New regulations for anti-CLDN18.2 antibodies

Abstract

Anti-CLDN18.2 antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions comprising same, and uses thereof are provided.

Description

[Technical Field]

[0001] The present disclosure relates generally to novel anti-CLDN18.2 antibodies that specifically bind to human CLDN18.2. [Background technology]

[0002] Claudin-18 (CLDN18) molecules (Genbank accession numbers: splice variant 1 (CLDN18A1 or CLDN18.1): NP_057453, NM_016369, and splice variant 2 (CLDN18A2 or CLDN18.2): NM_001002026, NP_001002026) are integral transmembrane proteins with molecular weights of approximately 27.9 / 27.72 kD. CLDN18 proteins are located in epithelial and endothelial tight junctions, which organize a network of interconnected strands of intramembrane particles between adjacent cells. CLDN18 and occludin are the most prominent transmembrane protein components in tight junctions. Due to their strong cell-cell adhesion properties, these tight junction proteins form a primary barrier to prevent and regulate paracellular transport of solutes and also restrict the lateral diffusion of membrane lipids and proteins, maintaining cell polarity. They are therefore crucially involved in organizing epithelial tissue architecture.

[0003] CLDN18 is a member of the tetraspanin family and has four hydrophobic regions. CLDN18 exhibits several different conformations that can be selectively processed by antibodies (see Sahin U, Koslowski M, Dhaene K, et al. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development [J]. Clinical Cancer Research, 2008, 14(23):7624-7634). CLDN18 conformation 1, as described for most CLDN family members, has all four hydrophobic regions that serve as transmembrane domains (TM), and two extracellular loops (loop 1 encompassed by hydrophobic regions 1 and 2; loop 2 encompassed by hydrophobic regions 3 and 4) are formed. The second conformation (CLDN18 conformation 2) shows that the portion between the first and fourth transmembrane domains (loop D3) is extracellular, as described for PMP22, because the second and third hydrophobic domains do not completely cross the plasma membrane. The third conformation (CLDN18 conformation 3) shows a large extracellular domain in which two internal hydrophobic regions are encompassed by the first and fourth hydrophobic regions. Due to the classical N-glycosylation site in loop D3, the CLDN18 topology variants CLDN18 topology 2 and CLDN18 topology 3 contain additional extracellular N-glycosylation sites.

[0004] CLDN18 exists in two splice variants in both mice and humans. The splice variants, CLDN18.1 and CLDN18.2, differ in the first 21 amino acids of the N-terminus, including the first TM and loop 1, but share the same C-terminal protein sequence (see Niimi T, Nagashima K, Ward JM, et al. Claudin-18, a novel downstream target gene for the T / EBP / NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing [J]. Molecular and cellular biology, 2001, 21(21):7380-7390).

[0005] CLDN18.1 is selectively expressed in normal lung and gastric epithelium, while CLDN18.2 is expressed exclusively in gastric cells. Most importantly, CLDN18.2 expression is restricted to differentiated, short-lived cells of the gastric epithelium and is absent from the gastric stem cell region. Even using highly sensitive RT-PCR, both variants are undetectable in any other normal human organs. However, they are highly expressed in several cancer types, including gastric, esophageal, pancreatic, and lung tumors, as well as in human cancer cell lines (see Matsuda Y, Semba S, Ueda J, et al. Gastric and intestinal claudin expression at the invasive front of gastric carcinoma [J]. Cancer science, 2007, 98(7):1014-1019).

[0006] There is a significant need for novel anti-CLDN18.2 antibodies that can be used for the treatment of diseases positive for CLDN18.2 expression, such as cancer. Summary of the Invention

[0007] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody.

[0008] The present disclosure provides, inter alia, novel monoclonal anti-CLDN18.2 antibodies, nucleotide sequences encoding same, and uses thereof.

[0009] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof against human CLDN18.2, capable of binding to an epitope comprising at least one, two, or three of the amino acid residues at positions D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of the amino acid sequence of SEQ ID NO: 30.

[0010] In certain embodiments, the epitope includes the amino acid residue at position E56. In certain embodiments, the epitope does not include at least one of the following residues: A42 or N45. In certain embodiments, the epitope includes amino acid residues at positions W30, L49, W50, R55, and E56. In certain embodiments, the epitope further includes one or more amino acid residues: T41, N45, Y46, R51, F60, E62, and R80. In certain embodiments, the epitope further includes one or more amino acid residues: D28, V43, N45, Y46, Y66, L72, L76, and V79.

[0011] In one aspect, the present disclosure provides an antibody capable of specifically binding to human CLDN18.2 and having the following characteristics: a) binds to cells expressing human CLDN18.2 with a Kd value of 2.5 nM or less (or 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or less) as measured by a KinExA assay; b) binds to cells expressing human CLDN18.2 with an EC50 value of 70 μg / ml or less (or 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml or less) as measured by flow cytometry; c) inducing complement-dependent cytotoxicity (CDC) on cells expressing human CLDN18.2 at an EC50 value of 1 μg / ml or less (or 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 μg / ml or less) as measured by a cytotoxicity assay; d) induce antibody-dependent cellular cytotoxicity (ADCC) on cells expressing human CLDN18.2 with an EC50 value of 2 μg / ml or less (or 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / ml or less) as measured by an ADCC reporter assay. The present invention provides an isolated antibody or antigen-binding fragment thereof, which can have at least one of:

[0012] In certain embodiments, the cells include NUGC4 cells, SNU-620 cells, SNU-601 cells, KATOIII cells, or comparable cells thereof having human CLDN18.2 protein expression levels comparable to or lower than those of NUGC4 cells, SNU-620 cells, SNU-601 cells, or KATOIII cells.

[0013] In certain embodiments, the cells comprise cells that highly express human CLDN18.2, cells that moderately express human CLDN18.2, or cells that lowly express human CLDN18.2.

[0014] In certain embodiments, cells that highly express human CLDN18.2 are those that express at least 40% (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 40–100%, 50–100%, 60–100%, 70–100%, 80–100%, 90–100%, 40–90%, 50–90%, 60–90%, 70–90%, 80–90%, 40–80%, 40–70%, 40–60%, 40–50%, 50–80%, 50–70%, 50–60%, 60–80%, 60–70%, or 70–80% were used for immunohistochemistry (IH) C), cells that moderately express human CLDN18.2 express human CLDN18.2 at a level where at least 30% (or at least 35%) to less than 40% of the cells stain positively by IHC, with an intensity of at least 1+ to less than 2+ as measured by IHC, and cells that lowly express human CLDN18.2 express human CLDN18.2 at a level where 0 to less than 30% of the cells (e.g., 5%, 10%, 15%, 20%, 25%, 5-25%, 10-25%, 15-25%, 20-25%, 5-20%, 5-15%, 5-10%, 10-20%, or 10-15%) stain positively by IHC, with an intensity of more than 0 but less than 1+ as measured by IHC.

[0015] In certain embodiments, the EC50 value for binding to NUGC4 cells is 70 μg / ml or less (or 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10 μg / ml or less).

[0016] In certain embodiments, ADCC on NUGC4 cells at an EC50 value of 2 μg / ml or less (or 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / ml or less) as measured by an ADCC reporter assay.

[0017] In one aspect, the present disclosure provides an antibody capable of specifically binding to human CLDN18.2 and having the following characteristics: a) binds to human CLDN18.2 with a Kd value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 15% or less of the Kd value of IMAB362 as measured by KinExA assay; b) binds to cells expressing human or mouse CLDN18.2 with an EC50 value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or 10% or less of the EC50 value of IMAB362 as measured by flow cytometry assay; c) induces complement-dependent cytotoxicity (CDC) on cells expressing human CLDN18.2 at an EC50 value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less of the EC50 value of IMAB362 as measured by a cytotoxicity assay; and d) induce antibody-dependent cellular cytotoxicity (ADCC) on cells expressing human CLDN18.2 at an EC50 value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% or less of the EC50 value of IMAB362 as measured by an ADCC reporter assay. and IMAB362 is an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73. An isolated antibody or antigen-binding fragment thereof is provided.

[0018] In certain embodiments, the cells include NUGC4 cells, SNU-620 cells, SNU-601 cells, KATOIII cells, or cell lines having human CLDN18.2 protein expression levels comparable to or lower than those of NUGC4 cells, SNU-620 cells, SNU-601 cells, or KATOIII cells. In certain embodiments, the cells include cells that highly express human CLDN18.2, cells that moderately express human CLDN18.2, or cells that lowly express human CLDN18.2.

[0019] In certain embodiments, cells that highly express human CLDN18.2 are those that express at least 40% (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-80%, 50-70%, 50-60%, 60-80%, 60-70%, or 70-80% were achieved by IHC. cells that moderately express human CLDN18.2 express human CLDN18.2 at a level where at least 30% (or at least 35%) to less than 40% of the cells stain positive by IHC at an intensity of at least 1+ to less than 2+ as measured by IHC; and cells that lowly express human CLDN18.2 express human CLDN18.2 at a level where 0 to less than 30% (e.g., 5%, 10%, 15%, 20%, 25%, 5-25%, 10-25%, 15-25%, 20-25%, 5-20%, 5-15%, 5-10%, 10-20%, or 10-15%) of the cells stain positive by IHC at an intensity of greater than 0 but less than 1+ as measured by IHC.

[0020] In certain embodiments, an isolated antibody or antigen-binding fragment thereof can bind to an epitope comprising at least one, two, or three of the amino acid residues at positions D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the epitope comprises the amino acid residue at position E56. In certain embodiments, the epitope does not comprise at least one of the following residues: A42 or N45. In certain embodiments, the epitope comprises amino acid residues at positions W30, L49, W50, R55, and E56. In certain embodiments, the epitope further comprises one or more amino acid residues: T41, N45, Y46, R51, F60, E62, and R80. In certain embodiments, the epitope further comprises one or more amino acid residues: D28, V43, N45, Y46, Y66, L72, L76, and V79.

[0021] In one aspect, the disclosure provides a polypeptide comprising heavy chain HCDR1, HCDR2, and HCDR3, and / or light chain LCDR1, LCDR2, and LCDR3 sequences, the HCDR1 sequence comprises GYNMN (SEQ ID NO: 1) or TYFIGVG (SEQ ID NO: 13) or a homologous sequence thereof with at least 80% sequence identity; the HCDR2 sequence comprises X1IDPYYX2X3TX4YNQKFX5G (SEQ ID NO: 32) or HIWWNDNKYYNTALKS (SEQ ID NO: 15) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity; the HCDR3 sequence comprises X6X7X8GNAFDY (SEQ ID NO: 33) or MGSGAWFTY (SEQ ID NO: 17) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence is KSSQX9LX 10 NX 11 GNX 12KNYLT (SEQ ID NO: 34) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity, LCDR2 sequence is WASTRX 13 S (SEQ ID NO: 35) or a homologous sequence thereof of at least 80% sequence identity, LCDR3 array is QNDYX 14 X 15 PX 16 T (SEQ ID NO: 36) or a homologous sequence thereof of at least 80% sequence identity; X1 is N or Y or H, X2 is G or V, X3 is A or G or T, X4 is R or T or S, X5 is K or R, X6 is S or M, X7 is Y or F, X8 is Y or H, X9 is S or N, and X 10 is L or F, and X 11 is S or N, and X 12 is Q or L, and X 13 is E or K, and X 14 is S or Y, and X 15 is F or Y and X 16 is F or L, Anti-CLDN18.2 antibodies or antigen-binding fragments thereof are provided.

[0022] In one aspect, the present disclosure provides a method for producing a heavy chain variable region comprising: a) an HCDR1 comprising a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 13; b) an HCDR2 comprising a sequence selected from SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 22, and c) HCDR3 comprising a sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, and SEQ ID NO: 21 and / or the light chain variable region comprises d) LCDR1 comprising the sequences of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 20; e) LCDR2 comprising the sequences of SEQ ID NO: 4 and SEQ ID NO: 16, and f) LCDR3 comprising a sequence selected from SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 18 The present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof comprising:

[0023] In certain embodiments, the heavy chain variable region comprises: a) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 3, and an HCDR3 comprising the sequence of SEQ ID NO: 5; b) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, and an HCDR3 comprising the sequence of SEQ ID NO: 5; c) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 9, and an HCDR3 comprising the sequence of SEQ ID NO: 11; d) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 15, and an HCDR3 comprising the sequence of SEQ ID NO: 17; e) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 19, and an HCDR3 comprising the sequence of SEQ ID NO: 21; and f) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 22, and an HCDR3 comprising the sequence of SEQ ID NO: 5 The antibody or antigen-binding fragment thereof provided herein is selected from the group consisting of:

[0024] In certain embodiments, the light chain variable region comprises: a) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; b) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 8; c) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; d) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 12; e) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 14, an LCDR2 comprising the sequence of SEQ ID NO: 16, and an LCDR3 comprising the sequence of SEQ ID NO: 18; and f) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6. The antibody or antigen-binding fragment thereof provided herein is selected from the group consisting of:

[0025] In certain embodiments, a) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 3, and an HCDR3 comprising the sequence of SEQ ID NO: 5; or the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; b) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, and an HCDR3 comprising the sequence of SEQ ID NO: 5; or the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 8; c) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 9, and an HCDR3 comprising the sequence of SEQ ID NO: 11; or the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; d) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 15, and an HCDR3 comprising the sequence of SEQ ID NO: 17; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 12; e) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 19, and an HCDR3 comprising the sequence of SEQ ID NO: 21; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 14, an LCDR2 comprising the sequence of SEQ ID NO: 16, and an LCDR3 comprising the sequence of SEQ ID NO: 18; or f) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 22, and an HCDR3 comprising the sequence of SEQ ID NO: 5; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; An antibody or antigen-binding fragment thereof provided herein.

[0026] In certain embodiments, the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47 and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity that still maintain specific binding affinity to CLDN18.2.

[0027] In certain embodiments, the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity that still maintain specific binding affinity to CLDN18.2.

[0028] In certain embodiments, a) the heavy chain variable region comprises the sequence of SEQ ID NO: 23 and the light chain variable region comprises the sequence of SEQ ID NO: 24; b) the heavy chain variable region comprises the sequence of SEQ ID NO: 25 and the light chain variable region comprises the sequence of SEQ ID NO: 26; c) the heavy chain variable region comprises the sequence of SEQ ID NO: 27 and the light chain variable region comprises the sequence of SEQ ID NO: 28; d) the heavy chain variable region comprises the sequence of SEQ ID NO: 29 and the light chain variable region comprises the sequence of SEQ ID NO: 26 or 28; e) the heavy chain variable region comprises the sequence of SEQ ID NO: 37 and the light chain variable region comprises the sequence of SEQ ID NO: 38; f) the heavy chain variable region comprises the sequence of SEQ ID NO: 39 and the light chain variable region comprises the sequence of SEQ ID NO: 40; g) the heavy chain variable region comprises the sequence of SEQ ID NO: 41 and the light chain variable region comprises the sequence of SEQ ID NO: 42; h) the heavy chain variable region comprises the sequence of SEQ ID NO: 43 and the light chain variable region comprises the sequence of SEQ ID NO: 44; i) the heavy chain variable region comprises the sequence of SEQ ID NO: 45 and the light chain variable region comprises the sequence of SEQ ID NO: 46; or j) the heavy chain variable region comprises the sequence of SEQ ID NO: 47 and the light chain variable region comprises the sequence of SEQ ID NO: 48; An antibody or antigen-binding fragment thereof provided herein.

[0029] In certain embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof provided herein further comprise one or more of heavy chains HFR1, HFR2, HFR3, and HFR4 and / or one or more of light chains LFR1, LFR2, LFR3, and LFR4; HFR1 is QVQLVQSGAEVKKPGASVKVSCKASGYX 17 FT (SEQ ID NO: 54) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity, HFR2 is WVX 18 QAPGQGLEWX 19 G (SEQ ID NO: 55) or a homologous sequence thereof with at least 80% (or at least 90%) sequence identity, The HFR3 sequence is RVTX 20TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 56) or a homologous sequence thereof with at least 80% (or at least 85%, 90%, 95%) sequence identity; HFR4 comprises WGQGTTVTVSS (SEQ ID NO: 57) or a homologous sequence thereof with at least 80% sequence identity; LFR1 is DIVMTQSPDSLAVSLGERATX 21 NC (SEQ ID NO: 58) or a homologous sequence thereof with at least 80% (or at least 85%, 90%, 95%) sequence identity, LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO: 59) or a homologous sequence thereof of at least 80% (or at least 85%, 90%) sequence identity; LFR3 is GVPDRFX 22 GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 60) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity, LFR4 comprises FGGGTKVEIK (SEQ ID NO: 61) or a homologous sequence thereof with at least 80% (or at least 90%) sequence identity; X 17 is T or S, and X 18 is R or K, and X 19 is M or I, and X 20 is M or L, and X 21 is I or M, and X 22 is either S or T.

[0030] In certain embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 62 and 63; HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 64 and 65; HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 66 and 67; HFR4 comprises the sequence of SEQ ID NO: 57, LFR1 comprises a sequence from the group consisting of SEQ ID NOs: 68 and 69; LFR2 comprises the sequence of SEQ ID NO: 59, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 70 and 71; LFR4 comprises the sequence of SEQ ID NO:61.

[0031] In certain embodiments, an antibody or antigen-binding fragment thereof provided herein that further comprises one or more amino acid residue substitutions or modifications still maintains specific binding affinity for CLDN18.2. In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences and / or one or more non-CDR regions of the VH or VL sequence.

[0032] In certain embodiments, the antibody binds to an epitope comprising at least one, two, or three of the amino acid residues at positions D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of human CLDN18.2 having the amino acid sequence of SEQ ID NO: 30.

[0033] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin constant region, optionally the constant region of a human Ig, or optionally the constant region of a human IgG. In certain embodiments, the constant region comprises the constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the constant region of human IgG1 comprises SEQ ID NO: 49 or a homologous sequence thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0034] In certain embodiments, the constant region comprises one or more amino acid residue substitutions or modifications that confer increased CDC or ADCC relative to the wild-type constant region. In certain embodiments, the constant region comprises one or more amino acid residue substitutions relative to SEQ ID NO: 49 selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof. In certain embodiments, the constant region comprises the sequence of SEQ ID NO: 51.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof is afucosylated.

[0036] In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. In certain embodiments, the antibody or antigen-binding fragment thereof is a camelized single-domain antibody, diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody, or bivalent domain antibody.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof is bispecific, and can specifically bind to a first epitope on CLDN18.2 and a second epitope on a second antigen that is either on CLDN18.2 or different from CLDN18.2. In certain embodiments, the second antigen is optionally an immune-related target selected from the group consisting of PD-L1, PD-L2, PD-1, CLTA-4, TIM-3, LAG3, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, ICAM-1, NKG2C, SLAMF7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, and CD83.

[0038] In certain embodiments, the second antigen comprises a tumor antigen. In certain embodiments, the tumor antigen is present on cells that express CLDN18.2.

[0039] In several embodiments, tumor antigens include CA-125, gangliosides G(D2), G(M2), and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptide, PSA, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFR (e.g., VEGFR3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, IL-2R, or CO17-1A.

[0040] In certain embodiments, the antibody or antigen-binding fragment thereof can specifically bind to mouse CLDN18.2. In certain embodiments, the antibody or antigen-binding fragment thereof does not bind to human CLDN18.1.

[0041] In certain embodiments, the antibody or antigen-binding fragment thereof is linked to one or more conjugate moieties, which in certain embodiments comprise a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer agent.

[0042] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment thereof provided herein for binding to CLDN18.2.

[0043] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein and one or more pharmaceutically acceptable carriers.

[0044] In one aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein. In one aspect, the present disclosure provides a vector comprising an isolated polynucleotide provided herein. In one aspect, the present disclosure provides a host cell comprising a vector provided herein.

[0045] In one aspect, the present disclosure provides a method of expressing an antibody or antigen-binding fragment thereof provided herein, the method comprising culturing a host cell provided herein under conditions in which a vector provided herein is expressed.

[0046] In one aspect, the present disclosure provides a method for treating a disease or condition in a subject in which modulation of CLDN18.2 activity would be beneficial, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein to the subject. In certain embodiments, the disease or condition is a CLDN18.2-associated disease or condition. In certain embodiments, the disease or condition is cancer, optionally a CLDN18.2-expressing cancer. In certain embodiments, the subject is identified as having cancer cells that express CLDN18.2. In certain embodiments, the subject is identified as having cancer cells that highly express CLDN18.2, intermediately express CLDN18.2, or lowly express CLDN18.2.In certain embodiments, cancer cells that highly express CLDN18.2 are those that express at least 40% (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%) of the cells at an intensity of at least 2+ as measured by IHC. At least 95%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-80%, 50-70%, 50-60%, 60-80%, 60-70%, or 70-80%) Cancer cells that moderately express CLDN18.2 express CLDN18.2 at a level where at least 30% (or at least 35%) to less than 40% of the cells stain positively by IHC, with an intensity of at least 1+ to less than 2+ as measured by IHC; and cancer cells that lowly express CLDN18.2 express CLDN18.2 at a level where more than 0 but less than 30% (e.g., 5%, 10%, 15%, 20%, 25%, 5-25%, 10-25%, 15-25%, 20-25%, 5-20%, 5-15%, 5-10%, 10-20%, or 10-15%) of the cells stain positively by IHC, with an intensity of more than 0 but less than 1+ as measured by IHC.

[0047] In certain embodiments, the method further comprises administering a therapeutically effective amount of a second therapeutic agent.In certain embodiments, the disease or condition is a CLDN18.2-related disease or condition.In certain embodiments, the disease or condition is cancer, optionally a cancer that expresses CLDN18.2.

[0048] In certain embodiments, the subject is a human.

[0049] In certain embodiments, administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0050] In certain embodiments, the method further comprises administering a therapeutically effective amount of a second therapeutic agent, in certain embodiments, the second therapeutic agent is selected from a chemotherapeutic agent, an anti-cancer agent, a radiation therapy, an immunotherapy agent, an anti-angiogenic agent, a targeted therapy agent, a cellular therapy agent, a gene therapy agent, a hormonal therapy agent, or a cytokine.

[0051] In one aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent.

[0052] In one aspect, the present disclosure provides a method for modulating CLDN18.2 activity in a cell that expresses CLDN18.2, the method comprising exposing the cell that expresses CLDN18.2 to an antibody or antigen-binding fragment thereof provided herein.

[0053] In one aspect, the present disclosure provides a method for detecting the presence or amount of CLDN18.2 in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of CLDN18.2 in the sample.

[0054] In one aspect, the present disclosure provides a method for diagnosing a CLDN18.2-associated disease or condition in a subject, the method comprising: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of CLDN18.2 in the sample; and c) correlating the presence or amount of CLDN18.2 with the presence or condition of a CLDN18.2-associated disease or condition in the subject.

[0055] In one aspect, the disclosure provides use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CLDN18.2-associated disease or condition in a subject.

[0056] In one aspect, the present disclosure provides use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a diagnostic reagent for diagnosing a CLDN18.2-associated disease or condition.

[0057] In one aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, useful for detecting CLDN18.2.

[0058] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CLDN18.2, including an antigen-binding fragment thereof provided herein.

[0059] In certain embodiments, the antigen-binding fragment is a Fab or scFv.

[0060] In certain embodiments, CAR is bispecific.In certain embodiments, CAR can specifically bind to a first epitope and a second epitope on CLDN18.2.In certain embodiments, the second epitope is on CLDN18.2.In certain embodiments, the second epitope is on a second antigen that is different from CLDN18.2.In certain embodiments, the second antigen comprises a tumor antigen.

[0061] In one aspect, the present disclosure provides a nucleic acid sequence encoding a chimeric antigen receptor (CAR) provided herein. In one aspect, the present disclosure provides a cell comprising the nucleic acid sequence provided herein. In one aspect, the present disclosure provides a cell genetically modified to express a CAR.

[0062] In one aspect, the present disclosure provides a vector comprising a nucleic acid sequence provided herein.

[0063] In one aspect, the present disclosure provides a method of stimulating a T cell-mediated immune response against cells or tissues expressing CLDN18.2 in a mammal, the method comprising administering to the mammal an effective amount of cells genetically modified to express a CAR provided herein.

[0064] In one aspect, the present disclosure provides a method of treating a mammal having a CLDN18.2-associated disease or condition, the method comprising administering to the mammal an effective amount of a cell provided herein, thereby treating the mammal.

