Combination of anti-claudin 18.2 / anti-4-1bb antibodies with second therapeutic agents in cancer treatment

Abstract

This article provides information on the combined use of bispecific and multispecific antibodies targeting tight junction protein 18.2 (CLDN18.2) and 4-1BB with secondary therapeutic agents in cancer treatment.

Description

Cross-reference of related applications

[0001] This application claims the benefit of International Application No. PCT / CN2023 / 122092, filed on September 27, 2023, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0002] Claudins are a family of proteins that form important components of cellular tight junctions. Claudin-18 splice variant 2 (CLDN18.2) is a gastric-specific membrane protein. In healthy tissues, CLDN18.2, as a component of tight junctions, is expressed only in short-lived differentiated cells of the gastric mucosa, limiting the accessibility of antibody therapies. However, it exhibits significant ectopic expression in primary and metastatic lesions of various epithelial-derived solid tumors, including gastric cancer, pancreatic cancer, esophageal cancer, and lung adenocarcinoma cells.

[0003] 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is a member of the TNF receptor superfamily (TNFRSF) and a co-stimulatory molecule expressed after activation of immune cells (both innate and adaptive immune cells). 4-1BB plays a crucial role in regulating the activity of various immune cells. Agonists of 4-1BB can enhance immune cell proliferation, survival, cytokine secretion, and the cytolytic activity of CD8 T cells. Numerous other studies have shown that activation of 4-1BB can enhance the immune response in mice to clear tumors.

[0004] Combination therapy is commonly used to treat cancer before and / or after surgery. Therefore, 4-1BB is considered a promising target molecule in cancer immunology and combination therapy. Summary of the Invention

[0005] This study found that the anti-CLDN18.2 / anti-4-1BB bispecific antibody TJ001 synergistically inhibited tumor growth when used in combination with first-line or second-line treatments for gastric cancer (such as FOLFOX, FOLFOX and nivolumab, CAPOX, paclitaxel, or ramucirumab). Furthermore, whether used alone or in combination, the treatment was effective not only against tumors with high or moderate CLDN18.2 expression, but also against tumors with low CLDN18.2 expression and against CLDN18.2-negative tumor cells adjacent to CLDN18.2-expressing tumor cells (bystander effect).

[0006] Therefore, according to one embodiment of this disclosure, a method for treating cancer in a patient in need is provided, comprising administering to the patient a combination of an anti-tight junction protein 18.2 (CLDN18.2) / anti-4-1BB antibody and a second therapeutic agent.

[0007] In some embodiments, the antibody comprises an anti-CLDN18.2 unit, the anti-CLDN18.2 unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VHCDR3, the light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 each comprise the amino acid sequences of SEQ ID NO: 3-5, and VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 6-8.

[0008] In some embodiments, the VH of the anti-CLDN18.2 unit comprises the amino acid sequence of SEQ ID NO: 1, and the VL of the anti-CLDN18.2 unit comprises the amino acid sequence of SEQ ID NO: 2.

[0009] In some embodiments, the antibody comprises an anti-4-1BB unit, the anti-4-1BB unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, the light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 each comprise the amino acid sequences of SEQ ID NO: 11-13, and VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 14-16.

[0010] In some embodiments, the VH of the anti-4-1BB unit comprises the amino acid sequence of SEQ ID NO: 9, and the VL of the anti-4-1BB unit comprises the amino acid sequence of SEQ ID NO: 10.

[0011] In some embodiments, the antibody comprises two heavy chains, each containing the amino acid sequence of SEQ ID NO: 17, and two light chains, each containing the amino acid sequence of SEQ ID NO: 18.

[0012] In some embodiments, the second therapeutic agent is selected from fluoropyrimidines, platinum-based drugs, taxanes, anti-PD1 or anti-PD-L1 antibodies, VEGFR2 antibodies, and any combination thereof.

[0013] In some embodiments, the second therapeutic agent is selected from 5-fluorouracil (5-FU), oxaliplatin, nivolumab, and any combination thereof.

[0014] In some embodiments, the second therapeutic agent comprises 5-FU and oxaliplatin. In some embodiments, the second therapeutic agent comprises 5-FU, oxaliplatin, and nivolumab.

[0015] In some embodiments, the second therapeutic agent includes paclitaxel and / or ramucirumab.

[0016] In some implementations, the antibody and the second therapeutic agent are administered to the patient simultaneously or sequentially.

[0017] One aspect of this disclosure provides a method for treating a tumor in a patient in need, comprising administering an anti-tight junction protein 18.2 (CLDN18.2) / anti-4-1BB antibody to the patient, wherein the tumor has a low CLDN18.2 expression score on immunohistochemical (IHC) staining.

[0018] In some implementations, the low expression indicates no grade 2 or higher staining and less than 80% of the cells have grade 1 staining.

[0019] In some implementations, the antibody is administered to the patient every 1, 2, 3, 4, 5, 6, 7, 14, 21 days, one month, two months, three months, four months, or six months.

[0020] In some implementations, the cancer is selected from stomach cancer, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0021] In some implementations, the cancer is stomach cancer. Attached Figure Description

[0022] Figures 1A-D illustrate in vitro T cell activation induced by the bispecific antibody TJ001 disclosed herein and tumor killing of CLDN18.2-positive tumor cells. A. Schematic diagram of the co-culture system. B. Immunohistochemical (IHC) staining results of CLDN18.2 in three human gastric cancer cell lines (MKN-45, MKN-45#14, and MKN-45#18). C. TJ001 (CD4B)-induced T cell activation and D. Tumor killing. CM: Negative control.

[0023] Figures 2A-C illustrate the bystander killing effect of TJ001. A. Schematic diagram of the co-culture system. B. Cell viability (%) of tumor cells in the co-culture system. C. Cell viability (%) of tumor cells in co-culture systems with different donors.

