Anti-claudin 18.2 multispecific antibodies and uses thereof

Abstract

To provide anti-claudin 18.2 multispecific antibodies and uses thereof.SOLUTION: The present disclosure relates generally to immunoglobulin-related compositions (e.g., multispecific antibodies or antigen-binding fragments thereof) capable of binding to a claudin 18.2 protein. The multispecific antibodies of the present technology are useful in methods for detecting and treating a claudin 18.2 related cancer in a subject in need thereof. Claudins are integral membrane proteins that form tight junctions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 061,895, filed August 6, 2020, U.S. Provisional Application No. 63 / 074,582, filed September 4, 2020, and U.S. Provisional Application No. 63 / 144,657, filed February 2, 2021, the disclosures of which are incorporated by reference herein in their entireties.

[0002] The present technology generally relates to the preparation and use of immunoglobulin-related compositions (e.g., multispecific antibodies or antigen-binding fragments thereof) that specifically bind to claudin 18.2 protein. In particular, the present technology relates to the preparation of claudin 18.2-binding multispecific antibodies and their use in the detection and treatment of cancer. [Background technology]

[0003] The following description of the background of the present technology is provided merely as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Claudins are integral membrane proteins that form tight junctions, which act as a physical barrier that prevents solutes and water from passing freely through the intercellular space between epithelial or endothelial cell sheets (Markov, A.G., et al., IUBMB Life 67:29-35 (2015); Furuse, M., et al., J Cell Biol 141:1539-1550 (1998); Nitta, T., et al., J Cell Biol 161:653-660 (2003); Deli, MA, Biochim Biophys Acta 1788:892-910 (2009)). Additionally, tight junctions also play an important role in maintaining cell polarity and signal transduction. Disruption of epithelial cell polarity is an early event in malignant transformation (Martin, TA and Jiang, WG, Biochim Biophys Acta 1788:872-891 (2009)). Claudin 18.2 is abundant in a significant proportion of primary gastric cancers and their metastases and plays an important role in their malignant transformation. For example, frequent ectopic activation of claudin 18.2 has been found in pancreatic, esophageal, ovarian, and lung tumors (Niimi et al., (2001) Mol Cell Biol 21(21):7380-7390, Tanaka et al. (2011) J Histochem Cytochem 59(10):942-952, Micke et al., (2014) Int J Cancer 135(9):2206-2214, Shimoba et al. (2016) Biochim Biophys Acta 1863(6 Pt A):1170-1178, Singh et al., (2017) J Hematol Oncol 10(1):105, Tokumitsu et al., (2017) Cytopathology 28(2):116-121). Therefore, there is an urgent need for novel anti-claudin 18.2 immunoglobulin-related compositions that are effective in treating claudin 18.2-associated malignancies. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Markov, AG, et al., IUBMB Life 67:29-35(2015) [Non-patent document 2] Furuse, M., et al., J Cell Biol 141:1539-1550(1998)

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Summary of the Invention

Means for Solving the Problems

[0006] In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprising a first antigen-binding portion that binds to a claudin18.2 epitope and at least a second antigen-binding portion that binds to a second epitope, wherein the first antigen-binding portion is a first heavy chain immunoglobulin variable domain (V H ) and the first light chain immunoglobulin variable domain (V L ), and the second antigen-binding portion comprises a second V H and the second V L (a) a first V H is selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, and 30 H - a CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 7, 13, 19, 25, and 31 H - CDR2 sequence and V selected from the group consisting of SEQ ID NOs: 8, 14, 20, 26, and 32 H and / or (b) a first V L is selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, and 33 L - a CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 155, and 156 L - a CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, and 35 L -CDR3 sequence.

[0007] In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprising a first antigen-binding portion that binds to a claudin18.2 epitope and at least a second antigen-binding portion that binds to a second epitope, wherein the first antigen-binding portion is a first heavy chain immunoglobulin variable domain (V H ) and the first light chain immunoglobulin variable domain (V L ), and the second antigen-binding portion comprises a second V H and the second V L (a) a first V H V of SEQ ID NO: 6 H - CDR1 sequence and V of SEQ ID NO: 7 H- CDR2 sequence and V of SEQ ID NO: 8 H and / or a first V L V of SEQ ID NO: 9 L - CDR1 sequence and V of SEQ ID NO: 10 or SEQ ID NO: 155 L - CDR2 sequence and V of SEQ ID NO: 11 L or (b) a first V H V of SEQ ID NO: 12 H - CDR1 sequence and V of SEQ ID NO: 13 H - CDR2 sequence and V of SEQ ID NO: 14 H and / or a first V L V of SEQ ID NO: 15 L - CDR1 sequence and V of SEQ ID NO: 16 or SEQ ID NO: 156 L - CDR2 sequence and V of SEQ ID NO: 17 L or (c) a first V H V of SEQ ID NO: 18 H - CDR1 sequence and V of SEQ ID NO: 19 H - CDR2 sequence and V of SEQ ID NO: 20 H and / or a first V L V of SEQ ID NO: 21 L - CDR1 sequence and V of SEQ ID NO: 22 L - CDR2 sequence and V of SEQ ID NO: 23 L and (d) a first V H V of SEQ ID NO: 24 H - CDR1 sequence and V of SEQ ID NO: 25 H - CDR2 sequence and V of SEQ ID NO: 26 H and / or a first V L V of SEQ ID NO: 27 L - CDR1 sequence and V of SEQ ID NO: 28 L - CDR2 sequence and V of SEQ ID NO: 29 L or (e) a first V H V of SEQ ID NO: 30 H - CDR1 sequence and V of SEQ ID NO: 31 H - CDR2 sequence and V of SEQ ID NO: 32 Hand / or a first V L is V of SEQ ID NO: 33 L - CDR1 sequence and V of SEQ ID NO: 34 L - CDR2 sequence and V of SEQ ID NO: 35 L -CDR3 sequence.

[0008] In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprising a first antigen-binding portion that binds to a claudin18.2 epitope and at least a second antigen-binding portion that binds to a second epitope, wherein the first antigen-binding portion is a first heavy chain immunoglobulin variable domain (V H ) and the first light chain immunoglobulin variable domain (V L ), and the second antigen-binding portion comprises a second V H and the second V L The first V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57; and / or (b) a first V L comprises an amino acid sequence selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, and 58-61.

[0009] Additionally or alternatively, in some embodiments of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments disclosed herein, the second V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; and / or (b) a second V L comprises an amino acid sequence selected from any one of SEQ ID NOs: 98, 103, or 158.

[0010] In any and all embodiments of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments disclosed herein, the multispecific (e.g., bispecific) antigen-binding fragments may be Fab, F(ab'), Fab', scF v , and F vmay be selected from the group consisting of:

[0011] Additionally or alternatively, in some embodiments, the multispecific (e.g., bispecific) antibody or antigen-binding fragment further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In certain embodiments, the multispecific (e.g., bispecific) antibody or antigen-binding fragment comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A. In other embodiments, the multispecific (e.g., bispecific) antibody or antigen-binding fragment comprises an IgG4 constant region comprising an S228P mutation.

[0012] In another aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody comprising a first antigen-binding portion that binds to a claudin18.2 epitope and a second antigen-binding portion that binds to a second epitope, wherein the first antigen-binding portion is a first heavy chain immunoglobulin variable domain (V H ) and the first light chain immunoglobulin variable domain (V L ), and the second antigen-binding portion comprises a second V H and the second V L (a) a first V L and / or (b) a first V H The sequence is at least 95% identical to the heavy chain immunoglobulin variable domain of any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57. Additionally or alternatively, in some embodiments, the second V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; and / or (b) a second V L comprises an amino acid sequence selected from any one of SEQ ID NOs: 98, 103, or 158.

[0013] In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody comprising a first antigen-binding portion that binds to a claudin18.2 epitope and a second antigen-binding portion that binds to a second epitope, wherein the multispecific antibody comprises a heavy chain (HC) amino acid sequence comprising SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:159, SEQ ID NO:161, or a variant thereof having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:160, SEQ ID NO:162, or a variant thereof having one or more conservative amino acid substitutions. In some embodiments, the multispecific (e.g., bispecific) antibody comprises an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:81 and SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:159 and SEQ ID NO:160, and SEQ ID NO:161 and SEQ ID NO:162, respectively.In another aspect, the disclosure provides a multispecific (e.g., bispecific) antibody comprising a first antigen-binding portion that binds to a claudin-18.2 epitope and a second antigen-binding portion that binds to a second epitope, wherein the multispecific antibody comprises (a) an LC sequence that is at least 95% identical to the LC sequence present in SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:160, or SEQ ID NO:162, and / or (b) an HC sequence that is at least 95% identical to the HC sequence present in SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:159, or SEQ ID NO: 161. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition contains an IgG4 constant region comprising an S228P mutation. In certain embodiments, the multispecific (e.g., bispecific) antibody or antigen-binding fragment comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A.

[0014] In any and all embodiments of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments disclosed herein, the multispecific (e.g., bispecific) antibodies or antigen-binding fragments bind to a CLDN18.2 polypeptide comprising an extracellular loop 1 (EL1) sequence. The extracellular loop 1 (EL1) sequence may comprise the amino acid sequence of SEQ ID NO: 2, or the CLDN18.2 polypeptide may comprise the amino acid sequence of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the multispecific (e.g., bispecific) antibodies of the present technology are monoclonal, chimeric, or humanized antibodies and / or lack α-1,6-fucose modifications.

[0015] In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and wherein (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3, and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of a second immunoglobulin or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, and (b) the light and heavy chain variable domains of the second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (b) the second polypeptide and the third peptide chain each comprise, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a second immunoglobulin variable domain. and a heavy chain constant domain of one immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, or 58-61.

[0016] Additionally or alternatively, in some embodiments, the heavy chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57; the light chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, or 58-61; the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; and the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 98, 103, or 158.

[0017] In other embodiments, the heavy chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157, the light chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 98, 103, or 158, the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57, and the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, or 58-61.

[0018] Additionally or alternatively, in some embodiments, the multispecific antibodies or antigen-binding fragments of the present technology also bind to T cells and / or CD3. In one aspect, the present disclosure provides T cells that have been armed ex vivo with a multispecific antibody or antigen-binding fragment of the present technology that also binds to T cells and / or CD3. In another aspect, the present disclosure provides an ex vivo method of generating therapeutic T cells, comprising arming T cells ex vivo with a multispecific antibody or antigen-binding fragment of the present technology that can bind to T cells and / or CD3, wherein the T cells are optionally human T cells, and the binding is non-covalent. In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of T cells that have been armed ex vivo with a multispecific antibody or antigen-binding fragment of the present technology that also binds to T cells and / or CD3.

[0019] In one aspect, the present disclosure provides a recombinant nucleic acid sequence encoding any of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments described herein. In another aspect, the present disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0020] In yet another aspect, the present disclosure provides pharmaceutical compositions comprising any of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments described herein and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. In some embodiments, the pharmaceutical composition further comprises an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0021] Additionally or alternatively, in some embodiments, the multispecific (e.g., bispecific) antibodies or antigen-binding fragments of the present technology bind to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in some embodiments, the second antigen-binding portion of the multispecific (e.g., bispecific) antibody or antigen-binding fragment binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.The small molecule DOTA hapten can be selected from the group consisting of DOTA, DOTA-Bn, DOTA-desferrioxamine, DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2, Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2, DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2, DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2, Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2, Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2, Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2, Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2, DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2, (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2, Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2, Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2, Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2, and Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

[0022] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments described herein, or any of the pharmaceutical compositions disclosed herein, wherein the multispecific (e.g., bispecific) antibody or antigen-binding fragment specifically binds to CLDN18.2. In some embodiments, the cancer is a solid tumor. Examples of cancer include, but are not limited to, gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. In some embodiments of the method, the multispecific (e.g., bispecific) antibody or antigen-binding fragment is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent. Examples of additional treatments include one or more of alkylating agents, platinum agents, taxanes, vincas, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapy, T cell, and immunomodulatory / stimulatory antibodies (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-TIM3 antibodies, anti-4-1BB antibodies, anti-CD73 antibodies, anti-GITR antibodies, or anti-LAG-3 antibodies).

[0023] In another aspect, the present disclosure provides a method for detecting cancer in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of a multispecific (e.g., bispecific) antibody or antigen-binding fragment of the present technology, wherein the multispecific (e.g., bispecific) antibody or antigen-binding fragment is configured to localize to cancer cells expressing CLDN18.2 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the multispecific (e.g., bispecific) antibody or antigen-binding fragment that is higher than a reference value. In certain embodiments, the cancer is a solid tumor. In some embodiments, the subject has been diagnosed with or is suspected of having cancer (e.g., gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer). The level of radioactivity emitted by the multispecific (e.g., bispecific) antibody or antigen-binding fragment can be detected using positron emission tomography or single-photon emission computed tomography. Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising a multispecific (e.g., bispecific) antibody or antigen-binding fragment of the present technology conjugated to a radionuclide.

[0024] In any and all embodiments of the methods disclosed herein, the subject is a human.

[0025] In yet another aspect, the present disclosure provides a method for detecting CLDN18.2 protein expression levels in a biological sample, the method comprising contacting the biological sample with any of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments disclosed herein and detecting binding to CLDN18.2 protein in the biological sample.

[0026] Also disclosed herein are kits for the detection and / or treatment of CLDN18.2-associated cancers, comprising at least one immunoglobulin-related composition of the present technology (e.g., any multispecific (e.g., bispecific) antibody or antigen-binding fragment described herein), or a functional variant (e.g., a substitution variant) thereof, and instructions for use. In certain embodiments, the immunoglobulin-related composition is conjugated to one or more detectable labels. In one embodiment, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-CLDN18.2 immunoglobulin-associated composition described herein. In some embodiments, the secondary antibody is conjugated to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.

[0027] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L (a) V H comprises any one of the amino acid sequences of SEQ ID NOs: 99 to 102, or SEQ ID NO: 157, and / or (b) V L In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 103 or SEQ ID NO: 158. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a heavy chain immunoglobulin variable domain (V) selected from the group consisting of SEQ ID NO: 101 and SEQ ID NO: 103, and SEQ ID NO: 157 and SEQ ID NO: 158, respectively. H ) and light chain immunoglobulin variable domains (V L ) amino acid sequence. Additionally or alternatively, in some embodiments, the anti-CD3 antibody or antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. Antigen-binding fragments include Fab, F(ab')2, Fab', scF v , and F v may be selected from the group consisting of:

[0028] Additionally or alternatively, in certain embodiments, the anti-CD3 antibody or antigen-binding fragment further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the anti-CD3 antibody further comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, and K322A. In other embodiments, the anti-CD3 antibody comprises an IgG4 constant region comprising an S228P mutation. Additionally or alternatively, in some embodiments, the anti-CD3 antibody lacks an α-1,6-fucose modification.

[0029] In one aspect, the disclosure provides a multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and wherein (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3, and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a light chain constant domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin. and (b) a heavy chain variable domain of a first immunoglobulin, wherein the light and heavy chain variable domains of a second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (b) the second polypeptide and the third peptide chain each comprise, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a heavy chain constant domain of the first immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 99-102, or 157, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin comprises SEQ ID NO: 103 or 158.

[0030] Additionally or alternatively, in some embodiments, the anti-CD3 multispecific antibody or antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in certain embodiments, the anti-CD3 multispecific antibody or antigen-binding fragment binds to CD3, GPA33, HER2 / neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr virus nuclear antigen) 1-6, LMP2, p53, lung resistance protein (LRP), Bcl-2, prostate-specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p(C SAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, PlGF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis Y (Le y) antigens, E-cadherin, V-cadherin, GPC3, EpCAM, CD4, CD8, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, CD56, DLL3, PD-1, PD-L1, CD28, CD137, CD99, GloboH, CD24, STEAP1, B7H3, polysialic acid, OX40, OX40-ligand, peptide-MHC complexes (with peptides derived from TP53, KRAS, MYC, EBNA1-6, PRAME, MART, tyronsinase, MAGEA1-A6, pmel17, LMP2, or WT1), or small molecule DOTA haptens.

[0031] In another aspect, the present disclosure provides a composition comprising any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0032] In yet another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein. In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0033] In one aspect, the present disclosure provides T cells that have been armed ex vivo with any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein. In another aspect, the present disclosure provides an ex vivo method of generating therapeutic T cells, the method comprising arming T cells ex vivo with any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein, wherein the T cells are optionally human T cells and the linkage is non-covalent. In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of T cells that have been armed ex vivo with any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein. [Brief explanation of the drawings]

[0034] [Figure 1] Splicing variants and schematic protein structure of claudin 18.2 are shown (adapted from Markov, A. Get al., IUBMB Life 67:29-35 (2015)). [Figure 2] The amino acid sequence alignment of hCLDN18.1-EL1 (SEQ ID NO: 1), hCLDN18.2-EL1 (SEQ ID NO: 2), and mCLDN18.2-EL1 (SEQ ID NO: 2) is shown, as well as the amino acid sequence alignment of hCLDN18.1-EL2 (SEQ ID NO: 3) and hCLDN18.2-EL2 (SEQ ID NO: 3). The amino acid sequences of hCLDN18.2-EL1 and mCLDN18.2-EL1 are identical. The amino acid sequences of hCLDN18.1-EL2 and hCLDN18.2-EL2 are identical. [Figure 3] CLDN18 RNA and protein expression in normal human tissues (adapted from Human Protein Atlas data: www.proteinatlas.org / ENSG00000066405-CLDN18 / tissue). [Figure 4]1 shows the expression of CLDN18 in human cancer tissues (adapted from Sahin U., et al., Clin Cancer Res 14:7624-7634 (2008)). [Figure 5] Cell lines (e.g., CHO, 3T3, and HEK293) stably expressing human CLDN18.2 are shown (analyzed using the benchmark antibody IMAB362, produced according to the sequence from imgt.org / 3Dstructure-DB / cgi / details.cgi?pdbcode=10473). [Figure 6] Virus-like particles (VLPs) expressing human CLDN18.2 EL1 are shown (analyzed using the benchmark antibody IMAB362, generated according to the sequence from imgt.org / 3Dstructure-DB / cgi / details.cgi?pdbcode=10473). Compared to the control (left), more than 90% of the purified VLPs (right) expressed hCLDN18.2 EL1. [Figure 7] Figure 1 shows the binding of five selected clones (32G4, 47D10, 29G4, 31A6, and 15B10) that showed specific binding to human CLDN18.2 as determined by FACS analysis. Upper panel: Binding of mouse chimeric antibody clones to cell surface-expressed CLDN18.1. Lower panel: Binding of mouse chimeric antibody clones to cell surface-expressed CLDN18.2. [Figure 8] 1 shows the binding affinities of murine anti-CLDN18.2 chimeric antibody clones 32G4, 47D10, 29G4, 31A6, and 15B10. [Figure 9A] 1 shows the binding affinity of exemplary humanized 32G4 antibody variants compared to the murine 32G4 chimeric control antibody. [Figure 9B] 1 shows the binding affinity of exemplary humanized 47D10 antibody variants compared to the murine 47D10 chimeric control antibody. [Figure 10] The amino acid sequence of human CLDN18.2 protein (SEQ ID NO: 4) is shown. [Figure 11] The amino acid sequence of human CLDN18.1 protein (SEQ ID NO: 5) is shown. [Figure 12] The VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences are shown for murine clones 32G4 (SEQ ID NOS: 6-11, respectively), 47D10 (SEQ ID NOS: 12-17, respectively), 29G4 (SEQ ID NOS: 18-23, respectively), 31A6 (SEQ ID NOS: 24-29, respectively), and 15B10 (SEQ ID NOS: 30-35, respectively). SEQ ID NO: 155 corresponds to the 32G4-huVL4 CDR2 sequence, and SEQ ID NO: 156 corresponds to the 47D10-huVL4 CDR2 sequence. [Figure 13] The amino acid sequences of the variable heavy immunoglobulin domain (VH) and variable light immunoglobulin domain (VL) are shown for murine clones 32G4 (SEQ ID NO:36 and SEQ ID NO:37, respectively), 47D10 (SEQ ID NO:38 and SEQ ID NO:39, respectively), 29G4 (SEQ ID NO:40 and SEQ ID NO:41, respectively), 31A6 (SEQ ID NO:42 and SEQ ID NO:43, respectively), and 15B10 (SEQ ID NO:44 and SEQ ID NO:45, respectively). The VH CDR1-3 and VL CDR1-3 amino acid sequences are underlined. [Figure 14] The amino acid sequences of four humanized VH variants (SEQ ID NOs: 46 to 49) and four humanized VL variants (SEQ ID NOs: 50 to 53) in clone 32G4 are shown. The amino acid sequences of VH CDR1 to 3 and VL CDR1 to 3 are underlined. [Figure 15] The amino acid sequences of four humanized VH variants (SEQ ID NOs: 54 to 57) and four humanized VL variants (SEQ ID NOs: 58 to 61) in clone 47D10 are shown. The amino acid sequences of VH CDR1 to 3 and VL CDR1 to 3 are underlined. [Figure 16] The heavy chain (HC) and light chain (LC) amino acid sequences of 32G4-huIgG1-V8 (SEQ ID NO: 62 and SEQ ID NO: 63) and 32G4-huIgG1-V9 (SEQ ID NO: 64 and SEQ ID NO: 65) are shown. The VHCDR1-3 and VLCDR1-3 amino acid sequences are underlined, and the VH and VL amino acid sequences are all in italics. [Figure 17]The heavy chain (HC) and light chain (LC) amino acid sequences of 47D10-huIgG1-V6 (SEQ ID NO: 66 and SEQ ID NO: 67) and 47D10-huIgG1-V7 (SEQ ID NO: 68 and SEQ ID NO: 69) are shown. The VHCDR1-3 and VLCDR1-3 amino acid sequences are underlined, and the VH and VL amino acid sequences are all in italics. [Figure 18] 1 shows exemplary antibody-dependent cellular cytotoxicity (ADCC) assay data for the 32G4 and 47D10 clones compared to the IMAB362 benchmark antibody and a negative isotype control. [Figure 19] 1 shows the cross-linking of exemplary humanized 32G4 and 47D10 antibody variants to the cynomolgus monkey and mouse claudin 18.2 target protein on the cell surface compared to the IMAB362 benchmark antibody and a negative isotype control. [Figure 20]

