Anti-CD40×CLDN18.2 bispecific antibody 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 cell membrane. It belongs to the tumor necrosis factor receptor (TNFR) family and is constitutively expressed on antigen-presenting cells (including dendritic cells, B cells, monocytes, and macrophages) and on several other cell types, including endothelial cells, fibroblasts, and thymic epithelial cells. CD40 is also expressed on several tumor cell types (e.g., chronic lymphocytic leukemia, multiple myeloma, renal carcinoma, and lung carcinoma). CD40 forms a trimer on the cell surface, and its corresponding ligand, CD40L (i.e., CD154), is expressed primarily on activated T cells. The interaction between CD40 and CD40L is a costimulatory signal for T cell activation. Binding of CD40 to CD40L on T cells can activate multiple pathways, including the NF-κB (nuclear factor κB) signaling pathway.

[0002] Agonistic antibodies targeting CD40, such as selicrelumab, dacetuzumab, and APX005, have demonstrated clinical activity in many indications in clinical trials. However, these agonistic antibodies are associated with dose-limiting toxicities (Hassan SB et al., Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials. Immunopharmacol Immunotoxicol 2014;36:96-104). The current major adverse event is hepatotoxicity. These agonistic antibodies nonspecifically activate immune cells expressing CD40 on the surface of hepatocytes, causing liver toxicity 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 differ only in their N-terminal sequences. Both CLDN18 subtype proteins consist of 261 amino acids and contain four transmembrane domains, but they are distributed in different tissues. CLDN18.1 is primarily expressed in lung tissue, while CLDN18.2 is expressed only in differentiated gastric mucosal epithelial cells and not in gastric stem cells (Sahin, Ugur, et al. "Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development." Clinical Cancer Research 14.23 (2008): 7624-7634.). CLDN18.2 is highly expressed in multiple tumor tissues, including non-small cell lung cancer (25%), gastric cancer (70%), pancreatic cancer (50%), and esophageal cancer (30%). However, it is rarely 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 differential expression between tumor cells and normal tissues, CLDN18.2 is currently a highly 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, thereby offering more possibilities for cancer treatment. Summary of the Invention

[0005] The present invention discloses a novel bispecific antibody that simultaneously targets CD40 and CLDN18.2, a polynucleotide encoding the bispecific antibody, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, a method for treating diseases associated with CD40 and / or CLDN18.2 using the bispecific antibody, and use of the bispecific antibody in the individual treatment, prevention, and / or diagnosis of diseases associated with CD40 and / or CLDN18.2. 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 achieving low agonist activity in sites such as hepatocytes where cross-linking effect cannot be formed.

[0006] In one aspect, the present invention provides a bispecific antibody that specifically binds CD40 and CLDN18.2 (anti-CD40×CLDN18.2 bispecific antibody), which comprises (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-CD40xCLDN18.2 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 to CD40, and the VHH forms a second antigen-binding portion that specifically binds to CLDN18.2, and optionally the VHH is linked to Fc via a linker.

[0009] In one embodiment, the invention provides an anti-CD40xCLDN18.2 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 invention provides an anti-CD40xCLDN18.2 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, 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 three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, 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; or The VH comprises three heavy chain CDRs shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:9, 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.

[0013] Furthermore, in another embodiment, the present invention provides an anti-CD40xCLDN18.2 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 is comprised of three heavy chain CDRs contained in SEQ ID NO: 11 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 11; the VL is comprised of three light chain CDRs contained in SEQ ID NO: 10 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 10; and the VHH is comprised of three CDRs contained in SEQ ID NO: 18 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 18; the VH is comprised of three heavy chain CDRs contained in SEQ ID NO: 12 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 12; the VL is comprised of three light chain CDRs contained in SEQ ID NO: 10 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 10; and the VHH is comprised of three CDRs contained in SEQ ID NO: 18 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with 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 is composed of three heavy chain CDRs contained in SEQ ID NO: 14 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 14; the VL is composed of three light chain CDRs contained in SEQ ID NO: 10 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 10; and the VHH is composed of three CDRs contained in SEQ ID NO: 18 and comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 18.

