Pharmaceutical composition of FAP / CD40 binding molecule and medical application thereof

Abstract

The invention relates to a pharmaceutical composition of FAP / CD40 binding molecules and medical application thereof. In particular, a pharmaceutical composition comprises a FAP / CD40 binding molecule and a buffering agent. The pharmaceutical composition has good biological activity and stability.

Description

A pharmaceutical composition of FAP / CD40 binding molecules and its medical use

[0001] This disclosure claims priority to Chinese patent application 202310174524.1 filed on February 24, 2023, and the entire contents of the aforementioned patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition comprising a FAP / CD40 binding molecule, and pharmaceutical uses thereof. Background Art

[0003] CD40 (TNFRSF5) is a transmembrane phosphorylated glycoprotein that belongs to the tumor necrosis factor receptor superfamily (TNFRS). CD40 is expressed in a variety of cell types, including B cells, follicular dendritic cells (DCs), epithelial cells, monocytes, macrophages, smooth muscle cells, and tumor cells. Its ligand CD40L is mainly expressed in activated T cells, activated B cells, platelets, and smooth muscle cells. CD40L binding causes CD40 multimerization, generating downstream activation, growth, and differentiation signals. CD40 signal transduction activates multiple downstream signaling pathways, such as NF-κB, MAPK, and STAT3 (Pype S, et al. J Biol Chem. 2000 Jun. 16; 275(24): 1858693). These pathways regulate gene expression by regulating the activating protein c-Jun, ATF2, and Rel transcription factors. The binding of CD40 to CD40L induces resting B cell proliferation, immunoglobulin switching, and antibody secretion, and plays an important role in tissue germinal center development and B cell survival, all of which are essential for humoral immune responses (Kehry M RJ Immunol 1996; 156: 2345-2348). CD40L binds to CD40 on DCs, inducing DC maturation, which is manifested by upregulation of the B7 family of co-stimulatory factors (CD80, CD86) and increased secretion of proinflammatory cytokines such as interleukin-12 (IL-12). The interaction between CD40 and CD40L provides co-stimulatory signals for T cell activation and promotes DC cells to present antigens to T cells.

[0004] With the clinical success of immune checkpoint inhibition (ICI) therapies targeting CD40, CTLA-4, and PD-L1 in cancer treatment, immunotherapy has become the most anticipated research area in the third generation of cancer treatment. Anti-tumor immunotherapies that activate CD40 to enhance antigen presentation and activation of T cells by DCs have demonstrated clinical efficacy. However, systemic CD40 activation can lead to target-specific toxicities such as cytokine storm, hepatotoxicity, hematotoxicity, and venous thrombosis caused by peripheral CD40 activation. These toxicities limit the therapeutic window of CD40 agonist antibodies. Clinical studies have been terminated for Pfizer's CD40 agonists CP-870 and 893, and for Medimmune's CD40L-Fc fusion protein MEDI5083 (AstraZeneca's novel fusion protein that activates the CD40 signaling pathway, consisting of three tandem CD40Ls and IgG4-Fc). In the development of second-generation CD40 agonist antibodies, researchers hope to mediate CD40 activation through tumor-associated antigen (TAA), so that CD40 is specifically activated inside the tumor, while reducing the activation of peripheral CD40, thereby increasing 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, but is selectively expressed in 93% of tumor tissues, 30% of which are highly expressed, such as in colon cancer, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, bladder cancer, and oral squamous cell carcinoma. WO2023025194 provides an anti-FAP antibody, an anti-CD40 antibody, and a bispecific antibody combining the two. The bispecific antibody can mediate tumor-specific CD40 activation through FAP, promote maturation and activation of APCs (e.g., dendritic cells (DCs), eliminate liver toxicity, peripheral blood toxicity, and other peripheral toxicities, and has an excellent dosing window and drugability.

[0006] Antibody drugs have large molecular weights and complex structures, making them susceptible to degradation, aggregation, and undesirable chemical modifications, leading to instability. To ensure that antibodies are suitable for administration and maintain stability during storage and subsequent use, leading to optimal efficacy, research into stable formulations of antibody drugs is crucial. For new FAP / CD40 binding molecules, the development of suitable pharmaceutical compositions remains a challenge.

[0007] Summary of the Invention

[0008] The present disclosure provides a pharmaceutical composition containing a FAP / CD40 binding molecule, wherein the composition has excellent stability.

[0009] The present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule and a buffer, wherein the buffer is selected from an acetate buffer, a succinate buffer, a histidine salt buffer, or a phosphate buffer. In some embodiments, the buffer is a histidine salt 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.

[0010] In some embodiments, the FAP / CD40 binding molecule comprises a first antigen binding domain that specifically binds FAP and a second antigen binding domain that specifically binds CD40, wherein the second antigen binding domain that specifically binds CD40 comprises (at least one) immunoglobulin single variable domain.

[0011] In some embodiments, the FAP / CD40 binding molecule comprises one immunoglobulin single variable domain that specifically binds CD40. In other embodiments, the FAP / CD40 binding molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunoglobulin single variable domains that specifically bind CD40, which can be the same or different.

[0012] In some embodiments, the FAP / CD40 binding molecule comprises at least one (e.g., 2, 3, 4) antigen binding domains that specifically bind to FAP.

[0013] 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 shown in SEQ ID NOs: 12, 13, and 14, respectively.

[0014] In some embodiments, in the aforementioned FAP / CD40 binding molecules, the immunoglobulin single variable domain that specifically binds to CD40 comprises or is as shown in any one of SEQ ID NOs: 11, 16 to 19, or has a sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% to any one of SEQ ID NOs: 23, 35 to 38.

[0015] In some embodiments, 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:

[0016] 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.

[0017] In some embodiments, in the aforementioned FAP / CD40-binding molecules, the heavy chain variable region of the first antigen-binding domain that specifically binds to FAP comprises an amino acid sequence as shown in SEQ ID NO: 1, or at least 80% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or at least 80% identical thereto.

[0018] In some embodiments, the FAP / CD40 binding molecule further comprises a human immunoglobulin Fc region (eg, a human IgG1 or IgG4 Fc region).

[0019] In some embodiments, in the aforementioned FAP / CD40 binding molecule, the first antigen-binding domain that specifically binds to FAP comprises a heavy chain (HC) and a light chain (LC);

[0020] For example, the heavy chain is of the IgG1 or IgG4 isotype, and the light chain is of the kappa isotype.

[0021] In some embodiments, the heavy chain is an amino acid sequence as set forth in SEQ ID NO: 24, or at least 80% identical thereto, and the light chain is an amino acid sequence as set forth in SEQ ID NO: 25, or at least 80% identical thereto.

[0022] In some embodiments, in the aforementioned FAP / CD40 binding molecules, the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region and a light chain variable region, wherein:

[0023] The immunoglobulin single variable domain that specifically binds to the second antigen-binding domain of CD40 is located at the N-terminus of the heavy chain variable region of the first antigen-binding domain that specifically binds to FAP;

[0024] The immunoglobulin single variable domain that specifically binds to the second antigen-binding domain of CD40 is located at the C-terminus of the heavy chain variable region of the first antigen-binding domain that specifically binds to FAP;

[0025] The immunoglobulin single variable domain that specifically binds to the second antigen-binding domain of CD40 is located at the N-terminus of the light chain variable region of the first antigen-binding domain that specifically binds to FAP; and / or

[0026] The immunoglobulin single variable domain that specifically binds to the second antigen-binding domain of CD40 is located at the C-terminus of the light chain variable region of the first antigen-binding domain that specifically binds to FAP.

[0027] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to the second antigen-binding domain of CD40 is connected to the first antigen-binding domain that specifically binds to FAP directly or through a linker; for example, the linker is a single variable domain having a structure such as (G4S) x The amino acid sequence shown, wherein x is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); for example, the linker is the amino acid sequence shown by (G4S)2, (G4S)3, or (G4S)4.

[0028] In some embodiments, the second antigen-binding domain that specifically binds to CD40 in the aforementioned FAP / CD40-binding molecule is multivalent (e.g., one aforementioned FAP / CD40-binding molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 second antigen-binding domains that specifically bind to CD40). In some specific embodiments, the second antigen-binding domain that specifically binds to CD40 is bivalent, tetravalent, or hexavalent. In some specific embodiments, the second antigen-binding domain that specifically binds to CD40 comprises 2, 3, 4, 5, or 6 of the aforementioned immunoglobulin single variable domains.

[0029] 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 as set forth in any one of SEQ ID NOs: 21-23, or at least 80% identical thereto, and the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 20, or at least 80% identical thereto. In some specific embodiments, the FAP / CD40 binding molecule comprises two first polypeptide chains and two second polypeptide chains; in some specific embodiments, the two first polypeptide chains are identical, and the two second polypeptide chains are identical.

[0030] In some embodiments, the aforementioned FAP / CD40 binding molecules are capable of inhibiting tumor growth (e.g., increase in size, increase in tumor weight) and / or metastasis (e.g., multi-organ or multi-tissue metastasis, distant metastasis) by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0031] In some embodiments, the aforementioned FAP / CD40 binding molecule is an anti-FAP / CD40 bispecific antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment includes but is not limited to: Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv), for example, scFv, Fv, Fab or Fab' fragment.

[0032] In some embodiments, the anti-FAP / CD40 bispecific antibody contains the aforementioned immunoglobulin single variable domain in the second antigen-binding domain that specifically binds to CD40, and the aforementioned heavy chain variable region (VH) and light chain variable region (VL) in the first antigen-binding domain that specifically binds to FAP.

[0033] In some embodiments, in the anti-FAP / CD40 bispecific antibody:

[0034] The first antigen-binding domain that specifically binds to FAP is a first antibody, which includes a heavy chain (HC) and a light chain (LC); and

[0035] The second antigen-binding domain that specifically binds to CD40 is a second antibody, which is a VHH and has CDR1, CDR2, and CDR3 of the aforementioned CD40-binding molecule.

[0036] In some embodiments, the VHH is located at the N-terminus and / or C-terminus of the heavy chain or light chain of the first antibody as the second antibody.

[0037] In some specific embodiments, the anti-FAP / CD40 bispecific antibody comprises a first antibody and a second antibody comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 2, 4, or 6) VHHs; the first antibody comprises two HCs and two LCs, the VH of one HC of the first antibody forms an antigen-binding site with the VL of one LC, and the VH of the other HC forms an antigen-binding site with the VL of the other LC.

[0038] In some specific embodiments, the first antibody of the anti-FAP / CD40 bispecific antibody or antigen-binding fragment thereof can be linked to 1, 2, 3, 4, 5, 6, 7, or 8 VHH second antibodies, and the VHH second antibodies can be the same or different, and can all be linked to the heavy chain N-terminus of the first antibody, or all be linked to the heavy chain C-terminus of the first antibody, or all be linked to the light chain N-terminus of the first antibody, or all be linked to the light chain C-terminus of the first antibody, or any combination of the heavy chain N-terminus, heavy chain C-terminus, light chain N-terminus, and light chain C-terminus.

[0039] In some embodiments, the anti-FAP / CD40 bispecific antibody comprises a first polypeptide chain and a second polypeptide chain:

[0040] (i) The first polypeptide chain, from N-terminus to C-terminus, is: [heavy chain of the first antibody] - linker 1 - [second antibody]; the second polypeptide chain is the light chain of the first antibody;

[0041] (ii) the first polypeptide chain, from N-terminus to C-terminus, is: [heavy chain of the first antibody]-linker 1-[second antibody] 1-linker 2-[second antibody] 2; the second polypeptide chain is the light chain of the first antibody;

[0042] (iii) the first polypeptide chain, from N-terminus to C-terminus, is: [heavy chain of the first antibody]-linker 1-[second antibody] 1-linker 2-[second antibody] 2-linker 3-[second antibody] 3; the second polypeptide chain is the light chain of the first antibody;

[0043] (iv) the first polypeptide chain, from N-terminus to C-terminus, is: [second antibody]-linker 1-[heavy chain of first antibody]; the second polypeptide chain is the light chain of the first antibody;

[0044] (v) the first polypeptide chain is, from N-terminus to C-terminus: [second antibody]2-linker2-[second antibody]1-linker1-[heavy chain of first antibody]; the second polypeptide chain is the light chain of the first antibody;

[0045] (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]; the second polypeptide chain is the light chain of the first antibody;

[0046] (vii) the first polypeptide chain is, from N-terminus to C-terminus, [second antibody] 1 - linker 1 - [heavy chain of first antibody] - linker 2 - [second antibody] 2; the second polypeptide chain is the light chain of the first antibody;

[0047] (viii) the first polypeptide chain is, from N-terminus to C-terminus, [second antibody] 1 - linker 1 - [second antibody] 2 - linker 2 - [heavy chain of first antibody] - linker 3 - [second antibody] 3; the second polypeptide chain is the light chain of the first antibody;

[0048] (ix) the first polypeptide chain is, from N-terminus to C-terminus, [second antibody] 1 - linker 1 - [heavy chain of first antibody] - linker 2 - [second antibody] 2 - linker 3 - [second antibody] 3; the second polypeptide chain is the light chain of the first antibody;

[0049] [Second antibody] 1, [Second antibody] 2, and [Second antibody] 3 may be the same or different.

[0050] In some embodiments, the second antibody of the VHH in the aforementioned anti-FAP / CD40 bispecific antibody is directly or via a linker connected to the first antibody. The linker (linker) is selected from: m S n ) x or (GGNGT) x or (YGNGT) x The amino acid sequence shown in FIG1 , wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and x is independently selected from an integer of 1-20 (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 the amino acid sequence shown in 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.

[0051] 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 can be a full-length antibody.

