Anti-CD40×CLDN18.2 bispecific antibodies and uses thereof

Abstract

The present invention relates to a bispecific antibody that simultaneously targets CD40 and CLDN18.2, which highly activates immune cells specifically at the site of a lesion and has few side effects. The present invention further relates to a pharmaceutical composition comprising said bispecific antibody, and its use and method for treating related diseases.

Description

[Background technology]

[0001] CD40 is a 50 kD costimulatory protein located on the surface of the cell membrane and belongs to the tumor necrosis factor receptor (TNFR) family. It is constitutively expressed on antigen-presenting cells (including dendritic cells, B cells, monocytes, and macrophages) and on several other cell types, such as endothelial cells, fibroblasts, and thymic epithelial cells. CD40 is also expressed on several tumor cells (e.g., chronic lymphocytic leukemia, multiple myeloma, renal carcinoma, lung carcinoma, etc.). CD40 forms a trimer on the cell surface, and its corresponding ligand, CD40L (i.e., CD154), is mainly expressed on the surface of activated T cells. The interaction of CD40 with CD40L is a costimulatory signal for T cell activation. Binding of CD40 to CD40L on T cells can activate several pathways, including the NF-κB (nuclear factor κB) signaling pathway.

[0002] Agonistic antibodies targeting CD40, such as selicrelumab, dacetuzumab, and APX005, have shown clinical activity in many indications in clinical trials, but these agonistic antibodies are associated with dose-limiting toxic adverse events (Hassan SB et al., Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials. Immunopharmacol Immunotoxicol 2014;36:96-104). The main adverse event currently is hepatotoxicity. These agonistic antibodies nonspecifically activate immune cells expressing CD40 on the surface of hepatocytes, causing hepatotoxicity and reducing the therapeutic index.

[0003] The human CLDN18 gene has two different first exons, and after transcription, alternative splicing ultimately generates two protein subtypes, CLDN18.1 and CLDN18.2, which have different sequences only at the N-terminus. Both CLDN18 subtype proteins consist of 261 amino acids and both have four transmembrane domains, but they are distributed in different tissues. CLDN18.1 is mainly expressed in lung tissue, while CLDN18.2 is expressed only on differentiated epithelial cells on the gastric mucosa, but not in gastric stem cells (Sahin, Ugur, et al. "Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development." ClinicalCancer Research14.23 (2008): 7624-7634.). CLDN18.2 is highly expressed in multiple types of tumor tissues, such as non-small cell lung cancer (25%), gastric cancer (70%), pancreatic cancer (50%), and esophageal cancer (30%). However, it is hardly expressed in normal tissues (Kumar, V., et al. (2018). "Emerging Therapies in the Management of Advanced-Stage Gastric Cancer." Front Pharmacol 9: 404). Due to the difference in expression between tumor cells and normal tissues, it is currently a very promising target for antitumor drugs.

[0004] Therefore, it is necessary to develop bispecific antibodies targeting CLDN18.2 and CD40 to specifically kill tumor cells and avoid adverse events such as liver toxicity, thus providing more possibilities for cancer treatment. Summary of the Invention

[0005] In the present invention, a bispecific antibody that simultaneously targets CD40 and CLDN18.2 is newly disclosed, and a polynucleotide encoding the bispecific antibody, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, a method for treating a disease associated with CD40 and / or CLDN18.2 using the bispecific antibody, and a use of the bispecific antibody in the individual treatment, prevention and / or diagnosis of a disease associated with CD40 and / or CLDN18.2 are also disclosed. The bispecific antibody can specifically kill tumor cells without causing adverse events such as hepatotoxicity by highly activating immune cells specifically at the site of the lesion and realizing low agonistic activity at sites such as liver cells where the cross-linking effect cannot be formed.

[0006] In a first aspect, the present invention provides a bispecific antibody that specifically binds CD40 and CLDN18.2 (anti-CD40×CLDN18.2 bispecific antibody), comprising (i) an anti-CD40 antibody or a fragment thereof, and (ii) an anti-CLDN18.2 antibody or a fragment thereof. In one embodiment, the anti-CLDN18.2 antibody or a fragment thereof is a single domain antibody (VHH).

[0007] In one embodiment, the invention provides an anti-CD40 x CLDN18.2 bispecific antibody, (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain whose structure is VL-CL.

[0008] wherein the VH and VL combine to form a first antigen-binding portion that specifically binds CD40, and the VHH forms a second antigen-binding portion that specifically binds CLDN18.2, and optionally the VHH is linked to Fc via a linker.

[0009] In one embodiment, the invention provides an anti-CD40 x CLDN18.2 bispecific antibody, (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain whose structure is VL-CL.

[0010] Where: the VH comprises the three heavy chain CDRs contained in SEQ ID NO:11, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:18; the VH comprises the three heavy chain CDRs contained in SEQ ID NO:12, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:18; the VH comprises the three heavy chain CDRs contained in SEQ ID NO:13, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:18; or The VH comprises the three heavy chain CDRs contained in SEQ ID NO:14, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:18.

[0011] In another embodiment, the present invention provides an anti-CD40 x CLDN18.2 bispecific antibody, (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain whose structure is VL-CL.

[0012] Where: the VH comprises three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; the VL comprises three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; and the VHH comprises three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17; the VH comprises three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7; the VL comprises three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and the VHH comprises three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; the VH comprises the three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, the VL comprises the three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and the VHH comprises the three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17; or The VH comprises the three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:9, the VL comprises the three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the VHH comprises the three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0013] Furthermore, in another embodiment, the present invention provides an anti-CD40×CLDN18.2 bispecific antibody, (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain whose structure is VL-CL.

[0014] Where: the VH comprises a sequence from the three heavy chain CDRs contained in SEQ ID NO:11 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:11; the VL comprises a sequence from the three light chain CDRs contained in SEQ ID NO:10 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10; and the VHH comprises a sequence from the three CDRs contained in SEQ ID NO:18 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:18; the VH comprises a sequence from the three heavy chain CDRs contained in SEQ ID NO:12 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12; the VL comprises a sequence from the three light chain CDRs contained in SEQ ID NO:10 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10; and the VHH comprises a sequence from the three CDRs contained in SEQ ID NO:18 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:18; the VH comprises a sequence from the three heavy chain CDRs contained in SEQ ID NO:13 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:13; the VL comprises a sequence from the three light chain CDRs contained in SEQ ID NO:10 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10; and the VHH comprises a sequence from the three CDRs contained in SEQ ID NO:18 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:18; or The VH comprises a sequence from the three heavy chain CDRs contained in SEQ ID NO:14 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:14; the VL comprises a sequence from the three light chain CDRs contained in SEQ ID NO:10 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10; and the VHH comprises a sequence from the three CDRs contained in SEQ ID NO:18 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:18.

[0015] In one embodiment, the invention provides an anti-CD40 x CLDN18.2 bispecific antibody, (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain whose structure is VL-CL.

[0016] Where: the VH comprises a sequence shown in SEQ ID NO:11, the VL comprises a sequence shown in SEQ ID NO:10, and the VHH comprises a sequence shown in SEQ ID NO:18; the VH comprises the sequence shown in SEQ ID NO: 12, the VL comprises the sequence shown in SEQ ID NO: 10, and the VHH comprises the sequence shown in SEQ ID NO: 18; the VH comprises the sequence shown in SEQ ID NO:13, the VL comprises the sequence shown in SEQ ID NO:10, and the VHH comprises the sequence shown in SEQ ID NO:18; or The VH comprises the sequence shown in SEQ ID NO:14, the VL comprises the sequence shown in SEQ ID NO:10, and the VHH comprises the sequence shown in SEQ ID NO:18.

[0017] In any one of the above embodiments, the single domain antibody (VHH) in the anti-CD40×CLDN18.2 bispecific antibody of the present invention is a camelid VHH, a partially humanized or fully humanized VHH, or a chimeric VHH.

[0018] In any one of the above embodiments, the VHH of the anti-CD40 x CLDN18.2 bispecific antibody of the present invention is linked to the Fc via a linker. The linker may be any universal flexible sequence already known in the art or to be obtained in the future, and is usually a short flexible amino acid sequence. In one specific embodiment, the linker is (G4S)n, where n is an integer equal to or greater than 1. For example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, such as GGGSG;GGSGG;GSGGGG;SGGGG;GGTGS;GTSPGG;GNGGGS;G4S-GGSGG-G4S-SGGGG;GGG;DGGGS;TGEKP;GGRR;EGKSSGSGSESKVD;KESGSVSSEQLAQFRSLD;GGRRGGGS;LRQRDGERP;LRQKDGGGSERP;GSTSGSGKPGSGEGSTKG. In one specific embodiment, the linker is selected from (G4S)3.

