP / 4-1BB / CD40 multispecific antibody or antigen-binding fragment thereof, including, but not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv).

[0160] In some embodiments, there is provided an anti-FAP / 4-1BB / CD40 multispecific antibody or antigen-binding fragment thereof that binds to or competitively binds to FAP, 4-1BB and / or CD40, or that binds to or competitively binds to the same epitope on FAP, 4-1BB and / or CD40 as the FAP / 4-1BB / CD40 binding molecule of the present disclosure.

[0161] In some embodiments, there are provided anti-FAP / 4-1BB / CD40 multispecific antibodies or antigen-binding fragments thereof that block binding of the FAP / 4-1BB / CD40 binding molecules of the present disclosure to FAP, 4-1BB and / or CD40.

[0162] In some embodiments, a protein or molecule is provided that comprises any of the FAP / 4-1BB / CD40 binding molecules of the present disclosure. For example, the protein or molecule is a complex, and the complex may include, for example, any detectable label.

[0163] The present disclosure provides an anti-FAP / 4-1BB / CD40 multispecific antibody that can mediate tumor-specific CD40 and 4-1BB activation by FAP, has maturation-promoting and activation effects on APCs (e.g., dendritic cells (DC)) and T cells, has low or no liver toxicity, peripheral blood toxicity, and other peripheral toxicities, has an excellent administration window, and has potential for drug discovery, providing a solution for the clinical treatment of tumors.

[0164] Polynucleotides and Vectors The present disclosure provides polynucleotides that encode any of the 4-1BB binding molecules, FAP / 4-1BB / CD40 binding molecules, FAP binding molecules, and CD40 binding molecules provided by the present disclosure.

[0165] In some embodiments, the polynucleotide may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the polynucleotide of the present disclosure is essentially an isolated nucleic acid.

[0166] The nucleic acids of the present disclosure may be in the form of, present in, and / or part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may, in particular, be an expression vector, i.e., a vector capable of providing expression of the FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, FAP binding molecule, or CD40 binding molecule in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vectors typically contain at least one nucleic acid of the present disclosure, operably linked to one or more suitable expression control elements (e.g., promoter, enhancer, terminator, etc.). The selection of these elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for expression of the FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules, FAP binding molecules, CD40 binding molecules of the disclosure are, for example, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.

[0167] The nucleic acids of the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of a polypeptide of the present disclosure, and / or may be isolated from a suitable natural source.

[0168] host cell The present disclosure provides recombinant host cells that express or are capable of expressing one or more binding molecules of the present disclosure, including at least one of a FAP / 4-1BB / CD40 binding molecule, a 4-1BB binding molecule, a FAP binding molecule, and a CD40 binding molecule.

[0169] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0170] Exemplary bacterial cells include, for example, cells of Gram-negative strains (e.g., Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0171] Exemplary fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0172] Illustratively, mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0173] The present disclosure may use amphibian cells, insect cells, plant cells and any other cells known in the art for expressing heterologous proteins.

[0174] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0175] Production or preparation method The present disclosure provides methods for preparing any of the protein-binding molecules of the present disclosure, including at least one of a FAP / 4-1BB / CD40 binding molecule, a 4-1BB binding molecule, a FAP binding molecule, and a CD40 binding molecule. In some embodiments, the methods include: - culturing a host cell of the present disclosure under conditions suitable for expression of said antibody; - recovering the protein of interest expressed in the host cells from the culture; - optionally further purifying and / or modifying the protein of interest of the present disclosure; Includes:

[0176] The FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules, FAP binding molecules, CD40 binding molecules, etc. of the present disclosure may be produced intracellularly in cells such as those described above (e.g., in the cytoplasm, periplasm or in inclusion bodies), then isolated from the host cells and optionally further purified, or may be produced extracellularly (e.g., in the medium in which the host cells are cultured), then isolated from the medium and optionally further purified.

[0177] Methods and reagents for recombinantly producing polypeptides, such as specific appropriate expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, target protein isolation and purification techniques applicable to the production of the binding molecules or antibodies of the present disclosure are known to those of skill in the art. Methods for producing and purifying antibodies are well known in the art and can be found, for example, in the Cold Spring Harbor Antibody Laboratory Techniques Manual (Chapters 5-8 and 15). Engineered antibodies of the present disclosure may be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains may be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be used to stably transfect cells. Mammalian expression systems result in glycosylation of antibodies, particularly 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 to produce antibodies. The culture medium into which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be concentrated by filtration using conventional methods. Soluble contaminants and polymers can be removed using conventional methods such as molecular sieving or ion exchange. The resulting product should be immediately frozen at, for example, -70°C or lyophilized.

[0178] However, the FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules, FAP binding molecules, CD40 binding molecules, etc. of the present disclosure may also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0179] composition The present disclosure provides compositions comprising any one or combination of any FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, polynucleotide, or vector encoding a FAP / 4-1BB / CD40 binding molecule or 4-1BB binding molecule provided by the present disclosure.

[0180] In some embodiments, the pharmaceutical composition contains an amount of the FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, or encoding nucleic acid or vector effective to treat, alleviate, or prevent cancer.

[0181] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0182] In some specific embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the FAP / 4-1BB / CD40 binding molecule, or the amount of the FAP / 4-1BB / CD40 binding molecule contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0183] In some specific embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the 4-1BB binding molecule, or the amount of the 4-1BB binding molecule contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0184] In some specific embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the FAP / 4-1BB / CD40 binding molecule and the 4-1BB binding molecule, or the amount of the FAP / 4-1BB / CD40 binding molecule and the 4-1BB binding molecule contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0185] In some embodiments, an article of manufacture or product is provided that includes the FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, polynucleotide, and / or vector. Optionally, the article of manufacture includes a container and a label. The container may be, for example, a vial, syringe, or test tube. The container holds a composition that is effective for treating a medical condition. A label on or associated with the container indicates that the composition is used to treat a selected medical condition. The composition contains the FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, polynucleotide, and / or vector.

[0186] Reagent Kits and Detection The present disclosure provides a reagent kit comprising the above-described FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, polynucleotide, vector, or composition. The present disclosure further provides a method, system, or device for detecting FAP, 4-1BB, or CD40 in vivo or in vitro, which comprises treating a sample with the above-described binding molecule, polynucleotide, vector, or composition of the present disclosure.

[0187] In some embodiments, the in vitro detection method, system, or device comprises, e.g., (1) contacting the sample with a FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, polynucleotide, vector, or composition; (2) detecting a complex formed between the binding molecule, polynucleotide, or vector and the sample; and / or (3) contacting a reference sample (e.g., a control sample) with a binding molecule, a polynucleotide; and (4) determining the extent of complex formation by comparison with a reference sample; For example, a change (e.g., a statistically significant change) in the formation of the complex in the sample or subject compared to that in a control sample or subject indicates the presence of FAP, 4-1BB, CD40 in the sample.

[0188] In some other embodiments, the in vivo detection method, system, or device comprises: (1) administering to a subject the binding molecule, polynucleotide, or vector; and (2) detecting the formation of a complex between the binding molecule, polynucleotide, or vector and the target; may include:

[0189] Detection may include determining the location or time of complex formation. Detection of the protein or nucleic acid capable of binding (e.g., FAP, 4-1BB, CD40) can be achieved by labeling the binding molecule or nucleic acid with a detectable substance and detecting the label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Formation of a complex between the binding molecule or nucleic acid and FAP, 4-1BB, or CD40 can be detected by measuring or visualizing the substance that does or does not bind to FAP, 4-1BB, or CD40. Conventional detection assays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry, can be used. For detection purposes, the binding molecules and nucleic acids of the present disclosure can be labeled with a fluorophore chromophore. In some embodiments, diagnostic reagents containing the nucleic acids or binding molecules are further provided, as well as related diagnostic uses.

[0190] In some embodiments, a reagent kit is further provided, which includes the binding molecule and polynucleotide, and may further include a diagnostic manual. The reagent kit may further include at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the binding molecule may be prepared as a pharmaceutical composition.

[0191] Methods for treating diseases and pharmaceutical uses The present disclosure provides methods and uses of FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules, encoding polynucleotides, vectors or pharmaceutical compositions in the prevention, treatment or amelioration of a disease or condition.

[0192] In some embodiments, the FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, or polynucleotide, vector, pharmaceutical composition provided by the present disclosure is: 1) Use in the preparation of a medicament for preventing, treating, or ameliorating a disease or condition; 2) For the prevention, treatment, or alleviation of a disease or condition; 3) For T cell activation, 4) Use in the preparation of a medicament for activating T cells; 5) To promote DC cell activation, 6) Use in the preparation of a medicament for promoting the activation of DC cells; There is at least one use.

[0193] In some embodiments, the present disclosure provides a method for preventing, treating, or alleviating a disease or condition, comprising administering to a patient or subject an effective amount of a FAP / 4-1BB / CD40 binding molecule, 4-1BB binding molecule, or encoding polynucleotide, vector, or pharmaceutical composition of the present disclosure to prevent, treat, or alleviate the disease or condition.

[0194] In some embodiments, the disease or condition is a tumor or cancer.

[0195] In some specific embodiments, the tumor or cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, and hematological cancer.

[0196] In some embodiments, the FAP / 4-1BB / CD40 binding molecules, 4-1BB binding molecules, or encoding polynucleotides, vectors, pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, and administration may be systemic or local.

[0197] In some embodiments, dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response), e.g., a single dose, multiple doses over a period of time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0198] Definition of Terms In order that this disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0199] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0200] "CD40" and "CD40 antigen" refer to an approximately 48 kD glycoprotein expressed on the surface of normal and neoplastic B cells, which acts as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al., 1987, J. Immunol. 138:788-785). Cells that endogenously express CD40 are any cells characterized by surface expression of CD40, including, but not limited to, normal and neoplastic B cells, interdigitating cells, basal epithelial cells, cancer cells, macrophages, endothelial cells, follicular dendritic cells, tonsillar cells, and bone marrow-derived plasma cells. Unless otherwise specified, "CD40" in the present disclosure refers to any native CD40 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). A cDNA molecule encoding CD40 was isolated from a library prepared from the Burkitt's lymphoma cell line Raji (Stamenkovic et al., 1989, EMBO J. 8:1403). In this disclosure, "CD40" encompasses full-length, unprocessed CD40 and any cell-derived processed form of CD40, as well as naturally occurring variants of CD40, such as splice variants and allelic variants. It also encompasses artificially expressed forms, functional variants, and the like.

[0201] "4-1BB" or "4-1BB antigen" refers to CD137, also known as tumor necrosis factor receptor superfamily 9, a member of the TNF receptor superfamily (TNFRSF), and CD8 + and CD4 +4-1BB is a costimulatory molecule expressed on the cell surface of T cells, regulatory T cells (Tregs), NK cells, NKT cells, B cells, neutrophils, and the like. 4-1BB belongs to the costimulatory molecules and is expressed after immune cell activation. The NCBI accession number for human 4-1BB is NP_001552.2. In the present disclosure, "4-1BB" may optionally include any of the proteins, fragments, or variants thereof, including (but not limited to) the known or wild-type 4-1BB described herein, and any naturally occurring splice variants, amino acid variants, or isoforms. It also includes artificially expressed forms, functional variants, and the like.

[0202] "Fibroblast activation protein (FAP)" and "FAP antigen," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refer to any naturally occurring FAP from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. "FAP" in this disclosure covers full-length, unprocessed FAPs and any cell-derived processed forms of FAPs, as well as naturally produced variants of FAPs, such as splice variants and allelic variants. It also includes artificially expressed forms, functional variants, and the like.

[0203] The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884, or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2, GeneBank Accession No. AAC51668. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to position 760.

[0204] The term "functional variant" includes, but is not limited to, homologs, fragments, truncations, mutants, modifications, etc. of a wild-type protein, where a functional variant of a protein has increased, decreased, or maintained activity of the protein compared to the wild-type protein.

[0205] "Binding molecule" covers any protein, polypeptide, or any molecule encompassing said protein or polypeptide, including, but not limited to, an antibody as defined in this disclosure, that is capable of specifically binding to an antigen (e.g., CD40, 4-1BB, or FAP).

[0206] In this disclosure, the terms "polypeptide," "peptide," or "protein" may be used interchangeably and refer to a polymer of amino acid residues or a collection of polymers of multiple amino acid residues. These terms are used to refer to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, and to naturally occurring and non-naturally occurring amino acid polymers. Polypeptide sequences are typically written such that the left-hand end of the polypeptide sequence is the amino-terminus (N-terminus) and the right-hand end of the polypeptide sequence is the carboxy-terminus (C-terminus).

[0207] The term "antibody" covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding domains), as long as they exhibit the desired antigen-binding activity.

[0208] An antibody may refer to an immunoglobulin, which has a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. The antigenicity of immunoglobulins varies due to differences in the amino acid composition and sequence of the heavy chain constant region. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ or λ chains based on differences in the constant region. Each of the five types of Ig may have either κ or λ chains. In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is highly variable and forms the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region (C region). The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0209] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly applicable in some embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" broadly refers to a product, e.g., a cell or a nucleic acid, protein, or vector, that has been modified by introducing a heterologous nucleic acid or protein or by modifying a naturally occurring nucleic acid or protein, or that the cell is derived from a cell that has been so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form, or express naturally occurring genes that are aberrantly expressed, under-expressed, or not expressed at all.

[0210] The term "antigen-binding domain" encompasses Fab, modified Fab, Fab', modified Fab', F(ab'), Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any one of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing and preparing these antigen-binding fragments are known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0211] A "multispecific antibody" refers to an antibody that binds to two or more different target antigens or different antigenic epitopes. The term "multispecific antibody" includes bispecific, trispecific, tetraspecific, pentaspecific, and hexaspecific. As used herein, the term "trispecific antibody" refers to an antibody that binds to three different antigens (e.g., FAP, CD40, or 4-1BB) or three different epitopes.

[0212] Determination or definition of CDRs can be achieved by solving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby accurately delineating the CDRs and identifying the residues that comprise the antibody's binding site. This can be accomplished by any one of a variety of techniques known to those of skill in the art, such as X-ray crystallography. Various analytical methods can be used to identify CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0213] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res, 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the location of certain structural loop regions (see, e.g., 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 from the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the base sequence (see, e.g., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).Additionally, the definition of the boundaries of other CDRs may not strictly follow one of the above methods, but may be shortened or extended based on predictions or experimental results that show that a particular residue or group of residues does not significantly affect antigen binding, while still overlapping with at least a portion of the Kabat CDRs. As used in this disclosure, CDR can refer to a CDR defined by any method (including a combination of methods) known in the art. The correspondence between each numbering system is well known to those skilled in the art and is illustratively shown in Table 1 below.