[0024] Figure 3A -D illustrates enhanced T-cell activation and tumor killing when used in combination with chemotherapy drugs. A. Schematic diagram of an in vitro co-culture system simulating the tumor microenvironment (TME). B. T-cell activation and tumor killing when CD4B is used in combination with first-line drugs; C. T-cell activation and tumor killing when CD4B is used in combination with second-line drugs; D. Tumor killing (SNU601) when CD4B is used in combination with first-line or second-line drugs. Ram: Ramucirumab. All drugs (as indicated) were added to each group at the same dose, whether used alone or in combination. CM: Control culture medium. Data are expressed as mean ± standard deviation (Mean ± SD), and were compared with control culture medium using one-way ANOVA followed by Dunnett's test.

[0025] Figures 4A-D illustrate the effect of CD4B in a gastric cancer PDX model. A. Schematic diagram of the dosing regimen. B. Tumor growth inhibition results in each treatment group. C. Percentage of tumor-infiltrating lymphocytes (TILs) on day 23 post-treatment (top left) and cell density of TILs (CD3+ / CD8+) and activated CD8+ T cells in tumor cells (top right). D. Correlation between CD3+ cell density and tumor volume. Zolbe: Zolbetuximab; BIW: twice weekly; TIW: three times weekly; QDx5 / week: once daily, five days a week. All drugs (as indicated) were added to each group at the same dose, whether used alone or in combination. Data are expressed as mean ± standard deviation and analyzed using unpaired t-tests. Detailed Implementation definition

[0026] It should be noted that the terms "an" or "a" entity refer to one or more of that entity; for example, "an antibody" should be understood to mean one or more antibodies. Therefore, the terms "an" (or "a"), "one or more," and "at least one" are used interchangeably in this document.

[0027] As used herein, the term "polypeptide" is intended to encompass both single "polypeptides" and multiple "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids, and does not imply a specific length of the product. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a single or multiple chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of any of the foregoing terms, or used interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence. They may be produced in any manner, including chemical synthesis.

[0028] The term "isolated" as used in this article regarding cells and nucleic acids, such as "isolated" DNA or RNA, refers to molecules that have been isolated from other DNA or RNA present in the natural source of macromolecules. The term "isolated" as used herein also refers to nucleic acids or peptides prepared using recombinant DNA technology that are substantially free of cellular material, viral material, or culture medium, or to chemically synthesized nucleic acids or peptides that are substantially free of chemical precursors or other chemical substances. Furthermore, "isolated nucleic acids" is intended to include nucleic acid fragments that are not naturally present in fragment form and would not be found in their natural state. The term "isolated" is also used herein to refer to cells or peptides isolated from other cellular proteins or tissues. "Isolated peptides" is intended to encompass both purified and recombinant peptides.

[0029] As used herein, the term “recombination” in relation to polypeptides or polynucleotides refers to a form of polypeptide or polynucleotide that is not naturally occurring, and non-limiting examples of which can be constructed by combining polynucleotides or polypeptides that do not normally co-occur.

[0030] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in the sequences that can be aligned for comparison purposes. When a position in the aligned sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of common matching or homologous positions in the sequences. An "irrelevant" or "non-homologous" sequence has less than 40% identity with one of the sequences disclosed herein, but preferably less than 25%.

[0031] A "sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence of a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that, when aligned, that percentage of bases (or amino acids) are identical in the comparison of the two sequences. This alignment and homology percentage or sequence identity can be determined using software programs known in the art, such as Ausubel. et al Those described in Current Protocols in Molecular Biology, eds. (2007). Preferably, alignment is performed using default parameters. One alignment procedure is BLAST, using default parameters. Specifically, the procedures are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = bidirectional; truncation value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are those polynucleotides that have the above-specified percentage of homology and encode polypeptides with the same or similar biological activities.

[0032] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any of its antigen-binding fragments or single chains. Therefore, the term "antibody" includes any molecule containing a protein or peptide that comprises at least a portion of an immunoglobulin molecule and has the biological activity of binding an antigen. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR) or its ligand-binding portion of the heavy or light chain, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region or any portion thereof, or at least a portion of a binding protein.

[0033] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody (such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc.). Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, specigelmers, and diaboses. The term "antibody fragment" also includes any synthetic or genetically engineered protein capable of forming a complex by binding to a specific antigen, similar to an antibody.

[0034] "Single-chain variable fragment" or "scFv" refers to the variable region (V) of the heavy chain of immunoglobulins.H ) and the variable region (V) of the light chain L These are fusion proteins. In some respects, these regions are linked by short linker peptides of 10 to 25 amino acids. For flexibility, the linker may be enriched with glycine, and for solubility, it may be enriched with serine or threonine. The linker can also transfer V... H N-terminus and V L The C-terminus is linked, or vice versa. Despite the removal of the constant region and the introduction of the adaptor, this protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019.

[0035] The term antibody encompasses a wide range of biochemically distinguishable classes of polypeptides. Those skilled in the art will understand that heavy chains can be classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and within these classes are further subclasses (e.g., γ1-γ4). The nature of this chain determines the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specificity. Given this disclosure, those skilled in the art can readily identify modifications of each of these classes and isotypes, and therefore these modifications are within the scope of this disclosure. All immunoglobulin classes are explicitly within the scope of this disclosure, and the following discussion generally pertains to IgG class immunoglobulin molecules. In the case of IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually connected by disulfide bonds in a "Y" configuration, where the light chain starts from the opening of the "Y" shape, extends along the variable region, and encloses the heavy chain.

[0036] The antibodies, antigen-binding peptides, variants, or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-derived antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing VL or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies of the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0037] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, light and heavy chains are covalently bonded to each other, while the "tail" regions of the two heavy chains are linked together via covalent disulfide bonds or non-covalent bonds, regardless of whether the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration branch to the C-terminus at the bottom of each chain.