[0065] In certain embodiments, the cells are autologous T cells. In certain embodiments, the CLDN18.2-associated disease or condition is cancer. In certain embodiments, the mammal is a human subject. In certain embodiments, the mammal is identified as having cancer cells that express CLDN18.2, and optionally, the mammal is identified as having cancer cells that highly express CLDN18.2, intermediately express CLDN18.2, or lowly express CLDN18.2. [Brief explanation of the drawings]

[0066] [Figure 1-1] Figure 1A is a scatter plot showing the binding affinity of 7C12, 11F12, 12E9, and 26G6 to HEK293-CLDN18.2 cells expressing human CLDN18.2, and Figure 1B is a scatter plot showing the binding affinity of 59A9, 18B10, 7C12, 12C12, and 11F12 to HEK293-CLDN18.2 cells expressing human CLDN18.2. [Figure 1-2]Figure 1C is a scatter plot showing the binding affinity of 7C12, 11F12, 12E9, and 26G6 to NUGC4 cells. Figure 1D is a scatter plot showing the binding affinity of 59A9, 18B10, 7C12, 12C12, and 11F12 to NUGC4 cells. Herein, the names of cell lines containing "CLDN18.2" described in the figures and examples refer to human CLDN18.2 unless otherwise specified. Mouse CLDN18.2 is abbreviated as "mCLDN18.2." [Figure 2-1] Figure 2A is a scatter plot showing that chimeric antibodies 7C12-C, 11F12-C, and 12E9-C bind to HEK293-CLDN18.2 cells with an EC50 of approximately 0.6 μg / ml, and 26G6-C binds to HEK293-CLDN18.2 cells with an EC50 of approximately 1 μg / ml. Figure 2B is a scatter plot showing that the CDC potency of antibodies 7C12-C, 11F12-C, 12E9-C, and 26G6-C is more than two-fold increased over IMAB362. [Figure 2-2] Figure 2C is a scatter plot showing that 59A9-C has a slightly higher EC50 (1.3 μg / ml) than 18B10-C (1.0 μg / ml). Figure 2D is a scatter plot showing that antibodies 59A9-C and 18B10-C had a greater than 3-fold increase in CDC potency compared to IMAB362. [Figure 3-1] Figure 3A is a scatter plot showing that 18B10-C binds to MKN45-CLDN18.2-high cells with significantly higher affinity than IMAB362. Figure 3B is a scatter plot showing that when using MKN45-CLDN18.2 cells, the two curves suggest that 18B10-C had better ADCC activity than IMAB362. [Figure 3-2] Figure 3C is a scatter plot showing that 18B10-C binds to MKN45-CLDN18.2-medium cells with significantly higher affinity than IMAB362. Figure 3D is a scatter plot showing that in MKN45-CLDN18.2-medium cells, 18B10-C had a greater than 50-fold increase in ADCC potency, as measured by EC50, than IMAB362. [Figure 4-1] Figure 4A is a scatter plot showing that three of the four chimeric antibodies, except for 26G6-C, bind to NUGC4 cells with an EC50 of approximately 10 μg / ml. Figure 4B is a scatter plot showing the ADCC activity of the 7C12-C, 11F12-C, 12E9-C, and 26G6-C chimeric antibodies against NUGC4 cells. [Figure 4-2] Figure 4C is a scatter plot showing that the 18B10-C chimeric antibody, but not 59A9-C, binds to NUGC4 cells with an EC50 of approximately 10 μg / ml. Figure 4D is a scatter plot showing the ADCC activity of the 59A9-C and 18B10-C chimeric antibodies against NUGC4 cells in a separate experiment. [Figure 5] FIG. 5 is a bar graph showing the selective binding of 18B10-C and IMAB362 to CLDN18.2- or CLDN18.1-expressing HEK293 cells. [Figure 6] 6A and 6B are scatter plots showing the binding affinity of hybridoma antibody 18B10 when competing with 10 μg / ml of 1MAB362 for MKN45-CLDN18.2-high cells and with 5 μg / ml of 18B10-C for MKN45-CLDN18.2-high cells, respectively. Hybridoma antibody 18B10 was able to competitively block the binding of 1MAB362 to MKN45-CLDN18.2-high cells. [Figure 7] Figures 7A-B are bar graphs showing the binding signals of chimeric antibodies to mutated hCLDN 18.2 variants using epitope mapping. Binding of 18B10-C was completely lost when E56 was mutated to Q. This change also applied to IMAB362 and other chimeric antibodies except for 59A9-C. Other amino acids, such as A42 and N45, also contributed to binding to some extent for IMAB362 and other antibodies, but not for 18B10-C. [Figure 8]Figure 8A is a scatter plot showing the binding affinity of all humanized variants and their chimeric counterparts. Figure 8B is a scatter plot showing the binding affinity of humanized antibody 18B10-HaLa compared to IMAB362 and the control hlgG1. 18B10-HaLa binds well to murine CLDN18.2, with better potency and a higher MFI than IMAB362. [Figure 9] 9 is a scatter plot showing the CDC effect of humanized antibody 18B10-HaLa on HEK293-CLDN18.2. 18B10-HaLa has more than 20-fold higher CDC activity than IMAB362. [Figure 10] Figure 10A is a scatter plot showing the binding affinity of humanized variants of 18B10 compared to chimeric 18B10 for MKN45 cells expressing intermediate levels of CLDN18.2 protein (MKN45-CLDN18.2-Medium). All humanized variants of 18B10 bind to MKN45-CLDN18.2-Medium cells with affinities comparable to those of chimeric 18B10. Figure 10B is a scatter plot showing ADCC reporter assays of 18B10-HaLa and IMAB362 in MKN45-CLDN 18.2-Medium cells. 18B10-HaLa has a significantly lower EC50 (0.05 μg / ml) than IMAB362, consistent with that of chimeric 18B10. [Figure 11] Figure 11A is a scatter plot showing the binding affinity of 18B10-HaLa to NUGC4 cells. Figure 11B is a scatter plot showing the ADCC effect of 18B10-HaLa compared to IMAB362. 18B10-HaLa has significantly better ADCC potency than IMAB362. [Figure 12] Figure 12 is a scatter plot showing the results of an ADCC assay using PBMCs (donor ID: A18Z017017) as effector cells. 18B10-HaLa shows significantly better ADCC potency than IMAB362. [Figure 13]Figure 13A is a bar graph showing the results of 18B10-HaLa epitope mapping (Ab concentration: 10 μg / ml), and Figure 13B is a bar graph showing the results of 59A9-C epitope mapping (Ab concentration: 10 μg / ml). [Figure 14-1] Figure 14A is a scatter plot showing the ADC cytotoxicity of both 18B10-HaLa-vcMMAE and IMAB362-vcMMAE in HEK293-CLDN18.2 cells. Both 18B10-HaLa-vcMMAE and IMAB362-vcMMAE induced cytotoxicity in HEK293-CLDN18.2 cells, whereas the control hIgG1-vcMMAE did not. Figure 14B is a scatter plot showing the results of the ADC cytotoxicity of 18B10-HaLa-MMAE and IMAB362-MMAE in NUGC-4 cells. 18B10-HaLa-vcMMAE demonstrated dose-dependent cell growth inhibition starting at a concentration of 0.03 μg / ml, whereas IMAB362-vcMMAE inhibited cell growth only at a higher concentration of 10 μg / ml. [Figure 14-2] Figure 14C is a scatter plot showing the ADC cytotoxicity of 18B10-HaLa-MMAE and IMAB362-MMAE in MKN45-CLDN18.2-high. 18B10-HaLa-vcMMAE reached 86% maximum cell killing, which was higher than IMAB362 (60%). [Figure 15] Figure 15 is a scatter plot showing the change in tumor volume over time for the isotype control, IMAB362, and 18B10-HaLa, which exhibits significantly better antitumor activity as measured by tumor size and TGI (tumor growth inhibition) than IMAB362 or the isotype control. [Figure 16] Figure 16 is a scatter plot showing the change in tumor volume over time for the model group (no PBMCs), isotype control, 18B10-HaLa 3 mg / kg (mpk), and 18B10-HaLa 10 mpk, respectively. Either 3 mpk or 10 mpk 18B10-HaLa had significant inhibition of tumor growth compared to the isotype control or PBMC control. [Figure 17] Figure 17 is a scatter plot showing the change in tumor volume over time for isotype control, 18B10-HaLa 0.1 mpk, 18B10-HaLa 0.3 mpk, and 18B10-HaLa 1 mpk, respectively. The results show that the antitumor activity of 18B10-HaLa is dose-dependent. [Figure 18-1] Figures 18A-I are scatter plots showing 18B10-HaLa hIgG1 binding to hFcγRI-his, hFcγRIIB-his, hFcγRIIIA(F176)-his, hFcγRIIIA(V176)-his, mouse FcγRI-his, mouse FcγRIIB-his, mouse FcγRIIIA-his, FcγRIV-his, and Cyno FcγRIII-his, mouse FcγRIIIA-his, FcγRIV-his, and Cyno FcγRIII-his. There was no significant difference between 18B10-HaLa_VLPYLL and 18B10-HaLa-wt in binding to human FcγRI or FcγRIIB. However, 18B10-Hala_VLPYLL showed a 10-fold increased binding to human FcγRIIIA(F176) and FcγRIIIA(V176) compared to its wild-type (wt) counterpart. Similar results were observed with mouse and cyno FcγRs. [Figure 18-2] See above. [Figure 18-3] See above. [Figure 19] Figures 19A-19B are scatter plots showing the binding affinity of 18B10 HaLa hlgG1 to huFcRn-biotin and human C1q, respectively. The results in Figure 19A show that there is no significant difference in FcRn binding between 18B10-HaLa_VLPYLL and 18B10-HaLa wt. The results in Figure 19B show that 18B10-HaLa_VLPYLL has a slightly better binding signal at lower C1q concentrations than 18B10-HaLa wt. [Figure 20]Figure 20A is a scatter plot showing the results of an 18B10-HaLa hlgG1 reporter assay for NUGC-4 using Jurkat-NFAT-luc-FcγRIIIA-V176 as effector cells (E / T ratio = 6:1). 18B10-HaLa-VLPYLL shows a 3-fold increase in ADCC potency (EC50 ∼0.0097 μg / ml) compared to that of 18B10-HaLawt (EC50 ∼0.032 μg / ml). Figure 20B is a scatter plot showing the results of a NUGC-4 ADCC assay using PBMCs (donor ID: A18Z017017) as effector cells. 18B10-HaLa-VLPYLL shows a 3-fold increase in ADCC potency compared to that of 18B10-HaLawt and a 100-fold increase in ADCC potency compared to that of IMAB362. [Figure 21] FIG. 21 shows a comparison of CLDN18.2 expression levels in different gastric cancer cell lines. [Figure 22-1] 22A-22D are scatter plots showing ADCC assays of different gastric cancer cell lines with different CLDN18.2 expression levels. [Figure 22-2] See above. [Figure 23] Figure 23 is a scatter plot showing the results of an ADCC reporter assay with NUGC4 (E / T ratio = 6:1). Antibodies produced using a process with the addition of 50 μM 2F-OF increased ADCC activity by more than 30-fold over that of a reference sample produced using a process without the addition of 2F-OF, or more than 1000-fold over that of IMAB362. [Figure 24] 24A-24C are scatter plots showing FACS binding of different gastric cancer cell lines using 18B10-HaLa low fucose. [Figure 25-1] 25A-25E are scatter plots showing the results of ADCC reporter assays in different gastric cancer cell lines using 18B10-HaLa low-fucose. [Figure 25-2] See above. [Figure 26-1]26A-26D are scatter plots showing the results of ADCC assays on different gastric cancer cell lines using PBMCs (donor ID: A19028011) as effector cells and a 2F-OF sample of 50 μM 18B10-HaLa. [Figure 26-2] See above. [Figure 27] FIG. 27 shows the specific cytotoxicity of 18B10-HaLa-low-fucose in an ADCC assay on NUGC-4 cells. [Figure 28] 28A-28B show tumor growth inhibition of different doses of 18B10-HaLa-low-fucose in an MKN45-CLDN18.2-high and hPBMC co-inoculated xenograft tumor model. [Figure 29] FIG. 29 shows tumor growth inhibition of 18B10-HaLaLowFucose in combination with oxaliplatin and 5-FU in the MKN45-CLDN18.2-high tumor model. [Figure 30] Figures 30A and 30B show tumor growth inhibition of antibodies in the MKN45-CLDN18.2-high xenograft tumor model. [Figure 31-1] Figures 31A, 31B, and 31C show the efficacy of 18B10-HaLa-Low-Fucose in combination with paclitaxel in the GC02-0004 PDX tumor model in nude mice. [Figure 31-2] Figures 31D and 31E show tumor growth inhibition of antibodies in the GC02-0004 PDX tumor model. [Figure 32] FIG. 32 shows tumor growth inhibition (TGI) of antibodies in the MKN45-CLDN18.2 xenograft model. [Figure 33] Figures 33A and 33B show FACS binding to pancreatic cancer cell lines using 18B10-HaLa low fucose. [Figure 34] Figures 34A and 34B show ADCC reporter assays in pancreatic cancer cell lines using Jurkat-NFAT-luc-FcγRIIIA-V176 as effector cells. [Figure 35] FIG. 35 shows tumor growth inhibition (TGI) of antibodies in the MIA PaCa-2-CLDN18.2 xenograft model. [Figure 36] FIG. 36 shows tumor growth inhibition (TGI) of antibodies in a BxPC-3-CLDN18.2 xenograft model. [Figure 37] Figures 37A and 37B show FACS binding to lung cancer cell lines using 18B10-HaLa low fucose. [Figure 38] FIG. 38 shows ADCC reporter assay in NCI-H146 using Jurkat-NFAT-luc-FcγRIIIA-V176 as effector cells. [Figure 39] FIG. 39 shows an ADCC assay with NCI-H460-CLDN18.2 using PBMCs as effector cells. [Figure 40] Figures 40A and 40B show tumor growth inhibition (TGI) of antibodies in the NCI-H146 and PBMC co-inoculation model. [Figure 41] FIG. 41 shows tumor growth inhibition (TGI) of antibodies in the NCI-H460-CLDN18.2 tumor model. [Figure 42] FIG. 42 shows FACS binding to colon cancer cell lines using 18B10-HaLa low fucose. [Figure 43] FIG. 43 shows ADCC reporter assay in colon cancer cell lines using Jurkat-NFAT-luc-FcγRIIIA-V176 as effector cells. DETAILED DESCRIPTION OF THE INVENTION

[0067] The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limitations on the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0068] definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of the present invention (especially in the context of the claims) are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0069] The term "antibody," as used herein, includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A naturally occurring, intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain contains a variable region (V H ) and the first, second, and third constant regions (C H1 , C H2 , C H3 mammalian light chains are classified as lambda or kappa, and each light chain consists of a variable region (V L) and constant regions. Antibodies are "Y" shaped, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three hypervariable loops called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding domains disclosed herein may be defined or specified by the rules of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J. Mol. Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901(1987); N.R. Whitelegg et al., Protein Engineering, v13(12), 819-824(2000); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83(1989), Kabat EAet al.,National Institutes of Health,Bethesda,Md.(1991), Marie-Paule Lefranc et al,Developmental and Comparative Immunology,27:55-77(2003), Marie-Paule Lefranc et al,Immunome Research, 1(3), (2005), Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)).The three CDRs are located between adjacent sections known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of the heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain). In certain embodiments, the antibodies provided herein include any antigen-binding fragment thereof.

[0070] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody comprising one or more CDRs, or any other antibody fragment formed from any other antibody portion that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab'), Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen as the parent antibody. In certain embodiments, antigen-binding fragments may comprise one or more CDRs from a particular human antibody.

[0071] "Fab," with respect to an antibody, refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) linked by a disulfide bond to the variable region and first constant region of a single heavy chain.

[0072] Fab can be obtained by papain digestion of an antibody at residues proximal to the N-terminus of the inter-heavy chain disulfide bond in the hinge region.

[0073] "Fab'" refers to a Fab fragment containing part of the hinge region, which can be obtained by pepsin digestion of an antibody at residues proximal to the C-terminus of the inter-heavy chain disulfide bond in the hinge region, and thus differs from Fab by a small number of residues in the hinge region (including one or more cysteines).

[0074] "F(ab')2" refers to a dimer of Fab' comprising two light chains and portions of two heavy chains.

[0075] "Fc" with respect to an antibody refers to the portion of the antibody consisting of the second and third constant regions of a first heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. IgG and IgM Fc regions contain three heavy chain constant regions (the second, third, and fourth heavy chain constant regions in each chain). The Fc region can be obtained by papain digestion of an antibody. The Fc portion of an antibody is responsible for various effector functions, such as ADCC, ADCP, and CDC, but does not function in antigen binding.

[0076] "Fv" in reference to an antibody refers to the smallest fragment of an antibody that retains a complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0077] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). An "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions together with a linker. In certain embodiments, an "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions, with a linker between the V of one moiety and the V of the other moiety. H The part of V of the other part L and another V to cooperate with the V portion to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L V dimerized with the moiety H -V L In another embodiment, the "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv) comprising V H1 and V L1 In cooperation with V H2 and V L2 and each cooperating pair has a different antigen specificity. L1 -V H2 V bound to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0078] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0079] "Camelized single domain antibodies," "heavy chain antibodies," "nanobodies," or "HCAbs" are antibodies that combine two V HIt refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38(1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302(2001); WO 94 / 04678; WO 94 / 25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally obtained from camelids (camels, dromedaries, and llamas). Camelized antibodies lack light chains but have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428):446-8(1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. April; 54(1):39-47(2002); Nguyen VK. et al. Immunology. May; 109(1):93-101(2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. November; 21(13):3490-8. Epub 2007 Jun 15(2007)). "Diabodies" include small antibody fragments with two antigen-binding sites, which are arranged in a single polypeptide chain as V L Domain and connected V H Domain (V H -V L or V L -V H) (see, for example, Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8(1993), EP 404097, WO 93 / 11161). The two domains on the same chain cannot pair because the linker is too short, and are therefore forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes).

[0080] A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain embodiments, two or more V H The domains are covalently linked with peptide linkers to form bivalent or multivalent domain antibodies. H The domains can target the same or different antigens.

[0081] In certain embodiments, a "(dsFv)2" is a compound comprising three peptide chains, linked by a peptide linker, and two V L Two V's bonded to the moiety H Includes parts.

[0082] In certain embodiments, a "bispecific ds diabody" is a V H1 and V L1 V via a disulfide bridge between L1 -V H2 V bound to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0083] In certain embodiments, a "bispecific dsFv" or "dsFv-dsFv'" comprises three peptide chains, the heavy chains of which are linked by a peptide linker (e.g., a long flexible linker) and each V is linked via a disulfide bridge. L1 and V L2Partially paired V H1 -V H2 Each disulfide paired heavy and light chain has a different antigen specificity.

[0084] The term "humanized," as used herein, means that an antibody or antigen-binding fragment comprises CDRs derived from a non-human animal, FR regions derived from a human, and, if applicable, a constant region derived from a human. In certain embodiments, amino acid residues in the variable region framework of a humanized CLDN18.2 antibody are substituted for sequence optimization. In certain embodiments, the variable region framework sequences of the humanized CLDN18.2 antibody chains are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the corresponding human variable region framework sequences.

[0085] The term "chimeric," as used herein, refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the remaining portion of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0086] The term "germline sequence" refers to a nucleic acid sequence encoding a variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other known variable region amino acid sequences encoded by germline immunoglobulin variable region sequences. A germline sequence can also refer to a variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. A germline sequence can be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that comprise variable regions. Sequence identity can be determined using the methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. A germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference variable region nucleic acid or amino acid sequence. Germline sequences can be determined, for example, from the publicly available International Immunogenetics Database (IMGT) and V-base.

[0087] An "anti-CLDN18.2 antibody" or "antibody to CLDN18.2," as used herein, refers to an antibody capable of specifically binding to CLDN18.2 (e.g., human or non-human CLDN18.2) with sufficient affinity to provide, for example, diagnostic and / or therapeutic uses.

[0088] The term "affinity," as used herein, refers to the strength of the non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen.

[0089] The terms "specific binding" or "specifically bind," as used herein, refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein bind to at least 10 -6 M or less (e.g., 5 × 10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, or 10 -9 Binding affinity (K D ) specifically binds to human and / or non-human CLDN18.2. D is the ratio of the dissociation rate to the association rate (k off / k on ) and can be determined by any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). In certain embodiments, K D The value can be suitably determined by using flow cytometry. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with a protein (see, e.g., Harlow & Lane, *Using Antibodies*, *A Laboratory Manual* (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction produces a signal that is at least twice the background signal, more typically at least 10-100 times the background signal.

[0090] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum correspondence. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available at the National Center for Biotechnology Information (NCBI) website; see also Altschul S F et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website; see also Higgins D Get al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tool or customize them as appropriate for alignment, for example, by selecting a suitable algorithm. In certain embodiments, non-identical residue positions can differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference.

[0091] As used herein, "homologous sequence" and "homologous sequence" are used interchangeably and refer to a polynucleotide sequence (or its complementary strand) or amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence, when aligned as necessary.

[0092] An "isolated" material has been altered by the hand of man from its natural state. When an "isolated" composition or material occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that has a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (e.g., ion exchange chromatography or reverse-phase HPLC).

[0093] The ability to "block binding" or "compete for the same epitope," as used herein, refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., between human CLDN18.2 and an anti-CLDN18.2 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In certain embodiments, this inhibition can be more than 60%, more than 70%, more than 80%, or more than 90%.

[0094] The term "antibody-drug conjugate," as used herein, refers to the linkage of an antibody or its antigen-binding fragment to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. The linkage may be a covalent bond or a non-covalent interaction, such as through electrostatic forces. Various linkers known in the art can be used to form antibody-drug conjugates. In addition, antibody-drug conjugates can be provided in the form of a fusion protein that can be expressed from a polynucleotide encoding the conjugate. As used herein, "fusion protein" refers to a protein created by the joining of two or more genes or gene fragments that originally encoded separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein possessing functional properties from each of the original proteins.

[0095] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, for example, non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein.

[0096] The term "anti-tumor activity" refers to a reduction in tumor cell proliferation, survival rate, or metastatic activity. For example, anti-tumor activity can be demonstrated by a reduction in the growth rate of abnormal cells or a stable or reduced tumor size during therapy, or a longer survival period resulting from therapy compared to a control not receiving therapy. Such activity can be assessed using any acceptable in vitro or in vivo tumor model, including, but not limited to, xenograft models, allograft models, mouse mammary tumor virus (MMTV) models, and other known models known in the art for examining anti-tumor activity.

[0097] As used herein, "effector function" or "antibody effector function" refers to a biological activity that is attributed to the binding of the Fc region of an antibody to its effector, such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is induced by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC), which is induced by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and antibody-dependent cellular phagocytosis (ADCP), in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently phagocytose the target cells. Effector functions include both those that act after antigen binding and those that act independently of antigen binding.

[0098] "Treating" a condition or "treatment" of a condition, as used herein, includes preventing or alleviating the condition, delaying the onset or slowing the rate of occurrence of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or eliminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0099] The term "vector," as used herein, refers to a vehicle into which a genetic element can be operably inserted to cause expression of the genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. A vector can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can contain an origin of replication. A vector can also contain substances that aid in cell entry, including, but not limited to, a viral particle, a liposome, or a protein coat. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0100] "Host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0101] The term "CLDN18.2" refers to Claudin-18 splice variant 2 derived from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include the human CLDN18.2 protein (NCBI Reference SEQ ID NO: NP_001002026.1, or SEQ ID NO: 30). Exemplary sequences of CLDN18.2 include the Mus musculus (mouse) CLDN18.2 protein (NCBI Reference SEQ ID NO: NP_001181852.1) and the Macaca fascicularis (cynomolgus monkey) CLDN18.2 protein (NCBI Reference SEQ ID NO: XP_015300615.1). CLDN18.2 is expressed in cancer cells. In one embodiment, CLDN18.2 is expressed on the surface of cancer cells.

[0102] The term "CLDN18.1" refers to Claudin-18 splice variant 1 derived from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, the CLDN18.1 is human CLDN18.1. Exemplary sequences of human CLDN18.1 include the human CLDN18.1 protein (NCBI Reference SEQ ID NO: NP_057453.1, or SEQ ID NO: 31), the Mus musculus (mouse) CLDN18.2 protein (NCBI Reference SEQ ID NO: NP_001181851.1), and the Macaca fascicularis (cynomolgus monkey) CLDN18.2 protein (NCBI Reference SEQ ID NO: XP_005545920.1).

[0103] As used herein, a "CLDN18.2-associated" disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of CLDN18.2. In some embodiments, the CLDN18.2-associated condition is, for example, cancer.

[0104] "Cancer," as used herein, refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal proliferation, invasion, or metastasis, and includes both solid tumors and non-solid cancers (e.g., hematological malignancies), such as leukemia. As used herein, a "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0105] Reference herein to a value or parameter "about" includes (and describes) embodiments directed to that value or parameter itself. For example, a statement referring to "about X" includes the statement "X." Numerical ranges include the numbers defining the range. Generally speaking, the term "about" refers to a stated value of a variable and all values ​​of that variable that are within experimental error of the stated value (e.g., within a 95% confidence interval of the mean) or within 10 percent of the stated value, whichever is greater. When the term "about" is used in the context of time (years, months, weeks, days, etc.), it refers to the period of time plus or minus one subquantity of time (e.g., about 1 year means 11-13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6-8 days, etc.), or within 10 percent of the stated value, whichever is greater.

[0106] Anti-CLDN18.2 antibody The present disclosure provides anti-CLDN18.2 antibodies and antigen-binding fragments thereof. The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein can specifically bind to CLDN18.2 (e.g., human CLDN18.2) or cells expressing CLDN18.2. "Specifically binding" as used herein means binding to a cell expressing CLDN18.2. -6 M or less (e.g., 5 × 10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8M or less, 2×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, or 10 -9 Binding affinity (e.g., K D ) means

[0107] i.Binding affinity The binding affinity of the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein is determined by the ratio of the dissociation rate to the association rate (k off / k on ) represents K D Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens more rapidly and tend to remain bound longer. Antigen binding affinity (e.g., K D ) can be suitably determined using any suitable method known in the art, including, for example, equilibrium binding exclusion assay (KinExA) or flow cytometry.

[0108] In certain embodiments, the "Kd" or "Kd value" of the present disclosure is measured by a KinExA assay, which, in certain embodiments, measures the solution binding affinity of an anti-CLDN18.2 antibody using the described anti-CLDN18.2 antibody and CLDN18.2. Generally, KinExA works by equilibrating a fixed amount of one binding partner (CBP) with various concentrations of the other binding partner (titrant), and then capturing a portion of the free CBP with a fluorescently labeled secondary antibody over a short contact time less than the time required for dissociation of the previously formed CBP-titrant complex. The fluorescent signal generated from the captured CBP is directly proportional to the concentration of free CBP in the equilibrium sample and, when measured continuously, is used to generate a binding curve (percent free CBP vs. total titrant concentration). Further details are available from Schreiber, G., Fersht, A.R. Nature Structural Biology. 1996, 3(5), 427-431. When an anti-CLDN18.2 antibody is used as a fixed amount of CBP, cells expressing CLDN18.2 can be used as a titrant, and vice versa. CLDN18.2 or cells expressing CLDN18.2 can be used to measure Kd by KinExA. In certain embodiments, the Kd of an anti-CLDN18.2 antibody or its antigen-binding fragment is determined according to the method described in Section 3 of Example 10 of the present disclosure.

[0109] Other suitable methods for measuring Kd can also be used where applicable, such as radiolabeled antigen binding assays (see, e.g., Chen, et al., (1999) J. Mol Biol 293:865-881) or surface plasmon resonance assays, such as BIAcore using immobilized CLDN18.2 CM5 chips in appropriate response units (RU).

[0110] In certain embodiments, the binding affinity of anti-CLDN18.2 antibody is measured by flow cytometry.Generally, CLDN18.2-expressing cells are incubated with a range of concentrations of anti-CLDN18.2 antibody, followed by incubation with a fluorescently labeled secondary antibody, and then analyzed for fluorescent signal intensity.In certain embodiments, the binding affinity of anti-CLDN18.2 antibody or its antigen-binding fragment is determined according to the method described in Example 5 of the present disclosure.

[0111] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein have a K of 2.5 nM or less (or 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or less) as measured by a KinExA assay. D The antibody specifically binds to human CLDN18.2 (or cells expressing human CLDN18.2) at a value of 0.1 to 1.0.

[0112] Alternatively, the binding affinity of the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein to human CLDN18.2 may be measured using the "half-maximal effective concentration" (EC), which refers to the concentration of antibody at which 50% of the maximal effect (e.g., binding) is observed. 50 ) value. 50 The EC value can be measured by methods known in the art, for example, sandwich assays such as ELISA, Western blot, flow cytometry assay, and other binding assays. In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein specifically bind to human CLDN18.2 (e.g., cells expressing human CLDN18.2) at an EC value of 70 μg / ml or less (or 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml or less) as measured by flow cytometry.

[0113] Binding affinity can be determined with respect to recombinant CLDN18.2 or a cell line expressing CLDN18.2. The antibodies and antigen-binding fragments provided herein can bind to cells expressing different levels of human CLDN18.2, particularly cells expressing relatively intermediate or low levels of human CLDN18.2.

[0114] In certain embodiments, binding affinity is determined using cells that express human CLDN18.2, such as NUGC4 cells, SNU-620 cells, SNU-601 cells, KATOIII cells, or comparable cells having human CLDN18.2 protein expression levels comparable to or lower than NUGC4 cells, SNU-620 cells, SNU-601 cells, or KATOIII cells.

[0115] NUGC4 cells are a cell line established from perigastric lymph nodes derived from cancer patients (see Akiyama S et al, Jpn J Surg. 1988 Jul;18(4):438-46). The NUGC4 cell line is available from the JCRB Cell Bank under accession number JCRB0834.

[0116] Both SNU-601 and SNU-620 cells are human gastric cancer cell lines established from the ascites of cancer patients by Seoul National University (SNU) (KU JL et al., Cancer Res Treat. 2005 Feb;37(1):1-19; Park et al., Int J Cancer. 1997 Feb 7;70(4):443-449). SNU-601 and SNU-620 cells are available from the Korean Cell Line Bank under accession numbers 00601 and 00620, respectively.