[0214] [Table 1]

[0215] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the known Kabat numbering system.

[0216] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of the protein and can often be added, removed, or transferred to other proteins without loss of function of the other parts and / or domains of the protein.

[0217] "Immunoglobulin domain" refers to the globular region of an antibody chain, which is characterized by maintaining the folding characteristics of the antibody molecule.

[0218] An "immunoglobulin variable domain" is an immunoglobulin domain that essentially consists of four "framework regions", namely "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", and three "complementarity-determining regions" or "CDRs", namely "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3". Thus, the general structure or sequence of an immunoglobulin variable domain may be shown as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain contains an antigen-binding site, thereby conferring specificity for an antigen.

[0219] "Antibody framework (FR)" refers to the part of a variable domain that serves as a support for the antigen binding loops (CDRs) of that variable domain.

[0220] The term "immunoglobulin single variable domain" is typically used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain and comprises a VH, VHH or VL domain) that is capable of forming a functional antigen-binding site when it does not interact with other variable domains (e.g., in the absence of the necessary VH / VL interactions between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" are nanobodies (including VHH, camelized VH such as humanized VHH and / or camelized human VH), IgNARs, domains, (single domain) antibodies (e.g., dAbs) as or derived from a VH domain. TM ) and (single domain) antibodies (e.g., dAbs) as or derived from the VL domain. TM ) Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. One specific example of an immunoglobulin single variable domain is a "VHH domain" (or abbreviated "VHH"), defined as follows:

[0221] "VHH domain" refers to a heavy chain single domain antibody, VHH, V HH These variable domains are also called "heavy-chain antibodies" (i.e., "antibodies lacking light chains"), and are the variable domains of antigen-binding immunoglobulins called "heavy-chain antibodies" (i.e., "antibodies lacking light chains") (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)). The term "VHH domain" is used to distinguish these variable domains from the VH and VL domains present in conventional antibodies with a tetrapeptide chain structure. VHH domains specifically bind to an epitope without the need for another antigen-binding domain (this is the opposite of the VH or VL domains in conventional antibodies with a tetrapeptide chain structure, where the epitope is recognized by both the VL and VH domains). VHH domains are small, stable, and efficient antigen-recognition units formed by a single immunoglobulin domain. "Heavy chain single domain antibody", "VHH domain", "VHH", "V HHThe terms "domain," "VHH antibody fragment," "VHH antibody," "Nanobody®," and "Nanobody® domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) may be used interchangeably. "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids and then humanized, or antibodies obtained by screening using phage display technology. The total number of amino acid residues in a VHH domain is usually in the range of 110-120, and often between 112-115. However, it should be noted that both relatively short and relatively long sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have previously been disclosed in the following documents: 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, 17 June 2009; and WO94 / 04678.

[0222] As is known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions based on the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows). This generally means that the Kabat numbering may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or conventions include Chothia, IMGT, and AbM.

[0223] A "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting nonhuman CDR sequences onto a human antibody variable region framework. This can overcome the strong immune response induced by chimeric antibodies containing a large amount of nonhuman protein components. To avoid a decrease in activity associated with reduced immunogenicity, activity can be maintained by performing minimal backmutations on the fully human antibody variable region. An example of "humanization" includes "humanizing" a VHH domain derived from a camelid by substituting one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a normal human tetrapeptide chain structure antibody. The humanized VHH domain may comprise one or more fully human framework region sequences, and in some specific embodiments, may comprise human framework region sequences of IGHV3. Another example of "humanization" includes antibodies produced by grafting mouse CDR sequences onto the variable region framework of a human antibody, i.e., the framework sequences of a different type of human germline antibody. This method can overcome the strong antibody response induced by chimeric antibodies containing a large amount of mouse protein components. Humanization methods include, for example, resurfacing of protein surface amino acids and CDR grafting to a universal framework, in which CDRs are "grafted" onto other "stents" (including, but not limited to, human stents or non-immunoglobulin stents). Suitable stents and techniques for CDR grafting are known in the art. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies of the present disclosure also include phage-displayed humanized antibodies that have undergone affinity maturation against their CDRs.Furthermore, in order to avoid a decrease in activity due to a decrease in immunogenicity, the activity can be maintained by performing minimal back mutations or reverse mutations on the framework sequences of the variable regions of the human antibody.

[0224] "Pre-ADA" (i.e., pre-ADA) is an ADA already present in a subject or individual to whom a drug (e.g., a protein or polypeptide-based drug, further, e.g., an antibody drug) is to be administered. Pre-ADA can be present in a subject or individual used for the first time in an experiment (i.e., a subject or individual to whom the drug has not been administered before).

[0225] Generally, "specific binding" or "selective binding" means that the binding molecule binds to an epitope on an antigen. The FAP / 4-1BB / CD40 binding molecules of the present disclosure bind to an epitope on an antigen, preferably at a concentration of 1000 kJ / µL, as measured in a Biacore, KinExA, or Fortibio assay. -7 ~10 -10 moles / liter (M), more preferably 10 -8 ~10 -10 moles / liter, more preferably 10 -9 ~10 -10 or lower dissociation constant (K D ), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably 10 -9 M, more preferably at least 10 -10 It binds to the antigen (i.e., FAP, CD40, 4-1BB) or its epitope with an association constant (KA) of M. -4 Any K greater than M D Values ​​are also generally considered to represent non-specific binding. Specific binding of a binding molecule to an antigen or epitope can be measured by any suitable method known in the art, including, for example, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry sorting (FACS), as described herein.

[0226] "Epitope" refers to the site on an antigen that binds to an immunoglobulin or antibody. An epitope may be formed from adjacent amino acids or from juxtaposed non-adjacent amino acids formed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. An epitope typically contains at least 3-15 amino acids in a unique spatial conformation. Methods for determining the epitope that binds to a given antibody are well known in the art and include Western blotting and immunoprecipitation detection assays. Methods for determining the spatial conformation of an epitope include techniques known in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0227] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules is determined by the dissociation constant (K D ) can be quantified by determining the K. For example, surface plasmon resonance (SPR) methods (Biacore) can be used to measure the kinetics of complex formation and dissociation. D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or k) and the dissociation rate constant kd (or koff), respectively. D is K D The dissociation constants ka and kd are related by the equation ka = kd / ka. The value of the dissociation constant can be determined directly by well-known methods and can also be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, the K can be calculated by double filtration nitrocellulose filter binding assays such as those disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). DOther standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and FACS, as well as other assays mentioned elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), e.g., Biacore. TM The K of each antibody / antigen complex may be evaluated by the KinExA system or KinExA. D By comparing values, one can compare the binding affinities associated with interactions with different molecules, for example, comparing the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by the K D value and the K of a non-target interaction (e.g., a known control antibody that does not bind to the FAP). D It can be evaluated by determining and comparing values.

[0228] "Conservative substitution" refers to a substitution of an amino acid residue with another amino acid residue having similar properties to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it is clear to those skilled in the art that even when amino acid residues in the above-mentioned group showing similar properties are substituted, it does not show a specific change in properties.

[0229] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two nucleic acid sequences or two polypeptides. When every position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, when each position in two DNA molecules is occupied by the same nucleotide, the molecules are homologous at that position. The percentage of 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 positions compared, multiplied by 100%. For example, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Typically, two sequences are compared when aligned to obtain the maximum percentage of homology.

[0230] "Nucleic acid" or "polynucleotide" may be used interchangeably herein and refer to any DNA or RNA molecule, single- or double-stranded, and, if single-stranded, to the molecule of its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the coding sequence.

[0231] A "host cell" includes an individual cell or cell culture that may be, or has been, a recipient of a vector for incorporating a nucleic acid insert. A host cell includes the progeny of a single host cell, and due to natural, accidental, or deliberate mutation, the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell. A host cell includes cells transfected and / or transformed in vivo with a nucleic acid according to the present disclosure. "Cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. It should also be understood that due to deliberate or unintentional mutation, all progeny may not be precisely identical in DNA content. Mutant progeny that have the same function or biological activity as screened for from the originally transformed cell are included.

[0232] The term "pharmaceutically acceptable additive" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate-buffered saline (PBS) or normal (0.9%) saline. Compositions containing such vectors are prepared by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and R. Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0233] "Inhibition" or "blocking" may be used interchangeably and covers both partial and complete inhibition / blocking. "Inhibition of growth" (e.g., of a cell) is intended to include any measurable decrease in cell growth.

[0234] "Inhibits the growth of" or "growth inhibition" means inhibiting the growth or proliferation of cells.

[0235] A "proliferative disorder" refers to a medical condition associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative condition refers to cancer. A "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative condition," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0236] "Cancer prevention" means delaying, inhibiting, or preventing the onset of cancer in a subject, where the onset of cancer or tumor development in the subject has not been confirmed, but a cancer susceptibility is identified, e.g., as determined by genetic screening or other methods. It further includes treating a subject with a precancerous condition, thereby halting the progression of the precancerous condition to a malignant tumor or causing its regression.

[0237] "Giving," "administration," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, e.g., therapeutic, drug metabolism, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving," "administration," and "treatment" also refer to treating, e.g., cells, ex vivo and in vitro, with a reagent, diagnostic, binding composition, or through another cell. When applied to a human, veterinary, or research subject, refer to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0238] "Treatment" refers to the administration of an internal or external therapeutic agent, e.g., one comprising any one of the binding molecules disclosed herein or a pharmaceutical composition thereof, to a subject suffering from, at risk of, or prone to one or more proliferative diseases or symptoms thereof, where the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the subject or population being treated in an amount that effectively alleviates one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms so that they do not progress to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, including the disease state, age, and weight of the subject, and the ability of the agent to produce the desired therapeutic effect in the subject. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating a target disease symptom in a subject, but should alleviate the target disease symptom in a statistically significant number of subjects, as determined by any statistical testing method known in the art, such as, for example, a Student's t-test, a chi-squared test, a Mann and Whitney U test, a Kruskal-Wallis test (H test), a Jonckheere-Terpstra test, and a Wilcoxon test.

[0239] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or pathology of a medical condition. An effective amount also refers to an amount sufficient to enable or facilitate diagnosis. The effective amount used in a subject can vary depending on factors such as the condition being treated, the subject's overall health, the 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. The subject of the present disclosure may be an animal or human subject.

[0240] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur. "And / or" should be considered to specifically indicate that each of the two specified features or components may or may not have the other. Thus, the term "and / or," as used in the phrase "A and / or B" in this disclosure, includes "A and B," "A or B," "A" (alone) and "B" (alone). Unless the context clearly indicates otherwise, throughout the specification and claims, words such as "comprise," "have," "include," and the like, should be understood to have an inclusive meaning, i.e., "including, but not limited to," rather than an exclusive or exhaustive meaning.

[0241] As used herein, a "subject" or "patient" refers to mammals, particularly primates, and especially humans. [Brief explanation of the drawings]

[0242] [Figure 1] This shows the results of a FACS binding test of antibodies to human 4-1BB antigen, awaiting measurement. [Figure 2] Binding of C5_V2, C5_V2-YTI, and C5_V2-YTIQ to human 4-1BB on the surface of HEK293 cells. [Figure 3] 3A and 3B are schematic diagrams of the structures of anti-FAP / 4-1BB / CD40 multispecific antibodies (FIG. 3 ae) and anti-4-1BB / CD40 (FIG. 3 f). [Figure 4A] This shows the results of FACS detection of the binding of the antibody to be measured to human FAP on the surface of CHOK1 cells. [Figure 4B] This shows the results of FACS detection of the binding of the antibody to be measured to human 4-1BB on the surface of HEK293 cells. [Figure 4C] This shows the results of FACS detection of the binding of the antibody to be measured to human CD40 on the surface of HEK293 cells. [Figure 5A] This shows the results of FACS detection of the binding of the antibody to be measured to human FAP on the surface of CHOK1 cells. [Figure 5B] This shows the results of FACS detection of the binding of the antibody to be measured to human 4-1BB on the surface of HEK293 cells. [Figure 5C] This shows the results of FACS detection of the binding of the antibody to be measured to human CD40 on the surface of HEK293 cells. [Figure 6A] This shows the results of FACS detection of the binding of the antibody to be measured to human FAP on the surface of CHOK1 cells. [Figure 6B] This shows the results of FACS detection of the binding of the antibody to be measured to human 4-1BB on the surface of HEK293 cells. [Figure 6C] This shows the results of FACS detection of the binding of the antibody to be measured to human CD40 on the surface of HEK293 cells. [Figure 7A] 7 shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where Figure 7A shows the results before cross-linking, Figure 7B shows the results after FAP cross-linking, and Figure 7C shows the results after CD40 cross-linking. [Figure 7B] 7 shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where Figure 7A shows the results before cross-linking, Figure 7B shows the results after FAP cross-linking, and Figure 7C shows the results after CD40 cross-linking. [Figure 7C] 7 shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where Figure 7A shows the results before cross-linking, Figure 7B shows the results after FAP cross-linking, and Figure 7C shows the results after CD40 cross-linking. [Figure 8A] 8A shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 8A shows the results before cross-linking, FIG. 8B shows the results after FAP cross-linking, and FIG. 8C shows the results after CD40 cross-linking. [Figure 8B] 8A shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 8A shows the results before cross-linking, FIG. 8B shows the results after FAP cross-linking, and FIG. 8C shows the results after CD40 cross-linking. [Figure 8C]8A shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 8A shows the results before cross-linking, FIG. 8B shows the results after FAP cross-linking, and FIG. 8C shows the results after CD40 cross-linking. [Figure 9A] 9A shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 9A shows the results before cross-linking, FIG. 9B shows the results after FAP cross-linking, and FIG. 9C shows the results after CD40 cross-linking. [Figure 9B] 9A shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 9A shows the results before cross-linking, FIG. 9B shows the results after FAP cross-linking, and FIG. 9C shows the results after CD40 cross-linking. [Figure 9C] 9A shows the results of FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 9A shows the results before cross-linking, FIG. 9B shows the results after FAP cross-linking, and FIG. 9C shows the results after CD40 cross-linking. [Figure 10A] 10A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 10A shows the results before cross-linking, FIG. 10B shows the results after FAP cross-linking, and FIG. 10C shows the results after 4-1BB cross-linking. [Figure 10B] 10A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 10A shows the results before cross-linking, FIG. 10B shows the results after FAP cross-linking, and FIG. 10C shows the results after 4-1BB cross-linking. [Figure 10C] 10A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 10A shows the results before cross-linking, FIG. 10B shows the results after FAP cross-linking, and FIG. 10C shows the results after 4-1BB cross-linking. [Figure 11A] 11A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 11A shows the results before cross-linking, FIG. 11B shows the results after FAP cross-linking, and FIG. 11C shows the results after 4-1BB cross-linking. [Figure 11B] 11A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 11A shows the results before cross-linking, FIG. 11B shows the results after FAP cross-linking, and FIG. 11C shows the results after 4-1BB cross-linking. [Figure 11C] 11A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 11A shows the results before cross-linking, FIG. 11B shows the results after FAP cross-linking, and FIG. 11C shows the results after 4-1BB cross-linking. [Figure 12A] 12A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 12A shows the results before cross-linking, FIG. 12B shows the results after FAP cross-linking, and FIG. 12C shows the results after 4-1BB cross-linking. [Figure 12B] 12A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 12A shows the results before cross-linking, FIG. 12B shows the results after FAP cross-linking, and FIG. 12C shows the results after 4-1BB cross-linking. [Figure 12C] 12A shows the results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection for the antibody being measured, where FIG. 12A shows the results before cross-linking, FIG. 12B shows the results after FAP cross-linking, and FIG. 12C shows the results after 4-1BB cross-linking. [Figure 13] These are the results of detecting the activation of antibodies awaiting measurement on T lymphocytes from donors 1 and 2. [Figure 14] These are the results of detecting the activation of antibodies awaiting measurement on T lymphocytes from donors 1 and 2. [Figure 15] These are the results of detecting the activation of CD3+ T lymphocytes from donors 1 and 2 by antibodies awaiting measurement, with RO7122290, MP0310, BNT312, and isotype IgG1 used as controls. [Figure 16] This is the detection result of the antibody awaiting measurement for promoting DC cell maturation. [Figure 17] This is the detection result of the antibody awaiting measurement for promoting DC cell maturation. [Figure 18] This is the detection result of the antibody awaiting measurement for promoting DC cell maturation. [Figure 19] This is the result of detecting the ability of the antibody to be measured to crosslink 4-1BB / CD40 expressing cells, BNT312, isotype IgG1, is used as a control. [Figure 20] Antibody MLR detection results awaiting measurement, with BNT312, isotype IgG1, used as a control. [Figure 21] This is the result of detecting the tumor-inhibitory effect of the antibody to be measured on MC38-mFAP transplanted tumors, and the solvent, RO7122290, and Ab10-A12V2-2 are used as controls. [Figure 22] On the 17th day after administration, the effect of the antibody on mouse ALT / AST was measured, and the solvent, Ab10-A12V2-2, and RO7211190 were used as controls. [Figure 23] The effect of the antibody on mouse body weight is to be measured, and the solvent, RO7122290, and Ab10-A12V2-2 are used as controls. [Figure 24] The effect of the antibody on the neutrophil and platelet counts in mice is to be measured. Vehicle, RO7122290, and Ab10-A12V2-2 are used as controls. [Figure 25] On the 8th day after administration, the effect of the antibody on mouse ALT / AST was measured, with the vehicle, Ab10-A12V2-2, and BNT312 used as controls. DETAILED DESCRIPTION OF THE INVENTION

