Anti-claudin 18.2 and Anti-4-1BB bispecific antibodies and use thereof

Abstract

To provide anti-claudin 18.2 and anti-4-1BB bispecific antibodies, and uses thereof.SOLUTION: There are provided bispecific and multi-specific antibodies that target both claudin 18.2 (CLDN18.2) and 4-1BB. These antibodies, in the absence of CLDN18.2-expressing cells, can bind to 4-1BB but are unable to activate 4-1BB signaling. In the presence of CLDN18.2-expressing cells, however, these antibodies can trigger CLDN18.2-dependent 4-1BB signaling, leading to potent immune response to the CLDN18.2-expressing tumor cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of PCT Application No. PCT / CN2019 / 100162, filed August 12, 2019, PCT / CN2019 / 104508, filed September 5, 2019, PCT / CN2020 / 071954, filed January 14, 2020, and PCT / CN2020 / 087968, filed April 30, 2020, the contents of each of which are hereby incorporated by reference in their entirety. [Background technology]

[0002] Claudins are a family of proteins that form important components of intercellular tight junctions. Claudin-18 splice variant 2 (CLDN18.2) is a gastric-specific membrane protein. In healthy tissues, CLDN18.2 is expressed exclusively in short-lived differentiated cells of the gastric mucosa as a component of tight junctions that are limited in accessibility to antibody treatment. However, CLDN18.2 is ectopically expressed at significant levels in various primary lesions and metastases of epithelial tumor entities, including adenocarcinoma cells of the stomach, pancreas, esophagus, and lung.

[0003] 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is a member of the TNF-receptor superfamily (TNFRSF) and a costimulatory molecule expressed in both innate and adaptive immune cells following immune cell activation. 4-1BB plays an important role in regulating the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8 T cells. Numerous other studies have shown that activation of 4-1BB enhances immune responses and eliminates tumors in mice. Therefore, 4-1BB is suggested to be a promising target molecule in cancer immunology. Summary of the Invention

[0004] Provided are bispecific and multispecific antibodies that target both claudin 18.2 (CLDN18.2) and 4-1BB. In some embodiments, the antibodies of the present technology include a "conditional agonist" anti-4-1BB moiety that cannot activate 4-1BB signaling without an anti-CLDN18.2 moiety that binds to the CLDN18.2 protein expressed on cells.

[0005] Activation of 4-1BB signaling is an expected mechanism for agonist antibodies such as utomilumab (PF-05082566) and urelumab (BMS-663513). However, the anti-4-1BB portion of the antibody disclosed herein does not require such activity. In fact, it is preferred that the anti-4-1BB portion of the antibody of the present invention cannot independently activate 4-1BB in the absence of CLDN18.2 binding. Interestingly, experimental examples have demonstrated that when the anti-CLDN18.2 portion binds to the CLDN18.2 protein on cells, such CLDN18.2 binding can induce 4-1BB signaling activation.

[0006] Compared to known anti-4-1BB agonist antibodies, which are typically associated with on-target, dose-limiting toxicity, the antibodies of the present disclosure are intended to be much safer. In tissues such as the liver where CLDN18.2 is not expressed, the antibodies of the present invention are not expected to induce a cytotoxic immune response because they are unable to activate 4-1BB signaling. In contrast, in tumor tissues where CLDN18.2 is expressed and / or accessible, the antibodies of the present invention can initiate a potent immune response against tumor cells. Thus, unlike anti-4-1BB antibodies currently in clinical development that cause on-target / inherent toxicity, the antibodies disclosed in the present invention may be both potent and safe for cancer treatment. [Brief explanation of the drawings]

[0007] [Figure 1]AC shows the three different bispecific formats tested in this disclosure.

[0008] [Figure 2] AD presents the results of ELISA for 4-1BB and CLDN18.2 binding.

[0009] [Figure 3] Cell-based 4-1BB binding is shown.

[0010] [Figure 4] AC Binding results for CLDN18.2 are shown.

[0011] [Figure 5] AD: CLDN18.2-dependent activation of 4-1BB signaling.

[0012] [Figure 6] A to E show PBMC responses in the presence of CLDN18.2-expressing cells.

[0013] [Figure 7] AD shows CD8+ T responses in the presence of CLDN18.2-expressing cells.

[0014] [Figure 8] AB shows the in vivo efficacy of anti-CLDN18.2-4-1BB antibody in a syngeneic mouse model and ex vivo analysis of its effect on tumor-infiltrating lymphocytes.

[0015] [Figure 9] AC shows the in vivo efficacy of the C-1A10 bispecific antibody in a syngeneic mouse model.

[0016] [Figure 10] 1 shows the dose-dependent antitumor efficacy of C-1A10 and D-1A10 in a syngeneic mouse model.

[0017] [Figure 11] AD: Pharmacokinetic and pharmacodynamic relationships of D-1A10 in a syngeneic mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0018] definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, for example, "an antibody" is understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0019] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "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 chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, "peptide," "dipeptide," "tripeptide," "oligopeptide," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. Polypeptides may be produced in any manner, including by chemical synthesis.

[0020] The term "isolated" as used herein with respect to cells, nucleic acids such as DNA or RNA, refers to molecules separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. As used herein, the term "isolated" refers to a nucleic acid or peptide that, when produced by recombinant DNA techniques, is substantially free of cellular material, viral material, or culture medium, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides that have been isolated from other cellular proteins or tissues. Isolated polypeptides are intended to encompass both purified and recombinant polypeptides.

[0021] As used herein, the term "recombinant" in reference to a polypeptide or polynucleotide means a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which is one that can be made by combining polynucleotides or polypeptides that do not normally occur together.

[0022] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which may be aligned for comparison purposes. If a position in the compared 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 matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity with one of the sequences of the present disclosure, although less than 25% identity is preferred.

[0023] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of sequence identity to another sequence, meaning that the percentage of bases (or amino acids) are the same in comparing the two sequences when aligned. This alignment and percentage homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, which uses the default parameters. Specifically, the program uses the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort BLASTN and BLASTP using by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are those that have the specified percentage homology referred to above and encode polypeptides having the same or similar biological activity.

[0024] 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 whole antibody, any antigen-binding fragment, or a single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, a heavy or light chain complementarity-determining region (CDR), or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0025] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as, for example, F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and bispecific antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex.

[0026] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L In some embodiments, the regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, serine or threonine for solubility, and V H N-terminus of V L The scFv molecule can be linked to the C-terminus of the original immunoglobulin, or vice versa. This protein maintains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. scFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019.

[0027] The term antibody encompasses a wide variety of biochemically distinguishable polypeptide classes. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and several subclasses thereof (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody: IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in light of the present disclosure and, therefore, are within the scope of the present disclosure. While all immunoglobulin classes are expressly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. For IgG, a typical immunoglobulin molecule contains two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons and two identical heavy polypeptide chains with a molecular weight of 53,000-70,000. These four chains are typically joined by disulfide bonds in a "Y" configuration, where the light chains bracket the heavy chains and begin at the beginning of the "Y" and extend through the variable region.