[0033] Figure 1 shows exemplary heavy chain (HC) and light chain (LC) amino acid sequences of the 32G4-huIgG1-V8xOKT3 (anti-CLDN18.2xCD3) bispecific antibody (BsAb) (SEQ ID NO: 81 and SEQ ID NO: 82) and the 32G4-huIgG1-V9xOKT3 (anti-CLDN18.2xCD3) BsAb (SEQ ID NO: 83 and SEQ ID NO: 84). The VHCDR1-3 and VLCDR1-3 amino acid sequences of the anti-CLDN18.2 immunoglobulin are underlined, all linkers are in bold, and the VH and VL amino acid sequences of the BsAb are all in italics. [Figure 21]

[0033] Figure 1 shows exemplary heavy chain (HC) and light chain (LC) amino acid sequences of the 47D10-huIgG1-V6xOKT3 (anti-CLDN18.2xCD3) bispecific antibody (BsAb) (SEQ ID NO:85 and SEQ ID NO:86) and the 47D10-huIgG1-V7xOKT3 BsAb (anti-CLDN18.2xCD3) (SEQ ID NO:87 and SEQ ID NO:88). The VHCDR1-3 and VLCDR1-3 amino acid sequences of the anti-CLDN18.2 immunoglobulin are underlined, all linkers are in bold, and the VH and VL amino acid sequences of the BsAbs are all in italics. [Figure 22]

[0033] Figure 1 shows exemplary heavy chain (HC) and light chain (LC) amino acid sequences of the 32G4-huIgG1-V8xhuSP34 (anti-CLDN18.2xCD3) bispecific antibody (BsAb) (SEQ ID NO:89 and SEQ ID NO:90), and the 32G4-huIgG1-V9xhuSP34 BsAb (anti-CLDN18.2xCD3) (SEQ ID NO:91 and SEQ ID NO:92). The VH and VL chain amino acid sequences of the anti-CLDN18.2 immunoglobulin are underlined, all linkers are in bold, and the VH and VL amino acid sequences of the BsAb are all in italics. [Figure 23]

[0033] Figure 1 shows exemplary heavy chain (HC) and light chain (LC) amino acid sequences of the 47D10-huIgG1-V6xhuSP34 (anti-CLDN18.2xCD3) bispecific antibody (BsAb) (SEQ ID NO:93 and SEQ ID NO:94) and the 47D10-huIgG1-V7xhuSP34 BsAb (anti-CLDN18.2xCD3) (SEQ ID NO:95 and SEQ ID NO:96). The VH and VL chain amino acid sequences of the anti-CLDN18.2 immunoglobulin are underlined, all linkers are in bold, and the VH and VL amino acid sequences of the BsAbs are all in italics. [Figure 24] 1 shows exemplary gastric cancer cell killing (TDCC) assay data for 32G4 anti-CD3 and 47D10 anti-CD3 bispecific antibody variants compared to IMAB362 anti-CD3 benchmark antibody and negative isotype control. [Figure 25] The VH and VL amino acid sequences of the anti-CD3 OKT3 antibody (SEQ ID NO: 97 and SEQ ID NO: 98), the VH amino acid sequences of humanized SP34 VH1 to 5 (SEQ ID NOs: 99 to 102 and 157), and the VL amino acid sequence of humanized SP34 VL (SEQ ID NOs: 103 and 158) are shown. [Figure 26]

[0033] Figure 1 shows exemplary heavy chain (HC) and light chain (LC) amino acid sequences of 32G4-V8xhuSP34-v5 (anti-CLDN18.2xCD3) bispecific antibody (BsAb) (SEQ ID NO:159 and SEQ ID NO:160) and 47D10-V7xhuSP34-v5 BsAb (anti-CLDN18.2xCD3) (SEQ ID NO:161 and SEQ ID NO:162). The VH and VL chain amino acid sequences of the anti-CLDN18.2 immunoglobulin are underlined, all linkers are in bold, and the VH and VL amino acid sequences of the BsAbs are all in italics. [Figure 27] 1 shows exemplary in vivo efficacy of 32G4-V8×huSP34-v5 in a mouse xenograft gastric cancer model. [Figure 28] 1 shows exemplary stability of 32G4-V8×huSP34-v5 under accelerated stress test conditions as assessed by SEC-HPLC. [Figure 29] 1 shows exemplary gastric cancer cell killing (TDCC) assay data for 32G4-V8×huSP34-v5 under accelerated stress test conditions. DETAILED DESCRIPTION OF THE INVENTION

[0035] It should be understood that certain aspects, modes, embodiments, variations and features of the present methods are described below at varying levels of detail in order to provide a substantial understanding of the present technology.

[0036] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) capable of specifically binding to claudin 18.2 polypeptides. The immunoglobulin-related compositions of the present technology are useful in methods for detecting or treating claudin 18.2-associated cancer in a subject in need thereof. Accordingly, various aspects of the present methods relate to the preparation, characterization, and manipulation of anti-claudin 18.2 antibodies. The immunoglobulin-related compositions of the present technology are useful, alone or in combination with additional therapeutic agents, to treat cancer. In some embodiments, the immunoglobulin-related composition is a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody.

[0037] In carrying out the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, series Methods in Enzymology (Academic Press, Inc., NY), MacPherson et al. (1991) PCR 1:A Practical Approach (IRL Press at Oxford University Press), MacPherson et al. al.(1995) PCR 2:A Practical Approach, Harlow and Lane eds.(1999)Antibodies,A Laboratory Manual;Freshney(2005)Culture of Animal Cells:A Manual of Basic Technique,5th edition, Gait ed.(1984)Oligonucleotide Synthesis, U.S. Patent No. 4,683,195, Hames and Higgins eds.(1984)Nucleic Acid Hybridization, Anderson (1999) Nucleic Acid Hybridization, Hames and Higgins eds. (1984) Transcription and Translation, Immobilized Cells and Enzymes (IRL Press (1986)), Perbal (1984) A Practical Guide to Molecular Cloning, Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory), Makrides ed. (2003) Gene Transfer and Expression See, for example, "Immunochemical Methods in Mammalian Cells," Mayer and Walker eds. (1987) "Immunochemical Methods in Cell and Molecular Biology" (Academic Press, London), and Herzenberg et al. eds. (1996) "Weir's Handbook of Experimental Immunology." Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, "Human Molecular Genetics," Second Edition (John Wiley and Sons, Inc., NY, 1999)). definition

[0038] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, etc. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry, and hybridization described below, are well known and commonly used in the art.

[0039] As used herein, the term "about" in reference to a number is generally interpreted as including numbers that fall within 1%, 5%, or 10% in either direction (greater or lesser) of the number (except where such number is less than 0% or greater than 100% of the possible value), unless otherwise stated or clear from the context.

[0040] As used herein, "administration" of an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be performed by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self-administration and administration by another.

[0041] "Adjuvant" refers to one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants can be administered to a subject before, during, or after administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and costimulatory factors.

[0042] As used herein, the term "antibody" refers collectively to immunoglobulin or immunoglobulin-like molecules, including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate, e.g., mammals such as humans, goats, rabbits, and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, "antibody" (including intact immunoglobulins) and "antigen-binding fragments" specifically bind to a molecule of interest (or a group of closely related molecules of interest) to the substantial exclusion of binding to other molecules (e.g., with a binding constant at least 10 times higher than that for other molecules in a biological sample). 3 M -1 Large, at least 10 4 M -1 Greater than or at least 10 5 M -1Antibodies and antibody fragments with large binding constants for molecules of interest. The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (bispecific antibodies, etc.). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.), Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.

[0043] More specifically, an antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody consists of a heavy chain and a light chain, each of which has a variable heavy (V H ) area and variable light (V L ) region. Together, they form the V H Area and V LThe regions involved in binding to the antigen recognized by the antibody are typically heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to antigens. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, the combined framework regions of the constituent light and heavy chains, primarily adopt a β-sheet conformation, with the CDRs forming loops that connect to, and in some cases form part of, the β-sheet structure. Thus, the framework regions function to form a scaffold that provides for the correct orientation of the CDRs through non-covalent interactions between the chains.

[0044] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are usually designated CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are usually identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to claudin 18.2 protein have a specific VH Area and V L Each CDR has a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that differ between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs). As used herein, "immunoglobulin-related composition" refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or an antigen-binding fragment thereof specifically binds to an antigen.

[0045] As used herein, the term "antibody-related polypeptide" refers to antigen-binding antibody fragments, including single-chain antibodies, which may include a variable region alone or in combination with all or part of the polypeptide elements of an antibody molecule: hinge region, CH1, CH2, and CH3 domains. The present technology also includes any combination of a variable region with a hinge region, CH1, CH2, and CH3 domains. Antibody-related molecules useful in the present methods include, for example, Fab, Fab', and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Examples include, but are not limited to, fragments containing any of the V domains. L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H"antibody fragments" or "antigen-binding fragments" may comprise a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0046] "Bispecific antibody" or "BsAb," as used herein, refers to an antibody that can simultaneously bind to two targets with different structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion in a bispecific antibody is a V H and / or V L In some such embodiments, V H and / or V L The V region is one found in a particular monoclonal antibody. In some embodiments, a bispecific antibody comprises two antigen-binding portions, each of which is a V region from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody comprises two antigen-binding moieties, one of which comprises a V region containing the CDRs from a first monoclonal antibody. H and / or V L The other antigen-binding portion comprises an immunoglobulin molecule having a V region containing CDRs from a second monoclonal antibody. H and / or V L These include antibody fragments having regions (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.).

[0047] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a mechanism of cell-mediated immune defense in which effector cells of the immune system actively lyse target cells, such as tumor cells, whose membrane surface antigens have been bound by an antibody, such as an anti-CLDN18.2 antibody.

[0048] As used herein, "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., a CLDN18.2 polypeptide). The antigen can also be administered to an animal to generate an immune response in the animal.

[0049] The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains the portion of the polypeptide that is involved in binding to the antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2. Any of the above antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as intact antibodies.

[0050] As used herein, "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, while high affinity complexes generally contain antibodies that tend to remain bound to the antigen for longer periods of time.

[0051] As used herein, the term "biological sample" refers to a sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsils, thymus, blood, hair, cheek, skin, serum, plasma, CSF, semen, prostatic fluid, seminal plasma, urine, feces, sweat, saliva, sputum, mucous membranes, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or cancers. Biological samples can be obtained from subjects for diagnostic or research purposes, or can be obtained from non-diseased individuals as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0052] As used herein, the term "CDR grafting" means replacing at least one CDR of an "acceptor" antibody with a CDR "graft" from a "donor" antibody having the desired antigen specificity.

[0053] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a murine Fc constant region) has been replaced, using recombinant DNA techniques, with the Fc constant region from an antibody of another species (e.g., a human Fc constant region). In general, Robinson et al., PCT / US86 / 02269, Akira et al., European Patent Application No. 184,187, Taniguchi, European Patent Application No. 171,496, Morrison et al., European Patent Application No. 173,494, Neuberger et al., WO86 / 01533, Cabilly et al. al. U.S. Patent Application No. 4,816,567, Cabilly et al., European Patent Application No. 0125,023, Better et al., Science 240:1041-1043, 1988, Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987, Liu et al., J. Immunol 139:3521-3526,1987, Sun et al. See, e.g., Nishimura et al., Proc. Natl. Acad. Sci. USA 84:214-218, 1987; Nishimura et al., Cancer Res 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559, 1988.

[0054] As used herein, the term "complement-dependent cytotoxicity" or "CDC" generally refers to the effector function of IgG and IgM antibodies that, when bound to surface antigens, trigger the classical complement pathway, inducing membrane attack complex formation and target cell lysis.

[0055] As used herein, the term "consensus FR" refers to the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0056] As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent in treating a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive the treatment or receives a placebo) are typically used.

[0057] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are separated by a light chain variable domain (V) within the same polypeptide chain. L ) connected to the heavy chain variable domain (V H ) including (V H V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al., Proc Natl Acad Sci USA, 90:6444-6448 (1993).

[0058] As used herein, the term "EC50," also known as the half maximal effective concentration, refers to the concentration of antibody that induces a response halfway between baseline and maximum after a specific exposure time.

[0059] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein, or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics such as general health, age, sex, weight, and tolerance to drugs. One of skill in the art will be able to determine the appropriate dosage depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, a therapeutic composition can be administered to a subject with one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount can be administered in one or more administrations.

[0060] As used herein, the term "effector cell" refers to an immune cell involved in the effector phase of an immune response, as opposed to the recognition and activation phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., neutrophils capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR are involved in the specific killing of target cells and present antigens to other components of the immune system or bind to cells that present antigens.

[0061] As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished by the fact that the binding to the former is lost in the presence of denaturing solvents, but not the latter. In some embodiments, the "epitope" of a CLDN18.2 protein is a region of the protein that an anti-CLDN18.2 antibody of the present technology specifically binds to. In some embodiments, the epitope is a conformational or nonconformational epitope. To screen for anti-CLDN18.2 antibodies that bind to an epitope, a routine cross-blocking assay, such as that described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine whether an anti-CLDN18.2 antibody binds to the same site or epitope as an anti-CLDN18.2 antibody of the present technology. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In a different method, peptides corresponding to different regions of the CLDN18.2 protein can be used in competition assays with a test antibody or with an antibody having a characterized or known epitope.

[0062] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA, splicing and other processing of the precursor mRNA to produce a mature mRNA, mRNA stability, translation of the mature mRNA into a protein (including codon usage and tRNA availability), and glycosylation and / or other modifications of the translation product if required for proper expression and function.

[0063] As used herein, the term "gene" means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0064] As used herein, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which can 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. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a particular percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for the alignment. One alignment program is BLAST, using default parameters. In particular, the programs BLASTN and BLASTP use the following default parameters: Genetic code=standard, filter=none, strand=both, cutoff=60, expect=10, Matrix=BLOSUM62, Descriptions=50 sequences, sort by -HIGH SCORE, Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percent homology and encode polypeptides having the same or similar biological activity.Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity, or less than 25% identity with each other.

[0065] As used herein, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Most frequently, humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are of human immunoglobulin consensus FR sequences, although the FR regions may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FRs will typically be no more than six in the H chain and no more than three in the L chain. The humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986), Reichmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0066] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally consist of amino acid sequences from the "complementarity determining regions" or "CDRs" (e.g., V L Around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V H Around 31-35B (H1), 50-65 (H2), and 95-102 (H3) in (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or those residues from the "hypervariable loops" (e.g., V L Residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) in V H These include 26-32 (H1), 52A-55 (H2), and 96-101 (H3) in (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0067] As used herein, the terms "identical" or percent "identity," when used in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a designated region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison width or designated region as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then said to be "substantially identical." The term also refers to or can be applied to the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0068] As used herein, "immunogen" refers to any antigen that is capable of inducing a humoral and / or cell-mediated immune response without immune tolerance.

[0069] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (herein abbreviated as LCVR or V LThe light chain constant region consists of one domain, C L It consists of V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0070] As used herein, the terms "individual," "patient," or "subject" can be an individual organism, vertebrate, mammal, or human. In some embodiments, the individual, patient, or subject is a human.

[0071] As used herein, the term "linker" refers to a functional group (e.g., a chemical or polypeptide) that covalently joins two or more polypeptides or nucleic acids such that they are linked together. As used herein, a "peptide linker" refers to a peptide linker that is used to link two proteins together (e.g., V H and V L A linker refers to one or more amino acids used to link (connect) domains. In certain embodiments, a linker comprises amino acids having the sequence GGGGSGGGSGGGGGS (SEQ ID NO: 79) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 80).

[0072] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, but is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0073] As used herein, the term "nucleic acid" or "polynucleotide" refers to any RNA or DNA, which may be unmodified or modified. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons.

[0074] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0075] As used herein, the term "polyclonal antibody" refers to an antibody preparation derived from at least two different antibody-producing cell lines. Use of this term includes at least two antibody preparations that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0076] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and longer chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as in a voluminous research literature.

[0077] As used herein, the term "recombinant," when used in reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0078] As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients by different routes, simultaneously or substantially simultaneously.

[0079] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration routes are the same or different. More specifically, sequential use refers to the total administration of one active ingredient followed by the other. Thus, one active ingredient can be administered over several minutes, hours, or days, followed by the administration of the other active ingredient. In this example, there is no simultaneous treatment.

[0080] As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route, at the same time, or substantially the same time.

[0081] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to a single chain antibody comprising two domains of an Fv fragment, V L and V H A single-chain antibody molecule may comprise a polymer having several individual molecules, for example, a dimer, trimer, or other polymer. v The two domains of the fragment are V L and V H are encoded by separate genes, but they can be synthesized using recombinant methods. L and V H The domains can be joined by a synthetic linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule (single-chain F v (scF v ) Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc Natl Acad Sci 85:5879-5883. Such single chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0082] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind to other molecules. The terms "specific binding," "specifically binds to," or "is specific for" a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, refer to, for example, a specific binding of about 10% to the molecule to which it binds. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10-10 M, 10 -11 M or 10 -12 K of M D The term "specifically binds" can refer to binding of a molecule (e.g., an antibody or antigen-binding fragment thereof) to a particular polypeptide (e.g., a CLDN18.2 polypeptide), or to an epitope on a particular polypeptide, without substantially binding to any other polypeptides or polypeptide epitopes.

[0083] As used herein, "sequence propensity" refers to any characteristic of a nucleic acid or amino acid sequence that can affect the heterogeneity of the immunoglobulin-related compositions of the present disclosure. Such sequence propensities include, but are not limited to, any sequence motifs that are prone to deamidation, isomerization, cleavage, oxidation, and glycosylation.

[0084] As used herein, the terms "subject," "patient," or "individual" can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient, or individual is a human.

[0085] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect in a subject in need thereof.

[0086] As used herein, "treating" or "treatment" encompasses the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., inhibiting its onset, (ii) alleviating the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that symptoms associated with the disease are, for example, alleviated, reduced, cured, or put into remission.

[0087] It should also be understood that the various modes of treatment for disorders described herein include total treatment, but are intended to mean "substantially" less than total treatment, where some biologically or medically relevant result is achieved. Treatment can be continuous long-term treatment for chronic illnesses, or single or several administrations for treatment of acute conditions.