[0243] Example 1. Screening and preparation of anti-FAP monoclonal antibodies 1.1 Sequence and preparation of screening antigens and detection antigens Human fibroblast activation protein (FAP, GeneBank accession number AAC51668) is a serine oligopeptidase with a molecular weight of approximately 170 kDa and is a homodimer. The sequence, origin, and use of the recombinant FAP protein used in this disclosure are shown in Table 2.

[0244] [Table 2]

[0245] 1.2 Screening of h-FAP-specific binding antibody fragments from a human natural Fab phage display library Using the h-FAP-biotin antigen as a target molecule, antigen-specific phages were captured by magnetic beads with avidin on the surface and enriched using a magnetic rack. The antigen-specific phages were eluted with a glycine solution at pH 2.2.

[0246] After two rounds of screening, 284 clones were randomly selected and positive clones were screened by phage ELISA. The specific steps were as follows: A total of 284 colonies were counted and inoculated into a 96-well plate. Each well contained 400 μL of culture medium (2YT + Amp + 0.2% glucose) and incubated at 37°C for 6 hours with shaking at 250 rpm. The plate was coated with the antigen h-FAP-His at 100 ng / 100 μL / well and left overnight at 4°C. The next day, after washing and blocking the 96-well plate, 100 μL of the overnight culture was added to each well. The plate was then incubated at 37°C for 1 hour, washed, and a secondary antibody (anti-human IgG-Fab antibody labeled with HRP) was added. The plate was incubated at 37°C for 40 minutes, washed, and a color development solution was added. The plate was then stored in the dark for 30 minutes, and the OD600 was read using a plate reader. A total of 122 positive clones obtained from two rounds of screening were sequenced, yielding 74 unique VHs (constituting the VH-enriched library), 48 unique κ light chains (constituting the KLC-enriched library), and 10 unique λ light chains (constituting the LLC-enriched library).

[0247] 1.3 Construction of a CHO cell surface full-length antibody display library 1) Construction of a full-length antibody CHO cell display gene library, including the following: PCR amplification of a VH-enriched library, a KLC-enriched library, and an LLC-enriched library using three sets of primers. The three enriched libraries were enzymatically digested and inserted into the corresponding assembly vectors to construct the corresponding assembly libraries. The KLC, LLC, and VH assembly libraries were enzymatically digested, and the enzymatically digested KLC fragment library, LLC fragment library, and VH fragment library were analyzed and purified by electrophoresis. The full-length antibody cell display vector was enzymatically digested, and the vector fragments were purified. The enzymatically digested and purified vector fragments, KLC fragment library, LLC fragment library, and VH fragment library were mixed and ligated. The ligation product was purified, electroporated, and transformed into E. coli, plated, and cultured overnight at 37°C. Colonies were counted. The library volume was detected to reach 3.6 × 10E6, more than 800-fold higher than the theoretical diversity (74 × 58 = 4292). All colonies were collected, vector DNA was extracted, and a full-length antibody cell display gene library was obtained.

[0248] 2) Construction of a full-length antibody cell display library specific to the h-FAP antigen. This involved the following: A full-length antibody cell display gene library was constructed by transforming vector DNA with the full-length antibody cell display gene library. The cell library was then screened by pressing with hygromycin. A stable transformed cell library was obtained, and the cell library was double-stained with a mouse anti-human kappa (or lambda) light chain antibody labeled with PE and an h-FAP antigen labeled with FITC. PE and FITC double-positive cells were selected by FACS, with one cell per well. Two 96-well plates were selected for the kappa light chain library, and one 96-well plate was selected for the lambda light chain library. The cells were then pressed with hygromycin and cultured.

[0249] 3) Analysis, identification, and display of single-cell clones expressing h-FAP antigen-specific antibodies by FACS. This included culturing under pressure with hygromycin for 14 days to obtain 153 stably transformed κ-chain single-cell clones and 50 λ-chain single-cell clones. The cells were digested with 0.5 mM EDTA-PBS buffer, and the single-cell clones were double-stained with PE-labeled mouse anti-human κ (or λ) light chain antibody and FITC-labeled h-FAP antigen. Analysis by FACS yielded 138 PE- and FITC-fluorescent double-positive single-cell clones.

[0250] 4) Antibody gene clones, including the following: The affinity of positive clones was detected and analyzed by FACS, and it was decided to perform PCR amplification of the antibody genes of 45 clones. The positive clones were collected by centrifugation, the supernatant was discarded, the cellular genomic DNA was extracted, and the VH and LC were subjected to PCR amplification. The amplified fragments were isolated by electrophoresis and sequenced for analysis. The variable region sequence of the anti-human FAP monoclonal antibody Ab10 is as follows:

[0251] >VH QVQLQQSGVEVKKPGASVTVSCRASGYSFADHFIHWVRQAPGQGFQWMGWINPNRGVTHHAQDFQGRVAMTRDMSTDTVYMELTSLRSDDTAVYYCARDASLTARPYYFYGFDVWGQGTLVTVSS (SEQ ID NO: 1), >VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPAFGQGTKVEIK (SEQ ID NO: 2), Among them, the CDR sequences are as follows:

[0252] [Table 3]

[0253] 1.4 Expression and purification of anti-FAP antibodies The heavy and light chain variable regions of the above anti-FAP antibody Ab10 sequence were linked to the human HC and LC constant regions, respectively, to form the full-length heavy chain (SEQ ID NO: 9) and full-length light chain (SEQ ID NO: 10). Plasmids were constructed, transiently transfected, expressed, and purified to obtain the target antibody. The full-length Ab10 sequence is as follows, with the underlined constant region sequence:

[0254] >Ab10 heavy chain full length QVQLQQSGVEVKKPGASVTVSCRASGYSFADHFIHWVRQAPGQGFQWMGWINPNRGVTHHAQDFQGRVAMTRDMSTDTVYMELTSLRSDDTAVYYCARDASLTARPYYFYGFDVWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:9 >Ab10 light chain full length DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPAFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 10 >Fab heavy chain QVQLQQSGVEVKKPGASVTVSCRASGYSFADHFIHWVRQAPGQGFQWMGWINPNRGVTHHAQDFQGRVAMTRDMSTDTVYMELTSLRSDDTAVYYCARDASLTARPYYFYG FDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV SEQ ID NO: 66 >Fab light chain DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPAFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 67

[0255] 1.5 ELISA binding assay The His-tagged FAP recombinant protein was directly coated, and after addition of the antibody, secondary antibody (anti-human IgG antibody coupled to HRP) and HRP substrate TMB were added to detect the antibody's antigen binding activity. The following procedure was performed: A 96-well microplate was coated with human FAP-His protein, and 100 μL of the antibody was added to each well at a concentration of 0.5 μg / mL. The plate was then incubated overnight at 4°C. After thorough washing, 200 μL of blocking solution was added per well and the plate was incubated at room temperature for 2 hours. After thorough washing, 100 μL of the anti-FAP antibody to be assayed, diluted with diluent, was added to each well. After incubation at room temperature for 1 hour, the plate was thoroughly washed, and 100 μL of HRP-labeled goat anti-human IgG secondary antibody, diluted 1:20,000 with diluent, was added to each well. After incubation at room temperature for 1 hour, the plate was thoroughly washed, and 100 μL of TMB was added to each well. The plate was then incubated in the dark for 15 minutes. 50 μL / well of 0.16 M sulfuric acid was added. The OD value at 450 nm was read using a Thermo MultiSkan Fc plate reader, and the binding EC 50 The value was calculated to be 0.0920 mg / mL.

[0256] 1.6 Surface plasmon resonance (SPR) binding assay A CM5 sensor chip was selected, and HBS-EP+ buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the mobile phase. Anti-human IgG (Fc) antibody was prepared at 30 μg / mL in 10 mM sodium acetate buffer (pH 5.0) and immobilized by amino coupling. Each antibody was prepared in HBS-EP+ buffer solution and captured by the anti-human IgG (Fc) antibody in the chip channel. h-FAP-His was prepared as the analyte and diluted two-fold in HBS-EP+ buffer solution. The diluted antibody was flowed through the experimental and reference channels at a flow rate of 30 μL / min, allowing 1 min for binding and 15 min for dissociation. 10 mM glycine at pH 1.5 was used as the regeneration buffer, and the run was performed for 30 s at a flow rate of 10 μL / min. After analyzing the data, the results in Table 4 show that Ab10 has a 5.3-fold higher affinity for the antigen FAP than the control anti-FAP antibody 28H1 (sequences 219 and 233 of patent US9266938B2). Meanwhile, other anti-FAP antibodies obtained by simultaneous screening according to the present disclosure, such as Ab2, Ab4, and Ab5 (sequences not shown), have lower binding affinities to the antigen than 28H1.

[0257] [Table 4]

[0258] 1.7 FACS binding assay The binding characteristics of anti-FAP antibodies in cells were detected by FACS experiments. The following was done: a CHO cell line overexpressing human FAP was constructed and plated (1E5 / well). 100 μL / well of the antibody to be measured was added, with the highest concentration being 100 nM, and diluted 5-fold to a total of 8 concentrations, and incubated at 4°C for 1 hour. Anti-hIgG Alexa Fluor-647 was added as a secondary antibody at a ratio of 1:500, incubated at 4°C for 30 minutes, and detected by FACS. Antibodies capable of binding to FAP can label cells. The EC binding of human FAP to antibody Ab10 was 50 The value was 0.0004320 μg / mL.

[0259] Example 2. Screening and preparation of anti-CD40 nanobodies 2.1 Sequence and preparation of immunizing antigens and screening antigens The following recombinant human CD40 proteins were selected: His-tagged human CD40 recombinant protein (h-CD40-His), C-terminal biotin-tagged human CD40 recombinant protein (h-CD40-biotin), and C-terminal His-tagged monkey CD40 recombinant protein (cyno-CD40-His). Their sequences and origins are shown in Table 5. The above protein reagents can be used in the experiments described in the following Examples.

[0260] [Table 5]

[0261] 2.2 Detection of alpaca immune processes and titers Alpacas were immunized with h-CD40-his every two weeks for a total of four immunizations. At the first immunization, 0.5 mg of antigen was mixed with 1 mL of complete Freund's adjuvant (CFA) and injected subcutaneously. For the following three immunizations, 0.25 mg of antigen was mixed with 1 mL of incomplete Freund's adjuvant (IFA) and injected subcutaneously. Blank serum was collected before immunization, and 50 mL of peripheral blood was collected one week after the third and fourth immunizations. Lymphocytes were isolated and serum titers were determined.

[0262] 2.3 Titer detection and phage library construction After immunization of alpacas four times, serum titers were measured. After the titers passed, PBMCs were isolated, total RNA was extracted, and the purity was checked. DNA was reverse transcribed. After two rounds of nested PCR, the purified vector was enzymatically cleaved and ligated to the VHH target fragment. After electroporation, clones were selected to obtain phage libraries A and B.

