Pharmaceutical compositions of FAP / CD40 binding molecules and their pharmaceutical uses

Abstract

A pharmaceutical composition of a FAP / CD40 binding molecule and its pharmaceutical use. Specifically, the pharmaceutical composition comprises a FAP / CD40 binding molecule and a buffering agent. The pharmaceutical composition has good biological activity and stability.

Description

[Technical Field]

[0001] This disclosure claims priority to Chinese Patent Application No. 202310174524.1, filed on February 24, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of drug formulations, and in particular to pharmaceutical compositions comprising FAP / CD40 binding molecules and their pharmaceutical uses. [Background technology]

[0003] CD40 (TNFRSF5) is a transmembrane phosphoglycoprotein belonging to the tumor necrosis factor receptor superfamily (TNFRS). CD40 is expressed on multiple cell types, including B cells, follicular dendritic cells (DCs), epithelial cells, monocytes, macrophages, smooth muscle cells, and tumor cells. Its ligand, CD40L, is expressed primarily on activated T cells, activated B cells, platelets, and smooth muscle cells. CD40-L binding induces CD40 multimerization, resulting in downstream activation, growth, and differentiation signals. CD40 signaling activates multiple downstream signaling pathways, including NF-κB, MAPK, and STAT3 (Pype S, et al. J Biol Chem. 2000 Jun. 16;275(24):1858-693). These pathways regulate gene expression by regulating the activating proteins c-Jun, ATF2, and Rel transcription factors. The binding of CD40 to CD40L induces resting B cell proliferation, immunoglobulin conversion, and antibody secretion, and plays a key role in the development of germinal centers in tissues and B cell survival, all of which are essential for humoral immune responses (Kehry M RJ Immunol 1996;156:2345-2348). CD40L binds to CD40 on DCs, inducing their maturation, which manifests as 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] Due to the clinical success of immune checkpoint blockade (ICI) therapy targeting CD40, CTLA-4, and PD-L1 in tumor treatment, immunotherapy has become one of the most promising research areas in third-generation tumor therapy. Among these, antitumor immunotherapies, which rely on CD40 activation to increase antigen presentation and activation by DC cells to T cells, have already demonstrated clinical efficacy. However, CD40 systemic activation can cause several target-specific toxicities, such as cytokine storm, hepatotoxicity, hematotoxicity, and venous thrombosis due to peripheral CD40 activation. These toxicities limit the therapeutic range of CD40 agonist antibodies. Pfizer's CD40 agonists CP-870 and CP-893 and Mediimmune's CD40L-Fc fusion protein MEDI5083 (a novel fusion protein from AstraZeneca that can activate the CD40 signaling pathway, consisting of three tandemly linked CD40L and IgG4-Fc fusion proteins) have been discontinued in clinical studies. Therefore, In developing second-generation CD40 agonist antibodies, researchers are attempting to mediate CD40 activation by tumor-associated antigens (TAAs), thereby specifically activating CD40 within tumors and reducing peripheral CD40 activation, thereby broadening the therapeutic window of CD40 agonist antibodies.

[0005] Fibroblast activation protein (FAP) α is a tumor-associated antigen. FAP is expressed at low levels in normal tissues of healthy adults and selectively expressed in 93% of tumor tissues, of which high expression is seen in 30% of tumor tissues, including colon cancer, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, bladder cancer, and oral squamous cell carcinoma. WO2023025194 provides anti-FAP antibodies, anti-CD40 antibodies, and bispecific antibodies thereof. The bispecific antibodies can mediate tumor-specific CD40 activation by FAP, have maturation-promoting and activation effects on APCs (e.g., dendritic cells, DCs), and can eliminate liver toxicity, peripheral blood toxicity, and other peripheral toxicities, offering an excellent administration window and potential for drug discovery.

[0006] Antibody drugs have large molecular weights and complex structures, making them prone to instability due to degradation, polymerization, or undesired chemical modifications. Research into stabilized formulations of antibody drugs is particularly important to ensure that antibodies are suitable for administration and maintain stability during storage and subsequent use, enabling them to achieve better efficacy. The development of suitable pharmaceutical compositions for new FAP / CD40 binding molecules remains necessary. Summary of the Invention

[0007] The present disclosure provides pharmaceutical compositions comprising FAP / CD40 binding molecules that have excellent stability.

[0008] The present disclosure provides pharmaceutical compositions comprising a FAP / CD40 binding molecule and a buffer, wherein the buffer is selected from an acetate buffer, a succinate buffer, a histidine buffer, or a phosphate buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer. In some embodiments, the buffer is a histidine-acetate buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer. In some embodiments, the buffer is a histidine-acetate buffer.

[0009] In some embodiments, the FAP / CD40 binding molecule comprises a first antigen-binding domain that specifically binds to a FAP and a second antigen-binding domain that specifically binds to CD40, wherein the second antigen-binding domain that specifically binds to CD40 comprises (at least one) immunoglobulin single variable domain.

[0010] In some embodiments, the FAP / CD40 binding molecule comprises one immunoglobulin single variable domain that specifically binds to CD40. In other embodiments, the FAP / CD40 binding molecule comprises two, three, four, five, six, seven, eight, nine, ten or more immunoglobulin single variable domains that specifically bind to CD40, and the immunoglobulin single variable domains can be the same or different.

[0011] In some embodiments, the FAP / CD40 binding molecule comprises at least one (eg, two, three, four) antigen binding domain that specifically binds to a FAP.

[0012] In some embodiments, in the FAP / CD40 binding molecule, the immunoglobulin single variable domain that specifically binds to CD40 comprises three complementarity determining regions, CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are set forth in SEQ ID NOs: 12, 13, and 14, respectively.

[0013] In some embodiments, in the FAP / CD40 binding molecule, the immunoglobulin single variable domain that specifically binds to CD40 is set forth in any one of SEQ ID NOs: 11, 16-19, or comprises at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 23, 35-38.

[0014] In some embodiments, the FAP / CD40 binding molecule comprises a first antigen-binding domain that specifically binds to a FAP and a second antigen-binding domain that specifically binds to CD40, wherein the first antigen-binding domain that specifically binds to a FAP comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein 1) the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 3, 4, and 5, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 6, 7, and 8, respectively; or 2) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 3, 4, and 5, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 6, 32, and 8, respectively.

[0015] In some embodiments, in the FAP / CD40 binding molecule, the heavy chain variable region of the first antigen-binding domain that specifically binds to the FAP comprises an amino acid sequence set forth in SEQ ID NO:1 or having at least 80% identity thereto, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:2 or having at least 80% identity thereto.

[0016] In some embodiments, the FAP / CD40 binding molecule further comprises a human immunoglobulin Fc region (eg, a human IgG1 or IgG4 Fc region).

[0017] In some embodiments, in the FAP / CD40 binding molecule, the first antigen-binding domain that specifically binds to the FAP comprises a heavy chain (HC) and a light chain (LC), For example, the heavy chain is an IgG1 or IgG4 isotype and the light chain is a Kappa isotype; In some specific embodiments, the heavy chain is an amino acid sequence set forth in SEQ ID NO: 24 or having at least 80% identity thereto, and the light chain is an amino acid sequence set forth in SEQ ID NO: 25 or having at least 80% identity thereto.

[0018] In some embodiments, in the FAP / CD40 binding molecule, the first antigen-binding domain that specifically binds to the FAP comprises a heavy chain variable region and a light chain variable region, the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds to CD40 is N-terminal to the heavy chain variable region of the first antigen-binding domain that specifically binds to FAP; the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds to CD40 is C-terminal to the heavy chain variable region of the first antigen-binding domain that specifically binds to FAP; the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds to CD40 is N-terminal to the light chain variable region of the first antigen-binding domain that specifically binds to FAP; and / or The immunoglobulin single variable domain of the second antigen-binding domain that specifically binds CD40 is C-terminal to the light chain variable region of the first antigen-binding domain that specifically binds FAP.

[0019] In some embodiments, the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds CD40 is linked to the first antigen-binding domain that specifically binds a FAP directly or via a linker, for example, the linker is (G4S) x wherein x is independently an integer selected from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). For example, the linker has an amino acid sequence represented by (G4S)2, (G4S)3, or (G4S)4.

[0020] In some embodiments, the second antigen-binding domain that specifically binds CD40 in the FAP / CD40 binding molecule is multivalent (e.g., one FAP / CD40 binding molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 second antigen-binding domains that specifically bind CD40). In some specific embodiments, the second antigen-binding domain that specifically binds CD40 is bivalent, tetravalent, or hexavalent. In some specific embodiments, the second antigen-binding domain that specifically binds CD40 comprises 2, 3, 4, 5, or 6 of the immunoglobulin single variable domains.

[0021] In some embodiments, the FAP / CD40 binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 21-23 or having at least 80% identity thereto, and the second polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO: 20 or having at least 80% identity thereto. In some specific embodiments, the FAP / CD40 binding molecule comprises two first polypeptide chains and two second polypeptide chains, and in some specific embodiments, the two first polypeptide chains are the same and the two second polypeptide chains are the same.

[0022] In some embodiments, the FAP / CD40 binding molecule can inhibit tumor growth (e.g., increase in volume, increase in tumor weight) and / or metastasis (e.g., multi-organ or multi-tissue metastasis, distant metastasis) 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%.

[0023] In some embodiments, the FAP / CD40 binding molecule is an anti-FAP / CD40 bispecific antibody or antigen-binding fragment thereof, and the antigen-binding fragment includes, but is 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), e.g., scFv, Fv, Fab, or Fab' fragments.

[0024] In some embodiments, the anti-FAP / CD40 bispecific antibody comprises an immunoglobulin single variable domain in the second antigen-binding domain that specifically binds CD40 as described above, and a heavy chain variable region (VH) and a light chain variable region (VL) in the first antigen-binding domain that specifically binds a FAP as described above.

[0025] In some embodiments, the anti-FAP / CD40 bispecific antibody: the first antigen-binding domain that specifically binds to FAP is a first antibody comprising a heavy chain (HC) and a light chain (LC); and The second antigen-binding domain that specifically binds to CD40 is a second antibody that is a VHH having CDR1, CDR2, and CDR3 of the above-mentioned CD40-binding molecule.

[0026] In some specific embodiments, the VHH is at the N-terminus and / or C-terminus of the heavy or light chain of the first antibody as the second antibody.

[0027] In some specific embodiments, the anti-FAP / CD40 bispecific antibody comprises one first antibody and one, two, three, four, five, six, seven, eight, nine, ten (e.g., two, four, six) VHHs of a second antibody, wherein the first antibody comprises two HCs and two LCs, and the VH of one HC of the first antibody forms an antigen-binding site together with the VL of one LC, and the VH of the other HC forms an antigen-binding site together with the VL of the other LC.

[0028] In some specific embodiments, the first antibody of the anti-FAP / CD40 bispecific antibody or antigen-binding fragment thereof may be linked to 1, 2, 3, 4, 5, 6, 7, or 8 VHH second antibodies, which may be the same or different, and which may all be linked to the N-terminus of the heavy chain of the first antibody, or all be linked to the C-terminus of the heavy chain of the first antibody, or all be linked to the N-terminus of the light chain of the first antibody, or all be linked to the C-terminus of the light chain of the first antibody, or any combination of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain.

[0029] In some specific embodiments, the anti-FAP / CD40 bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, (i) the first polypeptide chain is, from N-terminus to C-terminus, [heavy chain of the first antibody]-linker1-[second antibody], and the second polypeptide chain is the light chain of the first antibody; (ii) the first polypeptide chain, from N-terminus to C-terminus, is [first antibody heavy chain]-linker1-[second antibody]1-linker2-[second antibody]2, and the second polypeptide chain is the first antibody light chain; (iii) the first polypeptide chain, from N-terminus to C-terminus, is [heavy chain of first antibody]-linker1-[second antibody]1-linker2-[second antibody]2-linker3-[second antibody]3, and the second polypeptide chain is the light chain of the first antibody; (iv) the first polypeptide chain, from the N-terminus to the C-terminus, is [second antibody]-linker1-[heavy chain of the first antibody], and the second polypeptide chain is the light chain of the first antibody; (v) the first polypeptide chain, from N-terminus to C-terminus, is [second antibody]2-linker2-[second antibody]1-linker1-[heavy chain of first antibody], and the second polypeptide chain is the light chain of the first antibody; (vi) the first polypeptide chain is, from N-terminus to C-terminus, [second antibody]3-linker3-[second antibody]2-linker2-[second antibody]1-linker1-[heavy chain of first antibody], and the second polypeptide chain is the light chain of the first antibody; (vii) the first polypeptide chain, from N-terminus to C-terminus, is [second antibody]1-linker1-[first antibody heavy chain]-linker2-[second antibody]2, and the second polypeptide chain is the first antibody light chain; (viii) the first polypeptide chain, from N-terminus to C-terminus, is [second antibody]1-linker1-[second antibody]2-linker2-[heavy chain of first antibody]-linker3-[second antibody]3, and the second polypeptide chain is the light chain of the first antibody; (ix) the first polypeptide chain, from N-terminus to C-terminus, is [second antibody]1-linker1-[first antibody heavy chain]-linker2-[second antibody]2-linker3-[second antibody]3, and the second polypeptide chain is the first antibody light chain; Among them, [second antibody] 1, [second antibody] 2, and [second antibody] 3 may be the same or different.

[0030] In some embodiments, the second antibody of the VHH in the anti-FAP / CD40 bispecific antibody is linked to the first antibody directly or via a linker. The linker may be m S n ) x or (GGNGT) x or (YGNGT) x wherein 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 x 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). For example, the linker is an amino acid sequence represented by G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, or (G4S)6. In some embodiments, linker 1, linker 2, and linker 3 may be the same or different.

[0031] In some embodiments, the heavy chain of the first antibody of the anti-FAP / CD40 bispecific antibody comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The first antibody may be a full-length antibody.

[0032] In some embodiments, the anti-FAP / CD40 bispecific antibody is such that the heavy chain of the first antibody is an IgG isotype, e.g., IgG1, IgG2, IgG3, or IgG4, e.g., an IgG1 isotype, and / or the light chain of the first antibody is a Kappa isotype.

[0033] In some embodiments, the two HCs of the anti-FAP / CD40 bispecific antibody comprise the same CDRs and / or the two LCs comprise the same CDRs. In some specific embodiments, the two HCs of the first antibody comprise the same VH and / or the two LCs comprise the same VL. In some specific embodiments, the two HCs of the first antibody have the same amino acid sequence and / or the two LCs have the same amino acid sequence.

[0034] In some embodiments, the second antibodies of the two VHHs of the anti-FAP / CD40 bispecific antibody have the same or different amino acid sequences, for example, the second antibodies of the two VHHs have the same amino acid sequence.

[0035] In some embodiments, the anti-FAP / CD40 bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain: a) the first polypeptide chains each independently comprise a VHH of a second antibody and a heavy chain (HC) of the first antibody; and b) the second polypeptide chains each independently comprise a light chain (LC) of the first antibody, wherein the VHH is linked to the N-terminus and / or C-terminus of the HC of the second antibody via a linker; Alternatively, i) the first polypeptide chains each independently comprise a heavy chain (HC) of a first antibody, and ii) the second polypeptide chains each independently comprise a VHH of a second antibody and a light chain (LC) of the first antibody, wherein the VHH is linked to the N-terminus and / or C-terminus of the LC of the first antibody directly or via a linker.

