Antibodies and uses thereof

Abstract

The invention relates to an antibody specifically binding to CLDN6 and an application thereof.

Description

Antibodies and their applications Technical Field

[0001] The present invention relates to the field of tumor immunotherapy or diagnosis, and more specifically, to antibodies that specifically bind to CLDN6 and applications thereof. Background Art

[0002] The claudin (CLDN) gene family encodes membrane proteins that are essential components of tight junctions. CLDN proteins consist of four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2), with both their N- and C-termini located in the cytoplasm. The extracellular loops of CLDN proteins from adjacent cells interact with each other to seal the cell lamellae and regulate paracellular transport between the lumen and the basolateral space. CLDN proteins play a role in various human diseases and pathologies. CLDN6, a member of the CLDN gene family, is not expressed in normal adult tissues but is highly expressed in various solid tumor tissues, such as ovarian, testicular, and endometrial cancers. Therefore, CLDN6 is a potential target for cancer therapy within the Claudin family. The extracellular domain sequence of CLDN6 shares high homology with its family members, CLDN9 and CLDN4, making the development of antibodies that specifically bind to CLDN6 extremely challenging.

[0003] Summary of the Invention

[0004] The present invention aims to provide antibodies that specifically recognize CLDN6. The present invention also relates to methods for preparing anti-CLDN6-specific antibodies, including humanized antibody preparation techniques and phage library screening techniques. The present invention also relates to studies on the properties and specificity of anti-CLDN6 antibodies (including but not limited to scFv forms). The present invention provides chimeric antigen receptors (CARs) targeting CLDN6 and methods for preparing the same. The present invention also provides isolated nucleic acids encoding the anti-CLDN6 antibodies and chimeric antigen receptors targeting CLDN6 of the present invention. The present invention also provides host cells comprising the nucleic acids of the present invention. The methods further include culturing the host cells of the present invention to produce the antibodies or CARs. The antibodies and / or CARs of the present invention are used to treat or diagnose tumors.

[0005] In a first aspect, an antibody or antigen-binding fragment that recognizes CLDN6 is provided, selected from the group consisting of:

[0006] (1) the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the HCDR3 of the heavy chain variable region has at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR3 of SEQ ID NO: 41;

[0007] (2) the antibody or antigen-binding fragment comprises a heavy chain variable region comprising HCDR1 represented by GYYMN (SEQ ID NO: 35); and / or

[0008] HCDR2 shown in EINPATGSTTYNQKFKA (SEQ ID NO: 36); and / or

[0009] HCDR3 represented by RDYYX1GSX2X3YAX4DY (SEQ ID NO: 52), wherein X1 is Y or L or a conservative substitution of Y or L, X2 is G or N or a conservative substitution of G or N, X3 is F or S or a conservative substitution of F or S, and X4 is M or L or a conservative substitution of M or L;

[0010] (3) the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 35, 36 and 37;

[0011] (4) the antibody or antigen-binding fragment comprises a light chain variable region, wherein the LCDR3 of the light chain variable region has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the LCDR3 of SEQ ID NO: 42, 43 or 44;

[0012] (5) the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 represented by QASQSVSNNLN (SEQ ID NO: 38); and / or

[0013] LCDR2 shown in GASKLED (SEQ ID NO: 39); and / or

[0014] LCDR3 represented by X5QHRX6X7WT (SEQ ID NO: 53), wherein X5 is L or Q or a conservative substitution of L or Q, X6 is Y or F or a conservative substitution of Y or F, and X7 is L or M or a conservative substitution of L or M;

[0015] (6) the antibody or antigen-binding fragment comprises a light chain variable region, wherein LCDR1, LCDR2 and LCDR3 of the light chain variable region have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 38, 39 and 40;

[0016] (7) The antibody or antigen-binding fragment comprises the heavy chain variable region described in any one of (1) to (3) and the light chain variable region described in any one of (4) to (6);

[0017] (8) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), and the variant contains at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).

[0018] In a preferred example, the HCDR2 of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO: 36.

[0019] In one embodiment, the antibody or antigen-binding fragment is selected from the group consisting of:

[0020] (1) The antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 35, and / or HCDR2 set forth in SEQ ID NO: 36, and / or HCDR3 set forth in SEQ ID NO: 37 or 41;

[0021] (2) the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 38, and / or LCDR2 as shown in SEQ ID NO: 39, and / or LCDR3 as shown in SEQ ID NO: 40, 42, 43 or 44;

[0022] (3) The antibody or antigen-binding fragment comprises the heavy chain variable region described in (1) and the light chain variable region described in (2);

[0023] (4) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (3), wherein the variant comprises at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (3).

[0024] In one embodiment, the antibody or antigen-binding fragment is selected from the group consisting of:

[0025] (1) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; or

[0026] (2) the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 41, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; or

[0027] (3) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 42; or

[0028] (4) the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 43; or

[0029] (5) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 41, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 42; or

[0030] (6) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 41, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 43; or

[0031] (7) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 44; or

[0032] (8) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), wherein the variant comprises at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).

[0033] In a second aspect, an antibody or antigen-binding fragment that recognizes CLDN6 is provided, selected from the group consisting of:

[0034] (1) The antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or 5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0035] (2) The antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3, 7, 9 or 11, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0036] (3) The antibody or antigen-binding fragment comprises the heavy chain variable region described in (1) and the light chain variable region described in (2);

[0037] (4) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (3), wherein the variant comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in VH or VL, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (3).

[0038] In one embodiment, the aforementioned antibody or antigen-binding fragment is selected from the group consisting of:

[0039] (1) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0040] (2) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0041] (3) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0042] (4) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0043] (5) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0044] (6) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0045] (7) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0046] The antibody or antigen-binding fragment described in (8) is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), wherein the variant comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in VH or VL, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).

[0047] In a third aspect, an antibody or antigen-binding fragment that recognizes CLDN6 is provided, comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 3, 7, 9, or 11, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and

[0048] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37 or 41.

[0049] In one embodiment, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 1, 5, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequence.

[0050] In a fourth aspect, an antibody or antigen-binding fragment that recognizes CLDN6 is provided, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1, 5, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and

[0051] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40, 42, 43 or 44.

[0052] In one embodiment, the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:3, 7, 9 or 11, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequence.

[0053] In one embodiment, the antibody or antigen-binding fragment of any of the foregoing is selected from a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, dAb fragment, multifunctional antibody, IgG4 antibody, scFv-Fc antibody, hybridoma antibody, chimeric antibody, humanized antibody, fully human antibody or monoclonal antibody.

[0054] In one embodiment, the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18 or 19, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above sequence, or comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in the above sequence.

[0055] In one embodiment, the antibody or antigen-binding fragment binds to CLDN6 and does not significantly bind to CLDN4 or CLDN9; and / or the antibody or antigen-binding fragment binds to cells expressing CLDN6 and does not significantly bind to cells expressing CLDN4, CLDN9, or a combination thereof.

[0056] In a fifth aspect, an immunoconjugate is provided, comprising: the antibody or antigen-binding fragment according to any one of aspects 1 to 4, and a functional molecule linked thereto.

[0057] In one embodiment, the functional molecule is selected from: a molecule targeting a tumor surface marker, a molecule that inhibits tumors, a molecule targeting a surface marker of an immune cell, or a detectable marker.

[0058] In one embodiment, the molecule targeting a tumor surface marker is an antibody or ligand that binds to a tumor surface marker other than CLDN6.

[0059] In one embodiment, the tumor-suppressing molecule is an anti-tumor cytokine or an anti-tumor toxin.

[0060] In one embodiment, the cytokine is selected from IL-7, IL-12, IL-15, IL-18, IL-21, type I interferon or TNF-α.

[0061] In one embodiment, the molecule targeting the surface marker of immune cells is an antibody that binds to the surface marker of T cells, which forms a T cell-involved bifunctional antibody with the antibody or antigen-binding fragment described in any one of the first to fourth aspects.

[0062] In one embodiment, the surface marker of the targeted immune cell is selected from: CD3, CD16, and CD28.

[0063] In one embodiment, the antibody targeting a surface marker of an immune cell is an anti-CD3 antibody.

[0064] In one embodiment, the immunoconjugate further comprises a connecting peptide between the antibody or antigen-binding fragment described in any one of the first to fourth aspects and the functional molecule connected thereto.

[0065] In a sixth aspect, a chimeric receptor is provided, comprising an extracellular region, wherein the extracellular region comprises the antibody or antigen-binding fragment according to any one of the first to fourth aspects;

[0066] The chimeric receptor includes: a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), a synthetic polypeptide receptor (synNotch) or a combination thereof.

[0067] In one embodiment, the chimeric receptor is a chimeric antigen receptor (CAR), which comprises the antibody or antigen-binding fragment described in any one of the first to fourth aspects, a transmembrane region and an intracellular signaling region.

[0068] In one embodiment, the antibody or antigen-binding fragment is connected to the transmembrane region via a hinge domain.

[0069] In one embodiment, the transmembrane region of the chimeric receptor comprises a transmembrane region selected from the α, β, γ or ζ chain of TCR, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD134, CD137, CD152, CD154 or PD1.

[0070] In one embodiment, the transmembrane region is selected from the transmembrane domain of CD8 or CD28.

[0071] In one embodiment, the transmembrane region is selected from the sequence shown in SEQ ID NO: 25 or 22, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0072] In one embodiment, the intracellular signaling region of the chimeric receptor comprises a primary signaling domain.

[0073] In one embodiment, the primary signaling domain is selected from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), CD66d or CD3ζ.

[0074] In one embodiment, the intracellular signaling region of the chimeric receptor further comprises one or more costimulatory signaling domains.

[0075] In one embodiment, the costimulatory signaling domain is selected from the intracellular signaling region of CARD11, CD2, CD5, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB or 4-1BBL, or a combination thereof.

[0076] In one embodiment, the costimulatory signaling domain is selected from the intracellular signaling domains of CD28 and / or CD137.

[0077] In one embodiment, the intracellular signaling region of the chimeric receptor is selected from SEQ ID NO: 24, or the sequence shown in SEQ ID NO: 23 and 24, or the sequence shown in SEQ ID NO: 26 and 24, or the sequence of SEQ ID NO: 23, 26 and 24, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the above sequences.

[0078] In one embodiment, the hinge region of the chimeric receptor is derived from CD8, IgG4 or IgG1.

[0079] In one embodiment, the hinge region comprises the sequence shown in SEQ ID NO: 21, 27, 28, 29 or 30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0080] In one embodiment, the chimeric receptor comprises:

[0081] The antibody or antigen-binding fragment of any one of the first to fourth aspects, the transmembrane region of CD8 / CD28, and CD3ζ; or

[0082] The antibody or antigen-binding fragment of any one of the first to fourth aspects, the transmembrane region of CD8 / CD28, the intracellular signaling region of CD137, and CD3ζ; or

[0083] The antibody or antigen-binding fragment of any one of the first to fourth aspects, the transmembrane region of CD8 / CD28, the intracellular signaling region of CD28, and CD3ζ; or

[0084] The antibody or antigen-binding fragment of any one of the first to fourth aspects, the transmembrane region of CD8 / CD28, the intracellular signaling region of CD28, CD137 and CD3ζ.

[0085] In one embodiment, the chimeric receptor comprises an amino acid sequence as shown in any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18 or 19, linked to a sequence as shown in any one of SEQ ID NOs: 45, 46 or 47, respectively, or an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the above sequences.

[0086] In one embodiment, the chimeric receptor comprises the sequence as shown in SEQ ID NO: 48 or 49, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0087] In the seventh aspect, a nucleic acid encoding the antibody or antigen-binding fragment of any one of the first to fourth aspects, the immunoconjugate of the fifth aspect, or the chimeric receptor of the sixth aspect is provided.

[0088] In the eighth aspect, a vector is provided, which comprises the nucleic acid described in the seventh aspect.

[0089] In the ninth aspect, a cell is provided, comprising the antibody or antigen-binding fragment of any one of the first to fourth aspects, the immunoconjugate of the fifth aspect, the chimeric receptor of the sixth aspect, the nucleic acid of the seventh aspect, and / or the vector of the eighth aspect.

[0090] In one embodiment, the cells include T cells, natural killer cells, natural killer T cells, NK92 cells, cytotoxic T cells, dendritic cells, macrophages, CIK cells, pluripotent stem cells, stem cell-derived immune cells, or a combination thereof.

[0091] In one embodiment, the T cells include natural T cells and / or T cells induced from pluripotent stem cells.

[0092] In one embodiment, the T cells include autologous T cells and / or allogeneic T cells.

[0093] In one embodiment, the T cells are primary T cells.

[0094] In one embodiment, the T cells are derived from human autologous T cells.

[0095] In one embodiment, the cell binds to cells expressing CLDN6 and does not significantly bind to cells expressing CLDN4, CLDN9, or a combination thereof.

[0096] In one embodiment, the cell further carries an exogenous cytokine coding sequence; and / or it further expresses a chimeric receptor that does not target CLDN6; and / or it further expresses a chemokine; and / or it further expresses a chemokine receptor; and / or it further expresses a safety switch; and / or it further expresses an inhibitory molecule.

[0097] In one embodiment, the cells further carry exogenous cytokines including coding sequences for IL-7, IL-12, IL-15, IL-18, IL-21, type I interferon, or TNF-α.

[0098] In one embodiment, the cells further express a chemokine comprising CCL19 or CCL21.

[0099] In one embodiment, the cells further express a chemokine receptor including CCR2, CCR4, CCR5, CXCR2, CXCR4, or CXCR5.

[0100] In one embodiment, the cell further expresses a safety switch comprising iCaspase-9, Truancated EGFR, or RQR8.

[0101] In one embodiment, the cells further express an inhibitory molecule, including an siRNA that reduces PD-1 expression or a protein that blocks PD-L1.

[0102] In the tenth aspect, a pharmaceutical composition is provided, comprising the antibody or antigen-binding fragment of any one of claims 1 to 4, the immunoconjugate of the fifth aspect, the chimeric receptor of the sixth aspect, the nucleic acid of the seventh aspect, the vector of the eighth aspect and / or the cell of the ninth aspect, and a pharmaceutically acceptable adjuvant.

[0103] In the eleventh aspect, a combination drug is provided, wherein the antibody or antigen-binding fragment described in any one of the first to fourth aspects, the immunoconjugate described in the fifth aspect, the chimeric receptor described in the sixth aspect, the cell described in the ninth aspect, and the pharmaceutical composition described in the tenth aspect are administered in combination with an agent that enhances their function.

[0104] In one embodiment, the drug is administered in combination with a chemotherapeutic drug; and / or administered in combination with an agent that improves one or more side effects associated therewith; and / or administered in combination with a cell expressing a chimeric antigen receptor targeting a protein other than CLDN6; and / or administered in combination with an agent that treats a disease associated with the expression of CLDN6.

[0105] In one embodiment, the agent comprises an antibody or antigen-binding fragment, a cell, RNA, a vaccine, an oncolytic virus, a checkpoint inhibitor, a BKT inhibitor, a chemotherapy drug, a radiotherapy agent, a hormone therapy agent, a toxin, an immunotherapy agent, or a combination thereof.

[0106] In the twelfth aspect, a method for preparing the antibody or antigen-binding fragment described in any one of the first to fourth aspects, the immunoconjugate described in the fifth aspect, or the chimeric receptor described in the sixth aspect is provided, the method comprising culturing the cells described in the ninth aspect, and isolating the antibody or antigen-binding fragment, immunoconjugate or chimeric receptor expressed by the cells.

[0107] In the thirteenth aspect, a kit is provided, characterized in that it comprises the antibody or antigen-binding fragment described in any one of the first to fourth aspects, the immunoconjugate described in the fifth aspect, the chimeric receptor described in the sixth aspect, the nucleic acid described in the seventh aspect, the vector described in the eighth aspect, the cell described in the ninth aspect and / or the pharmaceutical composition described in the tenth aspect.

[0108] In the fourteenth aspect, uses of the antibody or antigen-binding fragment of any one of aspects one to four, the immunoconjugate of aspect five, the cell of aspect nine, the pharmaceutical composition of aspect ten, and / or the kit of aspect thirteen are provided for: (1) killing cells expressing CLDN6; (2) inhibiting the proliferation of cells expressing CLDN6; (3) mediating the alleviation of a disease or tumor; (4) preventing the formation or re-formation of a tumor; (5) inhibiting the metastasis of cells expressing CLDN6; and (6) preparing a drug for treating / diagnosing a disease.

[0109] In one embodiment, the disease expresses CLDN6.

[0110] In one embodiment, the disease is selected from an inflammatory disorder, an infection, an autoimmune disease, and a tumor.