[0263] 2.4 Affinity panning of phage libraries and ELISA identification Using the h-CD40 antigen as the target molecule, screening was performed using the Gly-HCl acid elution method, and specific phages were eluted. After two rounds of screening, 384 clones were randomly selected from the first and second rounds of titration plates, and positive clones were screened by phage ELISA, detecting the optical density at 450 nm. Based on the sequencing results, sequence alignment and phylogenetic tree analysis were performed on the sequences. The sequence of A297 is as follows, with the underlined CDRs according to the Kabat numbering convention:

[0264] >A297 QVQLVESGGGMVEPGGSLRLSCAASGFTFS RYGMK WVRQAPGKGPEWVS TINSHGDTTYYADSVKG RFTISRDNAQNTVYLQMNSLKPEDTALYYCLR IDYSDYSS RGQGTQVTVSS (SEQ ID NO: 11), Among them, the CDR sequences are as follows:

[0265] [Table 6]

[0266] 2.5 Humanization of anti-CD40 nanobodies Based on the typical VHH structure of the obtained Nanobody A297, the VHH variable region sequence was compared with an antibody germline database to obtain highly homologous human germline templates. The preferred human germline template for antibody A297 of the present disclosure is IGHV3-48*03. Furthermore, the CDRs were grafted into human FRs, and key amino acids that affect antibody structure and function were backmutated to restore binding affinity and activity. The sequence of A297V3 is as follows, with the underlined CDRs based on the Kabat numbering system:

[0267] >A297V3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYGMKWVRQAPGKGLEWVS TINSHGDTTYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCLR IDYSDYSS RGQGTLVTVSS (SEQ ID NO: 15)

[0268] 2.6 Detection of the antigen-binding ability of anti-CD40 nanobodies The above anti-CD40 nanobody sequence was linked to human IgG1-Fc (SEQ ID NO: 16) with C220A, S267E, and L328F mutations (numbered according to the Eu system). Plasmids were constructed, transiently transfected, expressed, and purified to obtain the target antibody. 9E5-SELFNS was used as a CD40 agonist positive control (see sequences 58 and 59 in WO2020108611A1).

[0269] >IgG1-Fc EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16) The binding ability of humanized antibodies to cells highly expressing the human antigen protein CD40 was detected by FACS. HEK293 cells were transiently transfected with a CD40 plasmid (Sino Biological, HG10774-CF) to obtain HEK293 cells highly expressing CD40. They were then resuspended in flow staining solution (PBS + 2% FBS) and gradient-diluted antibodies were added at different concentrations. After incubation on ice for 1 hour, the cells were washed with PBS and centrifuged at 400g for 5 minutes. A goat anti-human Fc antibody labeled with the fluorescent group Alexa Fluor 647 was added as the secondary antibody, and the cells were incubated on ice for 1 hour. After washing twice with PBS, the cell surface fluorescent signal was detected by FACS. EC 50The values ​​are shown in Table 7. The humanized antibody has relatively high affinity for any of the CD40-high expressing cell lines (A297V4 sequence not shown).

[0270] [Table 7]

[0271] The activation effect of HEK-Blue™ CD40L cells was observed when cross-linked with FcγRIIb. TM The in vitro agonist activity of CD40 antibodies was evaluated by detecting their activation effect on CD40L cells. The S267E / L328F mutation in the Fc region of the humanized CD40 antibody enhanced the affinity of IgG1 Fc to FcγRIIb, thereby enhancing FcγRIIb cross-linking of the antibody. FcγRIIb-mediated cross-linking of CD40 antibodies was also enhanced by HEK-Blue. TM The activation effect on CD40L cells was simulated by the agonist activity of fully cross-linked CD40 antibodies. HEK293 cells were transiently transfected with FcγRIIb plasmid (CD32B / Fcgr2b cDNA ORF Clone, Human, N-His tag, Sino Biological, HG10259-NH) to obtain HEK293 cells highly expressing FcγRIIb. HEK-Blue TM CD40L cells were seeded at 5E4 / well in a 96-well cell culture plate (medium: DMEM, 10% FBS, 100 μg / mL Normocin), and FcγRIIb-expressing HEK293 cells were added at 5E4 / well. 100 μL of gradient-diluted antibodies to be tested were added to each well and cultured overnight at 37°C. The cells were centrifuged, and 20 μL of cell supernatant was transferred to a new 96-well plate. 180 μL of QUANTI-Blue substrate solution was added, and the plate was incubated in the dark for 30 minutes. The absorbance at 620 nm was measured using an Envision plate reader, and EC 50 The EC value and Emax value (relative fluorescence intensity of antibody-free group) were calculated. 50 The in vitro cellular agonist activity of CD40 antibodies was evaluated using the EC50 The values ​​and Emax values ​​(relative fluorescence intensity) are shown in Table 8. The results showed that in the above reporter gene cell system, the humanized CD40 antibodies had relatively strong agonistic activity when cross-linked by FcγRIIb.

[0272] [Table 8]

[0273] Example 3. Preparation and screening of anti-4-1BB nanobodies 3.1 Alpaca immunization, titer detection, and affinity panning of phage libraries Alpacas were immunized with His-tagged human 4-1BB recombinant protein (Acrobiosystems, 41B-H5227) twice a week for a total of four immunizations. For the first immunization, 0.5 mg of antigen was mixed with 1 mL of complete Freund's adjuvant (CFA) and injected subcutaneously. For the following three immunizations, 0.25 mg of antigen was mixed with 1 mL of incomplete Freund's adjuvant (IFA) and injected subcutaneously. Blank serum was collected before immunization, and 50 mL of peripheral blood was collected one week after the third and one week after the fourth immunization. PBMCs were isolated, total RNA was extracted, and purity was confirmed. After two rounds of nested PCR, the nanobody target fragments were ligated into phage display vectors. Electroporation was performed to generate a phage library.

[0274] >Human 4-1BB protein sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ (SEQ ID NO: 17) To obtain anti-4-1BB nanobodies that simultaneously recognize both human and monkey antigens, we used a two-round cross-screening strategy of human and monkey antigens. The screening antigens for the first and second rounds were human 4-1BB and monkey 4-1BB, respectively. Phages specifically binding to 4-1BB were eluted using the Gly-HCl acid elution method in each round of screening. Ninety-six clones (192 clones in total) were randomly selected from the first and second round titration plates, and positive clones were screened by phage ELISA, detecting the optical density at 450 nm. The positive clones were sequenced. Based on the sequencing results, sequence alignment and phylogenetic tree analysis were performed, and 14 unique sequences were identified, including H27, C5, and C145. The sequence of C5 is as follows:

[0275] >C5 QVQLVESGGGLVQPGGSRLRLSCSASGTFFS SYAMS WVRQAPGKGLEWVS DINSGGESTFYEDSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAK HPLTFTIATMNDYDY WGQGTQVTVSS (SEQ ID NO: 18), Among them, the CDR sequences are as follows:

[0276] [Table 9]

[0277] The C5 sequence was linked to human IgG1-Fc with C220A, S267E, and L328F mutations (numbered according to the Eu system). The sequence of the linked VHH-Fc fusion protein is as follows: Furthermore, introduction of mutations such as L234A, L235A, and N297A (numbered according to the Eu system) into the Fc of human IgG1 completely eliminated antibody FcγR-mediated effector functions, and introduction of S228P (numbered according to the Eu system) into the Fc of human IgG4 stabilized the antibody molecule and prevented the formation of half molecules. Both of these are selectable IgG Fc.

[0278] An exemplary antibody sequence linked to C5 and IgG1-Fc (containing C220A, S267E, and L328F mutations) is shown in SEQ ID NO: 22, where the underlined portion is the IgG1-Fc sequence.

[0279] >C5-Fc QVQLVESGGGLVQPGGSLRLSCSASGTFFSSYAMSWVRQAPGKGLEWVSDINSGGESTFYEDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKHPLTFTIATMNDYDYWGQGTQVTVSS EPKSADKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGA (SEQ ID NO: 22) Plasmids were constructed, cells were transiently transfected, and the antibodies were expressed and purified. Target antibodies were obtained by detection.

[0280] 3.2 Detection of antigen-binding activity of anti-4-1BB antibody and its agonist activity (1) Detection of binding ability to 4-1BB antigen The binding activity of the anti-4-1BB single domain antibody to human 4-1BB protein was detected using a flow cytometer. HEK293-Hu4-1BB cells were obtained by transient transfection of HEK293 cells (ATCC CRL-1573) with a gene expressing human 4-1BB protein (CD137 cDNA ORF Clone, Human, C-OFPSpark tag, purchased from Sino Biological, Cat. #HG10041-ACR). The cell culture medium was DMEM (Gibco, Cat. #11995065) containing 10% fetal bovine serum. The experimental medium was sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum. HEK293-Hu4-1BB cells were washed twice with the experimental medium and cultured at a concentration of 1 × 10 cells. 5 HEK293-Hu4-1BB cells were seeded at 1 / well in a 96-well U-bottom plate, and various concentrations of anti-4-1BB antibody VHH-Fc samples were added. The cells were incubated at 4°C for 1 hour, then washed twice with experimental medium. Goat anti-human IgG (H+L) Alexa Fluor 488 antibody (Thermo, Cat# A11013) was then added, washed twice, and the fluorescence signal was read using a flow cytometer. Urelumab monoclonal antibody was used as a positive control, and the MFI values ​​of each antibody are shown in Figure 1. The results showed that the anti-4-1BB antibodies had different degrees of binding ability to 4-1BB on the HEK293-Hu4-1BB cell surface, with C5 exhibiting excellent cell membrane surface antigen binding activity.

[0281] (2) Detection of agonist activity against the 4-1BB signaling pathway The agonist activity of the anti-4-1BB antibody was evaluated using a 4-1BB / NF-κB reporter gene. HEK293 cells (ATCC CRL-1573) were transiently transfected with a gene expressing human 4-1BB (CD137 cDNA ORF Clone, Human, C-OFPSpark tag, purchased from Sino Biological, Cat. #HG10041-ACR) and an NF-κB reporter gene (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, purchased from Promega, Cat. #E849A). This resulted in HEK293-Hu4-1BB / NF-κB double-transfected cells. 4-1BB activation could be characterized by the activation of the NF-κB signaling pathway. HEK293 cells were transiently transfected with the FcγRIIb plasmid (CD32B / Fcgr2b cDNA ORF Clone, Human, N-His tag, purchased from Sino Biological, Cat# HG10259-NH) to obtain HEK293 cells highly expressing FcγRIIb. The cell culture medium was DMEM (Gibco, Cat# 11995065) containing 10% fetal bovine serum. HEK293-Hu4-1BB / NF-κB cells (2 × 10 6 50 μL of the solution (100 μg / mL) was seeded into a 96-well cell culture plate, and 40 μL of medium or FcγRIIb-expressing HEK293 cells (2.5 × 10 6 10 × 10 μL of gradient diluted anti-4-1BB antibody was added to each well and incubated at 37°C for 6 hours. The cells were removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat# G7940) was added to each well. The cells were incubated in the dark for 5 minutes, and the fluorescent signal was measured using an Envision plate reader (PerkinElmer, 2150). EC 50 The EC value and Emax value (fluorescence intensity relative to the antibody-free group) were calculated. 50The in vitro cellular agonist activity of the anti-4-1BB antibodies was evaluated using the β-gamma (β) values. The results are shown in Table 10. The results showed that without FcγRIIb (i.e., CD32b) cross-linking, the anti-4-1BB antibodies showed weaker activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway than the urelumab control, suggesting that the anti-4-1BB antibodies of the present disclosure have better safety. After FcγRIIb cross-linking, the anti-4-1BB antibodies showed significant activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, with C5 showing the strongest activation ability and comparable to the urelumab control. Based on the activity results, sequence C5, which had low background activation before FcγRIIb cross-linking and even stronger activation activity after FcγRIIb cross-linking, was screened and humanized.

[0282] [Table 10]

[0283] 3.3 Humanization The amino acid numbers of the CDR and FR regions (human framework regions, framework) of the C5 sequence were labeled using the Kabat numbering system. The FR1+CDR1, FR2+CDR2, and FR3+CDR3 sequences were aligned with an antibody germline database to obtain human germline FR templates with relatively high homology. FR1 was derived from IGHV3-64*04, FR2 from IGHV3-23*03, and FR3 from IGHV3-74*01. To reduce immunogenicity in humans, the above human germline FR regions were substituted and inserted into the original sequences. Key amino acids that affect antibody structure and function were backmutated to restore binding strength and activity. The humanized sequences are as follows:

[0284] >C5_V1 QVQLVESGGGLVQPGGSRLRLSCSASGTFFS SYMASWVRQAPGKGLEWVS DINSGGESTFYEDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR HPLTFTIATMNDYDY WGQGTLVTVSS (SEQ ID NO: 23), >C5_V2 QVQLVESGGGLVQPGGSRLRLSCSASGTFFS SYMAS WVRQAPGKGLEWVS DINSGGESTFYEDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAK HPLTFTIATMNDYDY WGQGTLVTVSS (SEQ ID NO: 24), >C5_V3 QVQLVESGGGLVQPGGSRLRLSCSASGTFFS SYMAS WVRQAPGKGLEWVS DINSGGESTFYEDSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAK HPLTFTIATMNDYDY WGQGTLVTVSS (SEQ ID NO: 25), (Note: The underlined parts are the CDR regions.) Each of the three humanized sequences was linked to human IgG1-Fc (SEQ ID NO: 16). Plasmids were constructed, transiently transfected, expressed, and purified.

[0285] 3.4 Detection of antigen-binding activity and agonistic activity of humanized anti-4-1BB antibody (1) Detection of binding ability to 4-1BB antigen The antigen-binding activity of the humanized antibodies was detected by the FACS detection method described in 3.2. The results are shown in Table 11. The results showed that the humanized anti-4-1BB antibodies had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and that C5_V1, C5_V2, and C5_V3 all maintained good binding activity.

[0286] [Table 11] (2) Detection of agonist activity against the 4-1BB signaling pathway The activation of the 4-1BB / NF-κB signal by the humanized antibodies was detected by the NF-κB luciferase reporter gene experiment as described in 3.2, and the results are shown in Table 12. As a result, after FcγRIIb cross-linking, the humanized anti-4-1BB antibodies showed activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, and C5, C5_V1, C5_V2, and C5_V3 showed activation abilities comparable to those of the urelumab control.

[0287] [Table 12]

[0288] 3.5 Removal of TCE sites Subsequently, T-cell epitope (TCE) prediction was performed on the C5_V2 sequence, and the CDR3 sequence of C5_V2 was modified based on the prediction results to reduce the number of TCEs. The F in the CDR3 region was mutated to Y (F99Y, according to the Kabat numbering system) to obtain a TCE-optimized version of the C5_V2 sequence, designated C5_V2-YTI. The sequence is as follows:

[0289] >C5_V2-YTI QVQLVESGGGLVQPGGSRLRLSCSASGTFFS SYMAS WVRQAPGKGLEWVS DINSGGESTFYEDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAK HPLTYTIATMNDYDY WGQGTLVTVSS (SEQ ID NO: 26), That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTI is shown in SEQ ID NO: 19, the amino acid sequence of CDR2 is shown in SEQ ID NO: 20, and the amino acid sequence of CDR3 is shown in HPLTYTIATMNDYDY (SEQ ID NO: 27).