[0015] In one embodiment, the invention provides an anti-CD40xCLDN18.2 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 the sequence shown in SEQ ID NO: 11, 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: 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×CLDN18.2 bispecific antibody of the present invention is linked to the Fc via a linker. The linker may be any commonly used flexible sequence already known in the art or developed in the future, and is typically a short, flexible amino acid sequence. In one specific embodiment, the linker is (G4S)n, where n is an integer of 1 or greater. For example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, or 9, such as GGGSG;GGSGG;GSGGGG;SGGGG;GGTGS;GTSPGG;GNGGGS;G4S-GGSGG-G4S-SGGGG;GGG;DGGGS;TGEKP;GGRR;EGKSSGSGSESKVD;KESGSVSSEQLAQFRSLD;GGRRGGGS;LRQRDGERP;LRQKDGGGSERP;GSTSSGKPGSGEGSTKG. 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 present invention may be selected from the native Fc regions of any IgG-based antibody already known in the prior art, such as consensus or embryonic IgG Fc regions. In one specific embodiment, the IgG Fc region may be an Fc region derived from a different IgG subclass, such as IgG1, IgG2, IgG3, or IgG4. In one specific embodiment, the IgG Fc region is the Fc sequence of IgG1. In another specific embodiment, the IgG Fc region may contain mutations / modifications to stabilize the antibody or reduce FcγRIIb-dependent cross-linking effects to avoid hepatotoxicity, such as modifications already known in the prior art. In one specific embodiment, the bispecific anti-CD40×CLDN18.2 antibody of the present invention has been modified to include a constant region that lacks 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-CD40xCLDN18.2 bispecific antibody of the invention is of kappa or lambda origin.

[0021] In one embodiment, the invention provides an anti-CD40xCLDN18.2 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 contained in SEQ ID NO: 19 and three VHH CDRs, 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 consisting of 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 consisting of 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 consisting of 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 consisting of 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-CD40xCLDN18.2 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., CD40-expressing tumor cells) and cells expressing CLDN18.2 (e.g., CLDN18.2-expressing tumor cells).

[0026] In a second aspect, the present invention provides polynucleotides (nucleic acids) encoding the anti-CD40×CLDN18.2 bispecific antibodies of the present invention and vectors comprising said polynucleotides, which are 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, the present invention provides a method for producing an anti-CD40×CLDN18.2 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-CD40×CLDN18.2 bispecific antibody of the invention, and preferably the step (ii) of recovering the anti-CD40×CLDN18.2 bispecific antibody of the invention.

[0029] In a fifth aspect, the present invention provides an immunoconjugate, reagent kit, pharmaceutical composition, combination product, or article of manufacture comprising the anti-CD40×CLDN18.2 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, the present invention provides use of an anti-CD40×CLDN18.2 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 abnormal. In one embodiment, the CLDN18.2-related disease is, for example, a cancer in which CLDN18.2 expression is abnormal.

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

[0032] In a seventh aspect, the present invention provides methods for treating CD40-related diseases and CLDN18.2-related diseases, comprising administering to a patient a therapeutically effective amount of an anti-CD40×CLDN18.2 bispecific antibody of the present invention, or an immunoconjugate, pharmaceutical composition, combination product, or article of the present 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 explanation of the drawings]

[0033] [Figure 1] FIG. 1 shows the schematic structure of the bispecific antibody of the present invention. [Figure 2] FIG. 2 shows the binding activity of the bispecific antibody of the present invention to the P17-His fusion protein. [Figure 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 the 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 activation of the CD40 signaling pathway by a bispecific antibody of the invention in the absence of a cross-linking agent (5B). [Figure 6A]FIG. 6A shows the activation of the CD40 signaling pathway by the bispecific antibody of the present invention when CLDN18.2 is expressed on the cell surface (6A). [Figure 6B] FIG. 6B shows that the CD40 signaling pathway is activated by the bispecific antibody of the present invention when CLDN18.2 is not expressed on the cell surface (6B). [Figure 7] FIG. 7 shows the inhibitory effect of bispecific antibodies of the present invention on tumor growth in vivo in humanized mice. [Figure 8A] FIG. 8A shows the results of whether the bispecific antibody of the present invention causes hepatotoxicity in humanized mice. [Figure 8B] FIG. 8B shows the results of whether the bispecific antibody of the present invention causes hepatotoxicity in humanized mice. [Figure 8C] FIG. 8C shows the results of whether the bispecific antibody of the present invention causes hepatotoxicity in humanized mice. [Figure 8D] FIG. 8D shows the results of whether the bispecific antibody of the present invention causes hepatotoxicity in humanized mice. DETAILED DESCRIPTION OF THE INVENTION

[0034] I. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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] For the purpose of describing this specification, the following definitions will be 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 value, is meant to cover a range of numbers or values ​​from a lower limit of 10% less than the specified number or value to an upper limit of 10% greater than the specified number or value.