[0111] In one embodiment, the tumor is a solid tumor.

[0112] In one embodiment, the tumor is ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonal tumors, epithelial ovarian cancer, non-small cell lung cancer, non-squamous non-small cell lung cancer, endometrial cancer, or a combination thereof.

[0113] In a fifteenth aspect, the present invention provides a medicament for the following uses and comprising the antibody or antigen-binding fragment of the first aspect, the immunoconjugate of the second aspect, the cell of the ninth aspect, and the pharmaceutical composition of the tenth aspect,

[0114] (1) Killing cells expressing CLDN6;

[0115] (2) inhibiting the proliferation of cells expressing CLDN6;

[0116] (3) mediate the remission of disease or tumor;

[0117] (4) prevent tumor formation or re-formation;

[0118] (5) inhibiting the metastasis of cells expressing CLDN6;

[0119] (6) For the treatment / diagnosis of diseases.

[0120] In the sixteenth aspect, a method for treating / diagnosing a disease is provided, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment as described in any one of the first to fourth aspects, or the immunoconjugate as described in the fifth aspect, or the cell as described in the ninth aspect, or the pharmaceutical composition as described in the tenth aspect, or the kit as described in the thirteenth aspect.

[0121] In one embodiment, the disease is selected from an inflammatory disorder, an infection, an autoimmune disease, and a tumor.

[0122] In one embodiment, the subject is a human.

[0123] In one embodiment, the cells are autologous or allogeneic T cells to the subject.

[0124] In the seventeenth aspect, provided is the antibody or antigen-binding fragment of any one of aspects one to four, the immunoconjugate of aspect five, the cell of aspect nine, the pharmaceutical composition of aspect ten, and / or the kit of aspect thirteen, for use in treating / diagnosing a disease comprising expression of CLDN6.

[0125] In one embodiment, the disease is selected from an inflammatory disorder, an infection, an autoimmune disease, and a tumor.

[0126] In one embodiment, the tumor is a solid tumor.

[0127] In one embodiment, the tumor is ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonal tumors, epithelial ovarian cancer, non-small cell lung cancer, non-squamous non-small cell lung cancer, endometrial cancer, or a combination thereof.

[0128] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 shows the flow cytometry results of 293T-CLDN4, 293T-CLDN6, and 293T-CLDN9 stably transfected cell lines;

[0130] Figure 2 shows that antibody H1 (scFv-huFc) specifically binds to 293T-CLDN6 cells;

[0131] Figure 3 shows the EC50 of antibody H1 binding to 293T-CLDN6 cells;

[0132] Figure 4 shows that antibodies P1, P2, P3, and P4 all specifically bind to 293T-CLDN6 cells;

[0133] Figure 5 shows the EC50 of binding of antibodies P1, P2, P3, and P4 to 293T-CLDN6 cells;

[0134] Figure 6 shows that antibodies H1, M1, and M2 all specifically bind to 293T-CLDN6 cells;

[0135] Figure 7 shows the EC50 of antibodies M1 and M2 binding to 293T-CLDN6 cells;

[0136] Figure 8 shows the positive rates of H1-28Z CAR T cells and P4-28Z CAR T cells;

[0137] Figure 9 shows the in vitro specific killing of target cells by H1-28Z CAR T cells and P4-28Z CAR T cells;

[0138] Figure 10 shows the in vitro killing of target cells by H1-28Z CAR T cells and P4-28Z CAR T cells;

[0139] Figure 11 shows the anti-tumor effects of H1-28Z CAR T cells and P4-28Z CAR T cells on subcutaneous transplanted tumors in NPG mice bearing human ovarian cancer cells (Figure 11A); changes in mouse body weight (Figure 11B); changes in tumor weight (Figure 11C); and survival of human T cells in the peripheral blood of mice (Figure 11D). DETAILED DESCRIPTION

[0140] The present invention provides humanized antibodies that specifically recognize CLDN6 (including fragments thereof, such as heavy chain variable regions (VH), light chain variable regions (VL), and scFv), chimeric receptors containing such antibodies, nucleic acids encoding such antibodies, and cells expressing such antibodies. The antibodies of the present invention can be used to prepare targeted anti-tumor drugs and drugs for tumor diagnosis. The present invention also provides methods for preparing and using such antibodies and cells expressing such antibodies.

[0141] the term

[0142] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics, and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, wherein suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail. In addition, unless otherwise specified, the materials, methods, and examples are illustrative only and are not intended to be limiting. In light of the present disclosure, it will be understood by those skilled in the art that many changes or modifications can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. The present invention is not limited in scope to the specific embodiments described herein (which are intended only as illustrations of various aspects of the invention), and functionally equivalent methods and components are within the scope of the present invention. The present invention includes modifications and adaptations of the subject matter of the present invention for various uses and conditions.

[0143] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art and are fully explained in the literature.

[0144] In the disclosure, various aspects of the claimed subject matter are presented in the form of ranges. It should be understood that the description in range form is merely for convenience and brevity and should not be interpreted as a hard limit to the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other stated or intermediate values ​​within the range are included in the claimed subject matter, and the upper and lower limits of the range also fall within the scope of the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller range, and they also fall within the scope of the claimed subject matter unless the upper and lower limits of the range are explicitly excluded. When a range is set to include one or two limits, the claimed subject matter also includes a range that excludes one or both of the limits. This applies regardless of the width of the range.

[0145] The term "about" refers to the usual error range for each value readily known to those skilled in the art. Reference herein to a value or parameter "about" includes embodiments referring to the value or parameter itself. For example, description of "about X" includes "X." "About" or "comprising" can mean a range of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ±17%, ±18%, ±19%, ±20%, ±25%, or ±30% of the value. Alternatively, particularly with respect to biological systems or methods, the term can refer to within an order of magnitude of a value, e.g., within about 5 times or about 2 times a value.

[0146] Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range described herein should be understood to include any integer within the range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of an integer).

[0147] In order to make the present invention easier to understand, some terms are first defined.

[0148] The term "Claudin 6 (CLDN6)" refers to a member of the CLDN family. The gene encoding the human CLDN6 protein is located on the p-arm of human chromosome 16 at 16p13.3 and is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, zebrafish, and frogs. A CLDN6 polypeptide has an amino acid sequence or fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% homologous or identical to the amino acid sequence encoded by the transcript expressed by the gene with NCBI GenBank Gene ID: 9074, and / or may optionally include up to one, two, or three conservative amino acid substitutions. Exemplary, the full-length amino acid sequence of human CLDN6 is set forth in SEQ ID NO: 32.

[0149] The term "Claudin 9 (CLDN9)" refers to the family member most closely related to CLDN6. The gene encoding the human CLDN9 protein is located on human chromosome 16, at 16p13.3, and consists of a single exon spanning approximately 2.1 kBp. Exemplarily, the full-length amino acid sequence of human CLDN9 is set forth in SEQ ID NO: 33.

[0150] The term "Claudin 4 (CLDN4)" is also a member of the CLDN family. The gene encoding the human CLDN4 protein spans approximately 1.82 kBp at chromosome location 17q11.23. Exemplary, the full-length amino acid sequence of human CLDN4 is shown in SEQ ID NO: 31.

[0151] The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably to refer to polymers of any length composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. The term refers to both short chains (commonly referred to in the art as peptides, oligopeptides and oligomers) and longer chains (commonly referred to in the art as proteins), and there are many types of proteins. Polypeptides include, for example, biologically active fragments, basic homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or combinations thereof. A polymer can be linear, cyclic, or branched, it can contain modified amino acids, particularly conservatively modified amino acids, and it can be interrupted by non-amino acids. The term also includes modified amino acid polymers, such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA-mediated amino addition, such as arginylation, ubiquitination, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0152] The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen and is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, multi-chain or single-chain, or complete immunoglobulins, and can be derived from natural sources or from recombinant sources, so long as it exhibits the desired antigen-binding activity.

[0153] The term "antigen-binding fragment" refers to antibodies and immunologically active portions of antibodies, ie, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.

[0154] The humanized antibodies H1, P1, P2, P3, P4, M1, and M2 of the present invention can specifically bind to CLDN6, but not to CLDN4 and CLDN9. In specific embodiments, the antigen-binding specificity of the antibodies H1, P1, P2, P3, P4, M1, and M2 of the present invention is superior to that of the hybridoma antibody SC27.105. Furthermore, the antigen-binding specificity of the antibodies H1, P1, P2, P3, P4, M1, and M2 of the present invention is superior to that of the control antibody C46-S (from patent WO2015150327A1).

[0155] The terms "specifically bind," "specifically recognize," or "are specific for" refer to a measurable and reproducible interaction, such as binding of a target and an antibody, where the binding is determinative of the presence of the target in the presence of other molecules. For example, an antibody that specifically binds to a target is one that binds to that target with greater affinity, more readily, and / or for a longer duration than it binds to other targets. In some embodiments, an antibody or ligand recognizes and binds to a cognate binding partner protein present in a sample, but does not substantially recognize or bind to other molecules in the sample. In some embodiments, specific binding can include but does not require exclusive binding. "Multispecificity" refers to an antibody having binding specificities for at least two different sites on an antigen.

[0156] "Antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and may refer to the antigen binding domain of an intact antibody, such as the antigenicity-determining variable region, which is sufficient to confer recognition and specific binding to a target, such as an antigen, on the antibody fragment. Examples of antibody fragments include, but are not limited to, (i) Fab fragments consisting of the VL, VH, CL, and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of the VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region; (v) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments; (vi) single-chain Fv molecule antigen-binding sites; (vii) bispecific single-chain Fv dimers; (viii) "dibodies" or "tribodies," multivalent or multispecific fragments constructed by gene fusion; (ix) scFv genetically fused to the same or different antibodies; (x) linear antibodies; (xi) camelid VHH domains; and (xii) single-domain antibodies such as sdAbs (VH or VL). Antibody fragments can be prepared by various techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells.

[0157] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are connected by a short flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Herein, scFv can have VL and VH in any order, for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or VH-linker-VL. The linker can be any amino acid of any length.

[0158] The term "linker" or "flexible polypeptide linker" refers to a peptide linker composed of amino acid (e.g., glycine and / or serine) residues, which is used alone or in combination to connect the heavy chain variable region and the light chain variable region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker comprising the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In one embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser). In one embodiment, the linker further comprises charged residues, such as lysine and / or glutamic acid, which can increase solubility. In one embodiment, the linker further comprises one or more proline.

[0159] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the native conformation of an antibody molecule and generally determines the antibody class.

[0160] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their native conformation. K and lambda light chains refer to the two major antibody light chain isotypes.

[0161] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (allotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0162] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that participates in antigen binding. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies typically have similar structures, with each domain comprising four conserved FRs and three CDRs. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to the antigen.

[0163] The term "hypervariable region" or "complementarity determining region" or "CDR" refers to each region of an antibody variable domain whose sequence is highly variable and / or forms structurally defined loops ("hypervariable loops") and / or contains residues that contact the antigen ("antigen contacts"), a non-contiguous amino acid sequence within an antibody variable region that confers specificity and / or binding affinity to the antibody. In certain embodiments, CDRs can be identified by a numbering system selected from the group consisting of Kabat, Chothia, IMGT, Gelfand, Aho, and AbM. For example, they can be identified by the Kabat numbering system. For example, an antibody can comprise six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3).

[0164] The term "Fc region" or "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0165] "Framework (FR)" refers to variable domain residues other than the hypervariable region (CDR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, in VH (or VL), CDR and FR sequences typically appear in the following order:

[0166] FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0167] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0168] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by a disulfide bond. From N-terminal to C-terminal, each heavy chain has a variable region (VH), which is also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), which is also referred to as a variable light chain domain or a light chain variable domain, followed by a light chain constant (CL) domain. The light chain of an antibody can be assigned to one of two types based on the amino acid sequence of its constant domain, referred to as κ (κ) and λ (λ).

[0169] The terms "whole antibody," "full-length antibody," and "intact antibody" are used interchangeably to refer to a full-length antibody having a structure substantially similar to a native antibody structure or having heavy chains containing an Fc region as defined herein or including an intact antigen-binding region.

[0170] The term "single domain antibody (sdAb)" refers to a type of antibody that lacks the antibody light chain and only has the heavy chain variable region. Due to its small molecular weight, it is also called a nanobody.

[0171] The term "single domain antibody" refers to an antibody comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody.

[0172] The terms "monoclonal antibody" and "single antibody" refer to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or generated during the preparation of the monoclonal antibody preparation, which variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the term "monoclonal" indicates that the antibody is obtained from a population of substantially homologous antibodies and is not to be construed as requiring that the antibody be prepared by any particular method. For example, it can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0173] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chains is derived from a specific source or species, while the remainder of the heavy and / or light chains is derived from antibodies of different sources or species. In certain embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In other embodiments, a chimeric antibody is a "type-switched" antibody in which the type or subclass has been changed by the type or subclass of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof. In certain embodiments, a chimeric antibody is a "humanized antibody."

[0174] The term "humanization" is used for non-human antibodies, such as rodents or primates, and is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof containing minimal sequences derived from non-human immunoglobulins. A "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one (generally two) variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. Various techniques for humanizing antibodies or antibody fragments well known in the art essentially substitute rodent CDRs or CDR sequences for corresponding human antibody sequences, i.e., CDR grafting. Humanized antibodies are often human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

[0175] In some embodiments, "humanized antibodies" may include mutations, such as mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo. In some embodiments, the human domain to be used for the preparation of humanized antibodies can be selected to reduce antigenicity. According to the so-called "best-fit" method, the variable domain sequences of rodent antibodies are screened relative to the complete library of known human variable domain sequences. Then, the human sequence that solves rodents can be accepted as human FR for humanized antibodies. In some embodiments, a specific FR derived from a consensus sequence is used, wherein the consensus sequence is the consensus sequence of all human antibodies of the light chain or heavy chain with a specific subgroup. The same FR can be used for several different humanized antibodies. In some embodiments, some FR residues of humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies derived from which CDR residues are derived), for example, in order to restore or improve antibody specificity or affinity. Or it refers to an antibody obtained by transplanting the amino acid sequence of the CDR of the VH and VL of a non-human animal antibody to the corresponding CDR of the VH and VL of a human antibody. The regions outside the CDRs of VH and VL are called framework regions (hereinafter referred to as FRs); in one example, a cDNA encoding the amino acid sequence of VH composed of the amino acid sequence of the CDRs of the VH of a non-human animal antibody and the amino acid sequence of the FRs of the VH of an arbitrary human antibody, and a cDNA encoding the amino acid sequence of VL composed of the amino acid sequence of the CDRs of the VL of a non-human animal antibody and the amino acid sequence of the FRs of the VL of an arbitrary human antibody are constructed, and a humanized antibody expression vector is constructed and introduced into animal or fungal or bacterial cells to thereby express the antibody.

[0176] Murine hybridoma antibodies produce immune side effects because their constant regions can be recognized by the human immune system, inducing human anti-mouse antibody responses (HAMA). Humanization can eliminate or reduce the immunogenicity of murine antibodies. Humanized antibodies have lower immunogenicity and are closer to natural antibodies in the human body, which can improve efficacy and safety. In one example, compared to non-humanized antibodies with the same specificity (e.g., mouse antibody precursors before humanization), the humanized antibodies disclosed herein have reduced immunogenicity in human subjects. The present invention humanized hybridoma antibody SC27.105 (from WO2016073649A1). In one example, the humanized antibodies H1, P1, P2, P3, P4, M1, M2, etc. of the present application have low immunogenicity in human subjects. Specifically, the antibodies H1, P1, P2, P3, P4, M1, and M2 of the present application have lower immunogenicity than the hybridoma antibody SC27.105. Low or reduced immunogenicity is characterized by the ability to treat patients long-term with measurable symptom relief and low and / or acceptable toxicity. Low or acceptable immunogenicity and / or high affinity, as well as other suitable characteristics, can contribute to achieving therapeutic outcomes. "Low or reduced immunogenicity" is defined herein as patients having a proportion of HAHA, HACA, or HAMA responses less than 90%, such as less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%.

[0177] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, for example, an antibody or antibody fragment expressed by a phage or yeast expression system or a mammalian cell expression system. The term should also be understood to refer to an antibody produced by: synthesizing a DNA molecule encoding the antibody and expressing the antibody protein from the DNA molecule; or synthesizing the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained by using recombinant DNA or amino acid sequence technology available and well known in the art.