[0028] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAI, and IgA2), or subclass of immunoglobulin molecule.

[0029] Light chains are classified as either kappa (κ) or lambda (λ). Each heavy chain class may be associated with either a kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom of each chain.

[0030] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in reference to function. In this context, it will be understood that the variable domains (VK, VH) of both the light and heavy chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CH1, CH2, or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding site or 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 CK domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0031] As noted above, the variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. In other words, the VK and VH domains, or a subset of complementarity-determining regions (CDRs), of an antibody combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some cases, for example, in certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule may consist of only heavy chains, without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0032] In naturally occurring antibodies, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, noncontiguous amino acid sequences that are specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domain, called the "framework" regions, exhibit less intermolecular variability. The framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect to, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that allows the CDRs to be positioned in the correct orientation by noncovalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the noncovalent binding of the antibody to its cognate epitope. The amino acids comprising each of the CDRs and framework regions are precisely defined and can therefore be readily identified by those skilled in the art for any heavy or light chain variable region (see "Sequences of Proteins"). of Immunological Interest,"Kabat, E.,et al., USDapartment of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987).

[0033] Where there is more than one definition for a term as used and / or accepted within the art, the definition of that term as used herein is intended to encompass all such meanings, unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of both the heavy and light chain polypeptides. This particular region is described by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immuno logical interest” (1983) and Chothia et al. , J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The definitions of CDRs by Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. However, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined by each of the above-cited references are set forth in the table below for comparison. The exact number of residues that encompass a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of an antibody. [Table 1]

[0034] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can undoubtedly assign this system of "Kabat numbering" to any variable domain sequence without reliance on experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., US Department of Health and Human Services, "Sequence of Proteins of Immunology." The numbering system described by "The National Interest" (1983) Refers to...

[0035] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues from the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue from the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about amino acid 33 from the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG, where X is any amino acid. CDR-L1 begins at about residue 24 (i.e., after the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at about residue 16 from the end of CDR-L1 and includes about 7 residues. CDR-L3 begins at about the 33rd residue from the end of CDR-L2 (i.e., after the cysteine ​​residue), includes about 7 to 11 residues, and ends with the sequence F or WGXG, where X is any amino acid.

[0036] The antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In another embodiment, the variable regions may be of chondricthoid origin (e.g., from sharks).

[0037] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of 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 used in the present disclosure may comprise a polypeptide chain having a CH1 domain; a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure comprises a polypeptide chain having a CH3 domain. Furthermore, an antibody used in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of the CH2 domain). As discussed above, it will be understood by those skilled in the art that the heavy chain constant region may be modified so that its amino acid sequence differs from that of naturally occurring immunoglobulin molecules.

[0038] The heavy chain constant regions of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may be derived from an IgG l In another example, the heavy chain constant region may comprise, in part, a CH1 domain derived from an IgG molecule and a hinge region derived from an IgG3 molecule. lIn another example, the heavy chain portion may be derived in part from an IgG molecule and may include a hinge region derived in part from an IgG3 molecule. l The antibody may comprise a chimeric hinge derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0039] As used herein, the term "light chain constant region" includes amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0040] A "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0041] As previously indicated, the subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0042] As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule extending from about residue 244 to residue 360 ​​of an antibody, using conventional numbering schemes, for example (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; Kabat et al., USDept. of Health and Human Services,"Sequences" of Proteins of Immunological Interest"( (See, e.g., Wang, 1983). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact, native IgG molecules. The CH3 domain, which extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues, is also well documented.

[0043] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. 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 divided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0044] By "specifically bind" or "having specificity," it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding involves a degree of 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 the epitope via its antigen-binding domain more readily than it would to a random, unrelated epitope. The term "specificity" is used herein to refer to the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with higher specificity than it has for related epitope "D."

[0045] As used herein, the term "treat" or "treatment" refers to both therapeutic and prophylactic or preventative treatment, where the purpose is to prevent or slow (attenuate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0046] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, zoo animals, sport animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0047] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of an antibody or composition of the present disclosure, e.g., used for detection, diagnostic procedures, and / or treatment. Anti-claudin-18.2 anti-4-1BB antibody

[0048] 4-1BB is an inducible costimulatory receptor expressed on activated T cells and natural killer (NK) cells. Clustering of 4-1BB trimers with 4-1BB ligand (41BBL) trimers on T cells triggers a signaling cascade that leads to the upregulation of antiapoptotic molecules, cytokine secretion, and enhanced effector function. On NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity. Agonistic monoclonal antibodies targeting 4-1BB have been developed to exploit 4-1BB signaling for cancer immunotherapy. Preclinical results in various induced and spontaneous tumor models suggest that targeting 4-1BB with agonistic antibodies can lead to tumor clearance and durable antitumor immunity.

[0049] Two agonist antibodies, urelumab and utomilumab, are currently undergoing clinical trials. Urelumab has strong efficacy but has demonstrated inflammatory liver toxicity. The liver toxicity appears to be on-target, making it difficult to separate from efficacy. Utomilumab is relatively safer than urelumab, but is also less effective.

[0050] In the present experimental example, we tested a few anti-4-1BB antibodies that were specifically selected for their inability to independently activate 4-1BB signaling. In their monospecific forms, the anti-4-1BB antibodies can bind to 4-1BB alone or to 4-1BB on the cell surface. However, binding of 4-1BB on the cell surface does not lead to 4-1BB signaling activation (see, for example, Example 3 and Figure 5).

[0051] Interestingly, when the binding fragment of one of these conditional agonist 4-1BB antibodies, 1A10, was incorporated into a bispecific antibody further comprising an anti-CLDN18.2 moiety, the resulting bispecific antibody was able to efficiently activate 4-1BB signaling in a CLDN18.2-binding-dependent manner (see Example 3 and Figure 5). It is worth noting that these tested bispecific antibodies abolished FcγR-mediated 4-1BB agonism using N297A IgG1 Fc. Therefore, 4-1BB signaling activation can be attributed solely to CLDN18.2 binding.

[0052] The on-target toxicity of the anti-4-1BB agonist antibody, urelumab, is attributed to the antibody's lack of selectivity in 4-1BB agonism.However, the antibody of the present technology can easily be recognized for its ability to overcome this limitation.In tissues such as the liver where CLDN18.2 is not expressed or is inaccessible, the antibody cannot activate 4-1BB-mediated cytotoxicity, making it safe.In contrast, in tumor tissues where CLDN18.2 is overexpressed or accessible, the antibody activates CLDN18.2 binding-dependent 4-1BB signal transduction, leading to 4-1BB-mediated immune cell activation, thereby treating tumors.