[0088] Amino acid sequence modifications of the anti-CLDN18.2 antibodies described herein are contemplated. Such modifications may be made to improve the binding affinity and / or other biological properties of the antibody, for example, to glycosylate encoded amino acids, to disrupt the antibody's ability to bind to C1q, Fc receptors, or to activate the complement system. Amino acid sequence variants of anti-CLDN18.2 antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, by peptide synthesis, or by chemical modification. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the desired antibody, as long as the resulting antibody possesses the desired properties. Modifications also include altering the glycosylation pattern of the protein. Sites of greatest interest for substitutional mutagenesis include hypervariable regions, although FR alterations are also contemplated.

[0089] Conservative amino acid substitutions are those that change a given amino acid for another amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, and size). "Conservative substitutions" are shown in the table below. [Table 1-1] [Table 1-2]

[0090] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution using the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein, and antibodies with similar or superior properties in one or more relevant assays can be selected for further development.

[0091] Claudine In humans, 27 claudin family members have been reported, including claudin 18. All claudins have four transmembrane domains and two extracellular loops, with N- and C-termini in the cytoplasm (Markov, A.G., et al., IUBMB Life 67:29-35 (2015); Furuse, M., et al., J Cell Biol 141:1539-1550 (1998); Turksen, K., and Troy, T.C., Biochim Biophys Acta 1816:73-79 (2011)).

[0092] The claudin family 18 gene consists of five exons. Two splicing variants, claudin 18.1 (CLDN18.1) and claudin 18.2 (CLDN18.2), exist. These variants are the product of alternative splicing that utilizes alternative DNA sequences in exon 1, which encodes the N-terminal portion of the protein, including the first extracellular loop (EL1) (Figure 1) (Mineta, K., et al., FEBS Lett 585:606-612 (2011); Suzuki, H., et al., Science 344:304-307 (2014)). CLDN18.1 and CLDN18.2 have different EL1 sequences but share the same EL2 sequence (Figure 2). CLDN18.2 is highly homologous among species, including humans, cynomolgus monkeys, and mice, all of which share the same EL1 amino acid sequence.

[0093] Expression of claudin 18 in normal human tissues is highly restricted, with CLDN18.1 found primarily in the lung and CLDN18.2 in the stomach (Figure 3) (see Sahin U., et al., Clin Cancer Res 14:7624-7634 (2008)). Cancerous expression of CLDN18.2 has been reported in gastric cancer, pancreatic cancer, and other cancers (Figure 4) (Sahin U., et al., Clin Cancer Res 14:7624-7634 (2008); Karanjawala, Z. E., et al., Am J Surg Pathol 32:188-196 (2008)). One study reported that 70% of gastric cancers, 50% of pancreatic cancers, 30% of esophageal cancers, and 25% of NSCLCs express CLDN18.2 (Sahin U., et al., Clin Cancer Res 14:7624-7634(2008)). CLDN18.2 is considered a specific gastric tumor-associated antigen (TAA).

[0094] The malignancy-associated expression of CLDN18.2 and its tissue-restricted expression make it an ideal target for antibody-based therapy (Sahin U., et al., Clin Cancer Res 14:7624-7634(2008)). Although there is no open access to normal tight junctions that form CLDN18.2 in the gastric mucosa, CLDN18.2 epitopes are exposed on the cell surface during malignant transformation, thereby making them accessible to therapeutic antibodies.

[0095] Immunoglobulin-related compositions of the present technology The present technology describes methods and compositions for the production and use of anti-CLDN18.2 immunoglobulin-related compositions (e.g., anti-CLDN18.2 antibodies or antigen-binding fragments thereof). The antibodies and antigen-binding fragments of the present technology selectively bind to CLDN18.2 polypeptide (FIG. 10) instead of CLDN18.1 polypeptide (FIG. 11). The anti-CLDN18.2 immunoglobulin-related compositions of the present disclosure may be useful for the diagnosis or treatment of CLDN18.2-associated cancers. Anti-CLDN18.2 immunoglobulin-related compositions within the scope of the present technology include, but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies, and diabodies that specifically bind to a target polypeptide, homolog, derivative, or fragment thereof. The present disclosure also provides antigen-binding fragments of any of the anti-CLDN18.2 antibodies disclosed herein, including Fab, F(ab)'2, Fab', scF, and the like. v , and F v The amino acid sequences of the anti-CLDN18.2 immunoglobulin-related compositions of the present technology are set forth in Figures 12-17 and 20-23.

[0096] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L (a) V H is selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, and 30 H- a CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 7, 13, 19, 25, and 31 H - CDR2 sequence and V selected from the group consisting of SEQ ID NOs: 8, 14, 20, 26, and 32 H and / or (b) V L is selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, and 33 L CDR1 and a V selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 155, and 156 L - a CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, and 35 L -CDR3 sequence.

[0097] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L (a) V H V of SEQ ID NO: 6 H - CDR1 sequence and V of SEQ ID NO: 7 H - CDR2 sequence and V of SEQ ID NO: 8 H and / or V L V of SEQ ID NO: 9 L - CDR1 sequence and V of SEQ ID NO: 10 or SEQ ID NO: 155 L - CDR2 sequence and V of SEQ ID NO: 11 L or (b) V H V of SEQ ID NO: 12 H - CDR1 sequence and V of SEQ ID NO: 13 H - CDR2 sequence and V of SEQ ID NO: 14 H and / or V L V of SEQ ID NO: 15 L - CDR1 sequence and V of SEQ ID NO: 16 or SEQ ID NO: 156 L - CDR2 sequence and V of SEQ ID NO: 17 L -CDR3 sequence, or (c) V H V of SEQ ID NO: 18 H - CDR1 sequence and V of SEQ ID NO: 19 H- CDR2 sequence and V of SEQ ID NO: 20 H and / or V L V of SEQ ID NO: 21 L - CDR1 sequence and V of SEQ ID NO: 22 L - CDR2 sequence and V of SEQ ID NO: 23 L -CDR3 sequence, or (d) V H V of SEQ ID NO: 24 H - CDR1 sequence and V of SEQ ID NO: 25 H - CDR2 sequence and V of SEQ ID NO: 26 H - CDR3 sequence and / or V L V of SEQ ID NO: 27 L - CDR1 sequence and V of SEQ ID NO: 28 L - CDR2 sequence and V of SEQ ID NO: 29 L -CDR3 sequence, or (e) V H V of SEQ ID NO: 30 H - CDR1 sequence and V of SEQ ID NO: 31 H - CDR2 sequence and V of SEQ ID NO: 32 H and / or V L is V of SEQ ID NO: 33 L - CDR1 sequence and V of SEQ ID NO: 34 L - CDR2 sequence and V of SEQ ID NO: 35 L -CDR3 sequence.

[0098] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L (a) V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57, and / or (b) V L comprises an amino acid sequence selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, and 58-61.

[0099] In any of the above embodiments, the antibody further comprises an Fc domain of any isotype, for example, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. Non-limiting examples of constant region sequences include the following:

[0100] Human IgD constant region, Uniprot:P01880 (SEQ ID NO: 70) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0101] Human IgG1 constant region, Uniprot:P01857 (SEQ ID NO:71) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0102] Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO:72) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0103] Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO:73) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0104] Human IgM constant region, Uniprot:P01871 (SEQ ID NO:74) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0105] Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO:75) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0106] Human IgA1 constant region, Uniprot:P01876 (SEQ ID NO:76) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0107] Human IgA2 constant region, Uniprot:P01877 (SEQ ID NO:77) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0108] Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 78) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0109] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NOs: 70-77. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 78.

[0110] Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment binds to the first extracellular loop of a CLDN18.2 polypeptide. In some embodiments, the CLDN18.2 polypeptide has the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the first extracellular loop comprises the amino acid sequence of SEQ ID NO: 2 (see Figure 2). In certain embodiments, the epitope is a conformational epitope or a non-conformational epitope.

[0111] In one aspect, the present disclosure provides an antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:159, SEQ ID NO:161, or a variant thereof with one or more conservative amino acid substitutions. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:160, SEQ ID NO:162, or a variant thereof with one or more conservative amino acid substitutions. In some embodiments, the immunoglobulin-related compositions of the present technology comprise an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:81 and SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:159 and SEQ ID NO:160, and SEQ ID NO:161 and SEQ ID NO:162, respectively.

[0112] In any of the above embodiments of the immunoglobulin-related composition, the HC and LC immunoglobulin variable domain sequences form an antigen-binding site that binds to the first extracellular loop of the CLDN18.2 polypeptide. In certain embodiments, the first extracellular loop comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the epitope is a conformational epitope or a nonconformational epitope.

[0113] In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0114] In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one CLDN18.2 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one CLDN18.2 polypeptide at a concentration of about 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Dissociation constant of M (K D ) In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0115] In certain embodiments, the immunoglobulin-related compositions comprise one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, or 58-61, and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the heavy chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a "conservative substitution," as defined herein.

[0116] In another aspect, the disclosure provides a multispecific (e.g., bispecific) antibody comprising: (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the LC sequence present in SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:160, or SEQ ID NO:162; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the HC sequence present in SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:159, or SEQ ID NO:161.

[0117] In certain embodiments, the immunoglobulin-related composition contains an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A. Additionally, or alternatively, in some embodiments, the immunoglobulin-related composition contains an IgG4 constant region comprising an S228P mutation.

[0118] In one aspect, the disclosure provides a multispecific (e.g., bispecific) antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and wherein (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3, and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of a second immunoglobulin or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, and (b) a light chain variable domain or a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a second epitope, linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (b) each of the second polypeptide and the third peptide chain comprises, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope. and a heavy chain constant domain of an immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, or 58-61.

[0119] Additionally or alternatively, in some embodiments, the heavy chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57; the light chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, or 58-61; the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; and the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 98, 103, or 158.

[0120] In other embodiments, the heavy chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157, the light chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 98, 103, or 158, the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57, and the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50-53, or 58-61.

[0121] In some embodiments, the anti-CLDN18.2 immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some embodiments, the anti-CLDN18.2 immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some embodiments, Fab fragments are used to promote rapid binding and cellular uptake and / or slow release. In some embodiments, F(ab)'2 fragments are used to promote rapid binding and cellular uptake and / or slow release.

[0122] In one aspect, the present technology provides nucleic acid sequences encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein.

[0123] In another aspect, the present technology provides host cells that express any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0124] The immunoglobulin-related compositions (e.g., anti-CLDN18.2 antibodies) of the present technology can be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of one or more CLDN18.2 polypeptides, or can be specific for both CLDN18.2 polypeptides and heterologous compositions such as heterologous polypeptides or solid supports. See, for example, WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al. See Kostelny et al., J. Immunol. 148:1547-1553 (1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized.

[0125] The immunoglobulin-related compositions of the present technology can also be recombinantly fused to heterologous polypeptides at the N- or C-terminus, or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugation). For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules useful as labels in detection assays, and effector molecules such as heterologous polypeptides, drugs, or toxins. See, for example, WO92 / 08495, WO91 / 14438, WO89 / 12624, U.S. Patent No. 5,314,995, and EP0396387.

[0126] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L (a) V H comprises any one of the amino acid sequences of SEQ ID NOs: 99 to 102, or SEQ ID NO: 157, and / or (b) V L In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 103 or SEQ ID NO: 158. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a heavy chain immunoglobulin variable domain (V) selected from the group consisting of SEQ ID NO: 101 and SEQ ID NO: 103, and SEQ ID NO: 157 and SEQ ID NO: 158, respectively. H ) and light chain immunoglobulin variable domains (V L ) amino acid sequence. Additionally or alternatively, in some embodiments, the anti-CD3 antibody or antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. Antigen-binding fragments include Fab, F(ab')2, Fab', scF v , and F v may be selected from the group consisting of:

[0127] Additionally or alternatively, in certain embodiments, the anti-CD3 antibody or antigen-binding fragment further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the anti-CD3 antibody further comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, and K322A. In other embodiments, the anti-CD3 antibody comprises an IgG4 constant region comprising an S228P mutation. Additionally or alternatively, in some embodiments, the anti-CD3 antibody lacks an α-1,6-fucose modification.

[0128] In one aspect, the disclosure provides a multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and wherein (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3, and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a light chain constant domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin. and (b) a heavy chain variable domain of a first immunoglobulin, wherein the light and heavy chain variable domains of a second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (b) the second polypeptide and the third peptide chain each comprise, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a heavy chain constant domain of the first immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 99-102, or 157, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin comprises SEQ ID NO: 103 or 158.

[0129] Additionally or alternatively, in some embodiments, the anti-CD3 multispecific antibody or antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in certain embodiments, the anti-CD3 multispecific antibody or antigen-binding fragment binds to CD3, GPA33, HER2 / neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr virus nuclear antigen) 1-6, LMP2, p53, lung resistance protein (LRP), Bcl-2, prostate-specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p(C SAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, PlGF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis Y (Le y) antigens, E-cadherin, V-cadherin, GPC3, EpCAM, CD4, CD8, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, CD56, DLL3, PD-1, PD-L1, CD28, CD137, CD99, GloboH, CD24, STEAP1, B7H3, polysialic acid, OX40, OX40-ligand, peptide-MHC complexes (with peptides derived from TP53, KRAS, MYC, EBNA1-6, PRAME, MART, tyronsinase, MAGEA1-A6, pmel17, LMP2, or WT1), or small molecule DOTA haptens.

[0130] In another aspect, the present disclosure provides a composition comprising any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0131] In yet another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein. In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising any and all embodiments of the anti-CD3 antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, dye, chromagen, imaging agent, drug, toxin, cytokine, enzyme, enzyme inhibitor, hormone, hormone antagonist, growth factor, radionuclide, metal, liposome, nanoparticle, RNA, DNA, or any combination thereof.

[0132] A. Methods of Preparing Anti-CLDN18.2 Antibodies of the Present Technology General Overview. First, a target polypeptide is selected against which antibodies of the present technology can be raised. For example, antibodies can be raised against the full-length CLDN18.2 protein or a portion of the first extracellular loop of the CLDN18.2 protein. Techniques for generating antibodies directed against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, those involving display libraries, xeno- or human-mice, hybridomas, etc. Target polypeptides within the scope of the present technology include any polypeptide derived from the CLDN18.2 protein containing the first extracellular loop that can elicit an immune response.

[0133] It is understood that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, directed against the CLDN18.2 protein and fragments thereof are suitable for use in accordance with the present disclosure.

[0134] Anti-CLDN18.2 antibodies that can be subjected to the techniques described herein include monoclonal and polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods useful for the high-yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab')2 antibody fragments, have been described. See U.S. Patent No. 5,648,237.

[0135] Generally, antibodies are obtained from a starting species. More specifically, the nucleic acid or amino acid sequences of the variable portions of the light chain, heavy chain, or both, of an antibody of the starting species having specificity for a target polypeptide antigen are obtained. The starting species can be any species that has been useful for generating antibodies or antibody libraries of the present technology (e.g., rat, mouse, rabbit, chicken, monkey, human, etc.).

[0136] Phage or phagemid display technology is a useful technique for inducing the antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of the sequence encoding the antibody of the present technology can be carried out in E. coli.

[0137] Due to the degeneracy of nucleic acid coding sequences, other sequences encoding substantially the same amino acid sequence as that of a naturally occurring protein can be used in the practice of this technology. These include, but are not limited to, nucleic acid sequences comprising all or part of the nucleic acid sequences encoding the above polypeptides, which are altered by substitution of different codons encoding functionally equivalent amino acid residues within the sequence, thus resulting in silent changes. It will be understood that the nucleotide sequences of immunoglobulins produced by this technology can tolerate up to 25% sequence homology variation, as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), as long as such variants form operational antibodies that recognize the CLDN18.2 protein. For example, one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of a similar polarity that functions as a functional equivalent, resulting in a silent change. Substitutions for amino acids within the sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins, or fragments or derivatives thereof, that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, binding to antibody molecules or other cellular ligands, etc.Additionally, immunoglobulin-encoding nucleic acid sequences can be mutated in vitro or in vivo to create and / or destroy translation, initiation, and / or termination sequences, or to create mutations in the coding region and / or create new restriction endonuclease sites or destroy existing ones to further facilitate in vitro modification. Any technique for mutagenesis known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, Tab linkers (Pharmacia), etc.

[0138] Preparation of Polyclonal Antisera and Immunogens. Methods for generating antibodies or antibody fragments of the present technology typically involve immunizing a subject (generally a non-human subject such as a mouse or rabbit) with purified CLDN18.2 protein or a fragment thereof, a nucleic acid encoding the CLDN18.2 protein or a fragment thereof, or a cell expressing the CLDN18.2 protein or a fragment thereof. Suitable immunogenic preparations can contain, for example, recombinantly expressed CLDN18.2 protein or a chemically synthesized CLDN18.2 peptide. Anti-CLDN18.2 antibodies that bind to the CLDN18.2 protein or a portion or fragment thereof can be generated using standard techniques for preparing polyclonal and monoclonal antibodies using the first extracellular loop of the CLDN18.2 protein, or a portion or fragment thereof, as an immunogen. In some embodiments, the antigenic CLDN18.2 peptide contains at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid residues. Longer antigenic peptides are sometimes preferable to shorter antigenic peptides, depending on the use, according to methods well known to those of skill in the art. Multimers of a given epitope are sometimes more effective than monomers.

[0139] By way of example, and not limitation, an immunogenic preparation can contain, for example, recombinantly expressed CLDN18.2 protein or a chemically synthesized CLDN18.2 peptide comprising the amino acid sequence of SEQ ID NO: 4. The first extracellular loop of the CLDN18.2 protein, or a portion or fragment thereof, for example, CLDN18.2-EL1 having the amino acid sequence of SEQ ID NO: 2, can be used as an immunogen to generate anti-CLDN18.2 antibodies that bind to the EL1 portion of the CLDN18.2 protein.

[0140] If necessary, the immunogenicity of the CLDN18.2 protein (or a fragment thereof) can be increased by fusion or conjugation with a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. The CLDN18.2 protein can also be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant, to increase the subject's immune response to the polypeptide. Various adjuvants used to increase immunological responses include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, Pluronic® polyol, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0141] Alternatively, nanoparticles, such as virus-like particles (VLPs), can be used to present antigens, such as CLDN18.2-EL1, to host animals. Virus-like particles are multiprotein structures that mimic the organization and three-dimensional structure of authentic native viruses and are not infectious because they do not carry any viral genetic material (Urakami A, et al., Clin Vaccine Immunol 24:e00090-17(2017)). When introduced into the host immune system, VLPs can elicit effective immune responses, making them attractive carriers of foreign antigens. A key advantage of VLP-based antigen presentation platforms is their ability to present antigens in a high-density, repetitive manner. Thus, antigen-carrying VLPs can induce strong B-cell responses by effectively cross-linking B-cell receptors (BCRs). VLPs can also be genetically engineered to refine their properties, such as immunogenicity. These techniques are standard in the art.

[0142] Isolating a protein or polypeptide sufficiently purified to raise antibodies can be time-consuming and sometimes technically challenging. Additional challenges associated with traditional protein-based immunization include concerns over the safety, stability, scalability, and consistency of protein antigens. Nucleic acid (DNA and RNA)-based immunization has emerged as a promising alternative. DNA vaccines are typically based on bacterial plasmids encoding the polypeptide sequence of a candidate antigen, e.g., CLDN18.2. Robust eukaryotic promoters are used to express the encoded antigen upon inoculation of the host with the plasmid, allowing for sufficient levels of transgene expression (Galvin TA, et al., Vaccine 2000, 18:2566-2583). Modern DNA vaccine production relies on DNA synthesis or one-step cloning into a plasmid vector and subsequent isolation of the plasmid, significantly reducing production time and costs. The resulting plasmid DNA is also highly stable at room temperature, avoiding refrigerated transport and resulting in substantially extended shelf life. These techniques are standard in the art.

[0143] Alternatively, a nucleic acid sequence encoding a target antigen, such as CLDN18.2, can be synthetically introduced into an mRNA molecule. The mRNA is then delivered to a host animal, where its cells recognize and translate the mRNA sequence into the polypeptide sequence of the candidate antigen, such as CLDN18.2, thus inducing an immune response against the foreign antigen. An attractive feature of mRNA antigens or mRNA vaccines is that mRNA is a non-infectious, non-integrating platform. There is no potential risk of infection or insertional mutagenesis associated with DNA vaccines. Additionally, mRNA is degraded by normal cellular processes and has an in vivo half-life that can be controlled through design and delivery method modifications (Kariko, K., et al., Mol Ther 16:1833-1840(2008); Kauffman, KJ, et al., J Control Release 240, 227-234(2016); Guan, S. & Rosenecker, J., Gene Ther 24, 133-143(2017); Thess, A., et al., Mol Ther 23, 1456-1464(2015)). These techniques are standard in the art.

