Anti-5t4 antibodies and uses

Abstract

The present invention describes anti-5T4 antibodies and antigen binding fragments thereof, as well as chimeric antigen receptors (CARs) comprising an anti-5T4 antigen binding domain. Also described are nucleic acids encoding the antibodies and CARs, compositions comprising the antibodies and CARs, methods of making the antibodies and CARs, and methods of using the antibodies and CARs to treat or prevent diseases, such as cancer and / or inflammatory diseases and / or autoimmune diseases.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 382,740, filed on November 8, 2022, the entire content of which is incorporated herein by reference. Field of the invention

[0003] The present invention relates to monoclonal anti - 5T4 antibodies, chimeric antigen receptors (CARs) comprising anti - 5T4 antigen - binding domains, nucleic acid fragments and expression vectors encoding said antibodies and CARs, recombinant cells comprising said vectors, and compositions comprising said antibodies, CARs and recombinant cells. In addition, the present invention also provides methods for preparing said antibodies and CARs, and methods for treating diseases including cancer, inflammation, autoimmune diseases and / or their complications using said antibodies and CARs.

[0004] Reference sequence listing submitted in electronic form

[0005] This application contains a sequence listing submitted in electronic form. The entire content of this electronic sequence listing (file name: 065799.40WO1Sequence Listing.xml; file size: 154,914 bytes; creation date: October 10, 2022) is incorporated herein by reference. Background of the invention

[0007] 5T4, also known as 5T4 carcino - fetal antigen, Waif1 or trophoblast glycoprotein (TPBG), is a highly N - glycosylated transmembrane protein with an apparent molecular weight of 72 kDa. In normal adult tissues, the expression level of 5T4 is usually relatively low, mainly expressed in tissues during fetal development. In addition, a certain degree of expression can also be detected in squamous epithelium, cervical endothelium and endometrial epithelium, gastric and large intestinal mucosal tissues, and pancreatic secretory duct epithelium.

[0008] The expression and upregulation of 5T4 are closely associated with poor prognosis in multiple cancers (Naganuma, H. et al., Anticancer Res. 2002, 22: 1033-1038; Wrigley, E. et al., Int. J. of Gynecologic Cancer. 1995, 5: 269-274; Starzynska, T. et al., British Journal of Cancer. 1994, 69: 899-902). Part of the reason is that it disrupts cell-cell contact, thereby further enhancing cell motility (Carsberg, C. J. et al., Int. J. Cancer. 1996, 68: 84-92). It is generally believed that 5T4 has the functions of promoting angiogenesis and epithelial-mesenchymal transition, thereby further enhancing the migration, invasion and anti-tumor drug resistance of cancer cells. (Spencer, H. L. et al., Arterioscler. Thromb. Vasc. Biol. 2019, 39: 1113-1124). 5T4 participates in Wnt / β-catenin signaling by activating the non-canonical Wnt signaling pathway, thereby enhancing cell migration and invasion abilities (He, P. et al., Molecular Medicine Reports. 2015, 12: 503-509; Kagermeier-Schenk, B. Developmental Cell. 2011, 21: 1129-1143.). In addition, since the site expressing CXCL12 may be relatively less affected by biotherapeutic drugs, and 5T4 is closely related to the expression of CXCR4 in tumor cells and subsequent chemotaxis towards the site expressing CXCL12, 5T4 can improve the survival rate of tumor patients (McGinn, O. J, et al., J. Cell Sci. 2012, 125: 5467-5478; Burger, J. A. and Kipps, T. J., Blood. 2006, 107: 1761-1767). In addition, 5T4 is highly expressed in multiple cancers such as bladder cancer, breast cancer, cervical cancer, lung cancer, gastric cancer and pancreatic cancer, making it an ideal tumor-associated antigen for targeted anti-cancer biologics against 5T4-positive tumors (Southall, P. J. et al., Br. J. Cancer. 1990, 61: 89-95).

[0009] Brief Description of the Invention

[0010] In one general aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to 5T4.

[0011] The present invention provides a separated monoclonal antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, and the light-chain variable region comprises light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, having the polypeptide sequences as shown below:

[0012] (1) SEQ ID NOs: 3, 4, 5, 6, 7, 8 respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, 14 respectively;

[0013] (2) SEQ ID NOs: 17, 18, 19, 20, 21, 22 respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, 28 respectively;

[0014] (3) SEQ ID NOs: 31, 32, 33, 34, 35, 36 respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, 42 respectively;

[0015] (4) SEQ ID NOs: 45, 46, 47, 48, 49, 50 respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, 56 respectively;

[0016] (5) SEQ ID NOs: 59, 60, 61, 62, 63, 64 respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, 70 respectively;

[0017] (6) SEQ ID NOs: 73, 74, 75, 76, 77, 78 respectively, or SEQ ID NOs: 79, 80, 81, 82, 83, 84 respectively;

[0018] (7) SEQ ID NOs: 87, 88, 89, 90, 91, 92 respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, 98 respectively;

[0019] (8) SEQ ID NOs: 101, 102, 103, 104, 105, 106 respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, 112 respectively;

[0020] (9) SEQ ID NOs: 115, 116, 117, 118, 119, 120 respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, 126 respectively;

[0021] (10) are SEQ ID NOs: 129, 130, 131, 132, 133, 134, or are SEQ ID NOs: 135, 136, 137, 138, 139, 140;

[0022] (11) are SEQ ID NOs: 143, 144, 145, 146, 147, 148, or are SEQ ID NOs: 149, 150, 151, 152, 153, 154;

[0023] wherein the antibody or antigen-binding fragment thereof specifically binds to 5T4, preferably human 5T4.

[0024] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127 or 141; or comprises a light chain variable region having a polypeptide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128 or 142.

[0025] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0026] (a) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 2;

[0027] (b) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 16;

[0028] (c) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 30;

[0029] (d) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 44;

[0030] (e) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58;

[0031] (f) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:71 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:72;

[0032] (g) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:85 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:86;

[0033] (h) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:99 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:100;

[0034] (i) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:113 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:114;

[0035] (j) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:127 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:128; or

[0036] (k) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:141 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:142.

[0037] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds to 5T4 and is capable of inducing effector-mediated tumor cell lysis by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC); and / or mediating the recruitment of conjugated drugs; and / or forming a bispecific antibody with another monoclonal antibody or antigen-binding fragment thereof having an anti-cancer effect.

[0038] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a chimeric.

[0039] In certain embodiments, the isolated monoclonal antibody or its antigen-binding fragment is human or humanized. In certain embodiments, the humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having a polypeptide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 155, 156, 159, 160, 161, 165, 166, 169, 170, 173, 174, 177, 178, or 179; or comprises a light chain variable region having a polypeptide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 157, 158, 162, 163, 164, 167, 168, 171, 172, 175, 176, 180, or 181.

[0040] In certain embodiments, the humanized monoclonal antibody or its antigen-binding fragment comprises:

[0041] (1) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0042] (2) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0043] (3) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0044] (4) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0045] (5) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0046] (6) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0047] (7) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0048] (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0049] (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0050] (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0051] (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0052] (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0053] (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0054] (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0055] (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0056] (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0057] (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0058] (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0059] (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0060] (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0061] (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0062] (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0063] (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0064] (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0065] (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0066] (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0067] (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0068] (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0069] (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0070] (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180; or

[0071] (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181.

[0072] The present invention also provides an isolated bispecific antibody or an antigen-binding fragment thereof, comprising the monoclonal antibody of the present invention or an antigen-binding fragment thereof.

[0073] The present invention also provides an isolated nucleic acid encoding the monoclonal antibody of the present invention or an antigen-binding fragment thereof or a bispecific antibody.

[0074] The present invention also provides a vector comprising an isolated nucleic acid encoding the monoclonal antibody of the present invention or an antigen-binding fragment thereof or a bispecific antibody or an antigen-binding fragment thereof.

[0075] The present invention also provides a host cell comprising the vector, the vector comprising an isolated nucleic acid encoding the monoclonal antibody of the present invention or an antigen-binding fragment thereof or a bispecific antibody or an antigen-binding fragment thereof.

[0076] In certain embodiments, a pharmaceutical composition is provided, the pharmaceutical composition comprising the isolated monoclonal antibody of the present invention or an antigen-binding fragment thereof, or an isolated bispecific antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0077] The present invention also provides a method for specifically targeting 5T4 on the surface of cancer cells in a subject in need thereof, the method comprising administering the pharmaceutical composition of the present invention to the subject.

[0078] The present invention also provides a method for treating cancer in a subject in need thereof, comprising administering the pharmaceutical composition of the present invention to the subject. The cancer can be any liquid tumor or solid tumor, for example, it can be selected from, but not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0079] The present invention also provides a method for treating an inflammatory disease and / or an autoimmune disease in a subject in need thereof, comprising administering the pharmaceutical composition of the present invention to the subject.