[0052] In some embodiments, the heavy chain of the first antibody of the anti-FAP / CD40 bispecific antibody is of IgG isotype, such as IgG1, IgG2, IgG3, or IgG4, for example, of IgG1 isotype; and / or the light chain of the first antibody is of Kappa isotype.

[0053] 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.

[0054] 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.

[0055] 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 chain independently comprises a VHH of the second antibody and the heavy chain (HC) of the first antibody; and b) the second polypeptide chain independently comprises a light chain (LC) of the first antibody; wherein the VHH is connected to the N-terminus and / or C-terminus of the HC of the second antibody via a linker;

[0056] Alternatively, i) the first polypeptide chains each independently comprise the heavy chain (HC) of the first antibody; and ii) the second polypeptide chains each independently comprise the VHH of the second antibody and the light chain (LC) of the first antibody; wherein the VHH is linked directly or via a linker to the N-terminus and / or C-terminus of the LC of the first antibody.

[0057] In some embodiments, the anti-FAP / CD40 bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0058] 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 remove or reduce the Fc effector function of IgG, including but not limited to: N297A or D265A / N297A of IgG1, or L234A / L235A, L234A / L235A / P329G, L234E, L234F, L234E / L235F, L234E / L235F / P329G on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N234A / L235A on IgG2 or IgG4 297A, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M on IgG1, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgG1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4.

[0059] In the context of mutations contained in the Fc region herein, " / " means "and", for example, "D265A / N297A" means "D265A and N297A", ​​that is, the Fc contains D265A and N297A mutations; the mutated amino acid positions are numbered according to the EU numbering system.

[0060] In some embodiments, the aforementioned FAP / CD40 binding molecules or anti-FAP / CD40 bispecific antibodies have one or more of the following features:

[0061] (a)≤10 -7 K D The value binds to human FAP or its epitope;

[0062] (b) ≤10 -7 K D The value binds to human CD40 or its epitope;

[0063] (c) inducing immune stimulation of CD40-expressing antigen-presenting cells (APCs);

[0064] (d) increasing the activation of APCs (e.g., dendritic cells) and / or promoting the proliferation of APCs (e.g., dendritic cells);

[0065] (e) stimulating tumor-specific T cell responses;

[0066] (f) causing or promoting apoptosis of tumor cells; and / or

[0067] (g) inhibiting tumor growth and / or metastasis.

[0068] In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 0.01 mg / mL - 500 mg / mL, such as 0.05 mg / mL - 450 mg / mL, 0.05 mg / mL - 400 mg / mL, 0.05 mg / mL - 350 mg / mL, 0.05 mg / mL - 300 mg / mL, 0.05 mg / mL - 250 mg / mL, 0.05 mg / mL - 200 mg / mL, 0.05 mg / mL - 150 mg / mL, 0.05 mg / mL - 140 mg / mL, 0.05 mg / mL - 130 mg / mL, 0.05 mg / mL - 120 mg / mL, 0.05 mg / mL - 110 mg / mL, 0.05 mg / mL - 100 mg / mL, 0.1 mg / mL - 400 mg / mL, 0.1 mg / mL - 350 mg / mL, 0.1 mg / mL - 300 mg / mL, 0.1 mg / mL - 250 mg / mL, 0.1 mg / mL - 200 mg / mL, 0.1 mg / mL - 150 mg / mL, 0.1 mg / mL - 140 mg / mL, 0.1 mg / mL - 130 mg / mL, 0.1 mg / mL - 120 mg / mL, 0.1 mg / mL - 110 mg / mL, 0.1 mg / mL - 100 mg / mL, 0.5 mg / mL - 350 mg / mL, 0.5 mg / mL - 300 mg / mL, 0.5 mg / mL - 250 mg / mL, 0.5 mg / mL - 200 mg / mL, 0.5 mg / mL - 150 mg / mL, 0.5 mg / mL - 140 mg / mL, 0.5 mg / mL - 130 mg / mL, 0.5 mg / mL - 120 mg / mL, 0.5 mg / mL - 110 mg / mL, 0.5 mg / mL - 100 mg / mL, 1 mg / mL - 300 mg / mL, 1 mg / mL - 250 mg / mL, 1 mg / mL - 200 mg / mL, 1 mg / mL - 150 mg / mL, 1 mg / mL - 140 mg / mL, 1 mg / mL - 130 mg / mL, 1 mg / mL - 120 mg / mL, 1 mg / mL - 110 mg / mL, 1 mg / mL - 100 mg / mL, 1 mg / mL - 95 mg / mL, 1 mg / mL - 90 mg / mL, 1 mg / mL - 85 mg / mL, 1 mg / mL - 80 mg / mL, 1 mg / mL - 75 mg / mL, 1 mg / mL - 70 mg / mL, 1 mg / mL - 65 mg / mL, 1 mg / mL - 60 mg / mL, 1 mg / mL - 55 mg / mL, 1 mg / mL - 50 mg / mL, 1 mg / mL - 45 mg / mL, 1 mg / mL - 40 mg / mL, 1 mg / mL - 35 mg / mL,1mg / mL-30mg / mL、1mg / mL-25mg / mL、1mg / mL-20mg / mL、1mg / mL-15mg / mL、5mg / mL-90mg / mL、5mg / mL-85mg / mL、5mg / mL-80mg / mL、5mg / mL-75mg / mL、5mg / mL-70mg / mL、5mg / mL-65mg / mL、5mg / mL-60mg / mL、5mg / mL-55mg / mL、5mg / mL-50mg / mL、5mg / mL-45mg / mL、5mg / mL-40mg / mL、5mg / mL-35mg / mL、5mg / mL-30mg / mL、5mg / mL-25mg / mL、5mg / mL-20mg / mL、5mg / mL-15mg / mL、8mg / mL-12mg / mL、30mg / mL-250mg / mL、30mg / mL-200mg / mL、30mg / mL-190mg / mL、30mg / mL-180mg / mL、30mg / mL-170mg / mL、30mg / mL-160mg / mL、30mg / mL-150mg / mL、30mg / mL-140mg / mL、30mg / mL-130mg / mL、30mg / mL-120mg / mL、30mg / mL-110mg / mL、30mg / mL-100mg / mL、50mg / mL-200mg / mL、50mg / mL-190mg / mL、50mg / mL-180mg / mL、50mg / mL-170mg / mL、50mg / mL-160mg / mL、50mg / mL-150mg / mL、50mg / mL-140mg / mL、50mg / mL-130mg / mL、50mg / mL-120mg / mL、50mg / mL-110mg / mL、50mg / mL-100mg / mL、70mg / mL-180mg / mL、70mg / mL-170mg / mL、70mg / mL-160mg / mL、70mg / mL-150mg / mL、70mg / mL-140mg / mL、70mg / mL-130mg / mL、70mg / mL-120mg / mL、70mg / mL-110mg / mL、70mg / mL-100mg / mL、90mg / mL-110mg / mL、In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 95 mg / mL-105 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 0.1 mg / mL-400 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 0.5 mg / mL-200 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 1 mg / mL-150 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 5 mg / mL-45 mg / mL. In some embodiments, the concentration of the FAP / CD40 binding molecule in the pharmaceutical composition is 50 mg / mL-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.

[0069] In some embodiments, the concentration of the buffer in the pharmaceutical composition is 0.1 mM-50 mM, for example, 0.1 mM-45 mM, 0.1 mM-40 mM, 0.1 mM-35 mM, 0.1 mM-30 mM, 0.1 mM-25 mM, 0.1 mM-20 mM, 0.1 mM-15 mM, 0.1 mM-10 mM, 0.5 mM-45 mM, 0.5 mM-40 mM, 0.5 mM-35 mM, 0.5 mM-30 mM, 0.5 mM-25 mM, 0.5 mM-20 In some embodiments, the concentration of the buffer in the pharmaceutical composition is 0.5 mM to 40 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is 1 mM to 30 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is 5mM-20mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM or about 30mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is at least 50mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is at least 10 mM.

[0070] In some embodiments, the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is selected from one or more of polysorbates and poloxamers. In some embodiments, the polysorbate is selected from one or more of polysorbate 20 and polysorbate 80. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80.

[0071] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.01 mg / mL-10 mg / mL, for example, 0.01 mg / mL-9 mg / mL, 0.01 mg / mL-8 mg / mL, 0.01 mg / mL-7 mg / mL, 0.01 mg / mL-6 mg / mL, 0.01 mg / mL-5 mg / mL, 0.01 mg / mL-4 mg / mL, 0.01 mg / mL-3 mg / mL, 0.01 mg / mL-2 mg / mL, 0.01 mg / mL-1 mg / mL, 0.01 mg / mL-0.5 mg / mL, 0.05 mg / mL-8 mg / 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 / mL-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-1mg / 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 0.6 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.

[0072] In some embodiments, the pharmaceutical composition further comprises a carbohydrate. In some embodiments, the carbohydrate is selected from one or more of sucrose, glucose, trehalose, and maltose. In some embodiments, the carbohydrate is sucrose.

[0073] In some embodiments, the concentration of the carbohydrate in the pharmaceutical composition is 1 mg / mL-200 mg / mL, for example, 1 mg / mL-150 mg / mL, 1 mg / mL-140 mg / mL, 1 mg / mL-130 mg / mL, 1 mg / mL-120 mg / mL, 1 mg / mL-110 mg / mL, 1 mg / mL-100 mg / mL, 1 mg / mL-95 mg / mL, 1 mg / mL-90 mg / mL, 1 mg / mL-85 mg / mL, 1 mg / mL-80 mg / mL, 10 mg / mL-150 mg / mL, 10 mg / mL-140 mg / mL, 10mg / mL-130mg / mL, 10mg / mL-120mg / mL, 10mg / mL-110mg / mL, 10mg / mL-100mg / mL, 10mg / mL-9 5mg / mL, 10mg / mL-90mg / mL, 10mg / mL-85mg / mL, 10mg / mL-80mg / mL, 20mg / mL-130mg / mL, 20mg / m L-120mg / mL, 20mg / mL-110mg / mL, 20mg / mL-100mg / mL, 20mg / mL-95mg / mL, 20mg / mL-90mg / mL, 2 0mg / mL-85mg / mL, 20mg / mL-80mg / mL, 30mg / mL-120mg / mL, 30mg / mL-110mg / mL, 30mg / mL-100mg / mL, 30mg / mL-95mg / mL, 30mg / mL-90mg / mL, 30mg / mL-85mg / mL, 30mg / mL-80mg / mL, 50mg / mL-11 0mg / mL, 50mg / mL-100mg / mL, 50mg / mL-95mg / mL, 50mg / mL-90mg / mL, 50mg / mL-85mg / mL, 50mg / m L-80mg / mL, 60mg / mL-100mg / mL, 60mg / mL-95mg / mL, 60mg / mL-90mg / mL, 60mg / mL-85mg / mL, 60m g / mL-80mg / mL, 70mg / mL-95mg / mL, 70mg / mL-90mg / mL, 70mg / mL-85mg / mL or 70mg / mL-80mg / mL. In some embodiments, the concentration of the saccharide in the pharmaceutical composition is 10 mg / mL to 150 mg / mL. In some embodiments, the concentration of the saccharide in the pharmaceutical composition is 30 mg / mL to 120 mg / mL. In some embodiments, the concentration of the saccharide in the pharmaceutical composition is 50 mg / mL to 100 mg / mL.In some embodiments, the concentration of carbohydrates 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 carbohydrates in the pharmaceutical composition is about 80 mg / mL. In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is at least 200 mg / mL. In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is at least 80 mg / mL.

[0074] In some embodiments, the pharmaceutical composition further optionally comprises one or more other excipients selected from a polyol and a metal chelator. In some embodiments, the polyol is selected from one or more of 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.

[0075] In some embodiments, the concentration of other excipients in the pharmaceutical composition is 0.001% w / v - 20% w / v, such as 0.005% w / v - 20% w / v, 0.005% w / v - 15% w / v, 0.005% w / v - 10% w / v, 0.005% w / v - 9% w / v, 0.005% w / v - 8% w / v, 0.005% w / v - 7% w / v, 0.005% w / v - 6% w / v, 0.005% w / v - 5% w / v, 0.005% w / v - 4% w / v, 0.005% w / v - 3% w / v, 0.005% w / v - 2% w / v, 0.005% w / v - 1% w / v, 0.005% w / v - 0.1% w / v, 0.005% w / v - 0.05% w / v, 0.008% w / v - 15% w / v, 0.008% w / v - 10% w / v, 0.008% w / v - 9% w / v, 0.008% w / v - 8% w / v, 0.008% w / v - 7% w / v, 0.008% w / v - 6% w / v, 0.008% w / v - 5% w / v, 0.008% w / v - 4% w / v, 0.008% w / v - 3% w / v, 0.008% w / v - 2% w / v, 0.008% w / v - 1% w / v, 0.008% w / v - 0.1% w / v, 0.008% w / v - 0.05% w / v, 0.01% w / v - 10% w / v, 0.01% w / v - 9% w / v, 0.01% w / v - 8% w / v, 0.01% w / v - 7% w / v, 0.01% w / v - 6% w / v, 0.01% w / v - 5% w / v, 0.01% w / v - 4% w / v, 0.01% w / v - 3% w / v, 0.01% w / v - 2% w / v, 0.01% w / v - 1% w / v, 0.01% w / v - 0.1% w / v, 0.01% w / v - 0.05% w / v, 0.1% w / v - 15% w / v, 0.1% w / v - 10% w / v, 0.1% w / v - 10% w / v, 0.1% w / v - 9% w / v, 0.1% w / v - 8% w / v, 0.1% w / v - 7% w / v, 0.1% w / v - 6% w / v, 0.1% w / v - 5% w / v, 1% w / v - 15% w / v, 1% w / v - 10% w / v, 1% w / v - 9% w / v, 1% w / v - 8% w / v, 1% w / v - 7% w / v, 1% w / v - 6% w / v, 1% w / v - 5% w / v, 0.001% w / v - 5% w / v, 0.001% w / v - 4% w / v, 0.001% w / v - 3% w / v, 0.001% w / v - 2% w / v, 0.001% w / v - 1% w / v, 0.001% w / v - 0.1% w / v or 0.001% w / v - 0.05% w / v.In some embodiments, the concentration of the other excipients in the pharmaceutical composition is 0.005% w / v to 15% w / v. In some embodiments, the concentration of the other excipients in the pharmaceutical composition is 0.008% w / v to 10% w / v. In some embodiments, the concentration of the other excipients in the pharmaceutical composition is 0.01% w / v to 8% w / v.