[0144] In describing the present technology, an immune response can be described as either a "primary" or a "secondary" immune response. A primary immune response, also described as a "protective" immune response, refers to an immune response that occurs in an individual as a result of several initial exposures (e.g., initial "immunization" or "priming") to a particular antigen, e.g., a CLDN18.2 protein. In some embodiments, immunity can occur as a result of inoculating an individual with a vaccine containing an antigen. For example, the vaccine can be a CLDN18.2 vaccine containing one or more CLDN18.2 protein-derived antigens. A primary immune response can weaken or decrease over time, and may even disappear or at least become so diminished that it is undetectable. Thus, the present technology also relates to a "secondary" immune response, also described herein as a "memory immune response." The term secondary immune response refers to an immune response induced in an individual in whom a primary immune response has already occurred.

[0145] Thus, a secondary immune response can be elicited to, for example, enhance (e.g., boost) a pre-existing immune response that has weakened or diminished, or to reconstitute a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response results from the stimulation of memory B cells generated upon initial presentation of the antigen. A delayed-type hypersensitivity (DTH) response is a response that activates the CD4 + It is a type of cellular secondary or memory immune response mediated by T cells. An initial exposure to an antigen primes the immune system, and additional exposures result in DTH.

[0146] After appropriate immunization, anti-CLDN18.2 antibodies can be prepared from the serum of the subject. If desired, antibody molecules directed against the CLDN18.2 protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain an IgG fraction.

[0147] Monoclonal antibody. In one embodiment of the present technology, the antibody is an anti-CLDN18.2 monoclonal antibody. For example, in some embodiments, the anti-CLDN18.2 monoclonal antibody may be a human or mouse anti-CLDN18.2 monoclonal antibody. For preparation of monoclonal antibodies directed against a CLDN18.2 protein, or a derivative, fragment, analog, or homolog thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975. Nature 256:495-497), the trioma technique, the human B cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol. Today 4:72), and the EBV hybridoma technique producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized in the practice of this technology and can be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of an antibody can be isolated. Using PCR with primers derived from sequences encoding conserved regions of the antibody, sequences encoding portions of the antibody can be amplified from the population, and DNA encoding the antibody or a fragment thereof, such as a variable domain, can then be reconstructed from the amplified sequences.Such amplified sequences can also be fused to DNA encoding other proteins—e.g., bacteriophage coat or bacterial cell surface proteins—for expression and display of the fusion polypeptide in phage or bacteria. The amplified sequences can be expressed and further selected or isolated, for example, based on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present in the CLDN18.2 protein. Alternatively, hybridomas expressing anti-CLDN18.2 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using conventional methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73:3-46). Screening the hybridomas using standard methods produces monoclonal antibodies of various specificities (i.e., against different epitopes) and affinities. Selected monoclonal antibodies with desired properties, e.g., CLDN18.2 binding, can be used as expressed by hybridomas; they can be conjugated to molecules such as polyethylene glycol (PEG) to alter their properties; or the cDNA encoding them can be isolated, sequenced, and manipulated in various ways. Synthetic dendrimer trees can be added to reactive amino acid side chains, such as lysine, to enhance the immunogenic properties of the CLDN18.2 protein. CPG-dinucleotide techniques can also be used to enhance the immunogenic properties of the CLDN18.2 protein. Other manipulations include substitution or deletion of specific aminoacyl residues responsible for antibody instability during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of the antibody for the CLDN18.2 protein.

[0148] Hybridoma technology. In some embodiments, the antibody of the present technology is an anti-CLDN18.2 monoclonal antibody produced by a hybridoma containing B cells obtained from a transgenic non-human animal, such as a transgenic mouse, whose genome includes a human heavy chain transgene and a human light chain transgene fused to an immortalized cell. Hybridoma technology is known in the art and is taught in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0149] Phage display techniques. As described above, the antibodies of the present technology can be produced by applying recombinant DNA and phage display technology. For example, anti-CLDN18.2 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Phage with desired binding properties are typically selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection using antigens bound or captured to solid surfaces or beads. The phages used in these methods are typically filamentous phage, including fd and M13, which have Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. In addition, the method can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., Science 246:1275-1281, 1989), allowing for the rapid and efficient identification of monoclonal Fab fragments with desired specificity for a CLDN18.2 polypeptide, e.g., a polypeptide, or a derivative, fragment, analog, or homolog thereof. Other examples of phage display methods that can be used to generate antibodies of the present technology include those described by Huston et al. al.,Proc.Natl.Acad.Sci USA,85:5879-5883,1988, Chaudhary et al.,Proc.Natl.Acad.Sci USA,87:1066-1070,1990, Brinkman et al.,J.Immunol.Methods 182:41-50,1995, Ames et al. al., J.Immunol.Methods 184:177-186,1995, Kettleborough et al.,Eur.J.Immunol.24:952-958,1994, Persic et al.,Gene 187:9-18,1997, Burton et al.,Advances in Immunology 57:191-280,1994, PCT / GB91 / 01134, WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, WO96 / 06213, WO92 / 01047(Medical Research Council et al. al.), WO97 / 08320 (Morphosys), WO92 / 01047 (CAT / MRC), WO91 / 17271 (Affymax), and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743. A useful method for displaying polypeptides on the surface of bacteriophage particles by linking them to the polypeptide through disulfide bonds is described by Lohning, U.S. Patent No. 6,753, 136. After phage selection, as described in the above references, the antibody coding regions from the phage can be isolated and used to produce whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.For example, techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be employed using methods known in the art, such as those disclosed in WO92 / 22324, Mullinax et al., BioTechniques 12:864-869, 1992, and Sawai et al., AJRI 34:26-34, 1995, and Better et al., Science 240:1041-1043, 1988.

[0150] Generally, antibodies or antibody fragments are presented on the surface of phage or phagemid particles, and hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against an appropriate antigen to identify variants that maintain superior binding activity. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector approaches can be used in this process, such as cloning antibody fragment libraries into lytic phage vectors (modified T7 or lambda Zap systems) for selection and / or screening.

[0151] Expression of Recombinant Anti-CLDN18.2 Antibodies. As described above, the antibodies of the present technology can be produced by applying recombinant DNA technology. Recombinant polynucleotide constructs encoding the anti-CLDN18.2 antibodies of the present technology typically include expression control sequences operably linked to the coding sequences of the anti-CLDN18.2 antibody chains, including naturally associated or heterologous promoter regions. Accordingly, another aspect of the present technology includes vectors containing one or more nucleic acid sequences encoding the anti-CLDN18.2 antibodies of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, a nucleic acid comprising all or part of the nucleotide sequence encoding the anti-CLDN18.2 antibody is inserted into an appropriate cloning vector or expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence) using recombinant DNA techniques well known in the art and described in detail below. Methods for generating diverse populations of vectors are described in Lerner et al., U.S. Patent Nos. 6,291,160 and 6,680,192.

[0152] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this disclosure, "plasmid" and "vector" can be used interchangeably, as plasmids are the most commonly used form of vector. However, this technology is intended to include other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions. Such viral vectors enable infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding the anti-CLDN18.2 antibody, and for collection and purification of the anti-CLDN18.2 antibody, e.g., a cross-reactive anti-CLDN18.2 antibody. See generally US2002 / 0199213. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors generally contain selectable markers, such as ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences. The vector can also encode a signal peptide, such as pectate lyase, useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0153] The recombinant expression vector of the present technology contains a nucleic acid encoding a protein having CLDN18.2-binding properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell to be used for expression. In a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of an expression vector can depend on factors such as the choice of host cell to be transformed and the desired expression level of the polypeptide. Typical regulatory sequences useful as promoters for recombinant polypeptide expression (e.g., anti-CLDN18.2 antibodies) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, a polynucleotide encoding the anti-CLDN18.2 antibody of the present technology is operably linked to an ara B promoter and expressible in a host cell. See U.S. Patent No. 5,028,530.The expression vectors of the present technology can be introduced into host cells to produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids (e.g., anti-CLDN18.2 antibodies) as described herein.

[0154] Another aspect of the present technology relates to anti-CLDN18.2 antibody-expressing host cells containing nucleic acids encoding one or more anti-CLDN18.2 antibodies. The recombinant expression vectors of the present technology can be designed for expression of anti-CLDN18.2 antibodies in prokaryotic or eukaryotic cells. For example, anti-CLDN18.2 antibodies can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells such as yeast, yeast cells, or mammalian cells. Suitable host cells are described in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS This is further discussed in J. IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vectors can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods useful for preparing and screening polypeptides with predetermined properties, such as anti-CLDN18.2 antibodies, through the expression of stochastically generated polynucleotide sequences have been previously reported. See U.S. Patent Nos. 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0155] Expression of polypeptides in prokaryotes is often carried out in E. coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add several amino acids to the polypeptide encoded thereby, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) increase the expression of the recombinant polypeptide, (ii) increase the solubility of the recombinant polypeptide, and (iii) aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction between the fusion moiety and the recombinant polypeptide, allowing separation of the recombinant polypeptide from the fusion moiety following purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0156] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of different active peptide or protein domains to obtain multifunctional polypeptides via polypeptide fusion are described by Pack et al., U.S. Pat. Nos. 6,294,353 and 6,692,935. One strategy for maximizing recombinant polypeptide expression in E. coli, e.g., anti-CLDN18.2 antibodies, is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. For example, see Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into expression vector so that each codon for each amino acid is preferentially used in expression host, for example, E. coli (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such modification of nucleic acid sequence of this technology can be carried out by standard DNA synthesis techniques.

[0157] In another embodiment, the anti-CLDN18.2 antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, Cell 30:933-943, 1982), pJRY88 (Schultz et al., Gene 54:113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, anti-CLDN18.2 antibodies can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors that can be used to express polypeptides, such as anti-CLDN18.2 antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3:2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

[0158] In yet another embodiment, the nucleic acid encoding the anti-CLDN18.2 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329:840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6:187-195, 1987). When used in mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells useful for expressing the anti-CLDN18.2 antibody of the present technology, see, for example, Chapters 16 and 17 of Sambrook,et al.,MOLECULAR CLONING:A LABORATORY MANUAL.2nd ed.,Cold See Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0159] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1:268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43:235-275, 1988), T-cell receptor (Winoto and Baltimore, EMBO J. 8:729-733, 1989), and immunoglobulin promoters (Banerji, et al., 1983. Cell 33:729-740; Queen and Baltimore, Cell 33:740-751, 1989). 33:741-748, 1983), neuron-specific promoters (e.g., neurofilament promoters, Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), pancreas-specific promoters (Edlund, et al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoters, U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166). Developmentally regulated promoters, such as murine hox promoters (Kessel and Gruss, Science 249:374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546, 1989), are also encompassed.

[0160] Another aspect of the present method relates to a host cell into which a recombinant expression vector of the present technology has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term as used herein.

[0161] The host cell can be any prokaryotic or eukaryotic cell. For example, anti-CLDN18.2 antibodies can be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. Winnacker, From See Genes To Clones, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences such as an origin of replication, a promoter, an enhancer, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Queen et al., Immunol. Rev. 89:49, 1986. Exemplary expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J. Immunol. 148:1149, 1992. Other suitable host cells are known to those skilled in the art.

[0162] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, gene guns, or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0163] Non-limiting examples of suitable vectors include those designed for propagation and expansion, or for expression, or both. For example, cloning vectors can be selected from the group consisting of the pUC series, pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as lambda-GT10, lambda-GT11, lambda-ZapII (Stratagene), lambda-EMBL4, and lambda-NM1149 can also be used. Non-limiting examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Non-limiting examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Calsbad, CA) can also be used according to the manufacturer's recommendations.

[0164] In certain embodiments, the vector is a mammalian vector. In certain embodiments, the mammalian vector contains at least one promoter element mediating signals necessary for initiation of mRNA transcription, the antibody coding sequence, and termination of transcription and polyadenylation of the transcript. In certain embodiments, the mammalian vector contains additional elements, such as an enhancer flanking donor and acceptor sites for RNA splicing, a Kozak sequence, and intervening sequences. In certain embodiments, highly efficient transcription can be achieved using, for example, the early and late promoters from SV40, the long terminal repeats (LTRS) from retroviruses such as RSV, HTLV-1, and HIV-1, and the early promoter of cytomegalovirus (CMV). Cellular elements can also be used (e.g., the human actin promoter). Non-limiting examples of mammalian expression vectors include vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), or pcDNA3.1 / Hygro(+ / -) (Invitrogen, Calsbad, Calif.), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), and pBC12MI (ATCC 67109). Non-limiting examples of mammalian host cells that can be used in combination with such mammalian vectors include human Hela293, HEK293, H9, and Jurkat cells, mouse 3T3, NIH3T3 and C127 cells, Cos1, Cos7, and CV1, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

[0165] In certain embodiments, the vector is a viral vector, e.g., a retroviral vector, a parvovirus-based vector, e.g., an adeno-associated virus (AAV)-based vector, an AAV-adenovirus chimeric vector, and an adenovirus-based vector, and a lentiviral vector, such as a herpes simplex (HSV)-based vector. In certain embodiments, the viral vector is engineered to be defective in viral replication. In certain embodiments, the viral vector is engineered to eliminate toxicity to the host. These viral vectors can be prepared using standard recombinant DNA techniques, see, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[0166] In certain embodiments, the vectors or polynucleotides described herein can be transferred into cells (e.g., ex vivo cells) by conventional techniques, and the resulting cells can be cultured by conventional techniques to produce the anti-CLDN18.2 antibodies or antigen-binding fragments described herein. Accordingly, provided herein are cells comprising a polynucleotide encoding an anti-CLDN18.2 antibody or antigen-binding fragment thereof operably linked to regulatory expression elements (e.g., a promoter) for expression of such sequences in a host cell. In certain embodiments, a vector encoding a heavy chain operably linked to a promoter and a vector encoding a light chain operably linked to a promoter can be coexpressed in a cell for expression of the whole anti-CLDN18.2 antibody or antigen-binding fragment. In certain embodiments, a cell comprises a vector comprising polynucleotides encoding both the heavy and light chains of an anti-CLDN18.2 antibody or antigen-binding fragment described herein operably linked to a promoter. In certain embodiments, the cell comprises two different vectors, a first vector comprising a polynucleotide encoding the heavy chain operably linked to a promoter, and a second vector comprising a polynucleotide encoding the light chain operably linked to a promoter. In certain embodiments, a first cell comprises a first vector comprising a polynucleotide encoding the heavy chain of an anti-CLDN18.2 antibody or antigen-binding fragment described herein, and a second cell comprises a second vector comprising a polynucleotide encoding the light chain of an anti-CLDN18.2 antibody or antigen-binding fragment described herein. In certain embodiments, a mixture of cells comprising the first cell and the second cell is provided herein. Examples of cells include, but are not limited to, human cells, human cell lines, E. coli (e.g., E. coli TB-1, TG-2, DH5a, XL-Blue MRF' (Stratagene), SA2821, and Y1090), B. subtilis, P. aeruginosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9, Ea4), etc.

[0167] It is known that in stable transfection of mammalian cells, only a small percentage of cells may integrate the foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cells on the same vector as that encoding the anti-CLDN18.2 antibody, or on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selectable marker gene will survive, while other cells die).

[0168] Host cells containing the anti-CLDN18.2 antibody of the present technology, such as prokaryotic or eukaryotic host cells in culture, can be used to produce (i.e., express) a recombinant anti-CLDN18.2 antibody. In one embodiment, the method includes culturing the host cells (into which a recombinant expression vector encoding the anti-CLDN18.2 antibody has been introduced) in a suitable medium such that the anti-CLDN18.2 antibody is produced. In another embodiment, the method further includes isolating the anti-CLDN18.2 antibody from the medium or the host cells. Once expressed, the anti-CLDN18.2 antibody, e.g., the anti-CLDN18.2 antibody or a collection of anti-CLDN18.2 antibody-related polypeptides, is purified from the culture medium and the host cells. The anti-CLDN18.2 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. In one embodiment, the anti-CLDN18.2 antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Typically, anti-CLDN18.2 antibody chains are expressed with a signal sequence and are therefore released into the culture medium. However, if the anti-CLDN18.2 antibody chains are not naturally secreted by host cells, they can be released by treatment with a mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, etc. (See generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0169] A polynucleotide encoding an anti-CLDN18.2 antibody, e.g., an anti-CLDN18.2 antibody coding sequence, can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. A suitable transgene includes a light chain and / or heavy chain coding sequence operably linked to a promoter and enhancer from a mammary gland-specific gene, such as casein or β-lactoglobulin. For the generation of transgenic animals, the transgene can be microinjected into a fertilized oocyte or integrated into the genome of an embryonic stem cell, and the nucleus of such a cell transferred into an enucleated oocyte.

[0170] Single-chain antibody. In one embodiment, the anti-CLDN18.2 antibody of the present technology is a single-chain anti-CLDN18.2 antibody. According to the technology of the present invention, techniques can be adapted to produce single-chain antibodies specific to CLDN18.2 protein (see, for example, U.S. Patent No. 4,946,778). Examples of techniques that can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Patent Nos. 4,946,778 and 5,258,498, Huston et al., Methods in Enzymology, 203:46-88, 1991, Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999, 1993, and Skerra et al., Science 240:1038-1040, 1988.

[0171] Chimeric and humanized antibodies. In one embodiment, the anti-CLDN18.2 antibody of the present technology is a chimeric anti-CLDN18.2 antibody. In one embodiment, the anti-CLDN18.2 antibody of the present technology is a humanized anti-CLDN18.2 antibody. In one embodiment of the present technology, the donor antibody and the acceptor antibody are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody.

[0172] Recombinant anti-CLDN18.2 antibodies, such as chimeric and humanized monoclonal antibodies, containing both human and non-human portions can be produced using standard recombinant DNA techniques and are within the scope of the present technology. For some uses, including in vivo use of the anti-CLDN18.2 antibodies of the present technology in humans and use of these agents in in vitro detection assays, chimeric or humanized anti-CLDN18.2 antibodies can be used. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.Such useful methods include, for example, those described in International Application No. PCT / US86 / 02269, U.S. Pat. No. 5,225,539, European Patent No. 184187, European Patent No. 171496, European Patent No. 173494, PCT International Publication No. 86 / 01533, U.S. Pat. Nos. 4,816,567, 5,225,539, and European Patent No. 125023, Better, et al., 1988. Science 240:1041-1043, Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84:3439-3443, Liu, et al., 1987. J. Immunol. 139:3521-3526, Sun, et al. al.,1987.Proc.Natl.Acad.Sci.USA 84:214-218, Nishimura,et al.,1987.Cancer Res.47:999-1005,Wood,et al.,1985.Nature 314:446-449,Shaw,et al.,1988.J.Natl.Cancer Inst.80:1553-1559, Morrison(1985)Science 229:1202-1207, Oi,et al.(1986)BioTechniques 4:214, Jones,et al.,1986.Nature 321:552-525, Verhoeyan,et al.,1988.Science 239:1534,Morrison,Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Patent No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141:4053-4060.For example, antibodies can be engineered using techniques including CDR grafting (EP 0239400, WO91 / 09967, U.S. Patent Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516, EP 460167), veneering or resurfacing (EP 0592106, EP 0519596, Padlan EA, Molecular Immunology, 28:489-498, 1991, Studnicka et al., Protein Engineering 7:805-814, 1994, Roguska et al., PNAS 91:969-973, 1994), and chain shuffling (U.S. Patent No. 5,565,332).In one embodiment, the cDNA encoding the murine anti-CLDN18.2 monoclonal antibody is digested with specifically selected restriction enzymes to remove the sequence encoding the Fc constant region and replace it with the equivalent portion of cDNA encoding a human Fc constant region (Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443, Liu et al. (1987) J Immunol 139:3521-3526, Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218, Nishimura et al. (1987) Cancer Res 47:999-1005, Wood et al. (1985) Nature 314:446-449, and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559, U.S. Patent Nos. 6,180,370, 6,300,064, 6,696,248, 6,706,484, and 6,828,422.