[0037] As used herein, the term "and / or" means any one of the alternatives or more than one 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 "comprising" 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 specific sequence is referred to, it is intended to also cover an antibody variable region consisting of that specific 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 same antigen as the intact antibody. 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 composed 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 of the Fc region (Lys447) may or may not be present. Unless otherwise specified, numbering of amino acid residues in an Fc region or constant region is 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, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. 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 antibody variable domains 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, and are 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), the Contact (University College London), and the International ImMunoGeneTics (ImmunoGeneTics). The database (IMGT) (http: / / imgt.cines.fr / ) and a North CDR definition based on affinity propagation clustering using multiple crystal structures.

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

[0045] 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 antibodies 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 via a single variable domain (e.g., a heavy chain variable domain (VH) or a light chain variable domain (VL), a heavy chain variable domain derived from a camelid heavy chain antibody, or a VH-like single domain (v-NAR) derived from a fish IgNAR). That is, the single variable domain can recognize a target antigen without requiring interaction with another variable domain. Examples of single domain antibodies include single domain antibodies derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks) ( WO 2005 / 035572 ). Camelid single domain antibodies, also referred to herein as VHHs, 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 currently known units capable of binding to a target antigen.

[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 specificities for at least two different antigen epitopes. In one embodiment, a multispecific antibody is provided herein, having binding specificities for a first antigen and a second antigen, and is also referred to as a "bispecific antibody."

[0049] An "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 an increase in 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 "cross-linking 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 cross-linking effect induces downstream intracellular signal transduction and further activates immune cells expressing the corresponding CD40.

[0052] Median Effective Concentration (EC 50 The term "maximum response" refers to the concentration of a drug, antibody, or toxin that induces a 50% response 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 percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into 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%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at 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 the corresponding position in the second sequence, the molecules are identical at this position.

[0056] A mathematical algorithm can be used to compare the sequences and calculate the percent identity between two sequences. In one preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com) is used to determine the percent identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used to determine the percent identity between two nucleotide sequences using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and the parameter set that 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 using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0) using a PAM120 weighted coset 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 or related sequences.

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

[0060] The term "prevention" includes the 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. Generally, in the context of cancer, the term "prevention" refers to the 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 an antibody, 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 an attending physician skilled in the art, taking into consideration several factors, such as the species of mammal, body weight, age, and general health of the mammal, 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 administered formulation, the selected dosing regimen, 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 necessary dosage and for the necessary duration. A therapeutically effective amount of an antibody or antibody fragment, or conjugate or composition thereof, will vary 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 assessed in an animal model system predictive of efficacy in human tumors.

[0063] A "prophylactically effective amount" refers to an amount that effectively achieves the desired prophylactic result, at dosages and for periods of time necessary. Because a prophylactic dose is typically 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 the 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.). Linking peptides typically comprise 5-50 amino acids in length, e.g., 10, 15, 20, 25, or 30 amino acids in length. It will be apparent to 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 pathological condition caused by, exacerbated by, 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 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" herein) exhibits significantly different expression in cancer tissues and normal tissues. This is thought to be due to the fact that 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 cancerous process, allowing CREB to activate the transcription of Claudin 18.2.

[0070] As used herein, the terms "anti-CD40 and CLDN18.2 bispecific antibody," "bispecific antibody specifically binding to CD40 and CLDN18.2," "anti-CD40×CLDN18.2 bispecific antibody," "CD40 / CLDN18.2 bispecific antibody," and similar terms refer to bispecific antibodies that can bind to targets CD40 and CLDN18.2 with sufficient affinity. The bispecific antibodies can recruit immune cells and redirect lysis of target cells. The bispecific antibodies can specifically activate immune cells at the site of the lesion with high potency and achieve low agonist activity in locations such as hepatocytes where cross-linking is not possible, 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 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 the 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 in the table below. TIFF0007772432000001.tif106170

[0074] In some embodiments, the substitutions occur in the CDR regions of the antibody. Typically, 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 substantially retains a particular biological property of the parent antibody. An exemplary substitutional 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, which 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] Studies have shown that the Fc region of a partial agonist anti-CD40 antibody cross-links with FcγRIIb, which is abundantly expressed on the surface of hepatocytes, nonspecifically activating CD40-expressing immune cells on the surface of hepatocytes and 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 at positions 234 and 235 (EU index) of the heavy chain. In a specific embodiment, the amino acid substitutions are L234A and L235A (LALA mutation).