[0178] The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having the desired one or more activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies having the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks, Meth. Mol. Biol., 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0179] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is then screened for phage binding to the antigen, as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, natural The library (e.g., by humans) thus provides a single source of antibodies to a variety of non-self antigens and self antigens without the need for any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom, J. Mol. Biol. 227:381-388 (1992). In certain embodiments, amino acid sequence variants of antibodies are provided herein. The term "parent antibody" refers to an antibody provided herein or an antibody obtained after mutation, or affinity maturation, etc., of an antibody provided herein. The parent antibody can be a naturally occurring antibody, or a variant or modified version of a naturally occurring antibody. A parent antibody can refer to the antibody itself, a composition comprising the parent antibody, or its encoded amino acid sequence.

[0180] The term "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (CDRs) compared to a parent antibody, which alterations result in an improvement in the affinity of the antibody for antigen.

[0181] The term "variant" refers to a polypeptide having substantially the same amino acid sequence as the sequence of an antibody provided herein, or having one or more activities encoded by substantially the same nucleotide sequence. The variant has the same or similar activities as the antibodies provided in the Examples of this application. For example, a variant can be a variant antibody or antibody variant based on the amino acid sequence of an antibody provided herein.

[0182] The term "variant antibody" or "antibody variant" includes antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent. The variant antibody sequence herein preferably has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with the parent antibody sequence. Antibody variants can refer to the antibody itself or to a composition comprising the antibody variant. Amino acid sequence variants of an antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. The term "amino acid modification" includes amino acid substitutions, additions and / or deletions. "Amino acid substitution" or "amino acid replacement" means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. "Amino acid insertion" means adding an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" means removing an amino acid at a specific position in the parent polypeptide sequence. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, eg, antigen binding.

[0183] The term "modification" refers to a change in the state or structure of a protein or polypeptide of the present invention. The modification can be chemical, structural or functional.

[0184] The term "conservative modification" or "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are replacements of an amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, as shown in Table 1.

[0185] Table 1: Families of amino acid residues with similar side chains

[0186] Thus, one or more amino acid residues in the CDR or framework regions of an antibody of the present invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody (variant antibody) can be tested for retained function.

[0187] Non-conservative substitutions entail exchanging a member of one of these groups for a member of another group.

[0188] A kind of substitution variant comprises one or more hypervariable regions or FR residues that replace parental antibody (for example, humanization or people's antibody).Usually, the variant of the gained being selected for further research will have the change (for example, improving) of certain biological properties (for example, the affinity of increase, the immunogenicity of reduction) relative to parental antibody and / or will substantially keep certain biological properties of parental antibody.Exemplary substitution variant is affinity maturation antibody, and it can for example, use the conventional preparation of affinity maturation technology (such as those described herein) based on phage display.In brief, one or more CDR or FR residues are mutated and variant antibody is displayed on phage and screens specific biological activity (for example, binding affinity).

[0189] Changes (e.g., substitutions) can be made in the CDR regions, for example, to improve antibody affinity. Such changes can be made in CDR "hot spots," i.e., residues encoded by codons that are mutated at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008), and / or residues that are antigen-depleted, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing and reselecting secondary libraries has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity includes CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized.

[0190] In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not significantly reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative modifications described herein) that do not significantly reduce binding affinity may be made in the CDRs. Such changes may, for example, be made to the outside of the residues that contact the antigen in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0191] In certain embodiments, amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusing the N-terminus or C-terminus of the antibody to an enzyme or polypeptide, which increases the serum half-life of the antibody.

[0192] The terms "anti-CLDN6 antibody," "antibody that binds to CLDN6," "CLDN6 antibody," and "antibody that recognizes CLDN6" refer to antibodies that bind to CLDN6 with sufficient affinity to be useful as diagnostic and / or therapeutic agents targeting CLDN6. In one embodiment, the extent of binding of the anti-CLDN6 antibody to an unrelated, non-CLDN6 protein (e.g., CLDN4, CLDN9) is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10% of the binding of the antibody to CLDN6, as determined by an enzyme-linked immunosorbent assay (ELISA). In one embodiment, the extent of binding of the anti-CLDN6 antibody to CLDN4 or CLDN9 is comparable to the level of binding detected in a negative control group. In certain embodiments, the anti-CLDN6 antibody binds to an epitope of CLDN6 that is conserved between CLDN6 species.

[0193] The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides that constitute the TCR, which are capable of binding to surface antigens on target cells and interacting with other polypeptides of the complete TCR complex, usually co-localized on the surface of T cells. A chimeric T cell receptor is composed of a TCR subunit and an antigen-binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least a portion of the TCR extracellular domain, a transmembrane domain, and a stimulatory domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit and the antibody domain are operatively linked, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the chimeric T cell receptor is integrated into the TCR expressed on the T cell.

[0194] The term "T cell antigen coupler (TAC)" includes three functional domains: (1) an antigen-binding domain, which can be a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and (3) a transmembrane domain and an intracellular domain of the CD4 co-receptor, which is linked to the protein kinase LCK and catalyzes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as the initial step in T cell activation.

[0195] The term "chimeric antigen receptor" (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0196] The term "transmembrane domain" is connected to the extracellular antigen binding domain. The transmembrane domain can be derived from natural or synthetic. When the transmembrane domain is of natural origin, it can be derived from any membrane-bound protein or transmembrane protein. On the one hand, whenever CAR binds to the target, the transmembrane domain can conduct the signal to the intracellular domain. The transmembrane domain includes: α, β, γ, δ or ζ chains of T cell receptors; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; IL2 receptor p55 (α chain), p75 (β chain) or γ chain; FcγRIII. When the transmembrane domain is of synthetic origin, it can include hydrophobic residues such as leucine and valine. On the one hand, phenylalanine, tryptophan and valine triplets can be present at both ends of the synthetic transmembrane domain.

[0197] In some cases, the membrane-spanning domain may include one or more additional amino acids adjacent to the membrane-spanning region, such as one or more amino acids associated with the extracellular region of the protein from which the membrane-spanning domain is derived, and / or one or more amino acids associated with the intracellular region of the protein from which the membrane-spanning domain is derived. In some cases, the membrane-spanning domain may be connected to the extracellular region of the CAR through a "hinge region". "Hinge region" is used to provide more flexibility and accessibility to the extracellular antigen-binding domain. The hinge region may include up to 300 amino acids, preferably 10-100 amino acids, most preferably 25 to 50 amino acids. The hinge region may be derived from all or part of the extracellular regions of naturally occurring molecules such as CD8, CD4, CD28, FcγRIII or IgG1, IgG4, or from all or part of an antibody constant region. The hinge region may also be a synthetic sequence.

[0198] The term "intracellular signaling region" or "intracellular signaling domain" refers to the intracellular portion of the CAR molecule, which is responsible for activating at least one normal effector function of the immune cell into which CAR has been introduced. The intracellular signaling domain produces cell lysis activity and auxiliary activity that can promote CAR-containing cells (e.g., CAR-T cells), including secretion of cytokines." Signaling domain" refers to a protein portion that transduces effector signal function signals and guides cells to perform specific functions. The signaling domain for CAR can be the cytoplasmic sequence of a T cell receptor, and the cytoplasmic sequence of a co-receptor that synergistically initiates signal transduction after antigen receptor engagement, and any derivatives or variants of these sequences, as well as any synthetic sequences with the same function.

[0199] Intracellular signaling domains can include functional signaling domains of stimulatory molecules (also referred to as stimulatory molecules, primary signal molecules) and / or co-stimulatory molecules. In one embodiment, the intracellular signaling domain can include primary signal molecules, exemplarily, primary signal molecules are derived from those domains of molecules responsible for the first stimulation or antigen-dependent stimulation. In one embodiment, the signal generated by TCR alone is not enough to fully activate T cells, and a second and / or co-stimulatory signal is also required. The intracellular signaling domain can include co-stimulatory intracellular signaling domains, exemplarily, co-stimulatory intracellular signaling domains include those domains derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain of the molecule, or a functional fragment thereof. For example, the stimulatory molecule can be a zeta chain that binds to a T cell receptor complex; for example, the cytoplasmic signaling domain further includes a functional signaling domain of one or more costimulatory molecules, such as intracellular sequences of 4-1BB (i.e., CD137), CD27, and / or CD28.

[0200] In the present invention, on the one hand, CAR comprises a chimeric fusion protein, and the protein comprises an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain contains a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein, and the protein comprises an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain contains a functional signal transduction domain derived from a costimulatory molecule and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein, and the protein comprises an extracellular antigen recognition domain, a transmembrane domain and an intracellular transduction domain, and the intracellular signal transduction domain comprises at least two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises an optional "leader sequence" at the amino acid (ND end) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0201] The term "leader sequence" (also known as a signal peptide) is operably linked to the CAR nucleic acid sequence and positioned so that the newly synthesized polypeptide is directed into the secretory pathway of the cell. The leader sequence is typically positioned at the 5' end of the nucleic acid sequence encoding the polypeptide. The signal peptide includes the signal sequence of a naturally occurring protein of the CAR or a synthetic non-naturally occurring signal sequence. In some embodiments, the signal peptide is selected from CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0202] The term "primary signal molecule" or "stimulatory molecule" regulates the initial activation of the TCR complex in a stimulating manner. Typically, the primary signal is triggered by the combination of, for example, a TCR / CD3 complex with an MHC molecule loaded with a peptide, thereby mediating a T cell response (including but not limited to, proliferation, activation, differentiation, etc.). The primary signal molecule that acts in a stimulating manner can include an immunoreceptor tyrosine activation motif or an ITAM signaling motif. Examples of functional signaling domains (primary signaling domains) of primary signaling molecules comprising ITAMs that are particularly useful in the present invention include but are not limited to, sequences derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), CD66d, DAP10, and DAP12. In the CAR of the present invention, the intracellular signaling domain includes an intracellular signaling sequence in any one or more CARs of the present invention, such as the primary signaling domain of CD3ζ.

[0203] The term "costimulatory signaling domain" or "costimulatory molecule" generally refers to an intracellular domain of a costimulatory molecule that can bind to a cell stimulatory signaling molecule, such as TCR / CD3, and the combination leads to the upregulation or downregulation of signals of T cell proliferation and / or key molecules. Costimulatory molecules are generally associated binding partners on T cells that specifically bind to costimulatory ligands, thereby mediating the co-stimulatory response of T cells, for example, including but not limited to proliferation. Costimulatory molecules are cell surface molecules or their ligands that are non-antigen receptors required for an effective immune response. The costimulatory intracellular signaling domain can be derived from the intracellular portion of a costimulatory molecule. Costimulatory molecules can appear in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins) and activating NK cell receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), GITR, CD30, CD40, ICOS, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83.

[0204] The term "CD3ζ (also known as CD3 Zeta)" or "TCRζ" or "ζ chain" is defined as the protein provided by GenBan Accession No. BAG36664.1, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. "CD3ζ domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signal required for T cell activation. The cytoplasmic domain of CD3ζ comprises residues 52 to 164 of GenBan Accession No. BAG36664.1, its functional orthologs - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. Exemplary, the amino acid sequence of CD3ζ is set forth in SEQ ID NO: 24.

[0205] The term "4-1BB (also known as CD137)" refers to a member of the TNFR superfamily having the amino acid sequence of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. The "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. Exemplary, the amino acid sequence of the 4-1BB costimulatory domain is set forth in SEQ ID NO: 26.

[0206] The term "chemokine" is a polypeptide with a molecular weight of 8 to 10 kDa. It is the largest cytokine family. Its main function is to recruit monocytes, neutrophils, lymphocytes, etc. in the blood into specific lymphoid organs and tissues as well as the site of infection.

[0207] The term "chemokine receptor" refers to a class of seven-transmembrane G protein-coupled receptors (GPCRs) that mediate the function of chemokines. They are typically expressed on the cell membranes of immune cells, neutrophils, endothelial cells, and other cells. Chemokines and chemokine receptors play an important role in mediating cell migration, proliferation, and defense against pathogen invasion, and are closely related to the development and progression of inflammation and cancer within the immune system.

[0208] The term "safety switch" was coined to improve the safety of CAR-T therapy by designing a rapid and reversible "off" or "on" safety switch for CAR-T cells to minimize treatment-related toxicities. Although CAR-T cell therapy has excellent clinical characteristics, when tumor burden is unpredictable and T cell activity is uncontrolled, it can trigger potentially fatal side effects such as severe CRS. To control toxicity, severe CRS requires appropriate monitoring using a CRS grading system as a guide and precise regulation using a small molecule-based safety switch.

[0209] Detailed Description of the Invention

[0210] Antibody

[0211] This article describes the generation of humanized anti-CLDN6-specific antibodies using conventional humanized antibody preparation techniques in the art, and the generation of mutants of these antibodies using CDR region randomization and phage screening. These molecules exhibit exquisite specificity. For example, this antibody only recognizes CLDN6 and 293T cells, OVCAR3 cells, and OV90 cells expressing CLDN6, but not cells expressing CLDN4, CLDN9, or a combination thereof. Unless otherwise specified, CLDN6 herein refers to human CLDN6.

[0212] In some embodiments, the present invention includes antibodies having scFv sequences fused to one or more heavy chain constant regions to form antibodies having human immunoglobulin Fc regions to produce bivalent proteins, thereby increasing the overall affinity and stability of the antibody. In addition, the Fc portion allows other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to be directly conjugated to antibodies, such as those used in antigen quantification studies, to immobilize antibodies for affinity measurements, for targeted delivery of therapeutics, for testing Fc-mediated cytotoxicity using immune effector cells, and many other applications.

[0213] The results presented herein highlight the specificity, sensitivity, and utility of the antibodies of the invention in targeting CLDN6.

[0214] The antibodies or antibody fragments of the present invention are based on single-chain antibody fragments (scFv) obtained using humanized antibody preparation technology and phage screening technology. The amino acid sequence of the scFv confers specificity to the antibody or antibody fragment for CLDN6 and forms the basis of all antibodies disclosed herein. Therefore, the scFv can be used to design a series of different "antibodies or antibody fragments", including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, multivalent antibodies, i.e., antibodies with more than one specificity for the same antigen or different antigens, for example, bispecific T cell-engaging antibodies (BiTEs), triabodies, etc. (see Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotechnology 28:355-362, 2010).

[0215] In a specific embodiment, the present invention provides full-length antibodies, whose heavy and light chains can be full-length (e.g., the antibody can include at least one, preferably two, complete heavy chains, and at least one, preferably two, complete light chains) or can include antigen-binding portions (Fab, F(ab')2, Fv or scFv). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE. The choice of antibody type will depend on the immune effector function that the designed antibody is intended to elicit. When constructing recombinant immunoglobulins, suitable amino acid sequences of the constant regions of various immunoglobulin isotypes and methods for producing a wide range of antibodies are known to those skilled in the art.

[0216] The present invention provides an antibody or antigen-binding fragment that recognizes CLDN6, comprising a heavy chain variable region comprising a HCDR1 set forth as GYYMN (SEQ ID NO: 35); and / or a HCDR2 set forth as EINPATGSTTYNQKFKA (SEQ ID NO: 36); and / or a HCDR3 set forth as RDYYX1GSX2X3YAX4DY (SEQ ID NO: 52), wherein X1 is Y or L, X2 is G or N, X3 is F or S, and X4 is M or L; and / or the antibody comprises a light chain variable region comprising a LCDR1 set forth as QASQSVSNNLN (SEQ ID NO: 38); and / or a LCDR2 set forth as GASKLED (SEQ ID NO: 39); and / or a LCDR3 set forth as X5QHRX6X7WT (SEQ ID NO: 53), wherein X5 is L or Q, X6 is Y or F, and X7 is L or M.

[0217] In certain embodiments, the antibody or antigen-binding fragment may comprise at least one CDR in the heavy chain variable region; and / or the antibody or antigen-binding fragment may comprise at least one CDR in the light chain variable region. In certain embodiments, the antibody or antigen-binding fragment may comprise one CDR, two CDRs, or three CDRs in the heavy chain variable region. In certain embodiments, the antibody or antigen-binding fragment may comprise one CDR, two CDRs, or three CDRs in the light chain variable region.

[0218] In certain embodiments, the antibody may comprise one CDR, two CDRs, or three CDRs in the heavy chain variable region, and one CDR, two CDRs, or three CDRs in the light chain variable region. For example, the antibody may comprise one CDR in the heavy chain variable region, and one CDR, two CDRs, or three CDRs in the light chain variable region; for example, the antibody may comprise two CDRs in the heavy chain variable region, and one CDR, two CDRs, or three CDRs in the light chain variable region; for example, the antibody may comprise three CDRs in the heavy chain variable region, and one CDR, two CDRs, or three CDRs in the light chain variable region; for example, the antibody may comprise three CDRs in the heavy chain variable region, and three CDRs in the light chain variable region.