[0076] In some embodiments, the concentration of mannitol in the pharmaceutical composition is 0.1% w / v-15% w / v, such as 0.5% w / v-12% w / v, 0.5% w / v-11% w / v, 0.5% w / v-10% w / v, 0.5% w / v-9% w / v, 0.5% w / v-8% w / v, 0.5% w / v-7% w / v, 0.5% w / v-6% w / v, 0.5% w / v-5% w / v, 1% w / v-10% w / v, 1% w / v-9% w / v, 1% w / v-8% w / v, 1% w / v-7% w / v, 1% w / v-6% w / v, or 1% w / v-5% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is 0.5% w / v-12% w / v. In some embodiments, the concentration of mannitol in the pharmaceutical composition is 1% w / v-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.

[0077] In some embodiments, the concentration of EDTA in the pharmaceutical composition is 0.001% w / v-5% w / v, for example, 0.001% w / v-4% w / v, 0.001% w / v-3% w / v, 0.001% w / v-2% w / v, 0.001% w / v-1% w / v, 0.001% w / v-0.1% w / v, 0.001% w / v-0.05% w / v, 0.005% w / v-4% w / v, 0.005% w / v-3% w / v, 0.005% w / v-2% w / v, 0.005% w / v-1% w / v, 0.005% w / v-0.1% w / v, or 0.005% w / v-0.05% w / v. In some embodiments, the concentration of EDTA in the pharmaceutical composition is between 0.001% w / v and 1% w / v. In some embodiments, the concentration of EDTA in the pharmaceutical composition is between 0.005% w / v and 0.1% w / v. In some embodiments, the concentration of EDTA 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 EDTA in the pharmaceutical composition is about 0.01% w / v. In some embodiments, the concentration of EDTA in the pharmaceutical composition is at least 5% w / v. In some embodiments, the concentration of EDTA in the pharmaceutical composition is at least 0.01% w / v.

[0078] In some embodiments, the pH of the buffer in the pharmaceutical composition is 3.5-9, e.g., 3.5-8.5, 3.5-8, 3.5-7.5, 3.5-7, 3.5-6.9, 3.5-6.8, 3.5-6.7, 3.5-6.6, 3.5-6.5, 3.5-6.4, 3.5-6.3, 3.5-6.2, 3.5-6.1, 3.5-6, 3.5-5.9, 3.5-5.8, 3.5-5.7, 3.5 -5.6, 3.5-5.5, 4-5.4, 4-5.3, 4-5.2, 4-5.1, 4-5.0, 4-8.5, 4-8, 4-7.5, 4-7, 4-6.9, 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, 4.8-6.2, 4.8-6.1, 4.8-6, 4.8-5.9, 4.8-5.8, 4.8-5.7, 4.8-5.6, 4.8-5.5, 5-6.In some embodiments, the pH of the buffer in the pharmaceutical composition is 3.5-7. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.0-6.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.2-6.2. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.5-6.0. 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.

[0079] In some embodiments, the pharmaceutical composition further comprises a pH adjusting agent, such as sodium hydroxide and / or hydrochloric acid.

[0080] In some embodiments, the pH of the pharmaceutical composition differs by no more than ±0.5 from the pH of the buffer it contains. In some embodiments, the pH of the pharmaceutical composition is 3.5-9, e.g., 3.5-8.5, 3.5-8, 3.5-7.5, 3.5-7, 3.5-6.9, 3.5-6.8, 3.5-6.7, 3.5-6.6, 3.5-6.5, 3.5-6.4, 3.5-6.3, 3.5-6.2, 3.5-6.1, 3.5-6, 3.5-5.9, 3.5-5.8, 3.5-5.7, 3.5-5.6, 3.5-5.5, 4-5.4, 4-5.3, 4-5.2, 4-5.1, 4-5.0, 4-8.5, 4-8, 4-7.5, 4-7, 4-6.9, 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, 4.8-6.2, 4.8-6.1, 4.8-6, 4.8.In some embodiments, the pH of the pharmaceutical composition is 3.5-7. In some embodiments, the pH of the pharmaceutical composition is 4.0-6.5. In some embodiments, the pH of the pharmaceutical composition is 4.2-6.2. In some embodiments, the pH of the pharmaceutical composition is 4.5-6.0. 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.

[0081] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule (e.g., Ab10-A297V3-4, whose heavy chain and light chain sequences are SEQ ID NOs: 22 and 20, respectively), comprising any one of the following groups 1) to 4):

[0082] 1) FAP / CD40 binding molecule;

[0083] Histidine salt buffers, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0084] 2) FAP / CD40 binding molecule;

[0085] Histidine buffer;

[0086] Polysorbates, such as polysorbate 80;

[0087] sucrose;

[0088] Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid;

[0089] 3) FAP / CD40 binding molecule;

[0090] Histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0091] Polysorbates, such as polysorbate 80;

[0092] sucrose;

[0093] Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid;

[0094] 4) FAP / CD40 binding molecule;

[0095] Histidine acetate buffers, such as histidine-acetate buffer;

[0096] Polysorbates, such as polysorbate 80;

[0097] sucrose;

[0098] Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid;

[0099] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule (e.g., Ab10-A297V3-4, whose heavy chain and light chain sequences are SEQ ID NOs: 22 and 20, respectively), which is any one of the following groups 1) to 4):

[0100] 1) FAP / CD40 binding molecule; histidine salt buffer; polysorbate; sucrose and water for injection.

[0101] 2) FAP / CD40 binding molecule; histidine hydrochloride buffer; polysorbate; sucrose and water for injection.

[0102] 3) FAP / CD40 binding molecule; histidine-histidine hydrochloride buffer; polysorbate; sucrose and water for injection.

[0103] 4) FAP / CD40 binding molecule; histidine-acetate buffer; polysorbate; sucrose and water for injection.

[0104] 5) The pharmaceutical composition according to any one of 1) to 4) above, wherein the polysorbate is polysorbate 80.

[0105] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule (e.g., Ab10-A297V3-4 whose heavy chain and light chain sequences are SEQ ID NOs: 22 and 20, respectively), comprising any one of the following groups 1) to 5):

[0106] 1) 0.01 mg / mL-500 mg / mL FAP / CD40 binding molecule;

[0107] 0.1 mM-50 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0108] 0.01mg / mL-10mg / mL polysorbate;

[0109] 1mg / mL-200mg / mL sucrose;

[0110] Optionally, the pharmaceutical composition further comprises 0.1% w / v-15% w / v mannitol or 0.001% w / v-5% w / v ethylenediaminetetraacetic acid;

[0111] and the pH of the pharmaceutical composition is 3.5-7;

[0112] 2) 0.1 mg / mL-400 mg / mL FAP / CD40 binding molecule;

[0113] 0.5 mM-40 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0114] 0.05mg / mL-5mg / mL polysorbate;

[0115] 10mg / mL-150mg / mL sucrose;

[0116] Optionally, the pharmaceutical composition further comprises 0.5% w / v-12% w / v mannitol or 0.001% w / v-1% w / v ethylenediaminetetraacetic acid;

[0117] and the pH of the pharmaceutical composition is 4-6.5;

[0118] 3) 0.5 mg / mL-200 mg / mL FAP / CD40 binding molecule;

[0119] 1 mM-30 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0120] 0.1mg / mL-3mg / mL polysorbate;

[0121] 30mg / mL-120mg / mL sucrose;

[0122] Optionally, the pharmaceutical composition further comprises 1% w / v-10% w / v mannitol or 0.005% w / v-0.1% w / v ethylenediaminetetraacetic acid;

[0123] and the pH of the pharmaceutical composition is 4.2-6.2;

[0124] 4) 1 mg / mL-150 mg / mL FAP / CD40 binding molecule;

[0125] 5 mM-20 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0126] 0.2mg / mL-2mg / mL polysorbate;

[0127] 50mg / mL-100mg / mL sucrose;

[0128] Optionally, the pharmaceutical composition further comprises 1% w / v-10% w / v mannitol or 0.005% w / v-0.1% w / v ethylenediaminetetraacetic acid;

[0129] and the pH of the pharmaceutical composition is 4.5-6;

[0130] 5) 1 mg / mL-40 mg / mL FAP / CD40 binding molecule;

[0131] 5 mM-20 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer;

[0132] 0.2mg / mL-2mg / mL polysorbate;

[0133] 50mg / mL-100mg / mL sucrose;

[0134] Optionally, the pharmaceutical composition further comprises 1% w / v-10% w / v mannitol or 0.005% w / v-0.1% w / v ethylenediaminetetraacetic acid;

[0135] The pH of the pharmaceutical composition is 4.5-6.

[0136] 6) 50 mg / mL-150 mg / mL FAP / CD40 binding molecule;

[0137] 5 mM-20 mM histidine acetate buffer, such as histidine-acetate buffer;

[0138] 0.2mg / mL-2mg / mL polysorbate;

[0139] 50mg / mL-100mg / mL sucrose;

[0140] Optionally, the pharmaceutical composition further comprises 1% w / v-10% w / v mannitol or 0.005% w / v-0.1% w / v ethylenediaminetetraacetic acid;

[0141] The pH of the pharmaceutical composition is 4.5-6.

[0142] 7) The pharmaceutical composition according to any one of 1) to 6) above, further comprising water for injection.

[0143] 8) The pharmaceutical composition according to any one of 1) to 4) above, wherein the polysorbate is polysorbate 80.

[0144] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a FAP / CD40 binding molecule (e.g., Ab10-A297V3-4, whose heavy chain and light chain sequences are SEQ ID NOs: 22 and 203, respectively), which comprises or is any one of the following groups 1) to 25):

[0145] 1) 1-150 mg / mL FAP / CD40 binding molecule;

[0146] about 10 mM histidine hydrochloride buffer or histidine acetate buffer, such as histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0147] 0.2mg / mL-2mg / mL polysorbate 80;

[0148] About 80 mg / mL sucrose;

[0149] and the pH of the pharmaceutical composition is 4.5-6;

[0150] 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 the FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0151] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0152] About 0.4 mg / mL polysorbate 80;

[0153] About 80 mg / mL sucrose;

[0154] and the pH of the pharmaceutical composition is about 5.5;

[0155] 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 the FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0156] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0157] About 0.4 mg / mL polysorbate 80;

[0158] About 80 mg / mL sucrose;

[0159] and the pH of the pharmaceutical composition is about 5.3;

[0160] 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 the FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0161] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0162] About 0.4 mg / mL polysorbate 80;

[0163] About 80 mg / mL sucrose;

[0164] and the pH of the pharmaceutical composition is about 5.2;

[0165] 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 the FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0166] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0167] About 0.4 mg / mL polysorbate 80;

[0168] About 80 mg / mL sucrose;

[0169] and the pH of the pharmaceutical composition is about 5;

[0170] 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 the FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0171] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0172] About 0.6 mg / mL polysorbate 80;

[0173] About 80 mg / mL sucrose;

[0174] and the pH of the pharmaceutical composition is about 5.5;

[0175] 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 FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0176] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0177] About 0.6 mg / mL polysorbate 80;

[0178] About 80 mg / mL sucrose;

[0179] and the pH of the pharmaceutical composition is about 5.3;

[0180] 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 FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0181] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0182] About 0.6 mg / mL polysorbate 80;

[0183] About 80 mg / mL sucrose;

[0184] and the pH of the pharmaceutical composition is about 5.2;

[0185] 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 FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL;

[0186] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0187] About 0.6 mg / mL polysorbate 80;

[0188] About 80 mg / mL sucrose;

[0189] and the pH of the pharmaceutical composition is about 5;

[0190] 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;

[0191] approximately 10 mM histidine-acetate buffer;

[0192] About 0.4 mg / mL polysorbate 80;

[0193] About 80 mg / mL sucrose;

[0194] and the pH of the pharmaceutical composition is about 5;

[0195] 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;

[0196] approximately 10 mM histidine-acetate buffer;

[0197] About 0.4 mg / mL polysorbate 80;

[0198] About 80 mg / mL sucrose;

[0199] and the pH of the pharmaceutical composition is about 5.2;

[0200] 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;

[0201] approximately 10 mM histidine-acetate buffer;

[0202] About 0.4 mg / mL polysorbate 80;

[0203] About 80 mg / mL sucrose;

[0204] and the pH of the pharmaceutical composition is about 5.3;

[0205] 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;

[0206] approximately 10 mM histidine-acetate buffer;

[0207] About 0.4 mg / mL polysorbate 80;

[0208] About 80 mg / mL sucrose;

[0209] and the pH of the pharmaceutical composition is about 5.5;

[0210] 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;

[0211] approximately 10 mM histidine-acetate buffer;

[0212] About 0.6 mg / mL polysorbate 80;

[0213] About 80 mg / mL sucrose;

[0214] and the pH of the pharmaceutical composition is about 5;

[0215] 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;