[0077] In one embodiment, the anti-CD40xCLDN18.2 bispecific antibody of the invention can enhance immune responses independent of antibody binding to Fc receptors, e.g., the bispecific anti-CD40xCLDN18.2 antibody of the invention can exhibit effective CD40 agonist properties 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 form 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 is desirable to generate antibodies that have been modified via cysteine ​​engineering, e.g., "thiomAbs," 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, nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions relative to the above nucleic acids, or nucleic acid sequences 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 containing the above-mentioned nucleic acid. In a preferred embodiment, the vector is an expression vector. It will 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 present 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 to 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 transfers. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as screened or selected from the originally transformed cell are included. 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 tissue.

[0084] IV. Compositions of the Invention In some embodiments, the present invention provides a composition comprising any of the anti-CD40×CLDN18.2 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, or the like, administered together with an active substance.

[0085] In one embodiment, a composition (e.g., a pharmaceutical composition) comprises a combination of an anti-CD40xCLDN18.2 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 specific indications and may be any effective substance for 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, such as chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective active agents, small molecule drugs, or immunomodulators. The therapeutic agents are present in an appropriate 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 present invention provides a method for producing an anti-CD40×CLDN18.2 bispecific antibody. The method comprises culturing a host cell containing a nucleic acid encoding the anti-CD40×CLDN18.2 bispecific antibody or an expression vector for said nucleic acid under conditions suitable for expression of the nucleic acid encoding the anti-CD40×CLDN18.2 bispecific antibody, and optionally isolating the anti-CD40×CLDN18.2 bispecific antibody. In one embodiment, the method further comprises recovering the anti-CD40×CLDN18.2 bispecific antibody from the host cell (or host cell culture medium).

[0088] To recombinantly produce an anti-CD40×CLDN18.2 bispecific antibody of the invention, nucleic acid encoding the anti-CD40×CLDN18.2 bispecific antibody of the invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced by routine procedures, for example, using oligonucleotide probes capable of specifically binding to nucleic acid encoding the anti-CD40×CLDN18.2 bispecific antibody of the invention.

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

[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 present invention further provides a reagent kit comprising an anti-CD40xCLDN18.2 bispecific antibody, pharmaceutical composition, immunoconjugate or combination product of the present invention, and an optional packaging insert with instructions for administration.

[0094] In some embodiments, the present invention further provides a pharmaceutical product comprising the anti-CD40xCLDN18.2 bispecific antibody, pharmaceutical composition, immunoconjugate, or combination product of the present invention. Optionally, the pharmaceutical product further comprises a packaging insert providing instructions for 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 an immune response in an individual, comprising 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 the anti-CD40×CLDN18.2 bispecific antibody, thereby allowing a CD40 antibody capable of activating B cells to be fixed to cells expressing CLDN18.2, activating B cells and downstream immune response cells such as T cells, thereby improving the killing effect and improving the relatively weak antibody-dependent cell-mediated cytotoxicity (ADCC) killing effect of current CLDN18.2 monoclonal antibodies. The principle of action is that the anti-CD40×CLDN18.2 bispecific antibody generates a cross-linking effect, which results in a significant immune activation response when the cross-linking effect is formed, and in the absence of the cross-linking effect, no 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 enhanced agonistic activity compared to when not bound to the cell surface, minimizes nonspecific agonistic activity, and improves the therapeutic index.

[0098] In one embodiment, the anti-CD40×CLDN18.2 bispecific antibody disclosed herein undergoes Fc fragment modification so that it no longer binds to Fc receptors on the liver. Furthermore, since the bispecific antibody of the present invention has a CLDN18.2 target-dependent activation effect and 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 cancers of various organ systems (including adenocarcinomas and squamous cell carcinomas), 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 of the 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 methods and compositions 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 myeloid 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. Refractory or recurrent malignancies (e.g., pancreatic cancer) can also 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 has 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 pharmaceutical 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) for preventing or treating 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 treating physician. The anti-CD40×CLDN18.2 bispecific antibody is appropriately administered to the patient as a single treatment or over a series of treatments. The dosage and treatment regimen of the anti-CD40×CLDN18.2 bispecific antibody may be determined by one skilled 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] Example Unless otherwise specified, the test methods used in the following examples are conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art. Materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0110] Example 1 Production 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 combined to form a bispecific antibody with the structure shown in Figure 1, where the sequences of each antibody are listed in Tables 1a-1d.