[0019] In any one of the above embodiments, the Fc region of the anti-CD40×CLDN18.2 bispecific antibody of the invention may be selected from the native Fc region of any IgG-based antibody already known in the prior art, e.g., consensus or embryonic IgG Fc region. In one specific embodiment, the IgG Fc region may be an Fc region from different IgG subclasses, e.g., from IgG1, IgG2, IgG3 and IgG4. In one specific embodiment, the IgG Fc region is an IgG1 Fc sequence. In another specific embodiment, the IgG Fc region may contain mutations / modifications to stabilize the antibody or to reduce FcγRIIb-dependent cross-linking effects so that liver toxicity can be avoided, e.g., modifications already known in the prior art. In one specific embodiment, the bispecific anti-CD40×CLDN18.2 antibody of the invention has been modified to contain a constant region that has no cross-linking effects. For example, the FcγRIIb-dependent cross-linking effect of the anti-CD40×CLDN18.2 bispecific antibody of the invention can be reduced or eliminated by mutating the glycosylation site N297 residue in the Fc region to Gly, Ala, Gln, Asp or Glu. In a preferred embodiment, the Fc region of the anti-CD40×CLDN18.2 bispecific antibody of the invention comprises the N297Q mutation to reduce binding to Fc receptors.

[0020] In one embodiment, the light chain constant domain CL of the anti-CD40×CLDN18.2 bispecific antibody of the invention is of kappa or lambda origin.

[0021] In one embodiment, the invention provides an anti-CD40 x CLDN18.2 bispecific antibody, (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain whose structure is VL-CL.

[0022] Where: the heavy chain comprises a sequence from three heavy chain CDRs and three VHH CDRs contained in SEQ ID NO:19 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:19; and the light chain comprises a sequence from three light chain CDRs contained in SEQ ID NO:23 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:23; the heavy chain comprises a sequence from three heavy chain CDRs contained in SEQ ID NO:20 and three VHH CDRs and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20; and the light chain comprises a sequence from three light chain CDRs contained in SEQ ID NO:23 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:23; the heavy chain comprises a sequence from three heavy chain CDRs contained in SEQ ID NO:21 and three VHH CDRs and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:21; and the light chain comprises a sequence from three light chain CDRs contained in SEQ ID NO:23 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:23; or The heavy chain comprises a sequence from three heavy chain CDRs and three VHH CDRs contained in SEQ ID NO:22 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:22, and the light chain comprises a sequence from three light chain CDRs contained in SEQ ID NO:23 and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:23.

[0023] In one embodiment, the invention provides an anti-CD40 x CLDN18.2 bispecific antibody, (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain whose structure is VL-CL.

[0024] Where: the heavy chain comprises the sequence shown in SEQ ID NO: 19 and the light chain comprises the sequence shown in SEQ ID NO: 23; the heavy chain comprises the sequence shown in SEQ ID NO:20 and the light chain comprises the sequence shown in SEQ ID NO:23; the heavy chain comprises the sequence shown in SEQ ID NO:21 and the light chain comprises the sequence shown in SEQ ID NO:23; or The heavy chain comprises the sequence shown in SEQ ID NO:22 and the light chain comprises the sequence shown in SEQ ID NO:23.

[0025] In one embodiment, the anti-CD40×CLDN18.2 bispecific antibody of the present invention can enhance the immune response to an antigen and induce antibody-dependent cellular cytotoxicity against cells expressing CD40 (e.g., tumor cells expressing CD40) and cells expressing CLDN18.2 (e.g., tumor cells expressing CLDN18.2).

[0026] In a second aspect, the invention provides polynucleotides (nucleic acids) encoding the anti-CD40xCLDN18.2 bispecific antibodies of the invention and vectors comprising said polynucleotides, preferably expression vectors.

[0027] In a third aspect, the present invention provides a host cell comprising the polynucleotide or vector of the present invention. The host cell may be any prokaryotic or eukaryotic cell commonly used in the art.

[0028] In a fourth aspect according to the invention there is provided a method for producing an anti-CD40xCLDN18.2 bispecific antibody of the invention, comprising the step (i) of culturing a host cell of the invention under conditions suitable for expression of the anti-CD40xCLDN18.2 bispecific antibody of the invention and, preferably, the step (ii) of recovering the anti-CD40xCLDN18.2 bispecific antibody of the invention.

[0029] In a fifth aspect, the present invention provides an immunoconjugate, a reagent kit, a pharmaceutical composition, a combination product or an article of manufacture comprising the anti-CD40xCLDN18.2 bispecific antibody of the present invention. In one embodiment, the pharmaceutical composition, combination product or article of manufacture provided by the present invention further comprises another therapeutic agent, and optionally an adjuvant. Preferably, the other therapeutic agent is selected from a chemotherapeutic agent or a cytotoxic agent.

[0030] In a sixth aspect of the present invention, there is provided a use of an anti-CD40xCLDN18.2 bispecific antibody, immunoconjugate, reagent kit, pharmaceutical composition, combination product or article of manufacture of the present invention for treating, preventing and / or diagnosing a CD40-related disease and a CLDN18.2-related disease. In one embodiment, the CD40-related disease is, for example, a cancer in which CD40 expression is aberrant. In one embodiment, the CLDN18.2-related disease is, for example, a cancer in which CLDN18.2 expression is aberrant.

[0031] In one embodiment, the invention provides the use of an anti-CD40xCLDN18.2 bispecific antibody, polynucleotide, vector, host cell, immunoconjugate, reagent kit, pharmaceutical composition, combination product or article as defined in the first to third and fifth aspects in a process for the manufacture of a medicament for treating, preventing and / or diagnosing a CD40-related or CLDN18.2-related disease.

[0032] In a seventh aspect of the present invention, there is provided a method for treating a CD40-related disease and a method for treating a CLDN18.2-related disease, comprising administering to a patient a therapeutically effective amount of an anti-CD40xCLDN18.2 bispecific antibody of the invention, or an immunoconjugate, pharmaceutical composition, combination product or article of the invention. In one embodiment, the CD40-related disease is, for example, a cancer in which CD40 expression is aberrant. In one embodiment, the CLDN18.2-related disease is, for example, a cancer in which CLDN18.2 expression is aberrant. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 shows the schematic structure of the bispecific antibody of the present invention. [Diagram 2] FIG. 2 shows the binding activity of the bispecific antibody of the present invention to a P17-His fusion protein. [Diagram 3] FIG. 3 shows the binding activity of a bispecific antibody of the present invention to huCD40-CHO-K cells. [Figure 4] FIG. 4 shows the binding activity of a bispecific antibody of the present invention to 18.2-HEK293 cells. [Figure 5A] FIG. 5A shows activation of the CD40 signaling pathway by a bispecific antibody of the invention in the presence of a cross-linking agent (5A). [Figure 5B] FIG. 5B shows that the CD40 signaling pathway is activated by a bispecific antibody of the present invention in the absence of a cross-linking agent (5B). [Figure 6A]FIG. 6A shows that the CD40 signal pathway was activated by the bispecific antibody of the present invention when CLDN18.2 was expressed on the cell surface (6A). [Figure 6B] FIG. 6B shows that the CD40 signal pathway was activated by the bispecific antibody of the present invention when CLDN18.2 was not expressed on the cell surface (6B). [Figure 7] FIG. 7 shows the inhibitory effect of the bispecific antibody of the present invention on tumor growth in vivo in humanized mice. [Figure 8A] FIG. 8A shows the results of whether or not the bispecific antibody of the present invention induces hepatotoxicity in humanized mice. [Figure 8B] FIG. 8B shows the results of whether or not the bispecific antibody of the present invention induces hepatotoxicity in humanized mice. [Figure 8C] FIG. 8C shows the results of whether or not the bispecific antibody of the present invention induces hepatotoxicity in humanized mice. [Figure 8D] FIG. 8D shows the results of whether the bispecific antibody of the present invention induces hepatotoxicity in humanized mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] I. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. It should be noted that the materials, methods and examples described herein are merely illustrative and are not intended to be limiting. Other features, objects and advantages of the present invention will become apparent from the specification and drawings, and from the claims.

[0035] In order to describe this specification, the following definitions are used. Where appropriate, terms used in the singular also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0036] The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values, from a lower limit of 10% less than the specified number or numerical value to an upper limit of 10% more than the specified number or numerical value.

[0037] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0038] As used herein, the term "comprising" or "including" means including the stated elements, integers, or steps without excluding any other elements, integers, or steps. When the term "comprising" or "including" is used herein, it also covers combinations of the stated elements, integers, or steps, unless otherwise specified. For example, when an antibody variable region "comprising" a particular sequence is referenced, it is intended to also cover an antibody variable region consisting of that particular sequence.

[0039] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen-binding site and covers natural and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, single domain antibodies, complete antibodies and antibody fragments. Preferably, the antibodies of the present invention are single domain antibodies or heavy chain antibodies.

[0040] The term "antibody fragment" refers to a molecule distinct from an intact antibody, which contains a portion of an intact antibody and is capable of binding to the antigen that the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single chain antibodies (e.g., scFv), single domain antibodies, bivalent or bispecific antibodies or fragments thereof, camelid antibodies (heavy chain antibodies), and bispecific or multispecific antibodies consisting of antibody fragments.