[0080] The present invention also provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof, or bispecific antibody or antigen-binding fragment thereof of the present invention. The method includes: culturing cells under conditions capable of producing the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, wherein the cells contain nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof from the cells or the culture.

[0081] The present invention also provides a method for preparing a pharmaceutical composition. The method for preparing the pharmaceutical composition containing the monoclonal antibody or antigen-binding fragment thereof, or bispecific antibody or antigen-binding fragment thereof of the present invention includes: combining the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0082] The present invention also provides a method for detecting the level of 5T4 in a subject. The method includes: (a) obtaining a sample from the subject; (b) co-incubating the sample with the antibody or antigen-binding fragment thereof of the present invention; and (c) detecting the level of 5T4 in the subject. In certain embodiments, the sample is a tissue sample. For example, the tissue sample can be a cancer tissue sample. In certain embodiments, the sample is a blood sample.

[0083] In another general aspect, the present invention also relates to a chimeric antigen receptor (CAR) construct for inducing T cell-mediated cancer killing. The CAR construct includes at least one antigen-binding domain, a hinge region, a transmembrane region, and an intracellular signaling domain that specifically binds to human 5T4.

[0084] The present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). The CAR may include (a) an extracellular domain, (b) a hinge region; (c) a transmembrane region, and (d) an intracellular signaling domain; the extracellular domain includes at least one antigen-binding domain that specifically binds to 5T4, preferably human 5T4.

[0085] In certain embodiments, the antigen-binding domain includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, and the light chain variable region includes light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences:

[0086] (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively;

[0087] (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively;

[0088] (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively;

[0089] (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively;

[0090] (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively;

[0091] (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, respectively, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively;

[0092] (7) SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively;

[0093] (8) SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively;

[0094] (9) SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively;

[0095] (10) SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively; or

[0096] (11) are SEQ ID NOs: 143, 144, 145, 146, 147 and 148, or are SEQ ID NOs: 149, 150, 151, 152, 153 and 154, respectively.

[0097] In certain embodiments, the antigen-binding domain comprises a heavy-chain variable region having a polypeptide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127 or 141; or comprises a light-chain variable region having a polypeptide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128 or 142.

[0098] In certain embodiments, the antigen-binding domain comprises:

[0099] (a) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 1 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 2.

[0100] (b) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 15 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 16;

[0101] (c) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 29 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 30;

[0102] (d) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 43 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 44;

[0103] (e) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 57 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 58;

[0104] (f) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 71 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 72;

[0105] (g) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 85 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 86;

[0106] (h) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100;

[0107] (i) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114;

[0108] (j) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128; or

[0109] (k) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141; a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142.

[0110] In certain embodiments, the antigen-binding domain is humanized and comprises variable chain regions, wherein the heavy chain variable region has a polypeptide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NOs: 155, 156, 159, 160, 161, 165, 166, 169, 170, 173, 174, 177, 178 or 179; or comprises a light chain variable region having a polypeptide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any of 157, 158, 162, 163, 164, 167, 168, 171, 172, 175, 176, 180 or 181.

[0111] In certain embodiments, the antigen-binding domain is humanized and comprises:

[0112] (1) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0113] (2) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0114] (3) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0115] (4) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0116] (5) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0117] (6) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0118] (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0119] (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0120] (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0121] (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0122] (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0123] (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0124] (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0125] (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165, and the light chain variable region may comprise a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0126] (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0127] (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0128] (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0129] (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0130] (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0131] (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0132] (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0133] (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0134] (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0135] (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0136] (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0137] (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0138] (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177; a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0139] (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178; a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0140] (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178; a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0141] (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179; a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180; or

[0142] (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179; a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181.

[0143] In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv).

[0144] In certain embodiments, the antigen-binding domain is a humanized single-chain variable fragment (scFv).

[0145] In certain embodiments, the chimeric antigen receptor (CAR) comprises one or more antigen-binding domains.

[0146] In certain embodiments, the intracellular signaling domain comprises one or more co-stimulatory domains and one or more activation domains.

[0147] The present invention also provides chimeric antigen receptors (CARs) encoded by the isolated polynucleotides.

[0148] The present invention also provides a vector comprising an isolated polynucleotide, the polynucleotide comprising a nucleic acid encoding the CAR of the present invention.

[0149] The present invention also provides a host cell comprising the vector of the present invention.

[0150] In certain embodiments, the host cell is a T cell, preferably a human T cell. In certain embodiments, the host cell is an NK cell, preferably a human NK cell. For example, in order to express the CAR of the present invention, T cells or NK cells can be genetically engineered for the treatment of diseases such as cancer.

[0151] The present invention also provides a method for preparing a host cell expressing the chimeric antigen receptor (CAR) of the present invention. The method includes transducing T cells or NK cells with a vector, the vector comprising an isolated nucleic acid encoding the CAR of the present invention.

[0152] The present invention also provides a method for preparing the CAR-T cells or CAR-NK cells of the present invention. The method includes culturing T cells or NK cells comprising an isolated polynucleotide under conditions for generating CAR-T cells or CAR-NK cells, the polynucleotide comprising a nucleic acid encoding the chimeric antigen receptor (CAR) of the present invention, and recovering the CAR-T cells or CAR-NK cells.

[0153] The present invention also provides a method for preparing a population of RNA-engineered cells comprising the chimeric antigen receptor (CAR) of the present invention. The method includes contacting the cells with an isolated polynucleotide comprising a nucleic acid encoding the chimeric antigen receptor (CAR) of the present invention, wherein the isolated polynucleotide is in vitro transcribed RNA or synthetic RNA.

[0154] The present invention also provides a method for treating cancer in a subject in need thereof, including administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention. The cancer can be any liquid or solid cancer, such as, but not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, bile duct cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0155] In certain embodiments, the method for treating cancer in a subject further includes administering to the subject in need thereof a drug that enhances the efficacy of cells expressing the CAR molecule.

[0156] In certain embodiments, the method for treating cancer in a subject in need thereof further includes administering to the subject in need thereof a drug that alleviates one or more side effects associated with administering cells expressing the CAR molecule.

[0157] In certain embodiments, the method for treating cancer in a subject in need thereof further includes administering to the subject a drug for treating 5T4-related diseases. Brief Description of the Drawings

[0159] In connection with the above summary and the following detailed description of the preferred embodiments of the present application and their accompanying drawings, the present application will be described in more detail. However, it should be understood that the present invention is not limited to the specific embodiments shown in the drawings.

[0160] Figure 1A-1C It shows that in an ELISA assay, the purified chimeric anti-5T4 monoclonal antibody (formed by fusing the VH and VL regions of a murine monoclonal antibody with the human IgG1 heavy chain constant region and the κ light chain constant region, respectively) binds to immobilized recombinant protein 5T4. The antigen is human 5T4 protein (32-355) with a polyhistidine tag at its C-terminus (ACROBiosystems, catalog number TPG-H52E5). L3F10-C is a chimera of L3F10; the remaining antibodies follow the same naming convention. The isotype control (IgG1) is a negative control antibody.

[0161] Figure 2 It shows the binding of the purified chimeric anti-5T4 monoclonal antibody to SK-OV-3 cells (ATCC, catalog number HTB-79) using FACS analysis. L3F10-C is a chimera of L3F10; the remaining antibodies follow the same naming convention. The isotype control (IgG1) is a negative control antibody.

[0162] Figure 3A-3G It shows the binding of the purified humanized anti-5T4 monoclonal antibody to SK-OV-3 cells (ATCC, catalog number HTB-79) using FACS analysis. L3D5-H1L1 is a humanized monoclonal antibody constructed from the L3D5-H1 heavy chain and the L3D5-L1 light chain; the remaining antibodies follow the same naming convention. The isotype control (IgG1) is a negative control antibody. Detailed Embodiments

[0163] Various publications, articles, and patents are cited or described in the Background section and throughout the specification; these references are hereby incorporated by reference in their entirety. The discussion of the documents, statutes, materials, devices, articles, or the like incorporated into this specification is intended to provide background information for the invention. Such discussion does not mean an admission that any or all of them constitute prior art relevant to any invention disclosed or claimed in this specification.

[0164] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Otherwise, certain terms used herein have the meanings as defined in the specification.

[0165] It should be noted in particular that, as used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents.

[0166] Unless otherwise indicated, any numerical values (such as the concentrations or concentration ranges described herein) should be understood to be modified by the term "about" in all cases. Thus, the numerical values generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As described herein, unless the context clearly indicates otherwise, the use of numerical ranges explicitly includes all possible sub-ranges and all individual numerical values within the range, including integers and fractions of the values within such ranges.

[0167] Unless otherwise indicated, when the term "at least" is used before a series of elements, it should be understood to refer to each element in the series. Those skilled in the art can identify or determine, based on conventional experimental means, many other embodiments equivalent to the specific embodiments of the invention described herein. Such equivalent embodiments should be considered to be included within the scope of the present invention.