[0053] Thus, according to one embodiment of the present disclosure, an antibody is provided, comprising an anti-claudin 18.2 (CLDN18.2) unit having binding specificity for a CLDN18.2 protein and an anti-4-1BB unit having binding specificity for a 4-1BB protein. In a preferred embodiment, the anti-4-1BB unit cannot activate 4-1BB signaling when bound to the 4-1BB protein in the absence of the anti-CLDN18.2 unit that binds to the CLDN18.2 protein.

[0054] The lack of 4-1BB agonism of the anti-4-1BB moiety can be achieved by many different means. It has been suggested that its activation requires 4-1BB clustering on the cell surface. Thus, in some embodiments, binding of the anti-4-1BB unit to the 4-1BB protein on a cell does not result in clustering of the 4-1BB protein in the absence of the anti-CLDN18.2 unit that binds to the CLDN18.2 protein.

[0055] The 4-1BB protein has four extracellular cysteine-rich pseudo-repeat domains (CRDs), CRD1, CRD2, CRD3, and CRD4 (see the amino acid sequence and CRD regions in the table below). Urelumab binds to the N-terminal CDR1 and activates 4-1BB cluster formation in a 4-1BB ligand (4-1BBL)-dependent manner. In some embodiments, the anti-4-1BB unit of the antibody disclosed in the present invention does not bind to CDR1. In some embodiments, the anti-4-1BB unit of the antibody disclosed in the present invention binds to CDR2. In some embodiments, the anti-4-1BB unit of the antibody disclosed in the present invention binds to CDR3. In some embodiments, the anti-4-1BB unit of the antibody disclosed in the present invention binds to CDR4. [Table 2]

[0056] In some cases, 4-1BB cluster formation may be mediated by an effector function of the anti-4-1BB antibody. Thus, in some embodiments, the antibodies of the present disclosure have an Fc fragment with reduced or no effector function. In some embodiments, such effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP).

[0057] The effector functions of antibodies can be modified using techniques known in the art, in some embodiments, the Fc fragment of the antibody is mutated or engineered in a way that reduces or eliminates its binding to FcγR. In some embodiments, the Fc fragment has reduced or no binding to FcγRI (CD64), in some embodiments, the Fc fragment has reduced or no binding to FcγRIIA (CD32), in some embodiments, the Fc fragment has reduced or no binding to FcγRIIB (CD32), in some embodiments, the Fc fragment has reduced or no binding to FcγRIIIA (CD16a), in some embodiments, the Fc fragment has reduced or no binding to FcγRIIIB (CD16b), in some embodiments, the Fc fragment has reduced or no binding to C1q (the first subcomponent of the C1 complex).

[0058] In some embodiments, the Fc fragment comprises one or more mutations that reduce or eliminate FcγR or C1q binding of the Fc fragment. Non-limiting examples of such mutations include an L235E mutation in an IgG1 Fc fragment, an L234A and / or L235A mutation in an IgG1 Fc fragment, a P329G or P329A mutation in an IgG1 Fc fragment, an F234A and / or L235A or L235E mutation in an IgG4 Fc fragment, an H268Q mutation, a V309L mutation, an A330S mutation, and / or a P331S mutation in an IgG2 Fc fragment, and a V234A mutation, a G237A mutation, a P238S mutation, an H268A mutation, a V309L mutation, an A330S mutation, and / or a P331S mutation (EU numbering) in an IgG2 Fc fragment.

[0059] Antibody effector functions can also be reduced by reducing or eliminating glycosylation of the Fc fragment (e.g., aglycosylated Fc fragments). In some embodiments, such reduction or inhibition can be achieved by using a cell line that is unable to glycosylate antibodies. In some embodiments, the Fc fragment is mutated.

[0060] Non-limiting examples of such mutations include mutations at N297 (such as N297A, N297G, and N297Q) (EU numbering). In some embodiments, the mutation is N297A. Antibody Types and Example Sequences

[0061] In the experimental examples, three different bispecific antibody formats were tested. Among them, format A in Figure 1 showed higher activity than those in Figure 1 B and Figure 1 C. These data suggest that a suitable format for the antibody of the present disclosure may use a Fab fragment for the anti-CLDN18.2 portion. In some embodiments, the format includes a single-chain fragment (scFv) for the anti-4-1BB portion. In some embodiments, the anti-4-1BB portion is not located between the Fab and Fc fragments.

[0062] In some embodiments, the anti-CLDN18.2 unit comprises a Fab fragment, preferably a pair of Fab fragments. In some embodiments, the anti-4-1BB unit comprises a Fab fragment, preferably a pair of Fab fragments. In some embodiments, the anti-4-1BB unit comprises an scFv, preferably a pair of scFv fragments.

[0063] In some embodiments, the anti-CLDN18.2 unit is located N-terminal to the anti-4-1BB unit, hi some embodiments, an Fc fragment is positioned between the anti-CLDN18.2 unit and the anti-4-1BB unit.

[0064] Exemplary CDR and VH / VL sequences are also provided for the anti-CLDN18.2 and anti-4-1BB units. In some embodiments, the anti-4-1BB unit comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with one or two amino acid substitutions from SEQ ID NO: 1, CDRH2 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence with one or two amino acid substitutions from SEQ ID NO: 2, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 3, 56, 57, 58, or 59, or an amino acid sequence with one or two amino acid substitutions from SEQ ID NO: 3, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 12 CDRL1 comprises the amino acid sequence of SEQ ID NO: 4 or 60 or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 4 or 60; CDRL2 comprises the amino acid sequence of SEQ ID NO: 5 or 61 or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 5 or 61; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 6 or 62 or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 6 or 62. [Table 3] [Table 4]

[0065] In some embodiments, CDRH1 comprises the amino acid sequence of SEQ ID NO: 1, CDRH2 comprises the amino acid sequence of SEQ ID NO: 2, CDRH3 comprises the amino acid sequence of SEQ ID NO: 3, 56, 57, 58 or 59, CDRL1 comprises the amino acid sequence of SEQ ID NO: 4 or 60, CDRL2 comprises the amino acid sequence of SEQ ID NO: 5 or 61, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 6 or 62.

[0066] In some embodiments, the anti-4-1BB unit comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 46-51, and 63-69, and a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 52-53, and 70-74. [Table 5]

[0067] In some embodiments, the anti-CLDN18.2 unit comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3, wherein (a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 7, CDRH2 comprises the amino acid sequence of SEQ ID NO: 8, CDRH3 comprises the amino acid sequence of SEQ ID NO: 9, CDRL1 comprises the amino acid sequence of SEQ ID NO: 10, CDRL2 comprises the amino acid sequence of SEQ ID NO: 11, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 12.