[0036] In some specific embodiments, the anti-FAP / CD40 bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0037] In some embodiments, mutations are introduced into the Fc region of the FAP / CD40 binding molecule or anti-FAP / CD40 bispecific antibody. The mutations are, for example, mutations that eliminate or reduce IgG Fc effector functions, such as N297A or D265A / N297A in IgG1, 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, These include, but are not limited to, V234A / G237A in IgG2, 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.

[0038] As used herein, in the context of mutations contained in the Fc region, " / " denotes "and", for example, "D265A / N297A" denotes "D265A and N297A", ​​i.e., the Fc contains the D265A and N297A mutations, and the amino acid positions of the mutations are numbered according to the EU numbering system.

[0039] In some embodiments, the FAP / CD40 binding molecule or anti-FAP / CD40 bispecific antibody has one or more of the following characteristics: (a)≦10 -7 K D binding to human FAP or an epitope thereof at a value (b)≦10 -7 K Dbinding to human CD40 or an epitope thereof at a value (c) inducing immune stimulation of CD40-expressing antigen-presenting cells (APCs); (d) increasing APC (e.g., dendritic cell) activation and / or promoting APC (e.g., dendritic cell) proliferation; (e) stimulating tumor-specific T cell responses; (f) causing or promoting apoptosis of tumor cells, and / or (g) inhibiting tumor growth and / or metastasis.

[0040] In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 0.01 mg / mL to 500 mg / mL, for example, 0.05 mg / mL to 450 mg / mL, 0.05 mg / mL to 400 mg / mL, 0.05 mg / mL to 350 mg / mL, 0.05 mg / mL to 300 mg / mL, 0.05 mg / mL to 250 mg / mL, 0.05 mg / mL to 200 mg / mL, 0.05 mg / mL to 150 mg / mL, 0.05 mg / mL to 140 mg / mL, 0.05 mg / mL to 130 mg / mL, 0.05 mg / mL to 120 mg / mL, 0.05 mg / mL to 110 mg / mL, 0.05 mg / mL to 100 mg / mL, 0.1 mg / mL to 400 mg / mL, 0.1 mg / mL to 350 mg / mL, 0.1 mg / mL to 300 mg / mL, 0.1 mg / mL to 250 mg / mL, 0.1 mg / mL to 200 mg / mL, 0.1 mg / mL to 150 mg / mL, 0.1 mg / mL to 140 mg / mL, 0.1 mg / mL to 130 mg / mL, 0.1 mg / mL to 120 mg / mL, 0.1 mg / mL to 110 mg / mL, 0.1 mg / mL to 100 mg / mL, 0.5 mg / mL to 350 mg / mL, 0.5 mg / mL to 300 mg / mL, 0.5 mg / mL to 250 mg / mL, 0.5 mg / mL to 200 mg / mL, 0.5 mg / mL to 150 mg / mL, 0.5 mg / mL to 140 mg / mL, 0.5 mg / mL to 130 mg / mL, 0.5 mg / mL to 120 mg / mL, 0.5 mg / mL to 110 mg / mL, 0.5 mg / mL to 100 mg / mL, 1 mg / mL to 300 mg / mL, 1 mg / mL to 250 mg / mL, 1 mg / mL to 200 mg / mL, 1 mg / mL to 150 mg / mL, 1 mg / mL to 140 mg / mL, 1 mg / mL to 130 mg / mL, 1 mg / mL to 120 mg / mL, 1 mg / mL to 110 mg / mL, 1 mg / mL to 100 mg / mL, 1 mg / mL to 95 mg / mL, 1 mg / mL to 90 mg / mL, 1 mg / mL to 85 mg / mL, 1 mg / mL to 80 mg / mL, 1 mg / mL to 75 mg / mL, 1 mg / mL to 70 mg / mL, 1 mg / mL to 65 mg / mL, 1 mg / mL to 60 mg / mL, 1 mg / mL to 55 mg / mL, 1 mg / mL to 50 mg / mL, 1 mg / mL to 45 mg / mL, 1 mg / mL to 40 mg / mL,1 mg / mL to 35 mg / mL, 1 mg / mL to 30 mg / mL, 1 mg / mL to 25 mg / mL, 1 mg / mL to 20 mg / mL, 1 mg / mL to 15 mg / mL, 5 mg / mL to 90 mg / mL, 5 mg / mL to 85 mg / mL, 5 mg / mL to 80 mg / mL, 5 mg / mL to 75 mg / mL, 5 mg / mL to 70 mg / mL, 5 mg / mL to 65 mg / mL, 5 mg / mL to 60 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 50 mg / mL, 5 mg / mL to 45 mg / mL, 5 mg / mL to 40 mg / mL, 5 mg / mL to 35 mg / mL, 5 mg / mL to 30 mg / mL, 5 mg / mL to 25 mg / mL, 5 mg / mL to 20 mg / mL, 5 mg / mL to 15 mg / mL, 8 mg / mL to 12 mg / mL, 30 mg / mL to 250 mg / mL, 30 mg / mL to 200 mg / mL, 30 mg / mL to 190 mg / mL, 30 mg / mL to 180 mg / mL, 30 mg / mL to 170 mg / mL, 30 mg / mL to 160 mg / mL, 30 mg / mL to 150 mg / mL, 30 mg / mL to 140 mg / mL, 30 mg / mL to 130 mg / mL, 30 mg / mL to 120 mg / mL, 30 mg / mL to 110 mg / mL, 30 mg / mL to 100 mg / mL, 50 mg / mL to 200 mg / mL, 50 mg / mL to 190 mg / mL, 50 mg / mL to 180 mg / mL, 50 mg / mL to 170 mg / mL, 50 mg / mL to 160 mg / mL, 50 mg / mL to 150 mg / mL, 50 mg / mL to 140 mg / mL, 50 mg / mL to 130 mg / mL, 50 mg / mL to 120 mg / mL, 50 mg / mL to 110 mg / mL, 50 mg / mL to 100 mg / mL, 70 mg / mL to 180 mg / mL, 70 mg / mL to 170 mg / mL, 70 mg / mL to 160 mg / mL, 70 mg / mL to 150 mg / mL, 70 mg / mL to 140 mg / mL, 70 mg / mL to 130 mg / mL, 70 mg / mL to 120 mg / mL, 70 mg / mL to 110 mg / mL, 70 mg / mL to 100 mg / mL, 90 mg / mL to 110 mg / mL, 95 mg / mL to 105 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 0.1 mg / mL to 400 mg / mL. In some embodiments,The concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 0.5 mg / mL to 200 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 1 mg / mL to 150 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 5 mg / mL to 45 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 50 mg / mL to 110 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is at least 500 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is at least 450 mg / mL, at least 400 mg / mL, at least 350 mg / mL, at least 300 mg / mL, at least 250 mg / mL, at least 200 mg / mL, at least 150 mg / mL, at least 100 mg / mL, at least 50 mg / mL, at least 10 mg / mL, at least 1 mg / mL, at least 0.1 mg / mL, or at least 0.01 mg / mL.

[0041] In some embodiments, the concentration of the buffer in the pharmaceutical composition is 0.1 mM to 50 mM, e.g., 0.1 mM to 45 mM, 0.1 mM to 40 mM, 0.1 mM to 35 mM, 0.1 mM to 30 mM, 0.1 mM to 25 mM, 0.1 mM to 20 mM, 0.1 mM to 15 mM, 0.1 mM to 10 mM, 0.5 mM to 45 mM, 0.5 mM to 40 mM, 0.5 mM to 35 mM, 0.5 mM to 30 mM, 0.5 mM to 25 mM, 0.5 mM In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is about 0.5 mM to 40 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is about 1 mM to 30 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is about 5 mM to 20 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is about 10 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is at least 50 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is at least 10 mM.

[0042] In some embodiments, the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is one or more selected from polysorbate and poloxamer. In some embodiments, the polysorbate is one or more selected from polysorbate 20 and polysorbate 80. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the surfactant is a polysorbate. In some embodiments, the surfactant is polysorbate 80.

[0043] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is from 0.01 mg / mL to 10 mg / mL, e.g., from 0.01 mg / mL to 9 mg / mL, from 0.01 mg / mL to 8 mg / mL, from 0.01 mg / mL to 7 mg / mL, from 0.01 mg / mL to 6 mg / mL, from 0.01 mg / mL to 5 mg / mL, from 0.01 mg / mL to 4 mg / mL, from 0.01 mg / mL to 3 mg / mL, from 0.01 mg / mL to 2 mg / mL, from 0.01 mg / mL to 1 mg / mL, from 0.01 mg / mL to 0.5 mg / mL, or from 0.05 mg / mL. ~8mg / mL, 0.05mg / mL~7mg / mL, 0.05mg / mL~6mg / mL, 0.05mg / mL~5mg / mL, 0.05mg / mL~4mg / mL, 0.05mg / mL~3mg / mL, 0.05mg / mL~2mg / mL, 0.05mg / m L~1mg / mL, 0.05mg / mL~0.5mg / mL, 0.1mg / mL~6mg / mL, 0.1mg / mL~5mg / mL, 0.1mg / mL~4mg / mL, 0.1mg / mL~3mg / mL, 0.1mg / mL~2mg / mL, 0.1mg / mL~1 mg / mL, 0.1mg / mL~0.5mg / mL, 0.2mg / mL~5mg / mL, 0.2mg / mL~4.5mg / mL, 0.2mg / mL~4mg / mL, 0.2mg / mL~3.5mg / mL, 0.2mg / mL~3mg / mL, 0.2mg / mL~ 2.5mg / mL, 0.2mg / mL~2mg / mL, 0.2mg / mL~1.5mg / mL, 0.2mg / mL~1mg / mL, 0.2mg / mL~0.5mg / mL, 0.3mg / mL~4.5mg / mL, 0.3mg / mL~4mg / mL, 0.3mg / m In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.05 mg / mL to 5 mg / mL.In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.1 mg / mL to 3 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.2 mg / mL to 2 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, or about 2 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.2 mg / mL, about 0.4 mg / mL, about 0.6 mg / mL, or about 0.8 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.4 mg / mL, about 0.6 mg / mL, or about 0.8 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.4 mg / mL or about 0.6 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.4 mg / mL or about 0.8 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is at least 10 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is at least 0.4 mg / mL, at least 0.6 mg / mL, or at least 0.8 mg / mL.

[0044] In some embodiments, the pharmaceutical composition further comprises a sugar. In some embodiments, the sugar is one or more selected from sucrose, glucose, trehalose, and maltose. In some embodiments, the sugar is sucrose.

[0045] In some embodiments, the concentration of the sugar in the pharmaceutical composition is 1 mg / mL to 200 mg / mL, for example, 1 mg / mL to 150 mg / mL, 1 mg / mL to 140 mg / mL, 1 mg / mL to 130 mg / mL, 1 mg / mL to 120 mg / mL, 1 mg / mL to 110 mg / mL, 1 mg / mL to 100 mg / mL, 1 mg / mL to 95 mg / mL, 1 mg / mL to 90 mg / mL, 1 mg / mL to 85 mg / mL, 1 mg / mL to 80 mg / mL, 10 mg / mL to 150 mg / mL, mg / mL, 10mg / mL~140mg / mL, 10mg / mL~130mg / mL, 10mg / mL~120mg / mL, 10mg / mL~110mg / mL, 10mg / mL~100mg / mL, 10mg / mL~95mg / mL, 1 0mg / mL~90mg / mL, 10mg / mL~85mg / mL, 10mg / mL~80mg / mL, 20mg / mL~130mg / mL, 20mg / mL~120mg / mL, 20mg / mL~110mg / mL, 20mg / mL~10 0mg / mL, 20mg / mL~95mg / mL, 20mg / mL~90mg / mL, 20mg / mL~85mg / mL, 20mg / mL~80mg / mL, 30mg / mL~120mg / mL, 30mg / mL~110mg / mL, 30m g / mL~100mg / mL, 30mg / mL~95mg / mL, 30mg / mL~90mg / mL, 30mg / mL~85mg / mL, 30mg / mL~80mg / mL, 50mg / mL~110mg / mL, 50mg / mL~100mg In some embodiments, the concentration of the sugar in the pharmaceutical composition is 10 mg / mL to 150 mg / mL. In some embodiments, the concentration of the sugar in the pharmaceutical composition is 30 mg / mL to 120 mg / mL.In some embodiments, the concentration of the sugar in the pharmaceutical composition is 50 mg / mL to 100 mg / mL. In some embodiments, the concentration of the sugar in the pharmaceutical composition is about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, or about 120 mg / mL. In some embodiments, the concentration of the sugar in the pharmaceutical composition is about 80 mg / mL. In some embodiments, the concentration of the sugar in the pharmaceutical composition is at least 200 mg / mL. In some embodiments, the concentration of the sugar in the pharmaceutical composition is at least 80 mg / mL.

[0046] In some embodiments, the pharmaceutical composition optionally further comprises one or more other additives selected from a polyol and a metal chelator. In some embodiments, the polyol is one or more selected from glycerol, mannitol, and sorbitol. In some embodiments, the metal chelator is selected from ethylenediaminetetraacetic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the polyol is mannitol. In some embodiments, the metal chelator is ethylenediaminetetraacetic acid.

[0047] In some embodiments, the concentration of the other additives in the pharmaceutical composition is 0.001% w / v to 20% w / v, for example, 0.005% w / v to 20% w / v, 0.005% w / v to 15% w / v, 0.005% w / v to 10% w / v, 0.005% w / v to 9% w / v, 0.005% w / v to 8% w / v, 0.005% w / v to 7% w / v, 0.005% w / v to 6% w / v, 0.005% w / v to 5% w / v, 0.005% w / v to 4% w / v, 0.005% w / v to 3% w / v, 0.005% w / v to 2% w / v, 0.005% w / v to 1% w / v, 0.005% w / v to 0.1% w / v, 0.005% w / v to 0.05% w / v, 0.008% w / v to 15% w / v, 0.008% w / v to 10% w / v, 0.008% w / v to 9% w / v, 0.008% w / v to 8% w / v, 0.008% w / v to 7% w / v, 0.008% w / v to 6% w / v, 0.008% w / v to 5% w / v, 0.008% w / v to 4% w / v, 0.008% w / v to 3% w / v, 0.008% w / v to 2% w / v, 0.008% w / v to 1% w / v, 0.008% w / v to 0.1% w / v, 0.008% w / v to 0.05% w / v, 0.01% w / v to 10% w / v, 0.01% w / v to 9% w / v, 0.01% w / v to 8% w / v, 0.01% w / v to 7% w / v, 0.01% w / v to 6% w / v, 0.01% w / v to 5% w / v, 0.01% w / v to 4% w / v, 0.01% w / v to 3% w / v, 0.01% w / v to 2% w / v, 0.01% w / v to 1% w / v, 0.01% w / v to 0.1% w / v, 0.01% w / v to 0.05% w / v, 0.1% w / v to 15% w / v, 0.1% w / v to 10% w / v, 0.1% w / v to 10% w / v, 0.1% w / v to 9% w / v, 0.1% w / v to 8% w / v, 0.1% w / v to 7% w / v, 0.1% w / v to 6% w / v, 0.1% w / v to 5% w / v, 1% w / v to 15% w / v, 1% w / v to 10% w / v, 1% w / v to 9% w / v, 1% w / v to 8% w / v, 1% w / v to 7% w / v, 1% w / v to 6% w / v, 1% w / v to 5% w / v, 0.001% w / v to 5% w / v, 0.001% w / v to 4% w / v, 0.001% w / v to 3% w / v, 0.001% w / v to 2% w / v, 0.001% w / v to 1% w / v, 0.001% w / v to 0.1% w / v or 0.001% w / v to 0.In some embodiments, the concentration of the other additives in the pharmaceutical composition is 0.005% w / v to 15% w / v. In some embodiments, the concentration of the other additives in the pharmaceutical composition is 0.008% w / v to 10% w / v. In some embodiments, the concentration of the other additives in the pharmaceutical composition is 0.01% w / v to 8% w / v.