[0216] approximately 10 mM histidine-acetate buffer;

[0217] About 0.6 mg / mL polysorbate 80;

[0218] About 80 mg / mL sucrose;

[0219] and the pH of the pharmaceutical composition is about 5.2;

[0220] 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;

[0221] approximately 10 mM histidine-acetate buffer;

[0222] About 0.6 mg / mL polysorbate 80;

[0223] About 80 mg / mL sucrose;

[0224] and the pH of the pharmaceutical composition is about 5.3;

[0225] 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;

[0226] approximately 10 mM histidine-acetate buffer;

[0227] About 0.6 mg / mL polysorbate 80;

[0228] About 80 mg / mL sucrose;

[0229] and the pH of the pharmaceutical composition is about 5.5;

[0230] 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;

[0231] approximately 10 mM histidine-acetate buffer;

[0232] About 0.8 mg / mL polysorbate 80;

[0233] About 80 mg / mL sucrose;

[0234] and the pH of the pharmaceutical composition is about 5;

[0235] 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;

[0236] approximately 10 mM histidine-acetate buffer;

[0237] About 0.8 mg / mL polysorbate 80;

[0238] About 80 mg / mL sucrose;

[0239] and the pH of the pharmaceutical composition is about 5.2;

[0240] 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;

[0241] approximately 10 mM histidine-acetate buffer;

[0242] About 0.8 mg / mL polysorbate 80;

[0243] About 80 mg / mL sucrose;

[0244] and the pH of the pharmaceutical composition is about 5.3;

[0245] 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;

[0246] approximately 10 mM histidine-acetate buffer;

[0247] About 0.8 mg / mL polysorbate 80;

[0248] About 80 mg / mL sucrose;

[0249] and the pH of the pharmaceutical composition is about 5.5;

[0250] 22) 1-45 mg / mL FAP / CD40 binding molecule, e.g., about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mg;

[0251] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0252] 0.2mg / mL-2mg / mL polysorbate 80;

[0253] About 80 mg / mL sucrose;

[0254] and the pH of the pharmaceutical composition is 4.5-6;

[0255] 23) 50-120 mg / mL FAP / CD40 binding molecule, such as about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL;

[0256] about 10 mM histidine acetate buffer, such as histidine-acetate buffer;

[0257] 0.2mg / mL-2mg / mL polysorbate 80;

[0258] About 80 mg / mL sucrose;

[0259] and the pH of the pharmaceutical composition is 4.5-6;

[0260] 24) The pharmaceutical composition of 1)-23), further comprising 4% w / v mannitol or 0.01% w / v ethylenediaminetetraacetic acid;

[0261] 25) is the pharmaceutical composition of 1)-24), wherein the final volume is 1 mL; when the volume needs to be fixed, the volume is fixed to 1 mL with water for injection.

[0262] In another aspect, the present disclosure provides a pharmaceutical composition containing a low concentration of a FAP / CD40 binding molecule (e.g., 1-45 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mg), or a method for preparing the same, wherein the pharmaceutical composition is prepared by diluting a pharmaceutical composition containing a high concentration of a FAP / CD40 binding molecule (e.g., 50-120 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL) with a diluent. In some embodiments, the diluent includes, but is not limited to, water, saline, or glucose solution.

[0263] The pharmaceutical composition disclosed herein has sufficient drug stability and can be stored stably for a long time.

[0264] In some embodiments, the pharmaceutical composition is stable at 2-8° C. 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. In some embodiments, the pharmaceutical composition is stable at 25° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the pharmaceutical composition is stable at 40° C. 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.

[0265] The present disclosure provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of dissolving the FAP / CD40 binding molecule.

[0266] To facilitate drug transportation, the pharmaceutical composition of the present disclosure can be further prepared into a lyophilized preparation.

[0267] In certain embodiments, the pharmaceutical composition as described in any one of the above items is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water, normal saline or glucose solution.

[0268] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the above pharmaceutical compositions after reconstitution.

[0269] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0270] The present disclosure provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized preparation. In certain embodiments, the reconstitution solution is selected from but not limited to water for injection, physiological saline or glucose solution.

[0271] The present disclosure also provides a product, comprising a container containing the aforementioned pharmaceutical composition, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution. In certain embodiments, the container is a neutral borosilicate glass injection vial. In certain embodiments, the product includes a package insert.

[0272] The present disclosure also provides a pharmaceutical composition or a lyophilized formulation or a reconstituted solution of a lyophilized formulation, which is used as a medicine for treating or alleviating a disease or condition.

[0273] Methods for treating diseases and pharmaceutical uses

[0274] 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 preventively and / or therapeutically effective amount of the aforementioned pharmaceutical composition or lyophilized formulation or a 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 responsive to immunotherapy.

[0275] In the above methods or uses, non-limiting examples of cancer or tumors include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus tumors, and osteosarcoma. Other examples of cancers that can be treated with the methods of the present disclosure include bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant 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, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, In some embodiments, the present invention includes but is not limited to: myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of said cancers. In some embodiments, the above-mentioned tumors or cancers are metastatic and / or advanced.

[0276] In addition, the present disclosure also provides a method for preventing and / or treating an infectious disease in a subject or patient, comprising administering the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of the lyophilized preparation to the subject or patient, so that the infectious disease of the subject is prevented and / or treated. Similar to the application for cancer or tumors as described above, it can be used alone or in combination with a vaccine to stimulate an immune response to pathogens, toxins and self-antigens. Examples of pathogens to which this treatment method can be particularly applied include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are not completely effective. These include but are not limited to HIV, hepatitis viruses (A, B, C), influenza viruses, herpes viruses, Giardia, malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0277] The pharmaceutical compositions containing the FAP / CD40 binding molecules disclosed herein can be used to treat patients in need of such treatment by parenteral administration, which can be administered by subcutaneous injection, intramuscular injection, or intravenous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0278] Figure 1 shows the binding of anti-FAP antibodies Ab9, Ab10, Ab14, and Ab15 to human FAP on the cell surface, using 28H1 as a positive control and IgG1 isotype as a negative control.

[0279] Figure 2 shows the 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.

[0280] Figure 3 shows the binding of anti-CD40 antibodies A12 and A297 to human CD40 on the surface of Raji cells, using 9E5-SELFNS as a positive control and IgG1 as a negative control.

[0281] Figure 4 shows the binding of anti-CD40 antibodies A12 and A297 to human CD40 on the surface of HEK293 cells, using 9E5-SELFNS as a positive control and IgG1 as a negative control. The ordinate represents the percentage of agonist activity relative to 200 nM 9E5-SELFNS.

[0282] Figure 5 shows the affinity flow cytometric test 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.

[0283] Figure 6 shows the humanized anti-CD40 antibodies A297_V1, A297_V2, A297_V3, and A297_V4 mediated by FcγRIIb in HEK-BlueTM For the activation of CD40L cells, 9E5-SELFNS was used as a positive control and hIgG was used as a negative control.

[0284] FIG7 is a schematic diagram of the structure of an anti-FAP / CD40 bispecific antibody.

[0285] Figure 8A shows the affinity flow cytometric test results of the anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 for the CHOK1 / FAP stably transformed cell that overexpresses the human FAP antigen; Figure 8B and Figure 8C show the affinity flow cytometric test results of the aforementioned anti-FAP / CD40 bispecific antibodies for the CHOK1 / FAP stably transformed cell that overexpresses the mouse FAP antigen and the cynomolgus macaque FAP antigen, respectively.

[0286] Figure 9A shows the affinity flow cytometric test results of the anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 for the HEK-Blue™ CD40L stably transfected cell that overexpresses the human CD40 antigen; Figure 9B shows the affinity flow cytometric test results of the aforementioned anti-FAP / CD40 bispecific antibodies for HEK293 cells that overexpress the cynomolgus macaque CD40 antigen.

[0287] FIG10 shows the flow cytometric detection results of the affinity of anti-FAP / CD40 bispecific antibodies Ab10-A297V3-2 and Ab10-A297V3-4 for CD40 on human immature DCs.

[0288] Figure 11 shows the DC maturation-promoting effect of concentration gradient anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 in the absence of CHOK1 / FAP stably transfected cell cross-linking, with LPC, hIgG1, and vehicle used as controls.

[0289] Figure 12 shows the DC maturation-promoting effect of concentration gradient anti-FAP / CD40 bispecific antibodies Ab10-A12V2-2, Ab10-A12V2-4, Ab10-A297V3-2, and Ab10-A297V3-4 in the presence of CHOK1 / FAP stably transfected cells cross-linked, with LPC, hIgG1, and vehicle used as controls.

[0290] Figure 13A shows the tumor growth curve of mice after a single dose of Ab10-A297V3-2 or Ab10-A297V3-4 was injected into mFAP-MC38 tumor-bearing hCD40 humanized mice; Figure 13B shows the activation of peripheral blood B cells in the mice; Figure 13C shows the body weight change curve of the mice; Figure 13D shows the changes in platelet counts in the mice; and Figure 13E shows the effects on liver function in the mice.

[0291] Figure 14A shows the tumor growth curve of mice after multiple doses of Ab10-A297V3-2 or Ab10-A297V3-4 were injected into mFAP-MC38 tumor-bearing hCD40 humanized mice; Figure 14B shows the activation of peripheral blood B cells in the mice; Figure 14C shows the body weight change curve of the mice; Figure 14D shows the changes in platelet counts in the mice; and Figure 14E shows the effects on liver function in the mice.

[0292] In Figures 13A to 14E , unless otherwise indicated, P values ​​are calculated relative to the hIgG1 control. The Student's t-test was used, where ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. DETAILED DESCRIPTION

[0293] 1. Terminology

[0294] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0295] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0296] "CD40" and "CD40 antigen" refer to an approximately 48 kD glycoprotein expressed on the surface of normal and neoplastic B cells that acts as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al., 1987, J. Immunol. 138:788-785). 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. "CD40" in this disclosure refers to any naturally occurring CD40 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. A cDNA molecule encoding CD40 has been isolated from a library prepared from the Burkitt's lymphoma cell line, Raji (Stamenkovic et al., 1989, EMBO J. 8:1403). Sequence information can be found in Table 8 of this disclosure. The term "CD40" as used herein encompasses full-length, unprocessed CD40 as well as any form of CD40 derived from processing in cells, and also encompasses naturally occurring variants of CD40, such as splice variants or allelic variants. In one embodiment, the CD40-binding molecules of this disclosure are capable of specifically binding to human, mouse, and / or cynomolgus monkey CD40.

[0297] "Fibroblast activation protein (FAP)" and "FAP antigen," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refer to any native FAP from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. "FAP" in the present disclosure encompasses full-length unprocessed FAP as well as any form of FAP derived from processing in cells, and also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. In one embodiment, the FAP-binding molecules of the present disclosure are capable of specifically binding to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO: 2), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2, GeneBank Accession NO. AAC51668. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid sequence of the human FAP ECD with His tag is shown in Table 2 of the present disclosure. The amino acid sequence of mouse FAP is shown in UniProt accession number 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 761. In some embodiments, the FAP-binding molecules of the present disclosure bind to the extracellular domain of FAP.

[0298] "Antibody" is used in the broadest sense to encompass various 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), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement order of their heavy chain constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, different subclasses can be formed based on 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 classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of both the heavy and light chains of antibodies vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region consists of three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. These three hypervariable regions determine the specificity of the antibody and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0299] "Bispecific antibodies" encompass antibodies (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) that specifically bind to two different antigens or at least two different 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, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11;2019:4516041).

[0300] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of 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.

[0301] 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 positions 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 produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," 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). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0302] Table 1. Relationships between CDR numbering systems

[0303] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0304] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0305] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a heavy chain antibody chain), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule, consisting of a two-layer sandwich of about seven antiparallel beta-sheet strands arranged in two beta-sheets, optionally stabilized by conserved disulfide bonds.

[0306] An "immunoglobulin variable domain" is an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0307] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0308] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains.TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0309] "VHH domain", also known as heavy chain single domain antibody, VHH, V H H domain, VHH antibody fragment, VHH antibody, nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., an "antibody lacking a light chain") (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 the light chain variable domain (referred to as "VL domain" in this disclosure) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domain in conventional tetrapeptide chain structure antibodies, in which case the epitope is recognized by both the VL domain and the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. H H domain", "VHH antibody fragment", "VHH antibody", as well as"" "VHH domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably. "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology. The total number of amino acid residues in a VHH domain will typically range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes of the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: 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.

[0310] As is well 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 numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0311] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the variable region framework of a human antibody. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. To avoid a decrease in activity while also reducing immunogenicity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity. Examples of "humanization" include "humanizing" a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide chain antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences. In addition, in order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0312] "Fully human antibodies" include antibodies with variable and constant regions of 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). "Fully human antibodies" do not include "humanized antibodies."

[0313] Typically, the CD40 binding molecules, FAP binding molecules of the present disclosure will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 - 8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen to be bound (i.e. CD40 or FAP). Any -4 M's K DValues ​​are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0314] When "competes" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., CD40 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); Solid phase direct labeling RIA with an I-125 label (see, e.g., Morel et 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 directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, the assay involves the use of purified antigen that binds to an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein (the antigen being on a solid surface or on the surface of a cell). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to an epitope that is sufficiently proximal to the epitope bound by the reference antigen binding protein that the two epitopes sterically interfere with each other's binding. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0315] Antibodies can be screened for competitive binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with or cross-compete for antigen binding. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Publication No. WO03 / 48731. Thus, antibodies that compete with the antibody molecules of the present disclosure for binding to the same epitope on CD40 or FAP can be obtained using conventional techniques known to those skilled in the art.