[0068] In some embodiments, the anti-CLDN18.2 unit comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3, wherein (b) CDRH1 comprises the amino acid sequence of SEQ ID NO: 13, CDRH2 comprises the amino acid sequence of SEQ ID NO: 14, CDRH3 comprises the amino acid sequence of SEQ ID NO: 15, CDRL1 comprises the amino acid sequence of SEQ ID NO: 16, CDRL2 comprises the amino acid sequence of SEQ ID NO: 17, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 18.

[0069] In some embodiments, the anti-CLDN18.2 unit comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3, wherein (c) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19, CDRH2 comprises the amino acid sequence of SEQ ID NO: 20, CDRH3 comprises the amino acid sequence of SEQ ID NO: 21, CDRL1 comprises the amino acid sequence of SEQ ID NO: 22, CDRL2 comprises the amino acid sequence of SEQ ID NO: 11, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 23. [Table 6]

[0070] In some embodiments, the VH of the anti-CLDN18.2 unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, and 30, and the VL of the anti-CLDN18.2 unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 29, and 31.

[0071] In some embodiments, provided is a bispecific antibody that adopts a format as illustrated in Figure 1A. In some embodiments, provided is an antibody comprising two first polypeptides and two second polypeptides, each first polypeptide having, from N-terminus to C-terminus, a heavy chain variable region (VH), a CH1, a CH2, a CH3, and a single-chain fragment (scFv) having specificity for a 4-1BB protein, each second polypeptide having a light chain variable region (VL) and a CL, each VH paired with one of the VLs and having specificity for a claudin 18.2 (CLDN18.2) protein, and the scFv cannot activate 4-1BB signaling when bound to the 4-1BB protein in the absence of a VH / VL pair that binds to the CLDN18.2 protein.

[0072] The VH / VL pair here constitutes the anti-CLDN18.2 unit, and the scFv constitutes the anti-4-1BB unit. The CH2-CH3 / CH2-CH3 pair constitutes the Fc fragment. The various embodiments described above regarding the anti-CLDN18.2 unit, the anti-4-1BB unit, and the Fc fragment may also be applicable here.

[0073] The following table provides non-limiting examples of first polypeptides (heavy components) and second polypeptides (light components): In some embodiments, each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 40, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 41.

[0074] In some embodiments, each of the first polypeptides comprises the amino acid sequence of SEQ ID NO:42 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO:43.

[0075] In some embodiments, each of the first polypeptides comprises the amino acid sequence of SEQ ID NO:44 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO:45. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

[0076] It will also be understood by those skilled in the art that the antibodies disclosed herein may be modified such that they differ in amino acid sequence from the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0077] In certain embodiments, the antibody comprises an amino acid sequence or one or more substructures not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure may comprise a flexible linker sequence or may be modified to add a functional substructure (e.g., PEG, a drug, a toxin, or a label).

[0078] The antibodies, variants, or derivatives thereof of the present disclosure include modified derivatives, i.e., derivatives modified by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, antibodies may contain one or more non-classical amino acids.

[0079] In some embodiments, the antibody may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0080] The antibody may be conjugated or fused to a therapeutic agent which may include a detectable label such as a radiolabel, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic agent, a photoactive diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound-enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0081] An antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0082] The antibody also 152 Fluorescence-emitting metals such as Eu or others of the lanthanide series can be used to detectably label the antibody. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for attaching various moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotherapy." notargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Ant ibodies For Drug Delivery",in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxi c Agents In Cancer Therapy:A Review",in Monoclonal Antibodies '84: Biological And Clinical Applications,Pinchera et al(eds.),pp.475-506(1985);"Analysis,Results,A nd Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy",in Monoclonal Antibodies For Ca Detection And Therapy,Baldwin et al.(eds.),Academic Press pp.303-16(1985),and Thorpe et al.,"The Preparation And Cy totoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58(1982)). I want to be illuminated. Polynucleotides encoding antibodies and methods for preparing antibodies

[0083] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0084] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be produced using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen administration, but whose endogenous gene locus has been disabled. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated by reference in their entirety. Treatment method

[0085] As described herein, the antibodies, variants, or derivatives of the present disclosure may be used in certain treatment and diagnostic methods.

[0086] The present disclosure is further directed to antibody-based therapies that involve administering antibodies of the present disclosure to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof described herein).

[0087] In some embodiments, provided is a method for treating cancer in a patient in need thereof. In one embodiment, the method involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the patient's cancer cells (e.g., stromal cells) overexpresses claudin 18.2.

[0088] Cell therapy, such as chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable cells can be used that are contacted with the antibody of the present disclosure (or alternatively, engineered to express the antibody of the present disclosure). After such contact or engineering, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient may have any of the types of cancer disclosed herein. Cells (e.g., T cells) can be, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0089] In some embodiments, the cells are isolated from the cancer patient themselves. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0090] Non-limiting examples of cancer include 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. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0091] Additional diseases or conditions that may be treated, prevented, diagnosed, and / or predicted with the antibodies or variants, or derivatives thereof, of the present disclosure that are associated with increased cell survival include leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, as well as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma ( lymphangioendotheliosarcoma), synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, sarcomas and carcinomas such as neuroblastoma and retinoblastoma.

[0092] The specific dosage and treatment regimen for any particular patient depends on various factors, including the specific antibody, variant or derivative thereof used, the patient's age, weight, overall health, sex, and dietary habits, as well as the administration time, excretion rate, drug combination, and the severity of the specific disease being treated.It is within the ordinary skill of medical caregivers to determine such factors.The amount also depends on the individual patient being treated, the administration route, the type of formulation, the characteristics of the compound being used, the severity of the disease, and the desired effect.The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0093] Methods of administration of antibodies, variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), or may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powders, ointments, drops, or transdermal patch), buccally, or as an oral or nasal spray.

[0094] As used herein, the term "parenteral" refers to modes of administration which include intravenous, intramuscular, intraperitoneal, substernal, subcutaneous and intraarticular injection and infusion.

[0095] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection may be facilitated by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir.Pulmonary administration can also be used, for example, by using an inhaler or nebulizer and formulation with an aerosolizing agent.

[0096] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area in need of treatment, which may be achieved, for example, but not by way of limitation, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, using a catheter, using a suppository, or using a placement agent, which may be a porous, non-porous, or gelatinous material, including a membrane such as a silastic membrane, or a fiber. Preferably, when administering proteins, including antibodies of the disclosure, care must be taken to use materials to which the protein does not absorb.

[0097] The amount of the antibody of the present disclosure that will be effective in treating, suppressing, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to facilitate the identification of optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective amounts can also be estimated from dose-response curves derived from in vitro or animal model test systems.