[0048] In some embodiments, the concentration of mannitol in the pharmaceutical composition is 0.1% w / v to 15% w / v, e.g., 0.5% w / v to 12% w / v, 0.5% w / v to 11% w / v, 0.5% w / v to 10% w / v, 0.5% w / v to 9% w / v, 0.5% w / v to 8% w / v, 0.5% w / v to 7% w / v, 0.5% w / v to 6% w / v, 0.5% w / v to 5% w / v, 1% w / v to 10% w / v, 1% w / v to 9% w / v, 1% w / v to 8% w / v, 1% w / v to 7% w / v, 1% w / v to 6% w / v, or 1% w / v to 5% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is 0.5% w / v to 12% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is 1% w / v to 10% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, or about 10% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is about 4% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is at least 15% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is at least 4% w / v.

[0049] In some embodiments, the concentration of ethylenediaminetetraacetic acid in the pharmaceutical composition is 0.001% w / v to 5% w / v, e.g., 0.001% w / v to 4% w / v, 0.001% w / v to 3% w / v, 0.001% w / v to 2% w / v, 0.001% w / v to 1% w / v, 0.001% w / v to 0.1% w / v, 0.001% w / v to 0.05% w / v, 0.005% w / v to 4% w / v, 0.005% w / v to 3% w / v, 0.005% w / v to 2% w / v, 0.005% w / v to 1% w / v, 0.005% w / v to 0.1% w / v, or 0.005% w / v to 0.05% w / v. In some embodiments, the concentration of ethylenediaminetetraacetic acid in the pharmaceutical composition is 0.001% w / v to 1% w / v. In some embodiments, the concentration of ethylenediaminetetraacetic acid in the pharmaceutical composition is 0.005% w / v to 0.1% w / v. In some embodiments, the concentration of ethylenediaminetetraacetic acid in the pharmaceutical composition is about 0.005% w / v, about 0.006% w / v, about 0.007% w / v, about 0.008% w / v, about 0.009% w / v, about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, or about 0.1% w / v. In some embodiments, the concentration of ethylenediaminetetraacetic acid in the pharmaceutical composition is about 0.01% w / v. In some embodiments, the concentration of ethylenediaminetetraacetic acid in the pharmaceutical composition is at least 5% w / v. In some embodiments, the concentration of ethylenediaminetetraacetic acid in the pharmaceutical composition is at least 0.01% w / v.

[0050] In some embodiments, the pH of the buffer in the pharmaceutical composition is 3.5 to 9, for example, 3.5 to 8.5, 3.5 to 8, 3.5 to 7.5, 3.5 to 7, 3.5 to 6.9, 3.5 to 6.8, 3.5 to 6.7, 3.5 to 6.6, 3.5 to 6.5, 3.5 to 6.4, 3.5 to 6.3, 3.5 to 6.2, 3.5 to 6.1, 3.5 to 6, 3.5 to 5.9, 3.5 to 5.8, 3.5 to 5.7, 3.5 to 5.6, 3.5 to 5.5, 4 to 5.4, 4 to 5.3, 4 to 5.2, 4 to 5.1, 4 to 5.0, 4 to 8.5, 4 to 8, 4 to 7.5, 4 to 7, 4 to 6.9, 4 to 6.8, 4 to 6.7, 4~6.6, 4~6.5, 4~6.4, 4~6.3, 4~6.2, 4~6.1, 4~6, 4~5.9, 4~5.8, 4~5.7, 4~5.6, 4~5.5, 4~5.4, 4~5.3, 4~5.2, 4~5.1, 4~5.0, 4.2~8, 4.2~7.5, 4.2~7, 4 .2~6.9, 4.2~6.8, 4.2~6.7, 4.2~6.6, 4.2~6.5, 4.2~6.4, 4.2~6.3, 4.2~6.2, 4.2~6.1, 4.2~6, 4.2~5.9, 4.2~5.8, 4.2~5.7, 4.2~5.6, 4.2~5.5, 4.2~5.4, 4.2~5.3, 4.2~5.2, 4.2~5.1, 4.2~5.0, 4.5~8, 4.5~7.5, 4.5~7, 4.5~6.9, 4.5~6.8, 4.5~6.7, 4.5~6.6, 4.5~6.5, 4.5~6.4, 4.5~6.3, 4.5~6.2, 4.5~6.1, 4 .5~6, 4.5~5.9, 4.5~5.8, 4.5~5.7, 4.5~5.6, 4.5~5.5, 4.5~5.4, 4.5~5.3, 4.5~5.2, 4.5~5.1, 4.5~5.0, 4.6~7.5, 4.6~7, 4.6~6.9, 4.6~6.8, 4.6~6.7, 4. 6~6.6, 4.6~6.5, 4.6~6.4, 4.6~6.3, 4.6~6.2, 4.6~6.1, 4.6~6, 4.6~5.9, 4.6~5.8, 4.6~5.7, 4.6~5.6, 4.6~5.5, 4.6~5.4, 4.6~5.3, 4.6~5.2, 4.6~5.1, 4 .6~5.0, 4.8~7, 4.8~6.9, 4.8~6.8, 4.8~6.7, 4.8~6.6, 4.8~6.5, 4.8~6.4, 4.8~6.3, 4.8~6.2, 4.8~6.1, 4.8~6, 4.8~5.9, 4.8~5.8, 4.8~5.7, 4.8~5.6, 4.In some embodiments, the pH of the buffer in the pharmaceutical composition is 8 to 5.5, 5 to 6.5, 5 to 6.4, 5 to 6.3, 5 to 6.2, 5 to 6.1, 5 to 6, 5 to 5.9, 5 to 5.8, 5 to 5.7, 5 to 5.6, or 5 to 5.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is 3.5 to 7. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.0 to 6.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.2 to 6.2. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.5 to 6.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.0, about 5.2, about 5.3, about 5.5, about 5.6, about 5.8, or about 6.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.0, about 5.2, about 5.3, or about 5.5.

[0051] In some embodiments, the pharmaceutical composition further comprises a pH modulator, such as sodium hydroxide and / or hydrochloric acid.

[0052] In some embodiments, the pH of the pharmaceutical composition differs by no more than ±0.5 compared to the pH of the buffer contained therein. In some embodiments, the pH of the pharmaceutical composition is 3.5 to 9, for example, 3.5 to 8.5, 3.5 to 8, 3.5 to 7.5, 3.5 to 7, 3.5 to 6.9, 3.5 to 6.8, 3.5 to 6.7, 3.5 to 6.6, 3.5 to 6.5, 3.5 to 6.4, 3.5 to 6.3, 3.5 to 6.2, 3.5 to 6.1, 3.5 to 6, 3.5 to 5.9, 3.5 to 5.8, 3.5 to 5.7, 3.5 to 5.6, 3.5 to 5.5, 4 to 5.4, 4 to 5.3, 4 to 5.2, 4 to 5.1, 4 to 5.0, 4 to 8.5, 4 to 8, 4 to 7.5, 4 to 7, or 4 to 6.9. , 4~6.8, 4~6.7, 4~6.6, 4~6.5, 4~6.4, 4~6.3, 4~6.2, 4~6.1, 4~6, 4~5.9, 4~5.8, 4~5.7, 4~5.6, 4~5.5, 4~5.4, 4~5.3, 4~5.2, 4~5.1, 4~5.0, 4.2~8, 4.2~7.5, 4.2~7, 4.2~6.9, 4.2~6.8, 4.2~6.7, 4.2~6.6, 4.2~6.5, 4.2~6.4, 4.2~6.3, 4.2~6.2, 4.2~6.1, 4.2~6, 4.2~5.9, 4.2~5.8, 4.2~5.7, 4.2~5.6, 4.2~5.5, 4.2~5.4, 4.2~5.3, 4.2~5.2, 4.2~5.1, 4.2~5.0, 4.5~8, 4.5~7.5, 4.5~7, 4.5~6.9, 4.5~6.8, 4.5~6.7, 4.5~6.6, 4.5~6.5, 4.5~6.4, 4.5~6.3, 4.5~6.2, 4.5~6.1, 4.5~6, 4.5~5.9, 4.5~5.8, 4.5~5.7, 4.5~5.6, 4.5~5.5, 4.5~5.4, 4.5~5.3, 4.5~5.2, 4.5~5.1, 4.5~5.0, 4 .6~7.5, 4.6~7, 4.6~6.9, 4.6~6.8, 4.6~6.7, 4.6~6.6, 4.6~6.5, 4.6~6.4, 4.6~6.3, 4.6~6.2, 4.6~6.1, 4.6~6, 4.6~5.9, 4.6~5.8, 4.6~5.7, 4.6~5.6, 4.6~5.5, 4.6~5.4, 4.6~5.3, 4.6~5.2, 4.6~5.1, 4.6~5.0, 4.8~7, 4.8~6.9, 4.8~6.8, 4.8~6.7, 4.8~6.6, 4.8~6.5, 4.8~6.4, 4.8~6.3, 4.In some embodiments, the pH of the pharmaceutical composition is 8 to 6.2, 4.8 to 6.1, 4.8 to 6, 4.8 to 5.9, 4.8 to 5.8, 4.8 to 5.7, 4.8 to 5.6, 4.8 to 5.5, 5 to 6.5, 5 to 6.4, 5 to 6.3, 5 to 6.2, 5 to 6.1, 5 to 6, 5 to 5.9, 5 to 5.8, 5 to 5.7, 5 to 5.6, or 5 to 5.5. In some embodiments, the pH of the pharmaceutical composition is 3.5 to 7. In some embodiments, the pH of the pharmaceutical composition is 4.0 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 4.2 to 6.2. In some embodiments, the pH of the pharmaceutical composition is 4.5 to 6.0. In some embodiments, the pH of the pharmaceutical composition is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH of the pharmaceutical composition is about 5.0, about 5.2, about 5.3, about 5.5, about 5.6, about 5.8, or about 6.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.0, about 5.2, about 5.3, or about 5.5.

[0053] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule (e.g., Ab10-A297V3-4, whose heavy and light chain sequences are SEQ ID NOs: 22 and 20, respectively), comprising any one of the following combinations 1) to 4): 1) FAP / CD40 binding molecule, histidine salt buffers, such as histidine hydrochloride buffers or histidine acetate buffers, histidine-histidine hydrochloride buffers or histidine-acetate buffers; 2) FAP / CD40 binding molecule, histidine salt buffer, Polysorbates, such as polysorbate 80, sucrose, Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid; 3) FAP / CD40 binding molecule, histidine hydrochloride buffers, such as histidine-histidine hydrochloride buffers; Polysorbates, such as polysorbate 80, sucrose, Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid; 4) FAP / CD40 binding molecule, histidine acetate buffers, e.g., histidine-acetate buffers; Polysorbates, such as polysorbate 80, sucrose, Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid.

[0054] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule selected from any one of the following combinations 1) to 4) (e.g., Ab10-A297V3-4, whose heavy and light chain sequences are SEQ ID NOs: 22 and 20, respectively): 1) FAP / CD40 binding molecule, histidine salt buffer, polysorbate, sucrose, and water for injection.

[0055] 2) FAP / CD40 binding molecule, histidine hydrochloride buffer, polysorbate, sucrose, and water for injection.

[0056] 3) FAP / CD40 binding molecule, histidine-histidine hydrochloride buffer, polysorbate, sucrose, and water for injection.

[0057] 4) FAP / CD40 binding molecule, histidine-acetate buffer, polysorbate, sucrose, and water for injection.

[0058] 5) The pharmaceutical composition according to any one of the above 1) to 4), wherein the polysorbate is polysorbate 80.

[0059] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule comprising any one of the following combinations 1) to 6) (e.g., Ab10-A297V3-4, whose heavy and light chain sequences are SEQ ID NOs: 22 and 20, respectively): 1) 0.01 mg / mL to 500 mg / mL of the FAP / CD40 binding molecule; 0.1 mM to 50 mM histidine hydrochloride buffer, for example, histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.01 mg / mL to 10 mg / mL of polysorbate, 1 mg / mL to 200 mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 0.1% w / v to 15% w / v of mannitol or 0.001% w / v to 5% w / v of ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 3.5 to 7. 2) 0.1 mg / mL to 400 mg / mL of the FAP / CD40 binding molecule; 0.5 mM to 40 mM histidine hydrochloride buffer, for example, histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.05 mg / mL to 5 mg / mL of polysorbate, 10 mg / mL to 150 mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 0.5% w / v to 12% w / v of mannitol or 0.001% w / v to 1% w / v of ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 4 to 6.5. 3) 0.5 mg / mL to 200 mg / mL of FAP / CD40 binding molecule; 1 mM to 30 mM histidine hydrochloride buffer, for example, histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.1 mg / mL to 3 mg / mL polysorbate, 30 mg / mL to 120 mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 1% w / v to 10% w / v mannitol or 0.005% w / v to 0.1% w / v ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 4.2 to 6.2; 4) 1 mg / mL to 150 mg / mL of FAP / CD40 binding molecule; 5 mM to 20 mM histidine hydrochloride buffer, for example, histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.2mg / mL to 2mg / mL polysorbate, 50mg / mL to 100mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 1% w / v to 10% w / v mannitol or 0.005% w / v to 0.1% w / v ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 4.5 to 6. 5) 1 mg / mL to 40 mg / mL of FAP / CD40 binding molecule; 5 mM to 20 mM histidine hydrochloride buffer, for example, histidine hydrochloride buffer; 0.2mg / mL to 2mg / mL polysorbate, 50mg / mL to 100mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 1% w / v to 10% w / v mannitol or 0.005% w / v to 0.1% w / v ethylenediaminetetraacetic acid; The pH of the pharmaceutical composition is 4.5 to 6.

[0060] 6) 50 mg / mL-150 mg / mL of FAP / CD40 binding molecule; 5 mM to 20 mM histidine acetate buffer, for example, histidine-acetate buffer; 0.2mg / mL to 2mg / mL polysorbate, 50mg / mL to 100mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 1% w / v to 10% w / v mannitol or 0.005% w / v to 0.1% w / v ethylenediaminetetraacetic acid; The pH of the pharmaceutical composition is 4.5 to 6.

[0061] 7) The pharmaceutical composition according to any one of the above 1) to 6) further comprises water for injection.