[0173] In one embodiment, the present technology provides for the construction of humanized anti-CLDN18.2 antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response, yet still have effective antibody effector functions. As used herein, the terms "human" and "humanized," with respect to antibodies, refer to any antibody that is expected to induce a therapeutically tolerable, weak immunogenic response in human subjects. In one embodiment, the present technology provides humanized anti-CLDN18.2 antibodies, heavy and light chain immunoglobulins.

[0174] CDR antibody. In some embodiments, the anti-CLDN18.2 antibody of the present technology is an anti-CLDN18.2 CDR antibody. Generally, the donor antibody and acceptor antibody used to generate the anti-CLDN18.2 CDR antibody are monoclonal antibodies from different species, and typically the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody. The graft is a single V of the acceptor antibody. H or V L or of a single CDR (or even part of a single CDR) within V H and V L The CDRs may be multiple CDRs (or portions thereof) in one or both of the variable domains. Often, all three CDRs in all variable domains of the acceptor antibody are replaced with the corresponding donor CDRs, but only as many as necessary to allow sufficient binding of the resulting CDR-grafted antibody to the CLDN18.2 protein. Methods for producing CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and Winter U.S. Patent No. 5,225,539, and EP 0682040. H and V L Methods useful for preparing polypeptides are taught by Winter et al., U.S. Patent Nos. 4,816,397, 6,291,158, 6,291,159, 6,291,161, 6,545,142, EP 0368684, EP 0451216, and EP 0120694.

[0175] After selecting suitable framework region candidates from the same family and / or same family members, either or both of the heavy and light chain variable regions are generated by grafting CDRs from the starting species into the hybrid framework regions. For any of the above embodiments, assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions can be achieved using conventional methods known to those of skill in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs from the starting species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding the CDR regions can also be isolated from the starting species antibody using suitable restriction enzymes and ligated to the target species framework by ligation with a suitable ligation enzyme. Alternatively, the framework regions of the variable chains of the starting species antibody can be altered by site-directed mutagenesis.

[0176] Because hybrids are constructed from a selection among multiple candidates corresponding to each framework region, there are many combinations of sequences that are suitable for construction according to the principles described herein. Thus, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0177] This process typically does not alter the FRs of the acceptor antibody adjacent to the grafted CDRs. However, one skilled in the art can replace specific residues in a given FR to make the FR more similar to the corresponding FR of the donor antibody, thereby improving the antigen-binding affinity of the resulting anti-CLDN18.2 CDR-grafted antibody. Suitable positions for substitution include amino acid residues adjacent to or capable of interacting with the CDRs (see, e.g., U.S. Pat. No. 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with a donor FR and modify it to make it more similar to the acceptor FR or human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR matches the human consensus FR at that position or is at least 90% identical to such consensus FR, doing so may not significantly increase the antigenicity of the resulting modified anti-CLDN18.2 CDR-grafted antibody compared to the same antibody with fully human FRs.

[0178] Bispecific antibodies (BsAbs). Bispecific antibodies are antibodies that can simultaneously bind to two targets with different structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. BsAbs can be generated, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, a bispecific binding agent, by molecular function, binds to one antigen (or epitope) with one of its two binding arms (one VH / VL pair) and to a different antigen (or epitope) with its second arm (a different VH / VL pair). By this definition, a bispecific binding agent has two different antigen-binding arms (both in specificity and CDR sequence) and is monovalent for each antigen to which it binds.

[0179] Multispecific antibodies, such as bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs), have, for example, at least one arm that specifically binds to CLDN18.2 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of B cells, T cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in certain embodiments, the second target antigen is selected from the group consisting of CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, and KIR. Exemplary Vs that bind to a second target antigen (e.g., CD3) include: H and V L The sequence is shown in Figure 25. In certain embodiments, the BsAb is capable of binding to tumor cells expressing the CLDN18.2 antigen on their cell surface. In some embodiments, the BsAb is engineered to promote tumor cell killing by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those with a first antigen-binding site specific for CLDN18.2 and a second antigen-binding site specific for a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, other DOTA-chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D A. et al., 1994, Cancer Res. 54(22):5937-5946).

[0180] Various bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed utilizing either the entire immunoglobulin framework (e.g., IgG), single-chain variable fragments (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprising an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs composed of two scFv units have been shown to be a clinically successful bispecific antibody format. In some embodiments, the BsAb comprises two single-chain variable fragments (scFvs) in tandem, designed such that the scFv that binds to a tumor antigen (e.g., CLDN18.2) is linked to the scFv that binds to T cells (e.g., by binding to CD3). In this manner, T cells are recruited to the tumor site so that they can mediate cytotoxic killing of tumor cells. See, for example, Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321:974-977 (2008)). In some embodiments, the BsAb of the present technology comprises two single-chain variable fragments (scFvs) in tandem, designed such that the scFv that binds to a tumor antigen (e.g., CLDN18.2) is linked to the scFv that binds to the small molecule DOTA hapten.

[0181] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues to cross-link more strongly than more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins to link two or more different single-chain antibody or antibody fragment segments with the required dual specificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0182] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in a similar manner. A variety of fusion proteins can be produced using recombinant methods. In certain embodiments, the BsAb of the present technology comprises an immunoglobulin, which comprises a heavy chain and a light chain, and an scFv. In certain embodiments, the scFv is linked to the C-terminus of the heavy chain of any CLDN18.2 immunoglobulin disclosed herein. In certain embodiments, the scFv is linked to the C-terminus of the light chain of any CLDN18.2 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for the in-frame connection of the heavy chain Fd with the scFv can be generated by PCR. L and V カッパ The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised to generate the V domain of the CLDN18.2 antibody. H The region is ligated into a vector containing a DNA sequence encoding the region, and the resulting vector can be used to transfect a suitable host cell, such as a mammalian cell, for expression of the bispecific fusion protein.

[0183] In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids in length. In some embodiments, the linker is characterized by not tending to adopt a rigid three-dimensional structure, but rather providing flexibility to the polypeptide (e.g., the first and / or second antigen-binding site). In some embodiments, linkers are used in the BsAbs described herein based on particular properties they confer to the BsAb, such as, for example, increased stability. In some embodiments, the BsAbs of the present technology comprise a G4S linker. In some specific embodiments, the BsAb of the present technology is (G4S) n linker, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more.

[0184] Fc Modifications. In some embodiments, the anti-CLDN18.2 antibodies of the present technology comprise a variant Fc region, which comprises at least one amino acid modification relative to a wild-type Fc region (or parent Fc region), such that the molecule has an altered affinity for an Fc receptor (e.g., FcγR), provided that the variant Fc region does not have substitutions at positions that make direct contact with an Fc receptor, based on crystallographic and structural analyses of Fc-Fc receptor interactions, such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make direct contact with an Fc receptor, such as FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G loop).

[0185] In some embodiments, the anti-CLDN18.2 antibodies of the present technology have altered affinity for activating and / or inhibitory receptors and have a variant Fc region with one or more amino acid modifications, where the one or more amino acid modifications are a substitution of N297 with alanine or a substitution of K322 with alanine. Additionally or alternatively, in some embodiments, the Fc region of the CLDN18.2 antibody disclosed herein contains two amino acid substitutions, Leu234Ala and Leu235Ala (referred to as LALA mutations), which eliminate FcγRIIa binding. LALA mutations are commonly used to attenuate cytokine induction from T cells, thus reducing antibody toxicity (Wines BD, et al., J Immunol 164:5313-5318 (2000)).

[0186] Glycosylation modifications. In some embodiments, the anti-CLDN18.2 antibodies of the present technology have an Fc region with variant glycosylation compared to the parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose, and in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells.

[0187] In some embodiments, the antibodies of the present technology may have modified glycosylation sites compared to a suitable reference antibody that binds to an antigen of interest (e.g., CLDN18.2) without altering the functionality of the antibody, e.g., its binding activity to the antigen. As used herein, a "glycosylation site" includes any particular amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more simple sugars linked together) can be specifically and covalently attached.

[0188] Oligosaccharide side chains are typically linked to the antibody backbone via either N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, such as serine or threonine. For example, an Fc-glycoform (hCLDN18.2-IgGln) lacking certain oligosaccharides containing fucose and terminal N-acetylglucosamine can be produced in specialized CHO cells and exhibits enhanced ADCC effector function.

[0189] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are encompassed within the present disclosure; see, e.g., U.S. Pat. No. 6,218,149, EP 0359096 B1, U.S. Patent Publication No. 2002 / 0028486, International Patent Application Publication No. 03 / 035835, U.S. Patent Publication No. 2003 / 0115614, U.S. Pat. No. 6,218,149, and U.S. Pat. No. 6,472,511, each of which is incorporated herein by reference in its entirety. In some embodiments, the carbohydrate content of an antibody (or a relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In certain specific embodiments, the present disclosure involves deleting a glycosylation site in the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0190] Engineered glycoforms can be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be produced by any method known to those of skill in the art, such as by using engineered or variant expression strains, by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase III (GnTIII), by expressing Fc region-containing molecules in various organisms or cell lines from various organisms, or by modifying carbohydrates after the Fc region-containing molecule has been expressed. Methods for producing engineered glycoforms are known in the art and are described in Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al. al., 2003, J. Biol. Chem. 278:3466-3473, U.S. Patent No. 6,602,684, U.S. Patent Application No. 10 / 277,370, U.S. Patent Application No. 10 / 113,929, International Patent Application Publication Nos. WO 00 / 61739 A1, WO 01 / 292246 A1, WO 02 / 311140 A1, WO 02 / 30954 A1, POTILLEGENT™ technology (Biowa, Inc. Princeton, NJ); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, for example, International Patent Application Publication No. WO 00 / 061739, US Patent Application Publication No. 2003 / 0115614, Okazaki et al., 2004, JMB, 336:1239-49.

[0191] Fusion Protein. In one embodiment, the anti-CLDN18.2 antibody of the present technology is a fusion protein. When fused to a second protein, the anti-CLDN18.2 antibody of the present technology can be used as an antigen tag. Examples of domains that can be fused to a polypeptide include heterologous signal sequences as well as other heterologous functional regions. The fusion does not necessarily have to be direct and can occur via a linker sequence. Furthermore, the fusion protein of the present technology can be engineered to improve the characteristics of the anti-CLDN18.2 antibody. For example, additional amino acids, particularly a region of charged amino acids, can be added to the N-terminus of the anti-CLDN18.2 antibody to improve stability and durability during purification from host cells or subsequent handling and storage. Peptide moieties can also be added to the anti-CLDN18.2 antibody to facilitate purification. Such regions can be removed before final preparation of the anti-CLDN18.2 antibody. The addition of peptide moieties to facilitate handling of polypeptides is a well-known and routine technique in the art. The anti-CLDN18.2 antibody of the present technology can be fused to a marker sequence, such as a peptide, that facilitates purification of the fused polypeptide. In selected embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif.), many of which are commercially available. For example, hexa-histidine provides convenient purification of the fusion protein, as described by Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37:767, 1984.

[0192] Thus, any of these above fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology, and in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo.

[0193] Fusion proteins with disulfide-bonded dimeric structures (as occurs with IgG) can be more efficient at binding and neutralizing other molecules than the monomeric secreted protein or protein fragment alone. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.

[0194] Similarly, EP-A-0464533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of an immunoglobulin molecule together with another human protein or fragment thereof. In many cases, the Fc portion in a fusion protein is beneficial for therapy and diagnosis and can therefore, for example, result in improved pharmacokinetic properties. See EP-A-0232262. Alternatively, it may be desirable to delete or modify the Fc portion after the fusion protein has been expressed, detected, and purified. For example, the Fc portion can interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug discovery, for example, human proteins such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8:52-58, 1995; Johanson et al., J. Biol. Chem., 270:9459-9471, 1995.

[0195] Labeled Anti-CLDN18.2 Antibody. In one embodiment, the anti-CLDN18.2 antibody of the present technology is conjugated with a labeling moiety, i.e., a detectable group. The particular label or detectable group conjugated to the anti-CLDN18.2 antibody is not a critical aspect of the present technology, as long as it does not significantly interfere with the specific binding of the anti-CLDN18.2 antibody of the present technology to the CLDN18.2 protein. The detectable group can be any material with a detectable physical or chemical property. Such detectable labels have been well developed in the fields of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, the label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g., 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 other imaging agents such as mTc), microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O. 89 Zr (for positron emission tomography), 99m T.C., 111Examples of suitable labels include In (for single photon emission computed tomography), enzymes (e.g., Radish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, which are incorporated herein by reference in their entireties for all purposes.

[0004] Handbook of Fluorescent Probes and Research Chemicals (6 th See also Molecular Probes, Inc., Eugene, OR).

[0196] The label can be attached directly or indirectly to the desired component of the assay according to methods well known in the art. As noted above, a wide variety of labels can be used, with the choice of label depending on factors such as the sensitivity required, ease of conjugation to the compound, stability requirements, available equipment, and disposal regulations.

[0197] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule that is inherently detectable or covalently bound to a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Many ligands and anti-ligands can be used. If the ligand has a natural anti-ligand, such as biotin, thyroxine, and cortisol, it can be used in conjunction with a labeled, naturally occurring anti-ligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, such as an anti-CLDN18.2 antibody.

[0198] Molecules can also be directly conjugated to signal-generating compounds, for example, by conjugation with an enzyme or fluorophore. Enzymes of interest as labels are primarily hydrolases, particularly phosphatases, esterases, and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, luciferin and 2,3-dihydrophthalazinediones, such as luminol. For a review of various labeling or signal-generating systems that can be used, see U.S. Pat. No. 4,391,904.

[0199] Means for detecting labels are well known to those skilled in the art. Thus, for example, if the label is a radioactive label, detection means include a scintillation counter or photographic film as in autoradiography. If the label is a fluorescent label, it can be detected by exciting the fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be detected visually, by photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers. Similarly, enzyme labels can be detected by providing the enzyme with an appropriate substrate and detecting the resulting reaction product. Finally, simple colorimetric labels can be detected by simply observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0200] Some assay methods do not require the use of labeled components.For example, agglutination assay can be used to detect the presence of target antibody, such as anti-CLDN18.2 antibody.In this case, antigen-coated particles are agglutinated by the sample containing target antibody.In this method, none of the components need to be labeled, and the presence of target antibody can be detected by simple visual inspection.

[0201] B. Identification and Characterization of Anti-CLDN18.2 Antibodies of the Present Technology Methods for Identifying and / or Screening Anti-CLDN18.2 Antibodies of the Present Technology. Methods useful for identifying and screening antibodies against a CLDN18.2 polypeptide for those with the desired specificity for the CLDN18.2 protein (e.g., those that bind to the first extracellular loop of a CLDN18.2 protein, such as a polypeptide comprising the amino acid sequence of SEQ ID NO: 2) include any immunologically mediated technique known in the art. Components of the immune response can be detected in vitro by a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells, and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2:373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen, and presentation of that antigen on MHC can be detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with a reagent that cross-links their Fc-epsilon receptors, and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4:432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0202] Similarly, the products of immune responses in either model organisms (e.g., mice) or human subjects can be detected by a variety of methods well known to those skilled in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing a sterile container over the scratched site to capture the migrating cells (Peters et al., Blood, 72:1310-5, 1988); and (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by immunohistochemistry. 3 (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72:1310-5, 1988); and (5) differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs that express these markers.

[0203] In one embodiment, the anti-CLDN18.2 antibody of the present technology is selected by displaying CLDN18.2 peptide on the surface of replicable genetic packaging.For example, see U.S. Patent Nos. 5,514,548, 5,837,500, 5,871,907, 5,885,793, 5,969,108, 6,225,447, 6,291,650, 6,492,160, EP585287, EP605522, EP616640, EP1024191, EP589877, EP774511, EP844306.A method useful for generating / selecting filamentous bacteriophage particles containing phagemid genomes encoding binding molecules with desired specificity has been described. See, for example, EP 774511, US 5,871,907, US 5,969,108, US 6,225,447, US 6,291,650, US 6,492,160.

[0204] In some embodiments, the anti-CLDN18.2 antibodies of the present technology are selected using display of CLDN18.2 peptides on the surface of yeast host cells. A method useful for isolating scFv polypeptides by yeast surface display is reported by Kieke et al., Protein Eng. 1997 Nov;10(11):1303-10.

[0205] In some embodiments, the anti-CLDN18.2 antibodies of the present technology are selected using ribosome display. A useful method for identifying ligands in a peptide library using ribosome display is reported by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-26, 1994, and Hanes et al., Proc. Natl. Acad. Sci. USA 94:4937-42, 1997.

[0206] In certain embodiments, the anti-CLDN18.2 antibody of the present technology is selected using tRNA display of CLDN18.2 peptide. A useful method for in vitro selection of ligands using tRNA display is reported by Merryman et al., Chem. Biol., 9:741-46, 2002.

[0207] In one embodiment, the anti-CLDN18.2 antibody of the present technology is selected using RNA display.A method useful for selecting peptides and proteins using an RNA display library is reported by Roberts et al.Proc.Natl.Acad.Sci.USA,94:12297-302,1997 and Nemoto et al.,FEBS Lett.,414:405-8,1997.A method useful for selecting peptides and proteins using a non-natural RNA display library is reported by Frankel et al.,Curr.Opin.Struct.Biol.,13:506-12,2003.

[0208] In some embodiments, the anti-CLDN18.2 antibodies of the present technology are expressed in the periplasm of Gram-negative bacteria and mixed with labeled CLDN18.2 protein. See WO02 / 34886. Clones expressing recombinant polypeptides with affinity for CLDN18.2 protein will increase the concentration of labeled CLDN18.2 protein bound to the anti-CLDN18.2 antibody, which can be isolated from the rest of the library as described in Harvey et al., Proc. Natl. Acad. Sci. 22:9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403.

[0209] After selection of a desired anti-CLDN18.2 antibody, it is contemplated that the antibody can be produced in large quantities by any technique known to those of skill in the art, such as, for example, expression in prokaryotic or eukaryotic cells. For example, but not limited to, an anti-CLDN18.2 antibody that is an anti-CLDN18.2 hybrid antibody or fragment can be produced using conventional techniques to construct an expression vector encoding an antibody heavy chain in which the CDRs and, if necessary, the minimal portion of the variable region framework required to retain the original species' antibody binding specificity (engineered according to the techniques described herein) are derived from a starting species antibody, and the remainder of the antibody is derived from a target species immunoglobulin that can be engineered as described herein, thereby creating a vector for expression of the hybrid antibody heavy chain.

[0210] Measuring CLDN18.2 Binding. In some embodiments, a CLDN18.2 binding assay refers to an assay format in which a CLDN18.2 protein and an anti-CLDN18.2 antibody are mixed under conditions suitable for determining binding between the CLDN18.2 protein and the anti-CLDN18.2 antibody and assessing the amount of binding between the CLDN18.2 protein and the anti-CLDN18.2 antibody. The amount of binding is compared to a suitable control, which can be the amount of binding in the absence of CLDN18.2 protein, the amount of binding in the presence of a nonspecific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assay methods include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for directly measuring CLDN18.2 protein binding to an anti-CLDN18.2 antibody include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of a candidate anti-CLDN18.2 antibody is at least 1% greater than the binding observed in the absence of the candidate anti-CLDN18.2 antibody, the candidate anti-CLDN18.2 antibody is useful as an anti-CLDN18.2 antibody of the present technology.

[0211] Use of the anti-CLDN18.2 antibody of the present technology General. The anti-CLDN18.2 antibodies of the present technology are useful in art-known methods related to the localization and / or quantification of CLDN18.2 protein (e.g., for use in measuring the level of CLDN18.2 protein in an appropriate physiological sample, for use in diagnostic methods, for use in polypeptide imaging). The antibodies of the present technology are useful for isolating CLDN18.2 protein by standard techniques such as affinity chromatography or immunoprecipitation. The anti-CLDN18.2 antibodies of the present technology can facilitate the purification of native immunoreactive CLDN18.2 protein from biological samples, such as mammalian serum or cells, as well as recombinantly produced immunoreactive CLDN18.2 protein expressed in a host system. Furthermore, anti-CLDN18.2 antibodies can be used to detect immunoreactive CLDN18.2 protein (e.g., in plasma, cell lysates, or cell supernatants) and assess the amount and pattern of expression of the immunoreactive polypeptide. The anti-CLDN18.2 antibody of the present technology can be used diagnostically to monitor the immunoreactive CLDN18.2 protein level in tissue as part of a clinical trial procedure, for example, to determine the effectiveness of a given treatment regimen.As mentioned above, detection can be facilitated by coupling (i.e., physically linking) the anti-CLDN18.2 antibody of the present technology to a detectable substance.