[0041] The term "Fc region" includes at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, numbering of amino acid residues in an Fc region or constant region is done according to the EU numbering system, also known as the EU index.

[0042] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies usually have a similar structure, where each domain contains four conserved framework regions (FR) and three complementarity determining regions (CDR) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0043] "Complementarity determining regions" or "CDR regions" or "CDRs" are regions in an antibody variable domain that are highly variable in sequence and form structure-defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy chain are usually referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. For a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined by any one or a combination of several known antibody CDR assignment systems, such as the Chothia system, which is based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), the Kabat system, which is based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), the AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics (ImmunoGeneTics), and the ELISA system (ImmunoGeneTics). The North CDR definition is based on the IMGT database (http: / / imgt.cines.fr / ) and affinity propagation clustering using multiple crystal structures.

[0044] Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined by any one of the above methods.

[0045] The CDRs may be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any one of the CDRs exemplified in the present invention). In one embodiment, the CDRs of the antibody of the present invention are positioned based on the proposed AbM numbering.

[0046] Unless otherwise specified, in the present invention, when residue positions in antibody variable regions and CDRs (including heavy chain variable region residues) are referred to, they refer to numbered positions based on the AbM numbering system.

[0047] The term "single domain antibody" refers to an antibody that can confer antigen binding by a single variable domain (e.g., a heavy chain variable domain (VH) or a light chain variable domain (VL), a heavy chain variable domain from a camelid heavy chain antibody, a VH-like single domain (v-NAR) from a fish IgNAR). That is, the single variable domain can recognize the target antigen without the need for interaction with another variable domain. Examples of single domain antibodies are single domain antibodies from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks) (WO 2005 / 035572). Single domain antibodies from camelids, also referred to herein as VHH, are antibodies that consist of only one heavy chain variable region and contain only one chain FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from the C-terminus to the N-terminus, and are also referred to as "nanobodies". Single domain antibodies are the smallest unit of a target antigen that can be bound currently known.

[0048] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity for at least two different antigen epitopes. In one embodiment, a multispecific antibody is provided herein, having binding specificity for a first antigen and a second antigen, also referred to as a "bispecific antibody."

[0049] "Immunoconjugate" refers to an antibody conjugated to one or more other substances, including, but not limited to, a cytotoxic agent or a label.

[0050] The term "agonist" refers to increasing some parameter (e.g., activity) of a given molecule (e.g., a costimulatory molecule). For example, the term includes substances that increase the activity of a given molecule (e.g., CD40) by at least 5%, 10%, 20%, 30%, 40% or more. Thus, agonism is not necessarily 100%.

[0051] In the context of "anti-CD40 antibodies," the term "bridging effect" refers to the phenomenon in which an anti-CD40 antibody binds to FcγRIIB via its Fc, promoting local aggregation (multimerization) of one or more anti-CD40 antibodies, and further promoting sufficient aggregation of CD40 molecules. The bridging effect induces the transmission of downstream intracellular signals, and further activates immune cells expressing the corresponding CD40.

[0052] "Median Effective Concentration (EC 50 The term "maximum maximal response" refers to the concentration of a drug, antibody, or toxin that induces a response of 50% between baseline and maximum after a given exposure time.

[0053] The term "therapeutic index (TI)" refers to the median lethal dose (LD 50 ) and median effective concentration (EC 50 ) and is an indicator of drug safety.

[0054] Calculations of sequence identity between sequences are performed as follows.

[0055] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison). In one preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as in the corresponding position in the second sequence, the molecules are identical at this position.

[0056] A mathematical algorithm can be used to achieve the sequence comparison and calculation of the percent identity between two sequences. In one preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated in the GAP program of the GCG software package (available at http: / / www.gcg.com), Blossum 62 matrix or PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 are used to determine the percent identity between two amino acid sequences. In another preferred embodiment, the GAP program of the GCG software package (available at http: / / www.gcg.com), NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6 are used to determine the percent identity between two nucleotide sequences. A particularly preferred set of parameters (and that which should be used unless otherwise specified) is a Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0057] Additionally, the percent identity between two amino acid or nucleotide sequences can be determined utilizing the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) implemented in the ALIGN program (version 2.0), using a PAM120 weighted covariance table, a gap length penalty of 12, and a gap penalty of 4).

[0058] Additionally or alternatively, the nucleic acid and protein sequences described herein may be used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.

[0059] The term "treatment" refers to alleviating, interrupting, slowing, alleviating, halting, reducing, or reversing the progression or severity of an already existing symptom, pathology, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, improving or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or slow the progression of a disease.

[0060] The term "prevention" includes inhibition of the onset or progression of a disease or condition, or a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. In general, in the context of cancer, the term "prevention" refers to administration of a drug before signs or symptoms of cancer appear, particularly in subjects at risk of cancer.

[0061] The term "effective amount" refers to the amount or dose of the antibody or conjugate or composition of the present invention that, after administration to a patient in one or more doses, produces the desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by the attending physician, who is skilled in the art, taking into consideration several factors, such as the species of mammal, body weight, age and general health status, the specific disease involved, the extent or severity of the disease, the response of the individual patient, the specific antibody administered, the mode of administration, the bioavailability characteristics of the formulation administered, the administration regimen selected, and the use of any combination therapy.

[0062] The term "therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result at the required dosage and for the required period of time. The therapeutically effective amount of an antibody or antibody fragment, or conjugate or composition thereof, varies depending on various factors, such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody fragment, or conjugate or composition thereof, are outweighed by the therapeutically beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and even more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that is predictive of efficacy in human tumors.

[0063] A "prophylactically effective amount" refers to an amount that, at dosages and for periods of time necessary, effectively achieves the desired prophylactic result. Because a prophylactic dose is usually administered prior to or at an earlier stage of disease in the subject, the prophylactically effective amount is less than the therapeutically effective amount.

[0064] The term "pharmaceutical composition" refers to a composition that is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain other ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0065] The terms "linking peptide", "linker" or "peptide linker" are used interchangeably herein and refer to a peptide comprising one or more consecutive amino acids, such as small or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). A linking peptide typically comprises 5-50 amino acids in length, such as 10, 15, 20, 25, 30 amino acids in length. It will be appreciated by those skilled in the art that many commonly used linkers can be used in embodiments of the present invention.

[0066] The term "CLDN18.2-associated disease" refers to any condition caused, exacerbated, or otherwise associated with aberrant expression (e.g., increased expression) or abnormal activity of CLDN18.2 (e.g., human CLDN18.2). In one embodiment, the CLDN18.2-associated disease is a cancer in which CLDN18.2 (e.g., human CLDN18.2) is aberrantly expressed (e.g., increased expression).

[0067] The term "CD40-associated disease" refers to any condition caused, exacerbated, or otherwise associated with aberrant expression (e.g., increased expression) or activity of CD40. In one embodiment, the CD40-associated disease is a cancer in which CD40 is aberrantly expressed (e.g., increased expression).

[0068] The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human.

[0069] II. Antibodies of the Invention Claudin 18.2 (also referred to as "CLDN18.2" in this specification) is significantly different in expression between cancer tissues and normal tissues. This is thought to be because the CREB binding site in the promoter region of Claudin 18.2 is highly methylated at CpG in normal tissues, whereas the CpG methylation level decreases during the canceration process, and CREB is involved in the transcriptional activation of Claudin 18.2.

[0070] The terms "anti-CD40 and CLDN18.2 bispecific antibody", "bispecific antibody that specifically binds to CD40 and CLDN18.2", "anti-CD40×CLDN18.2 bispecific antibody", "CD40 / CLDN18.2 bispecific antibody" and similar terms mentioned herein refer to a bispecific antibody that can bind to targets CD40 and CLDN18.2 with sufficient affinity. The bispecific antibody can recruit immune cells and redirect lyse target cells. The bispecific antibody can specifically activate immune cells at the lesion site with high intensity and achieve low agonist activity at sites such as liver cells that cannot form a cross-linking effect, thereby specifically killing tumor cells without causing adverse events such as liver toxicity.

[0071] In one embodiment, the bispecific anti-CD40xCLDN18.2 antibody of the invention comprises an amino acid modification, such as, for example, an amino acid substitution, addition, or deletion, preferably an amino acid substitution, and more preferably a conservative amino acid substitution.

[0072] In one embodiment, the amino acid modification of the present invention occurs in a region other than CDR (e.g., FR). In one embodiment, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same type. For example, one acidic amino acid is substituted with another acidic amino acid, one basic amino acid is substituted with another basic amino acid, or one neutral amino acid is substituted with another neutral amino acid.

[0073] Exemplary substitutions are set out in the table below. TIFF2025505928000001.tif106170

[0074] In some embodiments, the substitutions occur in the CDR regions of the antibody. Usually, the resulting variant has a modification (e.g., improvement) in a particular biological property (e.g., increased affinity) compared to the parent antibody and / or has a particular biological property of the parent antibody that is substantially retained. An exemplary substitution variant is an affinity matured antibody.