[0062] 8) The pharmaceutical composition according to any one of the above 1) to 6), wherein the polysorbate is polysorbate 80.

[0063] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one of the following combinations 1) to 25), or a FAP / CD40 binding molecule that is any one of the following combinations 1) to 25) (e.g., Ab10-A297V3-4, whose heavy and light chain sequences are SEQ ID NOs: 22 and 20, respectively): 1) 1 mg / mL to 150 mg / mL of FAP / CD40 binding molecule; about 10 mM histidine hydrochloride buffer or histidine acetate buffer, for example, histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.2mg / mL to 2mg / mL polysorbate 80, approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is 4.5 to 6. 2) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 3) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 4) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 5) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 6) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 7) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 8) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, for example, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 9) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule, for example, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 10) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 11) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 12) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 13) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.4 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 14) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 15) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 16) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 17) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.6 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 18) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.8 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 19) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.8 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 20) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.8 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 21) about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL of a FAP / CD40 binding molecule; approximately 10 mM histidine-acetate buffer, approximately 0.8 mg / mL polysorbate 80; approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 22) 1 mg / mL to 45 mg / mL of a FAP / CD40 binding molecule, for example, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, e.g., histidine-histidine hydrochloride buffer; 0.2mg / mL to 2mg / mL polysorbate 80, approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is 4.5 to 6. 23) 50 mg / mL to 120 mg / mL of a FAP / CD40 binding molecule, for example, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL; about 10 mM histidine acetate buffer, e.g., histidine-acetate buffer; 0.2mg / mL to 2mg / mL polysorbate 80, approximately 80 mg / mL sucrose, and the pH of the pharmaceutical composition is 4.5 to 6. 24) The pharmaceutical composition according to any one of 1) to 23), further comprising 4% w / v mannitol or 0.01% w / v ethylenediaminetetraacetic acid; 25) When a fixed volume is required, the pharmaceutical composition according to any one of 1) to 24) is adjusted to 1 mL using water for injection, and the final volume is adjusted to 1 mL.

[0064] In another aspect, the present disclosure provides a pharmaceutical composition comprising a low concentration of a FAP / CD40 binding molecule (e.g., 1 mg / mL to 45 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL), obtained by diluting a pharmaceutical composition comprising a high concentration of a FAP / CD40 binding molecule (e.g., 50 mg / mL to 120 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL) with a diluent, or a method for preparing the same. In some embodiments, the diluent includes, but is not limited to, water, saline, or glucose solution.

[0065] The pharmaceutical composition of the present disclosure already has sufficient drug stability and can be left stably for a long period of time.

[0066] In some embodiments, the pharmaceutical composition is stable for at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 36 months at 2° C. to 8° C. In some embodiments, the pharmaceutical composition is stable for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months at 25° C. In some embodiments, the pharmaceutical composition is stable for at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months at 40° C.

[0067] The present disclosure provides a method of preparing the pharmaceutical composition, comprising the step of dissolving the FAP / CD40 binding molecule.

[0068] For ease of drug delivery, the pharmaceutical compositions of the present disclosure can also be prepared as lyophilized formulations.

[0069] In some embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water, saline, or a glucose solution.

[0070] The present disclosure further provides a lyophilized formulation, which, after being reconstituted, can form any one of the pharmaceutical compositions described above.

[0071] The present disclosure further provides a lyophilized formulation obtained by lyophilizing any one of the pharmaceutical compositions described above.

[0072] The present disclosure further provides a reconstituted solution prepared by reconstituting the lyophilized formulation. In some embodiments, the reconstituted solution is selected from, but not limited to, water for injection, saline, or glucose solution.

[0073] The present disclosure further provides a product comprising a container containing the pharmaceutical composition, the lyophilized formulation, or the reconstituted solution. In some embodiments, the container is an injection vial made of neutral borosilicate glass tubing. In some embodiments, the product includes a pharmaceutical package insert.

[0074] The present disclosure further provides a pharmaceutical composition or a lyophilized formulation or a reconstituted solution of a lyophilized formulation that is a medicament for treating or alleviating a disease or condition.

[0075] Methods for treating diseases and pharmaceutical uses In some embodiments, the present disclosure provides a method for preventing and / or treating cancer or tumors, comprising administering to a patient or subject a prophylactically and / or therapeutically effective amount of the pharmaceutical composition or reconstituted solution of the lyophilized formulation, thereby inhibiting tumor cell growth in the patient or subject. In some specific embodiments, the cancer is preferably, but not limited to, a cancer that is responsive to immunotherapy.

[0076] In the above-described methods or uses, non-limiting examples of cancers or tumors include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, endometrial cancer, and osteosarcoma. Other examples of cancers treatable by the methods of the present disclosure include bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic ... Cancers include leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal column tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, and combinations of the above cancers. In some embodiments, the tumor or cancer is metastatic and / or advanced.

[0077] The present disclosure also provides a method for preventing and / or treating an infectious disease in a subject or patient by administering the pharmaceutical composition, lyophilized formulation, or reconstituted solution of the lyophilized formulation to the subject or patient. As with the use in treating cancer or tumors described above, the pharmaceutical composition can be used alone or in combination with a vaccine to stimulate an immune response against pathogens, toxins, and autoantigens. Examples of pathogens for which this treatment method can be used include those for which there is currently no effective vaccine or for which conventional vaccines are insufficiently effective. These include, but are not limited to, HIV, hepatitis viruses (types A, B, and C), influenza virus, herpes virus, giardia, malaria, leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0078] Pharmaceutical compositions containing the FAP / CD40 binding molecules of the present disclosure can be used to treat patients in need of such treatment by parenteral administration, which can be by subcutaneous, intramuscular, or intravenous injection. [Brief explanation of the drawings]

[0079] [Figure 1] Binding of anti-FAP antibodies Ab9, Ab10, Ab14, and Ab15 to cell surface human FAP, using 28H1 as a positive control and IgG1 isotype as a negative control. [Figure 2] Binding of anti-FAP antibodies Ab9, Ab10, Ab14, and Ab15 to cell surface mouse FAP, using 28H1 as a positive control and IgG1 isotype as a negative control. [Figure 3] Binding of anti-CD40 antibodies A12 and A297 to human CD40 on Raji cells, with 9E5-SELFNS as a positive control and IgG1 as a negative control. [Figure 4] Binding of anti-CD40 antibodies A12 and A297 to human CD40 on the surface of HEK293 cells. 9E5-SELFNS was used as a positive control, and IgG1 was used as a negative control. The vertical axis represents the percentage of agonist activity relative to 200 nM 9E5-SELFNS. [Figure 5] This shows the affinity flow detection results of humanized anti-CD40 antibodies A297_V1, A297_V2, A297_V3, and A297_V4 to HEK293 cells overexpressing human CD40, using 9E5-SELFNS as a positive control and hIgG as a negative control. [Figure 6] This shows the activation effect of humanized anti-CD40 antibodies A297_V1, A297_V2, A297_V3, and A297_V4 on HEK-Blue™ CD40L cells via FcγRIIb, using 9E5-SELFNS as a positive control and hIgG as a negative control. [Figure 7] FIG. 1 is a schematic diagram of the structure of an anti-FAP / CD40 bispecific antibody. [Figure 8A] This shows the results of affinity flow detection of anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 to a CHOK1 / FAP stable transformant that highly expresses the human FAP antigen. [Figure 8B] These are the results of affinity flow detection of the above anti-FAP / CD40 bispecific antibodies to CHOK1 / FAP stable transformants highly expressing mouse FAP antigen and cynomolgus monkey FAP antigen, respectively. [Figure 8C] These are the results of affinity flow detection of the above anti-FAP / CD40 bispecific antibodies to CHOK1 / FAP stable transformants highly expressing mouse FAP antigen and cynomolgus monkey FAP antigen, respectively. [Figure 9A] This shows the results of affinity flow detection of anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 on HEK-Blue™ CD40L stable transfectants that highly express human CD40 antigen. [Figure 9B] 1 shows the results of affinity flow detection of the above anti-FAP / CD40 bispecific antibody to HEK293 cells highly expressing cynomolgus monkey CD40 antigen. [Figure 10]1 shows the results of flow detection of the affinity of anti-FAP / CD40 bispecific antibodies Ab10-A297V3-2 and Ab10-A297V3-4 to CD40 on human immature DCs. [Figure 11] The effect of a concentration gradient of anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 on promoting DC maturation in the absence of crosslinking of CHOK1 / FAP stable transfectants, with LPC, hIgG1, and carrier used as controls. [Figure 12] The effect of a concentration gradient of anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 on promoting DC maturation in crosslinked CHOK1 / FAP stable transformed cells, with LPC, hIgG1, and carrier used as controls. [Figure 13A] 1 shows mouse tumor growth curves after injection of a single dose of antibody Ab10-A297V3-2 or Ab10-A297V3-4 into mFAP-MC38 tumor-bearing hCD40 humanized mice. [Figure 13B] Activation of corresponding peripheral blood B cells in mice. [Figure 13C] The corresponding weight change curves of the mice. [Figure 13D] Corresponding platelet count changes in mice. [Figure 13E] Corresponding effects on liver function in mice. [Figure 14A] 1 shows mouse tumor growth curves after injecting mFAP-MC38 tumor-bearing hCD40 humanized mice with multiple doses of antibody Ab10-A297V3-2 or Ab10-A297V3-4. [Figure 14B] Activation of corresponding peripheral blood B cells in mice. [Figure 14C] The corresponding weight change curves of the mice. [Figure 14D] Corresponding platelet count changes in mice. [Figure 14E]The results show the effects of IgG1 on the liver function of the corresponding mice. In Figures 13A to 14E, unless otherwise indicated, all P values ​​are calculated relative to the control hIgG1. Statistical analysis was performed using Student's t-test, with ns indicating no significant difference, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0080] 1. Terminology In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0081] 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).

[0082] "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). For sequence information, see Table 8 of the present disclosure. "CD40" as used herein 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. In one embodiment, a CD40 binding molecule of the present disclosure can specifically bind to human, mouse, and / or cynomolgus monkey CD40.

[0083] "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" of the present disclosure covers full-length, unprocessed FAPs and any cell-derived processed forms of FAPs, as well as naturally occurring variants of FAPs, such as splice variants and allelic variants. In one embodiment, a FAP-binding molecule of the present disclosure can specifically bind to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) Accession No. Q12884 (Version 149, SEQ ID NO: 2), 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. The amino acid sequence of human FAP ECD, including His-tagged, is shown in Table 2 of the present disclosure. The amino acid sequence of mouse FAP is shown in UniProt Accession No. P97321 (Version 126, SEQ ID NO: 143), or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to position 761. In some embodiments, the FAP-binding molecule of the present disclosure binds to the extracellular domain of FAP.

[0084] The term "antibody" is used in the broadest sense to refer to any antibody that exhibits the desired antigen-binding activity, and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments or antigen-binding portions). Antibodies may also refer to immunoglobulins, which have a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The antigenicity of immunoglobulins varies due to differences in the amino acid composition and sequence of the immunoglobulin heavy chain constant regions. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, and the 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 location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains and λ chains based on differences in the constant region. Each of the five types of Ig may have either κ chains 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 (CDRs) and four framework regions (FRs) with relatively conserved sequences. 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 from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, 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.

[0085] "Bispecific antibodies" covers antibodies (including antibodies or antigen-binding fragments thereof, e.g., single chain antibodies) that specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, and F(ab)4-CrossMAb (Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 1). 18, pages 585-608 (2019), see Chen S1 et al., J Immunol Res. 2019 Feb 11, 2019:4516041).

[0086] The 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 enabling accurate delineation of the CDRs and identification of the residues comprising the antibody's binding site. This can be accomplished by any one of a variety of techniques known to those skilled in the art, such as X-ray crystallography. Several 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.

[0087] 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, "AbMTM, 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 depending 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 various numbering systems is well known to those skilled in the art and is illustratively shown in Table 1 below.

[0088] [Table 1]

[0089] The CDR amino acid residues of the VL and VH regions of the antibodies of this disclosure conform in number and position to the known Kabat numbering system.

[0090] 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.

[0091] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy-chain antibody) or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain maintains the immunoglobulin fold characteristic of an antibody molecule and is characterized by being composed of a two-layer sandwich of about seven antiparallel β-strands arranged in two β-sheets, optionally stabilized by conserved disulfide bonds.

[0092] An "immunoglobulin variable domain" refers to an immunoglobulin domain that is essentially composed of four "framework regions," referred to in the art and below as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, which are spaced apart by three "complementarity-determining regions" or "CDRs," referred to in the art and below as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. The general structure or sequence of an immunoglobulin variable domain may therefore be depicted as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain contains the antigen-binding site and thus confers specificity for the antigen.

[0093] "Antibody framework (FR)" refers to the part of a variable domain that serves as a stent for the antigen binding loops (CDRs) of that variable domain.

[0094] 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) that are derived as or as a VH domain. TM ) and (single domain) antibodies (e.g., dAbs) as or derived from VL domains. 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:

[0095] "VHH domain" refers to a heavy chain single domain antibody, VHH, V H H domains, also known as VHH antibody fragments, VHH antibodies, or nanobodies, are variable domains of antigen-binding immunoglobulins called "heavy-chain antibodies" (i.e., "light-chain-depleted antibodies") (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 the variable domain from the heavy-chain variable domain (referred to as "VH domain" in this disclosure) and light-chain variable domain (referred to as "VL domain" in this disclosure) present in conventional tetrapeptide chain antibodies. 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 tetrapeptide chain antibodies, 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. HThe terms "H 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 in camelids, as well as antibodies produced in camelids and subsequently humanized, or antibodies screened by phage display technology. The total number of amino acid residues in a VHH domain is typically in the range of 110-120, and often between 112-115. However, it should be noted that both shorter and longer 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.

[0096] 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.

[0097] 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 immunogenicity and a concomitant decrease in activity, activity can be maintained by performing minimal back mutations on the fully human antibody variable region. An example of "humanization" includes a VHH domain derived from a camelid species, which can be "humanized" by replacing 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 (also referred to as "sequence optimization" in the present disclosure, which may also cover other modifications to the sequence, such as removal of potential post-translational modification sites, that confer improved properties to the VHH). A humanized VHH domain may contain one or more fully human framework region sequences. Furthermore, to avoid a decrease in immunogenicity and a concomitant decrease in activity, activity can be maintained by performing minimal back mutations or reverse mutations on the framework sequences of the human antibody variable regions.

[0098] A "fully human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). A "fully human antibody" does not include a "humanized antibody."

[0099] Typically, the CD40 binding molecules, FAP binding molecules of the present disclosure have a cytotoxicity of about 100%, preferably 100%, as measured in a Biacore or 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 a lower dissociation constant (K D ), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 It binds to the antigen to be bound (i.e., CD40 or FAP) with an association constant (KA) of M. -4 K is larger than M D Values ​​are also generally considered to represent non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any suitable known method, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays) described in this disclosure.