[0290] The C5_V2-YTI sequence was linked to human IgG1-Fc (SEQ ID NO: 16). The plasmid was constructed, transiently transfected, expressed, and purified to obtain the target antibody.

[0291] 3.6 Detection of the antigen-binding ability of the humanized 4-1BB antibody after TCE removal TCE removal was optimized for humanized antibody C5_V2, and the resulting antibody was C5_V2-YTI. The antigen-binding activities of C5_V2 and C5_V2-YTI were compared using the FACS detection method described in 3.2, and the results are shown in Table 13. The results showed that C5_V2-YTI maintained good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells and was equivalent to C5_V2.

[0292] [Table 13]

[0293] 3.7 Reducing the risk of antibody aggregation We found that the C5_V2-YTI antibody has a relatively large hydrophobic region on its C-terminal surface, which poses a risk of antibody aggregation. Mutating the hydrophobic amino acid Leu (L) in the TLVTVSS sequence to the hydrophilic amino acid Gln (Q) effectively reduced the area of ​​the C-terminal hydrophobic surface region of the antibody and reduced the risk of antibody aggregation. Furthermore, to optimize the pI value of the sequence, the two acidic amino acids Glu (E) in the sequence were mutated to the basic amino acid Gln (Q) and the neutral amino acid Ala (A), respectively. The modified antibody was named C5_V2-YTIQ and its sequence is as follows:

[0294] >C5_V2-YTIQ QVQLVESGGGLVQPGGSRLRLSCSASGTFFS SYMAS WVRQAPGKGLEWVS DINSGGQSTFYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAK HPLTYTIATMNDYDY WGQGTQVTVSS (SEQ ID NO: 28), That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTIQ is shown in SEQ ID NO: 19, the amino acid sequence of CDR2 is shown in DINSGGQSTFYADSVKG (SEQ ID NO: 29), and the amino acid sequence of CDR3 is shown in SEQ ID NO: 27.

[0295] That is, the anti-4-1BB nanobody has the following general structure:

[0296] CDR1 is SYAMS (SEQ ID NO: 19), CDR2 is DINSGGX1STFYX2DSVKG (SEQ ID NO: 30), wherein X1 is E or Q, and wherein X2 is E or A; CDR3 is HPLTX3TIATMNDYDY (SEQ ID NO: 31), in which X3 is F or Y.

[0297] 3.8 Detection of the antigen-binding ability of engineered anti-4-1BB nanobodies C5_V2-YTI and C5_V2-YTIQ were linked to human IgG1-Fc (SEQ ID NO: 16). Plasmids were constructed, transiently transfected, expressed, and purified to obtain the target antibodies.

[0298] The antigen-binding activities of C5_V2, C5_V2-YTI, and C5_V2-YTIQ were compared using FACS detection, and the results are shown in Figure 2 and Table 14. Urelumab was used as a positive control. The results showed that C5_V2, C5_V2-YTI, and C5_V2-YTIQ all maintained good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells.

[0299] [Table 14]

[0300] Example 4. Design and preparation of anti-FAP / 4-1BB / CD40 multispecific antibodies 4.1 Design, Expression, and Purification of Anti-FAP / 4-1BB / CD40 Polyspecific Antibodies A fusion protein of FAP, 4-1BB, and CD40 was constructed, and a schematic diagram of its molecular structure is shown in Figure 3.

[0301] In the molecule B977703 shown in Figure 3a, the C-terminus of the two heavy chains of the anti-FAP monoclonal antibody Ab10 was linked to the N-terminus of the anti-4-1BB nanobody C5V2-YTI via a (G4S)2 linker, and the C-terminus of the two anti-4-1BB nanobodies was linked to the N-terminus of the anti-CD40 nanobody A297V3 via a (G4S)2 linker.

[0302] In the molecule B977704 shown in Figure 3b, the C-terminus of the two heavy chains of the anti-FAP monoclonal antibody Ab10 was linked to the N-terminus of the anti-4-1BB nanobody C5V2-YTI via a (G4S)2 linker, and the C-terminus of the two light chains of the anti-FAP monoclonal antibody Ab10 was linked to the N-terminus of the anti-CD40 nanobody A297V3 via a (G4S)2 linker.

[0303] In molecule B977707 shown in Figure 3c, two anti-4-1BB nanobodies C5V2-YTI were linked sequentially via a (G4S)2 linker, and their N-termini were linked to the C-termini of two heavy chains of anti-FAP monoclonal antibody Ab10 via linkers (G4S)2, respectively, and the C-termini of two light chains of anti-FAP monoclonal antibody Ab10 were linked to the N-termini of anti-CD40 nanobody A297V3 via (G4S)2 linkers, respectively.

[0304] In the molecule B785502 shown in Figure 3(d), the C-terminus of the two heavy chains of the anti-FAP monoclonal antibody Ab10 was linked to the N-terminus of the anti-CD40 nanobody A297V3 via a (G4S)2 linker, and the C-terminus of the two anti-CD40 nanobodies A297V3 was linked to the N-terminus of the anti-4-1BB nanobody C5V2-YTI via a (G4S)2 linker.

[0305] In the molecule B785503 shown in Figure 3e, the C-terminus of the two heavy chains of the anti-FAP monoclonal antibody Ab10 was linked to the N-terminus of the anti-CD40 nanobody A297V3 via a (G4S)2 linker, and the C-terminus of the two light chains of the anti-FAP monoclonal antibody Ab10 was linked to the N-terminus of the anti-4-1BB nanobody C5V2-YTI via a (G4S)2 linker.

[0306] The molecule B785511 shown in Figure 3f is a control specific antibody 4-1BB / CD40, in which two anti-4-1BB nanobodies C5V2-YTI were linked to the N-terminus of the Fc domain via the hinge region of the IgG1 heavy chain, and the C-terminus of the Fc domain was linked to the N-terminus of the anti-CD40 nanobody A297V3 via a (G4S)2 linker.

[0307] In the molecules shown in Figure 3 a to f, the Fc is a human IgG1 Fc with L234A and L235A mutations to eliminate FcγR-mediated effector function.

[0308] 4.2 Design, Expression, and Purification of Anti-FAP / 4-1BB / CD40 Multispecific Antibodies To reduce binding to pre-anti-drug antibodies (pre-ADA) or anti-drug antibodies (ADA), modifications were made to the C-terminal nanobody of the anti-FAP / 4-1BB / CD40 multispecific antibody, changing the TVSS at the C-terminus of the nanobody to TVS. AA The optimized sequence for A297V3 is A297V3_AA, and the optimized sequence for C5_V2-YTI is C5_V2-YTI_AA.

[0309] >A297V3_AA EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMKWVRQAPGKGLEWVSTINSHGDTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLRIDYSDYSSRGQGTLVTVSAA (SEQ ID NO: 32) >C5_V2-YTI_AA QVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAMSWVRQAPGKGLEWVSDINSGGESTFYEDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAKHPLTYTIATMNDYDYWGQGTLVTVSAA (SEQ ID NO: 33) The anti-CD40 nanobody A297V3 located at the C-terminus of the heavy chain in B977703 was modified to A297V3_AA to obtain B21326101, the anti-CD40 nanobody A297V3 located at the C-terminus of the light chain in B977704 was modified to A297V3_AA, and the anti-4-1BB nanobody C5_V2-YTI located at the C-terminus of the heavy chain in B977704 was modified to C5_V2-YTI_AA to obtain B21326102. The specific molecular sequences are shown in Table 32.

[0310] 4.3 Modification design, expression and purification of anti-FAP / 4-1BB / CD40 multispecific antibodies to reduce antibody aggregation To reduce aggregation, further modifications were made to B21326101, replacing C5_V2-YTI with C5_V2-YTIQ (SEQ ID NO: 28), and the optimized molecule was designated B21546901.

[0311] 4.4 Control molecules BNT312 is a CD40 / 4-1BB bibody molecule, Ab10-A12V2-2 is a FAP / CD40 bibody molecule, RO7300490 is a FAP / CD40 bibody molecule, RO7122290 is a FAP / 4-1BBL bibody molecule, and MP0310 is a FAP / 4-1BB bibody molecule, all of which were used as control molecules.

[0312] The multispecific antibody sequences are shown in Table 32. The control molecule sequences are shown in Table 33.

[0313] 4.5 Expression and purification Transient transfection and expression: For example, using a 30 mL expression system, the culture medium was diluted with 60 μL of transfection reagent and then mixed uniformly with 15 μg of plasmid. The mixture was then incubated at 37°C for 15 minutes, and the transfection mixture was added dropwise to the cell suspension while shaking. The mixture was then placed on a shaker and cultured for one week to allow expression. The supernatant was then collected and centrifuged at 8000 rpm for 5 minutes.

[0314] Antibody purification: First, a Protein A affinity chromatography column was equilibrated with 20 mL of 1x PBS at a flow rate of 1 mL / min. The loading flow rate was 1 mL / min. 20 mL of 1x PBS was washed at a flow rate of 1 mL / min. The column was then eluted with citrate buffer (pH 3.4) at 1 mL / min. The fraction was collected and the absorbance at 280 nm was measured using a NanoDrop device. Finally, the concentrated protein was transferred to a dialysis bag and dialyzed in a beaker of 1x PBS. The target antibody was obtained by detection.

[0315] Example 5. Detection of antigen-binding activity of anti-FAP / 4-1BB / CD40 multispecific antibodies 5.1 Detection of antigen-binding activity of anti-FAP / 4-1BB / CD40 polyspecific antibodies The binding activity of anti-FAP / 4-1BB / CD40 polyspecific antibodies to human FAP / 4-1BB / CD40 protein was detected by FACS experiments. CHO-K1-HuFAP cells were obtained by stable transfection of CHO-K1 cells with a protein expressing human FAP. The cell culture medium was Ham's F12 + GlutaMAX™-I (Gibco, Cat. #31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-Hu4-1BB cells and HEK293-HuCD40 cells were HEK293 cells overexpressing human 4-1BB and human CD40 protein, respectively, and the cell culture medium was DMEM (Gibco, Cat. #11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. The experimental medium was sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum. CHO-K1-HuFAP, HEK293-Hu4-1BB, or HEK293-HuCD40 cells were washed twice with the experimental medium and then cultured at 1 × 10 5 Cells were seeded into a 96-well U-bottom plate at 100 cells / well, and various concentrations of samples to be measured were added. The cells were incubated at 4°C for 1 hour, washed twice with experimental medium, and then Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was added. After washing twice, the fluorescent signal was read using a flow cytometer.

[0316] The FACS detection results in Table 15, Figure 4A, showed that the FAP / 4-1BB / CD40 multispecific antibody had strong binding ability to FAP on the surface of CHO-K1-HuFAP cells, comparable to that of the control antibody RO7122290, demonstrating that the antibody's binding to FAP was not affected after preparation into a multispecific antibody. The results in Table 16, Figure 4B, showed that the FAP / 4-1BB / CD40 multispecific antibody had good binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells. The results in Table 17, Figure 4C, showed that the FAP / 4-1BB / CD40 multispecific antibody had relatively good binding ability to CD40 on the surface of HEK293-HuCD40 cells, demonstrating that the antibody's binding to CD40 was not affected after preparation into a multispecific antibody.

[0317] [Table 15] [Table 16] [Table 17]

[0318] 5.2 Detection of antigen-binding activity of anti-FAP / 4-1BB / CD40 polyspecific antibodies Using the detection method provided in Example 5.1, the antigen binding activity of FAP / 4-1BB / CD40 multispecific antibodies B977703, B977704, B21326101, and B21326102 was detected, and RO7122290, Ab10-A12V2-2, BNT312, and IgG1 were used as controls.

[0319] The FACS detection results (Tables 18-20, Figures 5A-5C) showed that the FAP / 4-1BB / CD40 multispecific antibody had strong binding ability to FAP on the surface of CHO-K1-HuFAP cells (Table 18, Figure 5A). Table 19, Figure 5B showed that the FAP / 4-1BB / CD40 multispecific antibody had good binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and the multispecific antibody after sequence modification of 4-1BB showed no difference in 4-1BB binding, demonstrating that the 4-1BB sequence modification had no effect on target binding. Table 20, Figure 5C showed that the FAP / 4-1BB / CD40 multispecific antibody had binding ability to CD40 on the surface of HEK293-HuCD40 cells, demonstrating that removal of the pre-ADA modification in the CD40 sequence had no obvious effect on target binding.

[0320] [Table 18] [Table 19] [Table 20]

[0321] 5.3 Detection of antigen-binding activity of anti-FAP / 4-1BB / CD40 multispecific antibodies The antigen binding activity of FAP / 4-1BB / CD40 multispecific antibodies B21546901 and B21326102 was detected using the detection method provided in Example 5.1, and RO7122290, RO7300490, BNT312, and IgG1 were used as controls.

[0322] The FACS detection results (Tables 21-23, Figures 6A-6C) showed that the FAP / 4-1BB / CD40 multispecific antibody had strong binding ability to FAP on the surface of CHO-K1-HuFAP cells, stronger than RO7300490 and comparable to the binding ability of the control antibody RO7122290. Table 22 showed that the FAP / 4-1BB / CD40 multispecific antibody had good binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, demonstrating that modifications in the 4-1BB sequence to reduce the risk of antibody aggregation had no obvious effect on binding to 4-1BB. Table 23 showed that the FAP / 4-1BB / CD40 multispecific antibody had good binding ability to CD40 on the surface of HEK293-HuCD40 cells.

[0323] [Table 21] [Table 22] [Table 23]

[0324] The above results showed that the anti-FAP / 4-1BB / CD40 multispecific antibody had strong binding ability to FAP on the CHO-K1-HuFAP cell surface, comparable to that of the control antibody RO7122290, demonstrating that the antibody's binding to FAP was not affected after preparation into a trispecific antibody. The anti-FAP / 4-1BB / CD40 multispecific antibody also had good binding ability to 4-1BB on the HEK293-Hu4-1BB cell surface, with no difference in 4-1BB binding between B977704 and the modified B21326102, demonstrating that the 4-1BB sequence modification had no effect on target binding. The anti-FAP / 4-1BB / CD40 multispecific antibody also had binding ability to CD40 on the HEK293-HuCD40 cell surface, demonstrating that the CD40 sequence modification had no significant effect on target binding.