[0075] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG11, IgG2, IgG3, or IgG4 Fc region) with an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0076] According to research, the Fc region of a partial agonist anti-CD40 antibody forms a cross-linking effect with FcγRIIB, which is highly expressed on the surface of hepatocytes, and non-specifically activates immune cells expressing CD40 on the surface of hepatocytes, causing hepatotoxicity. Therefore, to reduce or eliminate the cross-linking effect caused by the Fc region, the anti-CD40×CLDN18.2 bispecific antibody of the present invention may contain a modification in the Fc region that reduces the binding affinity of the antibody of the present invention to FcγRIIb. In one embodiment, the modification is located in the CH2 domain of the Fc region, for example, at position 329 (EU index) of the heavy chain (e.g., P329G). In one embodiment, the bispecific anti-CD40×CLDN18.2 antibody of the present invention contains amino acid substitutions located at positions 234 and 235 (EU index) of the heavy chain. In one specific embodiment, the amino acid substitutions are L234A and L235A (LALA mutation).

[0077] In one embodiment, the bispecific anti-CD40xCLDN18.2 antibodies of the invention can enhance immune responses independent of antibody binding to Fc receptors, for example, the bispecific anti-CD40xCLDN18.2 antibodies of the invention can exhibit effective CD40 agonist characteristics without cross-linking Fc receptors, e.g., FcγR.

[0078] In one embodiment, there is a disulfide bond between CH1 and CL of the anti-CD40xCLDN18.2 bispecific antibody of the invention. In one embodiment, the number of disulfide bonds varies depending on the IgG format from which the antibody constant domain is derived. In some embodiments, there are two or four disulfide bonds between the hinge regions.

[0079] In some embodiments, it may be desirable to generate antibodies that have been altered via cysteine ​​engineering, e.g., "thio mAbs," in which one or more residues of the antibody are replaced by cysteine ​​residues.

[0080] III. Nucleic Acids of the Invention and Vectors and Host Cells Containing Them The present invention provides nucleic acids encoding any of the above bispecific antibodies or antigen-binding fragments thereof. The present invention further covers nucleic acids that hybridize under stringent conditions to the above nucleic acids, that have one or more substitutions (e.g. conservative substitutions), deletions or insertions compared to the above nucleic acids, or that have at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the above nucleic acids.

[0081] Furthermore, the present invention provides a vector comprising the above-mentioned nucleic acid. In a preferred embodiment, the vector is an expression vector. It can be understood by those skilled in the art that vectors commonly used in this field can be applied to the present invention.

[0082] In one embodiment, the invention provides a host cell comprising the nucleic acid or the vector.

[0083] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced and the progeny of such a cell. Host cells include "transformants" and "transformed cells", including the primary transformed cell and its progeny, without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. As used herein, we include mutant progeny that have the same function or biological activity as screened or selected from the originally transformed cell. Host cells are any type of cell line used to produce the antibody molecules of the invention, including eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells, and prokaryotic cells, such as E. coli cells. Host cells include cultured cells, including cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0084] IV. Compositions of the Invention In some embodiments, the present invention provides a composition comprising any of the anti-CD40xCLDN18.2 bispecific antibodies or antigen-binding fragments thereof described herein. Preferably, the composition is a pharmaceutical composition. In one embodiment, the composition comprises a pharmaceutical adjuvant. The term "pharmaceutical adjuvant" refers to a diluent, adjuvant, carrier, excipient, stabilizer, etc., administered together with an active substance.

[0085] In one embodiment, a composition (e.g., a pharmaceutical composition) comprises a combination of an anti-CD40×CLDN18.2 bispecific antibody of the invention, or an antigen-binding fragment thereof, and one or more other therapeutic agents (e.g., a chemotherapeutic agent, a cytotoxic agent, a vaccine, another antibody, an anti-infective active agent, a small molecule drug, or an immunomodulatory agent, etc.).

[0086] The pharmaceutical composition of the present invention may further comprise one or more other therapeutic agents. The therapeutic agents are required for the treatment of a particular indication and are any active substances in the prevention or treatment of tumors (e.g., cancer) and infections (e.g., chronic infections). Preferably, the therapeutic agents do not adversely affect each other's activity. For example, it is preferable to further provide other anti-cancer active therapeutic agents. For example, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infection active agents, small molecule drugs or immunomodulators. The therapeutic agents are present in a suitable combination in an effective amount according to the target application.

[0087] V. Production of Anti-CD40×CLDN18.2 Bispecific Antibodies of the Invention In one embodiment, the invention provides a method for producing an anti-CD40xCLDN18.2 bispecific antibody, the method comprising culturing a host cell comprising a nucleic acid encoding an anti-CD40xCLDN18.2 bispecific antibody or an expression vector for said nucleic acid under conditions suitable for expression of the nucleic acid encoding said anti-CD40xCLDN18.2 bispecific antibody, and optionally isolating said anti-CD40xCLDN18.2 bispecific antibody. In one embodiment, the method further comprises recovering the anti-CD40xCLDN18.2 bispecific antibody from said host cell (or host cell culture medium).

[0088] To recombinantly produce an anti-CD40xCLDN18.2 bispecific antibody of the invention, one first isolates nucleic acid encoding the anti-CD40xCLDN18.2 bispecific antibody of the invention and inserts it into a vector for further cloning and / or expression in a host cell. Such nucleic acid is easy to isolate and sequence by standard procedures, for example using oligonucleotide probes capable of specifically binding to nucleic acid encoding the anti-CD40xCLDN18.2 bispecific antibody of the invention.

[0089] The anti-CD40×CLDN18.2 bispecific antibody of the present invention produced as described herein can be purified by known conventional techniques, such as, for example, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will further depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. The purity of the anti-CD40×CLDN18.2 bispecific antibody of the present invention can be determined by any one of several well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0090] VI. Combination Products or Reagent Kits In some embodiments, combination products are provided comprising an anti-CD40xCLDN18.2 bispecific antibody or Fab fragment of the invention, or an immunoconjugate thereof, and one or more other therapeutic agents (such as, for example, a chemotherapeutic agent, another antibody, a cytotoxic agent, an anti-infective active agent, a small molecule drug, or an immunomodulatory agent).

[0091] In some embodiments, the combination product is used to prevent or treat a tumor, hi some embodiments, the tumor is, for example, a cancer.

[0092] In some forms, two or more components of the combination product may be administered to a subject sequentially, separately or simultaneously.

[0093] In some embodiments, the invention further provides reagent kits comprising the anti-CD40xCLDN18.2 bispecific antibodies, pharmaceutical compositions, immunoconjugates or combination products of the invention, and optional packaging inserts providing instructions for administration.

[0094] In some embodiments, the invention further provides a pharmaceutical product comprising the anti-CD40xCLDN18.2 bispecific antibody, pharmaceutical composition, immunoconjugate or combination product of the invention. Optionally, the pharmaceutical product further comprises a packaging insert indicating administration.

[0095] VII. Uses and Treatment Methods of the Anti-CD40×CLDN18.2 Bispecific Antibodies of the Invention In one aspect, the present invention relates to a method for modulating immune response in an individual, which comprises administering to a subject an effective amount of the anti-CD40×CLDN18.2 bispecific antibody disclosed herein or a pharmaceutical composition, immunoconjugate or combination product comprising said anti-CD40×CLDN18.2 bispecific antibody, so that the CD40 antibody capable of activating B cells is fixed to cells expressing CLDN18.2, activating B cells and downstream immune response cells such as T cells, improving the killing effect and improving the relatively weak antibody-dependent cell-mediated cytotoxicity (ADCC) killing effect of the current CLDN18.2 monoclonal antibody. The principle of action is that the anti-CD40×CLDN18.2 bispecific antibody generates a cross-linking effect, whereby when the cross-linking effect is formed, a significant immune activation reaction occurs, and when the cross-linking effect is absent, no immune activation effect occurs or only a weak immune activation effect occurs.

[0096] In one embodiment, a therapeutically effective amount of an anti-CD40xCLDN18.2 bispecific antibody, pharmaceutical composition, immunoconjugate or combination product disclosed herein restores, enhances, stimulates or increases an immune response in a subject.

[0097] In one embodiment, the anti-CD40×CLDN18.2 bispecific antibody disclosed herein, when bound to CLDN18.2 on the cell surface, exhibits significantly improved agonistic activity compared to when not bound to the cell surface, minimizes non-specific agonistic activity, and improves the therapeutic index.

[0098] In one embodiment, the anti-CD40×CLDN18.2 bispecific antibody disclosed herein undergoes modification of the Fc fragment so that it no longer binds to Fc receptors on the liver. Furthermore, the bispecific antibody of the present invention has a CLDN18.2 target-dependent activation effect, and since the CLDN18.2 target is a tumor-specific target, the anti-CD40×CLDN18.2 bispecific antibody of the present invention can completely avoid the occurrence of hepatotoxicity.