[0316] "CD40 binding molecule" means any protein that can specifically bind to CD40 or any molecule comprising the protein. CD40 binding molecules may include antibodies or conjugates thereof directed against CD40 as defined herein. CD40 binding molecules also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The "CD40 binding molecules" of the present disclosure may comprise at least one immunoglobulin single variable domain (e.g., VHH) that binds to CD40. In some embodiments, the "CD40 binding molecules" 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, the CD40 binding molecules of the present disclosure may also comprise a linker and / or a moiety with effector function, such as 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 "CD40 binding molecules" of the present disclosure also encompass bispecific / multispecific antibodies comprising immunoglobulins that bind to different antigens (e.g., 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).

[0317] "FAP-binding molecule" means any protein that can specifically bind to FAP or any molecule comprising the protein. The FAP-binding molecule may include an antibody or a conjugate thereof directed against FAP as defined in the present disclosure.

[0318] "FAP / CD40 binding molecule" means any protein that can specifically bind to CD40 and FAP or any molecule comprising the protein. The FAP / CD40 binding molecule may include an antibody or conjugate thereof as defined in the present disclosure directed against CD40 and FAP.

[0319] "Binding to CD40" or "binding to CD40" means being able to interact with CD40 or its epitope, which may be of human origin. "Binding to FAP" or "binding to FAP" means being able to interact with FAP or its epitope, which may be of human origin. An "antigen binding site" of the present disclosure refers to a discrete, three-dimensional site on an antigen that is recognized by an antibody of the present disclosure.

[0320] "Antigen" refers to a molecule used to immunize an immunocompetent vertebrate to generate antibodies that recognize the antigen, or to screen an expression library (e.g., particularly a phage, yeast, or ribosome display library). In the present disclosure, antigen is defined more broadly to include a target molecule specifically recognized by an antibody, as well as a portion or mimetic of a molecule used in an immunization process for generating antibodies or in screening libraries for selecting antibodies. For example, with respect to the antibodies of the present disclosure that bind to human CD40, monomers and multimers (e.g., dimers, trimers, etc.) of human CD40, as well as truncated and other variants of human CD40, are referred to as antigens.

[0321] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0322] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. For example, when human CD40 or its epitope is used as an analyte and an antibody is used as a ligand, the antibody binds to the epitope at a specific binding rate of less than 10 -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen or an epitope thereof, and its affinity for binding to the predetermined antigen or an epitope thereof is at least twice that of its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. "Antibody that recognizes an antigen" may be used interchangeably with "specifically binding antibody" in this disclosure.

[0323] "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 by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). D Other 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 exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those of non-target interactions (e.g., control antibodies known not to bind CD40). D The value is evaluated.

[0324] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar 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. In addition, 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 non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0325] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0326] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0327] "Agonist activity," "agonist activity," or "agonism" refers to a substance's function as an agonist. Binding of an agonist to a cell receptor elicits a response or activity that is similar or identical to that elicited by the receptor's natural ligand. For example, a CD40 agonist or CD40 agonistic antibody can induce any or all of the following responses: cell proliferation and / or differentiation; upregulation of cell-to-cell adhesion through molecules such as ICAM-1, E-selectin, VCAM, etc.; secretion of proinflammatory cytokines, such as IL-1, IL-6, IL-8, IL-12, TNF, etc.; signal transduction through the CD40 receptor through the following pathways, such as TRAF (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 MEK / ERK pathway, PI3K / Akt pathway, P38MAPK pathway, etc.; transduction of anti-apoptotic signals through molecules such as XIAP, Mcl-1, BCLx, etc.; generation of B and / or T cell memory; B cell antibody production; B cell isotype switching; upregulation of cell surface expression of class II MHC and CD80 / 86, etc.

[0328] "CD40-associated disease" or "CD40-associated disorder" refers to a disorder in which cells expressing CD40 are modified or eliminated. These cells include CD40-expressing cells that exhibit abnormal proliferation or CD40-expressing cells that are associated with cancerous or malignant growth. More specific examples of cancers that exhibit abnormal expression of the CD40 antigen include B lymphoblasts, 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 kidney cancer. Such disorders include, but are not limited to, leukemias, lymphomas (including B-cell lymphomas and non-Hodgkin's lymphomas), multiple myeloma, Waldenstrom's macroglobulinemia; solid tumors, including sarcomas, such as osteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinomas (including ovarian adenocarcinoma), Kaposi's sarcoma / Kaposi's tumor, and squamous cell carcinoma.

[0329] "Proliferative disease" refers to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0330] "Cancer," "cancerous," "proliferative disorder," and "tumor" as referred to in this disclosure are not mutually exclusive.

[0331] "Preventing cancer" refers to delaying, inhibiting, or preventing the onset of cancer in a subject in whom the onset of cancer or tumorigenesis has not been demonstrated, but in whom a predisposition to cancer has been identified, for example, by genetic screening or other methods. This also includes treating a subject with a precancerous condition to halt the progression of the precancerous condition to a malignant tumor or to cause its regression.

[0332] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising mannitol or ethylenediaminetetraacetic acid" means that the pharmaceutical composition may but need not contain mannitol or ethylenediaminetetraacetic acid.

[0333] As used herein, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to ±30%, for example, a pH of about 5.5 means pH 5.5 ± 1.65. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0334] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include tris (Tris), acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0335] An "acetate buffer" is a buffer comprising 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, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

[0336] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0337] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Preferred are 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.

[0338] "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, and the like. Preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0339] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, facilitate administration to an organism, and promote absorption of the active ingredient to exert its biological activity.

[0340] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0341] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0342] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0343] The pharmaceutical compositions disclosed herein can achieve a stable effect in which the FAP / CD40 binding molecule substantially retains its physical stability and / or chemical stability and / or biological activity after storage. For example, the pharmaceutical composition substantially retains its physical and chemical stability as well as its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Various analytical techniques are currently available for measuring protein stability after storage at a selected temperature for a selected period of time.

[0344] A stable pharmaceutical antibody formulation is one in which no significant change is observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after storage at 25°C for a period of 1 month, 3 months, 6 months, or 1 month at 40°C. Typical acceptable standards for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of the antibody monomers degrade as measured by SEC-HPLC. By visual analysis, the pharmaceutical antibody formulation is colorless or clear to slightly opalescent. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, of truncation is observed, and typically no more than about 10%, preferably no more than about 5%, of aggregation is formed.

[0345] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured 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 protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0346] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0347] An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by antigen binding assays.

[0348] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0349] "Treatment" means administering an internal or external therapeutic agent, such as any of the antibodies disclosed herein or pharmaceutical compositions thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0350] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0351] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0352] The equipment and methods used in the testing process are as follows:

[0353] Appearance:

[0354] Using the visual method, wipe the sample bottle clean and observe the sample color, clarity and visible foreign matter on a clarity tester with a white background and a black background under a light intensity of 1000-1500lx.

[0355] Appearance inspection instrument: Jingtuo Instrument YB-2A clarity tester.

[0356] SEC size exclusion chromatography:

[0357] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0358] 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.)

[0359] SEC instrument: Agilent 1260-Bio; chromatographic columns: Waters, XBrige SEC (300×7.8mm 3.5μm)

[0360] NR-CE capillary gel electrophoresis:

[0361] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0362] Non-reduced CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of all peak areas.

[0363] CE measurement instrument: Sciex model PA800plus

[0364] icIEF imaging capillary isoelectric focusing electrophoresis:

[0365] A technique for separating proteins based on their isoelectric points (pI).

[0366] icIEF main peak content percentage = main peak area / total area*100% (total area is the sum of the areas of acidic peak, main peak and basic peak).

[0367] The instrument used for icIEF determination was manufactured by Protein Simple, model number: Muarice.

[0368] Protein concentration determination:

[0369] Protein concentration was determined using a UV-visible spectrophotometer (Nano Drop 2000) with a pathlength of 1 mm.

[0370] Exemplary Antibody Pharmaceutical Composition (Formulation) Preparation Process

[0371] Step 1: Mix a FAP / CD40 binding antibody (e.g., Ab10-A297V3-4, whose heavy and light chain sequences are SEQ ID NOs: 22 and 20, respectively) with the following excipients in the prescribed amounts to prepare a stock solution containing the FAP / CD40 binding molecule. After filtration, sample the solution for sterility testing. Filter the stock solution through a 0.22 μm filter cartridge and collect the filtrate.

[0372] Step 2: Adjust the filling volume to 1.15 mL, fill the filtrate into a 2 mL vial, add a stopper, and take samples at the beginning, middle, and end of filling to detect the difference in filling volume.

[0373] Step 3: Turn on the capping machine, add aluminum caps, and start capping.

[0374] Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling quantity. Print and apply vial labels; print carton labels, folding cartons, packing, and affixing box labels.

[0375] 2. Implementation and Test Examples

[0376] The present disclosure is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0377] Experimental methods in the examples disclosed herein that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the raw material or product manufacturers. Reagents without specific sources are commercially available.

[0378] Example

[0379] The preparation and purification methods of the FAP / CD40 binding molecules in this application have been described in patent document No. WO2023025194, and the entire contents of the aforementioned application document can be incorporated into the present disclosure.

[0380] Example 1. Screening and preparation of anti-FAP monoclonal antibodies

[0381] 1.1 Screening, sequencing, and preparation of antigens

[0382] 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, source, and use of the recombinant FAP protein used in the present disclosure are shown in Table 2.

[0383] Table 2. Sources of recombinant protein amino acid sequences

[0384] 1.2 Screening of h-FAP-specific binding antibody fragments from a human natural Fab phage display library

[0385] Using h-FAP-biotin as the target molecule, antigen-specific phages were captured using magnetic beads with avidin on their surface and enriched using a magnetic rack. Antigen-specific phages were eluted using a glycine solution at pH 2.2.

[0386] After two rounds of screening, 284 clones were randomly selected and screened for positive clones using phage ELISA. The following specific steps were performed: 284 colonies were counted and plated, each well of a 96-well plate containing 400 μL of culture medium (2YT+Amp+0.2% glucose) and incubated at 37°C with shaking at 250 rpm for 6 hours. The plate was coated with the antigen h-FAP-His at 100 ng / 100 μL / well and incubated at 4°C overnight. The next day, the 96-well plate was washed and blocked, and 100 μL of the overnight culture was added to each well. The plate was incubated at 37°C for 1 hour, washed, and a secondary antibody (HRP-labeled anti-human IgG-Fab antibody) was added. The plate was incubated at 37°C for 40 minutes, washed, and a colorimetric solution was added. The plate was stored in the dark for 30 minutes, and the OD600 value was read using a microplate reader. A total of 122 positive clones obtained after two screenings were sequenced, resulting in 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).

[0387] 1.3 Construction of a full-length antibody display library on the surface of CHO cells

[0388] 1) Construction of a full-length antibody CHO cell display gene library, including: PCR amplification of a VH-enriched library, a KLC-enriched library, and an LLC-enriched library using three sets of primers, respectively. Enzyme digestion of the three enriched libraries followed by insertion into corresponding component vectors to construct the corresponding component libraries. Enzyme digestion of the KLC, LLC, and VH component libraries was performed, and the digested KLC, LLC, and VH fragment libraries were purified by electrophoresis. The full-length antibody cell display vector was enzymatically digested and the vector fragments were purified. The purified vector fragments, KLC, LLC, and VH fragment libraries were mixed and ligated. The ligation products were purified, electroporated into Escherichia coli, plated, cultured overnight at 37°C, and colonies were counted. The library capacity reached 3.6 × 10E6, over 800 times the theoretical diversity (74 × 58 = 4292). All colonies were collected, and vector DNA was extracted to obtain the full-length antibody cell display gene library.

[0389] 2) Constructing a h-FAP antigen-specific full-length antibody cell display library, comprising: transforming the full-length antibody cell display library with the full-length antibody cell display gene library vector DNA to construct a full-length antibody CHO cell display library. Hygromycin pressure screening was used to screen the cell library. Stably transformed cell libraries were obtained, and the cell library was double-stained with PE-labeled mouse anti-human κ (or λ) light chain antibody and FITC-labeled h-FAP antigen. PE- and FITC-double-positive cells were sorted by FACS, with one cell per well. The κ light chain library was sorted across two 96-well plates, and the λ light chain library was sorted across one 96-well plate. Hygromycin pressure culture was then applied.

[0390] 3) FACS analysis was used to identify single-cell clones expressing h-FAP antigen-specific antibodies. The following steps were performed: 14 days of hygromycin-pressurized culture yielded 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 double-stained with PE-labeled mouse anti-human κ (or λ) light chain antibody and FITC-labeled h-FAP antigen. FACS analysis yielded 138 single-cell clones that were both PE- and FITC-positive.

[0391] 4) Cloning of antibody genes, including affinity analysis of positive clones by FACS, and selection of 45 clones for PCR amplification of the antibody genes. Positive clones were collected by centrifugation, the supernatant discarded, and genomic DNA extracted. VH and LC fragments were amplified by PCR. The amplified fragments were separated by electrophoresis and sequenced. Six unique VH and six unique κ fragments were identified. Combinations yielded 12 clones with unique sequences. The VH and VL sequences of one of these clones are shown in Table 3.

[0392] Table 3. Amino acid sequences of h-FAP antibody VH and VL (κ of KLC)

[0393] Note: The underlined CDRs are based on the Kabat numbering convention.

[0394] The present disclosure also provides the full-length heavy chain and full-length light chain sequences of AblO.