[0219] Thus, the antibodies or antigen-binding fragments of the present invention comprise a heavy chain variable region, wherein the HCDR3 of the heavy chain variable region has at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR3 of SEQ ID NO: 41; and / or

[0220] The antibodies or antigen-binding fragments of the present invention comprise a light chain variable region, wherein the LCDR3 of the light chain variable region has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the LCDR3 of SEQ ID NO: 42, 43 or 44.

[0221] In certain embodiments, the antibody may comprise the heavy chain variable region and / or the light chain variable region. For example, the heavy chain variable region may comprise a heavy chain CDR1 (HCDR1), wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), wherein the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 36. For example, the heavy chain variable region may comprise a heavy chain CDR3 (HCDR3), wherein the HCDR3 may comprise the amino acid sequence set forth in either SEQ ID NO: 37 or 41. For example, the heavy chain variable region may comprise HCDR1 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 35, and the HCDR3 may comprise the amino acid sequence set forth in either SEQ ID NO: 37 or 41. For example, the heavy chain variable region may comprise HCDR1 and HCDR2, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 35, and the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 36. For example, the heavy chain variable region may comprise HCDR2 and HCDR3, wherein the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 36, and the HCDR3 may comprise the amino acid sequence set forth in either SEQ ID NO: 37 or 41. For example, the heavy chain variable region may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 35, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 36, and the HCDR3 may comprise the amino acid sequence set forth in either SEQ ID NO: 37 or 41. For example, the light chain variable region may comprise a light chain CDR1 (LCDR1), wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 38. For example, the light chain variable region may comprise a light chain CDR2 (LCDR2), wherein the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 39. For example, the light chain variable region may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 40, 42, 43, or 44. For example, the light chain variable region may comprise LCDR1 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 38, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 40, 42, 43, or 44.For example, the light chain variable region may comprise LCDR1 and LCDR2, wherein LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 38, and LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 39. For example, the light chain variable region may comprise LCDR2 and LCDR3, wherein LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 39, and LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 40, 42, 43, or 44. For example, the light chain variable region may comprise LCDR1, LCDR2, and LCDR3, wherein LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 38, wherein LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 39, and wherein LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 40, 42, 43, or 44.

[0222] In certain embodiments, the antibody may comprise the heavy chain variable region and the light chain variable region. For example, the antibody comprises the HCDR1 of SEQ ID NO: 35, the HCDR2 of SEQ ID NO: 36, the HCDR3 of SEQ ID NO: 37, the LCDR1 of SEQ ID NO: 38, the LCDR2 of SEQ ID NO: 39, and the LCDR3 of SEQ ID NO: 40; or the antibody comprises the HCDR1 of SEQ ID NO: 35, the HCDR2 of SEQ ID NO: 36, the HCDR3 of SEQ ID NO: 41, the LCDR1 of SEQ ID NO: 38, the LCDR2 of SEQ ID NO: 39, and the LCDR3 of SEQ ID NO: 40; or the antibody comprises the HCDR1 of SEQ ID NO: 35, the HCDR2 of SEQ ID NO: 36, the HCDR3 of SEQ ID NO: 37, the LCDR1 of SEQ ID NO: 38, the LCDR2 of SEQ ID NO: 39, and the LCDR3 of SEQ ID NO: 42; or the antibody comprises the HCDR1 of SEQ ID NO: 35, the HCDR2 of SEQ ID NO: 36, the HCDR3 of SEQ ID NO: 41, the LCDR1 of SEQ ID NO: 38, the LCDR2 of SEQ ID NO: 39, and the LCDR3 of SEQ ID NO: 42. : The antibody comprises the HCDR1 of SEQ ID NO:35, the HCDR2 of SEQ ID NO:36, the HCDR3 of SEQ ID NO:41, the LCDR1 of SEQ ID NO:38, the LCDR2 of SEQ ID NO:39, and the LCDR3 of SEQ ID NO:42; or the antibody comprises the HCDR1 of SEQ ID NO:35, the HCDR2 of SEQ ID NO:36, the HCDR3 of SEQ ID NO:41, the LCDR1 of SEQ ID NO:38, the LCDR2 of SEQ ID NO:39, and the LCDR3 of SEQ ID NO:43; or the antibody comprises the HCDR1 of SEQ ID NO:35, the HCDR2 of SEQ ID NO:36, the HCDR3 of SEQ ID NO:41, the LCDR1 of SEQ ID NO:38, the LCDR2 of SEQ ID NO:39, and the LCDR3 of SEQ ID NO:43. LCDR2 shown in NO: 39, LCDR3 shown in SEQ ID NO: 44.In a specific embodiment, the HCDR2 of the antibody or antigen-binding fragment of the invention has the amino acid sequence shown in SEQ ID NO:36.

[0223] The CDRs can be identified using a numbering system selected from the group consisting of Kabat, Chothia, IMGT, Gelfand, Aho, and AbM. The antibodies recognizing CLDN6 provided herein can comprise a CDR sequence identified using any of the above numbering systems, or a combination thereof. The CDRs in the heavy and / or light chains comprising the antibody do not necessarily have to be identified using the same numbering system. For example, one or some CDRs in the antibody can be identified using the Kabat system, while the other CDRs can be identified using any one or a combination of the above numbering systems. In certain embodiments, the CDRs of the antibody can be identified using a single numbering system, such as the Kabat numbering system; the Chothia numbering system; the IMGT numbering system; the Gelfand numbering system; the Aho numbering system; or the AbM numbering system.

[0224] In certain embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1 or 5 or a variant thereof.

[0225] In certain embodiments, the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 3, 7, 9 or 11, or a variant thereof.

[0226] In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 1 or 5, or a variant thereof, and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 7, 9, or 11, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or a variant thereof, and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 7, 9, or 11, or a variant thereof; for example, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5, or a variant thereof, and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 7, 9, or 11, or a variant thereof.

[0227] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9. For example, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11.

[0228] In certain embodiments, the present invention provides an antibody that recognizes CLDN6, comprising a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 1 or 5, or a variant thereof.

[0229] In certain embodiments, the present invention provides an antibody that recognizes CLDN6, comprising a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3, 7, 9, or 11, or a variant thereof.

[0230] In certain embodiments, the present invention provides an antibody that recognizes CLDN6, comprising the above-mentioned heavy chain variable region and light chain variable region.

[0231] Given that these heavy chain variable region and light chain variable region sequences can each bind to CLDN6, heavy chain and light chain variable region sequences can be "mixed and matched" to generate anti-CLDN6 binding molecules of the present invention.

[0232] In certain embodiments, the present invention provides variants of antibodies or variants of fragments thereof that bind to CLDN6. Such variants comprise a heavy chain variable region whose HCDR1, HCDR2, and HCDR3 have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 35, 36, and 37. Such variants may also comprise a light chain variable region whose LCDR1, LCDR2, and LCDR3 have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 38, 39, and 40.

[0233] Furthermore, variants of the antibodies of the present invention or variants of their fragments comprise heavy and / or light chain variable regions that are at least 80% identical to the heavy or light chain variable region sequences of the antibodies of the present invention. Preferably, the amino acid sequence identity of the heavy and / or light chain variable regions is at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%, particularly 96%, more particularly 97%, even more particularly 98%, most particularly 99%, including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. Variants can be obtained by methods such as yeast library screening, phage library screening, and point mutation using the antibodies described herein as parent antibodies.

[0234] In certain embodiments, the present invention provides an antibody that specifically binds to CLDN6, wherein the antibody is a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, dAb fragment or multifunctional antibody.

[0235] In certain embodiments, the antibody is a hybridoma antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0236] In certain embodiments, the antibody is a monoclonal antibody.

[0237] Antibody assay

[0238] The anti-CLDN6 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art, including, for example, ELISA, biacore, Western blot, and flow cytometric analysis. Suitable assays are described in detail in the Examples.

[0239] The term "affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including assaying antibody affinity using Biacore. For example, herein, the "affinity" of an antibody for CLDN6 is expressed as the antibody's KD. The KD of an antibody refers to the equilibrium dissociation constant for an antibody-antigen interaction. The larger the KD value for an antibody binding to its antigen, the weaker its binding affinity for that particular antigen. For example, herein, the "affinity" of an antibody for a CLDN antigen is expressed as the antibody's EC50.

[0240] The term "EC50" refers to the concentration that can cause 50% of the maximal effect.

[0241] Various methods for determining antibody binding affinity are known in the art. In one embodiment, the method for determining binding affinity utilizes surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that can be used to analyze real-time biospecific interactions by, for example, detecting changes in protein concentration in a biochip matrix using a Biacore system.

[0242] "No significant binding" is characterized by reduced affinity for the antibody and antigen, a faster off-rate, and / or a lower binding signal, particularly a faster off-rate and / or a lower binding signal. In one example, "no significant binding" refers to a level of binding of the antibody to the antigenic protein or polypeptide that is not statistically significantly higher than background; background is the binding level detected in the absence of the antibody or in the presence of a negative control protein (e.g., an isotype control antibody). For example, the binding level of the antibody to the antigenic protein or polypeptide is detected using a biosensor assay (e.g., Biacore), or the binding level of the antibody to an antigenic protein or polypeptide expressed on the cell surface. For example, PBS is used instead of the antibody to detect the antigen binding level. In one example, the antibody of the present invention binds to CLDN6. In one example, the antibody of the present invention does not significantly bind to CLDN4 or CLDN9.

[0243] antigen

[0244] The term "antigen" or "Ag" refers to a substance that is recognized and specifically bound by an antigen binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. "Antigen" can also refer to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In addition, antigens can be derived from recombinant DNA or genomic DNA. Any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that can elicit an immune translation can encode an antigen. Those skilled in the art understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. Including but not limited to using partial nucleotide sequences of more than one gene, these nucleotide sequences can be arranged in various combinations to encode polypeptides that can elicit a desired immune response. In addition, antigens do not need to be encoded by a gene. Antigens can be produced synthetically, or can be derived from a biological sample, or can be macromolecules other than polypeptides. The biological sample may include, but is not limited to, tissue samples, tumor samples, cells or fluids, and other biological components.

[0245] The term "epitope" refers to an antigen or portion of an antigen that is recognized by an antibody, B cell, T cell, or engineered cell. For example, an epitope can be a tumor epitope or pathogen epitope recognized by an antibody; an antibody can recognize multiple epitopes within an antigen. Epitopes can also be mutated.

[0246] The term "antigenic determinant," also known as "antigenic epitope," "epitope," or "antigenic determinant," includes any determinant or region capable of being bound by an antibody. An antigenic epitope is a region of an antigen that is bound by an antibody targeting the antigen, including specific amino acids that are in direct contact with the antibody. For example, an antigenic epitope can consist of a continuous sequence of the CLDN6 protein sequence or a discontinuous three-dimensional structure of the CLDN6 protein sequence. For example, the antigen used herein is human CLDN6.

[0247] Immunoconjugates

[0248] The present invention also provides immunoconjugates comprising the antibodies described herein and one or more functional molecules linked thereto. The antibodies provided herein have been described above, and the conjugates provided herein encompass all of their technical solutions. The antibodies and functional molecules can be covalently linked, coupled, attached, cross-linked, or otherwise formed into immunoconjugates.

[0249] "Connection" or "fusion" are used interchangeably herein. It generally refers to the joining of two or more chemical elements or components by any means including chemical conjugation or recombinant methods. "In-frame fusion" refers to the joining of two or more ORFs to form a longer ORF that is continuous in a manner that maintains the correct reading frame of the original open reading frame (ORF). Thus, the resulting recombinant fusion protein is a single protein containing two or more fragments that correspond to the polypeptides encoded by the original ORFs (these fragments are generally not so connected in nature). Although the reading frame is therefore continuous throughout the fusion fragment, these fragments can be physically or spatially separated by, for example, in-frame connecting sequences (e.g., "flexons").

[0250] The functional molecule is selected from the group consisting of: a molecule targeting a tumor surface marker, a molecule that inhibits tumors, a molecule targeting a surface marker of an immune cell, or a detectable marker. In some embodiments, the molecule targeting a tumor surface marker can be an antibody or ligand that binds to a tumor surface marker and can act synergistically with the antibodies of the present invention to more precisely target tumor cells.

[0251] In some embodiments, the tumor-inhibiting molecule comprises a cytotoxic agent, including but not limited to radioisotopes, chemotherapeutic agents, growth inhibitors, enzymes and fragments thereof, antibiotics, and toxins. In some embodiments, the tumor-inhibiting molecule is an anti-tumor cytokine, including but not limited to IL-2, IL-7, IL-12, IL-15, type I interferon, and TNF-α.

[0252] In some embodiments, the molecule of the surface marker targeting immune cells is an antibody or ligand that binds to the surface marker of immune cells and can recognize immune cells. It carries the antibody of the present invention to the immune cells, and at the same time, the antibody of the present invention can target immune cells to tumor cells, thereby triggering immune cells to specifically kill tumor cells. The surface marker of the immune cells is selected from CD3, CD6, CD28, NKG2A, NKG2C, NKG2D, CD94, CD159a, CD159c, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, and the immune cells are selected from T cells, NK cells, and NKT cells. In one embodiment, the molecule of the surface marker targeting immune cells is an antibody that binds to the surface marker of T cells, which forms a bifunctional antibody with the antibody of the present invention that involves T cells.

[0253] In some embodiments, the detectable marker includes, but is not limited to, a fluorescent marker, a chromogenic marker, for example, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron-emitting metal, and a non-radioactive paramagnetic metal ion. The marker used to label the antibody for detection and / or analysis and / or diagnosis purposes depends on the specific detection / analysis / diagnosis technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc.

[0254] The immunoconjugates include antibody-drug conjugates (ADCs) in which an antibody is conjugated to one or more drugs, including but not limited to maytansinoids, auristatins such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF), dolastatin, calicheamicin or its derivatives, anthracyclines (e.g., daunomycin or doxorubicin), methotrexate, vindesine, taxanes (e.g., docetaxel, paclitaxel, leronate, tesetaxel, and oxtataxel), trichothecenes, and CC1065. Another aspect of the present invention provides nucleic acid molecules encoding the immunoconjugates of the present invention. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0255] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression. The host cells can be prokaryotes, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

[0256] Chimeric receptors

[0257] The present invention also provides a chimeric receptor, which generally refers to the expression product of a fusion molecule formed by connecting cDNA corresponding to DNA fragments or proteins from different sources using genetic recombination technology, and may include an extracellular domain, a transmembrane domain, and an intracellular domain. Wherein, the extracellular domain comprises an antigen binding domain. In one embodiment, the extracellular domain comprises an antibody provided by the present invention, and the antibody has been described in detail above, and the chimeric receptor provided by the present invention includes all its technical solutions. The chimeric receptor includes but is not limited to: a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), and a synthetic polypeptide receptor (synNotch).

[0258] In one embodiment, the cells expressing CAR can be targeted to bind to the antigen of interest by engineering an antigen binding domain that specifically binds to the antigen of interest into the CAR.

[0259] In some embodiments, the chimeric receptor of the present invention is a chimeric antigen receptor (CAR). The chimeric antigen receptor generally includes an extracellular antigen binding region or an antibody. In some embodiments, the extracellular antigen binding region can be completely human. In other cases, the extracellular antigen binding region can be humanized. In other cases, the extracellular antigen binding region can be mouse-derived, or the chimera in the extracellular antigen binding region is composed of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region can be non-human.

[0260] In certain embodiments, the chimeric antigen receptor can also be designed to include a variety of antigen binding regions, including single-chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fab) selected from libraries, single domain fragments, or natural ligands that bind to their cognate receptors. In some embodiments, the extracellular antigen binding region may include scFv, Fab, or natural ligands, and any derivatives thereof. The extracellular antigen binding region may refer to a molecule other than a complete antibody, which may include a portion of a complete antibody and may bind to the antigen bound by the complete antibody. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies, linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed by antibody fragments. The extracellular antigen binding region, such as scFv, Fab, or natural ligand, may be part of a CAR that determines antigen specificity. The extracellular antigen binding region may bind to any complementary target. The extracellular antigen binding region may be derived from an antibody with a known variable region sequence. The extracellular antigen binding region may be obtained from an antibody sequence obtained from an available mouse hybridoma. Alternatively, extracellular antigen-binding regions can be obtained from whole exosome cleavage sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TILs).

[0261] In some embodiments, the binding specificity of the extracellular antigen binding region of CAR can be determined by complementary determining region or CDR, such as light chain CDR and / or heavy chain CDR. In some embodiments, the binding specificity of the extracellular antigen binding region of CAR can be determined by light chain variable region and / or heavy chain variable region.