[0099] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) in a subject, said method comprising administering to the subject a therapeutically effective amount of an anti-CD40xCLDN18.2 bispecific antibody disclosed herein, or a pharmaceutical composition, immunoconjugate or combination product comprising same.

[0100] In some embodiments, the tumor is a cancer in which CLDN18.2 is abnormally expressed. In some embodiments, the cancer in which CLDN18.2 is abnormally expressed is, for example, bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, genital and reproductive organ cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumor, neuroectodermal cancer, spinal axis tumor, glioma, cerebrospinal meningioma and pituitary adenoma. Preferably, the cancer is gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer or lung cancer.

[0101] In some embodiments, the tumor is a cancer in which CD40 is abnormally expressed. In some embodiments, the cancer includes, but is not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant tumors, such as sarcomas and carcinomas of each organ system (including adenocarcinoma and squamous cell carcinoma), such as cancers invading the liver, lung, breast, lymph, gastrointestinal tract (e.g., colon), pancreas, genitourinary tract (e.g., kidney, bladder epithelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors, such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer in lung cancer, small intestine cancer, and esophageal cancer. Squamous cell carcinomas include malignant tumors, such as lung, esophagus, skin, head and neck region, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, such as advanced melanoma. In one embodiment, the cancer is lymphoma, renal cell carcinoma, non-small cell lung cancer, liver cancer, pancreatic cancer, colon adenocarcinoma, or breast cancer. The method and composition of the present invention can also be used to treat or prevent the above cancer lesions.

[0102] Non-limiting examples of preferred cancers for treatment include lymphoma (e.g., diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma), breast cancer (e.g., metastatic breast cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), lung cancer (e.g., non-small cell lung cancer (NSCLC), e.g., stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myeloma (e.g., multiple myeloma), leukemia (e.g., chronic myelogenous leukemia), skin cancer (e.g., melanoma (e.g., stage III or stage IV melanoma) or Merkel carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), myelodysplastic syndrome, bladder cancer (e.g., transitional cell carcinoma), kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal cell carcinoma, e.g., advanced or metastatic clear cell renal cell carcinoma), and colon cancer. In addition, refractory or recurrent malignancies (e.g., pancreatic cancer) can be treated using the anti-CD40 antibodies described herein, or pharmaceutical compositions, immunoconjugates or combination products comprising same.

[0103] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease described herein (e.g., a tumor described herein). In some embodiments, the subject is undergoing or has undergone other treatments, such as chemotherapy and / or radiation therapy. Alternatively or in combination, the subject is immunocompromised or at risk of becoming immunocompromised as a result of infection.

[0104] In some embodiments, the methods of prevention or treatment described herein comprise administering to the subject or individual in combination an anti-CD40xCLDN18.2 bispecific antibody, pharmaceutical composition, immunoconjugate or combination product disclosed herein and one or more other therapies (e.g., therapeutic methods and / or other therapeutic agents).

[0105] The anti-CD40xCLDN18.2 bispecific antibodies (and drug compositions or immunoconjugates comprising same) herein may be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, and, when necessary for localized treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Any suitable route (e.g., injection (e.g., intravenous or subcutaneous injection)) may be used, depending in part on whether the administration is short-term or chronic. The present specification covers a variety of dosing schedules, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration and pulse infusion.

[0106] The appropriate dose of the anti-CD40×CLDN18.2 bispecific antibody of the invention (when used alone or in combination with one or more other therapeutic agents) to prevent or treat a disease depends on the type of disease being treated, the type of anti-CD40×CLDN18.2 bispecific antibody, the severity and course of the disease, whether the anti-CD40×CLDN18.2 bispecific antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the anti-CD40×CLDN18.2 bispecific antibody, and the judgment of the attending physician. The anti-CD40×CLDN18.2 bispecific antibody is appropriately administered to the patient in one treatment or over a series of treatments. The dose and treatment regimen of the anti-CD40×CLDN18.2 bispecific antibody may be determined by one of skill in the art.

[0107] It should be understood that instead of the anti-CD40xCLDN18.2 bispecific antibody, the immunoconjugate, composition or combination product of the invention can be used to effect any of the above prevention or treatment.

[0108] The following examples are provided to enhance understanding of the present invention, but are not intended to limit, and should not be construed as limiting, the scope of protection of the present invention.

[0109] Working Example The test methods used in the following examples are all conventional methods within the scope of the art of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology, unless otherwise specified. Materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified.

[0110] Example 1: Preparation of anti-CD40×CLDN18.2 bispecific antibody Each of the anti-CD40 antibodies C8-WT, C8-2, C8-6, and C8-10 in patent application CN202110577432.9 and the VHH of the anti-CLDN18.2 antibody NA3SH1-T4-hVH6 in patent application CN202110795793.0 were constructed into a bispecific antibody having the structure shown in Figure 1, where the sequences of each antibody are shown in Tables 1a-1d.

[0111] [Table 1a] TIFF2025505928000002.tif35170

[0112] [Table 1b] TIFF2025505928000003.tif35170

[0113] [Table 1c] TIFF2025505928000004.tif88170

[0114] [Table 1d] TIFF2025505928000005.tif44170

[0115] Specifically, the nucleotide sequences encoding the heavy chain variable regions and the light chain variable regions of the above-mentioned four anti-CD40 antibodies obtained above were each joined to nucleotide sequences encoding heavy and light chain constant region fragments to obtain full-length heavy and light chain coding nucleotide sequences corresponding to the anti-CD40 antibodies. In order to reduce the binding of the antibody of the present invention to the Fc receptor on hepatocytes, the Fc of the bispecific antibody is of the hIgG1(N297Q) Fc subtype. The nucleotide sequences of the VHHs encoding the above-mentioned anti-CLDN18.2 antibodies were then linked to the C-terminus of the heavy chain constant regions of four different anti-CD40 antibodies via linker sequences to obtain the heavy chain sequences of the bispecific antibodies shown in FIG. 1. Here, the linker sequence encodes a flexible linker commonly used in the art for linking different peptide molecules, such as that shown in GGGGSGGGSGGGGS (SEQ ID NO:25). The obtained coding nucleotide sequences were then constructed into eukaryotic expression vector plasmid pcDNA3.4, and the heavy and light chains of the bispecific antibodies were co-expressed in CHO-K cells using the ExpiCHO transient expression system (Thermo Fisher, A29133) according to the manufacturer's instructions, and then affinity purified by COLUMN XK16 / 20 (purchased from Cytiva) to obtain anti-CD40 x CLDN18.2 bispecific antibodies C8-WT-NA3S-HC, C8-2-NA3S-HC, C8-6-NA3S-HC, and C8-10-NA3S-HC, respectively, whose heavy and light chain amino acid sequence numbers are shown in Table 2.

[0116] [Table 2] TIFF2025505928000006.tif38170

[0117] Example 2 Affinity Activity Analysis of Anti-CD40×CLDN18.2 Bispecific Antibodies 2.1 Binding ability of bispecific antibodies to CD40 protein The four resulting bispecific antibodies were tested for affinity to the His-tagged extracellular domain of human CD40 (Uniprot: P25942 positions 21-193) (also referred to herein as P17-His fusion protein) by ELISA.

[0118] 2μg / mL P17-His fusion protein was coated on 96-well ELISA plates with 30μL per well and left at 4℃ overnight. The next day, after blocking with 5% PBSM (PBS + 5%Milk) for 2 hours at room temperature, serial dilutions (20nM in the first well, 10-fold dilution in the second well, 3-fold dilution in the third to seventh wells, 4-fold dilution in the seventh to eighth wells), positive control APX005 (sequence derived from Apexigen Inc. US20120301488A1, self-prepared in the laboratory) or isotype IgG1 negative control were added. After incubation at room temperature for 1 hour, the plates were washed three times with PBST (PBS + 0.05% Tween-20) and then 30μL Goat-anti-human-Fc-HRP (abcam, ab97225) was added. After incubation at room temperature for 1 hour, the plates were washed three times with PBST (PBS + 0.05% Tween-20) and TMB color development solution was added for 5 to 30 minutes. After incubation, OD450 was measured using a microplate reader (Beckman Coulter), and the results were analyzed and graphed using Graphpad 7.0 to evaluate the binding between the bispecific antibody and the P17-His fusion protein.

[0119] The results are shown in Figure 2. As can be seen from Figure 2, the EC50 value of the positive control APX005 binding to the P17-His fusion protein was 0.06124 nM, and the EC50 values ​​of the bispecific antibodies C8-WT-NA3S-HC, C8-2-NA3S-HC, C8-6-NA3S-HC and C8-10-NA3S-HC binding to the P17-His fusion protein were 0.1098 nM, 0.1270 nM, 0.1015 nM and 0.1044 nM, respectively, showing affinity comparable to that of the positive control APX005.