[0038] Both the light and heavy chains are divided into regions that are structurally and functionally homologous. The terms "constant" and "variable" are used according to function. In this regard, it should be understood that the variable domains (VL) of the light chain and the variable domains (VH) of the heavy chain determine antigen recognition and specificity. Conversely, the constant domains (CL) of the light chain and the constant domains (CH1, CH2, or CH3) of the heavy chain confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of constant domains increases with their distance from the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0039] As described above, the variable region enables antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VL and VH domains, or complementarity-determining regions (CDRs), combine to form the variable region defining a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites at the ends of each arm of the Y-chain. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) for each of the VH and VL chains. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins, the complete immunoglobulin molecule may consist only of the heavy chain, without the light chain. See, for example, Hamers-Casterman. et al. , Nature 363:446-448 (1993).

[0040] In naturally occurring antibodies, the six “complementarity-determining regions” or “CDRs” present in each antigen-binding domain are short, discontinuous amino acid sequences that are specifically localized to form the antigen-binding domain when the antibody presents its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain (called “framework” regions) exhibit less intermolecular variability. Framework regions primarily adopt a β-sheet conformation, and CDRs form cyclic structures connecting these β-sheets and, in some cases, form part of a β-sheet. Thus, the framework regions, by forming a scaffold, position the CDRs in the correct orientation via interchain non-covalent interactions. The antigen-binding domain formed by the localized CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids containing the CDRs and framework regions, as they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al ., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol ., 196:901-917 (1987)).

[0041] If a term is used and / or has two or more definitions in the art, its definition as used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe discontinuous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. This specific region has been described by Kabat. et al ., US Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983) and Chothia et al ., J. MoI. BiolThe description in 196:901-917 (1987), the entire contents of which are incorporated herein by reference. The CDR definitions of Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. However, the application of either definition to refer to the CDR of an antibody or its variants is within the scope of the terminology defined and used herein. The appropriate amino acid residues covering the CDRs defined in the above-cited documents are listed in the table below for comparison. The exact residue numbering covering a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR based on the amino acid sequence of the variable region of the antibody.

[0042] Kabat et al. also defined a numbering system applicable to variable domain sequences of any antibody. Those skilled in the art can readily apply this "Kabat numbering" system to any variable domain sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat number" refers to Kabat... et al The numbering system proposed in US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0043] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately the 31st amino acid (i.e., approximately 9 residues after the first cysteine ​​residue), comprises approximately 5-7 amino acids, and terminates at the next tryptophan residue. CDR-H2 begins at the 15th residue after the termination of CDR-H1, comprises approximately 16-19 amino acids, and terminates at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the termination of CDR-H2; comprises 3-25 amino acids; and terminates at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately the 24th residue (i.e., after the cysteine ​​residue); comprises approximately 10-17 residues; and terminates at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the termination of CDR-L1 and comprises approximately 7 residues. CDR-L3 begins at approximately the 33rd residue after the termination of CDR-L2 (i.e., after the cysteine ​​residue); it consists of approximately 7-11 residues and terminates at sequence F or WGXG, where X is any amino acid.

[0044] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, alpaca, horse, or chicken antibodies. In another embodiment, the variable region can be derived from cartilaginous fish (e.g., from sharks).

[0045] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in this disclosure may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain containing a CH3 domain. Furthermore, an antibody for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified so that their amino acid sequences differ from those of naturally occurring immunoglobulin molecules.

[0046] The heavy chain constant regions of antibodies disclosed in this paper can be derived from different immunoglobulin molecules. For example, the heavy chain constant regions of peptides can contain components derived from IgG. l The molecule contains the CH1 domain and a hinge region derived from the IgG3 molecule. In another example, the heavy chain constant region may contain a portion derived from IgG. l The molecule is derived in part from the hinge region of the IgG3 molecule. In another example, the heavy chain portion may contain components partially derived from IgG. l The molecules are derived in part from the chimeric hinges of the IgG4 molecule.

[0047] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant κ domain or a constant λ domain.

[0048] "Light chain-heavy chain pair" refers to a set of light and heavy chains that can form dimers through disulfide bonds between the CL or CK domains of the light chain and the CH1 domain of the heavy chain.

[0049] As previously mentioned, the subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (closest to the amino terminus) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0050] As used herein, the term "CH2 domain" includes portions of the heavy chain molecule, for example, extending from approximately residue 244 to residue 360 ​​of the antibody using a conventional numbering scheme (residues 244-360, Kabat numbering system; residues 231-340, EU numbering system; see Kabat). et al. (US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983)). The unique feature of the CH2 domain is that it does not pair tightly with other domains. Instead, in the intact natural IgG molecule, two N-linked branched glycan chains interspersed between the two CH2 domains. It is well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, containing approximately 108 residues.

[0051] As used herein, the term "hinge region" refers to the heavy-chain portion of the molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further subdivided into three distinct domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain (Roux). et al. , J. Immunol 161:4083 (1998)).

[0052] "Specific binding" or "specific to" generally refers to an antibody binding to an epitope through its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to it more easily through its antigen-binding domain than to a random, unrelated epitope. In this paper, the term "specificity" is used to define the relative affinity of an antibody for a given epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with higher specificity than to related epitope "D."

[0053] As used herein, the term "treat" refers to therapeutic actions and preventative or protective measures aimed at preventing or slowing (mitigating) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to prolonged survival compared to expected survival without treatment. Subjects requiring treatment include those who already have a condition or disease, those who are susceptible to a condition or disease, or those who wish to prevent a condition or disease.

[0054] The terms "effective amount," "pharmaceutical effective amount," and "therapeutic effective amount" refer to the amount that effectively elicits the desired biological or medical response, including the amount of a compound or peptide sufficient to treat a disease when administered to a subject. Effective amounts will vary depending on the compound or peptide, the disease and its severity, and the age, weight, etc., of the subject being treated. Effective amounts can include a range of amounts. Pharmaceutically effective amounts include the effective dose when used in combination with other medications.