[0325] Example 6. Luciferase reporter gene detection of FAP / 4-1BB / CD40 multispecific antibodies 6.1 FAP / 4-1BB / CD40 multispecific antibody 4-1BB / NF-κB luciferase reporter gene detection experiment The agonist activity of the antibody was evaluated using a 4-1BB / NF-κB reporter gene detection experiment. HEK293 cells (ATCC CRL-1573) were transiently transfected with a gene expressing human 4-1BB (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; Sino Biological, Cat# HG10041-ACR) and the NF-κB genome (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, Promega, Cat# E849A) to obtain HEK293-Hu4-1BB / NF-κB double-transfected cells. Hu4-1BB activation was characterized by the activation level of the NF-κB signaling pathway. CHO-K1-HuFAP cells were obtained by stably transfecting CHO-K1 cells with a protein expressing human FAP, and the cell culture medium was Ham's F12 + GlutaMAX™-I (Gibco, Cat#31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-HuCD40 cells were obtained by overexpressing human CD40 protein in HEK293 cells, and the cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. 50 μL of HEK293-Hu4-1BB / NF-κB cells (2 × 10 6 10 cells / mL) were seeded in a 96-well cell culture plate, and 40 μL of medium, FAP-expressing CHO-K1-HuFAP cells or CD40-expressing HEK293-HuCD40 cells (2.5 × 10 6Cells / mL) were added to each well, and 10 × 10 μL of gradient diluted antibody was added to each well. The wells were then cultured at 37°C for 6 hours. The cells were removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat# G7940) was added to each well. The wells were incubated in the dark for 5 minutes. The fluorescent signal was measured using an Envision plate reader, and the EC50 and Emax values ​​(fluorescence intensity relative to the antibody-free group) were calculated. The in vitro cellular agonist activity of the anti-4-1BB antibody was also evaluated.

[0326] The results (Table 24, Figures 7A to 7C) showed that the uncrosslinked FAP / 4-1BB / CD40 multispecific antibodies B977703 and B977704 did not induce background activation of 4-1BB, whereas the other antibodies all exhibited some background activation. All FAP / 4-1BB / CD40 multifunctional antibodies possessed 4-1BB activity, crosslinking and activating FAP or CD40.

[0327] [Table 24]

[0328] 6.2 FAP / 4-1BB / CD40 multispecific antibody 4-1BB / NF-κB luciferase reporter gene detection experiment The 4-1BB agonist activity of the FAP / 4-1BB / CD40 multispecific antibody was detected using the method provided in Example 6.1. The results are shown in Figures 8A-8C and Table 25.

[0329] The results in Table 25 and Figure 8A indicate that the uncrosslinked FAP / 4-1BB / CD40 multispecific antibodies did not exhibit background 4-1BB activation. The results in Table 25 and Figure 8B indicate that the 4-1BB agonistic activity of antibodies B977703 and B977704 after crosslinking with FAP was comparable to that of B21326101 and B21326102, demonstrating that sequence modifications of 4-1BB did not affect 4-1BB activation activity. The results in Table 25 and Figure 8C indicate that all FAP / 4-1BB / CD40 multispecific antibodies possessed 4-1BB activity by crosslinking and activating CD40, and that the 4-1BB agonistic activity of the FAP / 4-1BB / CD40 multispecific antibodies after crosslinking with CD40 was stronger than that of BNT312.

[0330] [Table 25]

[0331] 6.3 4-1BB / NF-κB luciferase reporter gene detection experiment using FAP / 4-1BB / CD40 multifunctional antibody The 4-1BB agonist activity of the FAP / 4-1BB / CD40 multispecific antibody was detected using the method provided in Example 6.1, and the results are shown in Figures 9A-9C and Table 26.

[0332] Table 26 and Figure 9A show that the uncrosslinked B21546901 and B21326102 antibodies have no background activation activity. Table 26 and Figure 9B show that the agonistic activity of antibody 4-1BB after crosslinking with FAP is comparable to the activation ability of RO7122290. Table 26 and Figure 9C show that all FAP / 4-1BB / CD40 multispecific antibodies have 4-1BB activity to crosslink and activate CD40, and that the agonistic activity of antibody 4-1BB after crosslinking with CD40 is stronger than that of BNT312.

[0333] [Table 26]

[0334] The above results showed that the uncrosslinked anti-FAP / 4-1BB / CD40 multispecific antibodies B977703, B977704, B21326101, B21326102, and B21546901 antibodies did not cause background activation of 4-1BB, and that the agonistic activity of the multiple anti-FAP / 4-1BB / CD40 multispecific antibodies 4-1BB after crosslinking with FAP was not significantly different, indicating that sequence modifications of 4-1BB have no effect on 4-1BB activation activity. Furthermore, the agonistic activity of the multiple anti-FAP / 4-1BB / CD40 multispecific antibodies 4-1BB after crosslinking with CD40 was comparable, and that the agonistic activity of antibody 4-1BB after crosslinking with CD40 was stronger than that of BNT312.

[0335] Example 7. Luciferase reporter gene detection of FAP / 4-1BB / CD40 multispecific antibodies 7.1 CD40 / NF-κB luciferase reporter gene detection experiment of FAP / 4-1BB / CD40 multispecific antibody The agonist activity of the antibodies was assessed using the CD40 / NF-κB reporter gene. HEK-Blue TM For CD40L (InvivoGen, hkb-cd40) cells, the cell culture medium was DMEM (Gibco, Cat# 11965092) containing 10% heat-inactivated fetal bovine serum and 100 μg / mL Normocin® (InvivoGen, ant-nr-1). HuCD40 activation was characterized by the activation level of the NF-κB signaling pathway. CHO-K1-HuFAP cells were obtained by stable transfection of CHO-K1 cells with a protein expressing human FAP. The cell culture medium was Ham's F12 + GlutaMAX™-I (Gibco, Cat# 31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-Hu4-1BB cells were obtained by overexpressing human 4-1BB protein in HEK293 cells, and the cell culture medium was DMEM (Gibco, Cat# 11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. 100 μL of HEK-BlueTM CD40L (1 × 10 6 Cells / mL) were seeded in a 96-well cell culture plate, and 80 μL of medium, FAP-expressing CHO-K1-HuFAP cells or 4-1BB-expressing HEK293-Hu4-1BB cells (1.25 × 10 6 10 μL of gradient diluted antibody was added to each well and incubated overnight at 37°C. 20 μL of cells were removed from each well and 180 μL of QUANTI-Blue® was added. TM The cells were incubated in the dark at 37°C for 30 minutes, and the OD was measured using an Envision plate reader. The EC50 and Emax values ​​(relative to those in the antibody-free group) were calculated, and the in vitro cellular agonist activity of the anti-CD40 antibodies was evaluated. The results are shown in Figures 10A to 10C and Table 27.

[0336] The results in Table 27 and Figure 10B indicate that the CD40 agonist activity of the FAP / 4-1BB / CD40 multispecific antibody after crosslinking with FAP is comparable to the activation abilities of the RO7300490 and Ab10-A12V2-2 molecules. The results in Table 27 and Figure 10C indicate that both FAP / 4-1BB / CD40 multispecific antibodies have CD40 activating activity upon crosslinking with 4-1BB.

[0337] [Table 27]

[0338] 7.2 CD40 / NF-κB luciferase reporter gene detection experiment of FAP / 4-1BB / CD40 multispecific antibody The CD40 agonist activity of the FAP / 4-1BB / CD40 multispecific antibody was detected using the method provided in Example 7.1, and the results are shown in Figures 11A-11C and Table 28.

[0339] The results in Table 28 and Figure 11B show that the agonistic activity of the FAP / 4-1BB / CD40 multispecific antibody CD40 after crosslinking with FAP was comparable to that of Ab10-A12V2-2, while the activation activities of B977703 and B977704 were comparable to those of B21326101 and B21326102, demonstrating that the CD40 sequence modifications had no effect on CD40 activation activity. The results in Table 28 and Figure 11C show that all FAP / 4-1BB / CD40 multispecific antibodies crosslinked to 4-1BB and activated it, exhibiting activation activity comparable to that of BNT312.

[0340] [Table 28]

[0341] 7.3 CD40 / NF-κB luciferase reporter gene detection experiment of FAP / 4-1BB / CD40 multispecific antibody The CD40 agonist activity of the FAP / 4-1BB / CD40 multispecific antibody was detected using the method provided in Example 7.1, and the results are shown in Figures 12A-12C and Table 29.

[0342] The results in Table 29 and Figure 12B show that the agonist activity of the FAP / 4-1BB / CD40 multispecific antibodies after cross-linking with FAP was comparable to that of RO7300490 and Ab10-A12V2-2. The results in Table 29 and Figure 12C show that all FAP / 4-1BB / CD40 multispecific antibodies have the ability to cross-link and activate 4-1BB, with B21546901 being slightly weaker than BNT312, and B21326102 being comparable to BNT312.

[0343] [Table 29]

[0344] The above results indicate that there was no significant difference in the CD40 agonist activity of multiple anti-FAP / 4-1BB / CD40 multispecific antibodies after crosslinking with FAP, which was comparable to the activating ability of the Ab10-A12V2-2 molecule. This indicates that CD40 sequence modifications have no effect on CD40 activation activity, and that all anti-FAP / 4-1BB / CD40 multispecific antibodies after crosslinking with 4-1BB have CD40 activation activity.

[0345] Example 8. FAP / 4-1BB / CD40 multispecific antibody detection of T lymphocyte activation 8.1 T lymphocyte activation experiments with FAP / 4-1BB / CD40 polyspecific antibodies T lymphocyte activation experiments were performed to detect the stimulatory ability of antibodies to T cells in the presence or absence of FAP. Freshly isolated and purified PBMCs were resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum to a density of 1 × 10 6 The density of cells was adjusted to 1.25 × 10 cells / mL. CHO-K1-HuFAP cells were obtained by stably transfecting CHO-K1 cells with a protein expressing human FAP. The cell culture medium was Ham's F12 + GlutaMAX™-I (Gibco, Cat# 31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. After resuscitation, the cells were passaged to adjust the cell condition, and the density was adjusted to 1.25 × 10 cells / mL. 6 A 96-well plate was coated with 100 μL / well of 0.25 μg / mL of the anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85), and after incubation at 37°C for 2 hours, residual antibody was washed away with PBS. Then, 100 μL of PBMC cells (1 × 10 cells / well) and 80 μL of medium or CHO-K1-HuFAP cells (1 × 10 cells / well) were added to each well. 5Cells (cells / well) were seeded into an OKT3-coated 96-well plate, and 20 μL / well of samples to be measured at 10× different concentrations were added. The plates were then incubated at 37°C in a 5% CO2 incubator for 3 days. The cell culture plates were removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatants. IL-2 levels were detected using a human IL-2 detection reagent kit (Cisbio, Cat# 62HIL02PEG). The specific procedures were as described in the reagent's instruction manual. The results are shown in Figure 13.

[0346] The results in Figure 13 show that the FAP / 4-1BB / CD40 multispecific antibody B977707 had some background activation activity in two donors. After crosslinking with FAP, both FAP / 4-1BB / CD40 multispecific antibodies were able to activate IL-2 secretion and significantly increased the activation activity of T lymphocytes.

[0347] 8.2 T lymphocyte activation experiments with FAP / 4-1BB / CD40 polyspecific antibodies Using the experimental method provided in Example 8.1, the stimulatory ability of the FAP / 4-1BB / CD40 multispecific antibody on T cells was detected, and the results are shown in Figure 14.

[0348] The results in Figure 14 show that in two donors, both the FAP / 4-1BB / CD40 multispecific antibody B21326102 and the control antibody BNT312 had some background T cell activation activity. After crosslinking with FAP, both FAP / 4-1BB / CD40 multispecific antibodies were able to better activate T cells to secrete IL-2, demonstrating significantly improved T cell activation ability.

[0349] 8.3 T lymphocyte activation experiments with FAP / 4-1BB / CD40 polyspecific antibodies Freshly isolated and purified PBMCs were resuspended in MACS Buffer (Miltenyi Biotec, Cat#130-091-221) and CD3 T cells were isolated using the EasySep™ Human T Cell Isolation Kit (Stemcell, Cat#17951) according to the kit's instructions. The isolated and purified CD3 T cells were resuspended in RPMI 1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum to a density of 1 × 10 6 The cells were adjusted to a density of 1.25 × 10 cells / mL. CHO-K1-HuFAP cells were obtained by stably transfecting CHO-K1 cells with a protein expressing human FAP, and the cell culture medium was Ham's F12 + GlutaMAX™-I (Gibco, Cat#31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-HuCD40 cells were obtained by overexpressing human CD40 protein in HEK293 cells, and the cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. After resuscitation, the cells were passaged to adjust the cell condition and the density was adjusted to 1.25 × 10 cells / mL. 6 A 96-well plate was coated with 100 μL / well of 0.25 μg / mL of anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85), incubated at 37°C for 2 hours, and then washed off the residual antibody with PBS. Then, 100 μL of PBMC cells (1 × 10 5 cells / well) and 80 μL of medium or CHO-K1-FAP cells or HEK293-HuCD40 (1 × 10 5The cells (cells / well) were seeded into a 96-well plate coated with OKT3, and 10x different concentrations of samples to be measured were added at a volume of 20 μL / well. The plate was then incubated at 37°C in a 5% CO2 incubator for 3 days. The cell culture plate was removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. The IL-2 level was detected using a human IL-2 detection reagent kit (Cisbio, Cat#62HIL02PEG). The specific procedure was as per the instruction manual of the reagent. The results are shown in Figure 15.

[0350] The results in Figure 15 show that in two donors, after cross-linking of FAP, both FAP / 4-1BB / CD40 polyspecific antibodies were able to stimulate IL-2 secretion, with stronger potency than MP0310. After cross-linking of CD40, both FAP / 4-1BB / CD40 polyspecific antibodies were able to stimulate IL-2 secretion, with stronger potency than the control antibody BNT312.