[0098] As a general proposition, the dosage of an antigen-binding polypeptide of the present disclosure administered to a patient is typically between 0.1 mg / kg and 100 mg / kg of the patient's body weight, between 0.1 mg / kg and 20 mg / kg of the patient's body weight, or between 1 mg / kg and 10 mg / kg of the patient's body weight. Generally, due to the immune response to the foreign polypeptide, human antibodies have a longer half-life in the human body than antibodies derived from other species. Therefore, it is often possible to administer lower dosages of human antibodies and less frequently. Furthermore, the dosage and frequency of administration of antibodies of the present disclosure may be reduced by enhancing antibody uptake (e.g., into the brain) and tissue penetration by modifications such as lipidation.

[0099] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines, including, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0100] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. composition

[0101] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0102] In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0103] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol, if desired. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for regulating tonicity such as sodium chloride or dextrose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained-release formulations. The compositions can be formulated as suppositories with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are found in Remington's Pharmaceutical Sciences by E.W. Martin. ces, which is incorporated herein by reference. Such compositions contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to a patient. The formulation should suit the mode of administration. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0104] In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to humans.Typically, compositions for intravenous administration are sterile isotonic buffer solution.If necessary, the composition may also contain a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the injection site.Generally, the ingredients are supplied individually or mixed together in unit dosage form, for example, as lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of active ingredient.When the composition is to be administered by injection, it can be prepared using an infusion bottle containing pharmaceutical-grade sterile water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0105] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. [Example]

[0106] Example 1: Generation of anti-CLDN18.2 / 4-1BB bispecific antibodies Three previously identified anti-CLDN18.2 antibodies, 4F11E2, 72C1B6A3, and 120B7B2, and an anti-4-1BB antibody, 1A10 (sequences shown in the table below), were selected to generate an anti-CLDN18.2-4-1BB bispecific antibody in a full-length IgG x scFv format (structure illustrated in Figure 1A). The anti-claudin-18.2 moiety was placed in the full IgG site, while the anti-4-1BB moiety was an scFv placed at the C-terminus of the Fc fragment. The bispecific antibody contained an IgG1 backbone with an N297A mutation to abolish Fcγ function. [Table 12] [Table 13] [Table 14]

[0107] The sequences of 4F11E2 and 1A10 were also used to generate two different types of bispecific antibodies, as illustrated in Figure 1B and Figure 1C, respectively. In the type shown in Figure 1B, the anti-claudin-18.2 moiety also took the form of a Fab, while the anti-4-1BB moiety was presented as an scFab fragment inserted between the anti-claudin-18.2 Fab and Fc fragments. IgG1(N297A) was also used here. [Table 15]

[0108] In type C of Figure 1, the anti-claudin-18.2 moiety was presented on intact IgG1, while the anti-4-1BB moiety was an scFab fragment placed at the C-terminus of Fc. [Table 16]

[0109] In preliminary tests, the bispecific antibodies of the types in Figure 1B and Figure 1C were outperformed by those of the type in Figure 1A for 4-1BB binding. Therefore, only those of type in Figure 1A were subjected to further testing as described below. Furthermore, E-1A10 was slightly less potent than C-1A10 and D-1A10, and therefore was also not included in further consideration. Example 2. Antigen binding of anti-CLDN18.2 / 4-1BB bispecific antibodies

[0110] In this example, the binding activity of the bispecific antibody of Example 1 to CLDN18.2 and 4-1BB was evaluated using protein- and cell-based assays. 2.1 ELISA binding to 4-1BB

[0111] Briefly, microtiter plates were coated overnight at 4°C with 100 μl of human 4-1BB-His protein diluted at 0.5 μg / ml in PBS and then blocked with 100 μl / well of 1% BSA. Three-fold dilutions of antibody, starting at 100 nM, were added to each well and incubated for 2 hours at room temperature. The plates were washed with PBS / Tween and then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody for 30 minutes at room temperature. After washing, the plates were developed with TMB substrate and analyzed at OD 450 nm using a plate reader. As shown in Figure 2A, the anti-CLDN18.2-4-1BB antibodies (C-1A10 and D-1A10) showed binding comparable to that of the anti-4-1BB monoclonal antibodies (1A10 and urelumab (BMUR)). 2.2 ELISA binding to CLDN18.2

[0112] To test CLDN18.2 binding, the CLDN18.2 protein was expressed in virus-like particles (VLPs) that mimic the native conformation. Similarly, microtiter plates were filled with 100 μl of CLDN18.2 diluted in PBS at 3 μg / ml. Plates were coated with VLPs overnight at 4°C and then blocked with 300 μl / well of 3% BSA. Three-fold dilutions of antibody, starting from 100 nM, were added to each well and incubated at 37°C for 2 hours. The plates were washed with PBS / Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody for 30 minutes at room temperature. After washing, the plates were developed with TMB substrate and analyzed at OD 450 nm using a plate reader. As shown in Figure 2B, the anti-CLDN18.2-4-1BB antibodies (C-1A10 and D-1A10) showed stronger binding than the anti-CLDN18.2 monoclonal antibody (I-MAB362). Meanwhile, binding to CLDN18.1 protein was also tested using the same experimental setup. As shown in Figure 2C, none of the antibodies showed cross-reactivity with CLDN18.1. 2.3 ELISA binding to double antigens

[0113] To further demonstrate that the anti-CLDN18.2-4-1BB antibody can simultaneously bind to CLDN18.2 and 4-1BB, a DACE (Dual Antigen Capture ELISA) test was performed. Briefly, microtiter plates were coated overnight at 4°C with 100 μl of CLDN18.2 VLP diluted to 3 μg / ml in PBS. Three-fold dilutions of antibody, starting from 100 nM, were then added to each well and incubated at 37°C for 2 hours. After washing, the plates were then incubated with 100 μl of human 4-1BB biotin protein at a concentration of 1 μg / ml at 37°C for 1 hour, followed by streptavidin-HRP for an additional 30 minutes at room temperature. After washing, the plates were developed with TMB substrate and analyzed at OD 450 nm using a plate reader. As shown in Figure 2D, D-1A10 can simultaneously bind to hCLDN18.2 and 4-1BB. In contrast, the CLDN18.2 mAb 72C1B6A3 showed no signal in this assay. 2.4 Cell-based binding to 4-1BB

[0114] To assess antigen binding properties, anti-CLDN18.2-4-1BB antibodies were analyzed by FACS for binding to HEK293 cells expressing 4-1BB. 5 A total number of HEK293-4-1BB cells were incubated with serially diluted antibodies in FACS buffer (PBS with 2% FBS) for 30 minutes at 4°C. After washing with FACS buffer, PE-conjugated anti-human IgG antibody was added to each well and incubated for 30 minutes at 4°C. After washing, the MFI of PE was assessed using a FACS Calibrator. As shown in Figure 3, the tested anti-CLDN18.2-4-1BB antibodies (C-1A10 and D-1A10) showed concentration-dependent binding to 4-1BB, comparable to that of the monoclonal antibodies (1A10 and BMUR). 2.5 Cell-based binding to CLDN18.2

[0115] To evaluate the binding ability to CLDN18.2, a CHO-K1 cell line stably expressing human CLDN18.2 was generated. Then, CHO-C18.2 cell lines were sorted for high expression (CHO-K1C18.2 High) and low expression (CHO-K1C18.2 Low) using flow cytometry. CHO-K1-C18.2 cells were incubated with serially diluted antibodies in FACS buffer at 4°C for 30 minutes. After washing with FACS buffer, PE-conjugated anti-human IgG antibody was added and incubated at 4°C for 30 minutes. The MFI of PE was evaluated by FACS. As shown in Figures 4A and 4B, the anti-CLDN18.2-4-1BB antibody bound to CLDN18.2-expressed cells in a concentration-dependent manner, while both bispecific antibodies showed stronger binding than IMAB362, a reference monoclonal anti-CLDN18.2 antibody currently undergoing clinical trials.