[0117] KATO III cells are a cell line derived from a metastatic site of a gastric cancer patient (see Sekiguchi M, et al. Jpn. J. Exp. Med. 48:61-68, 1978). The KATO III cell line is available from the ATCC under accession number ATCC HTB-103.

[0118] Cell lines recombinantly expressing human CLDN18.2 protein can also be established by transfecting and expressing DNA encoding human CLDN18.2 into cell lines such as Chinese hamster ovary (CHO), HEK cells, or the MKN45 cell line (National Infrastructure of Cell Line Resource, Cat#3111C0001CCC000229), among others.

[0119] In certain embodiments, binding affinity is determined using cells that highly express human CLDN18.2, cells that moderately express human CLDN18.2, or cells that lowly express human CLDN18.2.

[0120] The expression level of human CLDN18.2 protein may vary among different cell lines. The expression level of CLDN18.2 protein in cells can be measured by any suitable method known in the art, such as quantitative fluorescence cytometry or immunohistochemistry (IHC). In certain embodiments, the expression level of human CLDN18.2 protein in a given cell is determined according to IHC. IHC involves detecting an antigen (e.g., CLDN18.2) in a cell or tissue by visualizing the antigen through antigen-antibody interaction. Typically, the antigen is detected using a primary antibody against the antigen. The primary antibody may be labeled to enable direct detection of the antigen. Alternatively, the primary antibody may be unlabeled and further contacted with a secondary antibody conjugated to a detectable label to enable indirect detection of the antigen. The primary antibody may be any antibody capable of specifically binding to human CLDN18.2, such as, but not limited to, any of the anti-CLDN18.2 antibodies provided herein or any anti-CLDN18.2 antibody known in the art. In certain embodiments, cells or tissues may be fixed using, for example, paraformaldehyde.

[0121] The term "high expression," as used herein with respect to cells expressing human CLDN18.2, refers to a cell expressing human CLDN18.2 in at least 40% of the cells (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 40-100%, 50-150%, 60-200%, 70-250%, 80-300%, 80-350%, 80-400%, 80-500%, 80-600%, 80-700%, 80-900%, 90-1500%, 90-2500), 90-3000, 90-4000, 90-5000, 90-6000, 90-7000, 90-8000, 90-9000, 90-1500, 90-1500, 90-2500, ...0, 90-15000, 90-25000, 90-30000, 9 "Intermediate expression" is intended to mean cells that express human CLDN18.2 at a level where at least 100%, 60-100%, 70-100%, 80-100%, 90-100%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-80%, 50-70%, 50-60%, 60-80%, 60-70%, or 70-80% of cells stain positive by IHC. Similarly, the term "intermediate expression," as used herein, means cells that express human CLDN18.2 at a level where at least 30% (or at least 35%) to less than 40% of cells stain positive by IHC, with an intensity of at least 1+ to less than 2+ as measured by IHC. Furthermore, the term "low expression," as used herein, refers to cells in which greater than 0 but less than 30% of cells (e.g., 5%, 10%, 15%, 20%, 25%, 5-25%, 10-25%, 15-25%, 20-25%, 5-20%, 5-15%, 5-10%, 10-20%, or 10-15%) express human CLDN18.2 at a level that stains positive by IHC at an intensity greater than 0 but less than 1+. Definitions are also provided in Table A below.

[0122] [Table A]

[0123] In certain embodiments, the human CLDN18.2 expression level is determined by IHC, as described in Sections 6 and 7 of Example 15. Briefly, cells expressing human CLDN18.2 are fixed with paraffin and detected via IHC using an anti-human CLDN18.2 antibody, followed by determining the relative proportion of positively stained cells and the staining intensity on the cell membrane. In certain embodiments, cells are stained in the IHC process using biotinylated anti-CLDN18.2 antibody GC182. Antibody GC182 has a heavy chain variable region sequence of SEQ ID NO: 74 and a light chain variable region sequence of SEQ ID NO: 75 (see also International Publication No. WO2013167259).

[0124] Based on the immunohistochemistry (IHC) determination results provided herein (Table 13), NUGC4 cells can be characterized as a cell line that moderately expresses human CLDN18.2, while SNU-620, SNU-601, and KATOIII cells can be characterized as cell lines that lowly express human CLDN18.2. In addition, recombinant cell lines can be generated that highly express human CLDN18.2. Examples of highly expressing cells include, but are not limited to, the MKN45-CLDN18.2 high cell line and the HEK293-CLDN18.2 cell line described in Section 3 of Example 1 herein.

[0125] Surprisingly, the present inventors have found that the anti-CLDN18.2 antibodies and fragments thereof provided herein have high affinity for cell lines that moderately express human CLDN18.2 (e.g., NUGC4 cells) and cell lines that lowly express human CLDN18.2 (e.g., SNU-620, SNU-601, and KATOIII cells). This distinguishes them from existing antibodies, such as IMAB362, which fail to exhibit specific or comparable binding to cells that lowly express human CLDN18.2. The chimeric IgG1 antibody IMAB362 is an anti-human CLDN18.2 antibody developed by Ganymed Pharmaceuticals AG, having the amino acid sequence disclosed in U.S. Patent Application Publication No. 2009169547 (the heavy and light chain variable region sequences of IMB362 are included herein as SEQ ID NOs:72 and 73) and CAS number 1496553-00-4. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 and does not bind to any other claudin family members, including the closely related splice variant 1 of Claudin18 (CLDN18.1).

[0126] In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein have a K of 2.5 nM or less (or 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or less) as measured by a KinExA assay. D Specifically binds to cells expressing human CLDN18.2 (e.g., NUGC4 cell line or KATOIII cell line) at a Kd value. In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein specifically bind to cells expressing human CLDN18.2 at a Kd value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or less of the Kd value of IMAB362 as measured by KinExA assay. In certain embodiments, the Kd value DThe value is determined using NUGC4 cells, KATOIII cells, SNU-601 cells, SNU-620 cells, or comparable cells having human CLDN18.2 protein expression levels comparable to or lower than those of NUGC4 cells, KATOIII cells, SNU-601 cells, or SNU-620 cells. In certain embodiments, the K D The value is determined using a cell line that highly expresses human CLDN18.2 or a cell line that moderately expresses human CLDN18.2.

[0127] In certain embodiments, the antibodies and antigen-binding fragments provided herein have an EC50 value of 70 μg / ml or less (or 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml or less) for binding to cells expressing human CLDN18.2 (or murine CLDN18.2), as measured by a flow cytometry assay. In certain embodiments, the antibodies and antigen-binding fragments provided herein specifically bind to cells expressing human CLDN18.2 with an EC50 value of 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 1%, or 0.1% or less of the EC50 value of IMAB362, as measured by a flow cytometry assay. In certain embodiments, EC50 is determined using a NUGC4 cell line, a KATOIII cell line, a SNU-601 cell line, a SNU-620 cell line, or a comparable cell line having a human CLDN18.2 protein expression level comparable to or lower than that of the NUGC4 cell line, the KATOIII cell line, the SNU-601 cell line, or the SNU-620 cell line, such as a cell line that expresses low or moderate human CLDN18.2. In certain embodiments, EC50 is determined using a cell line that expresses high levels of human CLDN18.2.

[0128] In certain embodiments, the antibodies and antigen-binding fragments provided herein have an EC50 value of 5, 4, 3, or 2 μg / ml or less for binding to a cell line that highly expresses human CLDN18.2 or a cell line that moderately expresses human CLDN18.2.

[0129] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein have an EC50 value of 70 μg / ml or less (or 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml or less) for binding to NUGC4 cells as measured by a flow cytometry assay.

[0130] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof do not bind to CLDN18.1 (eg, human CLDN18.1 or murine CLDN18.1).

[0131] In certain embodiments, the antibodies and antigen-binding fragments thereof can specifically bind to murine CLDN18.2 (e.g., cells expressing murine CLDN18.2) with an EC50 value of 1.5 μg / ml or less as measured by flow cytometry. In certain embodiments, the antibodies and antigen-binding fragments thereof bind to murine CLDN18.2 with an EC50 of 0.1 μg / ml to 1.5 μg / ml (e.g., 0.1 μg / ml to 1.2 μg / ml, 0.2 μg / ml to 1 μg / ml, 0.5 μg / ml to 1 μg / ml, 0.6 μg / ml to 1 μg / ml, 0.6 μg / ml to 0.8 μg / ml, or 0.67 μg / ml) as measured by flow cytometry.

[0132] 1. ADCC and CDC activity In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or CDC activity in cells expressing different levels of human CLDN18.2.

[0133] As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, subsequently causing lysis of the target cells. Target cell lysis is extracellular, requires direct cell-to-cell contact, and does not involve complement. ADCC can be viewed as a mechanism that directly induces varying degrees of immediate tumor destruction, resulting in antigen presentation and induction of tumor-specific T cell responses. In vivo induction of ADCC is thought to result in tumor-specific T cell responses and host-derived antibody responses.

[0134] Methods for performing ADCC are known in the art. Generally, target cells, such as cells expressing CLDN18.2, are incubated with a range of concentrations of an anti-CLDN18.2 antibody, washed, and then effector cells, such as cells expressing Fc receptors, are added to allow ADCC to occur. Cytotoxicity or cell viability is determined at a time point several hours after mixing of the target cells with the effector cells to quantify the level of ADCC. Cytotoxicity can be detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein such as lactate dehydrogenase (LDH)) from lysed target cells. In another embodiment, cell viability is determined by an indicator of metabolically active cells (e.g., ATP) using a luciferase reporter gene that generates a luminescent signal proportional to the number of viable cells in culture (i.e., an ADCC reporter assay) (see, e.g., Crouch, SP et al. (1993) J. Immunol. Methods 160, 81-8). Examples of effector cells are NK cells, PBMCs, or cells expressing FcγRIII. In certain embodiments, the ADCC activity of the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein is determined according to the method described in Section 2 of Example 7.

[0135] "Complement-dependent cytotoxicity" or "CDC" is another antibody-directed method of cell killing by target lysis in the presence of complement. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are also highly effective at directing CDC via the classical complement activation pathway. In this cascade, the formation of an antigen-antibody complex results in the exposure of multiple closely spaced Clq binding sites on the CH2 domain of participating antibody molecules, such as IgG molecules complexed with their cognate antigens (Clq is one of the three subcomponents of complement C1). These exposed Clq binding sites convert the previously low-affinity Clq-IgG interaction into a high-avidity interaction, which initiates a cascade of events involving a series of other complement proteins, leading to the proteolytic release of the effector cell chemotactic / activating agents C3a and C5a. The complement cascade culminates in the formation of the membrane attack complex (MAC), which forms pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0136] CDC activity can be determined by a method similar to that for ADCC activity discussed above, except that effector cells are not used and the presence of complement derived from human serum is required. Briefly, antibody samples are serially diluted in assay medium and incubated with target cells expressing CLDN18.2 in the presence of human serum complement. After incubation, cytotoxicity or cell viability is determined by the release of label from lysed target cells or an indicator of metabolically active cells (e.g., ATP). CellTiter-Glo reagent, which assays ATP in metabolically active cells, can be used, and the degree of cell lysis can be quantified by measuring the intensity of luminescence using an appropriate reader. In certain embodiments, the CDC activity of the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein is determined according to the method described in Section 1 of Example 7.

[0137] In certain embodiments, ADCC- or CDC-induced cell death mediated by the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein can be determined by loss of membrane integrity assessed by uptake of propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)), or 7AAD compared to untreated cells.

[0138] Surprisingly, the present inventors have found that the anti-CLDN18.2 antibodies and fragments thereof provided herein can induce ADCC and / or CDC in cell lines that moderately express human CLDN18.2 (e.g., NUGC4 cells) or that lowly express human CLDN18.2 (e.g., SNU-620, SNU-601, and KATOIII cells). This is distinct from existing antibodies such as IMAB362, which are unable to induce ADCC or CDC in such cell lines that moderately or lowly express human CLDN18.2.

[0139] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein can induce complement-dependent cytotoxicity (CDC) on cells expressing human CLDN18.2 at an EC50 value of 1 μg / ml or less (or 0.9, 0.8, 0.7, 0.6, 0.5 μg / ml or less) as measured by a cytotoxicity assay. In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein can induce CDC on cells expressing human CLDN18.2 at an EC50 value of 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less of the EC50 value of IMAB362 as measured by a cytotoxicity assay. In certain embodiments, CDC is determined using a cell line that moderately expresses human CLDN18.2 or a cell line that highly expresses human CLDN18.2.

[0140] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein are capable of inducing antibody-dependent cellular cytotoxicity (ADCC) on cells expressing human CLDN18.2 with an EC50 value of 2 μg / ml or less (or 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / ml or less) as measured by an ADCC reporter assay. In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein induce ADCC on cells expressing human CLDN18.2 at an EC50 value that is 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% or less of the EC50 value of IMAB362 as measured by an ADCC reporter assay, or at a total ADCC potency of at least 120%, 150%, 180%, or 200% of the total ADCC potency of IMAB362 (e.g., as indicated by the maximum level of ADCC activity observed in a plot of antibody concentration versus ADCC activity level). In certain embodiments, ADCC is determined using the NUGC4 cell line, the KATOIII cell line, the SNU-601 cell line, the SNU-620 cell line, or comparable cells having human CLDN18.2 protein expression levels comparable to or lower than the NUGC4 cell line, the KATOIII cell line, the SNU-601 cell line, or the SNU-620 cell line, such as a cell line that moderately expresses human CLDN18.2 or a cell line that lowly expresses human CLDN18.2.

[0141] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein are capable of inducing ADCC on NUGC4 cells with an EC50 value of 2 μg / ml or less (or 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / ml or less) as measured by an ADCC reporter assay.

[0142] epitope In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein binds to an epitope including at least one or more (e.g., one, two, three, or more) of the amino acid residues at positions D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of human CLDN18.2 having the amino acid sequence of SEQ ID NO: 30.

[0143] The term "epitope," as used herein, refers to a specific group of atoms or amino acids of an antigen to which an antibody binds. Epitopes can include specific amino acids, sugar side chains, phosphoryl or sulfonyl groups that directly contact the antibody. Those skilled in the art will recognize that, without undue experimentation, they can determine whether an antibody binds to the same, overlapping, or adjacent epitope as an antibody of the present disclosure (e.g., any of the hybridoma / chimeric or humanized antibodies 7C12, 11F12, 26G6, 59A9, 18B10, and their chimeric and humanized variants provided herein) by determining whether the two antibodies compete for binding to the CLDN18.2 antigen polypeptide.

[0144] The term "compete for binding," as used herein with respect to two antigen-binding proteins (e.g., antibodies), means that one antigen-binding protein prevents or reduces the binding of the other antigen-binding protein to an antigen (e.g., human / mouse CLDN18.2) as determined by a competitive binding assay. Competitive binding assays are well known in the art and include, for example, direct or indirect radioimmunoassays (RIA), direct or indirect enzyme immunoassays (EIA), and sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253). Typically, such assays involve the use of purified antigen or antigen-bearing cells bound to a solid surface, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess. When two antibodies compete for binding to CLDN18.2, they bind to the same or overlapping epitope, or to adjacent epitopes that are sufficiently close to each other that they sterically interfere with the epitope bound by the other antibody. Typically, when present in excess, a competing antibody inhibits (e.g., reduces) specific binding of a test antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, or more.

[0145] In certain embodiments, the epitope or amino acid residues of the epitope to which the antibody binds can be determined by mutating specific residues in the antigen, i.e., CLDN18.2. If the antibody binds to a mutant CLDN18.2 in which the amino acid residue has been mutated, for example, to alanine, at a level significantly reduced compared to the binding to wild-type CLDN18.2, this may indicate that the mutated residue is directly involved in the binding of the antibody to the CLDN18.2 antigen, or that the mutated residue is in close proximity to the antibody when the antibody binds to the antigen. Such a mutated residue is considered to be present within the epitope, and the antibody is considered to specifically bind to the epitope containing that residue. As used herein, a significantly reduced level of binding means that the binding affinity (e.g., EC50, Kd, ​​or binding ability) between the antibody and mutant CLDN18.2 is reduced by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to the binding between the antibody and wild-type CLDN18.2. Such binding measurements can be performed using any suitable method known in the art and disclosed herein, including, but not limited to, KinExA assays and flow cytometry.

[0146] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein exhibits significantly lower binding to mutant CLDN18.2 in which residues of wild-type CLDN18.2 are substituted with alanine, and the residues are selected from the group consisting of D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of human CLDN18.2. In certain embodiments, the residue is E56. In certain embodiments, the residue is selected from the group consisting of W30, L49, W50, R55, and E56. In certain embodiments, the residue is selected from the group consisting of T41, N45, Y46, R51, F60, E62, and R80. In certain embodiments, the residues are selected from the group consisting of D28, V43, N45, Y46, Y66, L72, L76, and V79.

[0147] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment provided herein exhibits at least an 80%, 90%, 95%, or 99% or greater reduction in binding to mutant CLDN18.2 comprising E56A of human CLDN18.2 compared to binding between the antibody and wild-type CLDN18.2.

[0148] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment provided herein exhibits at least a 50%, 60%, 70%, 80%, or 90% reduction in binding to mutant CLDN18.2 comprising one or more mutated residues selected from the group consisting of W30A, L49A, W50A, R55A, and E56A of human CLDN18.2 compared to binding between the antibody and wild-type CLDN18.2.

[0149] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment provided herein exhibits at least a 30%, 35%, 40%, 45%, or 50% reduction in binding to mutant CLDN18.2 comprising one or more mutated residues selected from the group consisting of D28, V43, N45, Y46, Y66, L72, L76, and V79 of human CLDN18.2 compared to binding between the antibody and wild-type CLDN18.2.

[0150] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein exhibits at least a 10%, 15%, 20%, 25%, or 30% reduction in binding to mutant CLDN18.2 comprising one or more mutated residues selected from the group consisting of T41A, N45A, Y46A, R51A, F60A, E62A, and R80A of human CLDN18.2 compared to binding between the antibody and wild-type CLDN18.2.

[0151] In certain embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof provided herein do not bind to A42 and / or N45.

[0152] In certain embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof provided herein bind to the epitopes provided herein and can induce ADCC or CDC activity in cell lines that moderately express human CLDN18.2 or that lowly express human CLDN18.2.

[0153] Antibody sequence In another aspect, the disclosure provides a polypeptide comprising heavy chain HCDR1, HCDR2, and HCDR3, and / or light chain LCDR1, LCDR2, and LCDR3 sequences, the HCDR1 sequence comprises GYNMN (SEQ ID NO: 1) or TYFIGVG (SEQ ID NO: 13) or a homologous sequence thereof of at least 80% sequence identity; the HCDR2 sequence comprises X1IDPYYX2X3TX4YNQKFX5G (SEQ ID NO: 32) or HIWWNDNKYYNTALKS (SEQ ID NO: 15) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity; the HCDR3 sequence comprises X6X7X8GNAFDY (SEQ ID NO: 33) or MGSGAWFTY (SEQ ID NO: 17) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence is KSSQX9LX 10 NX 11 GNX 12 KNYLT (SEQ ID NO: 34) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity, LCDR2 sequence is WASTRX 13 S (SEQ ID NO: 35) or a homologous sequence thereof of at least 80% sequence identity, LCDR3 array is QNDYX 14 X 15 PX 16T (SEQ ID NO: 36) or a homologous sequence thereof of at least 80% sequence identity; X1 is N or Y or H, X2 is G or V, X3 is A or G or T, X4 is R or T or S, X5 is K or R, X6 is S or M, X7 is Y or F, X8 is Y or H, X9 is S or N, and X 10 is L or F, and X 11 is S or N, and X 12 is Q or L, and X 13 is E or K, and X 14 is S or Y, and X 15 is F or Y and X 16 is F or L, Anti-CLDN18.2 antibodies or antigen-binding fragments thereof are provided.

[0154] In one aspect, the present disclosure provides a method for producing a heavy chain variable region comprising: a) an HCDR1 comprising a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 13; b) an HCDR2 comprising a sequence selected from SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 22, and c) an HCDR3 comprising a sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, and SEQ ID NO: 21; and / or d) LCDR1 comprising the sequences of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 20; e) LCDR2 comprising the sequences of SEQ ID NO: 4 and SEQ ID NO: 16, and f) LCDR3 comprising a sequence selected from SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 18 a light chain variable region comprising The present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof comprising:

[0155] In certain embodiments, the heavy chain variable region comprises: a) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 3, and an HCDR3 comprising the sequence of SEQ ID NO: 5; b) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, and an HCDR3 comprising the sequence of SEQ ID NO: 5; c) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 9, and an HCDR3 comprising the sequence of SEQ ID NO: 11; d) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 15, and an HCDR3 comprising the sequence of SEQ ID NO: 17; e) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 19, and an HCDR3 comprising the sequence of SEQ ID NO: 21; and f) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 22, and an HCDR3 comprising the sequence of SEQ ID NO: 5 The antibody or antigen-binding fragment thereof provided herein is selected from the group consisting of:

[0156] In certain embodiments, the light chain variable region comprises: a) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; b) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 8; c) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; d) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 12; e) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 14, an LCDR2 comprising the sequence of SEQ ID NO: 16, and an LCDR3 comprising the sequence of SEQ ID NO: 18; and f) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6. The antibody or antigen-binding fragment thereof provided herein is selected from the group consisting of:

[0157] In certain embodiments, a) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 3, and an HCDR3 comprising the sequence of SEQ ID NO: 5, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; or b) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, and an HCDR3 comprising the sequence of SEQ ID NO: 5, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 8; or c) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 9, and an HCDR3 comprising the sequence of SEQ ID NO: 11, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; or d) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 15, and an HCDR3 comprising the sequence of SEQ ID NO: 17, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 12; or e) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 19, and an HCDR3 comprising the sequence of SEQ ID NO: 21, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 14, an LCDR2 comprising the sequence of SEQ ID NO: 16, and an LCDR3 comprising the sequence of SEQ ID NO: 18; or f) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 22, and an HCDR3 comprising the sequence of SEQ ID NO: 5, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; An antibody or antigen-binding fragment thereof provided herein.

[0158] In certain embodiments, the antibodies provided herein comprise one or more (e.g., one, two, three, four, five, or six) CDR sequences of CLDN18.2 antibodies 7C12, 11F12, 26G6, 59A9, 18B10, and 12E9.

[0159] "7C12," as used herein, refers to a murine antibody having a heavy chain variable region of SEQ ID NO:37 and a light chain variable region of SEQ ID NO:38.

[0160] "11F12," as used herein, refers to a murine antibody having a heavy chain variable region of SEQ ID NO:39 and a light chain variable region of SEQ ID NO:40.

[0161] "26G6," as used herein, refers to a murine antibody having a heavy chain variable region of SEQ ID NO:41 and a light chain variable region of SEQ ID NO:42.

[0162] "59A9," as used herein, refers to a murine antibody having a heavy chain variable region of SEQ ID NO:43 and a light chain variable region of SEQ ID NO:44.

[0163] "18B10," as used herein, refers to a murine antibody having a heavy chain variable region of SEQ ID NO:45 and a light chain variable region of SEQ ID NO:46.

[0164] "12E9," as used herein, refers to a murine antibody having a heavy chain variable region of SEQ ID NO:47 and a light chain variable region of SEQ ID NO:48.

[0165] Table 1 shows the CDR sequences of these CLDN18.2 antibodies. The heavy and light chain variable region sequences are also provided in Table 2 below.

[0166] [Table 1]

[0167] [Table 2]

[0168] The anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody, or an anti-idiotype antibody. A recombinant antibody is an antibody prepared using recombinant methods in vitro rather than in an animal.

[0169] Although CDRs are known to be responsible for antigen binding, it has been found that not all six CDRs are necessarily essential or invariant. In other words, one, two, or three CDRs (corresponding to any one of SEQ ID NOS: 1-22) in anti-CLDN18.2 antibodies 7C12, 11F12, 26G6, 59A9, 18B10, or 12E9 can be replaced, changed, or modified while substantially maintaining specific binding affinity to CLDN18.2.

[0170] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence of one of the anti-CLDN18.2 antibodies 7C12, 11F12, 26G6, 59A9, 18B10, or 12E9. In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequences of SEQ ID NOs: 5, 11, 17, and 21. The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore thought to be in the most contact with the antigen and provide the most free energy for the antibody's affinity for the antigen. In addition, the heavy chain CDR3 is thought to be the most diverse CDR of the antigen-binding site in terms of length, amino acid composition, and conformation due to multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81). Diversity in the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) and desirable antigen binding affinities (Schier R, et al. J Mol Biol. 263:551-67).

[0171] In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein comprise all or part of the heavy chain variable domain and / or all or part of the light chain variable domain.In one embodiment, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein are single-domain antibodies consisting of all or part of the heavy chain variable domain provided herein.More information about such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0172] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof can specifically bind to CLDN18.2. The CDR sequences provided in Table 1 are obtained from mouse antibodies, but can be grafted to any suitable FR sequences of any suitable species, such as mouse, human, rat, or rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0173] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments are desirable because they have reduced immunogenicity in humans. Humanized antibodies have chimeric variable regions when non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can essentially be achieved by substituting non-human (e.g., mouse) CDR genes for the corresponding human CDR genes of human immunoglobulin genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0174] Suitable human heavy and light chain variable domains can be selected to this end using methods known in the art. In an illustrative example, a "best-fit" approach can be used in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain germline sequences to identify the human sequence that is closest to the non-human query sequence and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, frameworks derived from the consensus sequence of all human antibodies may be used for grafting non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0175] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein are composed substantially entirely of human sequences, with the exception of the non-human CDR sequences. In some embodiments, the variable region FRs and, if present, the constant region, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences may be derived from different human immunoglobulin genes, e.g., the FR sequences may be derived from one human antibody and the constant region may be derived from another human antibody. In some embodiments, the humanized antibodies or antigen-binding fragments comprise human heavy / light chain FRs 1-4.

[0176] In some embodiments, the human-derived FR region may contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or fragment thereof more similar to the non-human parent antibody structure in order to reduce or avoid immunogenicity and / or to improve or maintain binding activity or binding affinity.

[0177] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein contain no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the heavy or light chain variable domain. In some embodiments, such amino acid residue changes can occur only in the heavy chain FR region, only in the light chain FR region, or in both chains. In certain embodiments, one or more amino acid residues are mutated, for example, back-mutated to the corresponding residue found in the non-human parent antibody (e.g., mouse framework region) from which the CDR sequence is derived. Suitable positions for mutation can be selected by those skilled in the art according to principles known in the art. For example, mutation positions can be selected if: 1) the residue in the framework of the human germline sequence is rarely present (e.g., less than 20% or less than 10% in human variable region sequences), 2) the position is directly adjacent to one or more of the three CDRs in the primary sequence of the human germline sequence, where it is likely to interact with residues in the CDRs, or 3) the position is close to a CDR in a three-dimensional model and therefore may have a sufficient probability of interacting with an amino acid in the CDR. The residue at the selected position can be backmutated to the corresponding residue in the parent antibody, or to a residue that is neither the corresponding residue in the human germline sequence nor the corresponding residue in the parent antibody but is typical of human sequences, i.e., a residue that occurs more frequently at that position in known human sequences belonging to the same subgroup as the human germline sequence (see U.S. Pat. No. 5,693,762).

[0178] In certain embodiments, the humanized light and heavy chains of the present disclosure are substantially non-immunogenic in humans and maintain substantially the same or much higher affinity for CLDN18.2 than the parent antibody.