[0168] In this patent application, the terms "comprising", "comprises", "including", "includes", "having", "has", "containing", or "contains", or any other synonymous expressions of these terms, should be understood to include one integer or a group of integers stated, but not exclude any other integer or combination of integers, and are intended to express a non-exclusive or open-ended meaning. For example, a composition, mixture, process, method, article, or device comprising a series of elements is not necessarily limited to these elements, but may include other elements not explicitly listed or inherent to such composition, mixture, process, method, article, or device itself. In addition, unless clearly indicated to the contrary, "or" means inclusive rather than exclusive. For example, the condition A or B is satisfied in any of the following cases: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0169] In this patent application document, when the conjunction "and / or" is used between multiple recited elements, it should be understood to cover the cases where each element is applied alone and the cases where the elements are applied in combination. For example, when two elements are connected by "and / or", the first case means only the first element is applied and the second element is not applied; the second case means only the second element is applied and the first element is not applied; the third case means both the first element and the second element are applied. Any of the above cases should be understood to conform to the meaning of the term "and / or" in this patent application document and meet its requirements. In addition, the cases where multiple cases are applied simultaneously should also be understood to conform to the meaning of the term "and / or" and meet its requirements.

[0170] In this patent application, the term "consisting of", and its synonymous expressions such as "composed of", "formed of", etc., when used throughout the specification and claims, means including any integer or combination of integers recited, but no other integers or combinations of integers are added to the specified method, structure or composition.

[0171] In this patent application, the term "consisting essentially of", and its synonymous expressions such as "essentially composed of", "essentially formed of", etc., when used throughout the specification and claims, means including any integer or combination of integers recited, and optionally including any integer or combination of integers that do not materially alter the basic or novel characteristics of the specified method, structure or composition. See M.P.E.P. Section 2111.03.

[0172] In this patent application, the term "subject" refers to any animal, preferably a mammal, and most preferably a human. The term "mammal" as used herein encompasses all mammals. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc. Preferably, the subject is a human.

[0173] The terms "right", "left", "lower" and "upper" are used to indicate the directions of the said drawings.

[0174] It should also be understood that when terms such as "about", "approximate", "generally", "substantially", etc. are used in connection with the dimensions or features of a component in a preferred embodiment of the present invention, it indicates that the described dimensions or features do not have strict boundaries or parameters, and do not exclude minor variations that are functionally equivalent or similar thereto. This concept is well known to those skilled in the art. At least, according to the mathematical and industrial principles in the art (such as rounding, measurement errors or other systematic errors, processing errors, etc.), such expressions containing numerical parameters should cover the errors of the least significant digits.

[0175] The term "identical" or percentage "identity", in the context of two or more nucleic acid or amino acid sequences (e.g., an anti-5T4 antibody and the polynucleotide encoding the antibody, a chimeric antigen receptor (CAR) comprising a specific antigen-binding domain of 5T4 and the polynucleotide encoding the above CAR), means that two or more sequences or subsequences are identical, or have a specific percentage of identical amino acid residues or nucleotides, after alignment by one of the following sequence alignment algorithms or by visual inspection to achieve maximum match.

[0176] For sequence alignment, usually one sequence is taken as the reference sequence, and the sequence to be tested is aligned with it. When using a sequence alignment algorithm, the sequence to be tested and the reference sequence are input into a computer, and the program parameters of the sequence algorithm are set. If necessary, the coordinates of the subsequence are set. Subsequently, the sequence alignment algorithm calculates the percentage of sequence identity of the sequence to be tested relative to the reference sequence according to the set program parameters.

[0177] Optimal sequence alignment for comparison can be achieved by the following methods: for example, using the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 1981; 2: 482), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 1970; 48: 443), the similarity search method of Pearson and Lipman (Proc. Nat’l. Acad. Sci. USA 1988; 85: 2444), or by digital processing using these algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI)), or by visual inspection (see Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., Supplement 1995 (Ausubel)).

[0178] For example, BLAST and BLAST 2.0, as algorithms suitable for determining the percentage of sequence identity and sequence similarity, were initially described in two papers: Altschul et al., J. Mol. Biol. 1990; 215: 403 - 410 and Altschul et al., Nucleic Acids Res. 1997. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies short words of length W in the sequence to be tested. When these short words are aligned with words of the same length in the database sequence, they either match or reach a certain positive threshold T, thereby identifying high-scoring sequence pairs (HSPs). T is called the neighborhood word score threshold (Altschul et al., ibid.). These initial neighborhood words serve as seeds for the initial search to find longer HSPs that contain the initial neighborhood words. Then, the word matches are extended in both directions along each sequence as long as the cumulative alignment score can increase.

[0179] For nucleotide sequences, the cumulative score is calculated using parameters M (a score bonus for a matching residue pair; always > 0) and N (a score penalty for a non-matching residue pair; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the string in either direction is stopped when any of the following occurs: the cumulative alignment score drops by X from the maximum value it reached; the cumulative score ≤ 0 due to the presence of one or more negative residue pairs; or the end of either sequence has been reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. For the BLASTN program applied to nucleotide sequence alignment, the default parameter values are set as follows: word length (W) is 11, expectation value (E) is 10, M = 5, N = -4, and both strands are aligned. For the BLASTP program applied to amino acid sequences, the default parameter values are set as follows: word length (W) is 3, expectation value (E) is 10, and the BLOSUM62 scoring matrix is used (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 1989; 89: 10915).

[0180] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 1989; 89: 10915). One criterion for determining similarity by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability of a chance match between two nucleotide or amino acid sequences. For example, when a test nucleic acid is aligned for similarity with a reference nucleic acid, if its minimum sum probability is approximately less than 0.1, the two are considered to have sequence similarity, where preferably the minimum sum probability is approximately less than 0.01, and most preferably the minimum sum probability is approximately less than 0.001.

[0181] Another basis for determining that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid has immunological cross-reactivity with the polypeptide encoded by the second nucleic acid (as described below). Thus, for example, when two polypeptides differ only by conservative substitutions, the amino acid sequences of the first and the second are generally considered to be substantially identical. Another basis for determining that two nucleic acid sequences are substantially identical is whether the two molecules can hybridize to each other under stringent conditions.

[0182] As used herein, the term "isolated" refers to a biological component (such as a nucleic acid, peptide, or protein) that has been sufficiently separated, produced, or purified from another biological component in which it naturally occurs (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins). Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. If a composition of "isolated" nucleic acids, peptides, and proteins is not part of the natural environment of those nucleic acids, peptides, or proteins, then the nucleic acids, peptides, and proteins are considered to be in an "isolated" state even if they have not been separated from the composition. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0183] As used herein, the term "polynucleotide" (synonymous with "nucleic acid molecule", "nucleotide", or "nucleic acid") refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, single- and double-stranded hybrid DNA, and single-stranded and double-stranded RNA, single- and double-stranded hybrid RNA, and hybrid molecules composed of DNA and RNA, which can be single-stranded, double-stranded, or single- and double-stranded composite hybrid molecules, among which the more classical ones are double-stranded or single- and double-stranded composite hybrid molecules. In addition, the term "polynucleotide" also includes triple-stranded structures containing RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA with backbone modifications for stability or other reasons. For example, "modified" bases include tritylated bases and rare bases such as hypoxanthine. DNA and RNA can be modified in various ways; thus, "polynucleotide" includes both polynucleotides in various chemically, enzymatically, or metabolically modified forms commonly found in nature, as well as the chemical forms of DNA and RNA unique to viruses and cells. "Polynucleotide" also includes relatively short nucleic acid chains, commonly referred to as oligonucleotides.

[0184] As used herein, the term "vector" refers to a replicon into which other nucleic acid segments can be operably inserted so as to effect the replication or expression of those segments.

[0185] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the present invention. A "host cell" can be any type of cell, such as a primary cell, a cultured cell, or a cell derived from a cell line. In one embodiment, the "host cell" is a cell that has been transfected with the nucleic acid molecule of the present invention. In another embodiment, the "host cell" is a progeny cell or a potential progeny cell of the above transfected cell. The progeny cells of a cell may be the same as or different from the parental cell, which may be affected by the environment, mutations in the progeny cells, or due to the integration of exogenous nucleic acid molecules into the host cell genome.

[0186] As used herein, the term "expression" refers to the biosynthesis of a gene product. This term encompasses the process of gene transcription into RNA. This term also encompasses the process of RNA translation into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed antibody can be located within the cytoplasm of the host cell, secreted into the extracellular environment (such as the growth medium of cell culture), or anchored to the cell membrane.

[0187] The terms "peptide", "polypeptide", or "protein" as used in the present invention may refer to a molecule composed of amino acids, which is well-known to those skilled in the art. The present invention uses the conventional single-letter or three-letter codes for amino acid residues. The terms "peptide", "polypeptide", and "protein" refer to amino acid polymers of any length, and the three terms have the same meaning in the present invention. The above polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. These terms also encompass amino acid polymers modified by natural modifications or intervention modifications; for example, disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other modification method such as coupling with a labeled component. This definition also includes polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), and other modification methods known in the art.