[0100] "Competition," when used in reference to competition between antigen-binding proteins for the same epitope (e.g., neutralizing antigen-binding protein or neutralizing antibody), refers to competition between antigen-binding proteins, as measured by the following assay: the antigen-binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., CD40 or a fragment thereof). A variety of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahl et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assays, solid-phase direct label sandwich assays (see, e.g., Harlow & Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using an I-125 marker (see, e.g., Morel et al., 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), and the like. al., 1988, Molec. Immunol. 25:7-15), solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552), and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). These assays typically involve the use of purified antigens (the antigens are on a solid surface or cell surface) capable of binding to an unlabeled detection antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antigen-binding protein to be measured. The antigen-binding protein to be measured is typically present in excess.Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antigen-binding protein so that the two epitopes spatially interfere with each other's binding. Typically, when a competing antigen-binding protein is present in excess, specific binding between the reference antigen-binding protein and a common antigen is inhibited (e.g., reduced) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0101] Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be performed to obtain antibodies that bind to an antigen by competing with or cross-competing with each other. A high-throughput method for obtaining antibodies that bind to the same epitope by cross-competition is described in International Patent Publication WO 03 / 48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies that bind to the same epitope on CD40 or FAP in competition with the antibody molecules of the present disclosure.

[0102] The term "CD40 binding molecule" refers to any protein capable of specifically binding to CD40 or any molecule comprising such a protein. A CD40 binding molecule may include an antibody or conjugate thereof as defined in the present disclosure against CD40. The term also encompasses immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. A "CD40 binding molecule" of the present disclosure may comprise at least one immunoglobulin single variable domain (e.g., VHH) that binds to CD40. In some embodiments, a "CD40 binding molecule" may comprise two, three, four, or more immunoglobulin single variable domains (e.g., VHH) that bind to CD40. In addition to binding to an immunoglobulin single variable domain comprising CD40, a CD40 binding molecule of the present disclosure may also comprise a moiety with effector function, such as a linker and / or a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, the term "CD40 binding molecule" of the present disclosure also covers bi / multispecific antibodies, which are immunoglobulins that bind to different antigens (e.g., comprising a first antibody that binds to a first antigen (e.g., CD40) and a second antibody that binds to a second antigen (e.g., FAP), optionally comprising a third antibody that binds to a third antigen, and further optionally comprising a fourth antibody that binds to a fourth antigen).

[0103] "FAP-binding molecule" refers to any protein or any molecule containing such a protein that can specifically bind to a FAP. A FAP-binding molecule may include an antibody or conjugate thereof as defined in this disclosure against a FAP.

[0104] "FAP / CD40 binding molecule" refers to any protein or molecule containing said protein that is capable of specifically binding to CD40 and a FAP. FAP / CD40 binding molecules may include antibodies or conjugates thereof as defined in this disclosure against CD40 and a FAP.

[0105] "Binding to CD40" or "CD40 binding" refers to the ability to interact with CD40 or an epitope thereof, which may be of human origin. "Binding to FAP" or "FAP binding" refers to the ability to interact with a FAP or an epitope thereof, which may be of human origin. An "antigen-binding site" of the present disclosure refers to a non-contiguous portion of an antigen in three dimensional space that is recognized by an antibody of the present disclosure.

[0106] "Antigen" refers to a molecule used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen, or to screen expression libraries (e.g., phage, yeast, or ribosome display libraries, among others). In this disclosure, antigen is defined more broadly and includes the target molecule specifically recognized by an antibody, as well as portions or mimetics of molecules used in immunization processes to produce antibodies or library screening to select antibodies. For example, with respect to the human CD40-binding antibodies of the present disclosure, both monomers and multimers (e.g., dimers, trimers, etc.) of human CD40, and truncated and other variants of human CD40, are referred to as antigens.

[0107] "Epitope" refers to the site on an antigen that binds to an immunoglobulin or antibody. An epitope may be formed from contiguous amino acids or from juxtaposed non-contiguous amino acids formed by tertiary folding of a protein. Epitopes formed by contiguous 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 which epitopes are bound by a given antibody are well known in the art and include Western blotting and immunoprecipitation detection assays. Methods for determining the spatial conformation of epitopes include techniques known in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0108] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a given antigen. For example, when human CD40 or an epitope thereof is used as an analyte and an antibody is used as a ligand, the antibody binds to an epitope on an instrument using surface plasmon resonance (SPR) technology at a binding site of about 10 -7 The equilibrium dissociation constant (K D ) and the binding affinity to the predetermined antigen or its epitope is at least twice its binding affinity to a nonspecific antigen other than the predetermined antigen (or its epitope) or a closely related antigen (e.g., BSA, etc.). The term "antibody that recognizes an antigen" may be used interchangeably with "antibody that specifically binds" in the present disclosure.

[0109] "Binding affinity" or "affinity" is used in the present 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 can be quantified by determining the dissociation constant (KD). KD can be determined, for example, by measuring the kinetics of complex formation and dissociation using surface plasmon resonance (SPR) techniques (Biacore). The rate constants corresponding to the binding and dissociation of a monovalent complex are called the binding rate constant ka (or k) and the dissociation rate constant kd (or koff), respectively. K 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 by Caceci et al. (1984, Byte 9:340-362). For example, the K can be determined 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 flow cytometry analysis, 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 can be evaluated by the KinExA system or KinExA. D By comparing K values, it is possible to compare the binding affinities associated with interactions with different molecules, for example, to compare the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by comparing the K values ​​of the interaction of interest (e.g., the specific interaction between an antibody and an antigen). D value and the K of a non-target interaction (e.g., a known control antibody that does not bind CD40). D It can be evaluated by determining and comparing values.

[0110] "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.

[0111] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide 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.

[0112] "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 reduction in cell growth.

[0113] "Agonist activity," "agonist activity," or "agonism" refers to the function of a substance as an agonist, where the binding of an agonist to a cellular receptor elicits a response or activity similar or identical to that produced by the receptor's natural ligand. For example, CD40 agonists or CD40 agonistic antibodies can induce any or all of the following responses: cell proliferation and / or differentiation; upregulation of cell-cell adhesion via molecules such as ICAM-1, E-selectin, and VCAM; secretion of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, and TNF; transduction of signals via the CD40 receptor through TRAFs (e.g., TRAF2 and / or TRAF3), MAP kinases such as NIK (NF-κB-inducing kinase), 1-κB kinase (IKKα / β), transcription factor NF-κB, Ras, and pathways such as the MEK / ERK pathway, the PI3K / Akt pathway, and the p38 MAPK pathway; transduction of anti-apoptotic signals via molecules such as XIAP, Mcl-1, and BCLx; production of B and / or T cell memory; production of B cell antibodies; B cell isotype switching; and upregulation of cell surface expression of class II MHC and CD80 / 86.

[0114] A "CD40-associated disease" or "CD40-associated condition" refers to a condition in which cells expressing CD40 are modified or eliminated. These cells include CD40-expressing cells that exhibit abnormal proliferation or CD40-expressing cells associated with cancerous or malignant growth. More specific examples of cancers that exhibit abnormal expression of the CD40 antigen include B-lymphoblastic neoplasms, Burkitt's lymphoma, multiple myeloma, T-cell lymphoma, Kaposi's sarcoma, osteosarcoma, epidermal and endothelial tumors, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer (melanoma), bladder cancer, and renal cancer. Such disorders include, but are not limited to, leukemia, lymphoma (including B-cell lymphoma and non-Hodgkin's lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, sarcomas such as osteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinoma (including ovarian adenocarcinoma), solid tumors including Kaposi's sarcoma / Kaposi's tumor, and squamous cell carcinoma.

[0115] "Proliferative disease" refers to a medical condition associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative condition refers to cancer.

[0116] "Cancer," "cancerous," "proliferative condition," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0117] "Cancer prevention" refers to 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 has been determined, for example, by genetic screening or other methods, to identify a cancer susceptibility. 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.

[0118] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not. For example, "optionally including mannitol or ethylenediaminetetraacetic acid" means that it may be present in the pharmaceutical composition, but is not necessarily present.

[0119] As used herein, the terms "about" or "approximately" refer to a numerical value that is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, the numerical portion being determined by how it is measured or determined (i.e., the limitations of the measurement system). For example, in the practice of the art, "about" may mean within or more than 1 standard deviation. Alternatively, "about" or "essentially comprising" may mean a range of at most ±30%, e.g., a pH of about 5.5 refers to pH 5.5 ± 1.65. Furthermore, particularly for biological systems or processes, the terms may mean at most one order of magnitude or at most 5 times the numerical value. Unless otherwise indicated, when specific values ​​appear in this application and claims, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of the specific value.

[0120] "Buffer" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include trishydroxymethylaminomethane (Tris), acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0121] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate.

[0122] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium salt, succinic acid-potassium succinate, succinic acid-calcium salt, etc. A preferred succinate buffer is succinic acid-sodium salt. Illustratively, the succinic acid-sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate salt.

[0123] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, and are preferably histidine hydrochloride buffers or histidine acetate buffers. Histidine acetate buffers are prepared from histidine and acetic acid, and histidine hydrochloride buffers are prepared from histidine and histidine hydrochloride or histidine and hydrochloric acid.

[0124] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. Preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0125] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients, that maintain the stability of the active ingredient, facilitate administration to the body, and contribute to the absorption of the active ingredient to further exert its biological activity.

[0126] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0127] Unless otherwise specified, the solvent in any solution form of the pharmaceutical composition described in the present disclosure is water.

[0128] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after subjecting a liquid or solution formulation to a vacuum freeze-drying step.

[0129] The pharmaceutical compositions described herein can achieve stable efficacy, in which the FAP / CD40 binding molecule essentially retains its physical and / or chemical stability and / or biological activity after storage, e.g., the pharmaceutical composition essentially retains its physical and chemical stability and biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, several analytical techniques are available for measuring protein stability, which can be used to measure stability after storage at a selected temperature for a selected period of time.

[0130] A stable drug-antibody formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or even up to 2 years. Furthermore, stable liquid formulations include liquid formulations that exhibit desired characteristics after storage at temperatures including 25°C for periods including 1 month, 3 months, or 6 months, or at 40°C for periods including 1 month. A typical acceptable standard for stability is degradation of antibody monomers, typically not more than about 10%, preferably not more than about 5%, as measured by SEC-HPLC. Upon visual analysis, the drug-antibody formulation is colorless or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably not more than about 5%, of the antibody is cleaved, and typically no more than about 10%, preferably not more than about 5%, of the antibody is aggregated.

[0131] An antibody "retains its physical stability" in a drug formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation as determined by visual inspection of color and / or clarity or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of proteins) and by FTIR spectroscopy (which determines the secondary structure of proteins).

[0132] An antibody "retains its chemical stability" in a drug formulation if it does not undergo significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that constantly alter the chemical structure of proteins include hydrolysis or cleavage (assessed by methods such as size-exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, and isoaspartic acid measurement), and isomerization (assessed by isoaspartic acid content measurement, peptide mapping, etc.).

[0133] An antibody "retains its biological activity" in a drug formulation if the antibody's biological activity over a given period of time is within a given range of the biological activity exhibited at the time of preparation of the drug formulation. Antibody biological activity can be determined, for example, by antigen binding assays.

[0134] "Administration," "giving," and "treatment," when used with an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" may refer, for example, to therapeutic, pharmacokinetic, 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. "Administration," "giving," and "treatment" also refer to in vitro and ex vivo treatment of, for example, cells with a reagent, diagnostic, binding composition, or via another cell. "Treatment," when used with a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0135] "Treatment" refers to administering to a subject, for example, an internal or external therapeutic agent comprising any one of the antibodies or pharmaceutical compositions thereof disclosed herein as a therapeutic agent, where the subject already suffers from, is at risk of, or is prone to suffer from, one or more proliferative diseases or symptoms thereof, and the known therapeutic agent has 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 various factors, such as the subject's disease state, age and weight, 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.

[0136] 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.

[0137] As used herein, a "subject" or "patient" refers to mammals, particularly primates, and especially humans.

[0138] The instruments and methods used in the detection process are as follows. exterior: Using the visual inspection method, wipe the sample bottle clean and observe the color, transparency, and visible foreign matter of the sample under the white and black backgrounds of a transparency detector at an illumination intensity of 1000 to 1500 lx, respectively.

[0139] Appearance detection equipment: Seitakuki YB-2A transparency detector.

[0140] SEC molecular exclusion chromatography: This is an analytical method for isolating solutes based on the correlation between the pore size of gel pores and the coil size of polymer sample molecules.

[0141] SEC monomer content percentage = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas). SEC measurement equipment: Agilent 1260-Bio, column: Waters XBrige BEH200Å SEC (300 × 7.8 mm, 3.5 μm).

[0142] NR-CE Capillary Gel Electrophoresis: This is electrophoresis, which is carried out by moving a gel through a capillary as a support medium, and is a method of separating samples according to their molecular weight at a constant voltage.

[0143] Percent purity of non-reduced CE = A Main / A Total * 100% (A Main is the peak area of ​​the main peak in the sample, and A Total is the sum of all peak areas).

[0144] CE measuring equipment: Sciex model number PA800 plus.

[0145] icIEF Imaging Capillary Isoelectric Focusing: This is a technique for isolating proteins based on their differences in isoelectric point (pI).

[0146] icIEF main peak content percentage = peak area of ​​main peak / total area * 100% (total area is the sum of acidic peak, main peak and basic peak areas).

[0147] icIEF measurement equipment manufacturer: Protein Simple, model number Muarice.

[0148] Protein concentration determination: Protein concentration measurement equipment: UV-visible spectrophotometer, model number: Nano Drop 2000, light path 1 mm.

[0149] Exemplary antibody pharmaceutical composition (formulation) preparation process Step 1: A FAP / CD40-binding antibody (e.g., Ab10-A297V3-4, whose heavy and light chain sequences are SEQ ID NOs: 22 and 20, respectively) was mixed with the following additives in the prescribed amounts to prepare a stock solution containing the FAP / CD40-binding molecule. After filtration, the stock solution was sampled using the center console to check for sterility. The stock solution was filtered through a 0.22 μm filter cartridge, and the filtrate was collected.

[0150] Step 2: The filling volume was adjusted to 1.15 mL, and the filtrate was filled into a 2 mL vial, which was then stoppered. Sampling was performed at the start, middle, and end of filling, and differences in the filling volume were detected using the center console.

[0151] Step 3: The capping machine was turned on and the aluminum cap was applied and crimped on.

[0152] Step 4: Visually inspect the product for defects such as incorrect filling amounts. Print and attach labels to the vials, print labels for the cartons, fold the cartons, place the products in the cartons, and attach the carton labels.

[0153] II. Examples and Test Examples The present disclosure is further illustrated by the following examples, which are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0154] Experimental methods in the examples of this disclosure for which specific conditions are not specified generally follow conventional conditions or conditions recommended by raw material or commercial manufacturers. Reagents for which specific sources are not specified are conventional commercially available reagents.

[0155] Example The methods for preparing and purifying the FAP / CD40 binding molecules of the present application are described in the patent application number WO2023025194, the entire contents of which are incorporated herein by reference.

[0156] 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.

[0157] [Table 2]

[0158] 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 the phages were enriched using a magnetic rack. The antigen-specific phages were eluted with a glycine solution at pH 2.2.

[0159] 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 per 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).

[0160] 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.

[0161] 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 hygromycin pressure pressing. 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 cultured by hygromycin pressure pressing.