[0179] In certain embodiments, the humanized antibodies and antigen-binding fragments thereof provided herein comprise one or more light chain FR sequences of the human germline sequence framework sequence VK / 4-1, with or without backmutations, and / or one or more heavy chain FR sequences of the human germline sequence framework sequence VH / 1-46. Backmutations can be introduced into the human germline sequence framework sequences, if needed. In certain embodiments, the humanized antibody 18B10 may contain one or more backmutations in the heavy chain framework sequence VH / 1-46 selected from the group consisting of R71I, T73K, T28S, M69L, R38K, and M48I, all based on Kabat numbering. The humanized antibody 18B10 may contain one or more backmutations in the light chain framework sequence VK / 4-1 selected from the group consisting of S63T and I21M, all based on Kabat numbering.

[0180] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47 and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity that still maintain specific binding affinity to CLDN18.2, particularly human CLDN18.2.

[0181] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof, antibody, or antigen-binding fragment thereof provided herein comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity that still maintains specific binding affinity to CLDN18.2, particularly human CLDN18.2.

[0182] In certain embodiments, a heavy chain variable region comprising the sequence of SEQ ID NO: 25 and a light chain variable region comprising the sequence of SEQ ID NO: 26; a heavy chain variable region comprising the sequence of SEQ ID NO: 27 and a light chain variable region comprising the sequence of SEQ ID NO: 28; a heavy chain variable region comprising the sequence of SEQ ID NO: 29 and a light chain variable region comprising the sequence of SEQ ID NO: 26 or 28; a heavy chain variable region comprising the sequence of SEQ ID NO: 37 and a light chain variable region comprising the sequence of SEQ ID NO: 38; a heavy chain variable region comprising the sequence of SEQ ID NO: 39 and a light chain variable region comprising the sequence of SEQ ID NO: 40; a heavy chain variable region comprising the sequence of SEQ ID NO: 41 and a light chain variable region comprising the sequence of SEQ ID NO: 42; a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 44; a heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 46; or A heavy chain variable region comprising the sequence of SEQ ID NO: 47 and a light chain variable region comprising the sequence of SEQ ID NO: 48 The anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein comprises:

[0183] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein further comprises one or more of heavy chains HFR1, HFR2, HFR3, and HFR4 and / or one or more of light chains LFR1, LFR2, LFR3, and LFR4; HFR1 is QVQLVQSGAEVKKPGASVKVSCKASGYX 17 FT (SEQ ID NO: 54) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity, HFR2 is WVX 18 QAPGQGLEWX 19 G (SEQ ID NO: 55) or a homologous sequence thereof with at least 80% (or at least 90%) sequence identity, The HFR3 sequence is RVTX 20 TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 56) or a homologous sequence thereof with at least 80% (or at least 85%, 90%, 95%) sequence identity; HFR4 comprises WGQGTTVTVSS (SEQ ID NO: 57) or a homologous sequence thereof with at least 80% sequence identity; LFR1 is DIVMTQSPDSLAVSLGERATX 21 NC (SEQ ID NO: 58) or a homologous sequence thereof with at least 80% (or at least 85%, 90%, 95%) sequence identity, LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO: 59) or a homologous sequence thereof of at least 80% (or at least 85%, 90%) sequence identity; LFR3 is GVPDRFX 22 GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 60) or a homologous sequence thereof of at least 80% (or at least 85%, 90%, 95%) sequence identity; LFR4 comprises FGGGTKVEIK (SEQ ID NO: 61) or a homologous sequence thereof with at least 80% (or at least 90%) sequence identity; X 17 is T or S, and X 18 is R or K, and X 19 is M or I, and X 20 is M or L, and X 21 is I or M, and X 22 is either S or T.

[0184] In certain embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 62 and 63, HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 64 and 65, HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 66 and 67, HFR4 comprises the sequence of SEQ ID NO: 57, LFR1 comprises a sequence from the group consisting of SEQ ID NOs: 68 and 69, LFR2 comprises the sequence of SEQ ID NO: 59, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 70 and 71, and LFR4 comprises the sequence of SEQ ID NO: 61.

[0185] [Table 3-1]

[0186] Table 3-2 illustrates the sequences of the variable regions of the humanized 18B10 antibody.

[0187] [Table 3-2]

[0188] In certain embodiments, the humanized antibodies provided herein may comprise a heavy chain variable region fused to a constant region of a human IgG1 isotype and a light chain variable region fused to a constant region of a human kappa chain.

[0189] The humanized anti-CLDN18.2 antibodies provided herein maintain specific binding affinity for cells expressing CLDN18.2, and in this embodiment, it is at least comparable to or even better than the parent antibody. The humanized antibodies provided herein can also maintain functional interaction with cells expressing CLDN18.2, such as NUGC4 cells, SNU-620 cells, SNU-601 cells, or KATOIII cells, in that all antibodies can mediate cell killing by ADCC, CDC, and apoptosis induced by cross-linking targets on the tumor cell surface and direct inhibition of proliferation. In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein further comprise an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.

[0190] In certain embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein further comprise a constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of the IgG1 isotype. In certain embodiments, the constant region of human IgG1 comprises SEQ ID NO: 49 or a homologous sequence thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0191] The constant region of the IgG1 isotype can induce effector functions such as ADCC or CDC. The effector function of the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein can cause cytotoxicity against cells expressing CLDN18.2. Effector function can be evaluated using various assays, such as Fc receptor binding assays, C1q binding assays, and cytolytic assays, as well as any of the assays described above for determining ADCC or CDC.

[0192] Antibody variants The anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein also encompass various types of variants of the antibody sequences provided herein.

[0193] In certain embodiments, the variants comprise one or more modifications or substitutions in one, two, or three CDR sequences provided in Table 1, one or more FR sequences, the heavy or light chain variable region sequences provided herein, and / or the constant region (e.g., Fc region). Such antibody variants maintain the specific binding affinity of their parent antibody to CLDN18.2 but have one or more desirable properties imparted by the modifications or substitutions. For example, antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or improved effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues), to name a few.

[0194] The parent antibody sequence can be screened using methods known in the art, such as "alanine substitution mutagenesis," to identify residues that are suitable or preferred for modification or substitution (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are generated and screened for desired properties. If substitutions at a particular amino acid position demonstrate the desired functional change, that position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by substituting different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0195] 1. Affinity Variants The affinity variants maintain the specific binding affinity of the parent antibody to CLDN18.2, or even have improved specific binding affinity to CLDN18.2 compared to the parent antibody. Various methods known in the art can be used to achieve this goal. For example, a library of antibody variants (e.g., Fab or scFv variants) can be created and expressed using phage display technology and then screened for binding affinity to human CLDN18.2. In another example, computer software can be used to virtually simulate the binding of an antibody to human CLDN18.2 and identify the amino acid residues of the antibody that form the binding interface. Such residues can be avoided in substitution to prevent a decrease in binding affinity, or can be targeted for substitution to achieve stronger binding.

[0196] In certain embodiments, at least one (or all) substitution in the CDR sequence, FR sequence, or variable region sequence comprises a conservative substitution. "Conservative substitution" in relation to an amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitution can be made in amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), in residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), in residues with acidic side chains (e.g., Asp, Glu), in amino acids with basic side chains (e.g., His, Lys, and Arg), or in residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in protein conformation, and therefore can maintain the biological activity of the protein.

[0197] In certain embodiments, the antibodies or antigen-binding fragments provided herein contain one or more amino acid substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variants contain a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less substitutions in one or more CDR sequences and / or FR sequences.

[0198] In certain embodiments, anti-CLDN18.2 antibodies and antigen-binding fragments thereof comprise one, two, or three CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the CDR sequences listed in Table 1, while maintaining binding affinity for CLDN18.2 at a level comparable to or even higher than that of its parent antibody.

[0199] In certain embodiments, anti-CLDN18.2 antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the variable region sequences of SEQ ID NOs: 23-29 and 37-48, while maintaining a level of binding affinity for CLDN18.2 that is comparable to or even greater than that of the parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in a sequence selected from SEQ ID NOs: 25-29 and 37-48. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (i.e., FRs).

[0200] 2. Glycosylation variants The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein also encompass glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment. As used herein, the term "glycosylation" refers to the enzymatic process of attaching glycans, such as fucose, xylose, mannose, or GlcNAc phosphoserine glycans, to proteins, lipids, or other organic molecules. Depending on the carbon linked to the glycan, glycosylation can be divided into five classes, including N-linked glycosylation, O-linked glycosylation, phosphate glycosylation, C-linked glycosylation, and glypiation.

[0201] Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, such as in the tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine.

[0202] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein include glycosylation variants with improved effector function, eg, ADCC or CDC.

[0203] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is afucosylated.The term " afucosylation " or " afucosylated " refers to the reduction or removal of the core fucose of the N-glycan attached to the antibody.The majority of glycans of human IgG antibodies are known as G0, G1, and G2, which are complex biantennary molecules with 0, 1, or 2 terminal galactose core fucose residues.

[0204] Afucosylated antibody variants are described, for example, in U.S. Patent Application Publication No. 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, U.S. Patent Application Publication No. 2003 / 0115614, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. 2004 / 0132141, U.S. Patent Application Publication No. 2004 / 0132142, U.S. Patent Application Publication No. 2004 / 0132143, U.S. Patent Application Publication No. 2004 / 0132144, U.S. Patent Application Publication No. 2004 / 0132145, U.S. Patent Application Publication No. 2004 / 0132146 ... They can be produced using methods known in the art, such as those described in U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO 2005 / 053742, WO 2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0205] In certain embodiments, the antibody glycosylation variant is afucosylated at Asn297 in the CH2 region of the Fc of the antibody. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues), although due to minor sequence variations in antibodies, it may be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300.

[0206] In certain embodiments, antibody glycosylation variants can be obtained by removal of a native glycosylation site (e.g., N297A substitution), such that, for example, the tripeptide sequence for the N-linked glycosylation site or the serine or threonine residue for the O-linked glycosylation site is no longer present in the antibody or in the Fc sequence. Alternatively, in certain embodiments, antibody glycosylation variants can be obtained by producing the antibody in a host cell line that is incapable of adding a selected sugar group to the mature core carbohydrate structure of the antibody.

[0207] 3. Cysteine-engineered variants The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein also encompass cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0208] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugation with, for example, cytotoxic and / or imaging compounds, labels, or radioisotopes, among others, at the engineered cysteine ​​site, for example, via maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine ​​residues are known in the art, see, for example, WO 2006 / 034488.

[0209] 4. Fc variants The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein also encompass Fc variants comprising modification or substitution of one or more amino acid residues in the Fc region and / or hinge region.

[0210] In certain embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a constant region containing one or more amino acid residue substitutions or modifications that confer increased CDC or ADCC compared to the wild-type constant region. Certain amino acid residues in the CH2 domain of the Fc region can be substituted to achieve enhanced ADCC activity, for example, by increasing the affinity of the Fc domain for FcγRIIIA. Methods for altering ADCC activity by antibody engineering have been described in the art, e.g., Shields RL et al., J Biol Chem. 2001, 276(9):6591-604; Idusogie EE et al., J Immunol. 2000, 164(8):4178-84; Steurer W et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE et al., J Immunol. 2001, 166(4):2571-5; Lazar GA et al., PNAS, 2006, 103(11):4005-4010; Ryan MC et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards JO et al., Mol. Cancer Ther. 2008, 7(8):2517-27; Shields R. Let al., J. Biol. Chem. 2002, 277:26733-26740; Shinkawa T. et al., J. Biol. Chem. 2003, 278:3466-3473.

[0211] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment comprises one or more amino acid substitutions that alter complement dependent cytotoxicity (CDC), e.g., by improving or reducing C1q binding and / or CDC (see, e.g., WO 99 / 51642, Duncan & Winter Nature 322:738-40 (1988), U.S. Pat. No. 5,648,260, U.S. Pat. No. 5,624,821, and WO 94 / 29351 for examples of other Fc region variants).

[0212] In certain embodiments, the constant region of an antibody or antigen-binding fragment thereof provided herein comprises a substitution of one or more amino acid residues relative to SEQ ID NO:49 (i.e., the wild-type sequence) selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof. In certain embodiments, the constant region comprises the sequence of SEQ ID NO:51.

[0213] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants can have an extended pharmacokinetic half-life when bound to FcRn at an acidic pH that allows the variant to escape lysosomal degradation and then be translocated and released extracellularly. Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art; see, for example, Vaughn, D. et al, Structure, 6(1):63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al, J. Immunology, 176:346-356 (2006).

[0214] antigen-binding fragment Anti-CLDN18.2 antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-CLDN18.2 antibodies provided herein, including, for example, the exemplary antibodies whose CDR sequences are shown in Table 1 and various variants thereof (e.g., affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants, etc.).

[0215] In certain embodiments, an anti-CLDN18.2 antigen-binding fragment provided herein is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0216] Various techniques can be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., in the case of Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., in the case of ScFv), and chemical coupling of two Fab'-SH fragments to form an F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0217] In certain embodiments, the antigen-binding fragment is an scFv. The production of scFv is described, for example, in WO 93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458. The scFv can be fused to an effector protein at the amino or carboxyl terminus to produce a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck).

[0218] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. The term "valent" as used herein refers to the presence of a specified number of antigen-binding sites in a given molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites in an antigen-binding molecule, respectively. Any molecule with more than two valencies is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0219] A bivalent molecule can be monospecific when both binding sites are specific for binding to the same antigen or epitope. This, in certain embodiments, achieves stronger binding to the antigen or epitope than its monovalent counterpart. Similarly, a multivalent molecule can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences despite having the same specificity).

[0220] Bivalent can also be bispecific, where the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific, where two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0221] bispecific antibody In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are bispecific. As used herein, the term "bispecific" encompasses molecules with three or more specificities and molecules with three or more specificities, i.e., multispecific. In certain embodiments, the bispecific antibodies and antigen-binding fragments provided herein can specifically bind to the first and second epitopes of CLDN18.2, or to CLDN18.2 and a second antigen. In certain embodiments, the first and second epitopes of CLDN18.2 are separate or do not overlap. In certain embodiments, the bispecific antibodies and antigen-binding fragments thereof can simultaneously bind to both the first and second epitopes. In certain embodiments, the second antigen is different from CLDN18.2.

[0222] In certain embodiments, the second antigen is an immune-related target. In some embodiments, the bispecific antibody and its antigen-binding fragment specifically bind to CLDN18.2 and an immune-related target, and can target immune cells to cells expressing CLDN18.2 (e.g., tumor cells expressing CLDN18.2) and / or activate a CLDN18.2-specific immune response against target cells expressing CLDN18.2. As used herein, immune-related target encompasses biomolecules involved in the development or regulation of immune responses, and optionally, cellular immune responses. Examples of immune-related targets are immune checkpoint molecules and surface molecules of cytolytic immune cells such as T cells or natural killer (NK) cells.

[0223] Immune checkpoint molecules can mediate costimulatory signals to amplify immune responses, or mediate costimulatory signals to suppress immune responses. Examples of immune checkpoint molecules include, for example, PD-L1, PD-L2, PD-1, CLTA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, and CD83.

[0224] Cytolytic immune cells can be triggered by their surface molecules to attack and mediate the lysis of target cells, such as tumor cells. In certain embodiments, the second antigen is a T cell surface antigen. Examples of T cell surface antigens include, but are not limited to, antigens selected from the group consisting of CD3, CD2, CD4, CD5, CD6, CD8, CD28, CD40L, and / or CD44, preferably CD3. In certain embodiments, the second antigen is the epsilon chain of CD3. In certain embodiments, binding of the bispecific antibody to CD3 on a T cell results in proliferation and / or activation of the T cell, which induces the release of cytotoxic factors, such as perforin and granzymes, and cytolysis and apoptosis of the target cell. In certain embodiments, the second antigen is an NK cell surface antigen, such as CD16 (FcγRIII) or CD56. In certain embodiments, binding of the bispecific antibody to CD16 on an NK cell causes NK cell degranulation and perforin-dependent target cell lysis (ADCC) of the target cell.

[0225] In certain embodiments, the second antigen comprises a tumor antigen. "Tumor antigen," as used herein, refers to tumor-specific antigens (e.g., antigens that are unique to tumor cells and not normally found in non-tumor cells), tumor-associated antigens (e.g., found in both tumor and non-tumor cells, but differentially expressed in tumor cells), and tumor neo-antigens (e.g., expressed in cancer cells due to somatic mutations that alter the protein sequence or create a fusion protein between two unrelated sequences).

[0226] Examples of tumor antigens include, but are not limited to, EpCAM, HER2 / neu, HER3 / neu, C250, CEA, MAGE, proteoglycan, VEGF, EGFR, αVβ3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD6, CD7, CD8, CD11, CD13, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD37, CD40, CD41, CD47, CD52, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, Ganglio Sid GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4, Trp2, gp100(Pmel 17), tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125 MUC, Wue antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EpHA2, and cell surface targets GC182, GT468, or GT512, PD-L1, arbovirus E protein epitopes, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin, ART-l / melan-A (MART-1), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5;Ras, unique tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens, e.g., Epstein-Barr virus antigen EBVA, and human papillomavirus (HPV) antigens E6 and E7. Protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum- 1, p15, p16, 43-9F, 5T4(791Tgp72), alpha-fetoprotein (fetoprotem), beta-HCG, BCA225, BTAA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA-50, CAM43, CD68, CO-029, FGF-5, G250, Ga733VEpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0227] In certain embodiments, the tumor antigen is associated with gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof. Examples of such tumor antigens include, but are not limited to, CA-125, ganglioside G(D2), G(M2), and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptide, PSA, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFR (e.g., VEGFR3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, IL-2R, or CO17-1A, CA19-9, and CA72-4. In certain embodiments, the tumor antigen is present in a cell that expresses CLDN18.2, for example, a cancer cell that expresses CLDN18.2.

[0228] The bispecific antibodies and antigen-binding fragments thereof provided herein may be in any suitable format known in the art. For example, exemplary bispecific formats include bispecific diabodies, scFv-based bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), BiTEs, CrossMabs, CrossFabs, Duobodies, SEEDbodies, leucine zippers, dual acting Fab (DAF)-IgGs, and Mabs. 2 It may be in a bispecific format (see, e.g., Brinkmann et al. 2017, Mabs, 9(2):182-212). Bispecific molecules may be symmetric or asymmetric in structure.

[0229] The bispecific antibodies and antigen-binding fragments provided herein can be made using any suitable method known in the art.

[0230] In one embodiment, two immunoglobulin heavy chain-light chain pairs with specificities for different antigens are co-expressed in a host cell to recombinantly produce a bispecific antibody (see, e.g., Milstein and Cuello, Nature, 305:537 (1983)), followed by purification by affinity chromatography.

[0231] In another embodiment, sequences encoding antibody heavy chain variable domains for the two specificities are each fused to an immunoglobulin constant domain sequence and inserted into one or more expression vectors that are then co-transfected with an expression vector for the light chain sequences into a host cell suitable for recombinant expression of the bispecific antibody (see, e.g., WO 94 / 04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be recombinantly constructed and expressed from host cells (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)).

[0232] Alternatively, the leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. The linked antibodies are reduced at the hinge region to four half antibodies (i.e., monomers) and then re-oxidized to form heterodimers (Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)).

[0233] Two antigen-binding domains can also be conjugated or cross-linked to form bispecific antibodies or antigen-binding fragments. For example, one antibody can be coupled to biotin while the other antibody can be coupled to avidin, and the strong association between biotin and avidin can combine the two antibodies into a complex to form a bispecific antibody (see, for example, U.S. Pat. No. 4,676,980, WO 91 / 00360, WO 92 / 00373, and EP 03089). In another example, two antibodies or antigen-binding fragments can be cross-linked by conventional methods known in the art, for example, as disclosed in U.S. Pat. No. 4,676,980.

[0234] Bispecific antigen-binding fragments can be produced from bispecific antibodies, for example, by proteolytic cleavage or chemical linkage. For example, an antigen-binding fragment of an antibody (e.g., Fab') can be prepared and converted to a Fab'-thiol derivative, which can then be mixed and reacted with another converted Fab' derivative having a different specificity for an antigen to form a bispecific antigen-binding fragment (see, e.g., Brennan et al., Science, 229:81 (1985)).

[0235] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein may be engineered at the interface so that knobs-into-hole bonds can form to promote heterodimerization of the two distinct antigen-binding sites. This can maximize the percentage of heterodimers recovered from recombinant cell culture. "Knobs-into-hole," as used herein, refers to the interaction between two polypeptides (e.g., Fc) in which one polypeptide has a protrusion (i.e., "knob") due to the presence of an amino acid residue with a bulky side chain (e.g., tyrosine or tryptophan), and the other polypeptide has a recess (i.e., "hole") with a small side chain amino acid residue (e.g., alanine or threonine), and the protrusion can enter the recess to promote the interaction of the two polypeptides to form a heterodimer or complex. Methods for generating polypeptides with knobs-into-holes are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

[0236] Conjugates In some embodiments, anti-CLDN18.2 antibodies and antigen-binding fragments thereof are linked to one or more conjugate moieties. A conjugate is a moiety that can bind to an antibody or its antigen-binding fragment. It is contemplated that various conjugates can be linked to the antibodies or antigen-binding fragments provided herein (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates can be linked to the antibody or antigen-binding fragment by, among others, covalent bonding, affinity bonding, intercalation, coordinate bonding, complex formation, association, admixture, or addition. In certain embodiments, an antibody or antigen-binding fragment thereof is linked to one or more conjugates via a linker. In certain embodiments, the linker is a hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

[0237] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope-binding moiety that can be utilized for binding to one or more conjugates. For example, such sites may include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, that facilitate covalent linkage to a conjugate.

[0238] The conjugate can be a clearance modifier, a therapeutic agent (e.g., a chemotherapeutic agent), a toxin, a radioisotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, or an enzyme substrate label), a pharmacokinetic-modifying moiety, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, another anti-cancer agent, or a purification moiety (e.g., a magnetic bead or nanoparticle).

[0239] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase, or β-D-galactosidase), radioisotopes, other lanthanides, luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0240] Examples of radioisotopes include: 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 Examples include P. Radioisotope-labeled antibodies are useful in receptor-targeted imaging experiments.

[0241] In certain embodiments, the conjugate may be a pharmacokinetic-modifying moiety such as PEG, which serves to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like.

[0242] In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead or nanoparticle.

[0243] Antibody-drug conjugates In certain embodiments, the present disclosure provides antibody drug conjugates (ADCs) comprising any of the above-described anti-CLDN18.2 antibodies or antigen-binding fragments conjugated to a cytotoxic agent.

[0244] ADCs can be useful for the local delivery of cytotoxic agents, for example, in the treatment of cancer. This allows targeted delivery of cytotoxic agents to tumors and their intracellular accumulation therein, particularly when systemic administration of these unconjugated cytotoxic agents can result in unacceptable levels of toxicity not only to tumor cells sought to be eliminated but also to normal cells (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986): 603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506; Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278).

[0245] In certain embodiments, a cytotoxic agent can be any agent that is harmful to cells or can damage or kill cells. In certain embodiments, the cytotoxic agent is optionally a toxin, a chemotherapeutic agent (e.g., a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a growth inhibitor, or other anti-cancer agent), or a radioisotope.

[0246] Examples of toxins include bacterial and plant toxins, such as diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin, alvin, modeccin, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PARI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, restrictocin, phenomycin, enomycin, and trichothecenes (see, e.g., WO 93 / 21232). Such large molecule toxins can be conjugated to the antibodies or antigen-binding fragments provided herein using methods known in the art, for example, as described in Vitetta et al (1987) Science, 238:1098.

[0247] Cytotoxic agents also include small molecule toxins and chemotherapeutic drugs, such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581, Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansine and maytansinoids (EP 1391213, Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623, U.S. Pat. No. 5,208,020), calicheamicin (Lode et al (1998) Cancer Res. 58:2928, Hinman et al (1993) Cancer Res.53:3336-3342), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vindesine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thioepa The active ingredient may also be chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine), calicheamicin, maytansinoids, dolastatins, uristatins such as MMAE and MMAF (U.S. Pat. Nos. 5,635,483 and 5,780,588), dolostatin, trichothecenes, and CC1065, and derivatives thereof that have cytotoxic activity. .

[0248] The cytotoxic agent can also be a highly radioactive isotope. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 , Sm 153 , Bi 212 , P 32 , Pb 212Radioisotopes include , and radioisotopes of Lu. Methods for conjugating radioisotopes to antibodies via suitable ligand reagents are known in the art (see, e.g., WO 94 / 11026, Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pub. (1991)). The ligand reagent has a chelating ligand capable of binding, chelating, or otherwise complexing with the radioisotope metal and also has a functional group reactive with the thiol of a cysteine ​​on an antibody or antigen-binding fragment. Exemplary chelating ligands include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Tex.).

[0249] The cytotoxic agent can be linked to the antibody or antigen-binding fragment via any suitable linker known in the art, see, e.g., U.S. Pat. Nos. 5,208,020, 6,441,163, or European Patent No. 0 425 235, Chari et al., Cancer Research 52:127-131 (1992), and U.S. Patent Application Publication No. 2005 / 0169933, the disclosures of which are expressly incorporated herein by reference.

[0250] In certain embodiments, the linker is cleavable under certain physiological conditions, thereby facilitating the release of the cytotoxic drug in cells. For example, the linker can be an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker, a thioether linker, and an esterase-labile linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Pat. No. 5,208,020). In some embodiments, the linker can include amino acid residues such as dipeptides, tripeptides, tetrapeptides, or pentapeptides. The amino acid residues in the linker can be naturally occurring or non-naturally occurring amino acid residues. Examples of such linkers include valine-citrulline (ve or val-cit), alanine-phenylalanine (af or ala-phe), glycine-valine-citrulline (gly-yal-cit), glycine-glycine-glycine (gly-gly-gly), and valine-citrulline-p-aminobenzyloxycarbonyl ("vc-PAB"). Amino acid linker components can be designed and optimized for selectivity with respect to enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0251] In certain embodiments, the cytotoxic agent is N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-hexanediamine), or the like. The antibodies or antigen-binding fragments provided herein can be linked by bifunctional linker reagents, including but not limited to, ethylenediamine, diisocyanates (e.g., toluene 2,6-diisocyanate), bis-active fluorine compounds (e.g., 1,5-difluoro[difluom]-2,4-dinitrobenzene), BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPRH, SBAP, SIA, SIAB, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSG (succinimidyl-(4-vinylsulfone)benzoate). These linker reagents are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA) (see pages 467-498, 2003-2004 Applications Handbook and Catalog).

[0252] In certain embodiments, in the ADCs provided herein, the antibody (or antigen-binding fragment thereof) is conjugated to one or more cytotoxic agents at an antibody:drug ratio of about 1 to about 20, about 1 to about 6, about 2 to about 6, about 3 to about 6, about 2 to about 5, about 2 to about 4, or about 3 to about 4.

[0253] The ADCs provided herein can be prepared by any suitable method known in the art. In certain embodiments, a nucleophilic group on an antibody (or antigen-binding fragment thereof) is first reacted with a bifunctional linker reagent and then linked to a cytotoxic agent, or vice versa, i.e., a nucleophilic group on a cytotoxic agent is first reacted with a bifunctional linker and then linked to an antibody.