[0188] The peptide sequences described herein are written according to the conventional convention, that is, from left to right is the N-terminal region to the C-terminal region of the peptide chain. Although amino acids exist in isomeric forms, unless otherwise specifically stated, they are all represented by the L-form of amino acids.

[0189] Chimeric antigen receptor (CAR)

[0190] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide that includes at least one extracellular domain that specifically binds an antigen or target, a transmembrane domain, and an intracellular T cell receptor activation signal transduction domain. When the extracellular domain of the CAR binds to a target antigen on the surface of a target cell, the CAR aggregates and transmits an activation signal to the cell containing the CAR. The CAR can redirect the specificity of immune effector cells and trigger cell proliferation, cytokine production, phagocytosis, and / or the production of molecules that can mediate the death of cells expressing the target antigen in a major histocompatibility complex (MHC)-independent manner.

[0191] In one aspect, the CAR includes an antigen-binding domain, a hinge region, a co-stimulatory domain, an activation domain, and a transmembrane region. In one aspect, the CAR includes an antigen-binding domain, a hinge region, two co-stimulatory domains, an activation domain, and a transmembrane region. In one aspect, the CAR includes two antigen-binding domains, a hinge region, a co-stimulatory domain, an activation domain, and a transmembrane region. In one aspect, the CAR includes two antigen-binding domains, a hinge region, two co-stimulatory domains, an activation domain, and a transmembrane region.

[0192] As used herein, the term "signal peptide" refers to a leading sequence at the amino terminus (N-terminus) of the nascent CAR protein that directs the nascent protein into the endoplasmic reticulum by co-translational or post-translational means and subsequently enables cell surface expression.

[0193] As used herein, the terms "extracellular antigen-binding domain", "extracellular domain", or "extracellular ligand-binding domain" refer to the portion of the CAR that is located outside the cell membrane and is capable of binding an antigen, target, or ligand.

[0194] As used herein, the term "hinge region" refers to the portion of the CAR that is used to connect two adjacent domains of the CAR protein (e.g., the extracellular domain and the transmembrane domain).

[0195] As used herein, the term "transmembrane domain" refers to the portion of the CAR that spans the cell membrane and anchors the CAR to the cell membrane. Sometimes, this portion is also referred to as the "transmembrane region".

[0196] Co-stimulatory domain

[0197] In this text, chimeric antigen receptors can incorporate co-stimulatory (signal transduction) domains to enhance their potency. Co-stimulatory (signal transduction) domains can be derived from co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules required for an effective immune response in addition to antigen receptors or their ligands. Co-stimulatory domains derived from co-stimulatory molecules include, but are not limited to, CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, programmed death receptor-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1; CD11a and CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, immunoglobulin alpha (CD79a), DAP10, Fcγ receptor, major histocompatibility complex (MHC) class I molecule, tumor necrosis factor receptor (TNFR), integrin, signal transduction lymphocytic activation molecule, B and T lymphocyte attenuator (BTLA), Toll-like ligand receptor, intercellular adhesion molecule-1 (ICAM-1), CDS, glucocorticoid-induced tumor necrosis factor receptor (GITR), B cell activating factor receptor (BAFFR), LIGHT, herpesvirus entry mediator (HVEM, also known as LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, interleukin-2 receptor beta (IL-2Rβ), interleukin-2 receptor gamma (IL-2Rγ), interleukin-7 receptor alpha (IL-7Rα), integrin alpha4 (ITGA4), very late antigen 1 (VLA1), CD49a, IA4, CD49D, integrin alpha6 (ITGA6), VLA-6, CD49f, integrin alphaD (ITGAD), integrin alphaE (ITGAE), CD103, integrin alphaL (ITGAL), CD1a, CD1b, CD1c, CD1d, integrin alphaM (ITGAM), integrin alphaX (ITGAX), integrin beta1 (ITGB1), CD29, integrin beta2 (ITGB2, also known as CD18), integrin beta7, NKG2D, TNFR2, tumor necrosis factor-related activation-induced cytokine (TRANCE) / receptor activator of nuclear factor-kappa B ligand (RANKL), DNAX accessory molecule 1 (DNAM1, also known as CD226), SLAMF4 (CD244,2B4), CD84, CD96 (tactile receptor), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), T cell-like immunoglobulin and mucin domain molecule (CRTAM), lymphocyte antigen 9 (Ly9, also known as CD229), CD160 (BY55), P-selectin glycoprotein ligand 1 (PSGL1), CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), signaling lymphocyte activation molecule (SLAM, also known as SLAMF1, CD150, IPO-3), B lymphocyte activation-related molecule (BLAME, also known as SLAMF8), selectin P ligand (SELPLG, also known as CD162), lymphotoxin β receptor (LTBR), linker for activation of T cells (LAT), GRB2-related adaptor protein 2 (GADS), SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), phosphoprotein associated matrix adaptor molecule (PAG / Cbp), CD19a, CD83 ligand, cytokine receptor, activating natural killer (NK) cell receptor, or a fragment or any combination thereof.,

[0198] Activation domain

[0199] In the present disclosure, the chimeric antigen receptor may comprise an activation domain. The activation domain includes, but is not limited to, CD3. CD3 is a component of the T cell receptor on natural T cells, and it has been demonstrated that CD3 is an important intracellular activation component in CARs. In a preferred embodiment, the CD3 is CD3ζ.

[0200] Hinge region

[0201] In the present disclosure, the chimeric antigen receptor comprises a hinge region. This region is part of the extracellular domain and is sometimes also referred to as the "spacer" region. Various hinges can be used in the present invention, including the co-stimulatory molecules, immunoglobulin (Ig) sequences or other suitable molecules described above, to maintain a specific spacing from the target cell. In some embodiments, the entire extracellular region comprises the hinge region.

[0202] Transmembrane region

[0203] In this document, chimeric antigen receptors (CARs) may include a transmembrane domain / region. The CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. The transmembrane domain can similarly be fused to the intracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is utilized. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. The transmembrane domain can be natural or derived from an artificial source. If of natural origin, the domain can be derived from any membrane-bound or transmembrane protein.Particularly useful transmembrane regions of the present invention can be derived from (i.e., comprise or be engineered from), but are not limited to, the following proteins: CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, programmed cell death protein-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1; CD11a and CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, immunoglobulin alpha chain (CD79a), DAP10, Fcγ receptor, MHC class I molecule, TNFR, integrin, signaling lymphocyte activation molecule, BTLA, Toll-like ligand receptor, ICAM-1, CDS, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1a, CD1b, CD1c, CD1d, ITGAM, ITGAX, ITGB1, CD29, ITGB2 (CD18), ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, cytokine receptor, activating NK cell receptor, immunoglobulin protein, or fragments or any combination thereof.

[0204] Immune cell

[0205] According to certain aspects, the cells provided by the present invention are immune cells, which comprise an isolated polynucleotide or a vector comprising the isolated polynucleotide, and the polynucleotide comprises a nucleotide sequence encoding the CAR provided herein. An immune cell comprising the isolated polynucleotide and / or vector of the present invention is referred to as an "engineered immune cell". Preferably, the engineered immune cell is of human origin (i.e., a human cell before recombinant modification).

[0206] For example, the engineered immune cell can be a cell of the lymphoid lineage. Non-limiting examples of cells of the lymphoid lineage include T cells and natural killer (NK) cells. T cells express a T cell receptor (TCR), where most T cells express an α chain and a β chain, while a minority population expresses a γ chain and a δ chain. The T cells that can be used as the engineered immune cells of the present invention can be CD4 + or CD8 + , including but not limited to helper T cells (CD4 + ), cytotoxic T cells (also known as cytotoxic T lymphocytes, CTLs; CD8 + cells), memory T cells (including central memory T cells, stem cell-like memory T cells, and effector memory T cells), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Other classical immune cells include but are not limited to macrophages, antigen-presenting cells (APCs), or any immune cells expressing inhibitors of cell-mediated immune responses, such as immune checkpoint inhibitory pathway receptors (such as PD-1). Hematopoietic stem cells and / or progenitor cells are the immune precursor cells used in the present invention and can be obtained through various methods known in the art, such as from bone marrow, umbilical cord blood, and adult peripheral blood after cytokine treatment. After being engineered, the immune cells recombinantly express the chimeric antigen receptors (CARs) of the present invention.

[0207] Immune cells and their precursor cells can be isolated by methods known in the art (including commercially available methods) (Rowland Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, NY 1999). Sources of immune cells or their precursor cells include but are not limited to peripheral blood, umbilical cord blood, bone marrow, or other hematopoietic cells. A variety of techniques can be used to isolate cells and obtain and enrich them. For example, negative selection can be used to remove unwanted immune cells. In addition, positive selection can be used to isolate or enrich the desired immune cells or their precursor cells, or both of the above methods can be used simultaneously. If a specific type of cell (such as a specific T cell) needs to be isolated, the cells can be isolated by cell surface markers or a combination of markers (such as, CD3, CD4, CD8, CD34).