[0395] >Ab10 full length heavy chain

[0396] >Ab10 light chain full length

[0397] Table 4. CDRs of anti-FAP antibodies

[0398] 1.4 Construction of a full-length antibody display library on the surface of CHO cells

[0399] 1) Construction of soluble antibody expression vectors, including: Enzyme digestion and purification of unique sequence-positive VH and LC fragments. Insertion of the VH fragment into a soluble heavy chain expression vector (IgG1), and the LC fragment into a soluble light chain expression vector. Colony sequencing was performed to confirm the identity of the fragments and DNA was extracted.

[0400] 2) Expression and purification of soluble antibodies, including: amplifying Expi293 cells in suspension culture. According to the light and heavy chain pairings determined above, 18 μg of light chain expression vector and 12 μg of heavy chain expression vector were mixed. Vector DNA was mixed with PEI at a weight ratio of 1:2.5 and used to transform Expi293 cells. On the sixth day, the culture supernatant was collected and the antibody was purified using the Protein A method. Analysis by SDS-PAGE denaturing gel electrophoresis showed that the antibody purity was greater than 90%. The cells were stored at -80°C.

[0401] 1.5 ELISA binding test

[0402] Directly coat the plate with His-tagged FAP recombinant protein. After adding the antibody, antibody binding activity is detected by adding a secondary antibody (HRP-conjugated anti-human IgG antibody) and the HRP substrate TMB. The procedure involves coating a 96-well microtiter plate with human FAP-His protein at a concentration of 0.5 μg / mL (100 μL / well) and incubating overnight at 4°C. Wash thoroughly, add 200 μL / well of blocking buffer, and incubate at room temperature for 2 hours. Wash thoroughly, add 100 μL / well of the anti-FAP test antibody diluted in diluent, and incubate at room temperature for 1 hour. Wash thoroughly, then add 100 μL / well of HRP-conjugated goat anti-human IgG secondary antibody diluted 1:20,000 in diluent. Incubate at room temperature for 1 hour, wash thoroughly, and add 100 μL / well of TMB. Incubate in the dark for 15 minutes. Add 50 μL / well of 0.16 M sulfuric acid. The OD value at 450 nm was read using a Thermo MultiSkanFc microplate reader, and the binding EC of the anti-FAP antibody was calculated. 50 The results are shown in Table 5.

[0403] Table 5. Affinity EC of anti-FAP antibodies binding to human FAP antigen 50 value

[0404] 1.6 Surface plasmon resonance (SPR) combined with testing

[0405] A CM5 sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibody was prepared at a 30 μg / mL solution in 10 mM sodium acetate buffer (pH 5.0) and immobilized by amino coupling. Each test antibody was prepared in HBS-EP+ buffer and captured by the anti-human IgG (Fc) antibody on the chip channel. h-FAP-His was prepared in HBS-EP+ buffer and serially diluted 2-fold. The diluted antibody was passed through the experimental and reference channels at a flow rate of 30 μL / min, with binding for 1 minute and dissociation for 15 minutes. 10 mM Glycine, pH 1.5, was used as the regeneration buffer, and the flow rate was 10 μL / min for 30 seconds. The data were analyzed and the results in Table 6 showed that compared with the control anti-FAP antibody 28H1 (sequence 219 and sequence 233 of patent US9266938B2), the affinity of Ab10 to the antigen FAP was 3-6 times higher.

[0406] Table 6. SPR affinity Ka, Kd and K of anti-FAP antibodies D value

[0407] The heavy and light chain variable region sequences of the control antibody 28H1 are as follows:

[0408] >VH of 28H1

[0409] >28H1 VL

[0410] 1.7 FACS binding assay

[0411] FACS experiments were used to detect the binding properties of anti-FAP antibodies on cells. Including: constructing a CHO cell line that overexpresses human FAP and plating (1E5 / well). Add the antibody to be tested, 100 μL / well, the highest concentration is 100 nM, 5-fold dilution, a total of 8 concentrations, and incubate at 4°C for 1 hour. Add anti-hIgG Alexa Fluor-647 as a secondary antibody, incubate at 4°C for 30 minutes at a ratio of 1:500, and detect using FACS. Antibodies that can bind to FAP can label cells. The relationship between the number of labeled cells that overexpress human FAP as a percentage of all cells and the antibody concentration is shown in Figure 1. The EC value of human FAP binding to the antibody 50 The values ​​are shown in Table 7; the relationship between the percentage of labeled cells overexpressing mouse FAP in all cells and the antibody concentration is shown in Figure 2, where Ab9, Ab14, and Ab15 are three other anti-FAP antibodies screened in this application.

[0412] Table 7. Affinity EC of anti-FAP antibodies binding to human FAP antigen 50 value

[0413] Example 2. Screening and preparation of anti-CD40 nanobody (VHH)

[0414] 2.1 Immunization antigens, screening antigen sequences and preparation

[0415] Select human CD40 recombinant protein with a His tag (h-CD40-His), human CD40 recombinant protein with a C-terminal biotin tag (h-CD40-biotin), and monkey CD40 recombinant protein with a C-terminal His tag (cyno-CD40-His). See Table 8 for their sequences and sources. These protein reagents can be used in the experiments described in the following examples.

[0416] Table 8. Amino acid sequence and source of recombinant protein

[0417] 2.2 Alpaca immunization process and potency testing

[0418] Alpacas were immunized with h-CD40-his every two weeks for a total of four immunizations. For the first immunization, 0.5 mg of the antigen was mixed with 1 mL of complete Freund's adjuvant (CFA) and injected subcutaneously. For the next three immunizations, 0.25 mg of the antigen was mixed with 1 mL of incomplete Freund's adjuvant (IFA) and injected subcutaneously. Before immunization, blank serum was collected. One week after the third and fourth immunizations, 50 mL of peripheral blood was collected for lymphocyte isolation and serum titer testing.

[0419] 2.3 Titer detection and phage library construction

[0420] After four immunizations, the alpaca serum titer was tested. Once the titer reached a satisfactory level, PBMCs were isolated, total RNA was extracted, purity tested, and reverse transcribed into DNA. After two rounds of nested PCR, the purified vector was ligated with the target VHH fragment by enzyme digestion. Electroporation was performed, and clones were selected to generate phage libraries A and B.

[0421] 2.4 Phage library affinity panning and ELISA identification

[0422] Using the h-CD40 antigen as the target molecule, Gly-HCl acid elution was used to screen specific phage. After two rounds of screening, 384 clones were randomly selected from the first and second round titer plates. Positive clones were screened using phage ELISA and measured by optical density at 450 nm. Sequence alignment and phylogenetic tree analysis were performed based on the sequencing results, resulting in the identification of 16 sequences. The amino acid sequences of the highly functional anti-CD40 antibodies are shown in Tables 9 and 10.

[0423] Table 9. Anti-CD40 single domain antibody sequences

[0424] Note: The underlined CDRs are based on the Kabat numbering convention.

[0425] Table 10. CDRs of anti-CD40 antibodies

[0426] 2.5 Expression and purification of anti-CD40 single domain antibody-Fc fusion protein

[0427] The above two VHHs were respectively linked to human IgG1-Fc containing the N297A mutation. The sequences of the VHH-Fc fusion proteins after ligation are shown in Table 11. The underlined part is human IgG1-Fc, and the N297A mutation is in bold.

[0428] 9E5-SELFNS was used as a CD40 agonist positive control (see SEQ ID NO: 58 and SEQ ID NO: 59 of WO2020108611A1).

[0429] Table 11. Fusion protein sequences of anti-CD40 single domain antibodies and human IgG1-Fc

[0430] Construct plasmids, transiently transfect cells, express antibodies, and purify them. After testing, the target antibody is obtained.

[0431] 2.6 FACS binding assay

[0432] FACS assay was used to detect the binding properties of anti-CD40 antibodies on cells. This included: obtaining Raji single cells and plating them. Adding the antibody to be tested at 100 μL / well, with a maximum concentration of 100 nM, followed by 5-fold dilution to obtain a total of 8 gradient points, and incubating at 4°C for 1 hour. Adding anti-hIgG Alexa Fluor-647 as a secondary antibody, incubating at a ratio of 1:500 at 4°C for 30 minutes. FACS assay was performed. A CD40 agonist was used as a positive control, and the EC values ​​of each antibody were quantified. 50 See Figure 3 and Table 12. Among them, A12 is another anti-CD40 antibody screened and obtained in this application.

[0433] Table 12. Affinity EC of anti-CD40 antibodies binding to human CD40 antigen 50 value

[0434] For CD40 agonist antibodies used in FAP / CD40 bispecific antibodies, their affinity for CD40 is EC 50 The RI should not be too high, otherwise the bispecific antibody will preferentially bind to CD40. It is more desirable that the bispecific antibody preferentially bind to FAP and thus exert its effect. Therefore, A297 has the advantage of being more consistent with the above characteristics, which is also reflected in Table 13.

[0435] 2.7 Activity detection of anti-CD40 nanoantibodies

[0436] Detect the activation effect of anti-CD40 antibody on CD40 reporter gene cells according to EC 50 Evaluate the agonist activity of CD40 antibodies. Including: Preparation of HEK-Blue TM CD40L cells (purchased from Invivogen, Cat#hkb-cd40) are stably transfected with the human CD40 gene and the NF-κB-mediated SEAP gene. The activation level of the CD40 signaling pathway can be characterized by measuring the secreted SEAP content in the supernatant using the SEAP substrate QUANTI-Blue. The cells were cultured in DMEM supplemented with 10% FBS, 100 μg / mL normocin, 100 μg / mL zeocin, and 30 pg / mL blasticidin. Cells were plated at 5E4 / well in a 96-well plate in DMEM supplemented with 10% FBS and 100 μg / mL normocin and cultured overnight. After cell attachment, 100 μL of serially diluted test antibodies was added to each well and incubated overnight 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 incubated in the dark for 15 minutes. The absorbance at 620 nm was measured using an Envision microplate reader to calculate the EC 50Using CD40 agonist (9E5-SELFNS) as a positive control, the relationship between the relative agonist activity percentage (compared with 200 nM 9E5-SELFNS) and the antibody concentration EC 50 See Figure 4 and Table 13. Among them, A12 is another anti-CD40 antibody screened and obtained in this application.

[0437] Table 13. Agonistic activity EC of anti-CD40 antibodies 50 Values ​​and their relative activation activity percentages compared with the positive control

[0438] 2.8 Humanization of anti-CD40 nanobody

[0439] Based on the above results, A297 was humanized. Based on the obtained VHH canonical structure of Nanobody A297, the VHH variable region sequences were compared with an antibody germline database to obtain a human germline template with high homology. The preferred human germline template for the disclosed antibody A297 is IGHV3-48*03. The CDRs were then transplanted into human FRs, and key amino acids that affect antibody structure and function were backmutated to restore binding and activity. The humanized sequences are shown in Table 14.

[0440] Table 14. Anti-CD40 humanized antibody sequences

[0441] The four VHHs were ligated into human IgG1-Fc containing the S267E / L328F mutation (according to EU numbering). The sequences of the ligated VHH-Fc fusion proteins are shown in Table 15. The underlined portion represents human IgG1-Fc, and the S267E / L328F mutation is in bold.

[0442] Table 15. Fusion protein sequences of humanized anti-CD40 single domain antibodies and human IgG1-Fc (S267E / L328F)

[0443] The above four VHH-Fc proteins were transiently transfected into cells for expression, and the antibodies were purified and tested to obtain the target antibody proteins.

[0444] 2.9 Affinity of humanized CD40 antibodies for CD40-overexpressing cell lines

[0445] FACS was used to detect the binding ability of humanized antibodies to cells that highly expressed human antigen protein CD40. HEK293 cells were transiently transfected with 5% dapoxetine (Tag; Sino Biological; HG10774-CF) to obtain HEK293 cells with high CD40 expression. The cells were resuspended in flow cytometry staining buffer (PBS + 2% FBS) and serially diluted with the antibody to be tested at varying concentrations. After incubation on ice for 1 hour, the cells were washed with PBS and centrifuged at 400 g for 5 minutes. A goat anti-human Fc antibody labeled with the fluorescent group Alexa Fluor 647 was added as a secondary antibody. The cells were stained on ice for 1 hour, washed twice with PBS, and the cell surface fluorescence signal was detected by FACS. The results are shown in Figure 5. 50 The values ​​are shown in Table 16. The humanized antibodies disclosed herein have a high affinity for cell lines that highly express CD40.

[0446] Table 16. Affinity EC of each humanized anti-CD40 antibody to human CD40 high-expressing cell lines 50 value

[0447] 2.10 Humanized CD40 Antibody to HEK-Blue TM Activation of CD40L cells

[0448] By detecting the CD40 antibody against HEK-Blue TM The in vitro agonist activity of CD40 antibodies was evaluated by the activation of CD40L cells. The S267E / L328F mutation in the Fc of humanized CD40 antibodies enhances the affinity of IgG1Fc and FcγRIIb, thereby enhancing the cross-linking of FcγRIIb to the antibody. TM The activation of CD40L cells mimics the agonist activity of CD40 antibodies when they are fully cross-linked. 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 that highly express FcγRIIb. TMCD40L cells were plated at 5E4 / well in a 96-well cell culture plate (culture medium: DMEM, 10% FBS, 100 μg / mL Normocin). HEK293 cells expressing FcγRIIb were added at 5E4 / well. 100 μL of serially diluted test antibodies were added to each well and incubated at 37°C overnight. The cells were centrifuged, and 20 μL of the cell supernatant was transferred to a new 96-well plate. 180 μL of QUANTI-Blue substrate solution was added and incubated in the dark for 30 minutes. The absorbance at 620 nm was measured using an Envision microplate reader, and the EC values ​​were calculated. 50 The EC values ​​and Emax values ​​(relative to the fluorescence intensity of the group without antibody) were used. 50 The in vitro cell agonist activity of CD40 antibodies was evaluated by RT-PCR.