[0055] "Subject," "individual," "animal," "patient," or "mammal" refers to any subject requiring diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, livestock, farm animals, and zoo, racing, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0056] As used herein, phrases such as “patient in need of treatment” or “subject in need of treatment” include subjects who would benefit from the administration of the antibodies or compositions disclosed herein (e.g., for detection, for diagnostic procedures and / or for treatment), such as mammalian subjects. Combination therapy

[0057] This study found that the anti-CLDN18.2 / anti-4-1BB bispecific antibody TJ001, when used in combination with other first- or second-line agents (such as FOLFOX and paclitaxel, or FOLFOX and nivolumab), synergistically inhibits tumor growth. Therefore, one embodiment of this disclosure provides a method for treating cancer in patients of need, involving administering a combination of an anti-tight junction protein 18.2 (CLDN18.2) / anti-4-1BB antibody and a second therapeutic agent to the patient. The second therapeutic agent may, for example, be selected from 5-fluorouracil (5-FU), platinum-based drugs, taxanes, anti-PD1 or anti-PD-L1 antibodies, and combinations thereof.

[0058] Anti-tight junction protein 18.2 (CLDN18.2) / anti-4-1BB antibody, also known as "bispecific antibody", consists of an anti-CLDN 18.2 unit and an anti-4-1BB unit. Anti-CLDN 18.2 unit

[0059] Tight junction protein 18 exists in two isoforms, isoform 1 and isoform 2. Isoform 2 (tight junction protein 18.2, or CLDN18.2) is a highly selective cell lineage marker. In normal tissues, CLDN 18.2 is strictly expressed in differentiated epithelial cells of the gastric mucosa. However, CLDN 18.2 has been found to be significantly expressed in primary and metastatic gastric cancer tissues, as well as pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer tissues, suggesting that CLDN18.2 is an attractive therapeutic target with great potential in gastric cancer and other types of solid tumors.

[0060] Any anti-CLDN18.2 known in the art can be used for the bispecific antibody of this application. In some embodiments, the anti-CLDN18.2 unit is selected from full-length antibodies, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the anti-CLDN18.2 unit includes Fab.

[0061] In some embodiments, the anti-CLDN18.2 unit comprises heavy chain variable regions (VH) CDR1, VH CDR2, and VHCDR3, which have the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 1, respectively. In some embodiments, the anti-CLDN18.2 unit comprises: (1) VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 3 or has one or more substituted amino acid sequences compared to SEQ ID NO: 3; (2) VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 4 or has one or more substituted amino acid sequences compared to SEQ ID NO: 4; and (3) VHCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 5 or has one or more substituted amino acid sequences compared to SEQ ID NO: 5.

[0062] In some embodiments, the anti-CLDN18.2 unit comprises a heavy chain variable region (VH) containing an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1.

[0063] In some embodiments, the anti-CLDN18.2 unit comprises light chain variable regions (VL) CDR1, VL CDR2, and VLCDR3, which respectively comprise the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 2. In some embodiments, the anti-CLDN18.2 unit comprises: (1) VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6 or has one or more substituted amino acid sequences compared to SEQ ID NO: 6; (2) VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7 or has one or more substituted amino acid sequences compared to SEQ ID NO: 7; and (3) VLCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 8 or has one or more substituted amino acid sequences compared to SEQ ID NO: 8.

[0064] In some embodiments, the anti-CLDN18.2 unit comprises a light chain variable region (VL) containing an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:2.

[0065] In some embodiments, the anti-CLDN18.2 unit comprises: (1) VH CDR1, VH CDR2 and VH CDR3, each comprising the amino acid sequences of CDR1, CDR2 and CDR3 within the heavy chain variable region (VH) as shown in SEQ ID NO: 1, and (2) VL CDR1, VL CDR2 and VL CDR3, each comprising the amino acid sequences of CDR1, CDR2 and CDR3 within the light chain variable region (VL) as shown in SEQ ID NO: 2. In some embodiments, the anti-CLDN18.2 unit comprises: (1) VHCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 3 or has one or more substituted amino acid sequences compared to SEQ ID NO: 3; (2) VHCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 4 or has one or more substituted amino acid sequences compared to SEQ ID NO: 4; (3) VHCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 5 or has one or more substituted amino acid sequences compared to SEQ ID NO: 5; (4) VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6 or has one or more substituted amino acid sequences compared to SEQ ID NO: 6; (5) VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7 or has one or more substituted amino acid sequences compared to SEQ ID NO: 7; and (6) VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 8 or has one or more substituted amino acid sequences compared to SEQ ID NO: 8.

[0066] In some embodiments, the anti-CLDN18.2 unit comprises: (1) a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, and (2) a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. Anti-4-1BB unit

[0067] 4-1BB is an inducible costimulatory receptor expressed on activated T cells and natural killer (NK) cells. The 4-1BB ligand (41BBL) trimer on T cells induces 4-1BB trimer clustering, triggering a signaling cascade that leads to upregulation of anti-apoptotic molecules, cytokine secretion, and enhanced effector function. On NK cells, 4-1BB signaling can enhance antibody-dependent cell-mediated cytotoxicity. Agonistic monoclonal antibodies targeting 4-1BB have been developed to leverage 4-1BB signaling for cancer immunotherapy. Preclinical results from various induced and spontaneous tumor models demonstrate that targeting 4-1BB with agonist antibodies can achieve tumor clearance and generate durable anti-tumor immunity.

[0068] Any 4-1BB antibody known in the art can be used for the bispecific antibody of this application. In some embodiments, the anti-4-1BB unit is selected from full-length antibodies, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the anti-4-1BB unit includes scFv.