[0208] In the treatment methods of the present invention, the immune cells or their precursor cells can be autologous or non - autologous (i.e., allogeneic) for the subject being administered. Autologous cells are engineered immune cells obtained by isolating immune cells from a subject and further recombinantly expressing the above - mentioned chimeric antigen receptor (CAR). Such cells are also obtained by leukapheresis, i.e., selectively removing white blood cells from the withdrawn blood, recombinantly modifying them, and then reinfusing them into the donor. Optionally, the immune cells or their precursor cells can also use allogeneic cells from a non - autologous donor (i.e., not the subject). In the case of a non - autologous donor, the cells need to be typed and matched for human leukocyte antigen (HLA) to determine the degree of suitability of the match. For both autologous and non - autologous cells, they can be selectively cryopreserved until ready for use.

[0209] A variety of methods have been previously described for isolating immune cells to recombinantly express the CAR of the present invention, and any of these methods can be employed, including but not limited to: using peripheral donor lymphocytes (Sadelain et al., Nat. Rev. Cancer 2003; 3: 35 - 45; Morgan et al., Science 2006; 314: 126 - 9); using lymphocyte cultures derived from tumor - infiltrating lymphocytes (TILs) in tumor biopsy tissues (Panelli et al., J. Immunol. 2000; 164: 495 - 504; Panelli et al., J. Immunol. 2000; 164: 4382 - 92); and using antigen - specific peripheral blood leukocytes selectively expanded in vitro with the aid of artificial antigen - presenting cells (AAPCs) or dendritic cells (Panelli et al., J. Immunol. 2000; 164: 495 - 504; Panelli et al., J. Immunol. 2000; 164: 4382 - 92). If stem cells are used, the cells can be isolated by methods well - known in the art (e.g., see Panelli et al., J. Immunol. 2000; 164: 495 - 504; Panelli et al., J. Immunol. 2000; 164: 4382 - 92).

[0210] In a specific embodiment, the method for preparing the engineered immune cells comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs as described in the embodiments of the present invention. Methods for preparing immune cells for immunotherapy are described in WO2014 / 130635, WO2013 / 176916 and WO2013 / 176915, which are incorporated herein by reference. The individual steps for preparing the engineered immune cells are disclosed in WO2014 / 039523, WO2014 / 184741, WO2014 / 191128, WO2014 / 184744 and WO2014 / 184143, which are also incorporated herein by reference.

[0211] In one specific embodiment, immune effector cells (such as T cells) are genetically modified by the CAR of the present invention (for example, transduced by a viral vector containing nucleic acid encoding the CAR), and then activated and expanded in vitro. In different embodiments, T cells can be genetically modified before or after expressing the CAR, using the methods described in detail in US6352694, US6534055, US6905680, US6692964, US5858358, US6887466, US6905681, US7144575, US7067318, US7172869, US7232566, US7175843, US5883223, US6905874, US6797514, US6867041, US2006 / 121005, which are incorporated herein by reference. T cells can be expanded in vitro or in vivo. Generally, the T cells of the present invention can be expanded by contacting with a surface that is attached with substances that can stimulate signals associated with the CD3 / TCR complex and ligands that can stimulate co-stimulatory molecules on the T cell surface. In addition, a T cell population can be stimulated by the above methods, for example, by specifically binding to an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD3 antibody immobilized on the cell surface, or by co-acting with a protein kinase C activator (such as bryostatin) and a calcium ionophore, or by activating the CAR itself. And co-stimulate the co-stimulatory molecules by using ligands that bind to co-stimulatory molecules on the T cell surface. For example, to stimulate T cell proliferation, a T cell population is specifically bound to an anti-CD3 antibody and an anti-CD28 antibody under suitable conditions. Suitable culture conditions for T cells include using a suitable culture medium (such as using MEM medium or RPMI1640 medium or X-vivo 5 medium), which contains factors necessary for cell proliferation and survival, such as serum (such as fetal bovine serum or human serum), cytokines (such as IL-2, IL-7, IL-15, and / or IL-21), insulin, IFN-g, GM-CSF, TGFβ, and / or any other cell growth additives known to those skilled in the art. In other embodiments, methods described in US6040177, US5827642, and WO2012129514 can be used to activate and stimulate T cells with feeder layer cells, suitable antibodies, and cytokines to promote their proliferation, and the above-mentioned documents are incorporated herein by reference.

[0212] Antibody and antigen-binding domain

[0213] The present invention generally relates to isolated anti-5T4 antibodies, chimeric antigen receptors (CARs), nucleic acids encoding said antibodies and CARs, and expression vectors, recombinant cells comprising the vectors, and compositions comprising the antibodies, CARs, and recombinant cells capable of expressing CARs. The present invention also relates to methods for preparing said antibodies and CARs, and methods for using said antibodies and CARs to treat diseases including cancer, inflammation, and autoimmune diseases. The antigen-binding domains of the antibodies and CARs of the present invention have one or more excellent functional characteristics, including but not limited to: high-affinity binding to 5T4, high specificity for 5T4, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing 5T4, and the ability to inhibit in vivo tumor growth in subjects and animal models when used alone or in combination with other anti-cancer therapies.

[0214] In summary, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind 5T4.

[0215] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including humanized antibodies, bispecific antibodies, and chimeric antibodies, as well as monoclonal or polyclonal antibody fragments. Generally, an antibody is a protein or peptide chain capable of specifically binding to a specific antigen, and its structural characteristics are well known to those skilled in the art. Immunoglobulins can be classified into five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) according to the amino acid sequence of the heavy chain constant region. Among them, IgA and IgG can be further subdivided into subtypes: IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibodies of the present invention can belong to any one of the above five major classes or the corresponding subtypes. Preferably, the antibodies of the present invention are of the IgG1, IgG2, IgG3, or IgG4 type. According to the amino acid sequence of the light chain constant region, the antibody light chains of vertebrate species can be divided into two classes, namely, the κ type and the λ type. Therefore, the antibodies of the present invention should comprise a κ-type or λ-type light chain constant region. According to the specific embodiments, the antibodies of the present invention comprise heavy and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light chain constant regions, an antibody also comprises an antigen-binding region composed of a light chain variable region and a heavy chain variable region, and each variable region comprises three complementarity-determining regions (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The three complementarity-determining regions of the light chain variable region can be referred to as LCDR1, LCDR2, and LCDR3, respectively, and the three complementarity-determining regions of the heavy chain variable region can be referred to as HCDR1, HCDR2, and HCDR3, respectively.

[0216] "Complementary determining region" (CDR) is the region in an antibody that binds an antigen. CDRs can be defined by a variety of delineation methods, such as the Kabat method (Wu et al., J Exp Med 132:211-50, 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia method (Chothia et al., J Mol Biol 196:901-17, 1987), the International Immunogenetics Information System (IMGT) method (Lefranc et al., Dev Comp Immunol 27:55-77, 2003), and the AbM method (Martin and Thornton, J Bmol Biol 263:800-15, 1996). The correspondence between different delineation methods and variable region numbering has been described (e.g., Lefranc et al., Dev Comp Immunol 27:55-77, 2003; Honegger and Pluckthun, J Mol Biol 309:657-70, 2001; International Immunogenetics Information System (IMGT) database; web resource: http: / / www_imgt_org). Existing programs such as abYsis provided by UCL Business PLC can be used to delineate CDRs. Unless otherwise expressly stated in the specification, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCDR3" as used in the present invention include CDRs defined by any of the above methods (Kabat method, Chothia method, IMGT method, or AbM method). Those skilled in the art should be familiar with the correspondence between different numbering systems (e.g., Kabat numbering and the specific numbering system of IMGT) (see, e.g., Kabat; Chothia; Martin; Lefranc, etc.).

[0217] Table A Complementary determining regions (CDRs) delineated using different methods

[0218]

[0219]

[0220] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds 5T4 is substantially free of antibodies that do not bind the same 5T4). In addition, the isolated antibody is substantially free of other cellular materials and / or chemicals.

[0221] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., each antibody in the group is identical, except for possible minor mutations that may have occurred spontaneously. The monoclonal antibodies of the present invention can be prepared by hybridoma means, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, the monoclonal antibody can be prepared by hybridoma techniques, where the hybridoma is obtained from B cells in a non-human transgenic animal (such as a transgenic mouse or rat) whose genome contains a human transgenic heavy chain and light chain.

[0222] As used herein, the terms "antigen-binding fragment" and / or "antigen-binding domain" refer to antibody fragments such as diabodies, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-stabilized antibodies (dsFv), (dsFv)2 fragments, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bispecific diabodies), multispecific antibodies, antibody portions of antibodies having one or more complementarity-determining regions (CDRs), camelid single-domain antibodies, nanobodies, domain antibodies, bispecific domain antibodies, or any other antibody fragment that can specifically bind an antigen but does not have a complete structure. The antigen-binding fragment is capable of specifically binding to the parent antibody or parent antibody fragment. In a specific embodiment, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and the Fd segment of the heavy chain. In other specific embodiments, the antigen-binding fragment comprises Fab and F(ab'). The antigen-binding domain is capable of binding the same antigen as the parent antibody binds. In a specific embodiment, the antigen-binding domain comprises a single-chain antibody molecule (scFv).