[0212] Detection of CLDN18.2 protein. An exemplary method for detecting the presence or absence of immunoreactive CLDN18.2 protein in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample with an anti-CLDN18.2 antibody of the present technology that can detect immunoreactive CLDN18.2 protein, thereby detecting the presence of immunoreactive CLDN18.2 protein in the biological sample. Detection can be achieved by means of a detectable label attached to the antibody.

[0213] The term "labeled" with respect to an anti-CLDN18.2 antibody is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0214] In some embodiments, the anti-CLDN18.2 antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, the anti-CLDN18.2 antibodies may be detectably labeled by covalent or non-covalent attachment of a chromogenic, enzymatic, radioisotope, isotope, fluorescent, toxin, chemiluminescent, nuclear magnetic resonance imaging agent, or other label.

[0215] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast-alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0216] Examples of suitable radioisotope labels are: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU,90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd and others are included. 111 In is an exemplary isotope used for in vivo imaging, as it is involved in the liver 125 I or 131 This is to avoid the problem of dehalogenation of I-labeled CLDN18.2-binding antibodies. In addition, this isotope has a more favorable gamma emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, 1-(P-isothiocyanatobenzyl)-DPTA coupled to monoclonal antibodies 111 In shows little uptake in non-tumor tissues, especially the liver, enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Contains Fe.

[0217] Examples of suitable fluorescent labels include: 152 Examples of suitable toxin labels include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0218] Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron.

[0219] The detection method of the present technology can be used to detect immunoreactive CLDN18.2 protein in biological samples in vitro and in vivo. In vitro techniques for detecting immunoreactive CLDN18.2 protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detecting immunoreactive CLDN18.2 protein include introducing a labeled anti-CLDN18.2 antibody into a subject. For example, the anti-CLDN18.2 antibody can be labeled with a radioactive marker, and its presence and location in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains CLDN18.2 protein molecules from the test subject.

[0220] Immunoassays and Imaging. The anti-CLDN18.2 antibodies of the present technology can be used to assay immunoreactive CLDN18.2 protein levels in biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be examined by classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, and radioisotopes or other radioactive agents, such as iodine ( 125 I, 121 I, 131I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 mTc), and fluorescent labels such as fluorescein, rhodamine, and green fluorescent protein (GFP), and biotin.

[0221] In addition to assaying the immunoreactive CLDN18.2 protein level in biological samples, the anti-CLDN18.2 antibody of the present technology can be used for in vivo imaging of CLDN18.2. Antibodies useful for this method include those that can be detected by X-ray radiography, NMR, or ESR. For X-ray radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not obviously harmful to the subject. Suitable markers for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into anti-CLDN18.2 antibodies by labeling nutrients in related scFv clones.

[0222] Radioactive isotopes (e.g., 131 I, 112 In, 99 An anti-CLDN18.2 antibody labeled with a suitable detectable imaging moiety, such as mTc), a radiopaque substance, or a material detectable by nuclear magnetic resonance, is introduced into a subject (e.g., parenterally, subcutaneously, or intraperitoneally). It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is typically 99 The range is approximately 5 to 20 millicuries of mTc. The labeled anti-CLDN18.2 antibody then accumulates at the site of cells containing the specific target polypeptide. For example, the labeled anti-CLDN18.2 antibody of the present technology accumulates in the subject in cells and tissues where the CLDN18.2 protein is localized.

[0223] Thus, the present technology provides a method for diagnosing a medical condition, comprising: (a) assaying the expression of immunoreactive CLDN18.2 protein by measuring binding of an anti-CLDN18.2 antibody of the present technology in an individual's cells or body fluids; and (b) comparing the amount of immunoreactive CLDN18.2 protein present in the sample with a standard reference, wherein an increase or decrease in the level of immunoreactive CLDN18.2 protein compared to the standard is indicative of the medical condition.

[0224] Affinity purification. The anti-CLDN18.2 antibody of the present technology can be used to purify immunoreactive CLDN18.2 protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins, and polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology," 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)).

[0225] The simplest method for binding an antigen to an antibody support matrix is ​​to collect beads in a column and pass the antigen solution through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a slow flow rate. The immobilized antibody captures the antigen as it passes through. Alternatively, the antigen solution can be contacted with the antibody support matrix by mixing the antigen solution with a support (e.g., beads) and rotating or rocking the slurry, allowing for maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through a column for bead collection. The beads are washed using a suitable wash buffer, and then the pure or substantially pure antigen is eluted.

[0226] The antibody or polypeptide of interest can be conjugated to a solid support such as beads. In addition, the first solid support such as beads can also be conjugated to a second solid support, which can be a second bead or other support, by any suitable means, including those disclosed herein for conjugating a polypeptide to a support. Thus, any of the conjugation methods and means disclosed herein for conjugating a polypeptide to a solid support can also be applied to conjugating a first support to a second support, and the first and second solid supports can be the same or different.

[0227] Linkers suitable for use in conjugating polypeptides to solid supports include a variety of agents that can be crosslinkers and react with functional groups present on the surface of the support, with the polypeptide, or both. Reagents useful as crosslinkers include homobifunctional and, particularly, heterobifunctional reagents. Useful bifunctional crosslinkers include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Crosslinkers can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, photolabile crosslinkers such as 3-amino-(2-nitrophenyl)propionic acid can be used as a means of cleaving the polypeptide from the solid support. (Brown et al., Mol. Divers, pp. 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and U.S. Pat. No. 5,643,722.) Other crosslinking reagents are well known in the art. (See, e.g., Wong (1991), supra; and Hermanson (1996), ibid.).

[0228] Antibodies or polypeptides can be immobilized to solid supports, such as beads, through covalent amide bonds formed between carboxyl-functionalized beads and the amino terminus of the polypeptide, or conversely, through covalent amide bonds formed between amino-functionalized beads and the carboxyl terminus of the polypeptide. Additionally, a bifunctional trityl linker can be attached to a support, e.g., a 4-nitrophenyl active ester on a resin, such as a Wang resin, through an amino or carboxyl group on the resin. Using the bifunctional trityl approach, the solid support can require treatment with a volatile acid, such as formic acid or trifluoroacetic acid, to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide can be deposited as a bead-free patch on the bottom of a well or on the flat surface of a solid support. After addition of a matrix solution, the polypeptide can be desorbed by MS.

[0229] Hydrophobic trityl linkers can also be used as acid-labile linkers by cleaving the amine-linked trityl group from the polypeptide using a volatile acid or an appropriate matrix solution, such as a matrix solution containing 3-HPA. The degree of acid lability can also be varied. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be modified with appropriate p-substitutions or more acid-labile tritylamine derivatives of the polypeptide, i.e., trityl ether and tritylamine bonds can be created in the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker by, for example, disrupting the hydrophobic attraction, or, if desired, cleaving the trityl ether or tritylamine bond under acidic conditions, including typical MS conditions where a matrix such as 3-HPA acts as an acid.

[0230] Orthogonally cleavable linkers can also be useful for linking a first solid support, e.g., a bead, to a second solid support or for linking a polypeptide of interest to a solid support. Using such linkers, a first solid support, e.g., a bead, can be selectively cleaved from the second solid support without cleaving the polypeptide from the support, and the polypeptide can then be later cleaved from the bead. For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to link the bead to the second solid support, and an acid-cleavable bifunctional trityl group can be used to immobilize the polypeptide to the support. If desired, the bond between the polypeptide and the solid support can be cleaved first, for example, leaving the bond between the first support and the second support intact. The trityl linker can provide a covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl group is easily cleaved under acidic conditions.

[0231] For example, beads can be attached to a second support via a linking group whose length and chemical properties can be selected to facilitate high-density binding of beads to the solid support or high-density binding of polypeptides to the beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing a variety of functional groups per attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, pentaerythrol, and tris-hydroxyaminomethane.

[0232] Non-covalent binding. An antibody or polypeptide can be conjugated to a solid support, or a first solid support can be conjugated to a second solid support through a non-covalent interaction. For example, magnetic beads made from ferromagnetic materials can be magnetized, attracted to a magnetic solid support, and released from the support by removing the magnetic field. Alternatively, the solid support can be provided with ionic or hydrophobic moieties, respectively, which can allow interaction of the ionic or hydrophobic moieties with a polypeptide, such as a polypeptide containing an attached trityl group, or a second solid support with hydrophobic properties.

[0233] A solid support can also be provided with a member of a specific binding pair and thus conjugated to a polypeptide or second solid support containing the complementary binding moiety. For example, avidin or streptavidin coated beads can be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or a derivative of biotin such as iminobiotin.

[0234] It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, biotin, for example, can be incorporated into either the polypeptide or the solid support, and conversely, avidin or other biotin-binding moiety can be incorporated into the support or polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and the antigens with which they specifically interact, and other such pairs known to those of skill in the art.

[0235] A. Diagnostic Uses of the Anti-CLDN18.2 Antibodies of the Present Technology General. The anti-CLDN18.2 antibodies of the present technology are useful in diagnostic methods. Thus, the present technology provides methods for using antibodies in diagnosing CLDN18.2 activity in a subject. The anti-CLDN18.2 antibodies of the present technology can be selected to have any level of epitope binding specificity and very high binding affinity for the CLDN18.2 protein. Generally, the higher the binding affinity of the antibody, the more stringent washing conditions can be used in the immunoassay to remove nonspecifically bound substances without removing the target polypeptide. Therefore, the anti-CLDN18.2 antibodies of the present technology useful in diagnostic assays typically have a binding affinity of about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , or 10 12 M -1 Furthermore, it is desirable that anti-CLDN18.2 antibodies used as diagnostic reagents have binding kinetics sufficient to reach equilibrium in at least 12 hours, at least 5 hours, or at least 1 hour under standard conditions.

[0236] Anti-CLDN18.2 antibodies can be used to detect immunoreactive CLDN18.2 protein in a variety of standard assay formats, including immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assay. See, for example, Harlow & Lane, "Antibodies, A Laboratory Manual" (Cold Spring Harbor Publications, New York, 1988), U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,879,262, 4,034,074, 3,791,932, 3,817,837, 3,839,153, 3,850,752, 3, See US Pat. Nos. 850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876. The biological sample can be obtained from any tissue or bodily fluid of a subject. In certain embodiments, the subject is at an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of CLDN18.2 protein in a sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsy tissue.

[0237] Immunometric or sandwich assays are one type of diagnostic method of this technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one antibody, for example, an anti-CLDN18.2 antibody or a population of anti-CLDN18.2 antibodies immobilized on a solid phase, and another anti-CLDN18.2 antibody or a population of anti-CLDN18.2 antibodies in solution. Typically, the solution anti-CLDN18.2 antibody or a population of anti-CLDN18.2 antibodies is labeled. When an antibody population is used, the population can contain antibodies that bind to different epitope specificities within the target polypeptide. Therefore, the same population can be used for both the solid-phase antibody and the solution antibody. When anti-CLDN18.2 monoclonal antibodies are used, first and second CLDN18.2 monoclonal antibodies with different binding specificities are used in the solid and solution phases. The solid-phase (also referred to as "capture") and solution (also referred to as "detection") antibodies can be contacted with the target antigen either sequentially or simultaneously. If the solid-phase antibody is contacted first, the assay is called a forward assay. Conversely, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After contacting the CLDN18.2 protein with the anti-CLDN18.2 antibody, the sample is typically incubated for a period ranging from about 10 minutes to about 24 hours, but is typically about 1 hour. A wash step is then performed to remove sample components that are not specifically bound to the anti-CLDN18.2 antibody used as a diagnostic reagent. If the solid-phase and solution antibodies are bound in separate steps, washing can be performed after either or both binding steps. After washing, binding is typically quantified by detecting a label linked to the solid phase via binding of a labeled solution antibody. Typically, for a given pair of antibodies or a group of antibodies and given reaction conditions, a calibration curve is prepared from samples containing known concentrations. The concentration of immunoreactive CLDN18.2 protein in the test sample is determined by interpolation from the calibration curve (i.e., standard curve).The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of CLDN18.2 protein in the sample.

[0238] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, as well as particles such as agarose, dextran-based gels, dipsticks, particles, microparticles, magnetic particles, test tubes, microtiter wells, and SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway, NJ). Immobilization can be by absorption or covalent binding. Optionally, the anti-CLDN18.2 antibody can be conjugated to a linker molecule such as biotin for binding to a surface-bound linker such as avidin.

[0239] In some embodiments, the present disclosure provides anti-CLDN18.2 antibodies of the present technology conjugated to a diagnostic agent. The diagnostic agent may include a radioactive or non-radioactive label, an imaging agent (such as for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label can be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. A diagnostic agent is a molecule conjugated to an antibody portion, i.e., an antibody or antibody fragment or subfragment, and administered, which is useful for diagnosing or detecting disease by locating cells containing the antigen.

[0240] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancing agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancing agents and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. To load an antibody component with a radioactive metal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail to which multiple chelating groups for binding the ion are attached. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains bearing pendant groups to which can be attached chelating groups such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose. Chelates can be attached to the antibodies of the present technology using standard chemistries. Chelates are typically linked to antibodies by groups that allow for the formation of bonds with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes for radioimaging. When complexed with non-radioactive metals such as manganese, iron, and gadolinium, the same chelates are useful for MRI when used with the CLDN18.2 antibodies of the present technology. Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N',N''-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are used with a variety of metals and radiometals, such as radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes include:The details of the activity of the child were also revealed. (i) DOTA-Phe- Lys(HSG)-D-Tyr-Lys(HSG)-NH2、(ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys (Tscg-Cys)-NH2、(iii)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2、(iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG-(Dv)-NHDO)、 -D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、(vii)DOTA-D-Phe-D-Lys(HSG)-D-ysTyr -NH2、(viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2、(ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2、-D-(x)Bz-Ac- TPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2、(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2、(xii)DOTA-D-Phe-D-D-Lys-(THyrHSG)- (HSG)-D-Lys(Tscg-Cys)-NH2、(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2、(xiv)Tscg-D-D-G-luHS G)-D-Glu-D-Lys(HSG)-NH2、(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、(xvi)Ac-D-Cys-D-Lys(DOTA-D-L-D-A-laDO-Tyr TA)-D-Cys-NH2、(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2、(xviii)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-NH2C(cys、s、sand (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

[0241] For RAIT 223 Other ring-type chelates, such as macrocyclic polyethers, intended to stably bind nuclides such as Ra are also contemplated. B. Therapeutic Uses of Anti-CLDN18.2 Antibodies of the Present Technology

[0242] In one aspect, the immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology are useful for treating CLDN18.2-associated cancers, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, or any other neoplastic tissue that expresses CLDN18.2. In some embodiments, the CLDN18.2-associated cancer is a solid tumor. Such treatments can be used in patients identified as having pathologically elevated levels of CLDN18.2 (e.g., diagnosed by the methods described herein) or diagnosed with a disease known to be associated with such pathological levels.

[0243] The compositions of the present technology can be used in conjunction with other therapeutic agents useful in the treatment of CLDN18.2-associated cancers. For example, the antibodies or antigen-binding fragments of the present technology can be administered separately, sequentially, or simultaneously with at least one additional therapeutic agent selected from the group consisting of alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents, T cell, and targeted biological therapeutic agents (e.g., therapeutic peptides described in U.S. Pat. No. 6,306,832, WO2012 / 007137, WO2005 / 000889, WO2010 / 096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic agents include cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolumid, topotecan, vincristine, vinblastine, eribulin, mutagenesis inhibitors, and cyclophosphamide. mycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, or combinations thereof.

[0244] Additionally or alternatively, in some embodiments, the antibodies or antigen-binding fragments of the present technology may be administered separately, sequentially, or simultaneously with at least one additional immune modulatory / stimulatory antibody, including but not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-TIM3 antibodies, anti-4-1BB antibodies, anti-CD73 antibodies, anti-GITR antibodies, and anti-LAG-3 antibodies.

[0245] The compositions of the present technology can optionally be administered to a subject in need thereof as a single bolus. Alternatively, the administration regimen can include multiple administrations given at various times after the appearance of a tumor.

[0246] Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intracranial, intratumoral, intrathecal, or topical. Administration includes self-administration and administration by another. It is also understood that the various modes of treatment of the described medical conditions include total treatment, but are intended to mean "substantial" to mean less than total treatment, where some biologically or medically relevant result is achieved.

[0247] In some embodiments, the antibodies of the present technology comprise a pharmaceutical formulation and can be administered to a subject in need thereof in one or more doses. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response).

[0248] Typically, an effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect ranges from about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Typically, the dosage range is from about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For administration of anti-CLDN18.2 antibodies, the dosage is about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg, of the subject's body weight every week, every two weeks, or every three weeks. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight every week, every two weeks, or every three weeks, or within the range of 1 to 10 mg / kg every week, every two weeks, or every three weeks. In one embodiment, a single dose of the antibody ranges from 0.1 to 10,000 micrograms per kg of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The anti-CLDN18.2 antibody may be administered on multiple occasions. The interval between single doses can be hourly, daily, weekly, monthly, or yearly. The intervals can also be irregular, as indicated by measuring the subject's blood levels of the antibody. In some methods, the dosage is adjusted to achieve a serum antibody concentration in the subject of about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL, or about 150 μg / mL to about 200 μg / mL. Alternatively, the anti-CLDN18.2 antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the subject. The dosage and frequency can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. In therapeutic applications, relatively high dosages are sometimes required at relatively short intervals until the progression of the disease is reduced or terminated, or until the subject shows partial or complete improvement of the symptoms of the disease. Thereafter, the patient can be administered a prophylactic regime.

[0249] In another aspect, the present disclosure provides a method for detecting cancer in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology, wherein the antibody is configured to localize to cancer cells expressing CLDN18.2 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value can be calculated by measuring the radioactivity level present in non-tumor (normal) tissues and calculating the average radioactivity level present in the non-tumor (normal) tissues ± standard deviation. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0250] In some embodiments, the subject is diagnosed with or suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0251] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle-emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and67 Examples of alpha particle emitting isotopes include: 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, and 255 Examples of Auger emitters include Fm. 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, and 203 Pb. In some embodiments of the method, nonspecific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via an N297A mutation in the Fc region, resulting in aglycosylation). The therapeutic efficacy of such immunoconjugates can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the immunoconjugate has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0252] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-CLDN18.2 antibody described herein provides a therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the anti-CLDN18.2 antibodies described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (the dose lethal to 50% of the population) or LD 100The therapeutic index can be determined by determining the dose (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic for use in humans. The dosage of the anti-CLDN18.2 antibodies described herein lies within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0253] Formulation of Pharmaceutical Compositions. In accordance with the methods of the present technology, anti-CLDN18.2 antibodies can be incorporated into pharmaceutical compositions suitable for administration. Pharmaceutical compositions generally comprise a recombinant or substantially purified antibody in a form suitable for administration to a subject, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 th (See, e.g., J.D., 1990). Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all U.S. Food and Drug Administration Good Manufacturing Practice (GMP) regulations. The pharmaceutical composition may further comprise an agent selected from the group consisting of an isotope, a dye, a chromagen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0254] The terms "pharmaceutically acceptable" and "physiologically tolerable," and their grammatical variations, are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to or in a subject without producing undesirable physiological effects that would prohibit administration of the composition. For example, a "pharmaceutically acceptable excipient" generally refers to an excipient that is safe, non-toxic, and useful in preparing a desired pharmaceutical composition, including excipients that are acceptable for veterinary use and for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. "Pharmaceutically acceptable salts and esters" refer to salts and esters that are pharmaceutically acceptable and possess the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-CLDN18.2 antibody, e.g., C 1-6Alkyl esters are also included. When two acidic groups are present, the pharmaceutically acceptable salt or ester can be a monoacid-monosalt or ester, or a disalt or ester; similarly, when more than two acidic groups are present, some or all of such groups can be salified or esterified. The anti-CLDN18.2 antibodies named in the present technology can exist in unsalted or unesterified form, or salified and / or esterified form, and the naming of such anti-CLDN18.2 antibodies is intended to include both the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. In addition, certain embodiments of the present technology can exist in two or more stereoisomeric forms, and the naming of such anti-CLDN18.2 antibodies is intended to include all single stereoisomers and all mixtures (racemic or otherwise) of such stereoisomers. Those skilled in the art will have no difficulty in determining the appropriate timing, sequence, and dosage of administration of particular drugs and compositions of the present technology.