[0162] 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.

[0163] 4) Antibody gene clones, including: the affinity of positive clones was detected and analyzed by FACS, and PCR amplification of antibody genes for 45 clones was determined. Positive clones were collected by centrifugation, the supernatant was discarded, cellular genomic DNA was extracted, VH and LC were PCR amplified, the amplified fragments were isolated by electrophoresis, and analyzed by sequencing. Six unique VH and six unique κ sequences were identified. When combined, 12 clones with unique sequences were obtained, and the VH and VL sequences of one of the clones are shown in Table 3.

[0164] [Table 3]

[0165] Note: The underlined parts are CDRs based on the Kabat numbering convention.

[0166] The disclosure further provides the full-length heavy and light chain sequences of Ab10.

[0167] >Ab10 heavy chain full length [ka] >Ab10 light chain full length [ka] [Table 4]

[0168] 1.4 Construction of a CHO cell surface full-length antibody display library 1) Construction of soluble antibody expression vectors, including: positive VH and LC fragments with unique sequences were enzymatically digested and purified, respectively, the VH fragment was inserted into a soluble heavy chain expression vector (IgG1), and the LC fragment was inserted into a soluble light chain expression vector, and the colonies were subjected to sequencing to confirm and extract DNA.

[0169] 2) Expression and purification of soluble antibodies, including the following: Expi293 cells were cultured in suspension and amplified. According to the light and heavy chain pairs identified above, 18 μg of light chain expression vector and 12 μg of heavy chain expression vector were mixed. The vector DNA and PEI were mixed at a weight ratio of 1:2.5 and used to transform Expi293 cells. On day 6, the culture supernatant was collected and the antibodies were purified using the Protein-A method. Analysis by SDS-PAGE denaturing gel electrophoresis showed that the purity of all antibodies was over 90%. The antibodies were stored at -80°C.

[0170] 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 and thorough washing, 100 μL of TMB was added to each well and the plate was 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 of the anti-FAP antibody was determined. 50 The values ​​were calculated and the results are shown in Table 5.

[0171] [Table 5]

[0172] 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 2-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 pH 1.5 was used as the regeneration buffer, and the assay was run for 30 s at a flow rate of 10 μL / min. After analyzing the data, the results in Table 6 show that Ab10 has 3-6 times higher affinity to the antigen FAP than the control anti-FAP antibody 28H1 (sequences 219 and 233 of patent US9266938B2).

[0173] [Table 6]

[0174] The heavy and light chain variable region sequences of the control antibody 28H1 are as follows: >28H1 VH [ka] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS (SEQ ID NO: 9) >28H1 VL [ka] EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIK (SEQ ID NO: 10) 1.7 FACS binding assay The binding characteristics of anti-FAP antibodies in cells were detected by FACS experiments. The following procedure involved constructing a CHO cell line overexpressing human FAP and seeding it on a plate (1E5 / well). The antibody to be measured was added at 100 μL / well, with the highest concentration of 100 nM, and then diluted 5-fold to a total of 8 concentrations. The cells were 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 relationship between the percentage of labeled human FAP-overexpressing cells in the total cells and antibody concentration is shown in Figure 1, and the binding EC of human FAP with the antibody was calculated. 50 The values ​​are shown in Table 7. The relationship between the number of cells overexpressing labeled mouse FAP as a percentage of the total cells and the antibody concentration is shown in Figure 2, among which Ab9, Ab14, and Ab15 are three other anti-FAP antibodies obtained by the screening of this application.

[0175] [Table 7]

[0176] Example 2. Screening and preparation of anti-CD40 nanobodies (VHHs) 2.1 Sequence and preparation of immunizing antigens and screening antigens The following recombinant proteins were selected: His-tagged human CD40 protein (h-CD40-His), C-terminal biotin-tagged human CD40 protein (h-CD40-biotin), and C-terminal His-tagged monkey CD40 recombinant protein (cyno-CD40-His). Their sequences and sources are shown in Table 8. The above protein reagents can be used in the experiments described in the following examples.

[0177] [Table 8]

[0178] 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.

[0179] 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.

[0180] 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. Positive clones were screened by phage ELISA, and the optical density at 450 nm was detected. Based on the sequencing results, sequence alignment and phylogenetic tree analysis were performed on the sequences. Sixteen sequences were screened, and the amino acid sequences of the anti-CD40 antibodies with relatively good functionality are listed in Tables 9 and 10.

[0181] [Table 9]

[0182] Note: The underlined parts are CDRs based on the Kabat numbering convention.

[0183] [Table 10]

[0184] 2.5 Expression and purification of anti-CD40 single domain antibody-Fc fusion protein Each of the two VHHs described above was linked to human IgG1-Fc containing the N297A mutation, and the sequence of the linked VHH-Fc fusion protein is shown in Table 11. The underlined part is human IgG1-Fc, and the bold part is the N297A mutation.

[0185] 9E5-SELFNS was used as a CD40 agonist positive control (see sequences 58 and 59 of WO2020108611A1).

[0186] [Table 11]

[0187] Plasmids were constructed, cells were transiently transfected, and the antibodies were expressed and purified. Target antibodies were obtained by detection.

[0188] 2.6 FACS binding assay The binding characteristics of anti-CD40 antibodies on cells were detected by FACS experiments. Raji cells were obtained and plated. The antibodies to be tested were added at 100 μL / well, with the highest concentration point being 100 nM, and diluted 5-fold to obtain a total of 8 gradient points. The antibodies were then incubated at 4°C for 1 hour. Anti-hIgG Alexa Fluor-647 was added as a secondary antibody at a ratio of 1:500 and incubated at 4°C for 30 minutes. Detection was performed by FACS. A CD40 agonist was used as a positive control, and the EC values ​​of each antibody were compared. 50are shown in Figure 3 and Table 12. Among them, A12 is another anti-CD40 antibody obtained by the screening of the present application.

[0189] [Table 12]

[0190] In terms of being used as a CD40 agonist in FAP / CD40 bispecific antibodies, the affinity EC 50 should not be too high, otherwise the bispecific antibody will preferentially bind to CD40, and more preferably, the bispecific antibody will preferentially bind to and function with FAP. Thus, A297 has the advantage of better meeting the above characteristics, which are also shown in Table 13.

[0191] 2.7 Activity detection of anti-CD40 nanobodies EC was detected by detecting the activation effect of anti-CD40 antibody on CD40 reporter gene cells. 50 The agonist activity of CD40 antibodies was assessed based on the following: HEK-Blue TMCD40L cells (purchased from Invivogen Cat# hkb-cd40) were stably transfected with the human CD40 gene and the NF-κB-mediated SEAP gene. The level of CD40 signaling pathway activation was measured by detecting the secreted SEAP content in the supernatant using the SEAP substrate QUANTI-Blue. Cells were seeded at 5E4 / well in DMEM medium containing 10% FBS, 100 μg / mL Normocin, 100 μg / mL Zeocin, and 30 pg / mL Blasticidin. After overnight cell adhesion, 100 μL of gradient-diluted antibody was added to each well and incubated at 37°C. The cells were centrifuged, and the 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 15 minutes. The absorbance was measured at 620 nm using an Envision plate reader, and the EC 50 The CD40 agonist (9E5-SELFNS) was used as a positive control, and the EC values ​​were calculated as a function of antibody concentration and percentage relative agonist activity (compared to 200 nM 9E5-SELFNS). 50 are shown in Figure 4 and Table 13. Among them, A12 is another anti-CD40 antibody obtained by the screening of the present application.

[0192] [Table 13]

[0193] 2.8 Humanization of anti-CD40 nanobodies Based on these results, A297 was humanized. Based on the typical VHH structure of the resulting 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 humanized sequences are shown in Table 14.

[0194] [Table 14]

[0195] Each of the four VHHs was linked to human IgG1-Fc containing the S267E / L328F mutations (according to the EU numbering system). The VHH-Fc fusion protein sequences after linkage are shown in Table 15. The underlined part is human IgG1-Fc, and the bold part is the S267E / L328F mutation.

[0196] [Table 15]

[0197] The four VHH-Fc antibodies were transiently transfected into cells, and the antibodies were purified and detected to obtain the target antibody proteins.

[0198] 2.9 Affinity of humanized CD40 antibodies to CD40-high expressing cell lines 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 (CD40 cDNA ORF Clone, Human, C-DYKDDDDK (Flag®) Tag, Sino Biological, HG10774-CF) to obtain HEK293 cells highly expressing CD40. The cells were 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 a secondary antibody, and the cells were stained for 1 hour on ice. After washing twice with PBS, the cell surface fluorescent signal was detected by FACS. The results are shown in Figure 5. EC 50 The values ​​are shown in Table 16. The humanized antibodies of the present disclosure have relatively high affinity for any of the CD40-high expressing cell lines.

[0199] [Table 16]

[0200] 2.10 HEK-Blue TM Activation of CD40L cells by humanized CD40 antibodies HEK-Blue of CD40 antibody when cross-linked by FcγRIIb TM The in vitro agonistic activity of CD40 antibodies was evaluated by detecting their activating 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. TMThe 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 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 values.

[0201] FcγRIIb-mediated CD40 antibody HEK-Blue TM The activation effect on CD40L cells is shown in Figure 6. 50 The values ​​and Emax values ​​(relative fluorescence intensity) are shown in Table 17.

[0202] The results showed that in the reporter gene cell line, the humanized CD40 antibodies all exhibited relatively strong agonistic activity when cross-linked by FcγRIIb.

[0203] [Table 17]

[0204] Example 3. Design and preparation of anti-FAP / CD40 bispecific antibodies 3.1 Design, expression, and purification of anti-FAP / CD40 bispecific antibodies Based on the results of screening for humanized anti-CD40 antibodies, A297V3 was selected. Based on the results of screening for anti-FAP antibodies, Ab10 was selected to construct an anti-FAP / CD40 bispecific antibody. The bispecific antibody used Ab10 as an IgG scaffold, with one, two, or three anti-CD40 nanobodies tandemly linked to the C-terminus of each of Ab10's two heavy chains. The linker between the CD40 nanobodies and between the C-terminus of the Ab10 heavy chain was "GGGGSGGGGS." Each bispecific antibody contained bivalent, tetravalent, and hexavalent CD40 nanobodies, respectively, as shown in Figure 7. In the antibody name, for example, Ab10-A297V3-2, Ab10 indicates the anti-FAP antibody used, A297V3 indicates the humanized anti-CD40 antibody used, and the 2 indicates the bivalent CD40. All other antibodies followed this naming convention. The transient transfection, expression, and purification of the antibodies were carried out according to the method described in Section 2.5 of Example 2. Target bispecific antibody molecules were obtained by detection. The amino acid sequences of each bispecific antibody molecule are as follows:

[0205] >Ab10-A12V2-2 light chain [ka] >Ab10-A297V3-2 heavy chain [ka] >Ab10-A297V3-4 heavy chain [ka] >Ab10-A297V3-6 heavy chain [ka] The light chains of Ab10-A297V3-2, Ab10-A297V3-4, and Ab10-A297V3-6 are all as shown in SEQ ID NO: 20.

[0206] Note: The underlined parts of the heavy chain are the CH1 and Fc regions, the underlined parts of the light chain are the CL region, the italicized parts are the linker, and the bolded parts are LALA (L234A and L235A mutations).

[0207] 3.2 Detection of antigen-binding affinity of anti-FAP / CD40 bispecific antibodies The affinity of the anti-FAP / CD40 bispecific antibody to its antigens, human FAP protein (Acro, FAP-H5244) and human CD40 protein (Acro, CD0-H5228), was detected using SPR (GE Healthcare, Biacore 8K). 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 fluid phase. Anti-human IgG (Fc) antibody was immobilized by amine coupling. Each antibody to be measured was prepared as a ligand in HBS-EP+ buffer solution and captured by the anti-human IgG (Fc) antibody in the chip channel. Different species of antigen proteins were used as analytes. The results are shown in Tables 18 and 19, and indicate that after preparation into bispecific antibodies, the binding of the antibodies to FAP is not affected, and the bispecific antibodies have lower affinity for CD40, preferentially bind to FAP, and still function.

[0208] [Table 18] [Table 19]

[0209] 3.3 Detection of affinity of anti-FAP / CD40 bispecific antibodies to FAP-high expressing cell lines In this experiment, the binding of the anti-FAP / CD40 bispecific antibody to stable CHOK1 cell transfectants highly expressing human FAP, cynomolgus monkey FAP, and mouse FAP was detected by FACS. Figures 8A-8C show that the bispecific antibody can bind to the FAP antigen on the cell surface of human, mouse, and cynomolgus monkey cells, and its binding ability is similar to that of a monoclonal antibody. EC 50 The values ​​are shown in Table 20. Among them, Ab10-A12V2-2 and Ab10-A12V2-4 are two other anti-FAP / CD40 bispecific antibodies obtained by the screening of the present application.

[0210] [Table 20]

[0211] 3.4 Detection of affinity of anti-FAP / CD40 bispecific antibodies to CD40-high expressing cell lines The binding of the bispecific antibodies to cell surface CD40 was detected by FACS using HEK-Blue™ CD40L cells (Invivogen, Cat# hkb-cd40) that overexpress human CD40 and HEK293 cells that were overexpressed with cynomolgus monkey CD40 via transient transfection with a cynomolgus monkey CD40 plasmid (Sino Biological, Cat# CG90970-UT). The results showed that the bispecific antibodies could bind to cell surface CD40 of both human and cynomolgus monkeys. Details are shown in Figures 9A and 9B, Figure 11, and Table 21. Among these, Ab10-A12V2-2 and Ab10-A12V2-4 are two other anti-FAP / CD40 bispecific antibodies obtained through this screening study.

[0212] [Table 21]

[0213] 3.5 Detection of FAP-dependent DC-promoting maturation activity of humanized anti-FAP / CD40 bispecific antibodies To confirm the effect of anti-FAP / CD40 bispecific antibodies on dendritic cell maturation, mononuclear cells were isolated from fresh PBMCs using CD14-positive magnetic beads and cultured for 5 days in 1640 medium containing 50 ng / mL GM-CSF and 50 ng / mL hIL-4. Half of the medium was replaced every 2–3 days. On day 5, induced dendritic cells were added to a 96-well plate at a density of 1E5 / well. Additionally, CHOK1 cells and CHOK1 cells overexpressing human FAP in logarithmic growth phase were added. Simultaneously, gradient dilutions of the antibody to be assayed were added. 1 μg / mL LPS served as a positive control, and 100 nM hIgG1 or no antibody served as a negative control. After 48 h of culture, the expression of the dendritic cell surface molecule CD83 was detected by FACS.

[0214] As shown in Figure 11 (without CHOK1 / FAP) and Figure 12 (with CHOK1 / FAP), the results indicate that the bivalent molecule Ab10-A297V3-2 is unable to activate DCs in the absence of CHOK1 / FAP, indicating that its CD40 agonist activity is entirely dependent on FAP-mediated crosslinking. On the other hand, the tetravalent molecule Ab10-A297V3-4 can induce DC maturation in the absence of CHOK1 / FAP cells and further enhance DC activation in the presence of CHOK1 / FAP. Therefore, for both bivalent and tetravalent molecules, FAP expressed on the dendritic cell surface can provide an activation window for bispecific antibodies. Among these, Ab10-A12V2-2 and Ab10-A12V2-4 are two other anti-FAP / CD40 bispecific antibodies obtained by the screening of this application.