[0116] SNU620 is a gastric cancer cell line that expresses endogenous CLDN18.2. As shown in Figure 4C, the anti-CLDN18.2-4-1BB bispecific antibody was also able to bind to SNU620. Example 3. Functional activity of anti-CLDN18.2 / 4-1BB bispecific antibodies 3.1 Cell line-based functional characterization of CLDN18.2-4-1BB bispecific antibody

[0117] To test the ability of bispecific antibodies to enhance 4-1BB signaling, a commercially available 4-1BB assay was used. In this assay, GloResponse™ NFκB-luc2 / 4-1BB Jurkat cell line (Promega, cat#CS196004) was used as the effector cell, and CHO-K1 cells expressing or not expressing CLDN18.2 or SNU620 were used as the target cell. The GloResponse™ NFκB-luc2 / 4-1BB Jurkat cell line was genetically modified to stably express 4-1BB and luciferase downstream of the response element. Luciferase expression is induced when the antibody binds to the 4-1BB receptor. Briefly, 2.5 x 10 cells per well were cultured. 4 Effector cells were cultured at a density of 2.5 x 10 cells in a white 96-well plate. 4 The antibodies were mixed with target cells. Six-fold serially diluted antibodies were added to white 96-well assay plates at final concentrations ranging from 16.7 nM to 0.28 nM. After 6 hours of incubation at 37°C, luminescence was obtained by adding luciferase substrate and measured using a microplate reader (PHERAstar). Four-parameter logistic curve analysis was performed using GraphPad software.

[0118] As shown in Figure 5, the BMUR monoclonal antibody was able to enhance 4-1BB signaling in a dose-dependent manner, whereas the 1A10 monoclonal antibody had no agonist activity in the same experimental setting. The activity of the anti-CLDN18.2-4-1BB bispecific antibodies D-1A10 and C-1A10 was dependent on CLDN18.2 expression. 3.2 Activity of Bispecific Antibodies to Enhance Human Peripheral Blood Mononuclear Cell (PBMC) Immune Responses

[0119] To test the ability of bispecific antibodies to stimulate human PBMC responses, a cytokine production assay was used. Human PBMCs stimulated with 0.5 μg / ml of human anti-CD3 antibody were used as effector cells. CLDN18.2-expressing CHO-K1 cells were used as target cells. Human PBMCs (1 × 10 5 ) were incubated with CHO-K1-CLDN18.2 or control CHO-K1 cells (2.5 × 10 4 PBMCs were co-cultured with CLDN18.2-expressing cells. Serially diluted bispecific antibodies were added to the mixed cultures at final concentrations starting from 20 nM. After 48 h, IL-2 and IFN-γ levels in the culture medium were measured using the IL-2 (human) LANCE Ultra TR-FRET Detection Kit and the IFN-γ (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). As shown in Figure 6, only the bispecific antibodies can activate PBMC responses in the presence of CLDN18.2-expressing cells.

[0120] To further demonstrate that bispecific antibody activity correlates with CLDN18.2 levels, human PBMCs from one donor were cocultured with cells from different gastric adenocarcinoma (GA) PDXs, which were shown to express different levels of CLDN18.2. Serially diluted bispecific antibodies were added to the mixed cultures at final concentrations starting from 20 nM. After 48 hours, IL-2 levels in the culture medium were measured using the IL-2 (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). As shown in Figures 6D and 6E, the activity of the bispecific antibody D-1A10 correlated with CLDN18.2 levels. D-1A10 effectively activated T cells even in tumors with low to moderate CLDN18.2 expression. 3.3 Activity of bispecific antibodies to enhance human CD8+ T cell responses

[0121] To further test the ability of the bispecific antibody in activating human CD8+ T cells, human CD8+ T cells isolated from PBMCs were used as effector cells. CHO-K1 or SNU620 cells expressing CLDN18.2 were used as target cells. Isolated CD8+ T cells (7.5 × 10 4 cells) in the presence of human anti-CD3 antibody. 4 The cells were co-cultured with 1000 mAbs of 1000 mAbs (1000 mAbs) and 1000 mAbs of 1000 mAbs (1000 mAbs). Serially diluted bispecific antibodies were added to the mixed cultures at final concentrations starting from 20 nM. After 48 h of co-culture, IL2 and IFN-γ levels in the culture medium were measured using the IL-2 (human) LANCE Ultra TR-FRET Detection Kit and the IFN-γ (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). As shown in Figure 7, the bispecific antibodies C-1A10 and D-1A10 increased the production of both IL2 and IFN-γ by activated CD8+ T cells in the presence of CHO-K1 overexpressing C18.2 (Figures 7A-7B) or SNU620 cells endogenously expressing C18.2 (Figures 7C-7D). Example 4. Tumor growth inhibition by anti-CLDN18.2-4-1BB bispecific antibody

[0122] Humanized mice expressing the extracellular domain of human 4-1BB were used. Mouse colon adenocarcinoma cells (MC38) were engineered to express human CLDN18.2. MC38-hCLDN18.2 cells were subcutaneously implanted into humanized mice (h4-1BB). Mice were intraperitoneally administered the following antibodies five times every three days: isotype control (10 mg / kg), anti-CLDN18.2 antibody (10 mg / kg), anti-4-1BB antibody (10 mg / kg), a combination of anti-CLDN18.2 (10 mg / kg) and anti-4-1BB (10 mg / kg), and anti-CLDN18.2-4-1BB bispecific antibody (13.3 mg / kg). Tumor volume was monitored twice weekly throughout the experiment by caliper measurement. Tumor growth inhibition induced by the bispecific antibody was significantly greater than that induced by the combination of each target monoclonal antibody, as shown in Figure 8. To further understand the mechanism of the antibody, tumor-infiltrating lymphocytes and peripheral lymphocytes were collected and analyzed by flow cytometry to obtain the percentage of CD3+ T cells. The results showed that the bispecific antibody could specifically increase the number of CD3+ T cells in the tumor microenvironment, while having no effect on peripheral blood.