[0120] 2.2 Binding ability of bispecific antibodies to CD40 expressed on the cell surface Furthermore, to test the binding ability of the bispecific antibody to CD40 expressed on the cell surface, CHO-K cells (Thermo Fisher) expressing human CD40 (Uniprot: P25942) on the cell surface were produced. Specifically, the human CD40 coding sequence was cloned into the multicloning site of the pcDNA3.4 (Invitrogen) vector, and then the expression vector expressing this human CD40 was introduced into the CHO-K cells for eukaryotic expression to obtain CHO-K cells expressing human CD40 on the cell surface (hereinafter, also referred to as huCD40-CHO-K cells).

[0121] 1.0 × 10 huCD40-CHO-K cells 5 Cells / well were seeded into a 96-well plate and serially diluted (115.6 nM in the first well, 10-fold diluted in the second well, 3-fold diluted in the third to seventh wells, and 4-fold diluted in the seventh to eighth wells) anti-CD40 x CLDN18.2 bispecific antibody was added, where the positive control was APX005 and the negative control was isotype IgG1. After incubation at 4°C for 30 min, the cells were washed and 100 μL of 1:300 diluted Goat F(ab')2 Anti-human Fc (PE) (Abcam, ab98596) was added and incubated for 30 min. The cells were then washed and the binding of the bispecific antibody of the present invention to CD40 expressed on the cell surface was measured by flow cytometry (Beckman Coulter).

[0122] The measurement results are shown in Figure 3. Figure 3 shows the ability of anti-CD40 x CLDN18.2 bispecific antibody at different concentrations to bind to huCD40-CHO-K cells, and indicates that this binding ability is concentration-dependent in the bispecific antibody concentration range of 0.015 nM to 115.6 nM.

[0123] 2.3 Affinity of bispecific antibodies to CLDN18.2 expressed on the cell surface To test the affinity of the bispecific antibody to CLDN18.2 expressed on the cell surface, HEK293 cells expressing human CLDN18.2 on the cell surface were produced. Specifically, the DNA sequence of full-length human CLDN18.2 (see SEQ ID NO:15 in WO2020238730A1 for the amino acid sequence) was constructed in pLVX-puro plasmid (Clontech, Cat#632164), and the resulting plasmid was electroporated into HEK293 cells (ATCC® CRL-1573). TM ) was transformed into the HEK293 cell line. Puromycin resistance pressure screening was performed to identify clones using antibody IMAB362 (Ganymed, Germany, antibody that specifically binds to CLDN18.2), and finally, a HEK293 cell line overexpressing human CLDN18.2 (also referred to as "18.2-HEK293 cell" in this specification) was obtained.

[0124] 18.2-HEK293 cells at 1.0 × 10 5 Cells / well were seeded into a 96-well plate and serially diluted (115.6 nM in the first well, 10-fold diluted in the second well, 3-fold diluted in the third to seventh wells, and 4-fold diluted in the seventh to eighth wells) of the bispecific antibody of the invention, the positive control NA3SH1-T4-hVH6 (amino acid sequence shown in SEQ ID NO:24) or the isotype IgG1 negative control were added. After incubation at 4° C. for 30 min, the cells were washed and 100 μL of 1:300 diluted Goat F(ab')2 Anti-human Fc (PE) was added and incubated for 30 min. The cells were then washed and the binding of the bispecific antibody to CLDN18.2 expressed on the surface of HEK293 cells was measured by flow cytometry.

[0125] FIG. 4 shows that different concentrations of anti-CD40×CLDN18.2 bispecific antibody bind to 18.2-HEK293 cells, and that the binding affinity is bispecific antibody concentration-dependent within the bispecific antibody concentration range of 0.015 nM to 115.6 nM.

[0126] 2.4 Binding ability of bispecific antibodies to CLDN18.1 expressed on the cell surface To test the affinity of the bispecific antibody for CLDN18.1 expressed on the cell surface, HEK293 cells expressing human CLDN18.1 (for the amino acid sequence, see SEQ ID NO:16 in WO2020238730A1) on the cell surface (see above for the preparation method; hereinafter also referred to as "18.1-HEK293 cells") were prepared.

[0127] 18.2-HEK293 cells at 1.0 × 10 5 Cells / well were seeded into a 96-well plate and serially diluted (115.6 nM in the first well, 10-fold diluted in the second well, 3-fold diluted in the third to seventh wells, and 4-fold diluted in the seventh to eighth wells) of the bispecific antibody of the present invention or the positive antibody NA3SH1-T4-hVH6 was added. After incubation for 30 min at 4° C., the cells were washed and 100 μL of 1:300 diluted PE-labeled Goat F(ab')2 Anti-human Fc (PE) was added and incubated for 30 min at 4° C. The cells were then washed and the binding of the bispecific antibody to CLDN18.1 expressed on the surface of HEK293 cells was measured by flow cytometry.

[0128] The results showed that different concentrations of anti-CD40×CLDN18.2 bispecific antibody did not bind to 18.1-HEK293 cells, indicating that the bispecific antibody obtained in the present invention specifically binds to CLDN18.2.

[0129] Example 3 In vitro functional assay of anti-CD40×CLDN18.2 bispecific antibodies To determine whether the bispecific antibodies of the invention can activate the NF-κB signaling pathway downstream of CD40 under different conditions, the measurements were carried out in this example by employing a CD40-NF-κB-Jurkat luciferase reporter gene cell line stably expressing CD40 (Uniprot No. P25942).

[0130] 3.1 Construction of CD40-NF-κB-Jurkat luciferase reporter gene cell line The pGL4.30 plasmid (Promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated (Invitrogen, Neon TM Jurkat cells (ATCC® TIB-152) were transfected with the Transfection System (MP922947). TM ) were transformed into the . The resulting cells were then transferred to RPMI 1640 medium (Hyclone, SH30243.01) containing 10% FBS (Gibco, 15140-141) but no antibiotics, and the cells were inoculated into 6-well cell culture dishes and cultured for 48 hours. Next, the cells were dispensed into 96-well cell culture plates at an average density of 1500 cells / well, and screened by adding hygromycin B (Basalmedia, S160J7) at a final concentration of 500 μg / mL. The growth status of cell line clones was observed for about 2 to 3 weeks, and cell lines that formed clones were selected and transferred to 24-well plates. After culturing and expanding the cells, some clones were transferred to a 96-well white-bottom plate (Corning, 3610) and stimulated with phorbol ester (working concentration 10 ng / mL) and ionomycin (working concentration 1 nM). After culturing for 6 hours in a 37°C, 5% carbon dioxide incubator, Bright-Lite substrate (Vazyme, DD1204-03) was added and the signal value was read using a microplate reader (Molecular Devices: Spectramax i3x). The expression level of NF-κB in different clones was evaluated to obtain a Jurkat cell line with high expression of NF-κB gene (named NF-κB-Jurkat cell).

[0131] Based on this, the full-length expression gene sequence of human CD40 (Uniprot No. P25942) was stably introduced into the NF-κB-Jurkat cells to screen for monoclonal cell lines. CD40L recombinant protein (ACRO Biosystems, Catalog No.: CDL-H5248) was added to this cell line culture system. The transcription and expression of the intracellular NF-κB luciferase reporter gene was activated via the CD40-CD40L signal axis, and the corresponding CD40-expressing cell line was measured and obtained by adding the luciferase catalytic substrate to generate a fluorescent signal, and was named CD40-NF-κB-Jurkat luciferase reporter gene cell line.

[0132] 3.2 Activation of the NF-κB signaling pathway by bispecific antibodies in the presence of crosslinking agents In this section, we determine whether the binding of the bispecific antibodies of the present invention to CD40 in the presence of a cross-linking agent has the ability to activate the downstream NF-κB signaling pathway, where expression of a luciferase reporter gene indicates activation of the NF-κB signaling pathway.

[0133] The specific implementation method is as follows: The bispecific antibody, positive control APX005, or isotype IgG1 negative control, which were serially diluted (28.9 nM in the first well, 5-fold serial dilution), were uniformly mixed with a crosslinker (AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ fragment specific, Jackson Immunoresearch, Catalog No.: 109-006-098) at a final concentration of 5 μg / mL, plated on a 96-well cell culture plate, and cultured at room temperature for 30 minutes, after which 1.0 × 10 CD40-NF-κB-Jurkat cells were added. 5 The cells / well were added to a cell culture plate and incubated for 6 hours in an incubator at 37°C. After incubation was completed, 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the plate was shaken for 2 minutes before measuring the fluorescence value of the 96-well plate.

[0134] The results are shown in Figure 5A. In the presence of the cross-linking agent, all anti-CD40 × CLDN18.2 bispecific antibodies were able to undergo the corresponding cross-linking reaction, activate the NF-κB signaling pathway downstream of CD40, and generate fluorescence.

[0135] 3.3 Inactivation of the NF-κB signaling pathway by bispecific antibodies in the absence of crosslinking agents In this section, we determine whether the binding of the bispecific antibodies of the present invention to CD40 in the absence of a cross-linking agent has the ability to activate the downstream NF-κB signaling pathway, where expression of a luciferase reporter gene indicates activation of the NF-κB signaling pathway.