[0223] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art, in which the heavy chain variable region and the light chain variable region are linked by a short peptide of about 15 to 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region, or comprises only a heavy chain variable region.

[0224] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to that of an antibody produced by a human using techniques known in the art. The definition of a human antibody includes intact or full-length antibodies and fragments, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0225] As used herein, the term "humanized antibody" and / or "humanized antigen-binding domain" refers to the modification of a non-humanized antibody and / or non-humanized antigen-binding domain to increase its sequence homology, thereby retaining the antigen-binding properties of the antibody and / or antigen-binding domain while reducing its antigenicity in humans.

[0226] As used herein, the term "chimeric antibody" and / or "chimeric antigen-binding domain" refers to an antibody and / or antigen-binding domain in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of the light and heavy chains generally correspond to the variable regions of an antibody and / or antigen-binding domain derived from a mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and performance, while the constant regions correspond to the sequences of an antibody and / or antigen-binding domain derived from another mammalian species (e.g., human) to avoid inducing an immune response in that species.

[0227] As used herein, the term "multispecific antibody" refers to an antibody comprising multiple immunoglobulin variable region sequences, wherein the first immunoglobulin variable region sequence among the multiple sequences has binding specificity for a first epitope, and the second immunoglobulin variable region sequence among the multiple sequences has binding specificity for a second epitope. In one embodiment, both the first epitope and the second epitope are located on the same antigen, e.g., on the same protein (or the same subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope do not overlap or substantially do not overlap. In one embodiment, the first epitope and the second epitope are located on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable region. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetravalent antibody molecule.

[0228] The term "bispecific antibody" as used in the present invention refers to a multispecific antibody that binds to no more than two epitopes or two antigens. The bispecific antibody is characterized by a first immunoglobulin variable region sequence having binding specificity for a first epitope and a second immunoglobulin variable region sequence having binding specificity for a second epitope. In one embodiment, both the first epitope and the second epitope are located on the same antigen, such as on the same protein (or the same subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope are located on different antigens, such as on different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody comprises a heavy chain variable region sequence and a light chain variable region sequence having binding specificity for a first epitope, and a heavy chain variable region sequence and a light chain variable region sequence having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises a half antibody or a fragment thereof having binding specificity for a first epitope, and a half antibody or a fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises a scFv or a fragment thereof having binding specificity for a first epitope, and a scFv or a fragment thereof having binding specificity for a second epitope. In one embodiment, the first epitope is located on 5T4 of the present invention, and the second epitope is located on PD-1, PD-L1, TIM-3, LAG-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD3, CD73, CD47, TIP-1, GPC3, apelin, DLL3, folate receptor α, Claudin 18.2, MUC16, mesothelin, IL13Ra2, PSCA, EGFRvIII, p95HER2, ROR1, ROR2, NKp46 and / or other tumor-associated immunosuppressors or surface antigens. In one embodiment, the first epitope and the second epitope are located on the same 5T4 of the present invention.

[0229] The antibody and / or antigen-binding domain that "specifically binds to 5T4" as used in the present invention refers to an antibody and / or antigen-binding domain that binds to 55T4, preferably human 5T4, with a KD value of less than or equal to 1×10 -7 M, preferably less than or equal to 1×10 -8 M, more preferably less than or equal to 5×10 -9 M, less than or equal to 1×10 -9 M, less than or equal to 5×10 -10 M, or less than or equal to 1×10 -10M. The term "KD" refers to the dissociation constant, which is obtained by the ratio of the dissociation rate constant (Kd) to the association rate constant (Ka) (i.e., Kd / Ka) and is expressed in molar concentration (M). According to the disclosure of the present invention, those skilled in the art can determine the KD value of an antibody and / or antigen-binding domain using methods known in the art. For example, it can be determined by surface plasmon resonance technology (such as using a biosensor system such as the

[0230] system) or biolayer interferometry (such as using the Octet RED96 system). The smaller the KD value of an antibody and / or antigen-binding domain, the higher the affinity of the antibody and / or antigen-binding domain for binding to the target antigen.

[0231] The term "IC 50 " used in the present invention refers to the half-maximal inhibitory concentration of the monoclonal antibody or bispecific antibody of the present invention or their antigen-binding fragments. IC 50 is an index for evaluating the ability of the monoclonal antibody or bispecific antibody of the present invention or their antigen-binding fragments to inhibit the binding of the target antigen (i.e., receptor or ligand) to its natural ligand or receptor, or to inhibit the function of the target antigen in cells. In certain embodiments, the KD value of the monoclonal antibody or its antigen-binding fragment, or the bispecific antibody or its antigen-binding fragment is about less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M or less than 10 -13 M.

[0232] The term "EC 50 " used in the present invention refers to the half-maximal effective concentration of the monoclonal antibody or bispecific antibody of the present invention or their antigen-binding fragments. EC 50 refers to the concentration of the antibody or its antigen-binding fragment required for the monoclonal antibody or bispecific antibody or their antigen-binding fragments to induce a biological effect (i.e., cell death) to reach half of the effect between the baseline level and the maximum level within a specified exposure time. In certain embodiments, the EC 50a value of less than about 1 μM, or in the range of about 1000 nM - about 100 nM, or in the range of about 100 nM - about 10 nM, or in the range of about 10 nM - about 1 nM, or in the range of about 1000 pM - about 500 pM, or in the range of about 500 pM - about 200 pM, or less than about 200 pM, or in the range of about 200 pM - about 150 pM, or in the range of about 200 pM - about 100 pM, or in the range of about 100 pM - about 10 pM, or in the range of about 10 pM - about 1 pM.

[0233] According to certain aspects, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, or a chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the monoclonal antibody or the antigen-binding fragment thereof or the antigen-binding domain comprises: a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, the light chain variable region comprises light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, and these complementarity-determining regions have the following polypeptide sequences:

[0234] (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively;

[0235] (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively;

[0236] (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively;

[0237] (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively;

[0238] (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively;

[0239] (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, respectively, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively;

[0240] (7) are SEQ ID NOs: 87, 88, 89, 90, 91 and 92, respectively, or are SEQ ID NOs: 93, 94, 95, 96, 97 and 98, respectively;

[0241] (8) are SEQ ID NOs: 101, 102, 103, 104, 105 and 106, respectively, or are SEQ ID NOs: 107, 108, 109, 110, 111 and 112, respectively;

[0242] (9) are SEQ ID NOs: 115, 116, 117, 118, 119 and 120, respectively, or are SEQ ID NOs: 121, 122, 123, 124, 125 and 126, respectively;

[0243] (10) are SEQ ID NOs: 129, 130, 131, 132, 133 and 134, respectively, or are SEQ ID NOs: 135, 136, 137, 138, 139 and 140, respectively; or

[0244] (11) are SEQ ID NOs: 143, 144, 145, 146, 147 and 148, respectively, or are SEQ ID NOs: 149, 150, 151, 152, 153 and 154, respectively

[0245] Wherein, the antibody or its antigen-binding fragment or antigen-binding domain specifically binds to 5T4, preferably human 5T4.

[0246] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof or a chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the monoclonal antibody or the antigen-binding fragment thereof or the antigen-binding domain comprises a heavy-chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to any one of SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127 or 141; or comprises a light-chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128 or 142. According to a preferred embodiment, the isolated monoclonal antibody or the antigen-binding fragment thereof or the antigen-binding domain of the present invention comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to any one of SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127 or 141; and the light-chain variable region having a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128 or 142.

[0247] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof or an antigen-binding domain thereof, comprising:

[0248] (a) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 1 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 2.

[0249] (b) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 15 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 16;

[0250] (c) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 29 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 30;

[0251] (d) a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 43 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 44;

[0252] (e) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58;

[0253] (f) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72;

[0254] (g) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86;

[0255] (h) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100;

[0256] (i) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114;

[0257] (j) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128; or

[0258] (k) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142.

[0259] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having SEQ ID NO: 3, 4, 5, 6, 7 and 8, respectively, or having the polypeptide sequences shown in SEQ ID NO: 9, 10, 11, 12, 13 and 14, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 1; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 2. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 1; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 2.

[0260] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 17, 18, 19, 20, 21 and 22, respectively, or having the polypeptide sequences shown in SEQ ID NO: 23, 24, 25, 26, 27 and 28, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical polypeptide sequence to SEQ ID NO: 15; and comprises a light chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical polypeptide sequence to SEQ ID NO: 16. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 15; and comprises a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 16.

[0261] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 31, 32, 33, 34, 35 and 36, respectively, or having the polypeptide sequences shown in SEQ ID NO: 37, 38, 39, 40, 41 and 42, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical polypeptide sequence to SEQ ID NO: 29; and comprises a light chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical polypeptide sequence to SEQ ID NO: 30. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 29; and comprises a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 30.