[0254] In certain embodiments, the cytotoxic agent may contain (or be modified to contain) a thiol-reactive functional group that can react with a cysteine ​​thiol of a free cysteine ​​of an antibody or antigen-binding fragment provided herein. Exemplary thiol-reactive functional groups include, for example, maleimide, iodoacetamide, pyridyl disulfide, haloacetyl, succinimidyl ester (e.g., NHS, N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, or phosphoramidite (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0255] The cytotoxic agent or antibody may be reacted with a linking reagent and then conjugated to form an ADC. For example, an N-hydroxysuccinimidyl ester (NHS) of a cytotoxic agent may be prepared, isolated, purified, and / or characterized, or may be formed in situ and reacted with a nucleophilic group on an antibody. Typically, the carboxyl form of the conjugate is activated by reaction with some combination of a carbodiimide reagent, such as dicyclohexylcarbodiimide; diisopropylcarbodiimide, to provide an NHS ester, or a uronium reagent, such as TsTu (O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, HBTU (O-benzotriazol-1-yl)-N,N,N'N'-tetramethyluronium hexafluorophosphate), or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), an activating agent, such as 1-hydroxybenzotriazole (HOBt), and N-hydroxysuccinimide. In some cases, the cytotoxic agent and antibody may be linked by in situ activation and reaction to form an ADC in one step. Other activating and linking reagents include TBTU (2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluronium hexafluorophosphate), TFFH (N,N',N'',N'''-tetramethyluronium 2-fluoro-hexafluorophosphate), PyBOP (benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole), and arylsulfonyl halides such as triisopropylbenzenesulfonyl chloride.In another example, the antibody or antigen-binding fragment may be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin.

[0256] Chimeric Antigen Receptor (CAR) Compositions The present disclosure also provides a chimeric antigen receptor (CAR) comprising the anti-CLDN18.2 antigen-binding domain and a T cell activation domain provided herein. A chimeric antigen receptor (CAR) is an engineered chimeric receptor that combines the antigen-binding domain of an antibody with one or more signaling domains for T cell activation. Immune cells such as T cells and natural killer (NK) cells can be genetically engineered to express CARs. T cells expressing CARs are called CAR-T cells. CARs can mediate antigen-specific cellular immune activity in T cells, thereby enabling the CAR-T cells to eliminate cells expressing the target antigen (e.g., tumor cells). In one embodiment, binding of the CAR-T cells provided herein to CLDN18.2 expressed on cells such as cancer cells results in the proliferation and / or activation of the CAR-T cells, and the activated CAR-T cells can release cytotoxic factors, such as perforin, granzymes, and granulysin, which can initiate cytolysis and / or apoptosis of cancer cells.

[0257] In some embodiments, the T cell activation domain of a CAR comprises a costimulatory signaling domain and a TCR signaling domain, which can be linked to each other in a random or specified order, optionally by a short peptide linker (e.g., a glycine-serine doublet linker) having, for example, a length of between 2 and 10 amino acids.

[0258] In some embodiments, the CAR further comprises a transmembrane domain. When expressed in a cell, the anti-CLDN18.2 antigen-binding domain is extracellular and the T cell activation domain is intracellular.

[0259] In certain embodiments, the CAR comprises an anti-CLDN18.2 antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CLDN18.2 and comprises an antigen-binding fragment of an antibody provided herein.

[0260] 1. Antigen-binding domain In some embodiments, the anti-CLDN18.2 antigen-binding domain of the CAR comprises one or more CDR sequences provided herein, one or more heavy chain variable domains or light chain variable domains provided herein, or one or more antigen-binding fragments obtained from any of the anti-CLDN18.2 antibodies provided herein.

[0261] In some embodiments, it is beneficial to obtain the antigen-binding domain from the same species as the CAR will ultimately be used. For example, for human use, it may be beneficial to obtain the antigen-binding domain used in the CAR from a human antibody or a humanized antibody. In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain can exist in various other forms, such as Fv, Fab, and (Fab')2, and bifunctional (i.e., bispecific) hybrid antibody fragments (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In certain embodiments, the antigen-binding domain comprises Fab or scFv.

[0262] 2. Transmembrane domain In certain embodiments, the CAR comprises a transmembrane domain fused to the extracellular antigen-binding domain of the CAR. In one embodiment, the transmembrane domain can be selected so that it is naturally associated with one of the domains in the CAR. In some cases, the transmembrane domain can be selected or modified to avoid binding to the transmembrane domain of other members of the T cell receptor complex.

[0263] The transmembrane domain of the CAR provided herein can be derived from the transmembrane domain of any natural membrane-bound or transmembrane protein, such as the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain of the CAR can also use various human hinges, such as a human Ig (immunoglobulin) hinge.

[0264] Alternatively, the transmembrane domain of the CAR provided herein can be synthesized to mainly contain hydrophobic residues, such as leucine and valine.In one embodiment, a triplet of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain.Optionally, a short oligopeptide or polypeptide linker of 2 to 10 amino acids in length can form the link between the transmembrane domain of the CAR and the intracellular signaling domain.A glycine-serine doublet provides a particularly suitable linker.

[0265] 3. TCR signaling domain The T cell activation domain of CAR provided herein comprises a TCR signaling domain.TCR signaling domain can activate the T cell that expresses CAR, thereby exerting at least one of the normal effector functions of T cell, such as cytolytic activity or helper activity, including secretion of cytokines.TCR signaling domain can be either the full-length of natural intracellular signaling domain or its fragment that is sufficient to transmit TCR effector function signal.

[0266] Exemplary intracellular signaling domains useful in the CARs provided herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that function in concert to initiate signal transduction and subsequent antigen receptor ligation, as well as any derivatives or variants of these sequences, and synthetic sequences that have the same functional capabilities.

[0267] The TCR signaling domain that functions in stimulatory manner can comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAM.The examples of ITAM that comprise the TCR signaling domain useful in CAR provided herein include those obtained from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d.In certain embodiments, the TCR signaling domain comprises the cytoplasmic signaling domain obtained from CD3ζ.

[0268] 4. Costimulatory Signaling Domains The T cell activation domain of the CAR provided herein further comprises a costimulatory signaling region.The costimulatory signaling region can be obtained from a costimulatory molecule that functions in an antigen-dependent manner to regulate TCR activation and is necessary for the efficient response of lymphocytes to antigens.Exemplary costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and the ligand that specifically binds to CD83.

[0269] 5. Bispecific CAR In certain embodiments, the CAR is bispecific. In certain embodiments, the bispecific CAR provided herein can specifically bind to a first and a second epitope of CLDN18.2, or specifically bind to CLDN18.2 and a second antigen.

[0270] In one embodiment, the CAR binds to a native epitope of CLDN18.2 present on the surface of living cells.

[0271] 6. Polynucleotide sequence encoding CAR In one aspect, the present disclosure further provides a nucleic acid sequence encoding the CAR provided herein, comprising a first polynucleotide sequence encoding the antigen-binding domain of the CAR provided herein, and optionally a second polynucleotide sequence encoding the transmembrane domain and T cell activation domain provided herein.In some embodiments, the sequence encoding the antigen-binding domain is operably linked to the sequence encoding the transmembrane domain and T cell activation domain.The nucleic acid sequence encoding the desired molecule can be obtained using recombinant DNA methods known in the art, for example, by screening a library from cells that express the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues that contain the gene using standard techniques.Alternatively, the gene of interest can be produced synthetically instead of by cloning.

[0272] In one aspect, the present disclosure provides a vector comprising the nucleic acid sequence encoding the CAR provided herein.In some embodiments, the vector is a retroviral and lentiviral vector construct that expresses the CAR of the present disclosure, which can be directly transduced into cells, or an RNA construct that can be directly transfected into cells.

[0273] In one embodiment, the present disclosure provides an isolated cell that comprises a nucleic acid sequence encoding a CAR and / or expresses a CAR provided herein.

[0274] In certain embodiments, the cells that contain the nucleic acid encoding the CAR or express the CAR are selected from the group consisting of T cells, NK cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In one embodiment, the cells that contain the nucleic acid encoding the CAR or express the CAR exhibit anti-tumor immunity when the antigen-binding domain of the CAR binds to its corresponding antigen. The cytotoxic lymphocytes are preferably autologous cells, but xenogeneic or allogeneic cells can also be used. As used herein, "autologous" refers to any material obtained from the same individual that will later be reintroduced.

[0275] In one aspect, the present disclosure further provides a method of stimulating a T cell-mediated immune response against cells or tissues expressing CLDN18.2 in a subject, the method comprising administering to the subject an effective amount of cells genetically modified to express a CAR provided herein.

[0276] In one aspect, the present disclosure further provides a method for treating a mammal with a disease, disorder or condition associated with increased expression of CLDN18.2, comprising administering to the mammal an effective amount of cells genetically modified to express the CAR provided herein, thereby treating the mammal.In certain embodiments, the cells are autologous T cells.In certain embodiments, the mammal has been diagnosed with a disease, disorder or condition associated with increased expression of CLDN18.2.

[0277] Polynucleotides and Genetic Recombination Methods The present disclosure provides isolated polynucleotides encoding anti-CLDN18.2 antibodies and antigen-binding fragments thereof. As used herein, the terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specified, a particular polynucleotide sequence implicitly encompasses the explicitly indicated sequence as well as conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0278] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[0279] The present disclosure provides a vector (e.g., expression vector) comprising the isolated polynucleotide provided herein. In certain embodiments, the expression vector provided herein comprises a polynucleotide encoding the antibody or antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the polynucleotide sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, rod-shaped viruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pL Examples include exA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

[0280] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing vector DNA herein include prokaryotic cells, yeast cells, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive bacteria, including Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, and Serratia, e.g., Serratia marcescens. marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[0281] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for anti-CLDN18.2 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species, and strains are also suitable, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500); and Saccharomyces cerevisiae (ATCC 56,500). 0), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces ocdentalis; and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger, are commonly available and useful herein.

[0282] Suitable host cells for expressing the glycosylated antibodies or antigen-fragments provided herein are obtained from multicellular organisms, such as vertebrate cells, including plant and insect cells. Numerous baculovirus strains and mutants derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm) have been identified, along with corresponding permissive insect host cells. Various viral strains for transfection, such as the L-1 mutant of Autographa californica NPV and the Bm-5 strain of silkworm NPV, are publicly available, and such viruses can be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be utilized as hosts.

[0283] However, most attention has been focused on vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human uterine cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatoma line (Hep G2). In some preferred embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, and their derivatives.

[0284] To produce anti-CLDN18.2 antibodies, host cells are transformed with the expression or cloning vectors described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies can be produced by homologous recombination, as known in the art.

[0285] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), are suitable for culturing host cells. In addition, any medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. No. Re. 30,985 may be used as a culture medium for host cells. Any of these media may be supplemented, if necessary, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

[0286] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any preceding step to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0287] Anti-CLDN18.2 antibodies and antigen-binding fragments thereof prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0288] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is immobilized is most often agarose, although other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0289] After any preliminary purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer performed at a pH between about 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0290] composition In another aspect, the disclosure provides a composition comprising an anti-CLDN18.2 antibody or antigen-binding fragment thereof.

[0291] In another aspect, the present disclosure provides a composition comprising an afucosylated anti-CLDN18.2 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-CLDN18.2 antibody in the composition has an amount of fucose that is 60% or less (e.g., less than 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%) of the total amount of oligosaccharides (sugars) at Asn297 according to the EU numbering system. The amount of fucose attached to the CH2 domain of the Fc region can be determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297. The amount of fucose can be measured by methods known in the art, such as mass spectrometry. In an exemplary embodiment, the antibody is treated with N-glycosidase (PNGaseF) to hydrolyze N-glycosylated oligosaccharides from the antibody. The hydrolyzed oligosaccharides are labeled with a fluorescent marker, RapiFluor-MS reagent, separated by ultra-high performance liquid-phase hydrophilic interaction chromatography, and detected by a fluorescence detector (UPLC-HILIC-FLR). The relative area comparison method is used to calculate the proportions of various oligosaccharides. In another exemplary embodiment, the amount of fucose can be measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546.

[0292] Pharmaceutical Composition The present disclosure further provides pharmaceutical compositions comprising an anti-CLDN18.2 antibody or antigen-binding fragment thereof (optionally afucosylated) and one or more pharmaceutically acceptable carriers.

[0293] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary ingredients known in the art, or various combinations thereof.

[0294] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers, or stabilizers, such as sugars and cyclodextrins.Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate.As disclosed herein, by including one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments and conjugates provided herein, oxidation of the antibodies or antigen-binding fragments is reduced.This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life.Therefore, in certain embodiments, compositions are provided that include one or more antibodies or antigen-binding fragments disclosed herein and one or more antioxidants, such as methionine. Further provided are methods for preventing oxidation, extending shelf life, and / or improving efficacy of the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0295] To further illustrate, pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's solution, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; Ingredients may include topical anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers may be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting and emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0296] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, and magnesium carbonate.

[0297] In certain embodiments, pharmaceutical compositions are formulated into injectable compositions.Injectable pharmaceutical compositions can be prepared in any conventional form, for example, liquid solution, suspension, emulsion, or suitable solid form for forming liquid solution, suspension, or emulsion.Injectable preparations can include sterile and / or non-pyrogenic solution ready for injection, sterile dry soluble matter, for example, freeze-dried powder that can be combined with solvent immediately before use for hypodermic tablets, sterile suspension ready for injection, sterile dry insoluble matter that can be combined with solvent immediately before use, and sterile and / or non-pyrogenic emulsion.Solution can be either aqueous or non-aqueous.

[0298] In certain embodiments, unit dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile and non-pyrogenic, as known and practiced in the art.

[0299] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients to improve stability or other pharmacological components of the powder or a reconstitution solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those of skill in the art, in one embodiment at a neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial can contain a single dose or multiple doses of an anti-CLDN18.2 antibody or antigen-binding fragment thereof, or composition thereof. Overfilling the vial with a small amount (e.g., about 10%) beyond that required for a dose or series of doses is permissible to facilitate accurate sample retrieval and accurate dosing. The lyophilized powder can be stored under appropriate conditions, for example, at about 4°C to room temperature.

[0300] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount will vary depending on the selected therapy given and can be determined empirically.

[0301] How to use The present disclosure also provides therapeutic methods comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment (optionally afucosylated) provided herein and / or a pharmaceutical composition provided herein, thereby treating or preventing a CLDN18.2-associated disease or condition.

[0302] In another aspect, provided is a method for treating a disease or condition in a subject that will benefit from the regulation of CLDN18.2 activity, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment provided herein (optionally afucosylated) and / or the pharmaceutical composition provided herein.In certain embodiments, the disease or condition is a CLDN18.2-related disease or condition.In some embodiments, the CLDN18.2-related disease or condition is cancer.

[0303] In certain embodiments, the cancer is selected from gastric cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

[0304] Examples of cancer include, but are not limited to, non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (e.g., basal breast carcinoma, ductal carcinoma, and lobular breast carcinoma), and carcinoma), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myeloma, mycosis fungoides, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancies, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma carcinoma), papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatocellular carcinoma, biliary ductal carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytocyte-rich B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV-positive and -negative PTLD, and diffuse large B-cell lymphoma These include: DLBCL, plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary central nervous system lymphoma, spinal axis tumor, brain stem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinal germ cell tumor.

[0305] In certain embodiments, the cancer is a CLDN18.2-expressing cancer. "CLDN18.2-expressing cancer," as used herein, means any cancer or tumor involving cancer cells that express CLDN18.2.

[0306] In certain embodiments, a subject is identified as having cancer cells that express CLDN18.2. The presence and / or expression level of CLDN18.2 on cancer cells can be determined by various methods known in the art. A biological sample containing or suspected to contain cancer cells can be obtained from the subject. In some embodiments, the biological sample can be obtained from cancer cells or cancer tissue, or tumor-infiltrating immune cells. In certain embodiments, the biological sample can be further processed to isolate analytes, such as nucleic acids or proteins. The presence and / or expression level of CLDN18.2 can be determined by, for example, quantitative fluorescence cytometry, immunohistology (IHC), or nucleic acid-based methods. For example, a biological sample from a subject can be exposed to an anti-CLDN18.2 antibody or its antigen-binding fragment, which binds to and detects expressed CLDN18.2 protein. Alternatively, CLDN18.2 can be detected at the nucleic acid expression level using methods such as qPCR, reverse transcription PCR, microarray, SAGE, and FISH.

[0307] In certain embodiments, the expression of CLDN18.2 in biological samples or cancer cells is determined or measured by IHC. In certain embodiments, the expression level of human CLDN18.2 protein on cancer cells derived from a subject can be determined according to the methods described in Sections 6 and 7 of Example 15 provided herein.

[0308] In certain embodiments, a subject is identified as having cancer cells that highly express CLDN18.2, moderately express CLDN18.2, or lowly express CLDN18.2. In certain embodiments, cancer cells that highly express CLDN18.2 are those that express at least 40% (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 190%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 1000%, at least 1200%, at least 1400%, at least 1500%, at least 1600%, at least 1700%, at least 1800%, at least 1900%, at At least 95%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-80%, 50-70%, 50-60%, 60-80%, 60-70%, or 70-80%) cancer cells that moderately express CLDN18.2 express CLDN18.2 at a level where at least 30% (or at least 35%) to less than 40% of the cells stain positive by IHC, with an intensity of at least 1+ to less than 2+ as measured by IHC; cancer cells that lowly express CLDN18.2 express CLDN18.2 at a level where more than 0% but less than 30% of the cells (e.g., 5%, 10%, 15%, 20%, 25%, 5-25%, 10-25%, 15-25%, 20-25%, 5-20%, 5-15%, 5-10%, 10-20%, or 10-15%) stain positive by IHC, with an intensity of more than 0 but less than 1+ as measured by IHC.

[0309] Examples of cancers that express CLDN18.2 include, but are not limited to, gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), ovarian cancer, colon cancer, colorectal cancer, gastrointestinal mesenchymal tumor (GIST), gastrointestinal carcinoid tumor, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, appendix cancer; prostate cancer, kidney cancer (e.g., renal cell carcinoma), liver cancer, head and neck cancer, and gallbladder cancer and metastases thereof, e.g., gastric cancer metastasis, e.g., Krukenberg's tumor, peritoneal dissemination, and lymph node metastasis.

[0310] In certain embodiments, the cancer that expresses CLDN18.2 can be adenocarcinoma, for example, advanced adenocarcinoma.In certain embodiments, the cancer is selected from intragastric, esophageal, pancreatic, biliary, lung, and ovarian adenocarcinoma.In certain embodiments, the cancer that expresses CLDN18.2 includes gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer.

[0311] Without wishing to be bound by any theory, the molecular and functional properties of CLDN18 are believed to make it a very attractive target for antibody-based cancer therapy. These include (i) the absence of CLDN18 in the majority of toxicity-related normal tissues, (ii) the restriction of CLDN18.2 mutant expression to unwanted cell populations, such as differentiated gastric cells, which can be replenished by target-negative stem cells in the stomach, (iii) potential differential glycosylation between normal and neoplastic cells, and (iv) the existence of distinct conformational topologies.

[0312] The molecular weight of CLDN18 protein has been found to differ between tumor cells and adjacent normal cells. Higher molecular weight CLDN18 protein is observed in healthy tissues, which can be reduced to the same molecular weight observed in tumors by treating normal tissue lysates with the deglycosylating compound PNGase F. This suggests that CLDN18 is less glycosylated in tumors than its normal tissue counterparts. A classical N-glycosylation motif is at amino acid residue 116 within the loop D3 domain of the CLDN18 molecule. The difference in molecular weight and the predicted structural differences may represent an altered epitope for antibody binding.

[0313] Additionally, CLDN18 as a tight junction protein may contribute to a favorable therapeutic window. Although tumor cells express CLDN, they often do not form classical tight junctions through homotypic and heterotypic association of CLDN, as found in normal epidermal tissues. Therefore, they likely have a significant pool of free CLDN, amenable to extracellular antibody binding and immunotherapy. It is possible that the binding of CLDN to epitopes in healthy epithelial tissues is blocked within tight junctions from access to antibody binding.

[0314] The therapeutically effective amount of the antibody or antigen-binding fragment provided herein will vary depending on various factors known in the art, such as body weight, age, past medical history, current drug administration, the subject's health status and potential for cross-reactivity, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression. Dosages can be proportionally increased or decreased by a skilled artisan (e.g., a physician or veterinarian) as indicated by these or other circumstances or requirements.

[0315] In certain embodiments, the antibody or antigen-binding fragment provided herein can be administered at a therapeutically effective dosage of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the dosage can be changed during the course of treatment. In certain embodiments, the dosage can be changed during the course of treatment depending on the subject's response.

[0316] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over time.

[0317] The antibodies or antigen-binding fragments disclosed herein can be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, e.g., intravenous infusion, intramuscular, or intradermal injection), or parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0318] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein can be administered alone or in combination with one or more additional therapeutic means or drugs.For example, the antibody or antigen-binding fragment thereof disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapy drug, an anti-cancer drug, radiation therapy, immunotherapy, an anti-angiogenic agent, targeted therapy, cell therapy, gene therapy, hormone therapy, palliative care, surgery for cancer treatment (e.g., tumor removal), or one or more antiemetic drugs, or other treatments for complications resulting from chemotherapy.

[0319] The term "immunotherapy" as used herein refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or boosts the immune system in a general way.Immunotherapy includes passive immunotherapy (e.g., antibody therapy or CAR-T cell therapy) by delivering an agent (e.g., effector cells) with established tumor immune reactivity, which can directly or indirectly mediate anti-tumor effects and does not necessarily rely on an intact host immune system.Immunotherapy can also include active immunotherapy, in which the treatment relies on the in vivo stimulation of the endogenous host immune system to react against abnormal cells by administering an immune response modifier.

[0320] Examples of immunotherapies include, but are not limited to, checkpoint modulators, cell transfection, cytokines, oncolytic viruses, and therapeutic vaccines.

[0321] Checkpoint modulators can interfere with the ability of cancer cells to evade immune system attack and help the immune system respond more effectively to tumors. Immune checkpoint molecules can mediate costimulatory signals to either enhance the immune response or suppress it. Examples of checkpoint modulators include, but are not limited to, modulators of PD-1, PD-L1, PD-L2, CLTA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, and CD83.

[0322] Adoptive cell transfer is a treatment that attempts to enhance the natural ability of T cells to fight cancer. In this treatment, T cells are collected from patients, expanded, and activated in vitro. In certain embodiments, T cells are converted into CAR-T cells in vitro. The T cells or CAR-T cells that are most active against cancer are cultured in vitro in large batches for 2 to 8 weeks. During this period, the patient undergoes treatments such as chemotherapy and radiation therapy to reduce the body's immunity. After these treatments, the in vitro cultured T cells or CAR-T cells are returned to the patient. In certain embodiments, the immunotherapy is CAR-T therapy.

[0323] Cytokine therapy can also be used to enhance tumor antigen presentation to immune system.Two main types of cytokines used to treat cancer are interferon and interleukin.Examples of cytokine therapy include but are not limited to interferon, for example, interferon-α, -β and -γ, colony-stimulating factors, for example, macrophage CSF, granulocyte macrophage CSF and granulocyte CSF, insulin growth factor (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor β, fibroblast growth factor (FGF), interleukins, for example, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factors, for example, TNF-α and TNF-β, or any combination thereof.

[0324] Oncolytic viruses are genetically engineered viruses that can kill cancer cells. They can specifically infect tumor cells, causing tumor cell lysis and the subsequent release of large amounts of tumor antigens, which then trigger the immune system to target and eliminate cancer cells bearing those tumor antigens. Examples of oncolytic viruses include, but are not limited to, talimogene laherparepvec.

[0325] Therapeutic vaccines combat cancer by enhancing the immune system's response to cancer cells. They can contain nonpathogenic microorganisms (e.g., Mycobacterium bovis, Bacillus Calmette-Guerin, or BCG) with genetically engineered viruses that target tumor cells or one or more immunogenic components. For example, BCG can be inserted directly into the bladder via catheter to generate an immune response against bladder cancer cells.

[0326] Anti-angiogenic agents can inhibit the growth of blood vessels that support tumor growth.Some anti-angiogenic agents target VEGF or its receptor VEGFR.Examples of anti-angiogenic agents include but are not limited to axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and Ziv-aflibercept.

[0327] "Targeted therapy" is a type of therapy that acts on specific cancer-associated molecules, such as specific proteins, that are present in cancer cells but not in normal cells or that are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to cancer growth and survival. Targeted therapy directs therapeutic agents to the tumor, thereby sparing normal tissues from the therapeutic agent's effects.

[0328] Targeted therapy can target, for example, tyrosine kinase receptors and nuclear receptors. Examples of such receptors include erbB1 (EGFR or HER1), erbB2 (HER2), erbB3, erbB4, FGFR, platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor-1 receptor (IGF-1R), estrogen receptor (ER), nuclear receptor (NR), and PR.

[0329] Targeted therapies can target molecules in tyrosine kinase or nuclear receptor signaling cascades, such as Erk and PI3K / Akt, AP-2α, AP-2β, AP-2γ, mitogen-activated protein kinase (MAPK), PTEN, p53, p19ARF, Rb, Apaf-1, CD-95 / Fas, TRAIL-R1 / R2, caspase-8, forkhead, Box03A, MDM2, IAPs, NF-kB, Myc, PI3K, Ra, FLIP, heregulin (HRG) (also known as gp30), Bcl-2, Bcl-xL, Bax, Bak, Bad, Bok, Bik, Blk, Hrk, BNIP3, BimL, Bid, and EGL-1.

[0330] Targeted therapy can also target tumor-associated ligands, such as estrogens, estradiol (E2), progesterone, estrogen, androgens, glucocorticoids, prolactin, thyroid hormones, insulin, P70 S6 kinase protein (PS6), survivin, fibroblast growth factor (FGF), EGF, Neu-differentiation factor (NDF), transforming growth factor alpha (TGF-α), IL-1A, TGF-β, IGF-1, IGF-II, IGFBPs, IGFBP proteases, and IL-10.

[0331] In certain of these embodiments, antibodies or antigen-binding fragments provided herein that are administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents; in certain of these embodiments, the antibody or antigen-binding fragment and the additional therapeutic agent may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered "in combination with" another therapeutic agent need not be administered simultaneously with the agent by or in the same composition. An antibody or antigen-binding fragment administered before or after another agent is considered to be administered "in combination with" that agent, as that term is used herein, even if the antibody or antigen-binding fragment and the second agent are administered by different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to protocols known in the art, such as those in the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)).

[0332] The present disclosure further provides a method for using an anti-CLDN18.2 antibody or an antigen-binding fragment thereof. In some embodiments, the present disclosure provides a method for inhibiting the proliferation of cells expressing CLDN18.2 in vivo or in vitro, the method comprising contacting the cells expressing CLDN18.2 with an antibody or an antigen-binding fragment thereof provided herein. In some embodiments, the present disclosure provides a method for regulating the CLDN18.2 activity of cells expressing CLDN18.2, the method comprising exposing the cells expressing CLDN18.2 to an antibody or an antigen-binding fragment thereof provided herein.

[0333] In some embodiments, the present disclosure provides a method for detecting the presence or amount of CLDN18.2 in a sample obtained from a subject, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof, and determining the presence or amount of CLDN18.2 in the sample. In certain embodiments, the biological sample contains cancer cells.