[0111] [Table 1a] TIFF0007772432000002.tif35170

[0112] [Table 1b] TIFF0007772432000003.tif35170

[0113] [Table 1c] TIFF0007772432000004.tif88170

[0114] [Table 1d] TIFF0007772432000005.tif44170

[0115] Specifically, the nucleotide sequences encoding the heavy chain variable regions and light chain variable regions of the 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. To reduce the binding of the antibodies of the present invention to Fc receptors on hepatocytes, the Fc of the bispecific antibody employs the hIgG1(N297Q) Fc subtype. The nucleotide sequences of the VHHs encoding the anti-CLDN18.2 antibody 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 bispecific antibody heavy chain sequences shown in Figure 1. Here, the linker sequence encodes a flexible linker commonly used in the art for linking different peptide molecules, such as GGGGSGGGSGGGGS (SEQ ID NO:25). The resulting coding nucleotide sequences were then constructed into the 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. Subsequently, affinity purification was performed using COLUMN XK16 / 20 (purchased from Cytiva) to obtain the 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] TIFF0007772432000006.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 by ELISA for their affinity to the His-tagged extracellular domain of human CD40 (Uniprot: P25942 positions 21-193) (also referred to herein as P17-His fusion protein).

[0118] 96-well ELISA plates were coated with 2 μg / mL P17-His fusion protein (30 μL per well) and incubated overnight at 4°C. The next day, plates were blocked with 5% PBSM (PBS + 5% milk) for 2 hours at room temperature. Then, serially diluted anti-CD40 × CLDN18.2 bispecific antibody (20 nM in the first well, 10-fold dilution in the second well, 3-fold dilution in the third to seventh wells, and 4-fold dilution in the seventh to eighth wells), positive control APX005 (sequence derived from Apexigen Inc. US20120301488A1, prepared in-house), or isotype IgG1 negative control were added. After incubation for 1 hour at room temperature, plates were washed three times with PBST (PBS + 0.05% Tween-20) and then 30 μL of Goat-anti-human-Fc-HRP (Abcam, ab97225) was added. After incubation at room temperature for 1 hour, the cells were washed three times with PBST (PBS + 0.05% Tween-20) and then TMB color development solution was added for 5-30 minutes. OD450 was measured using a microplate reader (Beckman Coulter). The results were analyzed using Graphpad 7.0, and graphs were generated 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 for binding of the positive control APX005 to the P17-His fusion protein was 0.06124 nM, and the EC50 values ​​for binding of the bispecific antibodies C8-WT-NA3S-HC, C8-2-NA3S-HC, C8-6-NA3S-HC, and C8-10-NA3S-HC to the P17-His fusion protein were 0.1098 nM, 0.1270 nM, 0.1015 nM, and 0.1044 nM, respectively, demonstrating affinities comparable to those 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 generated. 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 CHO-K cells for eukaryotic expression, resulting in CHO-K cells expressing human CD40 on the cell surface (hereinafter referred to as huCD40-CHO-K cells).

[0121] 1.0 × 10 huCD40-CHO-K cells 5 Cells were seeded into a 96-well plate at 115.6 nM per well 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 anti-CD40 x CLDN18.2 bispecific antibody was added. The positive control was APX005, and the negative control was isotype IgG1. After incubation at 4°C for 30 minutes, 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 minutes. 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 binding ability of different concentrations of the anti-CD40 x CLDN18.2 bispecific antibody to huCD40-CHO-K cells, and demonstrates 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 for CLDN18.2 expressed on the cell surface, HEK293 cells expressing human CLDN18.2 on the cell surface were prepared. 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 the pLVX-puro plasmid (Clontech, Cat. #632164), and the resulting plasmid was transfected into HEK293 cells (ATCC® CRL-1573) by electroporation. TM ) were transformed into HEK293 cells. Puromycin resistance screening was performed, and clones were identified using the antibody IMAB362 (Ganymed, Germany, an antibody that specifically binds to CLDN18.2). Finally, a HEK293 cell line overexpressing human CLDN18.2 (also referred to as "18.2-HEK293 cells" herein) was obtained.

[0124] 18.2-HEK293 cells 1.0 × 10 5 Cells were seeded into a 96-well plate at 115.6 nM per well, 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, the positive control NA3SH1-T4-hVH6 (amino acid sequence shown in SEQ ID NO: 24), or the isotype IgG1 negative control was added. After incubation at 4°C for 30 minutes, 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 minutes. 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] Figure 4 shows that different concentrations of anti-CD40 x CLDN18.2 bispecific antibody bind to 18.2-HEK293 cells, and that this 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 (see above for the preparation method; hereinafter also referred to as "18.1-HEK293 cells") expressing human CLDN18.1 (for the amino acid sequence, see SEQ ID NO: 16 in WO2020238730A1) on the cell surface were prepared.