[0262] In some embodiments, the extracellular region of CAR includes a hinge or a spacer, and the hinge and the spacer can be used interchangeably. The hinge can be considered as a part of the CAR for providing flexibility to the extracellular antigen binding region. In some embodiments, the hinge can be used to detect CAR on the cell surface of the cell, particularly when the antibody detecting the extracellular antigen binding region does not work or is not available. In some embodiments, the hinge may not belong to an immunoglobulin, but to another molecule, such as the natural hinge of the CD8α molecule. The CD8α hinge may contain cysteine ​​and proline residues known to play a role in the interaction between CD8 auxiliary receptors and MHC molecules. The hinge can be adjusted according to the extracellular antigen binding region used. The hinge can be of any length. In some embodiments, the hinge can be the natural hinge region of IgG1, IgG4, or a mutated hinge region thereof, or other structural parts outside the hinge region. For example, the hinge can comprise an amino acid sequence as shown in SEQ ID NO: 21, 27, 28, 29 or 30.

[0263] The transmembrane domain (or structural region) of the CAR can anchor the CAR to the plasma membrane of the cell. The native transmembrane portion of CD28 can be used in the CAR. In other cases, the native transmembrane portion of CD8α can also be used in the CAR. "CD8" can be a protein that is at least 85, 90, 95, 96, 97, 98, 99 or 100% identical to NCBI Reference No.: NP_001759 or a fragment thereof having stimulatory activity. A "CD8 nucleic acid molecule" can be a polynucleotide encoding a CD8 polypeptide. In some cases, the transmembrane region can be the native transmembrane portion of CD28. "CD28" can refer to a protein that is at least 85, 90, 95, 96, 97, 98, 99 or 100% identical to NCBI Reference No.: NP_006130 or a fragment thereof having stimulatory activity. A "CD28 nucleic acid molecule" can be a polynucleotide encoding a CD28 polypeptide. In some embodiments, the transmembrane portion can include a CD8α region. For example, the transmembrane domain can include an amino acid sequence as shown in SEQ ID NO: 22 or 25.

[0264] The (thin) intracellular signaling region of CAR can be responsible for activating at least one of the effector functions of the immune response cells comprising the CAR. CAR can induce the effector functions of T cells, for example, the effector functions are cytolytic activity or auxiliary activity, including the secretion of cytokines, such as IL-2, TNF-α, γ-IFN, etc. Therefore, the term intracellular signaling region refers to a protein portion that transduces effector function signals and guides cells to perform specific functions. Although the entire intracellular signaling region can generally be used, in many cases, it is not necessary to use the entire chain of the signaling domain. In some embodiments, a truncated portion of the intracellular signaling region is used. In some embodiments, the term intracellular signaling region is therefore intended to include any truncated portion of the intracellular signaling region sufficient to transduce effector function signals.

[0265] Preferred examples of signaling domains (or structural regions) used in CARs may include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that cooperate to initiate signal transduction after target-receptor binding, as well as any derivative or variant sequences thereof and any synthetic sequences of these sequences having the same functionality.

[0266] An example of a T cell signaling domain containing one or more ITAM motifs is the CD3 ζ domain, also known as the T cell receptor CD3 ζ chain or CD247. This domain is part of the T cell receptor-CD3 complex and plays an important role in combining antigen recognition of several intracellular signal transduction pathways with primary effector activation of T cells. As used herein, CD3 ζ primarily refers to human CD3 ζ and its isoforms, as known from Swissprot entry P20963, including proteins with substantially the same sequence. As part of a chimeric antigen receptor, a full T cell receptor CD3 ζ chain is not required, and any derivative thereof comprising the signaling domain of the T cell receptor CD3 ζ chain is suitable, including any functional equivalent thereof. For example, the signaling domain of the CD3 ζ chain may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0267] In certain embodiments, the intracellular signaling domain (or structural region) of the CAR can be selected from any one of the costimulatory domains in Table 2. In some embodiments, the domain can be modified so that the identity with the reference domain can be about 50% to about 100%. Any one of the domains in Table 1 can be modified so that the modified form can contain about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or up to about 100% identity.

[0268] In certain embodiments, the intracellular signaling region of CAR may further include one or more costimulatory domains. The intracellular signaling region may include a single costimulatory domain, such as a ζ chain (first generation CAR) or thereof and CD28 or 4-1BB (second generation CAR). In other examples, the intracellular signaling region may include two costimulatory domains, such as CD28 / OX40 or CD28 / 4-1BB (third generation). For example, the costimulatory domain of the CD28 may include an amino acid sequence as shown in SEQ ID NO:23.

[0269] In certain embodiments, together with intracellular signaling domains such as CD8, these co-stimulatory domains can produce downstream activation of kinase pathways, thereby supporting gene transcription and functional cellular responses. The co-stimulatory domains of CARs can activate proximal signaling proteins associated with the CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB / OX40 (TNF-receptor-associated factor adaptor protein) pathways as well as MAPK and Akt activation.

[0270] In some cases, the signal produced by CAR may be combined with auxiliary or costimulatory signals.For costimulatory signal domains, chimeric antigen receptor-like complexes can be designed to include several possible costimulatory signal domains. As is well known in the art, in immature T cells, the combination of T cell receptors alone is not enough to induce the complete activation of T cells as cytotoxic T cells. Complete productive T cell activation requires a second costimulatory signal. It has been reported that several receptors for costimulation are provided for T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88 and 4-1BB. The signal transduction pathways used by these costimulatory molecules can all synergize with the main T cell receptor activation signal. The signal provided by these costimulatory signal transduction regions can synergize with the main effector activation signal derived from one or more ITAM motifs (such as CD3zeta signal transduction domains), and the requirements for T cell activation can be completed.

[0271] In some embodiments, the addition of a co-stimulatory domain to the chimeric antigen receptor-like complex can enhance the efficacy and durability of the engineered cells. In other embodiments, the T cell signaling domain and the co-stimulatory domain are fused to each other to form a signaling region.

[0272] Table 2. Costimulatory domains

[0273] The present invention provides cells (e.g., T cells) that are engineered to express CAR, wherein the cells expressing CAR (e.g., CAR-T cells) exhibit anti-tumor properties. On the one hand, cells are transduced with CAR, and CAR is expressed on the cell surface. In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding CAR. In some embodiments, the viral vector is a reverse transcription vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, cells can stably express CAR. In some embodiments, cells (e.g., T cells) are transfected with nucleic acids encoding CAR, such as mRNA, cDNA, or DNA.

[0274] In certain embodiments, the CAR comprises an antigen-binding domain that targets CLDN6. In certain embodiments, the CLDN6-binding portion of the CAR is a scFv, an antibody fragment that is functional and retains comparable binding affinity to the IgG antibody from which it is derived, e.g., it binds to the antigen with comparable efficacy; thereby providing a biochemical response, such as activating an immune response, inhibiting signaling initiation from its target antigen, inhibiting kinase activity, etc. Exemplary, the anti-CLDN6 antigen-binding domain of the CAR comprises an scFv sequence as set forth in any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, or 19.

[0275] In certain embodiments, the anti-CLDN6 antigen binding domain of the CAR is a humanized antibody or a fragment thereof. In certain embodiments, the anti-CLDN6 antigen binding domain of the CAR is a fully human antibody or a fragment thereof. In certain embodiments, the anti-CLDN6 antigen binding domain of the CAR is a murine antibody or a fragment thereof.

[0276] In certain embodiments, the CAR of the present invention combines the antigen binding domain of a specific antibody with an intracellular signaling molecule. For example, intracellular signaling molecules include, but are not limited to, CD3ζ chain, 4-1BB and CD28 signaling modules and combinations thereof.

[0277] In certain embodiments, CLDN6-CAR includes at least one intracellular signaling domain selected from CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3 ζ signaling domain, and any combination thereof. On the one hand, CLDN6-CAR includes at least one intracellular signaling domain from one or more non-CD137 (4-1BB) or CD28 costimulatory molecules.

[0278] As an example, the sequence of CLDN6-CAR comprises: an extracellular domain as shown in SEQ ID NO: 13, a hinge domain as shown in SEQ ID NO: 21, a transmembrane domain as shown in SEQ ID NO: 22, a costimulatory signal domain as shown in SEQ ID NO: 23, and a primary signal domain as shown in SEQ ID NO: 24 (H1-28Z); or an extracellular domain as shown in SEQ ID NO: 17, a hinge domain as shown in SEQ ID NO: 21, a transmembrane domain as shown in SEQ ID NO: 22, a costimulatory signal domain as shown in SEQ ID NO: 23, and a primary signal domain as shown in SEQ ID NO: 24 (P4-28Z).

[0279] Exemplarily, the amino acid sequence of the chimeric antigen receptor comprises a sequence as shown in any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, or 19 linked to a sequence as shown in any one of SEQ ID NOs: 45, 46, or 47, respectively. For example, the amino acid sequence of the chimeric antigen receptor comprises: the sequence set forth in SEQ ID NO: 13 linked to the sequence set forth in SEQ ID NO: 45; or the sequence set forth in SEQ ID NO: 13 linked to the sequence set forth in SEQ ID NO: 46; or the sequence set forth in SEQ ID NO: 13 linked to the sequence set forth in SEQ ID NO: 47; or the sequence set forth in SEQ ID NO: 14 linked to the sequence set forth in SEQ ID NO: 45; or the sequence set forth in SEQ ID NO: 14 linked to the sequence set forth in SEQ ID NO: 46; or the sequence set forth in SEQ ID NO: 14 linked to the sequence set forth in SEQ ID NO: 47; or the sequence set forth in SEQ ID NO: 15 linked to the sequence set forth in SEQ ID NO: 45; or the sequence set forth in SEQ ID NO: 15 linked to the sequence set forth in SEQ ID NO: 46; or the sequence set forth in SEQ ID NO: 15 linked to the sequence set forth in SEQ ID NO: 47; or the sequence set forth in SEQ ID NO: 16 linked to the sequence set forth in SEQ ID NO: 45; or the sequence set forth in SEQ ID NO: 16 linked to the sequence set forth in SEQ ID NO: 46; or the sequence set forth in SEQ ID NO: 16 linked to the sequence set forth in SEQ ID NO: 47. or the sequence set forth in SEQ ID NO:18 is linked to the sequence set forth in SEQ ID NO:45; or the sequence set forth in SEQ ID NO:19 is linked to the sequence set forth in SEQ ID NO:46; or the sequence set forth in SEQ ID NO:19 is linked to the sequence set forth in SEQ ID NO:47; or the sequence set forth in SEQ ID NO:19 is linked to the sequence set forth in SEQ ID NO:45; or the sequence set forth in SEQ ID NO:19 is linked to the sequence set forth in SEQ ID NO:46; or the sequence set forth in SEQ ID NO:19 is linked to the sequence set forth in SEQ ID NO:47.

[0280] Those skilled in the art can replace the transmembrane domain and intracellular domain of the chimeric antigen receptor with conventional transmembrane domains and intracellular domains, and all of them fall within the scope of protection of this application.

[0281] Nucleic acids, vectors, viruses, host cells

[0282] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, oligonucleotides, fragments generated by PCR, fragments generated by any one of connection, chain scission, endonuclease action and exonuclease action. Nucleic acid sequence comprises the natural nucleotide sequence, and also implicitly comprises conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, straight homologues, SNPs and complementary sequences. The modified nucleotides can have changes in sugar moieties and / or pyrimidine or purine base moieties. For example, sugar modifications include replacing one or more hydroxyl groups with halogens, alkyl groups, amines and azido groups, or sugars can be functionalized with ethers or esters.

[0283] The term "codon" refers to the three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by the ribosome into an amino acid residue.

[0284] The term "codon optimization" refers to the fact that the frequency of occurrence of synonymous codons (codons encoding the same amino acid) in coding DNA is biased in different species, and codons that are usually rare in highly expressed genes of a given species are replaced by common codons of highly expressed genes in this species. Codon degeneracy allows multiple nucleotide sequences to encode the same polypeptide. Degenerate codon replacement can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues.

[0285] The term "coding" refers to the intrinsic properties of specific nucleotide sequences in polynucleotides such as genes, DNA or mRNA, i.e., the sequence can serve as a template, to synthesize other polymers and macromolecules with the nucleotide sequence (such as rRNA, tRNA and mRNA) or the amino acid sequence of regulation and the biological properties resulting therefrom in biological processes. Therefore, when the transcription and translation corresponding to the mRNA of a gene in a cell or other biological system cause protein production, the gene, cDNA or RNA encodes the protein. Coding strand (its nucleotide sequence is identical to the mRNA sequence, is usually provided in the sequence table) and non-coding strand (transcribed as a template for a gene or cDNA) can both be referred to as coded protein or other products of the gene or cDNA.

[0286] The terms "nucleic acid molecule encoding," "coding DNA sequence," and "coding DNA" refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence. A "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. As used herein, the term "sequence" when referring to a nucleotide sequence includes DNA or RNA and can be single-stranded or double-stranded.

[0287] The term "target sequence" refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of the CRISPR complex. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

[0288] The term "identity" or "homology" refers to the subunit sequence identity between two nucleic acid molecules, or between two polypeptide molecules. When a subunit position is occupied by the same monomeric subunit in both molecules, for example, when two DNA molecules are both occupied by adenosine at a position, then they are homologous or identical at that position. The homology between two sequences is the number of matching or homologous positions divided by the total number of positions in the sequences, and the result is multiplied by 100 to produce the percentage of sequence identity. For example, when 80% of the positions in two sequences (e.g., 8 bp in a nucleic acid molecule 10 bp long) are homologous, then the two sequences are 80% homologous.

[0289] The term "transfection" or "transduction" or "transformation" refers to the introduction of exogenous nucleic acid into a host cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0290] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as plasmids, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). The plasmid vector may also comprise a selection marker that provides for identification and / or screening of cells that receive the vector.

[0291] The term "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. Non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, etc. Vectors can be associated with or combined with any cell penetration technology (such as sonoporation or electroporation or their derivatives). The choice of vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transfected with the vector. Each vector contains different components depending on the function of the vector (amplification and / or expression of heterologous polynucleotides) and the compatibility of the vector with the specific host cell in which it is located. Vector components generally include but are not limited to: an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence. Physical methods for introducing vectors into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Chemical methods for introducing vectors into cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes).

[0292] The term "slow virus" refers to the genus of the Retroviridae family. Retroviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most effective methods for gene delivery vectors. HIV, SIV, and FIV are all examples of slow viruses. Vectors derived from slow viruses provide a means of achieving significant levels of gene transfer in vivo. "Integrating slow virus vectors (LV)" refer to these vectors as non-limiting examples, which can integrate into the genome of target cells. "Non-integrating slow virus vectors (NILV)" relatively refer to effective gene delivery vectors that are not integrated into the genome of target cells by the action of viral integrase.

[0293] The term "endogenous" refers to any substance that originates from the organism, cell, tissue, or system itself, or any substance produced in the organism, cell, tissue, or system, such as a nucleic acid molecule or polypeptide.

[0294] The term "exogenous" refers to any substance, such as a nucleic acid molecule, polypeptide, cell, that is introduced into an organism, cell, tissue, or system, or that is produced outside of the organism, cell, tissue, or system.

[0295] The term "exogenous protein" may be a protein that recognizes a target antigen and is introduced into cells exogenously, such as an exogenous receptor (ie, the aforementioned "chimeric receptor" herein).

[0296] The term "host" refers to a recipient of a transplant, and in some embodiments, may be an individual, such as a human, into whom exogenous cells are implanted.

[0297] The term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the nucleic acid or peptide is "isolated" if it is partially or completely separated from its coexisting substances in its natural state. An isolated nucleic acid or protein can exist in a substantially pure form or can exist in a non-natural environment, such as a host cell. An "isolated" material can also be provided by artificial assembly methods, such as chemical synthesis or recombinant expression.

[0298] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0299] The term "promoter" is a DNA sequence that RNA polymerase recognizes, binds to, and begins to transcribe. It is an important component of a gene, and its main function is to regulate the start time and expression level of gene transcription. In some embodiments, the nucleic acid encoding CAR is operably connected to a promoter.

[0300] The present invention provides isolated nucleic acids encoding antibodies or fragments thereof that recognize CLDN6, vectors, and host cells comprising the nucleic acids or vectors. The nucleic acids can be in intact cells, in cell lysates, or in partially purified or substantially purified form.

[0301] The nucleic acids of the present invention can be obtained using standard molecular biology techniques, for example, cDNA encoding the light and heavy chains of an antibody or encoding the VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are generally known in the art and can vary depending on the host cell used.