[0334] In some embodiments, the present disclosure provides a method for diagnosing a CLDN18.2-related disease or condition in a subject, the method comprising: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of CLDN18.2 in the sample; and c) correlating the presence or amount of CLDN18.2 with the presence or status of a CLDN18.2-related disease or condition in the subject. In certain embodiments, the biological sample contains cancer cells. In some embodiments, the expression level of CLDN18.2 in the cancer cells is determined by IHC (e.g., according to the methods described in Sections 6 and 7 of Example 15 provided herein). In some embodiments, the subject is identified as having cancer cells that highly express CLDN18.2, intermediately express CLDN18.2, or lowly express CLDN18.2.

[0335] In some embodiments, the method further comprises administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof provided herein to a subject. In some embodiments, the subject has cancer cells that moderately express CLDN18.2 or that lowly express CLDN18.2.

[0336] In some embodiments, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety. The kit may be useful in detecting the presence or amount of CLDN18.2 in a biological sample, or may be useful in the diagnostic methods provided herein.

[0337] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent. The kit may be useful in treating, preventing, and / or ameliorating a CLDN18.2-associated disease.

[0338] In some embodiments, the present disclosure further provides an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CLDN18.2-associated disease or condition in a subject. [Example]

[0339] While the present disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred, it should be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

[0340] [Example 1] Preparation of CLDN18.2- or CLDN18.1-expressing cell lines 1. Generation of HEK293-human CLDN18.2, HEK293-human CLDN18.1, and HEK293-mouse CLDN18.2 cell lines HEK293-human CLDN18.2 cells (hereinafter referred to as HEK293-CLDN18.2) and HEK293-mouse CLDN18.2 cells (hereinafter referred to as HEK293-mCLDN18.2) were constructed by MabSpace Biosciences (Suzhou) Co., Limited. Briefly, HEK293 cells (Shanghai Institutes for Biological Sciences, catalog number GNhu43) were transfected with pcDNA3.1 / hCLDN18.2 or pcDNA3.1 / mCLDN18.2 plasmids and selected with G418 to obtain stable-expressing cell lines HEK293-CLDN18.2 or HEK293-mCLDN18.2. The expression levels of hCLDN18.2 or mCLDN18.2 were detected by the IMAB362 antibody, which can bind to both human and mouse CLDN18.2. IMAB362 was expressed according to the sequence disclosed in US2009169547A1. Single cell clones with the highest signal were selected and expanded for cell banking.

[0341] Heavy chain variable region of IMAB362 (SEQ ID NO: 72) QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS

[0342] Light chain variable region of IMAB362 (SEQ ID NO: 73) DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK

[0343] HEK293-human CLDN18.1 cells (hereinafter referred to as HEK293-CLDN18.1) were also constructed as described above. CLDN18.1 expression was detected by anti-CLDN18 antibody (Abcam, catalog number ab222513), which recognizes both CLDN18.1 and CLDN18.2.

[0344] 2. Generation of CHO-CLDN18.2 Transient-expressing Cells CHO-CLDN18.2-expressing cells were constructed as follows: CHO cells were transiently transfected with pcDNA3.1 / CLDN18.2 without a selection reagent. Cell membrane proteins were extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit and used for animal immunization boost.

[0345] 3. Generation of MKN45-CLDN18.2 Transient-expressing Cells MKN45-CLDN18.2 cells were constructed by MabSpace Biosciences (Suzhou) Co., Limited. Briefly, MKN45 cells (National Infrastructure of Cell Line Resource, catalog number 3111C0001CCC000229) were transfected with the pcDNA3.1 / CLDN18.2 plasmid and selected with G418 to obtain the stable expressing cell line MKN45-CLDN18.2. The expression level of CLDN18.2 was detected using the IMAB362 antibody using FACS. Monoclonal cells with the highest, medium, and low signals were selected and amplified for cell banking.

[0346] [Example 2] The above cell lines were used in the following experiments. antibody generation 1.Immunization Both DNA and cell immunogens were prepared for immunization. Six- to eight-week-old mice of various strains were divided into two groups. One group was initiated and boosted intravenously with 100 μg / mouse of pVAC2-mcs / CLDN18.2 plasmid and 100 μg / mouse of CpG. The other group was injected im with the same DNA and CpG. Both groups were injected on days 1 and 10, and antibody titers were detected on day 18 by FACS binding to HEK293-CLDN18.2 cells. 100 μl / well of diluted mouse serum was added to plates containing HEK293-CLDN18.2 or gastric cancer NUGC4 cells (JCRB, catalog number JCRBB0834) and then incubated at 4°C for 30 minutes. After washing with buffer, 100 μl / well of goat anti-mIgG-FITC (1:500 dilution) was added for further incubation at 4° C. for 30 minutes. Following washing with FACS wash buffer, cells were analyzed by flow cytometry. Mice with higher binding signals and titers were selected for the following fusion procedure.

[0347] 2.Fusion Four days before fusion, 5 × 10 7 Each mouse was intraperitoneally boosted with 100 HEK293-CLDN18.2 cells. On the day of fusion, spleens were aseptically harvested and then processed into single-cell suspensions. Viable log-phase myeloma cells (SP2 / 0) were mixed with mouse splenocytes in fusion medium at a 1:1 ratio and subsequently electrofused for 1 minute. Cells were resuspended and cultured in 96-well culture plates at 200 μl per well in a 37°C, 5% CO2 incubator. After 7 days of culture, the growth medium was replaced with fresh growth medium, and hybridoma supernatants were subsequently screened 2–3 days later.

[0348] [Example 3] Antibody screening 1. Screening of human CLDN18.2 positive binders by FACS assay Log-phase CLDN18.2-expressing HEK293-CLDN18.2 cells were cultured at 10 cells per well. 5The cells were resuspended in PBS at a density of 100 μl / 100 μl. After washing the cells three times with FACS wash buffer (PBS + 2% FBS), 100 μl / well of hybridoma supernatant was added to each well for 30 minutes of incubation at 4°C. The cells were again washed three times with FACS wash buffer and then incubated with 100 μl / well of goat anti-mIgG-FITC (1:400 dilution) for an additional 30 minutes at 4°C. After a final three washes with FACS wash buffer, the cells were analyzed by flow cytometry.

[0349] 2. Screening of CLDN18.1 negative binders by FACS assay 10 log-phase CLDN18.1-expressing HEK293-CLDN18.1 cells per well 5 The cells were resuspended in PBS at a density of 100 μl / 100 μl. After washing the cells three times with FACS wash buffer (PBS + 2% FBS), 100 μl / well of hybridoma supernatant was added to each well for 30 minutes of incubation at 4°C. The cells were again washed three times with FACS wash buffer and then incubated with 100 μl / well of goat anti-mIgG-FITC (1:400 dilution) for an additional 30 minutes at 4°C. After a final three washes with FACS wash buffer, the cells were analyzed by flow cytometry.

[0350] Clones with high signals of CLDN18.2 binding but no CLDN18.1 binding were selected for subsequent subcloning to generate monoclones including 7C12, 11F12, 12E9, 26G6, 59A9, 18B10, and 12C12.

[0351] [Example 4] Subcloning of positive hybridoma clones and small-scale antibody production 1. Subcloning of positive hybridoma clones Cells with the desired binding profile were selected from FACS-positive hybridoma wells by limiting dilution in a 96-well plate. These cells were grown for 7 days. When a sufficient cell mass was reached, the supernatant from each well was collected and rescreened using a cell binding assay (see Example 3).

[0352] Clones with the highest cell binding activity from each 96-well plate were expanded into 96-well plates containing 200 μl of hybridoma growth medium per well for the second round of limiting dilution. After 7 days, cell supernatants from the 96-well plates were analyzed by FACS assay. Subcloning was performed twice more until more than 90 / 96 wells showed positive binding signals. Clones with the highest binding activity were identified, further expanded, and cultured for antibody production. Isotypes were determined using standard methods.

[0353] 2. Small-scale Antibody Production Hybridoma cells were seeded and cultured for 14 days. CLDN18.2 monoclonal antibody (mAb) was purified from the hybridoma cell culture by affinity chromatography using a Protein A chromatography column (Protein A High Performance (Bio-Rad)).

[0354] After purification, CLDN18.2 mAb was formulated in PBS by dialysis using a 10,000 MWCO membrane (Pierce Slide-A-Lyzer or dialysis tubing) followed by a filtration step.

[0355] [Example 5] Cell binding analysis of purified CLDN18.2 hybridoma antibodies Log-phase HEK293-CLDN18.2 and NUGC4 cells were resuspended in PBS. After washing the cells three times with FACS wash buffer (PBS + 2% FBS), 100 μl / well of diluted hybridoma Ab ranging from 400 nM to 0.002 nM was added to each well for 30 min of incubation at 4°C. The cells were again washed three times with FACS wash buffer and then incubated with 100 μl / well of goat anti-mIgG-FITC (1:400 dilution) for an additional 30 min at 4°C. After a final three washes with FACS wash buffer, the cells were analyzed by flow cytometry.

[0356] Most of the hybridoma antibodies showed high-affinity binding to HEK293-CLDN18.2 cells but less to NUGC4 cells. The difference in binding is likely due to the different expression density, conformation, and / or glycosylation state of CLDN18.2 protein in these two cell lines. Interestingly, 7C12, 11F12, 59A9, and 18B10 had comparable binding affinities to both HEK293-CLDN18.2 and NUGC4 cells (Figures 1A-1D, Table 4). These hybridoma antibodies were selected for gene cloning and chimeric antibody expression for further functional ADCC / CDC characterization.

[0357] [Table 4]

[0358] [Example 6] Generation of chimeric antibodies The sequences of the light and heavy chain variable regions of the mouse anti-human CLDN18.2 antibody were obtained by polymerase chain reaction (PCR) amplification from candidate hybridoma cell lines. After sequencing analysis and confirmation, the above variable region genes, including the sequence of the light chain variable region (VL) fused to the human IgG kappa constant region and the sequence of the heavy chain variable region (VH) fused to the human IgG1 constant region, were cloned into the recombinant expression vector, pcDNA3.1(+), for antibody production and purification.

[0359] ExpiCHO cells were transfected with equal amounts of DNA from the heavy and light chain vectors using the ExpiCHO transfection kit. The transfected cells were cultured in shake flasks at 125 rpm in an incubator at 8% CO2 and 37°C. The cell cultures were harvested on day 10, and the recovered antibodies were purified by affinity chromatography. The resulting antibodies were analyzed to determine the level of purity using SDS-PAGE and size exclusion chromatography (TSKgel G3000SWXL, TOSOH). The chimeric antibodies were designated 7C12-C, 11F12-C, 12E9-C, 26G6-C, 59A9-C, 18B10-C, and 12C12-C.

[0360] [Example 7] Characterization of purified chimeric CLDN18.2 antibody 1. Binding and cytotoxic effects in HEK293-CLDN18.2 cells Cell binding of the chimeric antibody was detected according to the method described in Example 5.

[0361] As shown in Figure 2A, 7C12-C, 11F12-C, and 12E9-C, which have very similar CDRs (only differing by 2-3 amino acids), bound to HEK293-CLDN18.2 cells with an EC50 of approximately 0.6 μg / ml. 26G6-C had an EC50 of 1.1 μg / ml. 59A9-C and 18B10-C were produced later, and as a result, they were tested separately. As shown in Figure 2C, 59A9-C, which has a different germline and CDR, had a slightly higher EC50 (1.3 μg / ml) than 18B10-C (1.0 μg / ml).

[0362] CDC (complement-dependent cytotoxicity) is an important mechanism of immune defense. Therefore, the CDC assay was used here to evaluate the biological efficacy of antibodies. Briefly, log-phase HEK293-CLDN18.2 cells were resuspended in RPMI1640 with 10% FBS. These cells were cultured at 8 × 10 per well. 3 The cells were plated at 100 μl / well. The anti-CLDN18.2 chimeric antibody and the control antibody IMAB362 were diluted using RPMI 1640 with 60% 20 mM HEPES and 40% human serum, and then added to the cell plate at a final concentration of 10 to 0.0012 μg / ml, 100 μl / well. The plate was incubated at 37°C for 80 minutes. The cell culture plate was then equilibrated to room temperature for 30 minutes. For cell viability analysis at room temperature using a microplate reader (Thermo VARIOSKAN FLASH 3001), the CellTiter-Glo Luminescent Cell Viability Assay Kit was used.

[0363] As shown in Figures 2B and 2D, all six CLDN18.2 chimeric antibodies induced CDC effects at lower concentrations compared to IMAB362. The potency of four antibodies (7C12-C, 11F12-C, 12E9-C, and 26G6-C) was more than two-fold increased over IMAB362. 59A9-C and 18B10-C had more than a three-fold increase in potency compared to IMAB362.

[0364] 2. Binding and cytotoxic effects in MKN45-CLDN18.2 cells MKN45 is a poorly differentiated gastric adenocarcinoma suitable for evaluating antitumor efficacy in vivo. However, MKN45 cells do not express human CLDN18.2 unless transfected. We found that different expression levels of human CLDN18.2 in MKN45 cells confer different sensitivity to CLDN18.2 antibodies. Then, high and medium CLDN18.2-expressing MKN45 cells (see Figure 21) were selected for the following study.

[0365] Cell-binding assays of the chimeric antibodies were performed as described in Example 5 using high- and medium-CLDN18.2-expressing MKN45 cells. As shown in Figures 3A (high) and 3C (medium), 18B10-C bound to both high- and medium-hCLDN18.2-expressing cells with significantly higher affinity than IMAB362. In MKN45-CLDN18.2-high cells, the potency of 18B10-C was approximately two-fold greater than that of IMAB362. An even more significant difference was observed in MKN45-CLDN18.2-medium cells, where 18B10-C showed an EC50 of 0.96 μg / ml, whereas IMAB362 did not bind.

[0366] ADCC activity was evaluated using Jurkat-NFAT-luc-FcγRIIIA-V176 cells as effector cells and MKN45-CLDN18.2 cells as target cells. Jurkat-NFAT-luc-FcγRIIIA-V176 cells were constructed at Mabspace Biosciences (Suzhou) Co., Limited. Briefly, Jurkat cells (Shanghai Institutes for Biological Sciences, catalog number SCSP-513) were transfected with the pGL4.30-luc / NFAT-RE / Hygro plasmid and selected with hygromycin to obtain the stable Jurkat-NFAT-luc cell line. The Jurkat-NFAT-luc cell line was further transfected with the pcDNA3.1-FcγRIIIA-V176 plasmid and selected with the antibiotic G418 to obtain the stable Jurkat-NFAT-luc-FcγRIIIA-V176 cell line.

[0367] Log-phase target cells were then resuspended in RPMI 1640 with 10% FBS and plated at 1 x 10 cells per well for 30 min incubation at 37°C. 4 The anti-hCLDN18.2 chimeric antibody and the control antibody IMAB362 were diluted using RPMI 1640 with 10% FBS and then added to the target cell plate at a final concentration of 100-0.0017 μg / ml. Log-phase Jurkat-NFAT-luc-FcγRIIIA-V176 cells were also plated at 6 × 10 per well in the above plates. 4 The cells were added at 1000 x g for 6 hours at 37°C. The plate was then incubated at 37°C for 6 hours. The cell culture plate was then equilibrated to room temperature for 30 minutes. The CellTiter-Glo Luminescent Cell Viability Assay Kit was used for cell viability analysis at room temperature using a microplate reader (Thermo VARIOSKAN FLASH 3001).

[0368] Based on the reporter readout curve, an EC50 can be calculated and used to evaluate ADCC efficacy. As shown in Figure 3B, when using MKN45-CLDN18.2-high cells, IMAB362 had few points for calculating an EC50, but the two curves suggested that 18B10-C had better ADCC activity than IMAB362. As shown in Figure 3D, in MKN45-CLDN18.2-medium cells, 18B10-C had a more than 50-fold increase in ADCC potency, as measured by EC50, compared with IMAB362. These results suggested that cells with medium CLDN18.2 expression could distinguish the 18B10-C antibody from IMAB362 better than high-expressing cells. No CDC activity was observed in MKN45-hCLDN18.2 cells (data not shown).

[0369] 3. Binding and Cytotoxic Effects in NUGC4 Cells NUGC4 represents a gastric cell line with expression levels of hCLDN18.2 similar to those from gastric cancer patients.

[0370] Cell binding assays and ADCC reporter assays were performed according to the same methods described above (see Section 2 of this Example). As shown in Figures 4A and 4C, five of the six chimeric antibodies, except for 26G6-C and 59A9-C, bound to NUGC4 cells with an EC50 of approximately 10 μg / ml (see Table 5). 26G6-C bound to NUGC4 with a higher EC50 (67 μg / ml), indicating higher affinity. 59A9-C showed both a higher EC50 (19 μg / ml) and a lower maximum signal. Furthermore, Figures 4B and 4D showed similar trends in their ADCC activity against NUGC4 cells. Due to the two separate experiments, the EC50 and maximum signal may vary between Figures 4B and 4D. Importantly, all tested chimeric antibodies demonstrated better ADCC activity than IMAB362, and in particular, 18B10-C had a 40-fold increase over IMAB362, with no CDC activity observed in NUGC4 cells (data not shown).

[0371] Table 5 summarizes the FACS binding data for all chimeric antibodies and IMAB362 to HEK293-CLDN18.2 and NUGC4 cells.

[0372] [Table 5]

[0373] 4. Specificity of chimeric CLDN18.2 antibodies CLDN18.2 has only a few amino acid differences from CLDN18.1, which is present in many normal tissues and organs. The specificity of antibody binding to CLDN18.2 is extremely important. The cell binding assay was the same as described above (see section 1 of this example). Figure 5 shows the binding of 18B10-C and IMAB362 to CLDN18.2- or CLDN18.1-expressing HEK293 cells. Both antibodies bound only to CLDN18.2-expressing cells, but not to CLDN18.1-expressing cells. Other chimeric antibodies also had similarly good selectivity (data not shown).

[0374] [Example 8] Epitope binning Hybridoma antibodies compete with benchmark antibodies for binding to CLDN18.2-expressing cells Log-phase MKN45-CLDN18.2 cells were resuspended in FACS wash buffer (PBS with 2% BSA) and then plated at 1 x 10 per well. 5The cells were added to a 96-well V-bottom plate at a density of 1 cell per well. Diluted hybridoma antibodies or IMAB362-mIgG2a (final concentrations: 100-0.01 μg / ml) were added to the plate. The plate was incubated at 4°C for 1 hour to allow the antibodies to fully occupy the antigens on the cell surface. The cells were washed twice with FACS wash buffer, and 10 μg / ml of IMAB362 or 5 μg / ml of 18B10-C was added to the cells for an additional 1 hour of incubation at 4°C. The cells were then washed three times and incubated with goat anti-hIgG(H+L)-FITC (1:200 dilution). Finally, the cells were washed three times with FACS wash buffer and analyzed by flow cytometry.

[0375] As shown in Figures 6A and 6B, hybridoma antibody 18B10 could completely block the binding of IMAB362 to MKN45-CLDN18.2 cells, indicating that 18B10 may have a higher binding affinity than IMAB362 but is dependent on similar or nearby amino acids (Table 6).

[0376] [Table 6]

[0377] [Example 9] Epitope mapping of selected antibodies by site-directed mutagenesis at CLDN18.2 amino acids that differ from CLDN18.1 1. Generation of Human CLDN18.2-mRFP and Human CLDN18.1-mRFP Constructs cDNAs encoding human CLDN18.1 (amino acids 1-261, SEQ ID NO: 31)-mRFP1 (amino acids 1-225) and human CLDN18.2 (amino acids 1-261, SEQ ID NO: 30)-mRFP1 (amino acids 1-225) were synthesized in vitro (SEQ ID NO: 52 and SEQ ID NO: 53 are the amino acid sequences, respectively). The PCR products were then cloned into the pcDNA3.1(+) vector by homologous recombination using Syno Assembly Mix Reagent (Synbio) according to the manufacturer's instructions. The plasmids were purified using the QIAGEN Plasmide Mega kit (QIAGEN).

[0378] Human CLDN18.1 and CLDN18.2 (Genbank accession numbers: splice According to the sequences of variant 1 (CLDN18.1): NP_057453, NM_016369 and splice variant 2 (CLDN18.2): NM_001002026, NP_001002026, eight different amino acids are located between positions 28 and 70, which may be the determinant for specific binding to human CLDN18.2 but not to CLDN18.1. Using the wild-type human CLDN18.2-mRFP plasmid generated above as a template, two segments of the integrated sequence were generated using primers. Variants of human CLDN18.2-mRFP with single amino acids changed to those of human CLDN18.1 at the indicated positions were amplified by overlapping PCR using primers. The specific mutations are Q29M, N37D, A42S, N45Q, Q47E, E56Q, G65P, and L69I. Variants of human CLDN18.1-mRFP with single amino acid alterations at the indicated positions were amplified by overlapping PCR using primers. Specific mutations were M29Q, D37N, S42A, Q45N, E47Q, Q56E, P65G, and I69L. The PCR products were then cloned into the pcDNA3.1(+) vector by homologous recombination. Individual positive clones were sequenced to identify and confirm the human CLDN18.2-mRFP variants.

[0379] These plasmids of mutant and wild-type human CLDN18.2-mRFP or human CLDN18.1-mRFP were then transfected into HEK293 cells. 6HEK293 cells were seeded in a 60 mm dish at a 60%-80% transfection ratio. 10 μg of DNA in 400 μl of 1x HBS and 10 μl of 25 kDa linear PEI transfection reagent (1 mg / ml stock solution dissolved in 1x HBS) were mixed to achieve a DNA / PEI ratio of 1:2.5. The mixture was then added to the HEK293 cell culture droplets. After 6-8 hours, the transfected cells were replaced with complete DMRM overnight. 24 hours after transfection, cells were collected for FACS analysis using chimeric antibodies.

[0380] Amino acid sequence of human CLDN18.1 (SEQ ID NO: 31) MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0381] Amino acid sequence of human CLDN18.1-mRFP1 (SEQ ID NO: 52): MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTS GIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDG GARTEDEVQSYPSKHDYVMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

[0382] Amino acid sequence of human CLDN18.2 (SEQ ID NO: 30) MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0383] Amino acid sequence of human CLDN18.2-mRFP1 (SEQ ID NO: 53): MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTS GIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDG GARTEDEVQSYPSKHDYVMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

[0384] 2. Binding of CLDN18.2 chimeric antibodies to site-mutated HEK293-CLDN18.2 or HEK293-CLDN18.1 cells Transfected HEK293-CLDN18.2 or HEK293-CLDN18.1 cells were cultured in PBS with 2% BSA for 10 min. 5 The cells were resuspended in 100 μl per well at a density of 100 μl per well. The cells were washed three times with FACS wash buffer (PBS + 2% FBS) and incubated with 100 μl per well of 10 μg / ml chimeric antibody and IMAB362 at 4°C for 30 minutes. The cells were then washed three times with FACS wash buffer and incubated with 100 μl per well of goat anti-hIgG(H+L)-FITC (1:200 dilution) for an additional 30 minutes at 4°C. Finally, the cells were washed three times with FACS wash buffer and analyzed by flow cytometry. To analyze CLDN18.2 binding to transfected cells, RFP-positive cells were used for control gating.

[0385] The percentage of binding signals of these chimeric antibodies to the mutated CLDN18.2 variants compared to that of the wild-type was calculated and summarized in Table 7. As shown in Figures 7A-7B, the binding of 18B10-C was completely lost when E56 was mutated to Q. This change also applied to IMAB362 and other chimeric antibodies except for 59A9-C. Furthermore, we found that other amino acids, such as A42 and N45, also contributed to the binding of IMAB362 and other antibodies to some extent, but not 18B10-C.

[0386] [Table 7]

[0387] [Example 10] Generation and characterization of humanized antibodies 1. Generation, Expression, and Purification of Humanized Antibodies 18B10 The human germline framework sequences VK / 4-1 for the light chain and VH / 1-46 for the heavy chain, respectively, were used for CDR grafting.

[0388] Heavy chain (HC) variants 1, 2, and 3 were obtained by directly grafting the three CDRs onto the germline sequence (18B10 HC germline, SEQ ID NO: 23) and backmutating them to R71I, T73K for HC variant 1 (Hu18B10_Ha, SEQ ID NO: 25), R71I, T73K, T28S, M69L for HC variant 2 (Hu18B10_Hb, SEQ ID NO: 27), and R71I, T73K, T28S, M69L, R38K, M48I for HC variant 3 (Hu18B10_Hc, SEQ ID NO: 29), respectively.

[0389] (1) Germline sequence of 18B10 HC:

[0390] VH / 1-46 (18B10-germline, SEQ ID NO: 23): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR

[0391] VH / 1-46 variant 1 (Hu18B10_Ha, SEQ ID NO: 25): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTSYNQKFKGRVTMTIDKSTSTVYMELSSLRSEDTAVYYCARMYHGNAFDYWGQGTTVTVSS

[0392] VH / 1-46 variant 2 (Hu18B10_Hb, SEQ ID NO: 27): QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTSYNQKFKGRVTLTIDKSTSTVYMELSSLRSEDTAVYYCARMYHGNAFDYWGQGTTVTVSS

[0393] VH / 1-46 variant 3 (Hu18B10_Hc, SEQ ID NO: 29): QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVKQAPGQGLEWIGNIDPYYGGTSYNQKFKGRVTLTIDKSTSTVYMELSSLRSEDTAVYYCARMYHGNAFDYWGQGTTVTVSS

[0394] Light chain (LC) variants 1 and 2 were obtained by directly grafting the three CDRs onto the germline sequence (18B10 LC germline, SEQ ID NO: 24) and, respectively, no backmutations for variant 1 (Hu18B10_La, SEQ ID NO: 26), S63T, I21M for LC variant 2 (Hu18B10_Lb, SEQ ID NO: 28).

[0395] (2) Germline sequence of 18B10 LC:

[0396] VK / 4-1 (18B10 LC germline, SEQ ID NO: 24) DIVMTQSPDSLAVSLGERATINCKSSQNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

[0397] VK / 4-1 variant 1 (Hu18B10_La, SEQ ID NO: 26) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNLKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGGGTKVEIK

[0398] VK / 4-1 variant 2 (Hu18B10_Lb, SEQ ID NO: 28) DIVMTQSPDSLAVSLGERATMNCKSSQSLLNSGNLKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGGGTKVEIK

[0399] Combinations of the above heavy and light chain variable regions result in the following humanized 18B10 antibodies: 18B10-HaLa (having a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 26), 18B10-HbLa (having a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 26), 18B10-HcLa (having a VH of SEQ ID NO: 29 and a VL of SEQ ID NO: 26), 18B10-HaLb (having a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28), 18B10-HbLb (having a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 28), and 18B10-HcLb (having a VH of SEQ ID NO: 29 and a VL of SEQ ID NO: 28).

[0400] The humanized variants of the 18B10 heavy and light chains are linked to the human IgG1 heavy chain constant region and kappa light chain constant region as shown below:

[0401] Human IgG1 heavy chain constant region (SEQ ID NO: 49): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0402] Human kappa light chain constant region (SEQ ID NO: 50): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0403] The variable regions of the heavy and light chain cDNAs described above were synthesized and fused to the constant regions of human IgG1 and human kappa. The heavy and light chains of the selected antibody genes were cloned into expression vectors, and large-scale DNA preparation was performed using Qiagen's Plasmid Maxiprep System. Transfection was performed using Invitrogen's ExpiFectamine™ CHO Reagent according to the manufacturer's protocol. The supernatant was harvested when cell viability was approximately 60%. The cell culture supernatant was filtered through a 0.22 μm filtration capsule to remove cell debris. The supernatant was loaded onto a pre-equilibrated Protein A affinity column. The Protein A resin in the column was then washed with equilibration buffer (PBS), and the antibody was eluted using 25 mM citric acid (pH 3.5). The pH was adjusted to approximately 6.0-7.0 using 1 M Tris-base (pH 9.0). Endotoxin was controlled at less than 1 EU / mg. The purified antibodies were then characterized by SDS-PAGE and SEC-HPLC.