[0215] Example 4. In vivo efficacy of anti-FAP / CD40 bispecific antibody Ab10-A297V3-2 / 4 in mice B-hCD40 humanized mice were obtained from Biocytogen JiangSu Co., Ltd. (species: Mus musc μLus, race: C57BL / 6, female). A stable transfectant was constructed by transfecting mouse intestinal cancer MC38 cells with the mouse full-length FAP plasmid, and the cells were cultured at 5 × 10 5The mice were inoculated subcutaneously at the right armpit at 0.1 mL / mouse, and the average tumor volume was 80-100 mm 3 Mice with appropriate individual tumor volumes were selected and randomly divided into groups, six per group. The mice were administered intraperitoneally twice a week. 48 hours after the first administration, peripheral blood samples were collected to measure the number of B cells and the expression of CD86 on the cell surface. Eight days after grouping, the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood were measured. After four doses, administration was discontinued. The hIgG1 control group received IgG1 treatment when the tumor volume reached 2,000 mm. 3 When any mouse exceeded this number, the final dose was administered, and regular blood tests were performed 24 hours after the final dose.

[0216] 4.1 In vivo efficacy and toxicity of a single dose of Ab10-A297V3-2 or Ab10-A297V3-4 in mice The antitumor activity of Ab10-A297V3-2 or Ab10-A297V3-4 and the CD40 agonist monoclonal antibody 9E5-mIgG1 was compared at a single dose. 9E5-mIgG1 has the same variable regions as 9E5-SELFNS, except that its heavy and light chain constant regions use the mouse mIgG1 heavy chain constant region and the mouse kappa chain constant region. Because the activity of CD40 agonist monoclonal antibodies depends on cross-linking of their Fc domains with FcγRIIb, 9E5-mIgG1, which uses the mouse IgG1 heavy chain constant region, demonstrated better CD40 agonist activity in vivo in mice. In mouse in vivo efficacy experiments, 9E5-mIgG1 was a better control antibody than 9E5-SELFNS.

[0217] Ab10-A297V3-4 has stronger antitumor activity than an equimolar dose of the CD40 monoclonal antibody 9E5-mIgG1, in terms of both TGI (tumor growth inhibition) and CR (complete response), and has the following sequence: >9E5-mIgG1 heavy chain [ka] >9E5-mIgG1 light chain [ka] Even at twice the molar concentration, Ab10-A297V3-2 still had weaker antitumor activity than Ab10-A297V3-4 and comparable to 9E5-mIgG1.

[0218] The activation of peripheral CD40 by the bispecific antibody molecules was measured by detecting the activation of mouse peripheral blood B lymphocytes. The results were consistent with the in vitro experiments. Ab10-A297V3-4 partially activated peripheral B lymphocytes due to its FAP-independent CD40 activation activity, whereas Ab10-A297V3-2 completely dependent on FAP for CD40 activation, and therefore no significant peripheral B cell activation was detected (One-Way ANOVA). The information on the tested antibodies is shown in Table 22, and the in vivo efficacy results are shown in Figure 13A (P<0.0001 for each antibody group compared to the hIgG1 control group) and Figure 13B.

[0219] [Table 22]

[0220] Referring to FIG. 13C, in terms of mouse toxicity, neither Ab10-A297V3-2 nor Ab10-A297V3-4 caused any change in mouse body weight.

[0221] Referring to Figure 13D, routine blood tests showed that compared with the equimolar hIgG1 control group, 9E5-mIgG1 was able to induce a degree of platelet reduction similar to hepatotoxicity, Ab10-A297V3-2 did not induce a platelet reduction, and Ab10-A297V3-4 was able to induce a platelet reduction to the same extent as 9E5-mIgG1. Detailed statistics on the in vivo dose and tumor growth inhibition rate of Ab10-A297V3-2 / 4 in mice are shown in Table 23.

[0222] The formula for calculating TGI is as follows:

[0223] TGI = (tumor volume of blank group on the day of administration - tumor volume of administration group on the day of administration) / (tumor volume of blank group on the day of administration) × 100% [Table 23]

[0224] ALT and AST detection showed that 9E5-mIgG1 exhibited some hepatotoxicity on day 8 after administration due to CD32B (i.e., FcγRIIB)-mediated cross-linking. Referring to Figure 13E, Ab10-A297V3-4 also exhibited some hepatotoxicity due to some background activation, but because its activation was independent of CD32B, its hepatotoxicity was lower than that of 9E5-mIgG1, and Ab10-A297V3-2 did not exhibit any hepatotoxicity because it could not activate CD40 without relying on FAP.

[0225] 4.2 In vivo efficacy and toxicity of Ab10-A297V3-2 or Ab10-A297V3-4 at multiple doses in mice To determine the therapeutic window of Ab10-A297V3-2 or 4, the dose of Ab10-A297V3-2 was further increased while the dose of Ab10-A297V3-4 molecule was reduced, as detailed in Table 24. Further increases in the dose of the bivalent Ab10-A297V3-2 molecule failed to further improve its antitumor activity, and no activation of peripheral B lymphocytes was observed. When the dose of Ab10-A297V3-4 was reduced to 1.3 mg / kg (i.e., 1 / 3 the molar dose of 9E5-mIgG1), it still had a TGI of 92.6%, far superior to 9E5-mIgG1.

[0226] To evaluate the effect of FAP antibodies in tetravalent CD40 bispecific antibodies in in vivo experiments, we compared the antitumor activity of Ab10-A297V3-4 with that of another tetravalent CD40 bispecific antibody (isotype-A297V3-4) that lacks FAP-binding function. Isotype-A297V3-4 and Ab10-A297V3-4 are identical in structure except that Ab10 in the anti-FAP moiety is replaced with another isotype antibody that does not bind to any mouse protein.

[0227] 14A-14E, the tetravalent CD40 bispecific antibody had relatively good antitumor activity without FAP binding, but its antitumor activity was enhanced upon FAP binding. Regarding toxicity, Ab10-A297V3-2 showed no abnormalities in body weight, ALT / AST, or platelet count even with increasing doses. However, for Ab10-A297V3-4, the ALT / AST and platelet count abnormalities observed at higher doses disappeared after dose reduction, and the antibody still maintained its strong antitumor effect (P<0.0001), demonstrating that both Ab10-A297V3-2 and Ab10-A297V3-4 have a broader therapeutic window than the CD40 monoclonal antibody 9E5-mIgG1. Antibody information is shown in Table 24, and statistical data on the in vivo efficacy of multiple doses of Ab10-A297V3-2 or Ab10-A297V3-4 is shown in Table 25.

[0228] [Table 24] [Table 25]

[0229] The anti-FAP / CD40 bispecific antibody used in the following examples is the aforementioned Ab10-A297V3-4, and its heavy and light chain amino acid sequences are SEQ ID NOs: 22 and 20, respectively.

[0230] Example 5. Screening of buffer systems and pH values ​​for anti-FAP / CD40 bispecific antibody formulations The following buffer solutions were prepared, and antibody formulations with an anti-FAP / CD40 bispecific antibody concentration of 20 mg / mL were prepared and subjected to high temperature (40°C) stability studies.

[0231] 1) 10 mM acetic acid-sodium acetate, pH 5.5 2) 10 mM histidine-histidine hydrochloride, pH 5.5 3) 10 mM histidine-histidine hydrochloride, pH 6.0 4) 10 mM histidine-histidine hydrochloride, pH 6.5 5) 10 mM succinic acid-sodium succinate, pH 5.0 6) 10 mM succinic acid-sodium succinate, pH 5.5 7) 10 mM succinic acid-sodium succinate, pH 6.0 8) 10 mM phosphate buffer, pH 6.5 9) 10 mM phosphate buffer, pH 7.0 10) 10 mM phosphate buffer, pH 7.5 [Table 26] [Table 27]

[0232] Test Results: As shown by the appearance results, after 4 weeks of storage at 40°C for formulations F1 to F10, formulations F2 (histidine salt buffer, pH 5.5), F3 (histidine salt buffer, pH 6.0), and F5 (succinate buffer, pH 5.0) showed no obvious particles, while the remaining formulations showed particles. Histidine salt buffer (pH 5.5, pH 6.0) and succinate buffer (pH 5.0) are superior to other buffer systems.

[0233] As shown by the SEC results, after 4 weeks of high-temperature storage at 40°C, the SEC purity of all formulations decreased, of which the degree of decrease in the histidine salt system (F2-F4) and acetate salt system (F1) was smaller than that in the succinate salt system (F5-F7) and phosphate buffer system (F8-F10), and there was no obvious change in the monomer purity of formulations F2 and F3.

[0234] As shown by the CEX results, after 4 weeks of storage at 40°C, the main peaks of formulations F1 and F3 decreased by about 20%, the decrease in the main peak of formulation F2 was next to F1 and F3 at about 24%, and the decrease in the main peak of the other formulations was more obvious.

[0235] As shown by the NR-CE results, after 4 weeks of storage at 40°C, formulations F1, F2, F5, and F6 showed the smallest decrease in purity compared to TO, at about 6%, followed by formulation F3, while the remaining formulations showed more obvious purity loss.

[0236] As mentioned above, formulations F2 and F3 were superior to the other formulations in terms of appearance, SEC, CEX, and NR-CE detection results, and showed better stability. Therefore, a histidine salt buffer system was selected as the final buffer system. F2 (10 mM histidine salt buffer, pH 5.5) and F3 (10 mM histidine salt buffer, pH 6.0) were selected as buffer systems for the next pH fine screening.

[0237] Example 6. Screening of surfactants in anti-FAP / CD40 bispecific antibody formulations Buffer systems of 10 mM histidine-histidine hydrochloride, pH 5.0 and pH 5.5 were selected, and formulations containing 25 mg / mL of anti-FAP / CD40 bispecific antibody, 80 mg / mL of sucrose, and surfactants of different types and concentrations were prepared, and their stability under repeated freeze-thaw and shaking was examined: 15) 10 mM histidine-histidine hydrochloride pH 5.0, 0.2 mg / mL polysorbate 80; 16) 10 mM histidine-histidine hydrochloride pH 5.5, 0.4 mg / mL polysorbate 80; 17) 10 mM histidine-histidine hydrochloride pH 5.5, 0.8 mg / mL polysorbate 80; 18) 10 mM histidine-histidine hydrochloride pH 5.5, 2 mg / mL poloxamer 188.

[0238] [Table 28]

[0239] Note: LY = pale yellow, SO = slightly opalescent, PF = no visible particles, AFP = few particles. *: 1 indicates 1 particle.

[0240] Experimental results As shown by the appearance results, after five rounds of freeze-thawing, one particle was observed in formula F16, and a small amount of visible particles appeared in the appearance of formulas F15, F17, and F18. After three days of shaking, no visible particles were observed in formulas F15 to F17, and a small amount of visible particles appeared in formula F18.

[0241] As shown by the results of insoluble microparticles, after 3 days of shaking and 5 rounds of freezing and thawing, there was no obvious change in the particle count of formulations F15 to F18, and the difference between each formulation was not significant.

[0242] As shown by the SEC results, after 5 rounds of freezing and thawing and 3 days of shaking, there was no obvious change in the SEC monomer content of formulations F15 to F18, and the difference between the formulations was not significant.

[0243] As shown by the CEX results, after 3 days of shaking and 5 rounds of freezing and thawing, there were no obvious changes in the main peak, acidic peak, and basic peak of formulations F15 to F18, and the differences between the formulations were not significant.

[0244] As shown by the NR-CE results, after 5 rounds of freezing and thawing and 3 days of shaking, there was no obvious change in the purity content of formulations F15 to F18. The difference between the formulations was not significant.

[0245] As mentioned above, in the appearance test using a histidine salt buffer solution at a pH range of 5.0 to 5.5, after five cycles of freezing and thawing, visible particles were observed in all of the formulations F15 to F18, with only one particle in formulation F16 being superior to the other formulations. After three days of shaking, a small number of particles were observed in formulation F18, while no visible particles were observed in the other formulations. There were no significant differences in the insoluble particles and purity results for the different formulations. Therefore, polysorbate 80 at a content of 0.04% (w / v) was selected as the surfactant.

[0246] Example 7. Screening of additives in anti-FAP / CD40 bispecific antibody formulations A 10 mM histidine-histidine hydrochloride buffer system was selected to prepare anti-FAP / CD40 bispecific antibody formulations containing 0.4 mg / mL polysorbate 80, different additives, and different antibody concentrations: 19) 10 mM histidine-histidine hydrochloride pH 5.0, 8% sucrose, 25 mg / mL anti-FAP / CD40 bispecific antibody; 20) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose, 25 mg / mL anti-FAP / CD40 bispecific antibody; 21) 10 mM histidine-histidine hydrochloride pH 5.5, 1% sucrose + 4% mannitol, 25 mg / mL anti-FAP / CD40 bispecific antibody; 22) 10 mM arginine-glutamic acid pH 5.0, 8% sucrose, 25 mg / mL anti-FAP / CD40 bispecific antibody; 23) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose + 0.01% EDTA, 25 mg / mL anti-FAP / CD40 bispecific antibody; 24) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose + 0.01% EDTA, 5 mg / mL anti-FAP / CD40 bispecific antibody; 25) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose, 5 mg / mL anti-FAP / CD40 bispecific antibody; The above formulation was left to stand under conditions of 2 to 8°C, 25°C and 40°C to examine its stability, and also to examine its stability after repeated freezing and thawing.

[0247] [Table 29] JPEG2026507021000040.jpg240153

[0248] Notes: LY = Light Yellow, SO = Slightly Opalescent, O = Opalescent, PF = No Visible Particles, AFP = A Few Particles. N / A: Not Detected.

[0249] Experimental results As shown by the appearance results, after freeze-thawing, visible particles appeared in formula F21, and the degree of opalescence gradually increased with increasing freeze-thaw cycles, while the remaining formulas were free of visible particles. After 4 weeks of storage at 40°C, a small amount of visible particles appeared in formulas F19 and F21, while the remaining formulas were free of visible particles. After 4 weeks of storage at 2-8°C and 25°C, no visible particles were observed in any of the formulas.

[0250] As shown by the SEC results, there was no obvious change in the monomer content of all formulations after 4 weeks of storage at 40°C. After 5 rounds of freeze-thawing, there was no obvious change in the SEC monomer content.

[0251] As shown by the CEX results, after 4 weeks of storage at 40°C, the main peaks of formulas F22 to F24 showed a relatively small decrease, while the main peaks of the remaining formulas showed a more obvious decrease. After five rounds of freezing and thawing, the contents of the main peak, acidic peak, and basic peak in CEX showed no obvious changes.

[0252] As shown by the NR-CE data, after 4 weeks of storage at 40°C, the purity of formula F19 decreased the most, and the purity of the other formulas also decreased slightly, but to a similar extent. After 5 rounds of freezing and thawing, no obvious changes were observed in the purity content of any of the formulas, and the differences between the formulas were not significant.