[0255] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and compounds for pharmaceutically active substances is well known to those skilled in the art. Except to the extent or to the extent that any conventional media or compound is incompatible with anti-CLDN18.2 antibodies, their use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0256] The pharmaceutical compositions of the present technology are formulated to be compatible with their intended route of administration. The anti-CLDN18.2 antibody compositions of the present technology can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally, or intramuscularly, or as an inhalant. The anti-CLDN18.2 antibody can optionally be administered in combination with other drugs that are at least partially effective in treating various CLDN18.2-associated cancers.

[0257] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial compound such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate; and a compound for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0258] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be desirable to include isotonic compounds, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition a compound which delays absorption, for example, aluminum monostearate and gelatin.

[0259] Sterile injectable solutions can be prepared by incorporating the required amount of anti-CLDN18.2 antibody of the present technology into a suitable solvent with one or a combination of the ingredients listed above, and optionally followed by filtration sterilization. Generally, dispersions are prepared by incorporating anti-CLDN18.2 antibody into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients listed above. For sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient and any additional desired ingredients from the solution already sterile-filtered. The antibody of the present technology can be administered in the form of a depot injection or implant preparation, which can be formulated in a way that allows the active ingredient to be released continuously or in a pulsatile manner.

[0260] Oral compositions generally contain an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the anti-CLDN18.2 antibody can be incorporated with an excipient and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash; the compound in the fluid carrier is applied to the mouth, swished, and expectorated or swallowed. Pharmaceutically compatible binding compounds and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating compound such as alginic acid, Primogel, or corn starch; a glidant such as magnesium stearate or sterote; a lubricant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring; or ingredients of a similar nature.

[0261] For administration by inhalation, the anti-CLDN18.2 antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0262] Systemic administration can also be via transmucosal or transdermal routes. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, anti-CLDN18.2 antibodies are formulated into ointments, salves, gels, or creams generally known in the art.

[0263] Anti-CLDN18.2 antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0264] In one embodiment, anti-CLDN18.2 antibodies are formulated with carriers that protect the anti-CLDN18.2 antibodies against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0265] T cells bound to the multispecific binding molecules of the present technology. Without being bound by any theory, it is believed that when the anti-CD3 multispecific binding molecules provided herein (e.g., CLDN18.2xCD3) are bound to T cells by the procedures described herein, the anti-CD3 scFv of the multispecific binding molecule binds to CD3 on the surface of the T cell. Without being bound by any theory, it is believed that binding of the multispecific binding molecule to the T cell (i.e., binding of the anti-CD3 scFv to CD3 expressed on the T cell) activates the T cell, thereby bypassing MHC restriction and allowing T cell receptor-based cytotoxicity to be redirected to the desired tumor target.

[0266] Therefore, the present disclosure also provides T cells bound to the multispecific binding molecules of the present technology. In a specific embodiment, the T cells are non-covalently bound to the multispecific binding molecules. In a specific embodiment, the T cells are autologous to the subject to whom the T cells are administered. In a specific embodiment, the T cells are allogeneic to the subject to whom the T cells are administered. In a specific embodiment, the T cells are human T cells.

[0267] In a specific embodiment, T cells that bind to the multispecific binding molecules of the invention are used in accordance with the therapeutic methods described herein. In a specific embodiment, T cells that are bound to the multispecific binding molecules of the present disclosure are used as part of a combination therapy as described below.

[0268] In specific embodiments involving combination therapy with T cell infusion, provided herein are pharmaceutical compositions comprising (a) a multispecific binding molecule described herein, (b) T cells, and / or (c) a pharmaceutically effective carrier. In specific embodiments, the T cells are autologous to the subject to whom the T cells are administered. In certain embodiments, the T cells are allogeneic to the subject to whom the T cells are administered. In specific embodiments, the T cells are either bound to the multispecific binding molecule or are not bound. In specific embodiments, the binding of the T cells to the multispecific binding molecule is non-covalent. In specific embodiments, the T cells are human T cells. Methods that can be used to bind multispecific binding molecules to T cells are known in the art. See, e.g., Lum et al., 2013, Biol Blood Marrow Transplant, 19:925-33; Janeway et al., Immunobiology: The Immune System in Health and Disease, 5th edition, New York: Garland Science; Vaishampaian et al., 2015, Prostate Cancer, 2015:285193; and Stromnes et al., 2014, Immunol Rev. 257(1):145-164.

[0269] In specific embodiments, administration of a multispecific binding molecule, a polynucleotide, vector, or cell encoding a multispecific binding molecule, or a pharmaceutical composition comprising a multispecific binding molecule provided herein occurs after the patient has been treated with a T cell infusion. In specific embodiments, the T cell infusion occurs using T cells that are autologous to the subject to whom the T cells are administered. In specific embodiments, the T cell infusion occurs using T cells that are allogeneic to the subject to whom the T cells are administered. In specific embodiments, the T cells are capable of binding to a molecule identical to a multispecific binding molecule described herein. In specific embodiments, the binding of the T cells to a molecule identical to a multispecific binding molecule is non-covalent. In specific embodiments, the T cells are human T cells.

[0270] C. Kit The present technology provides kits for the detection and / or treatment of CLDN18.2-associated cancers, comprising at least one immunoglobulin-related composition (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant). Optionally, the above-mentioned components of the kits of the present technology are packaged in a suitable container and labeled for the diagnosis and / or treatment of CLDN18.2-associated cancers. The above-mentioned components may be stored in unit or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous, preferably sterile solutions or as lyophilized, preferably sterile, formulations for reconstitution. The kit may further comprise a second container holding a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients of the pharmaceutical composition and saline. The kit may further comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The container may be formed from a variety of materials such as glass or plastic, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit may further include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, media for one or more suitable hosts. The kit may optionally include instructions customarily included in commercial packaging of therapeutic or diagnostic products, containing, for example, information about the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic product.

[0271] The kit is useful for detecting the presence of immunoreactive CLDN18.2 protein in biological samples, including, but not limited to, serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites, or blood, or biopsy samples from body tissues. For example, the kit can include one or more humanized, chimeric, bispecific, or multispecific anti-CLDN18.2 antibodies (or antigen-binding fragments thereof) of the present technology capable of binding to CLDN18.2 protein in a biological sample, a means for determining the amount of CLDN18.2 protein in the sample, and a means for comparing the amount of immunoreactive CLDN18.2 protein in the sample with a standard. One or more of the anti-CLDN18.2 antibodies can be labeled. Kit components (e.g., reagents) can be packaged in suitable containers. The kit can further include instructions for using the kit to detect immunoreactive CLDN18.2 protein.

[0272] In an antibody-based kit, the kit may include, for example, 1) a first antibody of the present technology, e.g., a humanized, chimeric, bispecific, or multispecific CLDN18.2 antibody (or antigen-binding fragment thereof), bound to a solid support that binds to the CLDN18.2 protein, and optionally, 2) a second, different antibody that binds to either the CLDN18.2 protein or the first antibody, and that is conjugated to a detectable label.

[0273] The kit may also include, for example, a buffer, a preservative, or a protein-stabilizing agent. The kit may further include components necessary for detecting a detectable label, such as an enzyme or a substrate. The kit may also include a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package along with instructions for interpreting the results of assays performed using the kit. The kits of the present technology may include written material on or within the kit container. The written material may explain how to use the reagents included in the kit, for example, for detecting CLDN18.2 protein in vitro or in vivo, or for treating a CLDN18.2-associated cancer in a subject in need of such treatment. In certain embodiments, the use of the reagents may be in accordance with the methods of the present technology. [Example]

[0274] The technology of the present invention is further illustrated by the following examples, which should not be construed as limiting in any way, and which demonstrate the preparation, characterization, and use of exemplary anti-CLDN18.2 antibodies of the present technology.

[0275] Example 1: Materials and Methods Construction of hCLDN18.2 and hCLDN18.1 gene expression vectors. Human cDNA encoding the CLDN18.2 protein (SEQ ID NO: 4, shown in Figure 10) was cloned into the pCMV3 expression vector (Sino Biological US Inc., Chesterbrook, PA) and used for stable cell line generation and as a DNA immunogen for mouse immunization. Similarly, human CLDN18.1 cDNA (SEQ ID NO: 5, shown in Figure 11) was cloned into the pCMV3 expression vector and used for stable cell line generation. The human CLDN18.1 cell line was used for counterscreening for selectivity.

[0276] Generation of cell lines expressing hCLDN18.2 and hCLDN18.1. The constructed pCMV3-hCLDN18.2 and pCMV3-hCLDN18.1 expression plasmids were used to transfect cells for the development of the following stable or transient cell lines: 1) 3T3-hCLDN18.2, a mouse embryonic fibroblast cell line used to boost mouse immunization; 2) CHO-hCLDN18.2, used for antibody screening by ELISA and FACS; 3) HEK293-hCLDN18.2, used for antibody screening by ELISA and FACS; and 4) HEK293-hCLDN18.1, used for antibody counterscreening. In the stable cell lines, all clones finally selected showed high expression levels of the target proteins. As shown in Figure 5, all three cell lines transfected with pCMV3-hCLDN18.2 showed at least 100-fold higher hCLDN18.2 expression than the parental control cell line.

[0277] Expression of hCLDN18.2-EL1 in virus-like particles (VLPs). To raise anti-hCLDN18.2-specific antibodies, the EL1 region was targeted because CLDN18.2 and 18.1 share the same EL2 sequence. To drive an immune response against the EL1 region, a vector was constructed to express the hCLDN18.2 EL1 region in virus-like particles (VLPs). pEF6-CLDN18.2EL1 and a pEF6-vector (Thermo Fisher Scientific, Waltham MA) carrying a chimeric gene of CLDN18.2EL1 with the CD81-cytosolic domain (pEF6-CLDN18.2EL1-CD81cd) were transfected into Expi293 cells using the following protocol. Expi293 cells were co-transfected with pEF6-CLDN18.2EL1 or pEF6-CLDN18.2EL1-CD81cd and the VLP core-encoding vector in 4 mL of OptimMEM with 180 μl of Epifectamine added and rotated at 4°C for 24 hours. 24 hours after transfection, the cell suspension was added to 26 mL of Expi293 Expression Medium and cultured at 37°C with shaking at 125 rpm. Expression was performed using the EXPI293™ MembranePro Expression System (Thermo) for 24 hours in shaking culture. Enhancers 1 and 2 (Fisher Scientific, Waltham MA) were added at 150 μl and 1.5 mL, respectively, and cultured for an additional 24 hours. Cells were then centrifuged for FACS, and the supernatant was collected for VLP precipitation. Cells were probed with mouse anti-CEA Ab followed by anti-mouse-PE conjugate. Total mouse IgG was used as an isotype control. FACS analysis showed that more than 90% of the purified VLPs expressed hCLDN18.2EL1 (Figure 6). The purified VLPs were used for booster immunization in mice.

[0278] Human CLDN18.2- and CLDN18.1-Expressing Cancer Cell Lines. To facilitate antibody characterization, in vitro cell killing assay development, and animal xenograft model development, the following cancer cell lines were purchased from ATCC, Manassas, VA: 1) CLDN18.2-expressing gastric cancer cell lines KatoIII, NCI-N87, NUGC4, and SNU-16, and 2) CLDN18.1-expressing lung cancer cell line A529.

[0279] Mouse immunization. Balb / C mice were immunized with a eukaryotic expression vector encoding CLDN18.2. Briefly, 70 μg of pCMV3-hCLDN18.2 plasmid was injected intramuscularly using the HELIOS® Gene Gun System (Bio-Rad, Hercules, CA) every 2 weeks for up to 4 times, resulting in the production of 10 hCLDN18.2-EL1-expressing mice. 7 3T3-hCLDN18.2 cells and a final boost of 10 μg of VLPs were co-administered. Serum titers were monitored over the course of immunization using a CHO-hCLDN18.2 cell-based ELISA assay, using the benchmark IMAB362 antibody as a positive control.

[0280] Hybridoma fusion, screening, and subcloning. After the final boost immunization, three mice with high serum titers against the benchmark IMAB362 antibody were selected for hybridoma fusion experiments. Three days after the final boost, fresh mouse B cells collected from lymph nodes and spleens were pelleted with mouse NS0 myeloma cells by centrifugation and fused by electroporation. The fused cells were resuspended in HAT selection medium and distributed into 96-well microtiter plates (60 plates for each fusion). Hybridomas were grown to at least 50% confluence (10–14 days after fusion) and then screened for the production of CLDN18.2-specific antibodies using a CHO-hCLDN18.2 cell-based ELISA with IMAB362 as a positive control. Positive clones were then confirmed by FACS analysis using CLDN18.2- and CLDN18.1-expressing cells. Only clones with more specific and stronger binding signals than the benchmark IMAB362 antibody were advanced to subcloning, and clonality was confirmed through 2-3 rounds of limiting dilution cloning.

[0281] FACS cell binding assay. Cells were incubated with 5 μg / mL of primary anti-claudin 18.2 antibody in PBS for 30 minutes at 4°C. After washing away excess primary antibody, a secondary phycoerythrin-conjugated antibody specific for human Fc was added. Cells were fixed with 1% paraformaldehyde (PFA) and then analyzed using a FACSCalibur cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA). The control was cells treated with secondary antibody alone, with a mean fluorescence intensity (MFI) of 5.

[0282] Antibody purification and characterization. After screening approximately 4000 hybridoma clones, five clones that showed higher binding signals than the benchmark IMAB362 antibody were selected for subcloning. Cells from the five final subcloned hybridomas were cultured at approximately 10 cells / mL. 6The cells were expanded to 50-100 ml cultures at a density of 100 cells / ml, and the secreted antibodies were purified using standard Protein A or Protein G columns. The purified antibodies were subjected to characterization to further confirm their binding specificity and affinity with the recombinant and endogenous cell lines.

[0283] Antibody gene sequencing. The heavy and light chain variable genes of five selected lead murine antibodies were amplified by PCR using degenerate primers (targeting the leader sequence region) disclosed in Table 2, and the PCR products were used directly for sequencing as a first pass. To obtain a clean readout, a TA cloning / sequencing step was added as a final confirmation. H and V L The sequences were cloned into a human IgG1 constant region to form chimeric antibodies. Plasmids expressing the heavy and light chains of the selected anti-CLDN18.2 antibodies were transiently co-expressed in HEK293 cells. Co-transfection was performed using polyethyleneimine (PEI) as a transfection reagent. Supernatants were collected 6 to 8 days after transfection. The antibodies were purified by protein A chromatography. The amino acid sequences of the heavy and light chain variable regions of the five mouse clones (SEQ ID NOs: 36 to 45) are shown in Figure 13. [Table 2-1] [Table 2-2] R=AG, Y=CT, M=AC, K=GT, S=CG, W=AT, H=ACT, B=CGT, V=ACG, D= AGT, N=ACGT

[0284] Humanization of 32G4 and 47D10 clones. H and V LThe variable regions of murine clones 32G4 and 47D10, containing the CLDN18.2 antibody, were humanized using germline CDR grafting. Briefly, the original murine sequence was aligned against all human germline sequences. The original murine and most closely matched germline sequences were analyzed for sequence trends, and the most appropriate germline framework was selected. The complementarity-determining regions (CDRs) from the parent murine anti-CLDN18.2 antibody were grafted onto the human framework, and backmutations were introduced as needed. For both 32G4 and 47D10, four humanized V H and four humanized V L Sequences were generated for the four V sequences from each clone. H and V L Sequence variants can be combined to generate 16 humanized antibody variants of 32G4 or 47D10. The four humanized Vs of 32G4 and 47D10 H and V L The amino acid sequences of the variants are shown in Figures 14 and 15, respectively.

[0285] Construction of humanized full IgG1 32G4 and 47D10 expression constructs. Two humanized full IgG1 antibody variants were constructed for 32G4 and 47D10 and used for further characterization. The amino acid sequences of the two full humanized IgG1 antibodies from 32G4 and 47D10 are shown in Figure 16 and Figure 17, respectively.

[0286] Engineering and expression of anti-CLDN18.2 x CD3 bispecific antibodies. The variable heavy and light chain gene sequences of the humanized 32G4 variants V8 and V9 and the humanized 47D10 variants V6 and V7 anti-CLDN18.2 antibodies were codon-optimized, synthesized, and inserted into mammalian expression plasmids carrying the constant region gene of human IgG1 (containing LALA mutations: L234A and L235A). The humanized SP34 or OKT3 anti-CD3 scFv was conjugated to the C-terminus of the light chain of the anti-CLDN18.2 antibody. Ten anti-CLDN18.2xCD3 bispecific antibody constructs were generated: 32G4-V8xOKT3, 32G4-V9xOKT3, 47D10-V6xOKT3, 47D10-V7xOKT3, 32G4-V8xhuSP34, 32G4-V9xhuSP34, 47D10-V6xhuSP34, 47D10-V7xhuSP34, 32G4-V8xhuSP34-v5, and 47D10-V7xhuSP34-v5. The final sequences were confirmed by forward and reverse sequencing of the inserts. The amino acid sequences of the ten anti-CLDN18.2xCD3 bispecific antibodies are shown in Figures 20-23 and 26. Plasmids expressing the heavy and light chains of specific anti-CLDN18.2 × CD3 bispecific antibodies were transiently co-expressed in HEK293 cells. Co-transfection was performed using polyethyleneimine (PEI) as a transfection reagent. Supernatants were collected 6–8 days after transfection. The bispecific antibodies were purified by protein A chromatography.

[0287] Example 2: Characterization of anti-CLDN18.2 antibodies of the present technology Five clones (32G4, 47D10, 29G4, 31A6, and 15B10) were selected based on their binding affinity and selective binding to human CLDN18.2 protein. FACS data show MFI values ​​in the upper right panel of each plot.

[0288] As shown in Figure 7, the binding of murine clones 32G4, 47D10, 29G4, 31A6, and 15B10 to CLDN18.2 was at least 1,000-fold stronger than their respective binding to CLDN18.1, as determined by FACS analysis. The binding affinity of the five murine clones to human CLDN18.2 was further evaluated using FACS cell surface binding analysis. As shown in Figure 8, the EC50 values ​​for binding of 32G4, 47D10, 29G4, 31A6, and 15B10 to human CLDN18.2 were 0.502 nM, 1.973 nM, 1.260 nM, 10.903 nM, and 2.196 nM, respectively. The EC50 binding of 32G4-huIgG1-V8, 32G4-huIgG1-V9, 47D10-huIgG1-V6, and 47D10-huIgG1-V7 to human CLDN18.2 was 0.147 nM, 0.129 nM, 0.22 nM, and 0.361 nM, respectively (see Figures 9A-9B). As shown in Figure 19, the humanized 32G4 and 47D10 antibody variants showed enhanced binding to the cynomolgus monkey and mouse claudin18.2 target proteins compared to the IMAB362 positive control antibody.

[0289] ADCC Assay. Antibody-dependent cellular cytotoxicity (ADCC) assays were performed using a bioluminescence reporter assay (Promega Cat. No. 7015, Madison, WI) using engineered Jurkat cells with NFAT-luc and Fc-RIIIa as effector cells and NUGC4 gastric cancer cells as target cells. Briefly, PBMCs were cultured overnight (18 hours) in complete RPMI 1640 medium containing 50 ng / ml IL-2. ADCC assays were performed according to the manufacturer's instructions. Briefly, 2 x 10 4 NUGC4 cells (target cells) were seeded into each well of a 96-well plate and cultured overnight in 100 μl / well of complete growth medium. 50 μl of anti-CLDN18.2 antibody was added to each well (at concentrations of 0, 0.001, 0.01, 0.1, and 1 μg / ml). 6 × 10 cells in 50 μl of medium were cultured overnight. 4Jurkat effector cells were added to each well. The cells were gently mixed. After 21 hours, the plate was centrifuged at 1000 rpm for 5 minutes. 50 μl of cell culture medium was then collected from each well and assayed for lactate dehydrogenase (LDH) release as a measure of cytotoxicity. As shown in Figure 18, the 32G4 and 47D10 monoclonal antibodies of the present technology demonstrated superior antibody-dependent cellular cytotoxicity (ADCC) compared to the IMAB362 positive control antibody.