[0351] Example 9. Detection of DC maturation promotion by FAP / 4-1BB / CD40 multispecific antibodies 9.1 DC maturation promotion experiment of FAP / 4-1BB / CD40 multispecific antibody The ability of antibodies to promote DC maturation in the presence or absence of FAP was examined in a DC maturation promotion experiment. Freshly isolated and purified PBMCs were resuspended in MACS Buffer (Miltenyi Biotec, Cat#130-091-221) and purified using EasySep TM Monocytes were isolated using a Human Monocyte Isolation Kit (Stemcell, Cat. #19359) according to the kit's instructions. The isolated monocytes were resuspended in RPMI 1640 (Gibco, Cat. #10491A-01) containing 10% fetal bovine serum, 50 ng / mL GM-CSF (Peprotech, 200-04), and 50 ng / mL IL-4 (Peprotech, 300-03) to a density of 1 × 10. 6The cells were adjusted to a density of 1 × 10 cells / mL and plated in 10 mL of 100 mm TC-treated culture dishes (Corning, 430167). The medium was changed every 2–3 days and cultured for 5 days to obtain iDCs. iDCs were collected by centrifugation at 400 × g for 10 minutes and resuspended in RPMI 1640 (Gibco, Cat# 10491A-01) containing 10% fetal bovine serum at a density of 1 × 10 cells / mL. 6 The concentration of each cell was adjusted to 50 μL / mL, and 50 μL was added to each well. Samples to be measured at 3x different concentrations were added at a volume of 50 μL / well. CHO-K1-HuFAP cells were obtained by stably transfecting CHO-K1 cells with a protein expressing human FAP. The cell culture medium was Ham's F12 + GlutaMAX™-I (Gibco, Cat# 31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. For CHO-K1-WT, the cell culture medium was Ham's F12 + GlutaMAX™-I (Gibco, Cat# 31765035) containing 10% fetal bovine serum. CHO-K1-HuFAP and CHO-K1-WT were collected by centrifugation at 300 × g for 5 minutes, washed twice with sterile PBS (phosphate buffer solution, pH 7.40), and resuspended in RPMI 1640 (Gibco, Cat# 10491A-01) to a density of 1 × 10 6 The cells were adjusted to a density of 2 × 10 cells / mL, and 10 μg / mL Mitomycin C (Sigma, Cat#50-07-7) was added, followed by incubation at 37°C for 45 minutes. After incubation, the cells were washed twice with PBS and resuspended in sterile PBS (phosphate buffer, pH 7.40) to a density of 2 × 10 cells / mL. 6 The cells were adjusted to a density of 1 × 10 cells / mL, and 5 μM Cell Trace Violet (ThermoFisher, Cat# C345557 A) was added and incubated at 37°C in the dark for 10 minutes. After 10 minutes, an equal volume of RPMI1640 (Gibco, Cat# 10491A-01) containing 40% fetal bovine serum was added, and the reaction was stopped for 10 minutes in the dark. The cells were washed twice with sterile PBS and resuspended in RPMI1640 (Gibco, Cat# 10491A-01) containing 10% fetal bovine serum to a density of 1 × 10 cells / mL. 6The concentration was adjusted to cells / mL, 50 μL was added to each well to make the total volume 150 μL, and the mixture was incubated at 37° C. in a 5% CO 2 incubator for 48 hours.

[0352] After 48 hours of incubation, DCs were transferred to a new 96-well U-bottom plate and washed twice by centrifugation at 300 × g for 5 minutes. 100 μL of FACS buffer (sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum) containing 0.1 μL of the live / dead dye APC-efluor780 (BD Pharmingen, Cat# 565388) was added to each well. The plate was incubated for 15 minutes in the dark at room temperature, and then washed twice by centrifugation at 300 × g for 5 minutes. 50 μL of FACS buffer (sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum) containing 1 μL of Human Trustain FCX™ (Biolegend, Cat. #422302) was added to each well and incubated at room temperature in the dark for 10 minutes. 50 μL of FACS buffer (sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum) containing 2.5 μL of PE Mouse Anti-Human CD86 (BD Pharmingen, Cat. #555658) was added and incubated on ice in the dark for 30 minutes. After incubation, the cells were washed twice by centrifugation in FACS buffer, resuspended in FACS buffer, and the fluorescent signal was read using a flow cytometer. The results are shown in Figure 16.

[0353] The results showed that none of the FAP / 4-1BB / CD40 polyspecific antibodies had background DC cell maturation-promoting activity. After crosslinking with FAP, all of the FAP / 4-1BB / CD40 polyspecific antibodies were able to promote DC cell maturation, with activity comparable to that of Ab10-A12V2-2 and RO7300490.

[0354] 9.2 DC maturation promotion experiment of FAP / 4-1BB / CD40 multispecific antibody Using the experimental method provided in Example 8.1, the activity of the FAP / 4-1BB / CD40 multispecific antibody to promote DC maturation was detected, and the results are shown in Figure 17.

[0355] The results showed that none of the FAP / 4-1BB / CD40 multifunctional antibodies had background DC cell maturation activity. After crosslinking with FAP, all of the FAP / 4-1BB / CD40 multifunctional antibodies were able to promote DC cell maturation, with activity comparable to that of the Ab10-A12V2-2 molecule.

[0356] 9.3 DC maturation promotion experiment of FAP / 4-1BB / CD40 multispecific antibody Using the experimental method provided in Example 8.1, the activity of the FAP / 4-1BB / CD40 multispecific antibody to promote DC maturation was detected, and the results are shown in Figure 18.

[0357] The results showed that none of the FAP / 4-1BB / CD40 polyspecific antibodies had background DC maturation-promoting activity. After crosslinking with FAP, both FAP / 4-1BB / CD40 polyspecific antibodies promoted DC cell maturation, comparable to Ab10-A12V2-2 and RO7300490. After crosslinking with 4-1BB, both FAP / 4-1BB / CD40 polyspecific antibodies promoted DC cell maturation, with B21326102 promoting it more strongly than the control antibody BNT312, and B21546901 promoting it less strongly than the control antibody BNT312.

[0358] Example 10. Detection of the ability of FAP / 4-1BB / CD40 multispecific antibodies to crosslink 4-1BB / CD40-expressing cells Clinical results of BNT312 have shown that peripheral cross-linking activation of CD40-4-1BB leads to severe liver toxicity. Therefore, the triple antibody molecule achieves tumor targeting through its strong FAP binding ability while simultaneously reducing the cross-linking ability of CD40-4-1BB, thereby reducing peripheral activation and the occurrence of liver toxicity. FACS experiments were performed to detect the ability of the FAP / 4-1BB / CD40 multifunctional antibody to cross-link 4-1BB / CD40-expressing cells. HEK293-Hu4-1BB cells and HEK293-HuCD40 cells are HEK293 cells overexpressing human 4-1BB and human CD40 protein, respectively. The cell culture medium was DMEM (Gibco, Cat. #11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. The experimental medium was sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum. HEK293-Hu4-1BB and HEK293-HuCD40 cells were harvested by centrifugation at 300 × g for 5 min, washed twice with sterile PBS (phosphate buffer, pH 7.40), and resuspended in sterile PBS (phosphate buffer, pH 7.40) to a density of 2 × 10 6 The cells were adjusted to a density of 2 × 10 cells / mL, and 5 μM Cell Trace Violet (ThermoFisher, Cat# C345557 A) and 5 μM Cell Trace Far red (ThermoFisher, Cat# C34564 A) were added and incubated at 37°C in the dark for 10 minutes. After 10 minutes, an equal volume of RPMI1640 (Gibco, Cat# 10491A-01) containing 40% fetal bovine serum was added, and the reaction was stopped for 10 minutes in the dark. The cells were washed twice with sterile PBS and resuspended in FACS Buffer (sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum) to a density of 2 × 10 cells / mL. 6 The concentration of cells / mL was adjusted, and 50 μL of HEK293-Hu4-1BB and HEK293-HuCD40 cells were added to each well. 3x different concentrations of samples to be measured were added at a volume of 50 μL / well to bring the total volume to 150 μL. The mixture was then incubated at 4°C for 1 hour. After incubation, the fluorescent signal was read using a flow cytometer. The results are shown in Figure 19.

[0359] The results showed that all FAP / 4-1BB / CD40 polyspecific antibodies were able to crosslink 4-1BB / CD40-expressing cells, although their crosslinking ability was weaker than that of the control antibody BNT312.

[0360] Example 11. Mixed lymphocyte culture detection of FAP / 4-1BB / CD40 polyspecific antibodies The CD40 / 4-1BB cross-linking ability of the triple antibody molecule and the control molecule BNT312 was detected by culture using mixed lymphocytes. Cryopreserved PBMCs were resuspended in MACS Buffer (Miltenyi Biotec, Cat#130-091-221) and purified by EasySep TM Monocytes were isolated using a Human Monocyte Isolation Kit (Stemcell, Cat. #19359) according to the kit's instructions. The isolated monocytes were resuspended in RPMI 1640 (Gibco, Cat. #10491A-01) containing 10% fetal bovine serum, 50 ng / mL GM-CSF (Peprotech, 200-04), and 50 ng / mL IL-4 (Peprotech, 300-03) to a density of 1 × 10. 6 The cells were adjusted to a density of 2 × 10 cells / mL and plated in 10 mL of 100 mm TC-treated culture dishes (Corning, 430167). The culture medium was changed every 2–3 days at 37°C for 5 days to obtain iDCs. The iDCs were collected by centrifugation at 400 × g for 10 minutes and resuspended in RPMI 1640 (Gibco, Cat. #10491A-01) containing 10% fetal bovine serum and 1 ng / mL Staphylococcus aureus enterotoxin B (SEB, Toxin Technology, Cat. #BT202) to a density of 2 × 10 cells / mL. 5The concentration of cells / mL was adjusted to 50 μL per well. Cryopreserved PBMCs from another donor were resuspended in MACS Buffer (Miltenyi Biotec, Cat#130-091-221), and CD8 T cells were isolated using a human CD8 T cell isolation reagent kit (Miltenyi Biotec, Cat#130-096-495). For specific procedures, refer to the kit's instruction manual. The isolated human CD8 T cells were resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum to a density of 1 × 10 6 The concentration of each cell was adjusted to 50 μL / mL, and 50 μL was added to each well. 3× different concentrations of samples to be measured were added at a volume of 50 μL / well to make a total volume of 150 μL, and the plates were incubated at 37°C for 5 days. The cell culture plates were removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. IL-2 levels were detected using a human IL-2 detection reagent kit (Cisbio, Cat#62HIL02PEG). For specific procedures, please refer to the reagent's instruction manual. The results are shown in Figure 20.

[0361] The results showed that the amount of IL-2 secreted by the control antibody BNT312 was higher than that of the corresponding FAP / 4-1BB / CD40 multispecific antibody at both concentrations.

[0362] These results suggest that the FAP / 4-1BB / CD40 multispecific antibody crosslinks 4-1BB / CD40-expressing cells and stimulates IL-2 secretion, characterizing the FAP-independent molecule. Both FAP / 4-1BB / CD40 multispecific antibodies were weaker than the control antibody BNT312. This suggests that when the 4-1BB and CD40 ends function simultaneously, the background activation of the FAP / 4-1BB / CD40 multispecific antibody is low in the FAP-independent case. The FAP / 4-1BB / CD40-specific antibody possesses the ability to localize FAP-mediated tumors and, at the same time, reduces toxicity by reducing non-FAP-dependent peripheral activation.

[0363] Example 12. In vivo efficacy and toxicity studies of anti-FAP / 4-1BB / CD40 multispecific antibodies 12.1 In vivo efficacy of FAP / 4-1BB / CD40 polyspecific antibodies in the murine colon cancer model MC38-mFAP 2 × 10 MC38-mFAP cells (constructed in-house) 5 The cells were subcutaneously inoculated into B-h4-1BB / CD40 humanized mice (provided by Biocytogen) at 100 μL / mouse, and the tumors were approximately 110 mm 3 Once tumors reached the normal size, 48 mice were randomly assigned based on tumor volume. Six mice per group were assigned to eight groups, each receiving vehicle, B977703 (6 mg / kg), B977703 (2 mg / kg), B977704 (6 mg / kg), B977704 (2 mg / kg), B977707 (2.28 mg / kg), RO7122290 (1.76 mg / kg), or Ab10-A12V2-2 (1.72 mg / kg). Tumor volumes were measured twice weekly during the treatment and observation periods and recorded. Among them, B977703 (2 mg / kg), B977704 (2 mg / kg), B977707 (2.28 mg / kg), RO7122290 (1.76 mg / kg), and Ab10-A12V2-2 (1.72 mg / kg) were administered at equimolar doses.

[0364] Tumor volume (TV) was calculated using the formula TV=1 / 2×a×b2, where a and b represent the major and minor diameters of the measured tumor, respectively.

[0365] Relative tumor growth rate T / C%=(T-T0) / (C-C0)×100%, tumor inhibition rate TGI%=1-T / C%.

[0366] CR% (Complete Tumor Regression Rate) = Complete tumor regression (<110mm 3 ) Number of mice / Number of mice admitted to the group.

[0367] The results are shown in Figure 21 and Table 30. The results showed that the FAP / 4-1BB / CD40 multispecific antibodies B977703, B977704, and B977707 at different doses all had good antitumor activity, which was superior to that of the control antibodies RO7122290 and Ab10-A12V2-2.

[0368] [Table 30] 12.2 Hepatotoxicity of FAP / 4-1BB / CD40 Polyspecific Antibody in the Murine Colon Cancer Model MC38-mFAP In the 12.1 test, blood was collected on the 17th day to detect aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the results of which are shown in FIG. 22, and body weight was measured twice a week, the results of which are shown in FIG.

[0369] As a result, the mouse weights remained stable during administration, suggesting that the FAP / 4-1BB / CD40 multispecific antibodies had no obvious toxicity or side effects (Figure 23). Based on the results of ALT / AST detection on day 17 (Figure 22), none of the FAP / 4-1BB / CD40 multispecific antibodies showed any obvious hepatotoxicity.

[0370] 12.3 Hematotoxicity of FAP / 4-1BB / CD40 Multifunctional Antibody in the Mouse Colon Cancer Model MC38-mFAP In Study 12.1, blood was collected 24 hours after the first administration and general blood tests were performed.

[0371] The results (FIG. 24) showed that none of the FAP / 4-1BB / CD40 multispecific antibodies caused a decrease in neutrophils (NEUT#) and platelets (PLT), and had no hematotoxicity.