[0302] Preferably, the nucleic acid molecule of the present invention is selected from SEQ ID NO: 2 or 6 encoding the heavy chain variable region, and / or selected from SEQ ID NO: 4, 8, 10 or 12 encoding the light chain variable region. Preferably, the nucleic acid molecule comprises a heavy chain variable region sequence of SEQ ID NO: 2, and a light chain variable region sequence of SEQ ID NO: 4; or a heavy chain variable region sequence of SEQ ID NO: 2, and a light chain variable region sequence of SEQ ID NO: 8; or a heavy chain variable region sequence of SEQ ID NO: 2, and a light chain variable region sequence of SEQ ID NO: 10; or a heavy chain variable region sequence of SEQ ID NO: 6, and a light chain variable region sequence of SEQ ID NO: 4; or a heavy chain variable region sequence of SEQ ID NO: 6, and a light chain variable region sequence of SEQ ID NO: 8; or a heavy chain variable region sequence of SEQ ID NO: 6, and a light chain variable region sequence of SEQ ID NO: 10; or a heavy chain variable region sequence of SEQ ID NO: 2, and a light chain variable region sequence of SEQ ID NO: 12.

[0303] In one embodiment, one or more vectors (eg, expression vectors) comprising the above-described nucleic acids are provided.

[0304] The term "cell" refers to a cell of human or non-human animal origin.

[0305] The term "host cell" refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells," which include the primary cell transformed and the offspring derived therefrom (regardless of the number of passages). The nucleic acid content of the offspring may not be identical to that of the parent cell and may contain mutations. Mutant offspring having the same function or biological activity as that screened or selected for in the original transformed cell are included herein.

[0306] The term "positive" for a particular marker refers to the detectable presence of a particular marker (typically a surface marker) on or in a cell. When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is significantly higher than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, and / or is substantially similar to the level of cells known to be positive for the marker, and / or is significantly higher than the level of cells known to be negative for the marker.

[0307] The term "negative" for a particular marker refers to the substantial detectable absence of a particular marker (typically a surface marker) on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is significantly higher than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, and / or significantly lower than the level in cells known to be positive for the marker, and / or substantially similar to the level in cells known to be negative for the marker.

[0308] The term "CLDN6-positive host cells" refers to host cells that express CLDN6 on the cell surface, which can be detected by, for example, flow cytometry using antibodies that specifically recognize epitopes on CLDN6.

[0309] In some embodiments, the host cell is an immune cell.

[0310] The term "immune cell" refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, neutrophils, eosinophils, and / or basophils. In some embodiments, the immune cells are T cells, NK cells, and NKT cells. In some embodiments, the T cells can be autologous T cells, xenogeneic T cells, or allogeneic T cells. In some embodiments, the NK cells can be allogeneic NK cells. "Immune effector function or immune effector response" refers to immune cells, such as functions or reactions that enhance or promote an immune attack on target cells. For example, an immune function or response refers to a property of T cells or NK cells that promotes the killing of target cells or inhibits growth or proliferation.

[0311] The term "artificially modified cells with immune cell function" refers to cells or cell lines that lack immune function but have been artificially modified or stimulated to acquire immune cell function. For example, 293T cells have been artificially modified to acquire immune cell function; for example, stem cells have been induced to differentiate into immune cells in vitro. Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0312] In some cases, the cells are generally primary cells, such as those isolated directly from an individual and / or isolated and frozen from an individual. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as a full T cell population, CD4+ cells, CD8+ cells, and subsets thereof, such as those defined by function, activation state, maturation, differentiation potential, expansion, recycling, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion, and / or degree of differentiation. For the subject to be treated, the cells can be allogeneic and / or autologous.

[0313] In some cases, "T cells" can be pluripotent stem cells from bone marrow that differentiate and mature into mature T cells with immune activity in the thymus. In some cases, "T cells" can be a cell population with specific phenotypic characteristics, or a mixed cell population with different phenotypic characteristics, such as "T cells" can be cells comprising at least one T cell subset: memory stem cell-like T cells (stem cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs), effector memory T cells (Tem), naive T (TN) cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), cytotoxic T cells, and / or mucosal-associated non-variant T (MAIT) cells. In some cases, "T cells" can be T cells of a certain specific subtype, such as αβT cells and γδT cells.

[0314] T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, T cells can be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove plasma molecules and the cells placed in a suitable buffer or culture medium for subsequent processing steps. In one embodiment, T cells can be obtained from healthy donors or from cells derived from patients diagnosed with tumors. CAR-T cells are obtained by conventional methods for preparing CAR-T cells in the art, using T cells obtained by culturing PBMC cells after activation with magnetic beads containing anti-CD3 and CD28 antibodies, and then infected with lentivirus.

[0315] The term "peripheral blood mononuclear cell" (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc.

[0316] The terms "activation" and "activation" are used interchangeably and may refer to the process by which a cell transitions from a quiescent state to an active state. This process may include response to antigens, migration, and / or phenotypic or genetic changes in functional activity. For example, the term "activation" may refer to the gradual activation of NK cells or T cells.

[0317] The term "T cell activation" or "T cell activation" refers to a state of T cells that are sufficiently stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.

[0318] "Genetic engineering" generally involves introducing a nucleic acid encoding the recombinant or engineered moiety into cells, for example, by retroviral or lentiviral transduction, transfection, or transformation, or by transposons, electroporation. In some embodiments, this is accomplished by first stimulating the cells, for example, by combining them with a stimulator that induces a response, such as proliferation, survival, and / or activation, as detected, for example, by expression of cytokines or activation markers, and then transducing the activated cells and expanding them in culture to a sufficient number for clinical use.

[0319] In another embodiment, a host cell comprising a nucleic acid as described herein is provided. The host cell comprises (e.g., is transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. In one embodiment, the host cell is eukaryotic, e.g., a 293T cell.

[0320] In another embodiment, the host cell expresses the chimeric receptor described herein.

[0321] In another embodiment, the host cell comprises a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DN T cell, a regulatory T cell, a NK92 cell, and / or a stem cell-derived immune cell.

[0322] In another embodiment, the T cells are derived from natural T cells and / or T cells induced by pluripotent stem cells; preferably, the T cells are autologous / allogeneic T cells; preferably, the T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.

[0323] In another embodiment, the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), γδ T cells, or a combination thereof.

[0324] In another embodiment, the host cell binds to cells expressing CLDN6 and does not significantly bind to cells expressing CLDN4, CLDN9, or a combination thereof.

[0325] In another embodiment, the host cell further carries an exogenous cytokine coding sequence.

[0326] In another embodiment, the host cell may further express another chimeric receptor in addition to the above-mentioned antigen-binding receptor.

[0327] In another embodiment, the host cell may also express a chemokine receptor.

[0328] In another embodiment, the host cell may also express a safety switch.

[0329] In one embodiment, a method of producing an anti-CLDN6 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0330] In order to express proteins, nucleic acids encoding antibodies of the present invention can be integrated into expression vectors. A variety of expression vectors can be used for protein expression. Expression vectors can include self-replicating extrachromosomal vectors, or vectors that integrate into the host genome. Expression vectors used in the present invention include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of expression vectors are commercially available or otherwise obtainable. Can be used in the present invention to express antibodies.

[0331] In a preferred embodiment, the host cell is administered in combination with an agent that enhances its function, preferably, in combination with a chemotherapeutic drug; and / or the host cell is administered in combination with an agent that improves one or more side effects associated with the host cell; and / or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting a protein other than CLDN6.

[0332] Pharmaceutical compositions, combination therapy

[0333] The antibodies, immunoconjugates comprising the antibodies, chimeric receptors, and host cells of the present invention can be used to prepare pharmaceutical compositions or diagnostic reagents. In addition to an effective amount of the antibody, immunoconjugate, chimeric receptor, nucleic acid, or host cell, the composition may also include a pharmaceutically acceptable carrier.

[0334] The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose, mannose, sucrose, dextran; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; phosphate buffer; adjuvants, such as aluminum hydroxide; buffers; diluents; stabilizers or excipients, etc.

[0335] The pharmaceutical compositions described herein may contain one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0336] Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and salts derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0337] The pharmaceutical compositions described herein may also contain an antioxidant. Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0338] The compositions of the present invention can be prepared into various dosage forms as needed, and can be administered by a physician based on factors such as the patient's type, age, weight, general condition, and route of administration to determine the dosage that is beneficial to the patient. The route of administration can be, for example, parenteral administration (e.g., injection) or other therapeutic modalities. "Parenteral" administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques. It can also be administered to the patient transarterially, intradermally, intratumorally, intranodally, intramedullary, or intraperitoneally.

[0339] In some embodiments, compositions can be isotonic, that is, they can have the osmotic pressure identical with blood and tear fluid.The expectation isotonicity of the present composition can use sodium chloride or other pharmaceutically acceptable reagents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes to realize.If necessary, the viscosity of compositions can use pharmaceutically acceptable thickening agent to maintain selected level.Suitable thickening agent includes, for example, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer etc.The preferred concentration of thickening agent will depend on selected reagent.Obviously, the selection of suitable carrier and other additives will depend on the character of definite route of administration and specific dosage form, for example liquid dosage form.

[0340] In one embodiment, the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits of the present invention are administered in combination with other known active agents or treatments. "Combined administration" refers to the administration of two or more different treatments to an individual. In one embodiment, one treatment is still ongoing when the second treatment begins, thereby overlapping the administration. In one embodiment, after the administration of one treatment ends, the second treatment begins. In one embodiment, combined administration makes the treatment more effective. In some embodiments, the effects of the two treatments can be partially additive, fully additive, or greater than additive. The treatments described in "combination administration" include, but are not limited to, one or more of the following: surgery, chemotherapy, radiation, immunosuppressants (e.g., cyclosporin, azathioprine, methotrexate, mycophenolate mofetil, and FK 50 6), antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cyclophosphamide, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. The treatments described in "combination administration" also include immunomodulators such as interferon α, interferon β, TGF-β2 peptide inhibitors, or poly-ICLC.

[0341] In some embodiments, the composition comprises another therapeutic agent. In some embodiments, the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention is administered in combination with a pharmaceutical agent that enhances its function. In some embodiments, the other therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and / or a pharmaceutically acceptable salt or analog thereof. Exemplary chemotherapeutic agents include alkylating agents, platinum-based agents, angiogenesis inhibitors (e.g., VEGF pathway inhibitors, tyrosine kinase inhibitors, EGF pathway inhibitors), and mTOR inhibitors. In some embodiments, the therapeutic agent includes, but is not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibin(TM) (vinorelbine, 5'-dehydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL), and other compounds derived from camptothecin and its analogs; podophyllotoxin derivatives, such as etoposide, teniposide, and midoxetine; alkylating agents cisplatin, cyclopentane, and dapoxetine; Phosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, brequizine, uracil mustard, cloprofen and dacarbazine; antimetabolites, including cytarabine, 5-fluorouracil, methotrexate, mercaptopurine, azathioprine and procarbazine; antibiotics, including but not limited to doxorubicin, bleomycin, dactinomycin, daunorubicin, mycobacterium serotonin, mitomycin, sarcoma mycin C and daunomycin; and other chemotherapy drugs, including but not limited to anti-tumor antibodies, dacarbazine, azacytidine, amsacon, melphalan, ifosfamide and mitoxantrone. In some embodiments, the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin and 5-fluorouracil. In some embodiments, the additional therapeutic agents include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides) and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including α and β) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2, etc.

[0342] In one embodiment, the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention is administered in combination with an inhibitor of an inhibitory molecule. Inhibitory molecules include PD1, PD-L1, CTLA-4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFRβ. Inhibitory nucleic acids such as dsRNA, such as siRNA or shRNA, can be used to inhibit the expression of inhibitory molecules in CAR-expressing cells. In one embodiment, the inhibitor is shRNA. In one embodiment, the inhibitory molecule is inhibited in CAR-expressing cells. In these embodiments, the dsRNA molecule that inhibits the expression of the inhibitory molecule can be connected to the nucleic acid encoding the components (e.g., all components) of the CAR. In one embodiment, the inhibitor of the inhibitory signal can be, for example, an antibody or antibody fragment that binds to the inhibitory molecule. For example, the active agent can be an antibody or antibody fragment (e.g., ipilimumab, Tremelimumab) that binds to PD1, PD-L1, PD-L2 or CTLA4.

[0343] In one embodiment, the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits of the present invention are administered in combination with an agent that ameliorates one or more of the side effects associated therewith. Such side effects include, but are not limited to, CRS. Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia, and the like. The co-administered agents provided herein can manage elevated levels of soluble factors caused by the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits. Elevated soluble factors in individuals include IFN-γ, TNFα, IL-2, and / or IL-6. The agent administered to ameliorate side effects can be an active agent that neutralizes one or more of these soluble factors. Such active agents include, but are not limited to, steroids, TNFα inhibitors, and / or IL-6 inhibitors. TNFα inhibitors include, but are not limited to, etanercept. IL-6 inhibitors include, but are not limited to, tocilizumab (toc).

[0344] In one embodiment, the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits of the present invention are administered in combination with cells expressing a chimeric antigen receptor other than CLDN6. In one embodiment, they are administered in combination with an agent that treats a disease associated with CLDN6 expression. In one embodiment, the agent comprises an antibody, a cell, RNA, a vaccine, an oncolytic virus, a checkpoint inhibitor, a BKT inhibitor, a chemotherapy drug, a radiotherapy agent, a hormonal therapy agent, a toxin, an immunotherapy agent, or a combination thereof.

[0345] The anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention and the other therapeutic agent can be administered simultaneously, in the same composition or separate compositions, or sequentially. In one embodiment, the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention can be administered first, followed by the other therapeutic agent. In one embodiment, the other therapeutic agent can be administered first, followed by the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention.

[0346] Reagent test kit

[0347] The present invention also provides a kit comprising an antibody, immunoconjugate, chimeric receptor, nucleic acid or host cell described herein. In some embodiments, the kit may include a therapeutic or prophylactic composition comprising an effective amount of an antibody, chimeric receptor, nucleic acid or host cell described herein in one or more unit dosage forms. In some embodiments, the kit comprises a sterile container that may contain a therapeutic or prophylactic composition; such a container may be a box, ampoule, bottle, vial, tube, bag, blister pack or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil or other material suitable for holding drugs. In some embodiments, the kit comprises an antibody, immunoconjugate, chimeric receptor, nucleic acid or host cell described herein, and instructions for administering the antibody, immunoconjugate, chimeric receptor, nucleic acid or host cell described herein to an individual. The instructions generally include a method for using the antibody, immunoconjugate, chimeric receptor, nucleic acid or host cell described herein to treat or prevent cancer or tumors. In some embodiments, the kit comprises a host cell described herein and may include approximately 1×10 4 cells to about 1×10 6 In some embodiments, the kit may include at least about 1×10 5 cells, at least about 1×10 6 cells, at least about 1×10 7 cells, at least about 4 × 10 7 cells, at least about 5×10 7 cells, at least about 6×10 7 cells, at least about 6×10 7 cells, 8×10 7 cells, at least about 9×10 7 cells, at least about 1×10 8 cells, at least about 2×10 8 cells, at least about 3×10 8 cells, at least about 4 × 108 cells, at least about 5×10 8 cells, at least about 6×10 8 cells, at least about 6×10 8 cells, at least about 8 × 10 8 cells, at least about 9×10 8 cells, at least about 1 × 10 9 cells, at least about 2×10 9 cells, at least about 3×10 9 cells, at least about 4 × 10 9 cells, at least about 5×10 9 cells, at least about 6×10 9 cells, at least about 8×10 9 cells, at least about 9×10 9 cells, at least about 1×10 10 cells, at least about 2×10 10 cells, at least about 3×10 10 cells, at least about 4 × 10 10 cells, at least about 5×10 10 cells, at least about 6×10 10 cells, at least about 7×10 10 cells, at least about 8×10 10 cells, at least about 9×10 10 cells, at least about 1×10 11 cells, at least about 2×10 11 cells, at least about 3×10 11 cells, at least about 4 × 10 11 cells, at least about 5×10 11 cells, at least about 8×10 11 cells, at least about 9×10 11 cells, or at least about 1 × 10 12 For example, approximately 5×10 10 In another example, the kit may include 3×10 6 cells; cells can be expanded to about 5×10 10 cells and administered to a subject.

[0348] In some embodiments, the kit may include allogeneic cells. In some embodiments, the kit may include cells that may include genomic modifications. In some embodiments, the kit may include "off-the-shelf" cells. In some embodiments, the kit may include cells that can be expanded for clinical use. In some cases, the kit may include contents for research purposes.