[0069] In some embodiments, the anti-4-1BB unit comprises VH CDR1, VH CDR2, and VH CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the heavy chain variable region (VH) as shown in SEQ ID NO: 9. In some embodiments, the anti-4-1BB unit comprises: (1) VH CDR1, which comprises the amino acid sequence as shown in SEQ ID NO: 11 or has one or more substituted amino acid sequences compared to SEQ ID NO: 11; (2) VH CDR2, which comprises the amino acid sequence as shown in SEQ ID NO: 12 or has one or more substituted amino acid sequences compared to SEQ ID NO: 12; and (3) VH CDR3, which comprises the amino acid sequence as shown in SEQ ID NO: 13 or has one or more substituted amino acid sequences compared to SEQ ID NO: 13.

[0070] In some embodiments, the anti-4-1BB unit includes a heavy chain variable region (VH) containing an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9.

[0071] In some embodiments, the anti-4-1BB unit comprises VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of CDR1, CDR2, and CDR3 within the light chain variable region (VL) as shown in SEQ ID NO: 10. In some embodiments, the anti-4-1BB unit comprises: (1) VL CDR1, comprising the amino acid sequence as shown in SEQ ID NO: 14 or having one or more substituted amino acid sequences compared to SEQ ID NO: 14; (2) VL CDR2, comprising the amino acid sequence as shown in SEQ ID NO: 15 or having one or more substituted amino acid sequences compared to SEQ ID NO: 15; and (3) VL CDR3, comprising the amino acid sequence as shown in SEQ ID NO: 16 or having one or more substituted amino acid sequences compared to SEQ ID NO: 16.

[0072] In some embodiments, the anti-4-1BB unit comprises a light chain variable region (VL) containing an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10.

[0073] In some embodiments, the anti-4-1BB unit comprises: (1) VH CDR1, VH CDR2 and VH CDR3, each comprising the amino acid sequences of CDR1, CDR2 and CDR3 within the heavy chain variable region (VH) as shown in SEQ ID NO: 9, and (2) VL CDR1, VL CDR2 and VL CDR3, each comprising the amino acid sequences of CDR1, CDR2 and CDR3 within the light chain variable region (VL) as shown in SEQ ID NO: 10. In some embodiments, the anti-4-1BB unit comprises: (1) VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 11; (2) VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 12; (3) VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 13; (4) VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 14; (5) VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 15; and (6) VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having one or more substitutions compared to SEQ ID NO: 16. 16 compared to sequences with one or more substituted amino acids.

[0074] In some embodiments, the anti-4-1BB unit comprises: (1) a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9, and (2) a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Bispecific antibodies

[0075] In some embodiments, the bispecific antibody of this disclosure comprises: (1) an anti-tight junction protein 18.2 (CLDN18.2) unit having binding specificity to the CLDN18.2 protein; and (2) a second antibody unit. In some embodiments, the anti-CLDN 18.2 unit may be any anti-CLDN 18.2 unit as described herein. In some embodiments, the second antibody unit has binding specificity to targets selected from 4-1BB, PD-1, PD-L1, and CD3. In some embodiments, the second antibody comprises an anti-4-1BB unit having binding specificity to the 4-1BB protein. In some embodiments, the second antibody comprises an anti-4-1BB unit as described herein.

[0076] In some embodiments, the bispecific antibody of this application comprises: (1) an anti-tight junction protein 18.2 (CLDN18.2) unit that has binding specificity to the CLDN18.2 protein; and (2) an anti-4-1BB unit that has binding specificity to the 4-1BB protein. In some embodiments, the bispecific antibody of this application comprises: (1) an anti-tight junction protein 18.2 (CLDN18.2) unit as described herein that has binding specificity to the CLDN18.2 protein; and (2) an anti-4-1BB unit as described herein that has binding specificity to the 4-1BB protein.

[0077] In some embodiments, anti-4-1BB is an scFv and is fused to the C-terminus of a heavy chain of anti-CLDN 18.2 unit. In some embodiments, anti-4-1BB is an scFv and is fused to the N-terminus of a heavy chain of anti-CLDN 18.2 unit. In some embodiments, anti-4-1BB is an scFv and is fused to the C-terminus of a light chain of anti-CLDN 18.2 unit. In some embodiments, anti-4-1BB is an scFv and is fused to the N-terminus of a light chain of anti-CLDN 18.2 unit.

[0078] In some embodiments, the bispecific antibody comprises a heavy chain component containing an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17. In some embodiments, the bispecific antibody comprises a light chain component containing an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18. In some embodiments, the bispecific antibody comprises: (1) a heavy chain component comprising an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17, and (2) a light chain component comprising an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18.

[0079] In some embodiments, the bispecific antibody is selected from TJ001 (Table 1), IBI389 (Innovent), Q-1802 (QureBio), AMG-910 (Amgen), QLS31905 (Qilu Pharma), PM1032 (Biotheus), and HBM7022 (Harbour, AZ). In some embodiments, the bispecific antibody is TJ001. Second treatment agent

[0080] In one embodiment, the combination therapy disclosed herein includes the bispecific antibody provided herein and a second therapeutic agent.

[0081] In one embodiment, the second therapeutic agent includes FOLFOX, CAPOX, taxanes (e.g., paclitaxel), VEGF / VEGFR inhibitors or antibodies, anti-PD1 or anti-PD-L1 inhibitors or antibodies (e.g., nivolumab), or any combination thereof.

[0082] As shown in the accompanying experimental examples, TJ001 exhibits a synergistic antitumor effect when used in combination with certain second therapeutic agents (such as FOLFOX, which is a combination of 5-fluorouracil (5-FU) and platinum-based drugs (such as oxaliplatin)).

[0083] In some embodiments, FOLFOX includes 5-FU and oxaliplatin. In some embodiments, FOLFOX also includes leucovorin, such as tetrahydrofolate.