[0372] 12.4 In vivo efficacy of FAP / 4-1BB / CD40 polyspecific antibodies in the murine colon cancer model MC38-mFAP 2 × 10 MC38-mFAP cells (constructed in-house) 5The cells were subcutaneously inoculated into B-h4-1BB / CD40 humanized mice (provided by Biocytogen) at 100 μL / mouse, and the tumors were approximately 110 mm 3 Once tumors reached the normal size, 42 mice were randomly assigned based on tumor volume, with 6 mice per group for a total of 7 groups. Each group received vehicle, B21326101 (6 mg / kg), B21326101 (2 mg / kg), B21326101 (0.5 mg / kg), Ab10-A12V2-2 (0.43 mg / kg), BNT312 (4.47 mg / kg), and BNT312 (1.49 mg / kg). B21326101 (6 mg / kg), B21326101 (2 mg / kg), BNT312 (4.47 mg / kg), and BNT312 (1.49 mg / kg) were administered once every 3 days for a total of 3 doses. Among these, B21326101 (6 mg / kg) and BNT312 (4.47 mg / kg) were administered at equimolar doses, B21326101 (2 mg / kg) and NT312 (1.49 mg / kg) were administered at equimolar doses, and B21326101 (0.5 mg / kg) and Ab10-A12V2-2 (0.43 mg / kg) were administered at equimolar doses. B21326101 (0.5 mg / kg) and Ab10-A12V2-2 (0.43 mg / kg) were administered once every 3 days for a total of 4 doses. Tumor volume was measured twice weekly during the administration and observation period, and measurements were recorded. On day 46 after administration, MC38-mFAP cells (2 × 10 5 Each mouse was inoculated with a single dose of 100 μL of IgG (100 cells / 100 μL / mouse), and the tumor volume of the mice was then measured and recorded. The calculation methods for tumor volume (TV), relative tumor growth rate (T / C%), tumor inhibition rate (TGI%), and CR% (tumor complete regression rate) are described in Section 12.1.

[0373] The results are shown in Table 31. The results showed that the FAP / 4-1BB / CD40 multispecific antibody B21326101 at different doses had good anti-tumor activity, which was superior to the control antibodies BNT312 and Ab10-A12V2-2.

[0374] [Table 31] 12.5 Hepatotoxicity of FAP / 4-1BB / CD40 polyspecific antibody in the murine colon cancer model MC38-mFAP

[0375] In the 12.4 study, blood was collected on the 8th day to detect AST and ALT.

[0376] The results are shown in Figure 25. The results of ALT / AST detection on day 8 indicated that none of the FAP / 4-1BB / CD40 multifunctional antibodies were hepatotoxic and had better safety. BNT312 showed obvious hepatotoxicity at both high and low doses.

[0377] [Table 32-1] [Table 32-2] [Table 32-3] [Table 32-4] [Table 32-5] [Table 32-6] [Table 33-1] [Table 33-2] [Table 33-3] [Table 33-4]

Claims

1. A FAP / 4-1BB / CD40 binding molecule comprising a first antigen-binding domain that specifically binds to FAP, a second antigen-binding domain that specifically binds to CD40, and a third antigen-binding domain that specifically binds to 4-1BB, wherein the third antigen-binding domain comprises at least one immunoglobulin single variable domain; the immunoglobulin single variable domain in the third antigen-binding domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 28, 18, 23-26 and 33, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; FAP / 4-1BB / CD40 binding molecule.

2. the immunoglobulin single variable domain in said third antigen-binding domain comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 30 and 31, respectively; Preferably, the immunoglobulin single variable domain in said third antigen-binding domain comprises: a-1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 29 and 27, respectively; a-2) CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 19, 20 and 27, respectively; or a-3) comprising CDR1, CDR2 and CDR3 shown in any one of SEQ ID NOs: 19, 20 and 21, respectively; The FAP / 4-1BB / CD40 binding molecule of claim 1.

3. the immunoglobulin single variable domain in the third antigen-binding domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, reduced antibody aggregation, reduced antibody isomerization, reduced pre-anti-drug antibody (pre-ADA) binding, and / or reduced anti-drug antibody (ADA) binding; Preferably, the framework region templates of the humanized modified human germline genes are derived from IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01. The FAP / 4-1BB / CD40 binding molecule of claim 1 or 2.

4. the immunoglobulin single variable domain in the third antigen-binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 28, 18, 23-26 and 33, or an amino acid sequence having at least 90% sequence identity thereto; The FAP / 4-1BB / CD40 binding molecule of any one of claims 1 to 3.

5. the second antigen-binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain in the second antigen-binding domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence set forth in SEQ ID NO: 32, 11 or 15, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; 5. The FAP / 4-1BB / CD40 binding molecule of claim 1.

6. the immunoglobulin single variable domain in said second antigen-binding domain comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 12, 13 and 14, respectively; Preferably, the immunoglobulin single variable domain in said second antigen-binding domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, reduced antibody aggregation, reduced antibody isomerization, reduced pre-anti-drug antibody (pre-ADA) binding, and / or reduced anti-drug antibody (ADA) binding, Preferably, the humanized modified heavy chain framework region module of the human germline gene is derived from IGHV3-48*03. The FAP / 4-1BB / CD40 binding molecule of claim 5.

7. the immunoglobulin single variable domain in the second antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 32, 11 or 15, or an amino acid sequence having at least 90% sequence identity thereto; The FAP / 4-1BB / CD40 binding molecule of claim 5 or 6.

8. The first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), The VH comprises HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NOs: 3, 4 and 5, respectively; and The VL comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 6, 7 and 8, respectively; 8. The FAP / 4-1BB / CD40 binding molecule of any one of claims 1 to 7.

9. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity thereto; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the first antigen-binding domain is selected from Fab, Fab', Fv, or ScFv, more preferably Fab; Preferably, the first antigen-binding domain comprises a Fab heavy chain and a Fab light chain, wherein: the Fab heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence having at least 90% sequence identity thereto; and the Fab light chain comprises the amino acid sequence set forth in SEQ ID NO: 67, or an amino acid sequence having at least 90% sequence identity thereto; The FAP / 4-1BB / CD40 binding molecule of claim 8.

10. 1. A FAP / 4-1BB / CD40 binding molecule comprising a first antigen-binding domain that specifically binds to FAP, a second antigen-binding domain that specifically binds to CD40, and a third antigen-binding domain that specifically binds to 4-1BB, wherein the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NOs: 3, 4 and 5, respectively; the VL comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 6, 7 and 8, respectively; Preferably, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity thereto; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the first antigen-binding domain comprises a Fab heavy chain and a Fab light chain, wherein: the Fab heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence having at least 90% sequence identity thereto; and the Fab light chain comprises the amino acid sequence set forth in SEQ ID NO:67, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the third antigen-binding domain is as defined in any one of claims 1 to 4, Preferably, the second antigen-binding domain is as defined in any one of claims 5 to 7. FAP / 4-1BB / CD40 binding molecule.

11. 1. A FAP / 4-1BB / CD40 binding molecule comprising: a first antigen-binding domain that specifically binds to FAP; a second antigen-binding domain that specifically binds to CD40; and a third antigen-binding domain that specifically binds to 4-1BB, wherein the second antigen-binding domain comprises at least one immunoglobulin single variable domain, and the immunoglobulin single variable domain in the second antigen-binding domain comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; Preferably, the immunoglobulin single variable domain in the second antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 11, 15 or 32, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the third antigen-binding domain is as defined in any one of claims 1 to 4, Preferably, the first antigen-binding domain is as defined in any one of claims 8 to 9. FAP / 4-1BB / CD40 binding molecule.

12. further comprising an Fc region, Preferably, the Fc region is derived from a human IgG1, IgG2, or IgG4 Fc region; More preferably, the human IgG1 Fc region has at least one of the following mutations: 234A, 235A, 220A, 297A, or 297Q, 267E, 328F; More preferably, the human IgG4 Fc region has at least one of the following mutations: 228P, 234A, 235A, and 447A; The mutations are numbered according to the EU numbering rules.

12. The FAP / 4-1BB / CD40 binding molecule of any one of claims 1 to 11.

13. further comprising a linker positioned between any two of the first, second and third antigen-binding domains; Preferably, the linker is (G ｘ S) ｙ a linker, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6; More preferably, x is 4 and y is 1, 2 or 3.

10. A FAP / 4-1BB / CD40 binding molecule according to any one of the preceding claims.

14. the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain have a valency ratio of (1 to 2):(1 to 5):(1 to 5), preferably 1:2:2, 1:2:4, 1:1:1, or 1:1:2; 10. A FAP / 4-1BB / CD40 binding molecule according to any one of the preceding claims.

15. 1. A FAP / 4-1BB / CD40 binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein, from N-terminus to C-terminus, the FAP / 4-1BB / CD40 binding molecule comprises: (I) a first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[a third antigen-binding domain that specifically binds to 4-1BB]-[linker]b-[a second antigen-binding domain that specifically binds to CD40]; and Second polypeptide chain: [Fab light chain of the first antigen-binding domain that specifically binds to FAP], (II) a first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[a third antigen-binding domain that specifically binds to 4-1BB]; and Second polypeptide chain: [Fab light chain of a first antigen-binding domain that specifically binds to FAP]-[linker]b-[second antigen-binding domain that specifically binds to CD40]; (III) first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[third antigen-binding domain that specifically binds to 4-1BB]-[linker]b-[third antigen-binding domain that specifically binds to 4-1BB]; and Second polypeptide chain: [Fab light chain of a first antigen-binding domain that specifically binds to FAP]-[linker]c-[second antigen-binding domain that specifically binds to CD40]; (IV) a first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[second antigen-binding domain that specifically binds to CD40]-[linker]b-[third antigen-binding domain that specifically binds to 4-1BB]; and Second polypeptide chain: [Fab light chain of the first antigen-binding domain that specifically binds to FAP], (V) a first polypeptide chain: [Fab heavy chain of a first antigen-binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[a second antigen-binding domain that specifically binds to CD40]; and Second polypeptide chain: [Fab light chain of the first antigen-binding domain that specifically binds to FAP]-[linker]b-[third antigen-binding domain that specifically binds to 4-1BB]; any group or combination of: wherein - represents a peptide bond, any two linkers may be the same or different, and a, b, and c are each independently selected from 0 or 1; Preferably, the linker is (G ｘ S) ｙ linkers, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6; more preferably, the linkers are each independently selected from G ４ S, (G ４ S) ２ , (G ４ S) ３ or (G ４ S) ４ Selected from Preferably, the first antigen-binding domain that specifically binds to FAP is selected from the first antigen-binding domains according to any one of claims 8 to 9, Preferably, the second antigen-binding domain that specifically binds to CD40 is selected from the second antigen-binding domains according to any one of claims 5 to 7, Preferably, the third antigen-binding domain that specifically binds to 4-1BB is selected from the first antigen-binding domains according to any one of claims 1 to 4. FAP / 4-1BB / CD40 binding molecule.

16. The present invention relates to a method for producing a polypeptide comprising: the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 49 and 50, respectively; the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 34 and 35, respectively; the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 36 and 37, respectively; the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 38 and 39, respectively; the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively; the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 42 and 43, respectively; the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 45 and 46, respectively; the first polypeptide chain and the second polypeptide chain comprise the amino acid sequences set forth in SEQ ID NOs: 47 and 48, respectively; or The first polypeptide chain and the second polypeptide chain of any one of the groups each include a combination of amino acid sequences having at least 90% sequence identity with each other.

10. A FAP / 4-1BB / CD40 binding molecule according to any one of the preceding claims.

17. A 4-1BB binding molecule, which is i) a third antigen-binding domain that specifically binds to 4-1BB, wherein the third antigen-binding domain comprises CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 18, 23 to 26, 28, and 33, and the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, ii) a first antigen-binding domain that specifically binds to FAP; or iii) a second antigen-binding domain that specifically binds to CD40; Further comprising: More preferably, the first antigen-binding domain is as defined in any one of claims 8 to 9, More preferably, the second antigen-binding domain is as defined in any one of claims 5 to 7. 4-1BB binding molecule.

18. the third antigen-binding domain comprises CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 19, 30 and 31, respectively; Preferably, the third antigen-binding domain comprises: a-1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 19, 29 and 27, respectively; a-2) CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 19, 20 and 27, respectively; or a-3) CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 19, 20 and 21, respectively; and CDR1, CDR2 and CDR3 as set forth in any one of:

18. The 4-1BB binding molecule of claim 17.

19. the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, reduced antibody aggregation, reduced binding to pre-anti-drug antibodies (pre-ADA), and / or reduced antibody isomerization; Preferably, the humanized modified heavy chain framework region modules of human germline genes are derived from IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01. 4-1BB binding molecule according to claim 17 or 18.

20. comprising an immunoglobulin single variable domain as set forth in the amino acid sequence set forth in any one of SEQ ID NOs: 18, 23-26, 28 and 33, or an amino acid sequence having at least 90% sequence identity thereto; Optionally, The 4-1BB binding molecule comprises: The amino acid sequence set forth in SEQ ID NO: 44, or an amino acid sequence having at least 90% sequence identity thereto, 20. A 4-1BB binding molecule according to any one of claims 17 to 19.

21. Encoding a FAP / 4-1BB / CD40 binding molecule according to any one of claims 1 to 16 or a 4-1BB binding molecule according to any one of claims 17 to 20. Polynucleotide.

22. A vector comprising the polynucleotide of claim 21.

23. 23. A host cell comprising the polynucleotide of claim 21 or the vector of claim 22.

24. 1. A method for preparing a FAP / 4-1BB / CD40 binding molecule, or a 4-1BB binding molecule, comprising: expressing in a host cell a FAP / 4-1BB / CD40 binding molecule according to any one of claims 1 to 16 or a 4-1BB binding molecule according to any one of claims 17 to 20; and isolating the FAP / 4-1BB / CD40 binding molecule or isolating the 4-1BB binding molecule from the host cell; Optionally, the method further comprises purifying the FAP / 4-1BB / CD40 binding molecule or the 4-1BB binding molecule. method.

25. 21. A pharmaceutical composition comprising a FAP / 4-1BB / CD40 binding molecule according to any one of claims 1 to 16, a 4-1BB binding molecule according to any one of claims 17 to 20, a polynucleotide according to claim 21, or a vector according to claim 22, preferably said pharmaceutical composition further comprising one or more medicament excipients, diluents or additives. Pharmaceutical compositions.

26. Use of the FAP / 4-1BB / CD40 binding molecule according to any one of claims 1 to 16, the 4-1BB binding molecule according to any one of claims 17 to 20, the polynucleotide according to claim 21, the vector according to claim 22, or the pharmaceutical composition according to claim 25 in any one of the following (1) to (3): (1) For the prevention or treatment of cancer, or for the preparation of a medicament for the prevention or treatment of cancer, (2) For the activation of T cells or the preparation of a drug for activating T cells, (3) For the preparation of a drug for activating DC cells or promoting the activation of DC cells, Preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and blood system cancer; use.

27. 19. A method of treating or ameliorating cancer, the method comprising administering to a subject in need thereof a FAP / 4-1BB / CD40 binding molecule of any one of claims 1 to 16, a 4-1BB binding molecule of any one of claims 17 to 20, a polynucleotide of claim 21, a vector of claim 22, or a pharmaceutical composition of claim 25; Preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and blood system cancer; method.

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