[0127] 18.2-HEK293 cells 1.0 × 10 5 Cells were seeded into a 96-well plate at 115.6 nM / well, 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 at 4°C for 30 minutes, 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 at 4°C for 30 minutes. 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 the 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 present invention can activate the NF-κB signaling pathway downstream of CD40 under different conditions, this example was performed 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 ATCC Transfection System, MP922947. TM ) were transformed into the 1640 medium (Hyclone, SH30243.01) containing 10% FBS (Gibco, 15140-141) but no antibiotics. The resulting cells were then seeded into 6-well cell culture dishes and cultured for 48 hours. Next, the cells were distributed into 96-well cell culture plates at an average density of 1500 cells / well and screened with hygromycin B (Basalmedia, S160J7) at a final concentration of 500 μg / mL. The growth of cell line clones was monitored over a 2-3 week period, and cloned cell lines 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 incubation in a 37°C, 5% CO2 incubator for 6 hours, Bright-Lite substrate (Vazyme, DD1204-03) was added and the signal was read using a microplate reader (Molecular Devices: Spectramax i3x). The expression levels of NF-κB in the different clones were evaluated, and a Jurkat cell line with high NF-κB gene expression (designated NF-κB-Jurkat cells) was obtained.

[0131] Based on this, we stably transfected the full-length human CD40 (Uniprot No. P25942) gene sequence into the NF-κB-Jurkat cells and screened for monoclonal cell lines. CD40L recombinant protein (ACRO Biosystems, catalog number: CDL-H5248) was added to this cell line culture system. The CD40-CD40L signaling axis activated transcription and expression of the intracellular NF-κB luciferase reporter gene, and the addition of a luciferase catalytic substrate generated a fluorescent signal. Cell lines expressing the corresponding CD40 were identified and designated CD40-NF-κB-Jurkat luciferase reporter gene cell lines.

[0132] 3.2 Activation of the NF-κB signaling pathway by bispecific antibodies in the presence of cross-linking agents In this section, we determine whether 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 procedure is as follows: Serially diluted (28.9 nM in the first well, 5-fold serial dilutions) of the bispecific antibody, the positive control APX005, or the isotype IgG1 negative control were homogeneously 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 incubated at room temperature for 30 minutes. After incubation, 1.0 × 10 CD40-NF-κB-Jurkat cells were added. 5 The cells were added to a cell culture plate at 1000 cells / well and cultured for 6 hours in an incubator at 37°C. After the culture was completed, 30 μL of the luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the plate was shaken for 2 minutes, after which the fluorescence value of the 96-well plate was measured.

[0134] The results are shown in Figure 5A. In the presence of a cross-linking agent, all anti-CD40 × CLDN18.2 bispecific antibodies underwent corresponding cross-linking reactions, activated the NF-κB signaling pathway downstream of CD40, and produced fluorescence.

[0135] 3.3 Inactivation of the NF-κB signaling pathway by bispecific antibodies in the absence of cross-linking agents In this section, we determine whether 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 Figure 5B. In the absence of a cross-linking agent, the positive control APX005 activated the NF-κB signaling pathway downstream of CD40, whereas the bispecific antibodies of the present invention barely or only slightly activated the NF-κB signaling pathway. Some bispecific antibodies exhibited functionality equivalent to that of the negative control. In the absence of a cross-linking 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 are expected to have less toxicity and side effects than control antibodies while weakly stimulating the CD40 molecule.

[0138] In the presence or absence of cross-linking effects, the bispecific antibodies of the present 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: 1.0 × 10 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 dilutions in the second well, 2-fold serial dilutions in the third to seventh wells, and 4-fold dilutions in the seventh to eighth wells) of each bispecific antibody or an isotype IgG1 negative control were added to the inoculated cells and incubated for 6 hours in an incubator at 37°C. After incubation, 30 μL of the luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the plate was shaken for 2 minutes. The fluorescence intensity of the 96-well plate was then 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, clearly activating 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, we determined whether the binding of the bispecific antibodies of the present 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: 1.0 × 10 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, 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 an isotype IgG1 negative control were added to the inoculated cells and incubated for 6 hours in an incubator at 37°C. After incubation, 30 μL of the luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the plate was 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 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 in Figures 6A and 6B demonstrate 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, further promoting the immune response of APC cells and downstream T cells, thereby achieving tumor killing. Furthermore, the Fc region of the bispecific antibody is correspondingly modified, effectively preventing nonspecific binding of the antibody to hepatocytes and preventing or reducing the hepatotoxic side effects of the antibody.