[0262] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 45, 46, 47, 48, 49 and 50, respectively, or having the polypeptide sequences shown in SEQ ID NO: 51, 52, 53, 54, 55 and 56, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 43; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 44. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 43; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 44.

[0263] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 59, 60, 61, 62, 63 and 64, respectively, or having the polypeptide sequences shown in SEQ ID NO: 65, 66, 67, 68, 69 and 70, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 57; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 58. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58.

[0264] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having polypeptide sequences shown in SEQ ID NO: 73, 74, 75, 76, 77 and 78 respectively, or having polypeptide sequences shown in SEQ ID NO: 79, 80, 81, 82, 83 and 84 respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 71; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 72. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 71; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72.

[0265] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having polypeptide sequences shown in SEQ ID NO: 87, 88, 89, 90, 91 and 92 respectively, or having polypeptide sequences shown in SEQ ID NO: 93, 94, 95, 96, 97 and 98 respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 85; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 86. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 85; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86.

[0266] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 101, 102, 103, 104, 105 and 106, respectively, or having the polypeptide sequences shown in SEQ ID NO: 107, 108, 109, 110, 111 and 112, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 99; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 100. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100.

[0267] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 115, 116, 117, 118, 119 and 120, respectively, or having the polypeptide sequences shown in SEQ ID NO: 121, 122, 123, 124, 125 and 126, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 113; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 114. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114.

[0268] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 129, 130, 131, 132, 133 and 134, respectively, or having the polypeptide sequences shown in SEQ ID NO: 135, 136, 137, 138, 139 and 140, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 127; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 128. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128.

[0269] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences shown in SEQ ID NO: 143, 144, 145, 146, 147 and 148, respectively, or having the polypeptide sequences shown in SEQ ID NO: 149, 150, 151, 152, 153 and 154, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 141; and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 142. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142.

[0270] According to another specific aspect, the present invention relates to an isolated monoclonal antibody of the present invention, or an antigen-binding fragment or antigen-binding domain thereof, wherein the monoclonal antibody or its antigen-binding fragment or antigen-binding domain is a chimera.

[0271] According to another specific aspect, the present invention relates to an isolated monoclonal antibody of the present invention, or an antigen-binding fragment or antigen-binding domain thereof, wherein the monoclonal antibody or its antigen-binding fragment or antigen-binding domain is human or humanized.

[0272] According to another specific aspect, the humanized monoclonal antibody or its antigen-binding fragment or humanized antigen-binding domain comprises a heavy chain variable region having a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 155, 156, 159, 160, 161, 165, 166, 169, 170, 173, 174, 177, 178 or 179; or comprises a light chain variable region having a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 157, 158, 162, 163, 164, 167, 168, 171, 172, 175, 176, 180 or 181.

[0273] According to another specific aspect, the humanized monoclonal antibody or its antigen-binding fragment or humanized antigen-binding domain comprises:

[0274] (1) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0275] (2) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0276] (3) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0277] (4) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0278] (5) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0279] (6) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0280] (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0281] (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0282] (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0283] (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0284] (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0285] (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0286] (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0287] (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0288] (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0289] (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0290] (17) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0291] (18) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0292] (19) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0293] (20) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0294] (21) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0295] (22) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0296] (23) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0297] (24) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0298] (25) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0299] (26) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0300] (27) The heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and the light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0301] (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0302] (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0303] (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180; or

[0304] (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181.

[0305] According to another specific aspect, the antigen-binding domain is a single-chain variable fragment (scFv).

[0306] In certain embodiments, the encoded antigen-binding domain is a humanized single-chain variable fragment (scFv).

[0307] According to another specific aspect, the chimeric antigen receptor comprises one or more antigen-binding domains.

[0308] According to another specific aspect, the intracellular signaling domain comprises one or more co-stimulatory domains and one or more activation domains.

[0309] In another general aspect, the present invention relates to an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof and / or bispecific antibody or antigen-binding fragment thereof of the present invention. In another general aspect, the present invention relates to an isolated polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the antigen-binding domain of the present invention. Those skilled in the art know that the coding sequence of a protein can be changed (such as substitution, deletion, insertion, etc.) without changing the amino acid sequence. Therefore, those skilled in the art know that the nucleic acid sequence encoding these antibodies or antigen-binding fragments can be changed without changing the amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0310] In another general aspect, the present invention relates to a vector comprising a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof, a bispecific antibody or an antigen-binding fragment thereof, and / or a CAR of the present invention. Any vector well-known to those skilled in the art can be used in the present invention, such as plasmids, cosmids, phage vectors or viral vectors. In certain embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector contains various elements for realizing the basic functions of an expression vector, such as a promoter, a ribosome binding site element, a terminator, an enhancer, a selection marker and an origin of replication. The promoter can be a constitutive promoter, an inducible promoter or a repressor promoter. Many expression vectors capable of delivering nucleic acid molecules into cells are known in the art and can thus be used to produce an antibody or an antigen-binding fragment thereof intracellularly. According to various embodiments of the present invention, recombinant expression vectors can be generated by conventional cloning techniques or artificial gene synthesis techniques.

[0311] In another general aspect, the present invention relates to a host cell comprising a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof, and / or a bispecific antibody or an antigen-binding fragment thereof of the present invention. In view of the content of the present disclosure, any host cell known to those skilled in the art can be used for recombinantly expressing the antibody or an antigen-binding fragment thereof of the present invention. In certain embodiments, the host cell can be Escherichia coli TG1 or BL21 cells (e.g., for expressing scFv fragments or Fab fragments of an antibody), or CHO-DG44 cells or CHO-K1 cells or HEK293 cells (e.g., for expressing full-length IgG antibodies). In a specific embodiment, the recombinant expression vector is transformed into the host cell by conventional techniques such as chemical transfection, heat shock or electroporation and is stably integrated into the host cell genome so as to effectively express the recombinant nucleic acid.

[0312] In another general aspect, the present invention relates to a method for preparing a monoclonal antibody or an antigen-binding fragment thereof, and / or a bispecific antibody or an antigen-binding fragment thereof of the present invention, the method comprising: culturing a cell under conditions for producing a monoclonal antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof of the present invention, the cell comprising a nucleic acid encoding the monoclonal antibody or an antigen-binding fragment thereof, or the bispecific antibody or an antigen-binding fragment thereof, and recovering the antibody or an antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or an antigen-binding fragment thereof can be obtained from the cell and purified according to conventional techniques known in the art and described herein.

[0313] In another general aspect, the present invention relates to cells transduced with a vector, said vector comprising a nucleic acid isolated encoding the CAR of the present invention. The term "transduced" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transduced" cell is a cell that has received exogenous nucleic acid. The cells include the original cells and their progeny cells. In certain embodiments, the cells are CAR-T cells, preferably human CAR-T cells, wherein the T cells are engineered to express the CAR of the present invention for treating diseases, such as cancer. In certain embodiments, the cells are CAR-NK cells, preferably human CAR-NK cells, wherein the NK cells are engineered to express the CAR of the present invention for treating diseases, such as cancer.

[0314] In another general aspect, the present invention relates to a method for preparing CAR-T cells, which includes transducing T cells with a vector comprising a nucleic acid isolated encoding the CAR of the present invention.

[0315] In another general aspect, the present invention relates to a method for preparing the CAR-T cells of the present invention, comprising: culturing T cells comprising a nucleic acid encoding the CAR of the present invention under conditions for generating the CAR-T cells; and recovering the CAR-T cells.

[0316] In another general aspect, the present invention relates to a method for preparing CAR-NK cells, comprising transducing NK cells with a vector comprising a nucleic acid isolated encoding the CAR of the present invention.

[0317] In another general aspect, the present invention relates to a method for generating the CAR-NK cells of the present invention, comprising culturing NK cells comprising a nucleic acid encoding the chimeric antigen receptor (CAR) under conditions for generating the CAR-NK cells; and recovering the CAR-NK cells.

[0318] In another general aspect, the present invention relates to a method for preparing a population of RNA-engineered cells comprising the chimeric antigen receptor (CAR) of the present invention. The method comprises contacting a population of cells with an isolated polynucleotide comprising a nucleic acid encoding the CAR of the present invention, wherein the isolated polynucleotide is in vitro transcribed RNA or synthetically produced RNA.

[0319] Pharmaceutical composition

[0320] In another general aspect, the present invention relates to a pharmaceutical composition, said pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof, bispecific antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated polypeptide, host cell, and / or engineered immune cell of the present invention, and a pharmaceutically acceptable carrier.

[0321] As used herein, the term "pharmaceutical composition" refers to a product that comprises an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a host cell of the invention, an engineered immune cell of the invention, an anti-5T4 monoclonal antibody of the invention or an antigen-binding fragment thereof, and / or a bispecific antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. The polynucleotides, polypeptides, host cells, engineered immune cells, anti-5T4 monoclonal antibodies of the invention or antigen-binding fragments thereof, and / or bispecific antibodies or antigen-binding fragments thereof of the invention, and compositions comprising them are also used to prepare a medicament for the therapeutic applications described herein.