[0449] CD40 antibody is mediated by FcγRIIb in HEK-Blue TM The activation of CD40L cells is shown in Figure 6. 50 The values ​​and Emax values ​​(relative fluorescence intensity) are shown in Table 17.

[0450] The results showed that in the above-mentioned reporter gene cell system, the agonist activity of the humanized CD40 antibody was strong when it was cross-linked by FcγRIIb.

[0451] Table 17. Effects of various humanized anti-CD40 antibodies on HEK-Blue TM Activation of CD40L cells

[0452] Example 3. Design and preparation of anti-FAP / CD40 bispecific antibodies

[0453] 3.1 Design, expression and purification of anti-FAP / CD40 bispecific antibody

[0454] Based on the results of the humanized anti-CD40 antibody screening, A297V3 was selected; based on the results of the anti-FAP antibody screening, Ab10 was selected to construct an anti-FAP / CD40 bispecific antibody. The bispecific antibody uses Ab10 as the IgG backbone, with one, two, or three anti-CD40 nanobodies linked in series to the C-termini of each of Ab10's two heavy chains. The linker between the CD40 nanobodies and to the C-termini of the Ab10 heavy chains uses "GGGGSGGGGS." Each bispecific antibody contains bivalent, tetravalent, and hexavalent CD40 nanobodies, respectively, as shown in Figure 7. For example, in Ab10-A297V3-2, Ab10 represents the anti-FAP antibody used, A297V3 represents the humanized anti-CD40 antibody used, and the last 2 represents the bivalent CD40 valency. This naming convention was used for all other antibodies. Transient transfection, expression, and purification of the antibodies were performed using the method described in 2.5 of Example 2. After testing, the desired bispecific antibody molecule was obtained. The amino acid sequences of each bispecific antibody molecule are as follows.

[0455] >Light chain of Ab10-A12V2-2

[0456] >Heavy chain of Ab10-A297V3-2

[0457] >Heavy chain of Ab10-A297V3-4

[0458] >Heavy chain of Ab10-A297V3-6

[0459] The light chains of Ab10-A297V3-2, Ab10-A297V3-4, and Ab10-A297V3-6 are all shown in SEQ ID NO: 20.

[0460] Note: The underlined regions of the heavy chain are CH1 and Fc, the underlined region of the light chain is CL, the italics are the linker, and the bold is LALA (L234A and L235A mutations).

[0461] 3.2 Antigen Binding Affinity Detection of Anti-FAP / CD40 Bispecific Antibodies

[0462] SPR (GE Healthcare; Biacore 8K) was used to measure the affinity between the anti-FAP / CD40 bispecific antibody and its antigens, human FAP protein (Acro, FAP-H5244) and human CD40 protein (Acro, CD0-H5228). A CM5 sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibodies were immobilized via amino-coupling. Each test antibody was prepared in HBS-EP+ buffer as a ligand and captured by the anti-human IgG (Fc) antibody on the chip channel. Antigen proteins from different species served as analytes. The results are shown in Tables 18 and 19, demonstrating that the preparation of the bispecific antibody did not affect the binding of the antibody to FAP. In addition, the bispecific antibody had a lower affinity for CD40, preferentially binding to FAP and exerting its activity.

[0463] Table 18. Affinity of anti-FAP / CD40 bispecific antibodies to human FAP

[0464] Table 19. Affinity of anti-FAP / CD40 bispecific antibodies to human CD40

[0465] 3.3 Affinity detection of anti-FAP / CD40 bispecific antibody for FAP-overexpressing cell lines

[0466] This experiment used FACS to detect the binding of the anti-FAP / CD40 bispecific antibody to CHOK1 cells stably expressing human FAP, cynomolgus monkey FAP, and mouse FAP. This showed that the bispecific antibody could bind to the cell surface FAP antigens of human, mouse, and cynomolgus monkey with a binding capacity similar to that of the monoclonal antibody (see Figures 8A-8C, 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 screening in this application.

[0467] Table 20. EC of anti-FAP / CD40 bispecific antibodies binding to FAP antigens on human and mouse cell surfaces 50 value

[0468] 3.4 Affinity Detection of Anti-FAP / CD40 Bispecific Antibody for Cell Lines Highly Expressing CD40

[0469] FACS was used to examine the binding of the bispecific antibodies to cell surface CD40, including HEK-Blue™ CD40L cells (Invivogen, Cat#hkb-cd40) that overexpress human CD40 and HEK293 cells transiently transfected with a cynomolgus macaque CD40 plasmid (Sino Biological, Cat#CG90970-UT) to overexpress cynomolgus macaque CD40. The results showed that the bispecific antibodies were able to bind to both human and cynomolgus macaque cell membrane surface CD40. See Figures 9A and 9B, Figure 11, and Table 21 for details. Ab10-A12V2-2 and Ab10-A12V2-4 are two other anti-FAP / CD40 bispecific antibodies screened in this application.

[0470] Table 21. EC of bispecific antibodies binding to CD40 antigen on human cell surface 50 value

[0471] 3.5 Detection of FAP-dependent DC maturation-promoting activity of humanized anti-FAP / CD40 bispecific antibody

[0472] To validate the effect of the anti-FAP / CD40 bispecific antibody on dendritic cell maturation, monocytes were isolated from fresh PBMCs using CD14-positive magnetic beads and cultured for 5 days in 1640 medium supplemented with 50 ng / mL GM-CSF and 50 ng / mL hIL-4. Half the medium was replaced every 2-3 days. On day 5, the induced dendritic cells were plated into a 96-well plate at a density of 1E5 / well. CHOK1 cells in phase II and CHOK1 cells overexpressing human FAP were then added. Serial dilutions of the test antibodies were also added, along with 1 μg / mL LPS as a positive control, and 100 nM hIgG1 and a no-antibody group as negative controls. After 48 hours of culture, expression of the dendritic cell surface molecule CD83 was assessed by FACS.

[0473] As shown in Figure 11 (without CHOK1 / FAP) and Figure 12 (with CHOK1 / FAP), the results show that the bivalent molecule Ab10-A297V3-2 cannot activate DC in the absence of CHOK1 / FAP, and its CD40 agonist activity is completely dependent on FAP-mediated crosslinking. The quadrivalent molecule Ab10-A297V3-4 can induce the maturation of DC cells in the absence of CHOK1 / FAP cells, and the activation of DC cells can be further enhanced in the presence of CHOK1 / FAP. Therefore, regardless of the bivalent molecule or the quadrivalent molecule, FAP expressed on the surface of dendritic cells can provide an activation window for bispecific antibodies. Among them, Ab10-A12V2-2 and Ab10-A12V2-4 are two other anti-FAP / CD40 bispecific antibodies obtained by screening in this application.

[0474] Example 4. In vivo efficacy of the anti-FAP / CD40 bispecific antibody Ab10-A297V3-2 / 4 in mice

[0475] B-hCD40 humanized mice were obtained from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. (species Mus muscμLus, strain C57BL / 6, female). Mouse full-length FAP plasmid was transfected into mouse intestinal cancer MC38 cells to construct a stable transfectant strain. 5×10 cells were inoculated. 5 / 0.1mL / mouse subcutaneously in the right armpit of the mouse, when the average tumor volume grows to 80-100mm 3 At the same time, mice with appropriate tumor volumes were selected and randomly divided into groups of 6 per group. The mice were intraperitoneally injected with the drug twice a week. Peripheral blood was drawn 48 hours after the first administration to detect the number of B cells and the expression of CD86 on the cell surface. The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood were measured on the 8th day after grouping. The drug was stopped after 4 doses until the tumor volume of mice in the hIgG1 control group exceeded 2000 mm. 3 The blood routine test was performed 24 hours after the last administration.

[0476] 4.1 Efficacy and toxicity of Ab10-A297V3-2 or Ab10-A297V3-4 in mice after a single dose

[0477] The antitumor activity of Ab10-A297V3-2 or Ab10-A297V3-4 was compared with that of the CD40 agonist mAb 9E5-mIgG1 at a single dose. 9E5-mIgG1 shares the same variable regions as 9E5-SELFNS, except that the heavy and light chain constant regions utilize the constant region of the mouse mIgG1 heavy chain and the constant region of the mouse kappa chain, respectively. Because the activity of CD40 agonist mAbs depends on FcγRIIb cross-linking of their Fc domains, 9E5-mIgG1, which utilizes the mouse IgG1 heavy chain constant region, is better suited to demonstrate CD40 agonist activity in mice. In in vivo efficacy studies, 9E5-mIgG1 served as a better control antibody than 9E5-SELFNS.

[0478] Ab10-A297V3-4 showed stronger anti-tumor activity than the CD40 monoclonal antibody 9E5-mIgG1 at the same molar dose, both in terms of TGI (Tumor growth inhibition) and CR (Complete Response). The sequence is as follows:

[0479] >9E5-mIgG1 heavy chain

[0480] >9E5-mIgG1 light chain

[0481] The antitumor activity of Ab10-A297V3-2 at twice the molar concentration was still weaker than that of Ab10-A297V3-4, and was comparable to that of 9E5-mIgG1.

[0482] The activation of peripheral CD40 by the bispecific antibody molecules was tested by detecting the activation of mouse peripheral blood B lymphocytes, and the results were consistent with the in vitro experiments. Ab10-A297V3-4 was able to partially activate peripheral B lymphocytes because it had CD40 activation activity that was partially independent of FAP, while Ab10-A297V3-2 was completely dependent on FAP for CD40 activation, so no significant peripheral B cell activation was detected (One-Way ANOVA). The tested antibody information is shown in Table 22, and the in vivo efficacy results are shown in Figure 13A (P < 0.0001 for each antibody group relative to the hIgG1 control group) and Figure 13B.

[0483] Table 22. Information of tested antibodies

[0484] From the perspective of mouse toxicity, neither Ab10-A297V3-2 nor Ab10-A297V3-4 produced changes in mouse body weight, see Figure 13C.

[0485] Routine blood tests revealed that compared to the hIgG1 control group at the same molar dose, 9E5-mIgG1 caused a moderate decrease in platelets, similar to hepatotoxicity. Ab10-A297V3-2 did not cause thrombocytopenia, while Ab10-A297V3-4 induced the same degree of platelet decrease as 9E5-mIgG1 (see Figure 13D). Detailed in vivo mouse dosing and tumor growth inhibition statistics for Ab10-A297V3-2 / 4 are shown in Table 23.

[0486] The calculation formula of TGI is:

[0487] TGI = (tumor volume of blank group on the day of treatment - tumor volume of group on the day of treatment) / (tumor volume of blank group on the day of treatment) * 100%

[0488] Table 23. In vivo dosage and tumor growth inhibition rate of Ab10-A297V3-2 or 4 in mice

[0489] ALT and AST measurements indicate that 9E5-mIgG1 exhibited some hepatotoxicity on day 8 after administration, mediated by CD32B (i.e., FcγRIIB) cross-linking. Ab10-A297V3-4 exhibited some hepatotoxicity due to its background activation, but its activation was independent of CD32B, resulting in less hepatotoxicity than 9E5-mIgG1. Ab10-A297V3-2 exhibited no hepatotoxicity due to its dependence on FAP for CD40 activation (see Figure 13E).

[0490] 4.2 Efficacy and toxicity of Ab10-A297V3-2 or Ab10-A297V3-4 in mice at multiple doses

[0491] To explore 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 was reduced (see Table 24 for details). Further increases in the dose of the bivalent Ab10-A297V3-2 molecule did not further enhance its anti-tumor activity, nor was peripheral B lymphocyte activation 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 exhibited a TGI of 92.6%, significantly superior to that of 9E5-mIgG1.

[0492] To evaluate the role of FAP antibodies in the quadrivalent CD40 bispecific antibody in vivo, the anti-tumor activity of Ab10-A297V3-4 was compared with that of another quadrivalent CD40 bispecific antibody without FAP binding (isotype-A297V3-4). Isotype-A297V3-4 is identical to Ab10-A297V3-4, except that the anti-FAP portion of Ab10 was replaced with another isotype antibody that does not bind to any mouse protein.

[0493] The results of Figures 14A-14E show that the quadrivalent CD40 bispecific antibody has good anti-tumor activity even without binding to FAP, and the anti-tumor activity is even stronger when bound to FAP. In terms of toxicity, Ab10-A297V3-2 showed no abnormalities in body weight, ALT / AST, and platelet count after increasing the dose. For Ab10-A297V3-4, after reducing the dose, the abnormalities in ALT / AST and platelet count previously observed at high doses disappeared, while a strong anti-tumor effect was still retained (P<0.0001), indicating that compared with the CD40 monoclonal antibody 9E5-mIgG1, both Ab10-A297V3-2 and Ab10-A297V3-4 have a wider therapeutic window. Antibody information is shown in Table 24, and the in vivo efficacy statistics of Ab10-A297V3-2 or Ab10-A297V3-4 at multiple doses are shown in Table 25.

[0494] Table 24. Information of tested antibodies

[0495] Table 25. In vivo efficacy statistics of Ab10-A297V3-2 / 4 at multiple doses

[0496] The anti-FAP / CD40 bispecific antibody used in the following examples is the aforementioned Ab10-A297V3-4, whose heavy chain and light chain amino acid sequences are SEQ ID NOs: 22 and 20, respectively.

[0497] Example 5. Screening of buffer systems and pH values ​​for anti-FAP / CD40 bispecific antibody preparations

[0498] The following buffer was prepared to prepare an anti-FAP / CD40 bispecific antibody formulation with a concentration of 20 mg / mL for high temperature (40°C) stability study.