[0146] Example 4 Functional Measurement of Anti-CD40×CLDN18.2 Bispecific Antibody in Animal Models In this example, the tumor-suppressing ability and hepatotoxicity of the bispecific antibody of the present invention were tested in an animal model. MC-38 cells (mouse colon cancer cells, Shanghai Model Organisms Center, catalog number: NM-S13-TM10) overexpressing CLDN18.2 were used as tumor cells. These cells are 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 a separate, ventilated chamber at a constant temperature of 21-24°C and humidity of 30-53%. Resuspended huCLDN18.2-MC38 cells were implanted into the right dorsal region of each mouse at a dose of 2 × 10 6 The mice were subcutaneously injected at a density of 100-130 mm (day 0). 3 On day 7, mice with particularly large differences in tumor volume were removed and randomly assigned to groups (5 mice per group) based on tumor volume. They were then randomly assigned to PBS-treated groups, CC14-treated groups (a chemical reagent hepatotoxicity positive control), selicrelumab-treated groups (Abgenix, clinical phase I; heavy and light chain amino acid sequences are shown in SEQ ID NOs: 26 and 27, respectively), C8-WT monoclonal antibody-treated groups (heavy and light chain amino acid sequences are shown in SEQ ID NOs: 28 and 23, respectively), and bispecific antibodies C8-WT-NA3S-HC and C8-2-NA3S-HC. Each antibody treatment group had two dose groups: 1 mpk and 20 mpk. Treatment was administered intravenously (iv) twice weekly for three weeks.

[0147] 4.1 Measuring 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, tumor growth inhibition rate TGI (%) = (1 - mean tumor volume of treatment group / mean tumor volume of PBS-treated group) × 100%. The results are shown in Figure 7 and Tables 3 and 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 achieved complete tumor remission (5 mm ) on day 18, regardless of whether they were administered at a high dose or a low dose. 3 In the control group, tumor volume (less than 100%) was confirmed, whereas complete tumor remission was first confirmed by day 21 with control selicrelumab. In the groups administered high doses of the bispecific antibodies C8-WT-NA3S-HC and C8-2-NA3S-HC, tumors in all mice showed complete remission by day 25, whereas only three mice in the group administered high doses of control selicrelumab showed complete remission. Two mice died in the group administered low doses of control selicrelumab and bispecific antibody C8-WT-NA3S-HC. No deaths occurred in the groups administered bispecific antibody C8-2-NA3S-HC, regardless of whether they were administered high or low doses. The tumor inhibition results in Figure 7 and Table 4 demonstrate the following: Both bispecific antibodies C8-WT-NA3S-HC and C8-2-NA3S-HC exhibited favorable tumor inhibition effects in vivo in mice. Here, the low-dose bispecific antibody C8-2-NA3S-HC showed 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 cross-linking.

[0148] [Table 3] TIFF0007772432000007.tif58170

[0149] [Table 4] TIFF0007772432000008.tif74170

[0150] 4.2 Measuring the hepatotoxicity of bispecific antibodies During administration, drug toxicity and side effects on the liver are primarily determined by measuring the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels; the higher the levels, the greater the damage to liver cells. In this study, we used an alanine aminotransferase (ALT / GPT) test kit (Reitman method) microplate method (Nanjing Jiancheng Bioengineering Institute, catalog number: C009-2-1) and an aspartate aminotransferase (AST / GOT) test kit (microplate method) (Nanjing Jiancheng Bioengineering Institute, catalog number: C010-2-1). The specific measurement methods are as follows:

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

[0152] Blood samples were taken from the mice on days 21 and 27 to measure AST and ALT levels. The results of ALT and AST measurements on day 21 are shown in Figures 8A and 8B. The results of ALT and AST measurements on day 27 are shown in Figures 8C and 8D. The ALT and AST levels of the bispecific antibody groups C8-WT-NA3S-HC and C8-2-NA3S-HC were comparable to those of the PBS group, while the levels of the selicrelumab control group were 7 to 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 been shown to have superior tumor-inhibitory effects and better targeting, and are safer than selicrelumab, with no liver toxicity or side effects.