[0253] As mentioned above, after five freeze-thaw cycles, formulation F21 exhibited fewer particles and increased opalescence compared to the other formulations. After 4 weeks of incubation at 40°C, formulations F19 and F21 exhibited fewer particles, while the other formulations showed no significant changes. The purity of the SEC monomer in each formulation showed no significant change. CEX results indicated that after 4 weeks of incubation at 40°C, formulations F20–F24 showed a smaller decrease in the main peak content than the other formulations. NR-CE results indicated that after 4 weeks of incubation at 40°C, formulation F19 showed the greatest decrease, while the purity of the other formulations decreased slightly. Therefore, formulation F20 (10 mM histidine buffer, 8% sucrose, 0.04% PS80, pH 5.5, protein concentration 25 mg / mL) was selected.

[0254] Example 8. Screening of protein concentration in anti-FAP / CD40 bispecific antibody formulations A buffer system of 10 mM acetate-histidine pH 5.0 and pH 5.5 was selected, 8% sucrose, 0.8 mg / mL polysorbate 80, and anti-FAP / CD40 bispecific antibody preparations with different antibody concentrations were prepared: 26) 10 mM acetate-histidine pH 5.0, 50 mg / mL anti-FAP / CD40 bispecific antibody; 27) 10 mM acetate-histidine pH 5.0, 80 mg / mL anti-FAP / CD40 bispecific antibody; 28) 10 mM acetate-histidine pH 5.0, 100 mg / mL anti-FAP / CD40 bispecific antibody; 29) 10 mM acetate-histidine pH 5.5, 50 mg / mL anti-FAP / CD40 bispecific antibody; The stability of each formulation was examined at 5°C and 25°C.

[0255] [Table 30]

[0256] As can be seen from the appearance results, the formulations with different antibody concentrations showed no obvious changes after being left at 5°C and 25°C for 3 months, indicating that they have relatively good stability.

[0257] As shown by the SEC results, the monomer content of the high-concentration formulations F27 and F28 showed a tendency to decrease after being stored at 25°C for 3 months, and the decrease was more pronounced at higher concentrations. Of these, the monomer purity of formulation F28 (100 mg / mL) decreased to about 95% after being stored at 25°C for 3 months. After being stored at 5°C for 3 months, there was no significant change in any of the formulations.

[0258] As shown by the results of CEX, after being left at 5°C for 3 months or at 25°C for 3 months, the main peak content of each formulation decreased slightly, and the difference between the formulations was not significant.

[0259] As shown by the results of NR-CE, after being left at 25°C for 3 months, the main peak of the F29 formulation decreased by about 5%, while no clear tendency for decrease was observed in any of the other formulations.

[0260] As mentioned above, stability is relatively good when the protein concentration is up to 100 mg / mL and the pH is 5.0.

[0261] Example 9. Alternative formulation methods Furthermore, the present invention also provides anti-FAP / CD40 bispecific antibody drug formulations in other formulations, in which the anti-FAP / CD40 bispecific antibody is the present Ab10-A297V3-4 having the heavy and light chain sequences of SEQ ID NOs: 22 and 20, respectively, including, but not limited to: (1) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.5; (2) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.3; (3) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.2; (4) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.0; (5) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.5; (6) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.3; (7) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer pH 5.2; (8) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.0; (9) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.0; (10) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.5; (11) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.3; (12) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.2; (13) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.0; (14) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.5; (15) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.3; (16) 25 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.6 mg / mL polysorbate 80, and 10 mM histidine-acetate buffer pH 5.2; (17) 10 mg / mL FAP / CD40 bispecific antibody, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride buffer, pH 5.5; As shown by the experimental results, all of the FAP / CD40 bispecific antibody formulations prepared by the above formulation methods have good stability and can be used to prepare FAP / CD40 bispecific antibody drugs.

Claims

1. 1. A pharmaceutical composition comprising a FAP / CD40 binding molecule and a buffer, The FAP / CD40 binding molecule comprises a first antigen-binding domain that specifically binds to FAP and a second antigen-binding domain that specifically binds to CD40, wherein the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein: 1) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences represented by SEQ ID NOs: 3, 4, and 5, respectively, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or 2) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 3, 4, and 5, respectively, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 6, 32, and 8, respectively; Among them, the buffer is selected from acetate buffer, succinate buffer, histidine buffer or phosphate buffer, preferably histidine buffer, more preferably histidine-hydrochloride buffer or histidine-acetate buffer, most preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; Pharmaceutical compositions.

2. the second antigen-binding domain in the FAP / CD40 binding molecule that specifically binds to CD40 comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprising three complementarity-determining regions, CDR1, CDR2, and CDR3, each comprising the amino acid sequence set forth in SEQ ID NOs: 12, 13, and 14, respectively; The pharmaceutical composition of claim 1.

3. The first antigen-binding domain of the FAP / CD40 binding molecule that specifically binds to FAP comprises: The heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 1 or having at least 90% identity thereto, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 2 or having at least 90% identity thereto. The pharmaceutical composition according to claim 1 or 2.

4. the immunoglobulin single variable domain in the second antigen-binding domain that specifically binds to CD40 in the FAP / CD40 binding molecule comprises an amino acid sequence set forth in any one of SEQ ID NOs: 11, 16-19 or having at least 90% sequence identity to any one of SEQ ID NOs: 11, 16-19; The pharmaceutical composition according to claim 2 or 3.

5. the second antigen-binding domain that specifically binds to CD40 in the FAP / CD40 binding molecule comprises 2, 3, 4, 5, or 6 of the immunoglobulin single variable domains; The pharmaceutical composition according to any one of claims 2 to 4.

6. The first antigen-binding domain of the FAP / CD40 binding molecule that specifically binds to FAP comprises a heavy chain variable region and a light chain variable region, the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds CD40 is N-terminal to the heavy chain variable region of the first antigen-binding domain that specifically binds FAP; the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds CD40 is C-terminal to the heavy chain variable region of the first antigen-binding domain that specifically binds FAP; the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds CD40 is N-terminal to the light chain variable region of the first antigen-binding domain that specifically binds FAP; and / or the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds to CD40 is C-terminal to the light chain variable region of the first antigen-binding domain that specifically binds to FAP; The pharmaceutical composition according to any one of claims 2 to 5.

7. the immunoglobulin single variable domain of the second antigen-binding domain that specifically binds CD40 in the FAP / CD40 binding molecule is linked directly or via a linker to the first antigen-binding domain that specifically binds FAP; Preferably, the linker is (G ４ S) ｘ wherein x is independently selected from an integer of 1 to 20; More preferably, the linker is (G ４ S) ２ , (G ４ S) ３ or (G ４ S) ４ The amino acid sequence is The pharmaceutical composition according to any one of claims 2 to 6.

8. the FAP / CD40 binding molecule further comprises a human immunoglobulin Fc region; Preferably, the Fc region is a human IgG1 or IgG4 Fc region; More preferably, the human IgG1 comprises a mutation that eliminates or reduces an Fc effector function; Most preferably, the human IgG1 has mutations selected from N297A, D265A / N297A, L234A / L235A, L234A / L235A / P329G, L234E, L234F, L234E / L235F and L234E / L235F / P329G. The pharmaceutical composition according to any one of claims 1 to 7.

9. the first antigen-binding domain in the FAP / CD40 binding molecule that specifically binds to FAP comprises a heavy chain and a light chain; Preferably, the heavy chain is of the IgG1 or IgG4 isotype and the light chain is of the Kappa isotype; More preferably, the heavy chain has an amino acid sequence set forth in SEQ ID NO: 24 or having at least 90% identity thereto, and the light chain has an amino acid sequence set forth in SEQ ID NO: 25 or having at least 90% identity thereto. The pharmaceutical composition according to any one of claims 1 to 8.

10. The FAP / CD40 binding molecule comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 21-23 or having at least 90% identity thereto, and the second polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO: 20 or having at least 90% identity thereto; Preferably, the FAP / CD40 binding molecule comprises two identical first polypeptide chains and two identical second polypeptide chains. The pharmaceutical composition according to any one of claims 1 to 9.

11. the FAP / CD40 binding molecule is an anti-FAP / CD40 bispecific antibody; The pharmaceutical composition according to any one of claims 1 to 10.

12. The pharmaceutical composition further comprises a surfactant, preferably the surfactant is one or more selected from polysorbate and poloxamer, more preferably the surfactant is polysorbate, and most preferably the surfactant is polysorbate 80. The pharmaceutical composition according to any one of claims 1 to 11.

13. The composition further comprises a sugar, and preferably the sugar is one or more selected from sucrose, glucose, trehalose, and maltose, and more preferably the sugar is sucrose. The pharmaceutical composition according to any one of claims 1 to 12.

14. further comprising one or more additives selected from polyols and metal chelating agents; Preferably, the polyol is one or more selected from glycerol, mannitol, and sorbitol, and the metal chelating agent is selected from ethylenediaminetetraacetic acid or a pharmaceutically acceptable salt thereof; More preferably, the polyol is mannitol and the metal chelating agent is ethylenediaminetetraacetic acid. The pharmaceutical composition according to any one of claims 1 to 13.

15. The pH of the pharmaceutical composition is 3.5 to 7, preferably 4 to 6.5, more preferably 4.2 to 6.2, and most preferably 4.5 to 6. The pharmaceutical composition according to any one of claims 1 to 14.

16. the concentration of the FAP / CD40 binding molecule is 0.01 mg / mL to 500 mg / mL, preferably 0.1 mg / mL to 400 mg / mL, more preferably 0.5 mg / mL to 200 mg / mL, and most preferably 1 mg / mL to 150 mg / mL; The pharmaceutical composition according to any one of claims 1 to 15.

17. The concentration of the buffer is 0.1 mM to 50 mM, preferably 0.5 mM to 40 mM, more preferably 1 mM to 30 mM, and most preferably 5 mM to 20 mM. The pharmaceutical composition according to any one of claims 1 to 16.

18. The concentration of the surfactant is 0.01 mg / mL to 10 mg / mL, preferably 0.05 mg / mL to 5 mg / mL, more preferably 0.1 mg / mL to 3 mg / mL, and most preferably 0.2 mg / mL to 2 mg / mL; The pharmaceutical composition according to any one of claims 12 to 17.

19. the concentration of the sugar is 1 mg / mL to 200 mg / mL, preferably 10 mg / mL to 150 mg / mL, more preferably 30 mg / mL to 120 mg / mL, and most preferably 50 mg / mL to 100 mg / mL; The pharmaceutical composition according to any one of claims 13 to 18.

20. the concentration of the additive is 0.001% w / v to 20% w / v, preferably 0.005% w / v to 15% w / v, more preferably 0.008% w / v to 10% w / v, and most preferably 0.01% w / v to 8% w / v; The pharmaceutical composition according to any one of claims 14 to 19.

21. 1. A pharmaceutical composition comprising: A FAP / CD40 binding molecule according to any one of claims 1 to 11; a histidine salt buffer, preferably a histidine hydrochloride buffer or a histidine acetate buffer, more preferably a histidine-histidine hydrochloride buffer or a histidine-acetate buffer; Polysorbate and Sucrose and Including, Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid. Pharmaceutical compositions.

22. Any one of the following sets of 1) to 4): 1) 0.01 mg / mL to 500 mg / mL of the FAP / CD40 binding molecule of any one of claims 1 to 11; 0.1 mM to 50 mM histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.01 mg / mL to 10 mg / mL of polysorbate, 1 mg / mL to 200 mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 0.1% w / v to 15% w / v mannitol or 0.001% w / v to 5% w / v ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 3.5 to 7; 2) 0.1 mg / mL to 400 mg / mL of the FAP / CD40 binding molecule of any one of claims 1 to 11; 0.5 mM to 40 mM histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.05 mg / mL to 5 mg / mL of polysorbate, 10 mg / mL to 150 mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 0.5% w / v to 12% w / v mannitol or 0.001% w / v to 1% w / v ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 4 to 6.5; 3) 0.5 mg / mL to 200 mg / mL of the FAP / CD40 binding molecule of any one of claims 1 to 11; 1 mM to 30 mM histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.1 mg / mL to 3 mg / mL of polysorbate, 30 mg / mL to 120 mg / mL sucrose; Optionally, the pharmaceutical composition further comprises 1% w / v to 10% w / v mannitol or 0.005% w / v to 0.1% w / v ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 4.2 to 6.2; 4) 1 mg / mL to 150 mg / mL of the FAP / CD40 binding molecule of any one of claims 1 to 11; a 5 mM to 20 mM histidine salt buffer, preferably a histidine hydrochloride buffer or a histidine acetate buffer, more preferably a histidine-histidine hydrochloride buffer or a histidine-acetate buffer; 0.2 mg / mL to 2 mg / mL of polysorbate, 50 mg / mL to 100 mg / mL sucrose, Optionally, the pharmaceutical composition further comprises 1% w / v to 10% w / v mannitol or 0.005% w / v to 0.1% w / v ethylenediaminetetraacetic acid; and the pH of the pharmaceutical composition is 4.5 to 6; Including, 22. The pharmaceutical composition of claim 21.

23. Any one of the following groups 1) to 21): 1) 1 mg / mL to 150 mg / mL of the FAP / CD40 binding molecule of any one of claims 1 to 11; about 10 mM histidine hydrochloride buffer or histidine acetate buffer, preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.2 mg / mL to 1 mg / mL of polysorbate 80, about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is 4.5 to 6; 2) a FAP / CD40 binding molecule according to any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 3) a FAP / CD40 binding molecule according to any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 4) a FAP / CD40 binding molecule according to any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 5) A FAP / CD40 binding molecule according to any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 6) A FAP / CD40 binding molecule according to any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 7) A FAP / CD40 binding molecule according to any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 8) A FAP / CD40 binding molecule according to any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 9) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL, preferably at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL; about 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 10) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 11) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 12) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 13) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.4 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 14) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 15) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 16) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 17) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.6 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; 18) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.8 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5; 19) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.8 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.2; 20) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.8 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3; 21) The FAP / CD40 binding molecule of any one of claims 1 to 11 at about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. about 10 mM histidine-acetate buffer, about 0.8 mg / mL polysorbate 80; about 80 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.5; Including, 22. The pharmaceutical composition of claim 21.

24. A lyophilized formulation comprising: The lyophilized formulation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 23, or the lyophilized formulation can be obtained by lyophilizing the pharmaceutical composition according to any one of claims 1 to 23. Lyophilized formulation.

25. 25. The freeze-dried formulation according to claim 24, which is prepared by reconstituting the freeze-dried formulation. Reconstitution solution.

26. A container containing the pharmaceutical composition according to any one of claims 1 to 23, the lyophilized formulation according to claim 24, or the reconstitution solution according to claim 25. product.

27. Use of the pharmaceutical composition according to any one of claims 1 to 23, the lyophilized formulation according to claim 24, or the reconstituted solution according to claim 25 in the preparation of a medicament for treating or alleviating a disease or condition, comprising: Preferably, the disease or condition is a tumor or cancer; 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 are more preferred. use.

28. 1. A method of treating or alleviating a tumor or cancer, comprising: Administering a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 23, the lyophilized formulation according to claim 24, or the reconstituted solution according to claim 25 to a subject in need thereof, The tumor or cancer is preferably 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, or blood system cancer; method.

Images