[0404] 2. Binding to human and mouse CLDN18.2 The binding of the humanized antibodies was tested according to the same method as described in Example 5.

[0405] As shown in Figure 8A, all humanized variants were directly tested against the chimeric variants to screen for the best ones. All variants fully retained their binding. 18B10-HaLa, which had only one reverse mutation, was then tested for its binding to HEK293-mouse CLDN18.2 cells (Figure 8B). 18B10-HaLa could bind to mouse CLDN18.2 better than IMAB362, with better potency and higher MFI, indicating that 18B10-HaLa had good cross-reactivity with mouse.

[0406] 3. Affinity analysis of humanized CLDN18.2 antibody by KinExA 18B10-HaLa and IMAB362 were directly evaluated for their affinity binding to CLDN18.2-expressing cells by KinExA. According to the KinExA 4000 (Sapidyne Instruments Inc.) instructions, 200 mg of PMMA hard beads (Sapidyne, No. 440176) were coated with 30 μg of goat anti-human IgG Fc antibody for 2 hours and then blocked with 10 mg / ml BSA for 1 hour. Two gastric cell lines, NUGC4 and KATOIII (ATCC, Cat. No. HTB-103), were harvested in log phase and mixed with 0.2 nM 18B10-HaLa or IMAB362. The cell-antibody mixture was diluted 2-fold using 0.2 nM 18B10-HaLa or IMAB362 and incubated at room temperature for 3 hours. The amount of free antibody increased with dilution. These free antibodies were captured by goat anti-human IgG Fc coated beads and then labeled with 1 μg / ml Alexa Fluor 647-anti-human IgG for readout.

[0407] The binding affinity of each antibody is summarized in Table 8. The Kd for 18B10-HaLa binding to NUGC4 cells and KATOIII was approximately 0.3 nM, which was more than 8-fold higher than that of IMAB362, consistent with the FACS binding results above.

[0408] [Table 8]

[0409] 4. CDC Assay on HEK293-CLDN18.2 Cells Similar to the method described above (see Example 7, section 1), 18B10-HaLa was directly tested with IMAB362 in a CDC activity assay. As shown in Figure 9, 18B10-HaLa had more than 20-fold higher CDC activity than IMAB362. The percentage of 18B10-HaLa-dependent specific cell killing reached 86% at a concentration of 0.3 μg / ml, while IMAB362 had no cell killing at the same concentration.

[0410] 5. Binding and cytotoxic effects in MKN45-CLDN18.2 cells The cell binding assay was the same as described above. As shown in Figure 10A, all humanized variants of 18B10 bound to cells with affinities comparable to those of chimeric 18B10. 18B10-HaLa, which has only one reverse mutation, was selected for further ADCC activity studies.

[0411] ADCC activity was tested using Jurkat-NFAT-luc-FcγRIIIA-V176 cells as effector cells and MKN45-CLDN18.2 cells as target cells. The assay protocol was the same as described above (see section 2 of Example 7). As shown in Figure 10B, 18B10-HaLa had a lower EC50 (0.05 μg / ml) than IMAB362, consistent with that of chimeric 18B10.

[0412] 6. Binding and Cytotoxic Effects in NUGC4 Cells Cell binding and ADCC assays were the same as those described above. Figure 11A shows the binding affinity of 18B10-HaLa to NUGC4 cells. Figure 11B shows the better ADCC potency (EC50 approximately 0.59 μg / ml) of 18B10-HaLa compared with IMAB362.

[0413] 7. ADCC Assay Using NUGC4 as Target Cells and PBMCs as Effector Cells Log-phase NUGC4 cells were resuspended in RPMI 1640 with 10% FBS. 1 × 10 cells were plated per well. 4The cells were pre-seeded in a 96-well U-bottom plate at 40 × 10 cells per well. Anti-CLDN18.2 antibody and IMAB362 were gradient-diluted in RPMI 1640 containing 10% FBS and added to the plate at final concentrations of 200 to 0.2 μg / ml and incubated at 37°C for 30 minutes. Frozen PBMCs from Miao Shun (Shanghai) Biological & Technology Co., Ltd. were removed from liquid nitrogen and immediately placed in a 37°C water bath. After centrifugation, the cells were resuspended in RPMI 1640 and 10% FBS and plated at 40 × 10 cells per well in the 96-well U-bottom plate mentioned above. 4 The plates were then placed in a 37°C incubator for 5 hours.

[0414] After incubation, plates were equilibrated to 22°C. LDH was detected by using the Promega CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (G7892) or the CytoTox 96® Non-Radioactive Cytotoxicity Assay (G1780). After adding lysis reagent and stop solution according to the manufacturer's instructions, fluorescence was measured under an excitation wavelength of 560 nm and an emission wavelength of 590 nm (G7892), or at absorbance of 490 nm or 492 nm (G1780).

[0415] Figure 12 shows representative data using PBMCs as effector cells. 18B10-HaLa showed better ADCC potency than IMAB362. EC50 values ​​may not be calculated accurately due to poor fit to the regression curve.

[0416] 8. Epitope Mapping of Selected Antibodies Using Site-Directed Mutagenesis in Human CLDN18.2 Using the same method and human CLDN18.2-mRFP plasmid as in Example 9, 42 amino acids between 28 and 80 of human CLDN18.2 were substituted with alanine one at a time, as listed below. These variants were amplified by overlapping PCR using primers: Specific mutations are Q28A, Q29A, W30A, S31A, T32A, Q33A, D34A, L35A, Y36A, N37A, N38A, V40A, T41A, V43A, F44A, N45A, Y46A, Q47A, L49A, W50A, R51A, S52A, V54A, R55A, E56A, E56A, S57A, S58A, F60A, T61A, E62A, R64A, Y66A, F67A, T68A, L69A, L70A, L72A, M75A, L76A, Q77A, V79A, and R80A. The PCR product was then cloned into the pcDNA3.1(+) vector by the method of homologous recombination using Syno assembly mix reagent (Synbio) according to the manufacturer's instructions. The plasmid was purified using the QIAGEN Plasmide Mega kit (QIAGEN).

[0417] These mutant and wild-type CLDN18.2-mRFP plasmids were then transfected into HEK293 cells. As in Example 9, 24 hours after transfection, the cells were analyzed by flow cytometry.

[0418] As shown in Figure 13A, the binding of 18B10-HaLa was completely lost (binding percentage <10%) when W30, L49, W50, and E56 were mutated to A, indicating that these four amino acids are crucial for its binding to human CLDN18.2. In particular, E56 is the most important one constituting the binding epitope. In addition to these four critical ones, several other amino acids, such as R51, F60, E62, and R80, also conferred binding when substituted with alanine (binding percentage between 10% and 25%). Figure 13B shows the binding of 59A9-C to the site-mutated CLDN18.2, which was only partially dependent on E56 (binding percentage approximately 22%). The binding percentages of the mutated CLDN18.2 to the antibody compared to the wild-type antibody are summarized in Table 9.

[0419] [Table 9-1] [Table 9-2]

[0420] [Example 11] Antibody Drug Conjugate (ADC) Internalization and Cytotoxicity 18B10-HaLa and control hIgG1 were conjugated with vcMMAE using the MC-vc-PAB-MMAE KIT (Levena Biopharma, catalog number SET0201). The drug-to-antibody ratio (DAR) of chimeric 18B10-HaLa was 4.05, while those of IMAB362 and control hIgG1 were 2.9 and 4.96, respectively. The effect of 18B10-HaLa-vcMMAE on cell viability was assessed using a colorimetric assay that detects cellular metabolic activity.

[0421] Log-phase HEK293-CLDN18.2, NUGC4 or MKN45-CLDN18.2-high cells were resuspended in their corresponding culture medium and then plated at 1 x 10 cells per well in cell culture plates.4 50 μl of 100 cells / well were added and incubated overnight at 37°C. Ab-vcMMAE, control hIgG1-vcMMAE, and Ab were then gradient diluted and added to each well at 50 μl / well. vcMMAE at a final concentration of 4.75 nM was used as a positive control for cytotoxicity. After 72 h, 100 μl / well of detection reagent from the CellTiter-Glo Luminescent Cell Viability Assay Kit was added to each well for 10 minutes at room temperature, followed by readings using a microplate reader.

[0422] As shown in Figure 14A, both 18B10-HaLa-vcMMAE and IMAB362-vcMMAE induced cytotoxicity in HEK293-CLDN18.2 cells, but not the control hIgG1-vcMMAE, indicating that the cytotoxicity was specific to hCLDN18.2. 18B10-HaLa and IMAB362 alone had no cytotoxicity against target cells (data not shown), indicating that the observed cytotoxicity was mediated by vcMMAE. Figure 14B showed the cytotoxic effect in NUGC4 gastric cancer cells. 18B10-HaLa-vcMMAE demonstrated dose-dependent cell growth inhibition starting at a concentration of 0.03 μg / ml. In contrast, IMAB362-vcMMAE inhibited cell growth only at a higher concentration of 10 μg / ml. In another gastric cancer cell line, MKN-45, transfected with CLDN18.2 (high expression), 18B10-HaLa-vcMMAE reached a maximum cell killing of 86%, which was higher than that of IMAB362 (60%) (as shown in Figure 14C).

[0423] It has been well-researched that ADCs function through antigen binding and internalization into target cells. Drugs conjugated to antibodies will not be released and will be able to kill cells until they are internalized and transported to the lysosome for degradation. We used this assay as a preliminary estimate of the internalization properties of 18B10-HaLa. The results suggest that it has potential internalization activity and could be developed as an ADC therapeutic.

[0424] [Example 12] In vivo efficacy evaluation of humanized CLDN18.2 antibody in the MKN45-CLDN18.2-high xenograft model 1. Antitumor efficacy in a MKN45-CLDN18.2-high xenograft model using nude mice In vitro studies (Example 10) showed that the humanized CLDN18.2 antibody was able to induce ADCC effects in MKN45-CLDN18.2-high cells (Example 1). Therefore, an in vivo model was established and used to evaluate antitumor activity. Briefly, female Balb / c nude mice were each infected with 5 × 10 6 MKN45-CLDN18.2-high cells were inoculated into the right flank by sc injection with 50% Matrigel (BD). Twelve days after inoculation, approximately 70 mm 3 Twenty-four mice with tumor sizes of 0.01 mg / kg were selected and randomized into three groups (n=8). Mice were then treated with isotype control or humanized CLDN18.2 antibody at a dose of 0.3 mg / kg by ip injection twice weekly for three weeks. Animals were sacrificed at the end of the study using CO2 inhalation. Tumor size and volume were measured two to three times weekly. Results were analyzed using Prism GraphPad and presented as mean ± SEM.

[0425] As shown in Figure 15, 18B10-HaLa demonstrated slightly better antitumor activity than IMAB362, as measured by tumor size and TGI, but both were significantly better than the isotype control (Table 10).

[0426] [Table 10]

[0427] 2. Antitumor efficacy in a MKN45-CLDN18.2-high and hPBMC co-inoculated xenograft model using NOD-SCID mice Human PBMC cells were obtained from Allcells. Twenty-four female SPF grade NOD-SCID mice were randomized into three groups (n=8), with six mice receiving 5×10 PBMCs as the model group (no PBMCs). 6 MKN45-CLDN18.2-high cells and 50% Matrigel (BD) were inoculated into the right flank by sc injection, and 18 mice were treated with 5 × 10 6 MKN45-CLDN18.2-high cells and 5 × 10 6 Human PBMC cells were inoculated with 50% Matrigel (BD). Four hours after inoculation, mice were treated with 10 mg / kg isotype control, 3 mg / kg, and 10 mg / kg 18B10-HaLa by i.p. injection twice weekly for 4 weeks. Animals were sacrificed at the end of the study using CO2 inhalation. Tumor size and volume were measured two to three times weekly. Results were analyzed using Prism GraphPad and presented as mean ± SEM.

[0428] As shown in Figure 16, tumor growth in the 18B10-HaLa group was completely inhibited during the treatment period. After treatment, tumors from the 3 mg / kg group began to grow after 20 days, while those in the 10 mg / kg group did not. The lack of significant difference between the group without PBMC and the PBS group with PBMC suggested that PBMC alone as effector cells without antibody could not inhibit tumor growth. Tumor growth inhibition (TGI) is summarized in Table 11. 18B10-HaLa had no effect on animal body weight (data not shown).

[0429] [Table 11]

[0430] 3.18B10-HaLa dose-dependently inhibited tumor growth of MKN45-CLDN18.2-high xenografts in nude mice 5 × 10 female Balb / c nude mice each 6Cells were inoculated into the right flank by sc injection with 50% Matrigel (BD). Nine days after inoculation, approximately 100 mm 3 Thirty-two mice with tumor sizes of 0.1 mg / kg were selected and randomized into four groups (n=8). Mice were then treated with isotype control, 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg 18B10-HaLa by ip injection twice weekly for three weeks. Animals were sacrificed at the end of the study using CO2 inhalation. Tumor size and volume were measured two to three times weekly. Results were analyzed using Prism GraphPad and presented as mean ± SEM.

[0431] As shown in Figure 17, the antitumor activity of 18B10-HaLa was dose-dependent, with the 1 mg / kg group showing the best tumor growth inhibitory activity (Table 12).

[0432] [Table 12]

[0433] [Example 13] Generation, expression, purification, and characterization of 18B10-HaLa-VLPYLL mutants with enhanced ADCC efficacy 1. Generation of 18B10-HaLa-VLPYLL mutants According to a study by Futa Mimoto et al., the L235V / F243L / R292P / Y300L / P396L mutation can increase binding affinity to FcγRIIIA by 10-fold, without any change to the inhibitory FcγR isoform, FcγRIIB. To test this hypothesis, we constructed and generated the 18B10-HaLa-L235V / F243L / R292P / Y300L / P396L (18B10-HaLa-VLPYLL) mutant to enhance its ADCC effect. This Fc variant was transiently transfected, expressed, and purified according to the same method as in Section 1 of Example 12.

[0434] It has been reported that five mutations in the Fc domain (L235V / F243L / R292P / Y300L / P396L) can increase binding affinity to both alleles of human CD16A (FcγRIIIA) without any change in binding affinity to the inhibitory FcγR isoform, FcγRIIB (Futa Mimoto et al., Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant [C] / / MAbs. Taylor & Francis, 2013, 5(2):229-236). To test this hypothesis, these mutations were introduced into Hu18B10_Ha_hIgG1 by overlap extension PCR, and the new construct was named Hu18B10_Ha_hIgG1_L235V / F243L / R292P / Y300L / P396L. The final PCR product was characterized by agarose gel electrophoresis. The fragment of the correct size was extracted from the gel and cloned into an expression vector. The correct construction of Hu18B10_Ha_hIgG1_P330S was then confirmed by sequencing analysis. The Hu18B10_Ha_hIgG1_L235V / F243L / R292P / Y300L / P396L and Hu18B10_La_hKappa plasmids were prepared by using the Plasmid Maxiprep System from Qiagen. The heavy and light chain plasmids were then co-transfected into Expi-CHO cells for expression and purification, as previously described above, according to the same method as in Section 1 of Example 10.

[0435] Engineered Fc L235V / F243L / R292P / Y300L / P396L (SEQ ID NO: 51) sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0436] 2. Binding to Fcγ receptors Futa Mimoto et al. compared a VLPYLL Fc mutant with the wild-type and found that the mutation increased its binding affinity to both FcγRIIIA F176 (63-fold) and FcγRIIIA V176 (33-fold) without affecting other FcγRs. To confirm this finding, ELISA binding between the antibody and these FcγRs was tested. Briefly, 18B10-HaLa-VLPYLL or 18B10-HaLa-wt antibody was coated onto a plate at a concentration of 1 μg / ml. After blocking and washing, serially diluted (5 μg / ml to 0.02 μg / ml) His-tagged FcγRs were added and incubated for 1 hour. Anti-His-HRP and TMB were then added for detection of FcγR binding at OD 450 nm.

[0437] As shown in Figures 18A and 18B, there was no significant difference between 18B10-HaLa_VLPYLL and 18B10-HaLa-wt in binding to human FcγRI or FcγRIIB. However, 18B10-HaLa_VLPYLL showed a 10-fold increase in binding to human FcγRIIIA(F176) and FcγRIIIA(V176) compared to its wild-type (wt) counterpart (Figures 18C and 18D). Similar results were observed when using mouse FcγR and cyno FcγR (Figures 18E-18I).

[0438] 3. Binding to FcRn and C1q FcRn binding was assessed by ELISA. Briefly, 18B10-HaLa_VLPYLL or wt was immobilized on a plate. Biotinylated FcRn was serially diluted with pH 6.0 dilution buffer (1 μg / ml to 0.0002 μg / ml) and then added for 1 hour of incubation. Streptavidin-HRP and TMB were then added for binding detection at OD450 nm.

[0439] The C1q binding assay was performed as follows: two antibodies were immobilized on a plate. Serially diluted C1q (20 μg / ml to 0.31 μg / ml) was added for 1 hour of incubation. Anti-C1q-HRP and TMB were then added for detection at OD450 nm.

[0440] As shown in Figure 19A, there was no significant difference in FcRn binding between 18B10-HaLa_VLPYLL and wt, indicating that the VLPYLL mutation had no effect on FcRn binding. Figure 19B shows that 18B10-HaLa_VLPYLL reached the same binding signal at a lower C1q concentration than wt, which may lead to increased CDC efficacy.

[0441] 4. ADCC Assay on NUGC4 Cells Using Jurkat-NFAT-luc-FcγRIIIA-V176 as Effector Cells ADCC reporter assay was performed according to the same method described above (see Example 7, section 2). As shown in Figure 20A, 18B10-HaLa_VLPYLL had a 3-fold increase in ADCC potency (EC50 approx. 0.0097 μg / ml) compared to that of the wt (EC50 approx. 0.032 μg / ml).

[0442] 5. ADCC Assay on NUGC4 Cells Using Human PBMCs as Effector Cells ADCC assays using human PBMCs were performed according to the method described above (see Section 7 of Example 10). As shown in Figure 20B, 18B10-HaLa_VLPYLL also had a 3-fold increase in ADCC potency compared to that of the wt antibody, although the maximum cytotoxicity of both was similar (approximately 45%). Compared to IMAB362, 18B10-HaLa_VLPYLL had a 100-fold increase in potency.

[0443] 6. MESFs of CLDN18.2 Expression in a Panel of Gastric Cancer Cell Lines The Quantum™ MESF (Molecules of Equivalent Soluble Fluorochrome) microsphere kit allows for standardization of fluorescence intensity units for application in quantitative fluorescent cell counting. A panel of gastric cancer (GC) cells was stained using 30 μg / ml 18B10-HaLa and goat anti-human IgG-FITC. Cells were detected using Quantum™ MESF beads on a flow cytometer with fixed fluorescence settings. Briefly, one drop of reference blank "B" was added to 400 μL of suspension solution, and then one drop of each fluorescence intensity population was combined in 400 μL of the same buffer for analysis. The microspheres were analyzed on the flow cytometer. A downloaded Bangs Laboratories quantitative analysis template, QuickCal® v.2.3, was utilized for data analysis using calibration curves and regression coefficient (r2) values. For accurate MESF assignment, instrument linearity was ensured, reaching a regression coefficient of ≥ 0.9995, and appropriate controls (e.g., unstained cells, isotype controls) were run in parallel.

[0444] As shown in Figure 21, the two transfected cell lines, HEK293-CLDN18.2 and MKN45-CLDN18.2-high, had higher levels of CLDN18.2 expression than other cell lines that may not represent tumor cells derived from GC patients. Among GC cell lines, NUGC4 had the highest expression of CLDN18.2. SNU-601 (Cobioer, Catalog No. CBP60507) and SNU-620 (Cobioer, Catalog No. CBP60508) had intermediate levels, while KATOIII and OCUM-1 (Cobioer, Catalog No. CBP60494) had low expression. Thus, CLDN18.2 has various expression levels among gastric cancer cells.

[0445] 7. IHC detection of CLDN18.2 expression in a panel of gastric cancer cell lines Gastric cancer cell lines were harvested in the logarithmic growth phase, washed with phosphate-buffered saline (PBS), and then fixed in 4% neutral-buffered paraformaldehyde (PFA) at room temperature for 30 minutes. After centrifugation, approximately 2–5 × 10 cells were collected. 7 The cells were resuspended in PBS at a density of 100 μg / ml, then mixed with 200 μl of molten agar, dehydrated in gradient alcohols, cleared in xylene, and embedded in paraffin wax for sectioning. The CLDN18.2 expression levels of these cells were detected by immunohistochemistry (IHC) using 3 μg / ml of GC182-biotin, an available monoclonal antibody for CLDN18.2 IHC detection, produced by Mabspace Bioscience according to the sequence in WO2013167259 and biotinylated in-house. IHC results were evaluated by the relative percentage of positive cells and the intensity of staining on the cell membrane. These cell lines were scored and evaluated according to the scoring guidelines for IMAB362 in clinical trials (Table 13). Only patients with moderate (2+) and strong (3+) staining in at least 40% of tumor cells were eligible for inclusion in the IMAB362 FAST study. Therefore, NUGC4, MKN45-CLDN18.2-high, and HEK293-CLDN18.2 fulfill the criteria. The results were consistent with those in Example 13, section 6 (Quantum™ MESF method).

[0446] Heavy chain variable region of GC182 (SEQ ID NO: 74): QIQLVQSGPELKKFGETVKISCKASGYTFTDYSIHWVKQAPGKGLKWMGWINTETGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARRTGFDYWGQGTTLTVSS

[0447] Light chain variable region of GC182 (SEQ ID NO: 75): DIVMTQAAFSIPVTLGTSASISCRSSKNLLHSDGITYLYWYLQRPGQSPQLLIYRVSNLASGVPNRFSGSESGTDFTLRISRVEAEDVGVYYCVQVLELPFTFGGGGTKLEIK

[0448] [Table 13]

[0449] 8. ADCC assay in gastric cancer (GC) cell lines with various CLDN18.2 expression levels using human PBMCs as effector cells To further test the hypothesis that the ADCC activity of CLDN18.2 antibodies is regulated by the expression level of CLDN18.2 in GC cells, an ADCC assay using human PBMCs as effector cells was performed according to the same method described above (see Section 7 of Example 10). Four gastric cell lines with various expression levels of CLDN18.2 were used as target cells. As shown in Figures 22A-22D, NUGC4 cells contained the highest ADCC activity (maximum cytotoxicity approximately 40%) among all CLDN18.2 antibodies. Among the three tested antibodies, 18B10-HaLa-VLPYLL showed better efficacy than 18B10-HaLa-wt and IMAB362. SNU-601 and SNU-620 cells induced moderate ADCC activity (maximum cytotoxicity 30% and 15%, respectively), while OCUM-1 cells had the lowest cytotoxicity (less than 10%). These results suggested that ADCC activity correlated with the CLDN18.2 expression level in these cell lines.

[0450] [Example 14] Process optimization and characterization of ADCC efficacy of 18B10-HaLa 1.18B10-HaLa process optimization It is well known that non-fucosylation or defucosylation selectively and significantly increases binding affinity to FcγRIII, leading to enhanced ADCC function. The following describes process optimization to reduce fucose and enhance ADCC.

[0451] Briefly, the cell bank seed was recovered and cultured in CD-CHO medium (Gibco) for 3 days, after which the cells were expanded in basal medium (Hyclone, ActiPro + 4 mM Gln + 1xHT) for 6 days. Then, 0 (as a reference sample) or 50 μM 2F-OF (2-deoxy-2-fluoro-L-fucose) was added to the reactor to control dissolved oxygen (DO) at approximately 40%. Half of the feed medium (Hyclone, Cell Boost 7a, Cell Boost 7b) was added, and the cell suspension was harvested when the variable cell density (VCD) was less than 80% or on day 13.

[0452] After collecting the cell suspension, the antibody titers of both the reference sample and the 50 μM 2F-OF sample were measured by HPLC. On day 13, the titer of the 50 μM 2F-OF sample was 4.73 g / L, which was even higher than that of the reference sample, indicating that it was not affected by 2F-OF.

[0453] Antibody quality was measured by HPLC after purification, and the 50 μM 2F-OF sample had similar purity (98.3%) to the reference sample (98.2%). There was no significant effect of 2F-OF on antibody quality.

[0454] N-glycans were simultaneously analyzed by HPLC, and the results are shown in Table 14. Compared with the reference sample, the addition of 2F-OF reduced the percentage of G0F(FA2) (from 61.6% to 1.9%) and fucose (from 87.7% to 13.7%), while the percentage of G0(A2) increased (from 8.1% to 69.8%). Therefore, 50 μM of 2F-OF was sufficient to control fucose to less than 15%, which may result in enhanced ADCC effect. The product obtained through this process (using 50 μM of 2F-OF) was named 18B10-HaLa low fucose.

[0455] [Table 14]

[0456] To demonstrate that 18B10-HaLa low fucose enhances effective FcγIIIa receptor affinity while maintaining affinity for FcRn, we compared the affinities of 18B10-HaLa low fucose and IMAB362-analogue using bilayer interference (BLI) technology from the Fortebio system. IMAB362-analogue with human IgG1 isotype and normal glycosylation served as a control.

[0457] In this study, FcγRI, FcγRIIa-H167, FcγRIIa-R167, FcγRIIb, FcγRIIIa-V176, FcγRIIIa-F176, FcγRIIIb-NA1, FcγRIIIb-NA2, and FcRn were loaded onto biosensors and immersed in varying concentrations of IMAB362-analog and 18B10-HaLa-low-fucose in solution. All binding data were collected at 30°C. When measuring the affinity of 18B10-HaLa-low-fucose or IMAB362-analog for C1q, biotinylated antibodies were loaded onto the biosensors and then incubated with C1q in solution. When antibody affinity for FcRn was measured by BLI, the pH was 6.0, whereas for other Fc receptor binding assays, the pH was 7.4. The experiment included five steps: 1. baseline acquisition, 2. loading the biosensor with human Fc gamma receptors, 3. a second baseline acquisition, 4. association of 18B10-HaLa low fucose and IMAB362 analogs for measuring k and 5. dissociation of antibodies for measuring k. 18B10-HaLa low fucose and IMAB362 analogs have similar affinities for FcγRI, FcRn, and C1q, but for other receptors, 18B10-HaLa low fucose exhibits slightly higher affinity than IMAB362 analogs. These results demonstrate that 18B10-HaLa low fucose exhibits enhanced ADCC activity and a half-life similar to that of normal glycosylated antibodies in clinical trials.

[0458] [Table 15]

[0459] As shown in Table 15, the affinity of 18B10-HaLa-low-fucose to human FcγRIIIa-V176 and human FcγRIIIa-F176 proteins was slightly higher than that of IMAB362, which may be caused by lower fucosylation. As shown in Table 15, the affinity of 18B10-HaLa-low-fucose to human FcRn protein was not achieved by lower fucosylation and was slightly higher than that of IMAB362. As shown in Table 15, the affinity of 18B10-HaLa-low-fucose to human C1q protein was not completely similar to that of IMAB362.