[0084] In some instances, the second therapeutic agent includes taxanes (e.g., paclitaxel) and / or anti-PD1 or anti-PD-L1 inhibitors or antibodies (e.g., nivolumab).

[0085] In one embodiment, the combination therapy includes the bispecific antibody, 5-FU, and platinum-based drugs disclosed herein.

[0086] "Platinum-based drugs" or "platinum-based antitumor drugs," or simply "platinum-based," are chemotherapeutic agents having one or more active moieties, said active moieties being platinum coordination complexes. Non-limiting examples of platinum-based drugs include cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and phenanthriplatin. In one specific embodiment, the platinum-based drug is oxaliplatin.

[0087] In some implementations, the second therapeutic agent includes fluoropyrimidines. Examples of fluoropyrimidines include oral fluoropyrimidine S-1; 5-fluorouracil (5-FU) or capecitabine, wherein capecitabine is a prodrug of 5-FU.

[0088] In some embodiments, the second therapeutic agent includes FOLFOX, which involves 5-FU, oxaliplatin, and optionally, leucovorin. In some embodiments, the second therapeutic agent includes CAPOX, which involves capecitabine and oxaliplatin.

[0089] In some embodiments, the second therapeutic agent includes taxanes. Taxanes are a class of diterpenoids with a taxadiene core, first discovered in plants of the genus *Taxus*. Non-limiting examples include paclitaxel, 10-deacetylbaccatin III, baccatin III, paclitaxel C, and 7-epitaxol. In one specific embodiment, the taxane is paclitaxel (PTX).

[0090] In some embodiments, the second therapeutic agent includes fluoropyrimidines, platinum-based drugs, and taxanes. In some embodiments, the second therapeutic agent includes 5-FU, platinum-based drugs, and paclitaxel. In some embodiments, the second therapeutic agent includes 5-FU, oxaliplatin, and paclitaxel. In some embodiments, the second therapeutic agent includes FOLFOX and paclitaxel.

[0091] In some implementations, the second therapeutic agent includes an anti-VEGF / VEGFR inhibitor or antibody. Examples of pan-VEGFR1-3 inhibitors include, but are not limited to, pazopanib, sunitinib, sorafenib, regorafenib, cabozantinib, lenvatinib, ponatinib, axitinib, and tivozanib. Examples of VEGFR2 inhibitors include, but are not limited to, vandetanib and ramucirumab. Examples of VEGF inhibitors include, but are not limited to, bevacizumab and aflibercept.

[0092] In some implementations, the second therapeutic agent includes taxanes (e.g., paclitaxel) and VEGF / VEGFR inhibitors, particularly VEGFR2 inhibitors. In some implementations, the second therapeutic agent includes paclitaxel and ramucirumab.

[0093] In some implementations, the second therapeutic agent includes an anti-PD-1 or anti-PD-L1 inhibitor or antibody. Examples of PD-1 inhibitors include, but are not limited to, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189. Examples of PD-L1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, dostarlimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, and AMP-514. In one specific implementation, the PD-1 inhibitor is nivolumab.

[0094] In some embodiments, the second therapeutic agent includes 5-FU, a platinum-based drug, and an anti-PD-1 or anti-PD-L1 inhibitor or antibody. In some embodiments, the second therapeutic agent includes FOLFOX and an anti-PD-1 or anti-PD-L1 inhibitor or antibody.

[0095] In some embodiments, the second therapeutic agent includes 5-FU, platinum-based drugs, and nivolumab. In some embodiments, the second therapeutic agent includes 5-FU, oxaliplatin, and nivolumab. In some embodiments, the second therapeutic agent includes FOLFOX and nivolumab. Cancer patients

[0096] Patients for whom the methods described in this disclosure are suitable for treatment include those with cancers such as gastric cancer, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is gastric cancer or gastric adenocarcinoma. Gastric cancer is a gastrointestinal tumor with both high morbidity and mortality.

[0097] In some embodiments, the cancer is characterized by expression of the CLDN18.2 protein. CLDN18.2 expression can be identified by methods known in the art, such as immunohistochemical (IHC) staining. Conventionally, anti-CLDN18.2 antibodies, as well as bispecific or multispecific antibodies specific to CLDN18.2, are used for cancers with at least moderate CLDN18.2 expression.

[0098] However, unexpectedly, the inventors observed (Example 1) that this bispecific antibody not only effectively inhibited the growth of tumor cells with moderate or high CLDN18.2 expression, but also was effective against CLDN18.2-negative cells mixed with CLDN18.2-expressing tumor cells. It is hypothesized that this bystander effect enables this bispecific antibody to produce anti-tumor effects in patients with relatively few CLDN18.2-expressing tumor cells and in patients with relatively low CLDN18.2 expression.

[0099] The expression level of CLDN18.2 in cells or tissues can be quantified based on the staining intensity of CLDN18.2 (e.g., membrane-bound CLDN18.2) in a sample. One example of an intensity scoring system is the four-point HSC, which is calculated based on staining intensity ranging from 0 (no staining), 1+ (weak staining), 2+ (obvious staining), 3+ (strong staining) to 4+ (extremely strong / saturated signal), and multiplied by the percentage of stained cells at each intensity level (0-100%) (see McCarty, KS Jr, et al, Cancer Res. 46 (suppl 8): 4244s-4248s (1986)).

[0100] As shown in the examples, negative CLDN18.2 expression means that <10% of cells are stained with 1+, low CLDN18.2 expression means that 65% of cells are stained with 1+ and no 2+ / 3+ staining, and moderate CLDN18.2 expression means that 35% of cells are stained with 1+ and 65% of cells are stained with 2+.

[0101] Therefore, according to one embodiment of this disclosure, a method for treating a tumor in a patient in need is provided, which involves administering the bispecific antibody of this disclosure to the patient. In some embodiments, the tumor has a low expression score on immunohistochemical (IHC) staining for CLDN18.2 expression.