[0349] In some embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage regimen and administration for treating or preventing a tumor or its symptoms; precautions, warnings, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and / or references. The instructions can be printed directly on the container (if any), or as a label on the container, or as a separate sheet, brochure, card, or folder provided within or in the container. In some embodiments, the instructions provide methods for administering the antibodies of the present invention for treating or preventing tumors. In some cases, the instructions provide methods for administering the antibodies of the present invention before, after, or simultaneously with the administration of a chemotherapeutic agent.

[0350] Methods for diagnosis / detection / treatment

[0351] The term "modulate" refers to a positive or negative change. Examples of modulation include a 1%, 2%, 10%, 25%, 50%, 75%, or 100% change. In one embodiment, it refers to a negative change.

[0352] The term "treatment" refers to interventions intended to alter the course of a disease, including both preventative and clinical pathological interventions. Therapeutic benefits include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or relieving the condition, or alleviating or improving the prognosis. The term does not imply a complete cure for the disease, the complete elimination of any symptoms, or an effect on all symptoms or consequences.

[0353] The term "anti-tumor effect" refers to a biological effect that can be manifested in various forms, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in lifespan, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or an improvement in various physiological symptoms associated with cancer. An "anti-tumor effect" can also be manifested as the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent tumorigenesis from the outset.

[0354] The term "prevention" refers to the prevention or prophylactic treatment of a disease or disease state by providing a method for preventing the occurrence or recurrence of a disease in a subject who is predisposed to developing the disease but has not yet been diagnosed with the disease, or by attempting to intervene before the disease develops (such as rejection of a cell transplant).

[0355] The term "autologous" refers to any substance obtained from an individual and subsequently reintroduced into that same individual.

[0356] The term "allogeneic" refers to any substance introduced into an individual that originates from a different individual of the same species as the individual. When two or more individuals differ genetically at one or more loci, the individuals are said to be allogeneic. In some aspects, allogeneic substances from individuals of the same species can be sufficiently genetically different to cause antigenic interaction.

[0357] The term "xenogeneic" refers to a transplant that originates from an individual of a different species.

[0358] The term "tumor" or "cancer" refers to a disease characterized by rapid and uncontrolled abnormal cell growth. Tumor cells or cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system.

[0359] The term "disease associated with CLDN6 expression" or "disease associated with CLDN6 expression" includes, but is not limited to, diseases associated with CLDN6 expression, or conditions associated with cells expressing CLDN6, such as ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonal tumors, ovarian epithelial cancer, non-small cell lung cancer, non-squamous non-small cell lung cancer, endometrial cancer, etc.

[0360] The term "detection" includes quantitative or qualitative detection. The antibodies of the present invention can be used to detect the presence of CLDN6 in biological samples, including blood, serum, cells or tissues.

[0361] The term "tumor antigen" refers to an antigen that appears or is overexpressed during the development and progression of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present invention refers to a tumor.

[0362] The tumor antigen of the present invention may be a solid tumor antigen or a hematological tumor antigen.

[0363] The tumor antigens of the present invention include but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (P DGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 Related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain (kappa light chain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AChR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; oncofetal variant of tumor necrosis zone; G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1);Hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 ( MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytic leukemia viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (Rho C); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAI R1); Fc fragment of the IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily, member 2 (LILRA2); CD300 molecule-like family, member f (CD300LF); C-type lectin domain family 12, member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). Preferably, the tumor antigen is CS1, Claudin18.2, GPC3, BCMA, or CD19.

[0364] The pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.

[0365] The term "subject" is intended to include living organisms that can elicit an immune response, and refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0366] The term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit and is effective at a dosage and for a period of time necessary to achieve the desired therapeutic or prophylactic result. This amount can be determined by a physician based on individual patient conditions such as age, weight, tumor size, degree of infection, and degree of metastasis.

[0367] Any of the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits provided herein can be used in therapeutic methods.

[0368] Any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit provided herein, by eliciting an antigen-specific response against CLDN6, provides one or more of the following: targeting and destroying CLDN6-expressing tumor cells, reducing or eliminating tumors, promoting infiltration of immune cells into tumor sites, and enhancing / prolonging anti-tumor responses. Because CLDN6 is expressed at undetectable levels in normal (i.e., non-cancerous) tissues, any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit provided herein is believed to avoid targeting / destroying normal tissues and cells.

[0369] In one aspect, any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit is provided for use as a medicament. In another aspect, any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit is provided for use in treating a disease. In certain embodiments, any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit is provided for use in a method of treating a disease. In certain embodiments, the present invention provides any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit for use in a method of treating a subject suffering from a disease, the method comprising administering to the subject an effective amount of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit. In one embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. The "subject" is preferably a human.

[0370] In another aspect, the present invention provides the use of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit in the preparation or formulation of a medicament. In one embodiment, the medicament is used to treat a disease. In another embodiment, the medicament is used in a method for treating a disease, the method comprising administering an effective amount of the medicament to a subject suffering from the disease. In one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the subject. The "subject" is preferably a human.

[0371] In another aspect, the present invention provides a method for treating a disease. In one embodiment, the method comprises administering an effective amount of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit to an individual suffering from a disease expressing CLDN6. In one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. The "individual" is preferably a human.

[0372] In another aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits provided herein, for example for use in any of the above-described treatment methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits provided herein and at least one additional therapeutic agent.

[0373] In another aspect, the pharmaceutical formulation is for use in treating a disease. In one embodiment, the pharmaceutical formulation is administered to a diseased individual. The "individual" according to any of the above embodiments is preferably a human.

[0374] In another aspect, the present invention provides a method for preparing a medicament or pharmaceutical formulation, comprising mixing any of the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions, or kits provided herein with a pharmaceutically acceptable carrier, e.g., for use in any of the above-described therapeutic methods. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0375] Any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit of the present invention can be used for treatment alone or in combination with other agents. Alternatively, any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit of the present invention can be co-administered with at least one additional therapeutic agent.

[0376] Such combination therapy described above includes combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration. In such cases, administration of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit of the present invention can occur before, simultaneously with, and / or after administration of the additional therapeutic agent or reagent. In one embodiment, administration of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit of the present invention and administration of the additional therapeutic agent occur within about one month, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days of each other.

[0377] Any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral administration, intrapulmonary administration or intranasal administration, and, if necessary for treatment, intralesional administration. Parenteral infusion includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term. Various dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0378] A formulation comprising a population of immunoreactive cells administered to an individual comprises a plurality of immunoreactive cells effective for treating and / or preventing a particular indication or disease. Thus, a therapeutically effective population of immunoreactive cells can be administered to an individual. Typically, administration comprises approximately 1×10 4 to about 1×10 10 In most cases, the preparation will contain approximately 1 × 10 5 to about 1×10 9 Immunoreactive cells, about 5×10 5 to about 5×10 8 immunoreactive cells, or approximately 1×10 6 to about 1×10 7 However, the number of CAR-immunoreactive cells administered to an individual will vary widely depending on the location, origin, identity, extent, and severity of the tumor, the age and physical condition of the individual to be treated, etc. The physician will ultimately determine the appropriate dose to use.

[0379] In some embodiments, chimeric receptors are used to stimulate a host cell-mediated immune response. For example, a T cell-mediated immune response is an immune response involving T cell activation. Activated antigen-specific cytotoxic T cells are able to induce apoptosis in target cells displaying foreign antigen epitopes on their surfaces, such as cancer cells displaying tumor antigens. In other embodiments, chimeric antigen receptors are used to provide anti-tumor immunity in mammals. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.

[0380] In some cases, a method for treating a subject with a tumor may involve administering one or more host cells of the present invention to a subject in need of treatment. The host cells can bind to tumor target molecules and induce cancer cell death. As previously described, the present invention also provides a method for treating a pathogen infection in an individual, comprising administering to the individual a therapeutically effective amount of a host of the present invention.

[0381] The frequency of administration of the immunoreactive cells of the present invention will depend on factors including the disease being treated, the elements of the specific immunoreactive cells, and the mode of administration. For example, administration may be 4 times, 3 times, 2 times daily, or daily, every other day, every three days, every four days, every five days, every six days, once a week, every eight days, every nine days, every ten days, once a week, or twice a month. As described herein, because the immune response cells of the present application have improved viability, they can be administered not only in a therapeutically effective amount lower than that of similar immune response cells that do not express exogenous type I interferon, but also in a less frequent manner to achieve at least similar, and preferably more significant, therapeutic effects.

[0382] Advantages of the present invention:

[0383] The present invention provides a humanized antibody that specifically recognizes CLDN6. CAR T cells prepared from the antibody exhibit good killing effects on target cells in vivo and in vitro.

[0384] It will be appreciated that certain features of the present invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are described for clarity in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of embodiments of the present invention are expressly included in the present invention and disclosed herein, just as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also expressly included in the present invention and disclosed herein, just as if each and every such subcombination were individually and expressly disclosed herein.

[0385] Example 1. Preparation of humanized CLDN6 antibodies

[0386] In this example, mouse anti-SC27.105 (from WO2016073649A1) was used as the parent antibody. The parent antibody sequence was compared with the germline sequence of the IMGT database to screen the SC27.105 antibody heavy chain CDR transplantation template 1 and light chain CDR transplantation template 2. The LCDR region of the SC27.105 antibody was used to replace the CDR region of the antibody template 2 to form the light chain variable region VL of the humanized antibody H1 (amino acid sequence shown in SEQ ID NO: 3). The 5th serine (Serine, S) in the heavy chain CDR2 of the SC27.105 antibody was mutated to alanine (Alanine, A), and then the parent antibody HCDR1, mutated HCDR2, and HCDR3 replaced the CDR region of the transplantation template 2 to form the heavy chain variable region VH of the humanized antibody H1 (amino acid sequence shown in SEQ ID NO: 1). The framework region in the VH amino acid sequence of the humanized antibody H1 is amino acids 1-30, 36-49, 67-98, and 113-123 in SEQ ID NO: 1; the framework region in the VL amino acid sequence of the humanized antibody H1 is amino acids 1-23, 35-49, 57-88, and 97-106 in SEQ ID NO: 3.

[0387] H1 heavy chain variable region (SEQ ID NO: 1), the CDR region is underlined, and the framework region is the sequence other than the CDR.

[0388] The CDR regions of the H1 light chain variable region (SEQ ID NO: 3) are underlined, and the framework regions are sequences other than the CDRs.

[0389] Example 2. Specificity and affinity detection of humanized antibody H1

[0390] 1. Establish cell lines

[0391] Human CLDN6 (SEQ ID NO: 32), CLDN4 (SEQ ID NO: 31), and CLDN9 (SEQ ID NO: 33) were transfected into 293T cells via lentivirus using conventional molecular biology techniques. Positive clones were selected by limiting dilution to construct 293T-CLDN6, 293T-CLDN4, and 293T-CLDN9 stable cell lines ( Figure 1 ).

[0392] 2. Detect the EC50 of the binding of antibody H1 to cell lines

[0393] Take 2×10 cells of 293T-CLDN6, 293T-CLDN4, and 293T-CLDN9 5 cells / well in a 96-well round-bottom culture plate and washed with PBS; antibody H1 was added, incubated at 4°C, centrifuged, the supernatant discarded, and washed with PBS; Goat-anti-Mouse FITC was added, incubated at 4°C, centrifuged, the supernatant discarded, and washed. The cells were analyzed by flow cytometry, and the results were statistically analyzed using FlowJo vX0.7 and graphed using GraphPad Prism8.0.

[0394] Test results showed that antibody H1 (scFv-huFc, 10 μg / ml) specifically bound to the 293T-CLDN6 cell line with an EC50 value of 474.9 nM (Figures 2, 3), but did not bind to the 293T-CLDN4 and 293T-CLDN9 cell lines (Figure 2). The control antibody C46-S (scFv-huFc, 10 μg / ml, from patent WO2015150327A1) significantly bound to 293T-CLDN4, 293T-CLDN6, and 293T-CLDN9 cells.

[0395] Example 3. Screening and identification of mutants of antibody H1

[0396] To improve the affinity of antibody H1, randomized mutations were performed on both the light and heavy chain CDR3s of antibody H1 using conventional molecular biology techniques. Two phage libraries, H1-VH and H1-VL, were constructed, each with a library capacity of 1E+9. The phage libraries were first incubated with 293T cells for 1-2 hours, centrifuged, and incubated with 293T-CLDN6 cells for 1-2 hours, followed by washing. The phages were then eluted from the cells, neutralized, and infected with Escherichia coli TG1. After expansion, the phages were purified and used in the next round of screening.

[0397] After repeating the above screening process for 2-3 rounds, approximately 1,000 single clones were selected for ELISA testing, resulting in over 70 clones with strong binding to 293T-CLDN6 cells. Sequencing yielded 23 sequences. These 23 clones were expressed in prokaryotes and purified to produce single-chain Fv (scFv). Flow cytometry revealed that four of these clones specifically bound to 293T-CLDN6 cells (as shown in Figure 4; the primary antibody in the blank control (NA) group was PBS). These clones were designated P1, P2, P3, and P4 (antibody concentration was 10 μg / ml).

[0398] The amino acid sequence of VH of antibody P1 is shown in SEQ ID NO: 5, the amino acid sequence of VL is shown in SEQ ID NO: 3; the amino acid sequence of HCDR3 is shown in SEQ ID NO: 41; and the amino acid sequence of scFv is shown in SEQ ID NO: 14.

[0399] The amino acid sequence of VH of antibody P2 is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 7; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 42; and the amino acid sequence of scFv is shown in SEQ ID NO: 15.

[0400] The amino acid sequence of VH of antibody P3 is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 9; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 43; and the amino acid sequence of scFv is shown in SEQ ID NO: 16.

[0401] The amino acid sequence of VH of antibody P4 is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 11; the sequence of LCDR3 is shown in SEQ ID NO: 44; and the amino acid sequence of scFv is shown in SEQ ID NO: 17.

[0402] The flow cytometry results (as shown in FIG5 ) showed that antibodies P1, P2, P3, and P4 all had good cell binding affinity to 293T-CLDN6 cells, with EC50 values ​​of 62.66 nM, 144.2 nM, 554.8 nM, and 183.2 nM, respectively.

[0403] Example 4. Specificity and affinity testing of H1 mutants

[0404] Using molecular cloning techniques, the heavy chain variable region of antibody P1 from Example 3 was combined with the light chain variable regions of antibodies P2 and P3, respectively, to generate antibodies M1 and M2. After prokaryotic expression and purification, purified single-chain Fvs (scFvs) were obtained. M1 and M2 were co-incubated with 293T-CLDN6, 293T-CLDN4, and 293T-CLDN9 cell lines, respectively. Cell-bound scFvs were fluorescently labeled and analyzed by flow cytometry. The mean fluorescence intensity (MFI) of the experimental data was calculated using FlowJo analysis software.

[0405] The detection results are shown in FIG6 . M1 and M2 (scFv, 5 ug / ml) specifically bind to 293T-CLDN6 cells, but do not bind to 293T-CLDN4 and 293T-CLDN9 cells.

[0406] Figure 7 shows that both M1 and M2 significantly bound to 293T-CLDN6 cells, with EC50 values ​​of 23.04 nM and 24.71 nM, respectively, which are nearly 20 times more affinities than antibody H1.

[0407] Example 5. Preparation of CLDN6 CAR-T cells

[0408] 1. Construction of CAR vector

[0409] Using PRRLSIN-cPPT.EF-1α as a vector, lentiviral plasmids PRRLSIN-cPPT.EF-1α-H1-28Z and PRRLSIN-cPPT.EF-1α-P4-28Z were constructed to express the second-generation chimeric antigen receptors for antibodies H1 and P4. The amino acid sequence of H1-28Z is shown in SEQ ID NO: 48, and the amino acid sequence of P4-28Z is shown in SEQ ID NO: 49.

[0410] The amino acid sequence of the control group C46-S-28Z is shown in SEQ ID NO:51.

[0411] 2. Preparation of CAR-T Cells

[0412] The conventional method for preparing CAR-T in the art is used. PBMC cells are activated by magnetic beads with anti-CD3 and CD28 antibodies and then cultured to obtain T cells, which are then infected with lentivirus to obtain CAR-T cells.

[0413] Lentivirus was packaged using the calcium phosphate method, and the viral supernatant was purified using PEG8000 / NaCl. The purified virus was then used to infect T cells activated for 48 hours using CD3 / CD28 magnetic beads at an MOI of 20 to generate CAR-T cells expressing H1-28Z, P4-28Z, and C46-S-28Z. Untransfected T cells were considered untransfected cells (UTDs). Five days after infection, the CAR-positive rate was determined by FACS using Biotin-anti-F(ab')2-488 (Jackson ImmunoResearch) as the primary antibody and SA-PE (eBioscience) as the secondary antibody. The results are shown in Figure 8.