[0136] The specific implementation method is almost the same as that disclosed in 3.2, except that no crosslinking agent is added.

[0137] The results are shown in FIG. 5B. In the absence of a crosslinking agent, the positive control APX005 activated the NF-κB signaling pathway downstream of CD40, while the bispecific antibodies of the present invention hardly activated or only slightly activated the NF-κB signaling pathway. Some bispecific antibodies showed functions equivalent to those of the negative control. In the absence of a crosslinking agent, the bispecific antibodies of the present invention have been shown to be weak agonist antibodies. Based on the weak activation ability of the bispecific antibodies, the bispecific antibodies of the present invention can be expected to have less toxicity and side effects compared to the control antibodies while weakly stimulating the CD40 molecule.

[0138] In the presence or absence of cross-linking effects, the bispecific antibodies of the invention exhibit clear differences in agonist activity, which is highly effective in reducing non-specific agonist activity and improving the therapeutic index.

[0139] 3.4 Activation of the NF-κB signaling pathway by bispecific antibodies in the presence of 18.2-HEK293 cells In this section, we determine whether binding of a bispecific antibody of the invention to CD40 has the ability to activate the downstream NF-κB signaling pathway in the presence of 18.2-HEK293 cells, where expression of a luciferase reporter gene indicates activation of the NF-κB signaling pathway.

[0140] The specific implementation method is as follows. CD40-NF-κB-Jurkat cells were cultured at 1.0 × 10 5 Seed into cell culture plates at 1.0 x 10 cells / well. 5 18.2-HEK293 cells were added at 18.2 cells / well, and serial dilutions (5.8 nM in the first well, 10-fold dilution in the second well, 2-fold serial dilutions in the third to seventh wells, and 4-fold dilution in the seventh to eighth wells) of each bispecific antibody or isotype IgG1 negative control were added to the inoculated cells and incubated for 6 hours in an incubator at 37°C. After incubation was completed, 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well and shaken for 2 minutes, after which the fluorescence value of the 96-well plate was measured.

[0141] The results are shown in Figure 6A. In the presence of 18.2-HEK293 cells, each bispecific antibody of the present invention exhibited a cross-linking effect and significantly activated the NF-κB signaling pathway downstream of CD40, and this activation ability was concentration-dependent within the bispecific antibody concentration range of 0.0045 nM to 5.8 nM.

[0142] 3.5 Inactivation of the NF-κB signaling pathway by bispecific antibodies in the presence of negative cells that do not express CLDN18.2 In this section, it was determined whether binding of the bispecific antibodies of the invention to CD40 has the ability to activate the downstream NF-κB signaling pathway in the presence of HEK293 cells that do not express CLDN18.2.

[0143] The specific implementation method is as follows. CD40-NF-κB-Jurkat cells were cultured at 1.0 × 10 5Seed into cell culture plates at 1.0 x 10 cells / well. 5 HEK293 cells (ATCC® CRL-1573) were cultured at 100 cells / well. TM ) was added to the inoculated cells, and serial dilutions (5.8 nM in the first well, 10-fold dilution in the second well, 2-fold dilution in the third to seventh wells, and 4-fold dilution in the seventh to eighth wells) of each bispecific antibody or isotype IgG1 negative control were added to the inoculated cells and incubated for 6 hours in an incubator at 37°C. After incubation was completed, 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well and shaken for 2 minutes, after which the fluorescence value of the 96-well plate was measured.

[0144] The results are shown in Figure 6B. In the case of HEK293 cells that do not express CLDN18.2, none of the bispecific antibodies of the present invention activated the NF-κB signaling pathway downstream of CD40.

[0145] The results of Figures 6A and 6B show that the ability of the bispecific antibody of the present invention to activate the NF-κB signaling pathway downstream of CD40 is dependent on the presence of CLDN18.2 and is concentration-dependent. The bispecific antibody of the present invention forms a cross-linking effect through binding to CLDN18.2 on 18.2-HEK293 cells, thereby effectively activating the NF-κB signaling pathway downstream of CD40 in CD40-NF-κB-Jurkat cells, and further promoting the immune response of APC cells and downstream T cells to achieve the effect of killing tumors. Furthermore, the Fc region of the bispecific antibody is correspondingly modified, which effectively prevents the non-specific binding of the antibody to hepatocytes, and prevents or reduces the hepatotoxic side effects of the antibody.

[0146] Example 4 Functional Determination of Anti-CD40×CLDN18.2 Bispecific Antibody in Animal Models In this example, the tumor suppression ability and hepatotoxicity of the bispecific antibody of the present invention were tested in an animal model. In this example, MC-38 cells (mouse colon cancer cells, Shanghai Model Organisms Center, catalog number: NM-S13-TM10) overexpressing CLDN18.2 were used as tumor cells (hereinafter referred to as huCLDN18.2-MC38 cells (see Example 2.3 of the present application for the construction method)). The experimental animals were 6-8 week-old (20-22 g) female CD40 humanized mice C57BL / 6-Cd40 tm1(CD40) The mice were housed in separate, ventilated, thermo-controlled chambers at temperatures between 21 and 24°C and humidity levels between 30 and 53%. Resuspended huCLDN18.2-MC38 cells were implanted at 2 × 10 in the right dorsal region of each mouse. 6 The mice were subcutaneously injected at a density of 100-130 mm3 on day 0. 3 When the tumor volume reached 100% (day 7), the mice with the largest difference in tumor volume were removed and divided into groups based on tumor volume (5 mice per group). They were randomly divided into PBS-treated group, CCl4-treated group (chemical reagent hepatotoxicity positive control), Selicrelumab-treated group (Abgenix, clinical phase I, heavy and light chain amino acid sequences are shown in SEQ ID NO:26 and SEQ ID NO:27, respectively), C8-WT monoclonal antibody-treated group (heavy and light chain amino acid sequences are shown in SEQ ID NO:28 and SEQ ID NO:23, respectively), bispecific antibody C8-WT-NA3S-HC, C8-2-NA3S-HC-treated group. Two dose groups, 1 mpk and 20 mpk, were set up for each antibody-treated group. The drugs were administered intravenously (iv) twice a week for 3 weeks.

[0147] 4.1 Measurement of the tumor-suppressing ability of bispecific antibodies The length (mm) and width (mm) of the tumor were observed and recorded at regular intervals, and the tumor volume (V) was calculated using the formula: V = (length × width) 2) / 2, and tumor growth inhibition rate TGI (%)=(1-mean tumor volume of treatment group / mean tumor volume of PBS treatment group)×100%. The results are shown in FIG. 7 and Tables 3-4. The analysis results of the complete tumor remission rate in Table 3 show the following. The groups administered with the bispecific antibodies C8-WT-NA3S-HC and C8-2-NA3S-HC showed complete tumor remission (5 mm) on day 18, regardless of whether they were administered at a high or low dose. 3 In the group administered high doses of bispecific antibodies C8-WT-NA3S-HC and C8-2-NA3S-HC, tumors in all mice showed complete remission on day 25, whereas in the group administered high doses of control Selicrelumab, only three mice showed complete remission. In the group administered low doses of control Selicrelumab and bispecific antibody C8-WT-NA3S-HC, two mice died. In the group administered bispecific antibody C8-2-NA3S-HC, no deaths occurred in either the high or low dose groups. The tumor inhibition results in Figure 7 and Table 4 show the following. Both bispecific antibodies C8-WT-NA3S-HC and C8-2-NA3S-HC showed good tumor inhibition effects in mice. Here, the low-dose bispecific antibody C8-2-NA3S-HC had a much better tumor inhibition rate than Selicrelumab, and the bispecific antibody showed a stronger tumor inhibition effect than the maternal monoclonal antibody C8-WT, and the bispecific antibody specifically killed CLDN18.2-positive tumor cells in vivo through the cross-linking action.

[0148] [Table 3] TIFF2025505928000007.tif58170

[0149] [Table 4] TIFF2025505928000008.tif74170

[0150] 4.2 Measurement of hepatotoxicity of bispecific antibodies During the administration process, the toxicity and side effects of drugs on the liver are mainly judged by measuring the content of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum; the higher the content, the greater the damage to liver cells. In this study, alanine aminotransferase (alanine aminotransferase / ALT / GPT) test kit (Reitman method) microplate method (Nanjing Jiancheng Bioengineering Institute, catalog number: C009-2-1) and aspartate aminotransferase (aspartate aminotransferase / AST / GOT) test kit (microplate method) (Nanjing Jiancheng Bioengineering Institute, catalog number: C010-2-1) were used. The specific measurement method is as follows:

[0151] First, 20 μL of substrate solution preheated at 37 ° C was added to the assay wells of the 96-well plate, and 5 μL of mouse serum collected at the end of administration was added and gently shaken to mix uniformly. Only 20 μL of substrate solution preheated at 37 ° C was added to the control wells and gently shaken to mix uniformly. The 96-well plate was placed in a 37 ° C incubator and bathed for 30 minutes, after which the 96-well plate was removed and 20 μL of 2,4-dinitrophenylhydrazine solution was added to the assay wells and gently shaken to mix uniformly. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the target wells, and 5 μL of distilled water was added, and the mixture was gently shaken to mix uniformly and develop color. The 96-well plate was then placed in a 37 ° C incubator and bathed for 20 minutes, after which 200 μL of 0.4 mol / L NaOH was added to all wells of the 96-well plate and gently shaken to mix uniformly to stop color development. Finally, the plate was left at room temperature for 10 minutes and then measured on a microplate reader (λ=510 nm).