[0499] 1) 10 mM acetic acid-sodium acetate, pH 5.5

[0500] 2) 10 mM histidine-histidine hydrochloride, pH 5.5

[0501] 3) 10 mM histidine-histidine hydrochloride, pH 6.0

[0502] 4) 10 mM histidine-histidine hydrochloride, pH 6.5

[0503] 5) 10 mM succinic acid-sodium succinate, pH 5.0

[0504] 6) 10 mM succinic acid-sodium succinate, pH 5.5

[0505] 7) 10 mM succinic acid-sodium succinate, pH 6.0

[0506] 8) 10 mM phosphate buffer, pH 6.5

[0507] 9) 10 mM phosphate buffer, pH 7.0

[0508] 10) 10 mM phosphate buffer, pH 7.5

[0509] Table 26. Buffer system and pH value screening results-1

[0510] Table 27. Buffer system and pH value screening results-2

[0511] Experimental results:

[0512] Appearance results showed that after four weeks of storage at 40°C, no obvious particles were observed in formulations F2 (histidine buffer, pH 5.5), F3 (histidine buffer, pH 6.0), and F5 (succinate buffer, pH 5.0). Visible particles were observed in all other formulations, indicating that histidine buffer (pH 5.5, pH 6.0) and succinate buffer (pH 5.0) outperformed other buffer systems.

[0513] SEC results showed that after 4 weeks of storage at 40°C, the SEC purity of all formulations decreased. The degree of decrease in the histidine salt system (F2-F4) and acetate system (F1) was smaller than that in the succinate system (F5-F7) and phosphate buffer system (F8-F10), and the monomer purity of formulations F2 and F3 did not change significantly.

[0514] CEX results showed that after being stored at 40°C for 4 weeks, the main peaks of prescriptions F1 and F3 decreased by about 20%, and the main peak of prescription F2 decreased to a degree second only to F1 and F3, at about 24%. The main peaks of other prescriptions decreased more significantly.

[0515] NR-CE results showed that after storage at 40°C for 4 weeks, the purity of formulations F1, F2, F5, and F6 decreased the least compared to T0, at about 6%; formulation F3 was second; and the purity of the remaining formulations decreased more significantly.

[0516] In summary, formulations F2 and F3 outperformed the other formulations in terms of appearance, SEC, CEX, and NR-CE testing, demonstrating improved stability. Therefore, a histidine buffer was selected as the final buffer system. F2 (10 mM histidine buffer, pH 5.5) and F3 (10 mM histidine buffer, pH 6.0) were selected as the buffer systems for the next round of pH screening.

[0517] Example 6. Screening of surfactants in anti-FAP / CD40 bispecific antibody formulations

[0518] A 10 mM histidine-histidine hydrochloride buffer system at pH 5.0 and pH 5.5 was used to prepare formulations containing 25 mg / mL anti-FAP / CD40 bispecific antibody, 80 mg / mL sucrose, and different types and concentrations of surfactants. The stability after repeated freeze-thaw and shaking was investigated.

[0519] 15) 10 mM histidine-histidine hydrochloride pH 5.0, 0.2 mg / mL polysorbate 80;

[0520] 16) 10 mM histidine-histidine hydrochloride pH 5.5, 0.4 mg / mL polysorbate 80;

[0521] 17) 10 mM histidine-histidine hydrochloride pH 5.5, 0.8 mg / mL polysorbate 80;

[0522] 18) 10 mM histidine-histidine hydrochloride pH 5.5, 2 mg / mL poloxamer 188.

[0523] Table 28. Screening results of surfactant types and concentrations Notes: LY = light yellow; SO = slightly opalescent; PF = no visible particles; AFP = few particles. *: 1 indicates one particle.

[0524] Experimental results

[0525] Appearance results showed that after five freeze-thaw cycles, one particle was observed in formulation F16, while a small amount of visible particles appeared in formulations F15, F17, and F18. After three days of shaking, no visible particles were observed in formulations F15 to F17, while a small amount of visible particles appeared in formulation F18.

[0526] The results of insoluble particulate matter showed that after shaking for 3 days and 5 cycles of freeze-thaw, the number of particles in prescriptions F15 to F18 did not change significantly, and there was little difference between the prescriptions.

[0527] The SEC results showed that after 5 cycles of freeze-thaw and 3 days of shaking, the SEC monomer content of formulations F15 to F18 did not show significant changes, and there was little difference between the formulations.

[0528] The CEX results showed that after shaking for 3 days and 5 cycles of freeze-thaw, the main peak, acid peak and base peak of prescriptions F15 to F18 had no obvious changes, and there was little difference between the prescriptions.

[0529] NR-CE results showed that after five cycles of freeze-thaw and three days of shaking, the purity and content of formulations F15 to F18 did not change significantly. There was little difference between the formulations.

[0530] In summary, in a histidine salt buffer system within a pH range of 5.0–5.5, visible particles were observed in formulations F15–F18 after five freeze-thaw cycles, with only one particle in formulation F16 being significantly better than the other formulations. After three days of shaking, a small amount of particles was observed in formulation F18, while none were observed in the other formulations. No significant differences were observed in the insoluble particulate matter and purity results among the different formulations. Therefore, polysorbate 80 was selected as the surfactant at a concentration of 0.04% (w / v).

[0531] Example 7. Screening of excipients in anti-FAP / CD40 bispecific antibody formulations

[0532] A 10 mM histidine-histidine hydrochloride buffer system was used to prepare anti-FAP / CD40 bispecific antibody formulations containing 0.4 mg / mL polysorbate 80, different excipients, and different antibody concentrations:

[0533] 19) 10 mM histidine-histidine hydrochloride pH 5.0, 8% sucrose, 25 mg / mL anti-FAP / CD40 bispecific antibody;

[0534] 20) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose, 25 mg / mL anti-FAP / CD40 bispecific antibody;

[0535] 21) 10 mM histidine-histidine hydrochloride pH 5.5, 1% sucrose + 4% mannitol, 25 mg / mL anti-FAP / CD40 bispecific antibody;

[0536] 22) 10 mM arginine-glutamate pH 5.0, 8% sucrose, 25 mg / mL anti-FAP / CD40 bispecific antibody;

[0537] 23) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose + 0.01% EDTA, 25 mg / mL anti-FAP / CD40 bispecific antibody;

[0538] 24) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose + 0.01% EDTA, 5 mg / mL anti-FAP / CD40 bispecific antibody;

[0539] 25) 10 mM histidine-histidine hydrochloride pH 5.5, 8% sucrose, 5 mg / mL anti-FAP / CD40 bispecific antibody;

[0540] The above formulation was placed at 2-8°C, 25°C and 40°C to investigate its stability, and the repeated freeze-thaw stability was also investigated.

[0541] Table 29. Anti-FAP / CD40 bispecific antibody excipient screening results Notes: LY = pale yellow; SO = slightly opalescent; O = opalescent; PF = no visible particles; AFP = few particles. N / A: not tested.

[0542] Experimental results

[0543] Appearance results showed that after freeze-thaw cycles, visible particles appeared in formulation F21, and the opalescence gradually increased with the number of freeze-thaw cycles. The remaining formulations showed no visible particles. After storage at 40°C for four weeks, formulations F19 and F21 showed a small amount of visible particles, while the remaining formulations showed no visible particles. After storage at 2-8°C and 25°C for four weeks, all formulations showed no visible particles.

[0544] SEC results showed that after 4 weeks of storage at 40°C, the monomer content of all formulations did not change significantly. After 5 cycles of freeze-thaw, the SEC monomer content did not change significantly.

[0545] CEX results showed that after four weeks of storage at 40°C, the main peaks of formulations F22 to F24 decreased slightly, while the main peaks of the remaining formulations decreased more significantly. After five cycles of freeze-thaw, the contents of the CEX main peak, acid peak, and base peak remained unchanged.

[0546] NR-CE results showed that after four weeks of storage at 40°C, the purity of formulation F19 decreased the most, while the purity of the other formulations also decreased slightly, but the decline was comparable. After five cycles of freeze-thaw, the purity content of all formulations remained unchanged, with little difference between the formulations.

[0547] In summary, based on the appearance results, after 5 cycles of freeze-thaw, formulation F21 showed a small amount of particles and an enhanced opalescence compared to the other formulations. After 4 weeks of incubation at 40°C, formulations F19 and F21 showed a small amount of particles, while the other formulations showed no significant changes. The SEC monomer purity of each formulation did not show significant changes. Based on the CEX results, after 4 weeks of incubation at 40°C, the main peak content of formulations F20 to F24 decreased less than that of the other formulations. Based on the NR-CE results, after 4 weeks of incubation at 40°C, formulation F19 showed the largest 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.

[0548] Example 8. Screening of protein concentration in anti-FAP / CD40 bispecific antibody preparations

[0549] A 10 mM acetate-histidine pH 5.0 and pH 5.5 buffer system was selected to prepare 8% sucrose, 0.8 mg / mL polysorbate 80, and anti-FAP / CD40 bispecific antibody formulations at different antibody concentrations:

[0550] 26) 10 mM acetate-histidine pH 5.0, 50 mg / mL anti-FAP / CD40 bispecific antibody;

[0551] 27) 10 mM acetate-histidine pH 5.0, 80 mg / mL anti-FAP / CD40 bispecific antibody;

[0552] 28) 10 mM acetate-histidine pH 5.0, 100 mg / mL anti-FAP / CD40 bispecific antibody;

[0553] 29) 10 mM acetate-histidine pH 5.5, 50 mg / mL anti-FAP / CD40 bispecific antibody;

[0554] The stability of each formulation at 5°C and 25°C was investigated respectively.

[0555] Table 30. Anti-FAP / CD40 bispecific antibody concentration screening results

[0556] The appearance results showed that the formulations with different antibody concentrations did not undergo significant changes after being stored at 5°C and 25°C for 3 months, indicating good stability.

[0557] SEC results showed that the monomer content of high-concentration formulations F27 and F28 showed a downward trend after 3 months at 25°C, with the decrease becoming more pronounced with increasing concentration. For formulation F28 (100 mg / mL), the monomer purity dropped to approximately 95% after 3 months at 25°C. No significant changes were observed in any of the formulations after 3 months at 5°C.

[0558] The CEX results showed that the main peak content of each prescription decreased slightly after being stored at 5℃ for 3 months or at 25℃ for 3 months, and the differences among the prescriptions were not significant;

[0559] The NR-CE results showed that after being placed at 25°C for 3 months, the main peak of F29 formulation decreased by about 5%, while other formulations showed no obvious downward trend.

[0560] In summary, the protein had good stability when the concentration was as high as 100 mg / mL and the pH was 5.0.

[0561] Example 9. Other optional formulations

[0562] In addition, the present invention also provides anti-FAP / CD40 bispecific antibody pharmaceutical preparations of other formulations, wherein the anti-FAP / CD40 bispecific antibody is Ab10-A297V3-4 of the present application, whose heavy chain and light chain sequences are SEQ ID NOs: 22 and 20, respectively, including but not limited to:

[0563] (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;

[0564] (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;

[0565] (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;

[0566] (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;

[0567] (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;

[0568] (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;

[0569] (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;

[0570] (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;

[0571] (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;

[0572] (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;

[0573] (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;

[0574] (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;

[0575] (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;

[0576] (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;

[0577] (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;

[0578] (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;

[0579] (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;

[0580] The experimental results show that the FAP / CD40 bispecific antibody preparations of the above formulations have good stability and can be used in the preparation of FAP / CD40 bispecific antibody drugs.

Claims

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

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

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

4. The pharmaceutical composition of claim 2 or 3, wherein 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 as shown in any one of SEQ ID NOs: 11, 16 to 19 or having at least 90% sequence identity with any one of SEQ ID NOs: 11, 16 to 19.

5. The pharmaceutical composition of any one of claims 2 to 4, wherein 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.

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

7. The pharmaceutical composition of any one of claims 2 to 6, wherein the immunoglobulin single variable domain that specifically binds to the second antigen binding domain of CD40 in the FAP / CD40 binding molecule is directly or through a linker connected to the first antigen binding domain that specifically binds to FAP; Preferably, the linker is a (G4S) x The amino acid sequence shown, wherein x is independently selected from an integer of 1-20; More preferably, the linker is an amino acid sequence represented by (G4S)2, (G4S)3 or (G4S)4.

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

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

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

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

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

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

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

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

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

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

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

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

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

21. A pharmaceutical composition comprising: The FAP / CD40 binding molecule according to any one of claims 1 to 11; Histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; Polysorbates; sucrose; Optionally, the composition further comprises mannitol or ethylenediaminetetraacetic acid.

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

23. The pharmaceutical composition according to claim 21, comprising any one of the following 1)-21): 1) 1-150 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; 0.2mg / mL-1mg / mL polysorbate 80; About 80 mg / mL sucrose; And the pH of the pharmaceutical composition is 4.5-6; 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; preferably 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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) 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 the FAP / CD40 binding molecule of any one of claims 1 to 11; 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.

24. A lyophilized preparation, which can form the pharmaceutical composition of any one of claims 1 to 23 after being reconstituted, or which is obtained by freeze-drying the pharmaceutical composition of any one of claims 1 to 23.

25. A reconstituted solution, wherein the reconstituted solution is prepared by reconstituted the lyophilized preparation according to claim 24.

26. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 23, the lyophilized preparation of claim 24, or the reconstituted solution of claim 25.

27. Use of the pharmaceutical composition according to any one of claims 1 to 23, the lyophilized preparation according to claim 24, or the reconstituted solution according to claim 25 in the preparation of a drug for treating or alleviating a disease or condition; The disease or condition is preferably a tumor or cancer; More preferably, it is 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.

28. A method for treating or alleviating a tumor or cancer, the method comprising: Administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 23, the lyophilized formulation of claim 24, or the reconstituted solution of 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.

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