[0414] Example 6. Specific killing of target cells by CLDN6 CAR-T cells in vitro

[0415] First, the target cells 293T-CLDN4 cells, 293T-CLDN6 cells, 293T-CLDN9 cells, OVCAR3 cells (human ovarian cancer cells, ATCC) and OV90 cells (human ovarian cancer cells, ATCC) were adjusted to a density of 0.2x10^6 / mL using 1640 culture medium. 50 μl was added to each well of a 96-well cell culture plate, i.e., 10,000 target cells per well. Then, 50 μl of effector cells (CAR-T cells, UTD as a control) were added according to a 1:1 effector-target ratio. After incubation at 37°C for 16 hours, the supernatant was collected and detected using an LDH kit. Finally, the OD490 value was read using a microplate reader.

[0416] As shown in Figure 9, H1CAR-T cells and P4CAR-T cells showed no cytotoxicity against 293T-CLDN4 and 293T-CLDN9 cells. However, they exhibited significant cytotoxicity against CLDN6-expressing 293T-CLDN6, OVCAR3, and OV90 cells, with a killing rate of approximately 60%-90% at an effector-target ratio of 3:1. These results demonstrate that H1CAR-T and P4CAR-T cells exhibit specific cytotoxicity against CLDN6-expressing cells in vitro. C46-S CAR-T cells exhibited nonspecific cytotoxicity.

[0417] Example 7. RTCA Detection and Analysis of CLDN6 CAR-T Cell Cytotoxicity to Ovarian Cancer Cells in Vitro

[0418] Target OV90 ovarian cancer cells were seeded at 1E+04 cells / pore in RTCA assay plates. Twenty hours later, UTD, H1-28Z-CAR T, and P4-28Z-CAR T cells were added at a 1:1 effector-target ratio. Target cell lysis rates were measured after 8, 12, 16, 24, 48, 72, and 80 hours of co-culture. As shown in Figure 10, both H1-28Z-CAR T and P4-28Z-CAR T cells showed significant cytotoxicity against OV90 cells, reaching over 90% after 48 hours and approaching 100% after 72 hours.

[0419] Example 8. Anti-tumor effect of CLDN6-CAR T cells on subcutaneous transplanted tumors in NPG mice bearing human ovarian cancer cells

[0420] 5×10 6 The average tumor volume of OV90 cells in NPG mice was approximately 234 mm 13 days after inoculation. 3 The mice were divided into 4 groups and injected with UTD (3x10 6 )、H1-28Z(1x10 6 )、H1-28Z(3x10 6 )、P4-28Z(3x10 6 The results are shown in Figure 11A. 22 days after CAR-T injection, H1-28Z (1x10 6 ) group had a tumor inhibition rate of 74.11%, H1-28Z (3x10 6 ) group had an inhibition rate of 88.26%, and the P4-28Z group had an inhibition rate of 70.81%. As shown in Figure 11B, there was no significant change in the body weight of mice in each CAR-T group. Figure 11C shows that according to the change in tumor weight, H1-28Z (1x10 6 ) group had a tumor inhibition rate of 73.46%, H1-28Z (3x10 6 ) group had a tumor inhibition rate of 92.41%, and the P4-28Z group had a tumor inhibition rate of 71.39%. Figure 11D shows the survival of human T cells in the peripheral blood of mice in each group 11 days after CAR-T cell infusion.

[0421] Sequence Listing

Claims

1. An antibody or antigen-binding fragment that recognizes CLDN6, selected from the group consisting of: (1) the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the HCDR3 of the heavy chain variable region has at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR3 of SEQ ID NO: 41; (2) the antibody or antigen-binding fragment comprises a heavy chain variable region comprising HCDR1 represented by GYYMN (SEQ ID NO: 35); and / or HCDR2 shown in EINPATGSTTYNQKFKA (SEQ ID NO: 36); and / or HCDR3 represented by RDYYX1GSX2X3YAX4DY (SEQ ID NO: 52), wherein X1 is Y or L or a conservative substitution of Y or L, X2 is G or N or a conservative substitution of G or N, X3 is F or S or a conservative substitution of F or S, and X4 is M or L or a conservative substitution of M or L; (3) the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 35, 36 and 37; (4) the antibody or antigen-binding fragment comprises a light chain variable region, wherein the LCDR3 of the light chain variable region has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the LCDR3 of SEQ ID NO: 42, 43 or 44; (5) the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 represented by QASQSVSNNLN (SEQ ID NO: 38); and / or LCDR2 shown in GASKLED (SEQ ID NO: 39); and / or LCDR3 represented by X5QHRX6X7WT (SEQ ID NO: 53), wherein X5 is L or Q or a conservative substitution of L or Q, X6 is Y or F or a conservative substitution of Y or F, and X7 is L or M or a conservative substitution of L or M; (6) the antibody or antigen-binding fragment comprises a light chain variable region, wherein LCDR1, LCDR2 and LCDR3 of the light chain variable region have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 38, 39 and 40; (7) The antibody or antigen-binding fragment comprises the heavy chain variable region described in any one of (1) to (3) and the light chain variable region described in any one of (4) to (6); (8) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), and the variant contains at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).

2. The antibody or antigen-binding fragment according to claim 1, wherein Select from the following groups: (1) The antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 35, and / or HCDR2 as shown in SEQ ID NO: 36, and / or SEQ ID NO: 37 or 41. HCDR3 as shown; (2) the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 38, and / or LCDR2 as shown in SEQ ID NO: 39, and / or LCDR3 as shown in SEQ ID NO: 40, 42, 43 or 44; (3) The antibody or antigen-binding fragment comprises the heavy chain variable region described in (1) and the light chain variable region described in (2); (4) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (3), wherein the variant comprises at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (3).

3. The antibody or antigen-binding fragment according to claim 2, wherein Select from the following groups: (1) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; or (2) the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 41, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; or (3) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 42; or (4) the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 43; or (5) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 41, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 42; or (6) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 41, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 43; or (7) The antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 44; or (8) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), wherein the variant comprises at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).

4. An antibody or antigen-binding fragment that recognizes CLDN6, selected from the group consisting of: (1) The antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or 5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (2) The antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3, 7, 9 or 11, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (3) The antibody or antigen-binding fragment comprises the heavy chain variable region described in (1) and the light chain variable region described in (2); (4) The antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (3), wherein the variant comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in VH or VL, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (3).

5. The antibody or antigen-binding fragment according to claim 4, wherein Select from the following groups: (1) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (2) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (3) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (4) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (5) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (6) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (7) the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or The antibody or antigen-binding fragment described in (8) is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), wherein the variant comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in VH or VL, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).

6. An antibody or antigen-binding fragment that recognizes CLDN6, comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 3, 7, 9, or 11, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37 or 41.

7. The antibody or antigen-binding fragment according to claim 6, wherein The heavy chain variable region comprises the sequence shown in SEQ ID NO: 1, 5, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequence.

8. An antibody or antigen-binding fragment that recognizes CLDN6, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1 or 5, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40, 42, 43 or 44.

9. The antibody or antigen-binding fragment according to claim 8, wherein The light chain variable region comprises the sequence shown in SEQ ID NO:3, 7, 9 or 11, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequence.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein Selected from whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, dAb fragment, multifunctional antibody, IgG4 antibody, scFv-Fc antibody, hybridoma antibody, chimeric antibody, humanized antibody, fully human antibody or monoclonal antibody.

11. The antibody or antigen-binding fragment according to claim 10, wherein The invention also provides an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18 or 19, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or comprising at least one and not more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid alterations in the above sequence.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 11, wherein: The antibody or antigen-binding fragment binds to CLDN6 and does not significantly bind to CLDN4 or CLDN9; and / or the antibody or antigen-binding fragment binds to cells expressing CLDN6 and does not significantly bind to cells expressing CLDN4, CLDN9, or a combination thereof.

13. An immunoconjugate comprising: The antibody or antigen-binding fragment according to any one of claims 1 to 12, and a functional molecule linked thereto.

14. A chimeric receptor comprising an extracellular region, wherein the extracellular region comprises the antibody or antigen-binding fragment of any one of claims 1 to 12; The chimeric receptor comprises: Chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), synthetic polypeptide receptor (synNotch), or a combination thereof.

15. The chimeric receptor according to claim 14, wherein The chimeric receptor is a chimeric antigen receptor (CAR), which comprises the antibody or antigen-binding fragment of any one of claims 1-12, a transmembrane region and an intracellular signaling region; preferably, the antibody or antigen-binding fragment is connected to the transmembrane region via a hinge domain.

16. The chimeric receptor according to claim 15, wherein The transmembrane region comprises a transmembrane region selected from the group consisting of the α, β, γ or ζ chain of TCR, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD134, CD137, CD152, CD154 or PD1; Preferably, the transmembrane region is selected from the transmembrane domain of CD8 or CD28; More preferably, the transmembrane region is selected from the sequence shown in SEQ ID NO: 25 or 22, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

17. The chimeric receptor according to claim 15 or 16, wherein The intracellular signaling region comprises a primary signaling domain; Preferably, the primary signaling domain is selected from TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), CD66d or CD3ζ.

18. The chimeric receptor according to any one of claims 15 to 17, wherein The intracellular signaling region further comprises one or more costimulatory signaling domains; Preferably, the costimulatory signal domain is selected from the intracellular signaling region of CARD11, CD2, CD5, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB or 4-1BBL, or a combination thereof; More preferably, the costimulatory signaling domain is selected from the intracellular signaling domains of CD28 and / or CD137.

19. The chimeric receptor according to claim 17 or 18, wherein The intracellular signaling region is selected from SEQ ID NO: 24, or a sequence including SEQ ID NOs: 23 and 24, or a sequence including SEQ ID NOs: 26 and 24, or a sequence including SEQ ID NOs: 23, 26 and 24, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the above sequences.

20. The chimeric receptor of claim 15, wherein The hinge region is derived from CD8, IgG4 or IgG1; Preferably, the hinge region comprises the sequence shown in SEQ ID NO: 21, 27, 28, 29 or 30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the above sequence.

21. The chimeric receptor according to any one of claims 14 to 20, wherein The chimeric receptor comprises: The antibody or antigen-binding fragment of any one of claims 1 to 12, the transmembrane region of CD8 / CD28, and CD3ζ; or The antibody or antigen-binding fragment of any one of claims 1 to 12, the transmembrane region of CD8 / CD28, the intracellular signaling region of CD137, and CD3ζ; or The antibody or antigen-binding fragment of any one of claims 1 to 12, the transmembrane region of CD8 / CD28, the intracellular signaling region of CD28, and CD3ζ; or The antibody or antigen-binding fragment of any one of claims 1 to 12, the transmembrane region of CD8 / CD28, the intracellular signaling region of CD28, CD137, and CD3ζ.

22. The chimeric receptor according to claim 21, wherein The chimeric receptor comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, or 19 linked to a sequence as set forth in any one of SEQ ID NOs: 45, 46, or 47, respectively, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; Preferably, the chimeric receptor comprises the sequence shown in SEQ ID NO: 48 or 49, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

23. A nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 12, the immunoconjugate of claim 13, or the chimeric receptor of any one of claims 14 to 22.

24. A vector comprising the nucleic acid of claim 23.

25. A cell comprising the antibody or antigen-binding fragment of any one of claims 1-12, the immunoconjugate of claim 13, the chimeric receptor of any one of claims 14-22, the nucleic acid of claim 23, and / or the vector of claim 24.

26. The cell according to claim 25, wherein The cells include T cells, natural killer cells, natural killer T cells, NK92 cells, cytotoxic T cells, dendritic cells, macrophages, CIK cells, pluripotent stem cells, stem cell-derived immune cells, or a combination thereof.

27. The cell according to claim 26, wherein The T cells include natural T cells and / or T cells induced by pluripotent stem cells; Preferably, the T cells include autologous T cells and / or allogeneic T cells; Preferably, the T cells are primary T cells; Preferably, the T cells are derived from human autologous T cells.

28. The cell according to any one of claims 25 to 27, wherein The cells bind to cells expressing CLDN6 and do not significantly bind to cells expressing CLDN4, CLDN9, or a combination thereof.

29. The cell according to any one of claims 25 to 28, wherein It also carries the coding sequence of an exogenous cytokine; and / or It also expresses a chimeric receptor that does not target CLDN6; and / or It also expresses chemokines; and / or It also expresses chemokine receptors; and / or It also expresses a safety switch; and / or It also expresses inhibitory molecules.

30. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-12, the immunoconjugate of claim 13, the chimeric receptor of any one of claims 14-22, the nucleic acid of claim 23, the vector of claim 24 and / or the cell of any one of claims 25-29, and a pharmaceutically acceptable adjuvant.

31. A combination therapy, characterized in that: The antibody or antigen-binding fragment of any one of claims 1 to 12, the immunoconjugate of claim 13, the chimeric receptor of any one of claims 14 to 22, the cell of any one of claims 25 to 29, or the pharmaceutical composition of claim 30 is administered in combination with an agent that enhances their function, Preferably, used in combination with chemotherapy drugs; and / or co-administered with an agent that ameliorates one or more side effects associated therewith; and / or co-administered with cells expressing chimeric antigen receptors targeting proteins other than CLDN6; and / or administered in combination with an agent for treating a disease associated with CLDN6 expression; Preferably, the agent comprises an antibody, a cell, an RNA, a vaccine, an oncolytic virus, a checkpoint inhibitor, a BKT inhibitor, a chemotherapeutic agent, a radiotherapeutic agent, a hormonal therapeutic agent, a toxin, an immunotherapeutic agent, or a combination thereof.

32. A method for preparing the antibody or antigen-binding fragment of any one of claims 1-12, the immunoconjugate of claim 13, or the chimeric receptor of any one of claims 14-22, the method comprising culturing the cells of any one of claims 25-29 and isolating the antibody, immunoconjugate, or chimeric receptor expressed by the cells.

33. A kit, characterized in that It includes the antibody or antigen-binding fragment of any one of claims 1-12, the immunoconjugate of claim 13, the chimeric receptor of any one of claims 14-22, the nucleic acid of claim 23, the vector of claim 24, the cell of any one of claims 25-29 and / or the pharmaceutical composition of claim 30.

34. Use of the antibody or antigen-binding fragment of any one of claims 1 to 12, the immunoconjugate of claim 13, the cell of any one of claims 25 to 29, the pharmaceutical composition of claim 30, and / or the kit of claim 33, (1) Killing cells expressing CLDN6; (2) inhibiting the proliferation of cells expressing CLDN6; (3) mediate the remission of disease or tumor; (4) prevent tumor formation or re-formation; (5) inhibiting the metastasis of cells expressing CLDN6; (6) Preparation of drugs for the treatment / diagnosis of diseases; It is characterized by: The disease expresses CLDN6; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors; more preferably, the tumor is a solid tumor; more preferably, the tumor is ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonal tumors, ovarian epithelial cancer, non-small cell lung cancer, non Squamous non-small cell lung cancer, endometrial cancer, or a combination thereof.

35. A pharmaceutical agent for the following use, comprising the antibody or antigen-binding fragment of any one of claims 1 to 12, the immunoconjugate of claim 13, the cell of any one of claims 25 to 29, and the pharmaceutical composition of claim 30, (1) Killing cells expressing CLDN6; (2) inhibiting the proliferation of cells expressing CLDN6; (3) mediate the remission of disease or tumor; (4) prevent tumor formation or re-formation; (5) inhibiting the metastasis of cells expressing CLDN6; (6) For the treatment / diagnosis of diseases.

36. A method for treating / diagnosing a disease, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 12, or the immunoconjugate of claim 13, or the cell of any one of claims 25 to 29, or the pharmaceutical composition of claim 30, or the kit of claim 33; Preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors; Preferably, the subject is a human; Preferably, the cells are autologous or allogeneic T cells to the subject.

37. The antibody or antigen-binding fragment of any one of claims 1-12, the immunoconjugate of claim 13, the cell of any one of claims 25-29, the pharmaceutical composition of claim 30, and / or the kit of claim 33, for use in treating / diagnosing a disease comprising expression of CLDN6; preferably, the disease is selected from an inflammatory disorder, an infection, an autoimmune disease, and a tumor; more preferably, the tumor is a solid tumor; more preferably, the tumor is ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonal tumors, epithelial ovarian cancer, non-small cell lung cancer, non-squamous non-small cell lung cancer, endometrial cancer, or a combination thereof.

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