[0123] In another repeat experiment using the same animal model, in vivo efficacy was further demonstrated. Figure 9A shows the tumor growth curves for each animal in each group. Tumor growth inhibition of the anti-CLDN18.2-4-1BB antibody (C-1A10) reached 105% at the end of the study. Six of seven mice in the bispecific group were tumor-free by day 25 after the first treatment.

[0124] Furthermore, in this example, 35 days after the first tumor inoculation, all anti-CLDN18.2-4-1BB antibody (C-1A10)-treated animals were re-challenged with a second dose of MC38-hCLDN18.2 tumor cells in the opposite flank, and tumor growth was monitored without further treatment. Results showed that, while tumor cells continued to grow in naive mice, all BsAb-treated mice were resistant to tumor re-challenge and were considered tumor-free until the end of the study (Figure 9B), suggesting that the bispecific antibody of the present invention can induce long-term protective immune memory against MC38 tumors. Similarly, in a satellite group (N=3 / group) in which mice received the same treatment, tumors were extracted and tumor-infiltrating lymphocytes (TILs) were quantified 3 days after the second dose of antibody. CD45 + TILs and CD8 + The percentage of TILs was significantly higher in the BsAb-treated group, whereas there was no effect on peripheral lymphocytes (Fig. 9C).

[0125] To further evaluate the anti-tumor efficacy of the anti-CLDN18.2-4-1BB bispecific antibody, the bispecific antibody was administered at different concentrations to 4-1BB-humanized mice transplanted with MC38-hCLDN18.2 antibody. The following antibodies were intraperitoneally administered to the mice every three days for four doses: isotype control (10 mg / kg), C-1A10 (13.3 mg / kg), C-1A10 (2.6 mg / kg), C-1A10 (0.5 mg / kg), D-1A10 (13.3 mg / kg), D-1A10 (2.6 mg / kg), and D-1A10 (0.5 mg / kg). Tumor volume was monitored twice weekly throughout the experiment by caliper measurement. As shown in Figure 10, both C-1A10 and D-1A10 were able to inhibit tumor growth in a dose-dependent manner.

[0126] To understand the pharmacokinetic (PK) and pharmacodynamic relationships of anti-CLDN18.2-4-1BB in a humanized mouse model, a single dose of D-1A10 at three different concentrations was administered to tumor-bearing humanized mice. Serum concentrations were measured at various time points. As shown in Figure 11, the overall PK profile was dose-dependent, and in vivo efficacy correlated with dose level. Ex vivo TIL analysis suggested a dose-dependent increase in CD8+ and CD45+ cells. Example 5. Testing of additional conditional agonist fully human anti-4-1BB antibodies

[0127] The following anti-4-1BB antibodies were identified for their ability to bind 4-1BB with high affinity and not activate 4-1BB signaling upon binding. [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] 5.1 Antigen binding measured by ELISA

[0128] To evaluate antigen-binding activity, ELISA tests were performed on the antibodies. Briefly, microtiter plates were coated overnight at 4°C with 100 μl / well of 0.1 μg / ml human 4-1BB-Fc protein in PBS, followed by blocking with 100 μl / well of 5% BSA. Five-fold dilutions of antibody, starting at 10 μg / ml, were added to each well and incubated for 1-2 hours at room temperature. The plates were washed with PBS / Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody for 1 hour at room temperature. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630 nm. The tested anti-4-1BB antibodies demonstrated 4-1BB binding ability. 5.2 Cell binding measured by FACS

[0129] To evaluate the antigen-binding properties, candidate antibodies were analyzed by FACS for binding to mammalian cells expressing 4-1BB. Briefly, 4-1BB-Jurkat cells were incubated with the antibody. After washing with FACS buffer (1% BSA in PBS), FITC anti-human IgG antibody was added to each well and incubated at 4°C for 1 hour. The MFI of FITC was evaluated using FACS Caliber. The tested anti-4-1BB antibodies demonstrated the ability to bind to 4-1BB expressed on the cell surface and could efficiently bind to 4-1BB expressed on mammalian cells. 5.3 Protein dynamics related to 4-1BB

[0130] To examine the binding kinetics of the antibodies, in this example, affinity ranking was performed using Octet Red 96. As shown in Table 5, the tested anti-4-1BB antibodies had high 4-1BB binding affinity. [Table 22]

[0131] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure; any compositions or methods that are functionally equivalent are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure, provided that the modifications and variations come within the scope of the appended claims and their equivalents.