[0290] These results demonstrate that the anti-CLDN18.2 immunoglobulin-related compositions of the present technology are useful in methods for detecting CLDN18.2 polypeptides in biological samples.

[0291] Example 3: Characterization of the in vivo and in vitro cytotoxic activity of anti-CLDN18.2 antibodies of the present technology In vitro cancer cell killing assay (TDCC: T cell-dependent cytotoxicity). The in vitro cancer cell killing assay was performed using the CellTiter-Glo luminescent cell viability assay, which monitors viable cells by measuring ATP released by viable cells. Briefly, target cells (claudin 18.2-HEK293) were seeded at 10k cells / well in RPMI 1640 medium one day prior to the assay. Cells were cultured until they reached 50% confluence. Anti-CLDN18.2 x anti-CD3 antibodies, 32G4-V8 x OKT3, 32G4-V9 x OKT3, 47D10-V6 x OKT3, and 47D10-V7 x OKT3 (see Figures 20-21), were serially diluted 1:10. IMAB-362-CD3 (OKT3) was used as a positive benchmark control, and isotype-CD3 (OKT3) was used as a negative control. After removing the supernatant from the cultured target cells, claudin18.2-HEK293, diluted antibody was added at 50 μl / well, followed by PBMC at 50 μl / well, and the culture was maintained at 37°C for 48 hours. ATP assays were performed using a CellTiter-Glo kit according to the manufacturer's instructions (Promega, Madison, WI). Figure 24 shows exemplary gastric cancer cell killing (TDCC) assay data for the 32G4 anti-CD3 and 47D10 anti-CD3 bispecific antibody variants compared to the IMAB362 anti-CD3 benchmark antibody and negative isotype control. As shown in Figure 24, the CLDN18.2 bispecific antibody of the present technology exhibited superior TDCC activity at concentrations of 0.001 nM or less compared to the IMAB-362-CD3 positive control antibody.

[0292] Development of an in vivo mouse xenograft model. BRG (Balb / c Rag2 - / - , IL2Rγ - / - A gastric cancer cell line xenograft (CDX) model was developed in mice using Kato-III and NUGC4. This mouse strain lacks adaptive immune cells and NK cells and was used for cancer cell transplantation. 25 million cancer cells were implanted subcutaneously, and tumor volume was measured twice weekly using calipers. When tumors reached approximately 1500 mm 3Once the animals reached 100 mg / kg, they were randomized for efficacy testing. Animals were divided into three groups: Group 1: 5 experimental mice (experimental group with 32G4-V8×huSP34-v5 anti-CLDN18.2×anti-CD3, dose concentration: 5 mg / kg, injection schedule: every other day, route: IV); Group 2: 5 control mice (PBS / saline, injection schedule: every other day; route: IV); Group 3: benchmark antibody (IMAB362×anti-CD3, 5 mg / kg, injection schedule: every other day, route: IV). Tumor measurements were performed twice weekly for up to 40 days after implantation. Figure 27 shows exemplary in vivo efficacy of 32G4-V8×huSP34-v5 in a mouse xenograft gastric cancer model compared to the negative control (PBS). As shown in Figure 27, the 32G4-V8×huSP34-v5 bispecific antibody demonstrated potent inhibition of gastric tumor growth in the mouse model.

[0293] The anti-CLDN18.2 immunoglobulin-related compositions of the present technology have demonstrated potent in vitro and / or in vivo cytotoxic activity against CLDN18.2-associated cancers. Thus, the immunoglobulin-related compositions of the present technology are useful for treating claudin18.2-associated cancers in subjects in need thereof.

[0294] Example 4: Stability of anti-CLDN18.2 antibodies of the present technology The stability of the 32G4-V8×huSP34-v5 molecule was evaluated by freeze / thaw (-80°C) / RT cycles and at 4°C, room temperature (25°C), and 40°C for 1, 3, 7, and 14 days, followed by SEC-HPLC and TDCC cancer cell killing activity assessment. Samples were tested in three buffer conditions (Buffer 1: 20 mM sodium citrate, 0.02% PS80, pH 6.0; Buffer 2: 20 mM sodium citrate, 5.8% sucrose, 0.02% polysorbate 80, pH 7.2; Buffer 3: PBS pH 7.4). As shown in Figure 28, the 32G4-V8×huSP34-v5 bispecific antibody remained stable under the tested conditions, and as shown in Figure 29, the 32G4-V8×huSP34-v5 bispecific antibody maintained its cancer cell killing activity under the tested conditions.

[0295] These results demonstrate that the anti-CLDN18.2 immunoglobulin-related compositions of the present technology are suitable for use in methods for detecting CLDN18.2 polypeptides in biological samples or methods for treating claudin 18.2-associated cancer in a subject in need thereof.

[0296] equivalent The present technology should not be limited with respect to the specific embodiments described in this application, which are intended as an illustration of one particular aspect of the present technology. Many modifications and variations of the present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the present technology. It is to be understood that the present technology is not limited to particular methods, reagents, compounds, compositions, or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0297] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0298] As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations thereof. Any recited range can be readily recognized as fully indicative and allowing for the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into lower thirds, middle thirds, upper thirds, etc. Also, as will be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc., are inclusive of the recited numbers and refer to ranges that can subsequently be divided into subranges as described above. Finally, as will be understood by those skilled in the art, ranges are inclusive of each individual number. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc., and so on.

[0299] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification. The present invention provides, for example, the following items. (Item 1) A bispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding portion that binds to a claudin 18.2 epitope and a second antigen-binding portion that binds to a second epitope, wherein the first antigen-binding portion is a first heavy chain immunoglobulin variable domain (V H ) and the first light chain immunoglobulin variable domain (V L ), wherein the second antigen-binding portion comprises a second V H and the second V L Including, (a) the first V H V of SEQ ID NO: 6 H - CDR1 sequence, V of SEQ ID NO: 7 H -CDR2 sequence, and V of SEQ ID NO: 8H - comprising a CDR3 sequence, and / or said first V L V of SEQ ID NO: 9 L - CDR1 sequence, V of SEQ ID NO: 10 or SEQ ID NO: 155 L -CDR2 sequence, and V of SEQ ID NO: 11 L - comprising a CDR3 sequence, (b) the first V H V of SEQ ID NO: 12 H - CDR1 sequence, V of SEQ ID NO: 13 H -CDR2 sequence, and V of SEQ ID NO: 14 H - comprising a CDR3 sequence, and / or said first V L V of SEQ ID NO: 15 L -CDR1 sequence, V of SEQ ID NO: 16 or SEQ ID NO: 156 L -CDR2 sequence, and V of SEQ ID NO: 17 L - comprising a CDR3 sequence, (c) the first V H V of SEQ ID NO: 18 H - CDR1 sequence, V of SEQ ID NO: 19 H -CDR2 sequence, and V of SEQ ID NO: 20 H - comprising a CDR3 sequence, and / or said first V L V of SEQ ID NO: 21 L - CDR1 sequence, V of SEQ ID NO: 22 L - CDR2 sequence, and V of SEQ ID NO: 23 L - comprising a CDR3 sequence, (d) the first V H V of SEQ ID NO: 24 H - CDR1 sequence, V of SEQ ID NO: 25 H - CDR2 sequence, and V of SEQ ID NO: 26 H - comprising a CDR3 sequence, and / or said first V L V of SEQ ID NO: 27 L - CDR1 sequence, V of SEQ ID NO: 28 L - CDR2 sequence, and V of SEQ ID NO: 29 L - comprising a CDR3 sequence, or (e) the first V H V of SEQ ID NO: 30 H - CDR1 sequence, V of SEQ ID NO: 31 H- CDR2 sequence, and V of SEQ ID NO: 32 H - comprising a CDR3 sequence, and / or said first V L V of SEQ ID NO: 33 L - CDR1 sequence V of SEQ ID NO: 34 L - CDR2 sequence, and V of SEQ ID NO: 35 L - a bispecific antibody or an antigen-binding fragment thereof, comprising the CDR3 sequence. (Item 2) a first antigen-binding moiety that binds to a claudin 18.2 epitope and a second epitope and a second antigen-binding portion that binds to a first heavy chain immunoglobulin variable domain (V H ) and the first light chain immunoglobulin variable domain (V L ), wherein the second antigen-binding portion comprises a second V H and the second V L and the first V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57, and / or (b) the first V L a bispecific antibody or an antigen-binding fragment thereof, comprising an amino acid sequence selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50 to 53, and 58 to 61. (Item 3) The second V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; and / or (b) said second V L 3. The bispecific antibody or antigen-binding fragment of item 1 or 2, wherein the bispecific antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 98, 103, or 158. (Item 4) 4. The bispecific antibody or antigen-binding fragment according to any one of items 1 to 3, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. (Item 5) 5. The bispecific antibody of item 4, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A, or comprising an IgG4 constant region comprising a S228P mutation. (Item 6) The antigen-binding fragment may be Fab, F(ab')2, Fab', scF v , and F v 4. The bispecific antigen-binding fragment of any one of items 1 to 3, selected from the group consisting of: (Item 7) 1. A bispecific antibody comprising a first antigen-binding portion that binds to a claudin 18.2 epitope and a second antigen-binding portion that binds to a second epitope, wherein the bispecific antibody comprises a heavy chain (HC) amino acid sequence comprising SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:159, SEQ ID NO:161, or a variant thereof having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:160, SEQ ID NO:162, or a variant thereof having one or more conservative amino acid substitutions. (Item 8) 8. The bispecific antibody of item 7, comprising an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:81 and SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:159 and SEQ ID NO:160, and SEQ ID NO:161 and SEQ ID NO:162, respectively. (Item 9) A bispecific antibody comprising a first antigen-binding portion that binds to a claudin 18.2 epitope and a second antigen-binding portion that binds to a second epitope, wherein the first antigen-binding portion is a first heavy chain immunoglobulin variable domain (V H ) and the first light chain immunoglobulin variable domain (V L ), wherein the second antigen-binding portion comprises a second V H and the second V L (a) a first V L The sequence is at least partially identical to any one of the light chain immunoglobulin variable domain sequences of SEQ ID NOs: 37, 39, 41, 43, 45, 50 to 53, and 58 to 61. and / or (b) a first V H the sequence is at least 95% identical to a heavy chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46-49, or 54-57, and optionally the second V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; and / or (b) said second V L comprises an amino acid sequence selected from any one of SEQ ID NOs: 98, 103, or 158. (Item 10) A bispecific antibody comprising a first antigen-binding portion that binds to a claudin 18.2 epitope and a second antigen-binding portion that binds to a second epitope, (a) an LC sequence that is at least 95% identical to an LC sequence present in SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 160, or SEQ ID NO: 162; and / or (b) a bispecific antibody comprising an HC sequence that is at least 95% identical to the HC sequence present in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 159, or SEQ ID NO: 161. (Item 11) 11. The bispecific antibody according to any one of items 7 to 10, wherein the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A. (Item 12) a bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other; (a) each of the first polypeptide chain and the fourth polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin; and (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; (b) each of the second polypeptide chain and the third polypeptide chain is, in an N-terminal to C-terminal direction, (i) a heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; (ii) a heavy chain constant domain of the first immunoglobulin; The heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46 to 49, or 54 to 57, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50 to 53, or 58 to 61. A bispecific antibody selected from one of: (Item 13) 13. The bispecific antibody or antigen-binding fragment of item 12, wherein the heavy chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46 to 49, or 54 to 57; the light chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50 to 53, or 58 to 61; the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; and the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 98, 103, or 158. (Item 14) 13. The bispecific antibody or antigen-binding fragment according to item 12, wherein the heavy chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 97, 99, 100, 101, 102, or 157; the light chain variable domain of the first immunoglobulin is selected from any one of SEQ ID NOs: 98, 103, or 158; the heavy chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46 to 49, or 54 to 57; and the light chain variable domain of the second immunoglobulin is selected from any one of SEQ ID NOs: 37, 39, 41, 43, 45, 50 to 53, or 58 to 61. (Item 15) 15. The bispecific antibody or antigen-binding fragment of any one of items 1 to 14, wherein the antibody or antigen-binding fragment binds to a CLDN18.2 polypeptide comprising an extracellular loop 1 (EL1) sequence. (Item 16) 16. The bispecific antibody or antigen-binding fragment of item 15, wherein the extracellular loop 1 (EL1) sequence comprises the amino acid sequence of SEQ ID NO: 2 or the CLDN18.2 polypeptide comprises the amino acid sequence of SEQ ID NO: 4. (Item 17) 17. The bispecific antibody or antigen-binding fragment according to any one of items 1 to 16, wherein the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody. (Item 18) 18. The bispecific antibody according to any one of items 1 to 5 or 7 to 17, wherein the antibody lacks an α-1,6-fucose modification. (Item 19) 19. The bispecific antibody or antigen-binding fragment according to any one of items 1 to 18, wherein the bispecific antibody or antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. (Item 20) 20. The bispecific antibody or antigen-binding fragment of any one of items 1 to 19, wherein the second epitope is CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or a small molecule DOTA hapten. (Item 21) 21. A recombinant nucleic acid sequence encoding the bispecific antibody or antigen-binding fragment according to any one of items 1 to 20. (Item 22) 22. A host cell or vector comprising the recombinant nucleic acid sequence of item 21. (Item 23) A bispecific antibody or antigen-binding fragment according to any one of items 1 to 20, and a pharmaceutically acceptable carrier. and an acceptable carrier. (Item 24) 24. The pharmaceutical composition according to item 23, further comprising an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. (Item 25) A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the bispecific antibody or antigen-binding fragment of any one of Items 1 to 20, or the pharmaceutical composition of Items 23 or 24, wherein the bispecific antibody or antigen-binding fragment specifically binds to CLDN18.2. (Item 26) 26. The method of claim 25, wherein the cancer is a solid tumor. (Item 27) 27. The method of item 25 or 26, wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. (Item 28) 28. The method of any one of items 25 to 27, wherein the bispecific antibody or antigen-binding fragment is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent. (Item 29) 29. The method of item 28, wherein the additional therapeutic agent is one or more of an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, a T cell, and a bisphosphonate therapy. (Item 30) 29. The method of claim 28, wherein the additional therapeutic agent is an immunomodulatory / stimulatory antibody. (Item 31) 31. The method of item 30, wherein the immunomodulatory / stimulatory antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4-1BB antibody, an anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody. (Item 32) 1. A method for detecting cancer in a subject in vivo, comprising: (a) administering to the subject an effective amount of the bispecific antibody or antigen-binding fragment according to any one of items 1 to 20, wherein the bispecific antibody or antigen-binding fragment is configured to localize to cancer cells that express CLDN18.2 and is labeled with a radioisotope; (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the bispecific antibody or antigen-binding fragment that is higher than a reference value. (Item 33) 33. The method of claim 32, wherein the subject has been diagnosed with or is suspected of having cancer. (Item 34) 34. The method of claim 32 or 33, wherein the level of radioactivity emitted by the bispecific antibody or antigen-binding fragment is detected using positron emission tomography or single photon emission computed tomography. (Item 35) 21. The method according to any one of items 1 to 20, wherein the target is a radionuclide conjugated 35. The method of any one of items 32 to 34, further comprising administering an effective amount of an immunoconjugate comprising the bispecific antibody or antigen-binding fragment. (Item 36) 36. The method according to any one of items 32 to 35, wherein the cancer is a solid tumor. (Item 37) 37. The method according to any one of items 32 to 36, wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. (Item 38) 38. The method according to any one of items 25 to 37, wherein the subject is a human. (Item 39) 21. A kit comprising the bispecific antibody or antigen-binding fragment according to any one of items 1 to 20 and instructions for use. (Item 40) 40. The kit of item 39, wherein the bispecific antibody or antigen-binding fragment is conjugated to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label. (Item 41) 41. The kit according to item 39 or 40, further comprising a secondary antibody that specifically binds to the bispecific antibody or antigen-binding fragment according to any one of items 1 to 20. (Item 42) 21. A method for detecting a CLDN18.2 protein expression level in a biological sample, comprising contacting the biological sample with the antibody or antigen-binding fragment according to any one of items 1 to 20, and detecting binding to the CLDN18.2 protein in the biological sample. (Item 43) Heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L an anti-CD3 antibody or antigen-binding fragment thereof comprising: (a) said V H(b) comprises any one of the amino acid sequences of SEQ ID NOs: 99 to 102, or SEQ ID NO: 157, and / or (b) the V L an anti-CD3 antibody or antigen-binding fragment thereof, comprising the amino acid sequence of SEQ ID NO: 103 or SEQ ID NO: 158. (Item 44) a heavy chain immunoglobulin variable domain (V) selected from the group consisting of SEQ ID NO: 101 and SEQ ID NO: 103, and SEQ ID NO: 157 and SEQ ID NO: 158, respectively; H ) and light chain immunoglobulin variable domains (V L 44. The anti-CD3 antibody or antigen-binding fragment of item 43, comprising the amino acid sequence: (Item 45) 45. The anti-CD3 antibody or antigen-binding fragment of item 43 or 44, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. (Item 46) 46. ​​The anti-CD3 antibody or antigen-binding fragment according to any one of Items 43 to 45, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. (Item 47) 47. The anti-CD3 antibody of item 46, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, and K322A. (Item 48) 47. The anti-CD3 antibody of item 46, comprising an IgG4 constant region comprising an S228P mutation. (Item 49) 49. The antibody according to any one of items 43 to 48, wherein the antibody lacks α-1,6-fucose modifications. Anti-CD3 antibody. (Item 50) The antigen-binding fragment may be Fab, F(ab')2, Fab', scF v , and F v 50. The anti-CD3 antigen-binding fragment of any one of items 43 to 45 or 49, selected from the group consisting of: (Item 51) an anti-CD3 multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other; (a) each of the first polypeptide chain and the fourth polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin; and (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; (b) each of the second polypeptide chain and the third polypeptide chain is, in an N-terminal to C-terminal direction, (i) a heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; (ii) a heavy chain constant domain of the first immunoglobulin; 1. An anti-CD3 multispecific antibody, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 99 to 102, or SEQ ID NO: 157, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin comprises SEQ ID NO: 103 or SEQ ID NO: 158. (Item 52) 52. The anti-CD3 multispecific antibody according to any one of Aspects 45 to 51, wherein the multispecific antibody or antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. (Item 53) The multispecific antibodies or antigen-binding fragments may be selected from the group consisting of CD3, GPA33, HER2 / neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr virus nuclear antigen) 1-6, LMP2, p53, lung resistance protein (LRP), Bcl-2, prostate-specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, PlGF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL-6, CD20 , CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis Y (Le y53. The anti-CD3 multispecific antibody or antigen-binding fragment of any one of paragraphs 45 to 52, which binds to a phospho- or phospho-specific antigen, E-cadherin, V-cadherin, GPC3, EpCAM, CD4, CD8, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, CD56, DLL3, PD-1, PD-L1, CD28, CD137, CD99, GloboH, CD24, STEAP1, B7H3, polysialic acid, OX40, OX40-ligand, peptide-MHC complexes (with peptides derived from TP53, KRAS, MYC, EBNA1 to 6, PRAME, MART, tyronsinase, MAGEA1 to A6, pmel17, LMP2, or WT1), or a small molecule DOTA hapten. (Item 54) 54. A composition comprising the antibody or antigen-binding fragment of any one of items 43 to 53 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. (Item 55) 54. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the multispecific anti-CD3 antibody or antigen-binding fragment thereof according to any one of items 45 to 53 or the composition according to item 54. (Item 56) 54. T cells that have been armed ex vivo with the anti-CD3 multispecific antibody or antigen-binding fragment according to any one of items 45 to 53. (Item 57) 21. The bispecific antibody or antigen-binding fragment according to any one of items 1 to 20, wherein the bispecific antibody or antigen-binding fragment binds to T cells and / or CD3. (Item 58) 58. T cells armed ex vivo with the bispecific antibody or antigen-binding fragment of item 57. (Item 59) 57. An ex vivo method for gener...

Claims

[Claim 1] The invention described in the specification.

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