[0460] 2. ADCC Reporter Assay on NUGC4 Using Jurkat-NFAT-luc-FcγRIIIA-V176 as Effector Cells ADCC testing was performed according to the same protocol described above (see Example 7, section 2). As shown in Figure 23, the antibody (18B10-HaLa low fucose) produced using a process with the addition of 50 μM 2F-OF had ADCC activity that was increased by more than 30-fold over that of a reference sample produced using a process without the addition of 2F-OF. Transiently expressed 18B10-HaLa was also included in this comparison, and due to process optimization, the maximum signal corresponding to ADCC activity was also increased.

[0461] 3. FACS binding to various gastric cancer cell lines using 18B10-HaLa low fucose FACS binding was performed according to the same protocol as in Section 2 of Example 7. As shown in Figures 24A-24C, 18B10-HaLaLowFucose was able to bind to these cell lines with higher potency than that of IMAB362. The EC50 of 18B10-HaLaLowFucose was 0.5-1.6 μg / ml, while IMAB362 had almost no binding signal at a concentration of approximately 1 μg / ml.

[0462] 4. ADCC reporter assay in various gastric cancer cell lines using Jurkat-NFAT-luc-FcγRIIIA-V176 as effector cells ADCC testing was performed according to the same protocol described above (see Section 2 of Example 7). As shown in Figures 25A-E, the EC50 of ADCC activity of 18B10-HaLaLowFucose was approximately 0.008 μg / ml using gastric cancer cell lines with various levels of CLDN18.2 expression. Compared with IMAB362, 18B10-HaLaLowFucose had at least 100-fold higher ADCC potency.

[0463] 5. ADCC reporter assay in various gastric cancer cell lines using PBMCs as effector cells ADCC tests were performed according to the same protocol described above (see Section 7 of Example 10). As shown in Figures 26A-26D, 18B10-HaLa low fucose induced significantly higher ADCC effects than IMAB362 in various gastric cancer cell lines. IMAB362 induced little cytotoxicity at low concentrations (0.01-0.1 μg / ml). However, at a concentration of 0.1 μg / ml, the cytotoxicity of 18B10-HaLa low fucose was almost saturated.

[0464] 6. Optimized ADCC Assay with NUGC4 Using PBMCs as Effector Cells For further studies, an optimized ADCC assay was developed using human PBMCs as effector cells. Briefly, prior to use, frozen PBMCs were recovered from liquid nitrogen and diluted to 5 × 10 6The cells were resuspended in RPMI1640 + 10% FBS at a density of 1000 cells / ml and incubated for 5 hours in a 37°C, 5% CO2 incubator before use. Target cells, NUGC4 cells, were labeled with CellTrace™ Far Red (Invitrogen, Catalog No. C34564) according to the manufacturer's instructions. The labeled NUGC4 cells and diluted antibody were added to a 96-well plate and incubated for 30 minutes in a 37°C, 5% CO2 incubator. PBMC cells were then added to the corresponding wells and incubated for 15 hours in the incubator. At the end of the culture, propidium iodide (PI) staining solution was added to mark dead NUGC4 cells. The percentage of PI-positive cells among CellTrace™ Far Red-positive cells was analyzed by flow cytometry. Specific cytotoxicity was calculated by subtracting the nonspecific killing percentage.

[0465] As shown in Figure 27, representative data for 18B10-HaLaLowFucose, the maximum specific cytotoxicity reached over 60% at a concentration of 1.2 μg / ml, with an EC50 of 0.014 μg / ml, whereas the maximum for the IMAB362-analogue was only 40% at the highest concentration (30 μg / ml), with an EC50 of 0.54 μg / ml, more than 30-fold that of 18B10-HaLaLowFucose.

[0466] [Example 15] Antitumor activity of 18B10-HaLa low in fucose in vivo 1. Antitumor efficacy in a MKN45-CLDN18.2-high and hPBMC co-inoculated xenograft model using NOD-SCID mice Human PBMC cells were obtained from Allcells. Sixty female SPF grade NOD-SCID mice were randomized into six groups (n=10), with 10 mice receiving 5×10 PBMCs as the model group (no PBMCs). 6 MKN45-CLDN18.2-high cells and 50% Matrigel (BD) were inoculated into the right flank by sc injection, and 50 mice were treated with 5 × 10 6MKN45-CLDN18.2-high cells and 5 × 10 6 Human PBMC cells were inoculated with 50% Matrigel (BD). Four hours after inoculation, mice were treated with 10 mg / kg isotype control, 1 mg / kg, 3 mg / kg, and 10 mg / kg 18B10-HaLa low fucose by i.p. injection twice weekly for 5 weeks. Animals were sacrificed at the end of the study using CO2 inhalation. Tumor size and volume were measured two to three times weekly. Results were analyzed using Prism GraphPad and presented as mean ± SEM.

[0467] As shown in Figure 28A, tumor growth was significantly inhibited by 18B10-HaLalow fucose in a dose-dependent manner. Notably, with 10 mg / kg of 18B10-HaLalow fucose, most tumors (7 / 10) disappeared by the end of the study (Figure 28B). The tumor growth inhibition rate of 18B10-HaLalow fucose was dose-dependent, with the 10 mg / kg TGI group reaching 95.86% (Table 16). Compared with the IMAB362-analogue, 18B10-HaLalow fucose had even more potent antitumor activity. Meanwhile, 18B10-HaLalow fucose had no effect on animal body weight (data not shown).

[0468] [Table 16]

[0469] 2. Efficacy of 18B10-HaLa-Low-Fucose in Combination with Oxaliplatin and 5-Fu in the MKN45-CLDN18.2-Large Tumor Model in Nude Mice Female SPF-grade nude mice were treated with 5 × 10 6 MKN45-CLDN18.2-high cells were inoculated. Approximately 90 mm 3At tumor size of 100 mg / kg, tumor-bearing mice were selected and randomized into four groups (n=8). Animals were treated with 10 mg / kg isotype control and vehicle, 10 mg / kg 18B10-HaLaLowFucose, 2.5 mg / kg oxaliplatin and 30 mg / kg 5-FU, and 10 mg / kg 18B10-HaLaLowFucose in combination with 2.5 mg / kg oxaliplatin and 30 mg / kg 5-FU, where 18B10-HaLaLowFucose was administered by IP injection twice weekly for 4 weeks, and oxaliplatin and 5FU were administered by IV injection once weekly for 4 weeks. Tumor size was measured two or three times weekly using calipers (INSIZE), and volume was calculated using the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively. 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test, and p < 0.05. * <0.05 and ** Differences were considered significant if <0.01.

[0470] As shown in Figure 29 and Table 17, the single-agent groups of 18B10-HaLa low fucose and oxaliplatin + 5-FU alone without PBMCs had mild tumor growth inhibition, with TGIs of 47% and 52%, respectively. However, the combination had a significant difference compared to the single-agent groups, with an enhanced tumor inhibition of 69%.

[0471] [Table 17]

[0472] 3. Efficacy of 18B10-HaLa-Low-Fucose in Combination with Paclitaxel in the MKN45-CLDN18.2-Large Tumor Model in Nude Mice MKN45-CLDN18.2-high cells were maintained in vitro as monolayer cultures in RPMI 1640 medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, and 100 μg / ml streptomycin (Hyclone) at 37°C with 5% CO2. For tumor inoculation, cells in the logarithmic growth phase were harvested and counted. Female Balb / c nude mice were inoculated with 5 × 10 6 Cells were seeded into the right flank by sc injection with 50% Matrigel (BD). 8–11 days after inoculation, approximately 100 mm 3 Twenty-four mice with tumor size were selected and randomized into three groups (n=8). Mice were then treated with isotype control or 18B10-HaLa low fucose at a dose of 10 mg / kg by ip injection twice a week for three weeks. Paclitaxel at 5 mg / kg was injected iv once a week. Animals were sacrificed at the end of the study using CO2 inhalation. Tumor size was measured in two dimensions using a vernier caliper (INSIZE), and volume was calculated in mm using the formula: V=0.5a×b2, where a and b represent the length and width of the tumor, respectively. 3 The tumor growth inhibition rate (TGI%) was calculated using the formula: TGI% = (1 - (TVDt (treated group) / TVDt (control group)) × 100%, where TVDt represents the tumor volume at each subsequent measurement. Histograms were generated using Prism GraphPad (mean ± SEM), and T analysis was used for statistical analysis. p < 0.05 represents a significant difference between groups; p < 0.01 represents a highly significant difference between groups.

[0473] As shown in Figure 30A, compared with the isotype control, 18B10-HaLa low fucose significantly inhibited tumor growth from day 5, with a TGI of approximately 43%. Similarly, paclitaxel, a commonly used second-line chemotherapy agent for gastric cancer, also had a TGI of approximately 45%. However, when they were combined, the tumor inhibition rate reached 61%, which was significantly different from the single-agent group (Figure 30B, Table 18). However, without human PBMCs inoculated with the tumor, the tumor volume was significantly larger than when human PBMCs were used. No significant changes in body weight were observed in any of the groups.

[0474] [Table 18]

[0475] 4. Efficacy of 18B10-HaLa-Low-Fucose in Combination with Paclitaxel in the GC02-0004 PDX Tumor Model in Nude Mice Tumor tissue from a gastric cancer patient-derived xenograft (PDX) model was derived from an adenocarcinoma / gastric cancer patient (number: GC-02-004) at Beijing Cancer Hospital and analyzed after six passages in nude mice. CLDN18.2 expression was detected by immunohistochemistry (IHC) using 3 μg / ml of GC182-biotin, a recognized IHC antibody for CLDN18.2 detection. GC182 was generated by MabSpace Bioscience according to the sequence in WO2013167259. The relative percentage of positive cells in this tumor tissue ranged from 40% to 70% (Figure 31A, 200x magnification). HER2 and PD-L1 expression were also detected by IHC using rabbit mAbs D8F12 (Cell Signaling Technology, catalog number 4290) and SP263 (generated by MabSpace Bioscience according to the sequence in WO2016124558), respectively. As a result, both tumor tissues were HER2-negative (Figure 31B) and PD-L1-negative (Figure 31C).

[0476] Each mouse was subcutaneously inoculated with a small tumor tissue block approximately 3 mm in diameter, sheared from the integrated tumor dissection, to form a tumor-bearing mouse. Two weeks after inoculation, approximately 50 mm 3 Animals with tumor sizes of 1000-10 ... 2 The tumor growth inhibition rate (TGI%) was calculated using the formula: TGI% = (1 - (TV) / (1 / 2)) (where a and b represent the length and width of the tumor, respectively). Dt (Treatment group) / TV Dt Calculated using (control group) × 100%. Dt represents tumor volume at each subsequent measurement. Histograms were generated using Prism GraphPad (mean ± SEM), and T analysis was used for statistical analysis. p<0.05 indicates a significant difference between groups; p<0.01 indicates a highly significant difference between groups.

[0477] As shown in Figure 31D, 18B10-HaLa low fucose led to a 48% tumor inhibition rate. Similarly, paclitaxel, a commonly used second-line chemotherapy agent for gastric cancer, also had only a 45% tumor inhibition rate. However, when they were combined, the tumor inhibition rate reached 68%, which was significantly different from the single-agent group (Table 19). As shown in Figure 31E, 5 mg / kg paclitaxel seemed to have a slight toxic effect on mouse body weight, but other treatments had no effect.

[0478] [Table 19]

[0479] 5. MKN45-CLDN18.2-Combination with DC101 in a High-Level Xenograft Tumor Model DC101 is a monoclonal antibody that reacts with murine VEGFR-2 (vascular endothelial growth factor receptor 2), also known as CD309, KDR, and Flk-1. VEGFR-2 is a member of the tyrosine protein kinase family. Upon binding to its ligand VEGF, VEGFR-2 plays an important role in vascular development and permeability. DC101 was found to competitively block the binding of VEGF and VEGFR-2, leading to a reduction in tumor microvascular density and tumor growth. This antibody was produced by MabSpace Biosciences (Suzhou) Co., Limited according to the sequence in US5840301.

[0480] Female SPF-grade nude mice were treated with 5 × 10 6 MKN45-CLDN18.2-high cells were inoculated. Approximately 90 mm 3 At tumor size of 1000 mg / kg, tumor-bearing mice were selected and randomized into four groups (n=8). Animals were treated with 10 mg / kg isotype control, 10 mg / kg 18B10-HaLaLowFucose, 3 mg / kg DC101, and 10 mg / kg 18B10-HaLaLowFucose combined with 3 mg / kg DC101. All antibodies were administered by ip injection twice weekly for four weeks. Tumor size was measured two or three times weekly in two dimensions using calipers (INSIZE), and volume was calculated using the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively. 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test, and p < 0.05. * <0.05 and ** Differences were considered significant if <0.01.

[0481] As shown in Figure 32 and Table 20, without PBMC, the single-agent groups (18B10-HaLa low fucose or DC101) had some tumor inhibition, with TGI of 47% and 35%, respectively. When they were combined, tumor growth was almost stopped, with an inhibition rate of 75%, which is a significant difference compared to the single-agent groups.

[0482] [Table 20]

[0483] [Example 18] Antitumor activity of 18B10-HaLa low-fucose in pancreatic cancer cells in vitro 1. Generation of MIA PaCa-2-CLDN18.2 and BxPC-3-CLDN18.2 Cell Lines The MIA PaCa-2-CLDN18.2 and BxPC-3-CLDN18.2 cell lines were constructed by MabSpace Biosciences (Suzhou) Co., Limited. Briefly, MIA PaCa-2 cells (Shanghai Institutes for Biological Sciences, Catalog No. SCSP-568) and BxPC-3 cells (Shanghai Institutes for Biological Sciences, Catalog No. TCHu12) were transfected with the pcDNA3.1 / hCLDN18.2 plasmid and selected with G418 to obtain the stable-expressing cell lines MIA PaCa-2-CLDN18.2 and BxPC-3-CLDN18.2. The expression level of CLDN18.2 was detected with the 18B10-HaLa low-fucose antibody. The single-cell clone with the highest signal was selected and amplified for cell banking.

[0484] 2. FACS binding to pancreatic cancer cell line using 18B10-HaLa low fucose FACS binding was performed according to the same protocol as in Section 2 of Example 7. As shown in Figures 33A-33B, 18B10-HaLalow fucose was able to bind to the two cell lines with higher potency than that of IMAB362. The maximum signal of 18B10-HaLalow fucose was significantly higher than that of IMAB362. The EC50 of 18B10-HaLalow fucose was approximately 0.53 μg / ml, which was also significantly lower than that of IMAB362.

[0485] 3. ADCC reporter assay in pancreatic cancer cell lines using Jurkat-NFAT-luc-FcγRIIIA-V176 as effector cells ADCC testing was performed according to the same protocol described above (see Example 7). As shown in Figures 34A-34B, the EC50 of ADCC activity of 18B10-HaLaLowFucose was approximately 0.001 μg / ml. Compared with IMAB362, 18B10-HaLaLowFucose had approximately four-fold higher ADCC potency.

[0486] [Example 19] Antitumor activity of 18B10-HaLa low-fucose in pancreatic cancer cells in vivo 1. Efficacy in a nude mouse MIA PaCa-2-CLDN18.2 xenograft model 5 × 10 female Balb / c nude mice aged 5–6 weeks were each 6 MIA PaCa-2-CLDN18.2 cells were inoculated into the right flank by sc injection with 50% Matrigel (BD). Twelve days after inoculation, approximately 70 mm 3Twenty-four mice with tumor sizes of 1000-10 ...

[0487] As shown in Figure 35, 18B10-HaLaLowFucose demonstrated better antitumor activity than IMAB362 as measured by tumor size and TGI, both of which were significantly better than the isotype control (Table 21). No significant body weight changes were observed in any group.

[0488] [Table 21]

[0489] 2. Efficacy in a BxPC-3-CLDN18.2 xenograft model using nude mice A BxPC-3-CLDN18.2 xenograft model was established and treated with antibodies following the same procedure as the MIA PaCa-2-CLDN18.2 model (Example 19, section 1).

[0490] As shown in Figure 36, IMAB362 failed to inhibit tumor growth at all, while 18B10-HaLa-low-fucose exhibited some antitumor activity (Table 22). No significant changes in body weight were observed in any of the groups.

[0491] [Table 22]

[0492] [Example 20] Antitumor activity of 18B10-HaLa low-fucose in lung cancer cells in vitro FACS binding to lung cancer cell line using 1.18B10-HaLa low fucose NCI-H146 was purchased from ATCC (catalog number: ATCC® HTB-173). NCI-H460-CLDN18.2 was purchased from Kyinno (catalog number: KC-1450) and stably transfected with CLDN18.2. FACS binding was performed according to the same protocol as in section 2 of Example 7. As shown in Figures 37A-37B, 18B10-HaLa-low-fucose could bind to the two cell lines in a dose-dependent manner. Due to the higher expression level of CLDN18.2 in NCI-H460-CLDN18.2 cells than in NCI-H146 cells, the maximum binding signal of the former cells was also significantly higher than that of the latter.

[0493] 2. ADCC reporter assay in NCI-H146 using Jurkat-NFAT-luc-FcγRIIIA-V176 as effector cells ADCC testing was performed according to the same protocol described above (see Section 2 of Example 7). As shown in Figure 38, 18B10-HaLalow fucose was able to induce ADCC in NCI-H146 cells, with an EC50 of approximately 0.003 μg / ml. Compared with IMAB362, 18B10-HaLalow fucose had approximately 150-fold higher ADCC potency.

[0494] 3. ADCC Assay with NCI-H460-CLDN18.2 Using PBMCs as Effector Cells ADCC tests mediated by primary PBMCs were performed according to the same protocol desc...

Claims

1. An anti-CLDN18.2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region comprises light chain LCDR1, LCDR2, and LCDR3 sequences; the HCDR1 sequence comprises GYNMN (SEQ ID NO: 1) or TYFIGVG (SEQ ID NO: 13); The HCDR2 sequence is X １ IDPYYX ２ X ３ TX ４ YNQKFX ５ G (SEQ ID NO: 32) or HIWWNDNKYYNTALKS (SEQ ID NO: 15), The HCDR3 sequence is X ６ X ７ X ８ GNAFDY (SEQ ID NO: 33) or MGSGAWFTY (SEQ ID NO: 17), The LCDR1 sequence is KSSQX ９ LX １０ NX １１ GNX １２ KNYLT (SEQ ID NO: 34), The LCDR2 sequence is WASTRX １３ S (SEQ ID NO: 35), The LCDR3 sequence is QNDYX １４ X １５ PX １６ T (SEQ ID NO: 36), X １ is N or Y or H, and X ２ is G or V, and X ３ is A or G or T, and X ４ is R or T or S, and X ５ is K or R, and X ６ is S or M, and X ７ is Y or F, and X ８ is Y or H, and X ９ is S or N, and X １０ is L or F, and X １１ is S or N, and X １２ is Q or L, and X １３ is E or K, and X １４ is S or Y, and X １５ is F or Y, and X １６ is F or L, a) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 19, and an HCDR3 comprising the sequence of SEQ ID NO: 21, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 14, an LCDR2 comprising the sequence of SEQ ID NO: 16, and an LCDR3 comprising the sequence of SEQ ID NO: 18; or b) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 3, and an HCDR3 comprising the sequence of SEQ ID NO: 5, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; or c) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, and an HCDR3 comprising the sequence of SEQ ID NO: 5, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 8; or d) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 9, and an HCDR3 comprising the sequence of SEQ ID NO: 11, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; or e) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 15, and an HCDR3 comprising the sequence of SEQ ID NO: 17, and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 2, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 12; or f) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 22, and an HCDR3 comprising the sequence of SEQ ID NO: 5; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 4, and an LCDR3 comprising the sequence of SEQ ID NO: 6; The antibody or antigen-binding fragment thereof.

2. further comprising one or more of heavy chains HFR1, HFR2, HFR3, and HFR4 and / or one or more of light chains LFR1, LFR2, LFR3, and LFR4; The HFR1 is QVQLVQSGAEVKKPGASVKVSCKASGYX １７ FT (SEQ ID NO: 54), The HFR2 is WVX １８ QAPGQGLEWX １９ G (SEQ ID NO: 55), The HFR3 sequence is RVTX ２０ TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 56), the HFR4 comprises WGQGTTVTVSS (SEQ ID NO: 57); The LFR1 is DIVMTQSPDSLAVSLGERATX ２１ NC (SEQ ID NO: 58), the LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO: 59); The LFR3 is GVPDRFX ２２ GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 60), the LFR4 comprises FGGGTKVEIK (SEQ ID NO: 61); X １７ is T or S, and X １８ is R or K, and X １９ is M or I, and X ２０ is M or L, and X ２１ is I or M, and X ２２ is S or T; The antibody or antigen-binding fragment thereof according to claim 1.

3. the HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 62 and 63; said HFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 64 and 65; said HFR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 66 and 67; said HFR4 comprising the sequence of SEQ ID NO: 57; said LFR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 68 and 69; said LFR2 comprising the sequence of SEQ ID NO: 59; said LFR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 70 and 71; The LFR4 comprises the sequence of SEQ ID NO:

61. The antibody or antigen-binding fragment thereof according to claim 2.

4. the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47; and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48; The antibody or antigen-binding fragment thereof according to claim 1.

5. the heavy chain variable region comprises the sequence of SEQ ID NO:25 and the light chain variable region comprises the sequence of SEQ ID NO:26; the heavy chain variable region comprises the sequence of SEQ ID NO:27 and the light chain variable region comprises the sequence of SEQ ID NO:28; the heavy chain variable region comprises the sequence of SEQ ID NO: 29 and the light chain variable region comprises the sequence of SEQ ID NO: 26 or 28; the heavy chain variable region comprises the sequence of SEQ ID NO: 37 and the light chain variable region comprises the sequence of SEQ ID NO: 38; the heavy chain variable region comprises the sequence of SEQ ID NO: 39 and the light chain variable region comprises the sequence of SEQ ID NO: 40; the heavy chain variable region comprises the sequence of SEQ ID NO:41 and the light chain variable region comprises the sequence of SEQ ID NO:42; the heavy chain variable region comprises the sequence of SEQ ID NO:43 and the light chain variable region comprises the sequence of SEQ ID NO:44; the heavy chain variable region comprises the sequence of SEQ ID NO:45 and the light chain variable region comprises the sequence of SEQ ID NO:46; or the heavy chain variable region comprises the sequence of SEQ ID NO: 47 and the light chain variable region comprises the sequence of SEQ ID NO: 48; The antibody or antigen-binding fragment thereof according to claim 1.

6. The antibody or antigen-binding fragment thereof of claim 1, further comprising an immunoglobulin constant region, further comprising a constant region of a human immunoglobulin, or further comprising a constant region of a human IgG.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the constant region comprises one or more amino acid residue substitutions relative to SEQ ID NO: 49 selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the constant region comprises the sequence of SEQ ID NO:

51.

9. The antibody or antigen-binding fragment thereof of claim 1, which is humanized or afucosylated.

10. Diabody, Fab, Fab', F(ab') ２ , Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ２ , a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), or a multispecific antibody.

11. The antibody or antigen-binding fragment thereof of claim 1, which is bispecific and can specifically bind to a first antigen, CLDN18.2, and a second antigen.

12. 12. The antibody or antigen-binding fragment thereof of claim 11, wherein the second antigen is an immune-associated target, and the immune-associated target is selected from the group consisting of PD-L1, PD-L2, PD-1, CLTA-4, TIM-3, LAG3, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, ICAM-1, NKG2C, SLAMF7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, and CD83.

13. 12. The antibody or antigen-binding fragment thereof of claim 11, wherein the second antigen comprises a tumor antigen, and the tumor antigen comprises CA-125, gangliosides G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptide, PSA, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFR (e.g., VEGFR3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, IL-2R, or CO17-1A.

14. The antibody or antigen-binding fragment thereof of claim 1, which is linked to one or more conjugate moieties, wherein the conjugate moieties comprise a clearance modifier, a chemotherapeutic drug, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent (e.g., vcMMAE), or other anti-cancer drug.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in claim 1 and one or more pharmaceutically acceptable carriers.

16. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1.

17. A vector comprising the isolated polynucleotide of claim 16.

18. A host cell comprising the vector of claim 17.

19. A method for expressing the antibody or antigen-binding fragment thereof described in claim 1, comprising culturing a host cell containing a vector comprising an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof described in claim 1 under conditions in which the vector is expressed.

20. 16. The pharmaceutical composition of claim 15, for use in a method for treating a disease or condition in a subject in which modulation of CLDN18.2 activity would be beneficial, wherein the method comprises administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of claim 1. The pharmaceutical composition.

21. 21. The pharmaceutical composition of claim 20, wherein the disease or condition is cancer, and optionally the cancer is a CLDN18.2-expressing cancer.

22. The pharmaceutical composition of claim 21, wherein the CLDN18.2-expressing cancer is a cancer cell that highly expresses CLDN18.2, a cancer cell that moderately expresses CLDN18.2, or a cancer cell that lowly expresses CLDN18.

2.

23. The cancer cells that highly express CLDN18.2 express CLDN18.2 at a level where at least 70% of the cells stain positive by IHC, with an intensity of at least 2+ as measured by IHC; The cancer cells that moderately express CLDN18.2 express CLDN18.2 at a level where at least 40% to less than 70% of the cells stain positive by IHC, with an intensity of at least 1+ as measured by IHC; The cancer cells that lowly express CLDN18.2 express CLDN18.2 at a level where more than 0 but less than 40% of the cells stain positive by IHC, with an intensity of at least 1+ as measured by IHC. The pharmaceutical composition of claim 22.

24. 22. The pharmaceutical composition of claim 21, wherein the cancer is gastric cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain tumor, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, digestive cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

25. 21. The pharmaceutical composition of claim 20, wherein the subject is a human.

26. 21. The pharmaceutical composition of claim 20, wherein the method further comprises administering a therapeutically effective amount of a second therapeutic agent.

27. A kit comprising the antibody or antigen-binding fragment thereof of claim 1 and a second therapeutic agent.

28. 1. A method for obtaining information for diagnosing cancer in a subject, comprising: a) contacting a sample obtained from a subject with the antibody or antigen-binding fragment thereof of claim 1; b) determining the expression level of CLDN18.2 in the sample as follows: Cancer cells that highly express CLDN18.2 express CLDN18.2 at a level where at least 70% of the cells stain positively by IHC, with an intensity of at least 2+ as measured by IHC; Moderately expressing CLDN18.2 cancer cells express CLDN18.2 at a level where at least 40% to less than 70% of the cells stain positively by IHC, at an intensity of at least 1+ as measured by IHC; Cancer cells that express low levels of CLDN18.2 express CLDN18.2 at an intensity of at least 1+ as measured by IHC, at a level where more than 0% but less than 40% of the cells stain positively by IHC. and; c) determining that administering the antibody or antigen-binding fragment thereof described in claim 1 to the subject is beneficial when the cancer cells expressing CLDN18.2 in the sample are cancer cells that highly express CLDN18.2, cancer cells that moderately express CLDN18.2, or cancer cells that lowly express CLDN18.

2.

29. A kit comprising the antibody or antigen-binding fragment thereof described in claim 1, useful for detecting CLDN18.

2.

30. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CLDN18.

2. The chimeric antigen receptor (CAR) comprises the antigen-binding fragment of claim 1.

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