Claims

1. an anti-CD40xCLDN18.2 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 and three light chain CDRs contained in SEQ ID NO: 10; the anti-CD40 antibody or fragment thereof comprises three heavy chain CDRs contained in SEQ ID NO: 12 and three light chain CDRs contained in SEQ ID NO: 10; the anti-CD40 antibody or fragment thereof comprises the three heavy chain CDRs contained in SEQ ID NO: 13 and the three light chain CDRs contained in SEQ ID NO: 10; or The anti-CD40 antibody or fragment thereof is an anti-CD40xCLDN18.2 bispecific antibody comprising three heavy chain CDRs contained in SEQ ID NO: 14 and three light chain CDRs contained in SEQ ID NO:

10.

2. The bispecific antibody of claim 1 , wherein the anti-CLDN18.2 antibody or fragment thereof is a single domain antibody (VHH), and the single domain antibody comprises three CDRs contained in SEQ ID NO:

18.

3. (i) a heavy chain having the structure VH-CH1-Fc-VHH; and (ii) comprises a light chain whose structure is VL-CL; where: the VH comprises three heavy chain CDRs contained in SEQ ID NO: 11, the VL comprises three light chain CDRs contained in SEQ ID NO: 10, and the VHH comprises three CDRs contained in SEQ ID NO: 18; the VH comprises three heavy chain CDRs contained in SEQ ID NO: 12, the VL comprises three light chain CDRs contained in SEQ ID NO: 10, and the VHH comprises 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 comprised in SEQ ID NO: 14, the VL comprises the three light chain CDRs comprised in SEQ ID NO: 10, and the VHH comprises the three CDRs comprised in SEQ ID NO:

18.

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

5. (i) a heavy chain having the structure VH-CH1-Fc-VHH; and (ii) comprises a light chain whose structure is VL-CL; where: the VH comprises the 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 the 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 the 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 the sequence set forth in SEQ ID NO: 12 or a sequence having at least 90% sequence identity with SEQ ID NO: 12, the VL comprises the 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 the 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 the sequence set forth in SEQ ID NO: 13 or a sequence having at least 90% sequence identity with SEQ ID NO: 13, the VL comprises the 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 the sequence set forth in SEQ ID NO: 18 or a sequence having at least 90% sequence identity with SEQ ID NO: 18; or 3. The bispecific antibody of claim 2, wherein the VH comprises the sequence shown in SEQ ID NO: 14 or a sequence having at least 90% identity to SEQ ID NO: 14, the VL comprises the sequence shown in SEQ ID NO: 10 or a sequence having at least 90% sequence identity to SEQ ID NO: 10, and the VHH comprises the sequence shown in SEQ ID NO: 18 or a sequence having at least 90% sequence identity to SEQ ID NO:

18.

6. The bispecific antibody of claim 3 , wherein the VHH is linked to an Fc via a linker.

7. The bispecific antibody according to claim 3 , wherein the Fc region is an Fc region of an IgG antibody.

8. 4. The bispecific antibody of claim 3, 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 whose structure is VL-CL; where: the heavy chain comprises the sequence set forth in SEQ ID NO: 19 or a sequence with at least 90% identity to SEQ ID NO: 19, and the light chain comprises the sequence set forth in SEQ ID NO: 23 or a sequence with at least 90% identity to SEQ ID NO: 23; the heavy chain comprises the sequence set forth in SEQ ID NO: 20 or a sequence with at least 90% identity to SEQ ID NO: 20, and the light chain comprises the sequence set forth in SEQ ID NO: 23 or a sequence with at least 90% identity to SEQ ID NO: 23; the heavy chain comprises the sequence set forth in SEQ ID NO: 21 or a sequence with at least 90% identity to SEQ ID NO: 21, and the light chain comprises the sequence set forth in SEQ ID NO: 23 or a sequence with at least 90% identity to SEQ ID NO: 23; or 3. The bispecific antibody of claim 2, wherein the heavy chain comprises the sequence set forth 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 set forth 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 claim 1 or 2.

11. A vector comprising the polynucleotide of claim 10.

12. A host cell comprising the polynucleotide of claim 10.

13. 3. A method for producing a bispecific antibody according to claim 1 or 2, comprising the step (i) of culturing a host cell of the invention under conditions suitable for expression of the bispecific antibody of the invention, and the step (ii) of recovering the bispecific antibody of the invention.

14. An immunoconjugate, reagent kit, pharmaceutical composition, combination product or article of manufacture comprising the bispecific antibody of claim 1 or 2.

15. 15. The reagent kit, pharmaceutical composition, combination product or article of manufacture of claim 14, further comprising other therapeutic agents, and optional pharmaceutical adjuvants.

16. Use of the bispecific antibody according to claim 1 or 2 in the manufacture of a drug or a reagent kit for treating, preventing and / or diagnosing a CD40-related disease or a CLDN18.2-related disease.

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