[0132] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In certain embodiments, for example, the following items are provided: (Item 1) an anti-claudin 18.2 (CLDN18.2) unit having binding specificity for the CLDN18.2 protein; an anti-4-1BB unit having binding specificity for the 4-1BB protein, the anti-4-1BB unit is unable to activate 4-1BB signaling when bound to a 4-1BB protein in the absence of the anti-CLDN18.2 unit that binds to a CLDN18.2 protein; antibody. (Item 2) 2. The antibody of item 1, wherein the binding of the anti-4-1BB unit to the 4-1BB protein on a cell does not result in clustering of the 4-1BB protein in the absence of an anti-CLDN18.2 unit that binds to the CLDN18.2 protein. (Item 3) 3. The antibody of item 1 or 2, further comprising an Fc fragment with reduced effector function. (Item 4) 4. The antibody of item 3, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP). (Item 5) 5. The antibody of item 4, wherein the Fc fragment has reduced or no binding to FcγR or C1q. (Item 6) 6. The antibody of item 5, wherein the Fc fragment has one or more mutations that reduce or eliminate FcγR or C1q binding. (Item 7) The Fc fragment an IgG1 Fc fragment with the L235E mutation; an IgG1 Fc fragment having an L234A mutation and / or an L235A mutation; an IgG1 Fc fragment having a P329G mutation or a P329A mutation; an IgG4 Fc fragment having an F234A mutation and / or an L235A mutation or an L235E mutation; an IgG2 Fc fragment having a H268Q mutation, a V309L mutation, an A330S mutation, and / or a P331S mutation; and IgG2 Fc fragment with V234A, G237A, P238S, H268A, V309L, A330S, and / or P331S mutations (EU numbering) 7. The antibody according to item 6, selected from the group consisting of: (Item 8) 4. The antibody of item 3, wherein the Fc fragment is aglycosylated. (Item 9) 9. The antibody of item 8, wherein the Fc fragment comprises a mutation that eliminates glycosylation. (Item 10) 10. The antibody of item 9, wherein the mutation is at N297, optionally at N297A, N297G, or N297Q. (Item 11) 4. The antibody of item 3, wherein the Fc fragment comprises an N297A mutation or a combination of L234A and L235A mutations. (Item 12) 12. The antibody of any one of items 1 to 11, wherein the anti-CLDN18.2 unit comprises a Fab fragment. (Item 13) 13. The antibody of item 12, wherein the anti-CLDN18.2 unit comprises a pair of Fab fragments. (Item 14) 14. The antibody of any one of items 1 to 13, wherein the anti-4-1BB unit comprises a Fab fragment or a single chain fragment (scFv). (Item 15) 15. The antibody of item 14, wherein the anti-4-1BB unit comprises a pair of scFv fragments. (Item 16) 16. The antibody of item 14 or 15, wherein the scFv is C-terminal to the Fc fragment of the anti-CLDN18.2 unit. (Item 17) the anti-4-1BB unit comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3; the CDRH1 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 1; the CDRH2 comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO:2; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 3, 56, 57, 58, or 59, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 3, 56, 57, 58, or 59; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 4 or 60, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 4 or 60; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 5 or 61, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 5 or 61; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 6 or 62, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 6 or 62; 17. The antibody according to any one of items 1 to 16. (Item 18) the anti-4-1BB unit comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3; the CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; said CDRH2 comprising the amino acid sequence of SEQ ID NO:2; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 3, 56, 57, 58, or 59; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 4 or 60; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 5 or 61; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 6 or 62; 17. The antibody according to any one of items 1 to 16. (Item 19) 19. The antibody according to any one of Items 1 to 18, wherein the anti-4-1BB unit comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 46 to 51, and 63 to 68, and a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 52 to 53, and 69 to 74. (Item 20) the anti-CLDN18.2 unit comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3; (a) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 7; said CDRH2 comprising the amino acid sequence of SEQ ID NO: 8; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 9; said CDRL1 comprising the amino acid sequence of SEQ ID NO: 10; said CDRL2 comprising the amino acid sequence of SEQ ID NO: 11; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 12; (b) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 13; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 14; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 15; said CDRL1 comprising the amino acid sequence of SEQ ID NO: 16; said CDRL2 comprising the amino acid sequence of SEQ ID NO: 17; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 18; or (c) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 19; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 20; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 21; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 22; said CDRL2 comprising the amino acid sequence of SEQ ID NO: 11; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 23; 20. The antibody according to any one of items 1 to 19. (Item 21) 21. The antibody of item 20, wherein the VH of the anti-CLDN18.2 unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, and 30, and the VL of the anti-CLDN18.2 unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 29, and 31. (Item 22) An antibody comprising two first polypeptides and two second polypeptides, each said first polypeptide having, from N-terminus to C-terminus, a heavy chain variable region (VH), a CH1, a CH2, a CH3, and a single-chain fragment (scFv) having specificity for a 4-1BB protein; each said second polypeptide having a light chain variable region (VL), and a CL; each VH is paired with one of the VLs and has specificity for a claudin 18.2 (CLDN18.2) protein; the scFv is unable to activate 4-1BB signaling when bound to a 4-1BB protein in the absence of the VH / VL pair that binds to a CLDN18.2 protein; antibody. (Item 23) 23. The antibody of item 22, wherein the CH2 and the CH3 constitute an Fc fragment with reduced effector function. (Item 24) The Fc fragment an IgG1 Fc fragment with the L235E mutation; an IgG1 Fc fragment having an L234A mutation and / or an L235A mutation; an IgG1 Fc fragment having a P329G mutation or a P329A mutation; an IgG4 Fc fragment having an F234A mutation and / or an L235A mutation or an L235E mutation; an IgG2 Fc fragment having a H268Q mutation, a V309L mutation, an A330S mutation, and / or a P331S mutation; and an IgG2 Fc fragment having a V234A mutation, a G237A mutation, a P238S mutation, a H268A mutation, a V309L mutation, an A330S mutation, and / or a P331S mutation (EU numbering); 24. The antibody of item 23. (Item 25) 24. The antibody of item 23, wherein the Fc fragment comprises a mutation at N297, optionally at N297A, N297G, or N297Q. (Item 26) the scFv comprises a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3; the CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; said CDRH2 comprising the amino acid sequence of SEQ ID NO:2; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 3, 56, 57, 58, or 59; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 4 or 60; the CDRL2 comprises the amino acid sequence of SEQ ID NO: 5 or 61; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 6 or 62; The antibody according to any one of Items 22 to 25. (Item 27) 27. The antibody according to any one of Aspects 22 to 26, wherein the scFv comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 46 to 51, and 63 to 68, and a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 52 to 53, and 69 to 74. (Item 28) the VH of the VH / VL pair comprises CDRH1, CDRH2, and CDRH3, and the VL of the VH / VL pair comprises CDRL1, CDRL2, and CDRL3; (a) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 7; said CDRH2 comprising the amino acid sequence of SEQ ID NO: 8; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 9; said CDRL1 comprising the amino acid sequence of SEQ ID NO: 10; said CDRL2 comprising the amino acid sequence of SEQ ID NO: 11; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 12; (b) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 13; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 14; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 15; said CDRL1 comprising the amino acid sequence of SEQ ID NO: 16; said CDRL2 comprising the amino acid sequence of SEQ ID NO: 17; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 18; or (c) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 19; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 20; the CDRH3 comprises the amino acid sequence of SEQ ID NO: 21; the CDRL1 comprises the amino acid sequence of SEQ ID NO: 22; said CDRL2 comprising the amino acid sequence of SEQ ID NO: 11; the CDRL3 comprises the amino acid sequence of SEQ ID NO: 23; 28. The antibody according to any one of Items 22 to 27. (Item 29) 28. The antibody of claim 27, wherein the VH of the VH / VL pair comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, and 30, and the VL of the VH / VL pair comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 29, and 31. (Item 30) Item 31. The antibody of Item 22, wherein each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 40 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 41. 32. The antibody of claim 22, wherein each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 42 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 43. 33. The antibody of claim 22, wherein each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 44 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 45. 33. One or more polynucleotides encoding the antibody of any one of items 1 to 32. (Item 34) 33. A composition comprising the antibody of any one of items 1 to 32 and a pharmaceutically acceptable carrier. (Item 35) 33. Use of the antibody of any one of items 1 to 32 for the preparation of a medicament for treating cancer. (Item 36) 33. A method for treating cancer in a patient in need thereof, comprising administering to said patient the antibody of any one of items 1 to 32. (Item 37) 37. The use of item 35 or the method of item 36, wherein the cancer is characterized by cancer cells that overexpress CLDN18.2 compared to corresponding normal cells. (Item 38) 38. The use according to item 35 or item 37, or the method according to item 36 or item 37, wherein the cancer is an epithelial tumor or the cancer is selected from the group consisting of 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. (Item 39) 38. The use according to item 35 or item 37, or the method according to item 36 or item 37, wherein the cancer is selected from the group consisting of gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, and ovarian cancer.

[0133]

Claims

[Claim 1] The invention as described in the drawings.

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