[0102] In some embodiments, low expression indicates no grade 2 or higher staining, and less than 80% of cells have grade 1 staining. In some embodiments, low expression indicates no grade 2 or higher staining, and 20-80% of cells have grade 1 staining. In some embodiments, low expression indicates no grade 2 or higher staining, and 40-75% of cells have grade 1 staining. In some embodiments, low expression indicates no grade 2 or higher staining, and 60-70% of cells have grade 1 staining.

[0103] In some embodiments, the treatment comprises a bispecific antibody and a second therapeutic agent as disclosed herein. In some embodiments, the cancer is gastric cancer. Composition

[0104] In some embodiments, the bispecific antibody is administered intravenously to the subject. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1 to about 30 mg / kg body weight. In some embodiments, the bispecific antibody is administered intravenously to the subject at a dose of about 0.1 to about 30 mg / kg body weight.

[0105] The antibodies and second therapeutic agents described herein can be administered to patients simultaneously or separately. In some embodiments, the combined administration of the second therapeutic agent includes co-administration (simultaneous administration), administration with independent formulations or single-drug formulations, and sequential administration in any order, wherein preferably there is a time period during which both (or both) of the antibody and the second therapeutic agent exert their biological activity simultaneously.

[0106] The methods of administration of the antibodies or variants thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding peptides or compositions may be administered via any convenient route, such as by infusion or bolus injection, absorption through an epithelial or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), and may be co-administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides disclosed herein may be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., via powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0107] As used in this article, the term "parenteral" refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0108] Administration can be systemic or localized. Furthermore, it may be necessary to introduce the antibodies of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be assisted by an intraventricular catheter (e.g., connected to a reservoir, such as the Ommaya reservoir). Pulmonary administration may also be used, for example, by using an inhaler or nebulizer and formulating with an aerosol.

[0109] It may be necessary to apply the antibodies or compositions of this disclosure topically to the site of treatment; this can be achieved (e.g., but not limited to) local infusion during surgery, topical application (e.g., postoperative use in conjunction with wound dressings), injection, via catheter, via suppository, or via implantation, said implant being a porous, non-porous, or gel-like material, including membranes (such as silicone rubber membranes) or fibers. Preferably, when administering the proteins (including antibodies) of this disclosure, care must be taken to use materials that do not adsorb to the protein.

[0110] This disclosure also provides pharmaceutical compositions. Such compositions comprise a pharmaceutically effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent as provided herein.

[0111] In one specific implementation, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeias for animal (especially human) use. Furthermore, "pharmaceutical acceptable carriers" are generally non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of pharmaceutical excipient.

[0112] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils (including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a preferred carrier when the drug composition is administered intravenously. Saline solutions, as well as aqueous solutions of dextran and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. It is also envisioned that the composition include antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and osmolarity regulators such as sodium chloride or dextran. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions would contain a therapeutically effective amount of an antigen-binding polypeptide (preferably in a purified form) and a suitable amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the route of administration. Parenteral formulations can be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0113] In one embodiment, the composition is formulated according to conventional methods to be a pharmaceutical composition suitable for intravenous administration to a human. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic (such as lidocaine) to relieve pain at the injection site. Typically, the components are supplied separately or mixed together in unit dosage forms, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet), with the amount of active substance indicated. In the case of administration by infusion, the composition may be provided in an infusion bottle containing sterile pharmaceutical-grade water or saline. In the case of administration by injection, ampoules of sterile water or saline for injection may be provided to allow the components to be mixed prior to administration.

[0114] The compounds disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions (such as anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.) and those formed with cations (such as cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.). Example Example 1. TJ001 in the treatment of gastric cancer

[0115] TJ001 is a bispecific antibody against -CLDN18.2 X 4-1BB, comprising two heavy chain components, each having a sequence of SEQ ID NO: 17, and two light chain components, each having a sequence of SEQ ID NO: 18 (Table 1). TJ001 recognizes cells with high, intermediate, and low expression of CLDN18.2 and enhances T cell activation by activating 4-1BB signaling. This study investigated the mechanism of action of TJ001 and explored its combination with first-line (1L) or second-line (2L) therapeutic agents for gastric cancer. Table 1. Sequence of TJ001

[0116] CLDN18.2 expression in formalin-fixed paraffin-embedded (FFPE) tumors from three human gastric cancer cell lines (MKN-45, MKN-45#14, and MKN-45#18, obtained from Genomeditech) was determined by immunohistochemical (IHC) staining. T cell activation and tumor killing mediated by the disclosed bispecific antibody (TJ001, also known as "CD4B") alone or in combination with other therapies were investigated using a co-culture system of tumor cells expressing different levels of CLDN18.2 and PBMCs. T cell activation was assessed by the production of IFNγ, IL2, and soluble 4-1BB, and tumor killing was assessed by CellTiter-Glo® luminescent cell viability (CTG) assay. The antitumor activity and pharmacodynamic effects of the combination therapy were also examined using an in vivo patient-derived xenograft (PDX) model of gastric cancer. In vitro drug efficacy

[0117] IHC staining revealed that MKN-45, MKN-45#18, and MKN-45#14 cells were negative (<10% = 1+), had low CLDN18.2 expression (65% = 1+, none = 2+ / 3+), and moderate CLDN18.2 expression (35% = 1+, 65% = 2+), respectively (see Figure 1B). Detection of IFNγ levels secreted by activated T cells showed that TJ001 induced T cell activation in a dose- and CLDN18.2 expression-dependent manner (see Figure 1C). Similar results were found in measurements of IL-2 and soluble 4-1BB (s4-1BB) levels. TJ001 induced tumor killing in CLDN18.2-positive MKN-45#14 and MKN-45#18 cells, but not in CLDN18.2-negative MKN-45 cells (see Figure 1D).