[0152] The mice were bled on the 21st and 27th days to measure the levels of AST and ALT. The results of ALT and AST measurements on the 21st day are shown in Figures 8A-8B. The results of ALT and AST measurements on the 27th day are shown in Figures 8C-8D. The ALT and AST values ​​of the bispecific antibody groups C8-WT-NA3S-HC and C8-2-NA3S-HC were equivalent to those of the PBS group, while the values ​​of the Selicrelumab control group were 7-8 times higher than those of the bispecific antibody group. The bispecific antibodies C8-WT-NA3S-HC and C8-2-NA3S-HC obtained in the present invention have better tumor suppression effects and better targeting, and furthermore, they have no toxicity or side effects on the liver and are safer than Selicrelumab.

Claims

1. An anti-CD40×CLDN18.2 bispecific antibody, comprising: (i) an anti-CD40 antibody or a fragment thereof; and (ii) an anti-CLDN18.2 antibody or a fragment thereof; the anti-CD40 antibody or fragment thereof comprises three heavy chain CDRs contained in SEQ ID NO:11 or variants thereof, and three light chain CDRs contained in SEQ ID NO:10 or variants thereof; the anti-CD40 antibody or fragment thereof comprises three heavy chain CDRs contained in SEQ ID NO:12 or variants thereof, and three light chain CDRs contained in SEQ ID NO:10 or variants thereof; the anti-CD40 antibody or fragment thereof comprises the three heavy chain CDRs contained in SEQ ID NO:13 or variants thereof, and the three light chain CDRs contained in SEQ ID NO:10 or variants thereof; or the anti-CD40 antibody or fragment thereof comprises the three heavy chain CDRs contained in SEQ ID NO:14 or variants thereof, and the three light chain CDRs contained in SEQ ID NO:10 or variants thereof; wherein said variant has one or two amino acid substitutions, additions and / or deletions compared to the sequence from which it is derived, said anti-CD40 x CLDN18.2 bispecific antibody.

2. The anti-CLDN18.2 antibody or fragment thereof is a single domain antibody (VHH), the single domain antibody comprising three CDRs contained in SEQ ID NO: 18 or variants thereof; The bispecific antibody of claim 1 , wherein the variant has one or two amino acid substitutions, additions and / or deletions compared to the sequence from which it is derived.

3. (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain having the structure VL-CL; Where: the VH comprises the three heavy chain CDRs contained in SEQ ID NO:11, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:18; the VH comprises the three heavy chain CDRs contained in SEQ ID NO:12, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:18; the VH comprises the three heavy chain CDRs contained in SEQ ID NO:13, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:18; or 3. The bispecific antibody of claim 1 , wherein the VH comprises the three heavy chain CDRs contained in SEQ ID NO:14, the VL comprises the three light chain CDRs contained in SEQ ID NO:10, and the VHH comprises the three CDRs contained in SEQ ID NO:

18.

4. (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain having the structure VL-CL; Where: the VH comprises three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; the VL comprises three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and the VHH comprises three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; the VH comprises three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7; the VL comprises three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and the VHH comprises three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; the VH comprises the three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, the VL comprises the three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and the VHH comprises the three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or 4. The bispecific antibody of claim 1 , wherein the VH comprises the three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, the VL comprises the three light chain CDRs shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and the VHH comprises the three CDRs shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:

17.

5. the VH comprises a sequence set forth in SEQ ID NO:11 or a sequence having at least 90% sequence identity with SEQ ID NO:11, the VL comprises a sequence set forth in SEQ ID NO:10 or a sequence having at least 90% sequence identity with SEQ ID NO:10, and the VHH comprises a sequence set forth in SEQ ID NO:18 or a sequence having at least 90% sequence identity with SEQ ID NO:18; the VH comprises a sequence set forth in SEQ ID NO:12 or a sequence having at least 90% sequence identity to SEQ ID NO:12, the VL comprises a sequence set forth in SEQ ID NO:10 or a sequence having at least 90% sequence identity to SEQ ID NO:10, and the VHH comprises a sequence set forth in SEQ ID NO:18 or a sequence having at least 90% sequence identity to SEQ ID NO:18; the VH comprises a sequence set forth in SEQ ID NO:13 or a sequence having at least 90% sequence identity to SEQ ID NO:13, the VL comprises a sequence set forth in SEQ ID NO:10 or a sequence having at least 90% sequence identity to SEQ ID NO:10, and the VHH comprises a sequence set forth in SEQ ID NO:18 or a sequence having at least 90% sequence identity to SEQ ID NO:18; or 5. The bispecific antibody of claim 1 , wherein the VH comprises a sequence as shown in SEQ ID NO:14 or a sequence with at least 90% sequence identity to SEQ ID NO:14, the VL comprises a sequence as shown in SEQ ID NO:10 or a sequence with at least 90% sequence identity to SEQ ID NO:10, and the VHH comprises a sequence as shown in SEQ ID NO:18 or a sequence with at least 90% sequence identity to SEQ ID NO:

18.

6. The VHH is linked to the Fc via a linker, preferably the linker is (G 4 S), where n is an integer equal to or greater than 1, for example, n is an integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, and more preferably, the linker is (G 4 S) 3 The bispecific antibody according to any one of claims 1 to 5, which is selected from the group consisting of

7. 7. The bispecific antibody of claim 1 , wherein the Fc region is an Fc region of an IgG based antibody, such as an IgG1 Fc region, such as an IgG1 Fc region having a modification, such as an IgG1 Fc region having the N297Q mutation.

8. The bispecific antibody of any one of claims 1 to 7, wherein the light chain constant domain CL of the antibody is of kappa or lambda origin.

9. (i) a heavy chain having the structure VH-CH1-Fc-VHH, and (ii) comprises a light chain having the structure VL-CL; Where: the heavy chain comprises a sequence set forth in SEQ ID NO:19 or a sequence having at least 90% identity to SEQ ID NO:19, and the light chain comprises a sequence set forth in SEQ ID NO:23 or a sequence having at least 90% identity to SEQ ID NO:23; the heavy chain comprises a sequence set forth in SEQ ID NO:20 or a sequence having at least 90% identity thereto, and the light chain comprises a sequence set forth in SEQ ID NO:23 or a sequence having at least 90% identity thereto; the heavy chain comprises a sequence set forth in SEQ ID NO:21 or a sequence having at least 90% identity to SEQ ID NO:21, and the light chain comprises a sequence set forth in SEQ ID NO:23 or a sequence having at least 90% identity to SEQ ID NO:23; or 9. The bispecific antibody of claim 1 , wherein the heavy chain comprises the sequence shown in SEQ ID NO:22 or a sequence with at least 90% identity to SEQ ID NO:22 and the light chain comprises the sequence shown in SEQ ID NO:23 or a sequence with at least 90% identity to SEQ ID NO:

23.

10. A polynucleotide encoding the bispecific antibody of any one of claims 1 to 9.

11. A vector, preferably an expression vector, comprising the polynucleotide of claim 10.

12. A host cell comprising the polynucleotide of claim 10 or the vector of claim 11.

13. 10. A method for producing a bispecific antibody according to any one of claims 1 to 9, comprising the step (i) of culturing a host cell of the invention under conditions suitable for the expression of the bispecific antibody of the invention, and optionally the step (ii) of recovering the bispecific antibody of the invention.

14. An immunoconjugate, a reagent kit, a pharmaceutical composition, a combination product or an article of manufacture comprising a bispecific antibody according to any one of claims 1 to 9.

15. The reagent kit, pharmaceutical composition, combination product or article of manufacture according to claim 14, further comprising other therapeutic agents and any pharmaceutical adjuvants, preferably said other therapeutic agents being selected from chemotherapeutic agents, cytotoxic agents.

16. Use of the bispecific antibody according to any one of claims 1 to 9, or the immunoconjugate, reagent kit, pharmaceutical composition, combination product or product according to claim 14 or 15 in a drug or reagent kit for treating, preventing and / or diagnosing a CD40-related disease or a CLDN18.2-related disease, preferably the CLDN18.2-related disease is a cancer in which CLDN18.2 is abnormally expressed, and the CD40-related disease is a cancer in which CD40 is abnormally expressed.

17. A method for treating a CD40-related disease or for treating a CLDN18.2-related disease comprising administering to a patient in need thereof a therapeutically effective amount of a bispecific antibody according to any one of claims 1-9, or an immunoconjugate, pharmaceutical composition, combination product or product according to claim 14 or 15.

Images