[0118] Interestingly, in co-culture systems simulating the tumor microenvironment, TJ001 exhibited a bystander tumor-killing effect, as observed in mixtures of MKN-45#14 and MKN-45 cells at varying ratios in the presence of TJ001 (see Figures 2B-C). Specifically, TJ001 killed neighboring CLDN18.2-negative tumor cells through T cell activation mediated by CLDN18.2-positive tumor cells, suggesting its therapeutic potential in treating solid tumors with broad CLDN18.2 expression levels, including low CLDN18.2 expression.

[0119] Furthermore, TJ001-mediated T cell activation and tumor killing are similar to chemotherapy regimens used in first-line treatment of gastric cancer (see...). Figure 3B 5-fluorouracil (5-FU) combined with oxaliplatin (Oxa) (FOLFOX) or second-line chemotherapy regimens (see [link to relevant treatment]). Figure 3C The effect was further enhanced when combined with paclitaxel (PTX). Similar results were also observed in tumor-killing activity assays performed in SNU601 cells when CD4B was combined with first- or second-line drugs. Figure 3D Therefore, when CD4B is used in combination with chemotherapy regimens used in first-line treatment of gastric cancer (including nivolumab (Nivo) combined with 5-fluorouracil and oxaliplatin (5-FU+Oxa or FOLFOX)) or chemotherapy regimens used in second-line treatment (ramucirumab (Ram) combined with paclitaxel (PTX)), its induced tumor-killing effect is significantly enhanced. In vivo effects when used in combination with chemotherapy drugs

[0120] In a gastric cancer PDX model with moderate CLDN18.2 expression (2+ percentage 25-50%), the triple therapy of TJ001 combined with nivolumab (Nivo) and FOLFOX showed better tumor growth inhibition (TGI, see Figure 4B) (40%) compared with TJ001 alone (25 mpk) (8%) or nivolumab (10 mpk) combined with FOLFOX (5-FU, 8 mpk + oxaliplatin, 3 mpk) (8%), and on day 23 post-treatment, tumor-infiltrating T cells (TILs (CD3+)) were significantly increased. + / CD8 + )) with activated CD8 + T cells (CD69) + / Ki67 + (See Figure 4C)

[0121] As shown in Figure 4B, the PDX model was resistant to Nivo + FOLFOX treatment (8% TGI), belonging to the "cold tumor" category (TIL percentage <1%, see Figure 4C left, expressed as percentage of hCD45 cells). The TGI of CD4B + Nivo + FOLFOX (40%) was observed to be superior to either Nivo + FOLFOX or CD4B + FOLFOX (27%). The combination therapy of CD4B + Nivo + FOLFOX showed a synergistic effect, with a Q value >1.15. As shown in Figure 4D, after CD4B + Nivo + FOLFOX treatment, CD3... + Increased T-cell infiltration suggests a correlation between T-cell infiltration and tumor growth inhibition.

[0122] The scope of this disclosure is not limited to the specific embodiments described, which are merely individual examples of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

[0123] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated as incorporated by reference.

Claims

1. A method of treating cancer in a patient in need, comprising administering to the patient a combination of an anti-tight junction protein 18.2 (CLDN18.2) / anti-4-1BB antibody and a second therapeutic agent.

2. The method according to claim 1, wherein the antibody comprises an anti-CLDN18.2 unit, the anti-CLDN18.2 unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 each comprise the amino acid sequences of SEQ ID NO: 3-5, and VL CDR1, VLCDR2 and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 6-8.

3. The method of claim 2, wherein the VH of the anti-CLDN18.2 unit comprises the amino acid sequence of SEQ ID NO: 1, and the VL of the anti-CLDN18.2 unit comprises the amino acid sequence of SEQ ID NO:

2.

4. The method according to any one of the preceding claims, wherein the antibody comprises an anti-4-1BB unit, the anti-4-1BB unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 each comprise the amino acid sequence of SEQ ID NO: 11-13, and VL CDR1, VLCDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 14-16.

5. The method of claim 4, wherein the VH of the anti-4-1BB unit comprises the amino acid sequence of SEQ ID NO: 9, and the VL of the anti-4-1BB unit comprises the amino acid sequence of SEQ ID NO:

10.

6. The method according to any one of the preceding claims, wherein the antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 17, and two light chains each comprising the amino acid sequence of SEQ ID NO:

18.

7. The method according to any one of the preceding claims, wherein the second therapeutic agent is selected from fluoropyrimidines, platinum-based drugs, taxanes, anti-PD1 or anti-PD-L1 antibodies, VEGFR2 antibodies, and combinations thereof.

8. The method of claim 7, wherein the second therapeutic agent is selected from 5-FU, oxaliplatin, nivolumab, and any combination thereof.

9. The method of claim 8, wherein the second therapeutic agent comprises 5-FU and oxaliplatin.

10. The method of claim 8, wherein the second therapeutic agent comprises 5-FU, oxaliplatin, and nivolumab.

11. The method of claim 7, wherein the second therapeutic agent comprises paclitaxel and / or ramucirumab.

12. The method according to any one of the preceding claims, wherein the antibody and the second therapeutic agent are administered to the patient simultaneously or sequentially.

13. A method of treating a tumor in a patient in need, comprising administering an anti-tight junction protein 18.2 (CLDN18.2) / anti-4-1BB antibody to the patient, wherein the tumor has a low expression score for CLDN18.2 expression by immunohistochemical (IHC) staining.

14. The method of claim 13, wherein the low expression indicates no grade 2 or higher staining and less than 80% of the cells have grade 1 staining.

15. The method according to any one of the preceding claims, wherein the antibody is administered to the patient once every 1, 2, 3, 4, 5, 6, 7, 14 or 21 days, or once a month, once every two months, once every three months, once every four months or six months.

16. The method according to any one of the preceding claims, wherein the cancer is selected from gastric cancer, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

17. The method of claim 16, wherein the cancer is gastric cancer.