[0322] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It should be understood that the properties of the carrier, excipient or diluent depend on the corresponding route of administration. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy or biological activity of the compositions described herein. Depending on the specific embodiment, any pharmaceutically acceptable carrier suitable for use in antibody pharmaceutical compositions can be used in the present invention.

[0323] The formulation of a pharmaceutically active ingredient with a pharmaceutically acceptable carrier is well known in the art, for example, see Remington: The Science and Practice of Pharmacy (e.g., 21st Edition (2005) and subsequent editions). Other non-limiting ingredients include, for example, buffers, diluents, solvents, isotonicity regulators, preservatives, stabilizers, and chelating agents, etc. The pharmaceutical compositions described herein can be prepared using one or more pharmaceutically acceptable carriers.

[0324] In one embodiment, the pharmaceutical composition is a liquid formulation. Preferred examples of liquid formulations are aqueous formulations (i.e., water-containing formulations). The liquid formulations can include various types such as solutions, suspensions, emulsions, microemulsions, gels, etc. Aqueous formulations generally contain at least 50% (w / w) water, or contain at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% (w / w) water.

[0325] In one embodiment, the pharmaceutical composition can be formulated as an injectable formulation, which can be injected by an injection device (such as a syringe or an infusion pump). The injectable formulation can be used for, for example, subcutaneous, intramuscular, intraperitoneal, intravitreal or intravenous delivery.

[0326] In another embodiment, the pharmaceutical composition is a solid preparation, such as a freeze-dried preparation or a spray-dried composition, which can be used directly or, before use, a solvent and / or diluent is added thereto by a physician or a patient. Solid dosage forms include tablets (such as compressed tablets and / or coated tablets) and capsules (such as hard capsules or soft capsules). The pharmaceutical composition can also be in the form of cachets, pills, powders, granules, lozenges or powders for reconstitution.

[0327] The dosage form can be of the immediate-release type and, in this case, may contain a water-soluble or dispersible carrier, or they can be of the sustained-release, controlled-release or modified-release type and, in this case, contain a water-insoluble polymer capable of regulating the dissolution rate of the dosage form in the gastrointestinal tract or subcutaneously.

[0328] In other embodiments, the pharmaceutical composition can be delivered by routes such as intranasal, oral or sublingual.

[0329] The pH value of the aqueous preparation is in the range of pH 3 to pH 10. In one embodiment of the present invention, the pH value of the preparation is about in the range of 7.0 to 9.5. In another embodiment of the present invention, the pH value of the preparation is about in the range of 3.0 to 7.0.

[0330] In another embodiment of the present invention, the pharmaceutical composition contains a buffering agent. Non-limiting examples of buffering agents include, but are not limited to: arginine, aspartic acid, glycylglycine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tris(hydroxymethyl)aminomethane, and mixtures of the above components. The buffering agent can be present alone or in the form of a mixture, and its concentration is about in the range of 0.01 mg / ml to 50 mg / ml, for example, about in the range of 0.1 mg / ml to 20 mg / ml. Pharmaceutical compositions containing each of the buffering agents constitute alternative embodiments of the present invention.

[0331] In another embodiment of the present invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include, but are not limited to, benzalkonium chloride, benzoic acid, benzyl alcohol, bronopol, butylparaben, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethylparaben, imidurea, methylparaben, phenol, 2-phenoxyethanol, 2-phenylethanol, propylparaben, sodium dehydroacetate, thimerosal, and mixtures thereof. The preservative may be present alone or in the form of a mixture, and its concentration is in the range of about 0.01 mg / ml to 50 mg / ml, for example, in the range of about 0.1 mg / ml to 20 mg / ml. The pharmaceutical composition containing each of the preservatives constitutes an alternative embodiment of the present invention.

[0332] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of the isotonic agent include, but are not limited to, salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), alditols (such as glycerol, 1,2-propanediol, 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (such as PEG400), and mixtures thereof. Carbohydrates are another example of isotonic agents. Non-limiting examples of the carbohydrates include, but are not limited to, monosaccharides, disaccharides or polysaccharides, or water-soluble dextrans, including, for example, fructose, glucose, mannose, sorbose, xylitol, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, α- and β-hydroxypropyl-β-cyclodextrin, soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. Sugar alcohols are another example of isotonic agents, where the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at least one hydroxyl group. Non-limiting examples of sugar alcohols include, but are not limited to, mannitol, sorbitol, inositol, galactitol, galactitol, xylitol, and arabitol. The isotonic agent may be present alone or in the form of a mixture, and its concentration is in the range of about 0.01 mg / ml to 50 mg / ml, for example, in the range of about 0.1 mg / ml to 20 mg / ml. The pharmaceutical composition containing each of the isotonic agents constitutes an alternative embodiment of the present invention.

[0333] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of the chelating agent include, but are not limited to, citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) salts, and mixtures thereof. The chelating agent may be present alone or in the form of a mixture, and its concentration is in the range of about 0.01 mg / ml to 50 mg / ml, for example, in the range of about 0.1 mg / ml to 20 mg / ml. The pharmaceutical composition containing each of the chelating agents constitutes an alternative embodiment of the present invention.

[0334] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of the stabilizer include, but are not limited to, one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0335] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer, which is carboxyl or hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-(methylthio)ethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing compounds (such as thioglycerol) or mercaptoacetic acid. The stabilizer can exist alone or in the form of a mixture, and its concentration is about in the range of 0.01 mg / ml to 50 mg / ml, for example, about in the range of 0.1 mg / ml to 20 mg / ml. The pharmaceutical composition containing each of the stabilizers constitutes an alternative embodiment of the present invention.

[0336] In other embodiments of the present invention, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant or two different surfactants. The term "surfactant" is a molecule or ion that contains both water-soluble (hydrophilic) components and lipid-soluble (lipophilic) components. For example, the surfactant includes anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactant can exist alone or in the form of a mixture, and its concentration is about in the range of 0.1 mg / ml to 20 mg / ml. The pharmaceutical composition containing each of the surfactants constitutes an alternative embodiment of the present invention.

[0337] In another embodiment of the present invention, the pharmaceutical composition comprises one or more protease inhibitors, such as ethylenediaminetetraacetic acid (EDTA) and / or benzamidine hydrochloride. The protease inhibitor can exist alone or in the form of a mixture, and its concentration is about in the range of 0.1 mg / ml to 20 mg / ml. The pharmaceutical composition containing each of the protease inhibitors constitutes an alternative embodiment of the present invention.

[0338] Another general aspect of the present invention relates to a method for preparing a pharmaceutical composition, which comprises a monoclonal antibody or an antigen-binding fragment thereof of the present invention, and / or a bispecific antibody or an antigen-binding fragment thereof. The preparation method includes binding the monoclonal antibody or an antigen-binding fragment thereof, and / or the bispecific antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0339] Method of use

[0340] In another general aspect, the present invention relates to a method of targeting 5T4 on the surface of cancer cells in a subject to effect cell killing, the method comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof that specifically binds 5T4, or a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof and / or the bispecific antibody or antigen-binding fragment thereof of the present invention. Binding of the 5T4 monoclonal antibody or bispecific antibody or antigen-binding fragment thereof can mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP) and / or antibody-dependent cytotoxicity (ADCC) or other effects leading to the death of the targeted cancer cells. For example, the monoclonal antibody or bispecific antibody or antigen-binding fragment thereof can recruit a conjugate drug, and / or can form a bispecific antibody with another monoclonal antibody to mediate the death of the targeted cancer cells.

[0341] Methods known in the art and described in the present invention are generally used to characterize the functional activity of antibodies and antigen-binding fragments thereof that bind 5T4. Methods for characterizing antibodies and antigen-binding fragments thereof that bind 5T4 include, but are not limited to, affinity and specificity assays, such as Biacore, ELISA, OctetRed and GatorBio assays, and detection of the binding of antibodies and antigen-binding fragments thereof to 5T4 on cells (5T4-transfected cells or cells that naturally express 5T4) by FACS. According to specific embodiments, methods for characterizing antibodies and antigen-binding fragments thereof that bind 5T4 are described below.

[0342] In another general aspect, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof that specifically binds 5T4, or administering the pharmaceutical composition of the present invention. The cancer can be, for example, selected from, but not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0343] In another general aspect, the present invention relates to a method for treating inflammation and / or autoimmune diseases in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to 5T4, and / or a bispecific antibody or an antigen-binding fragment thereof, or administering a pharmaceutical composition of the present invention.

[0344] In another general aspect, the present invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention. The cancers include, but are not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0345] According to an embodiment of the present invention, the CAR-T cells or CAR-NK cells comprise a therapeutically effective amount of the CAR of the present invention expressed, and the pharmaceutical composition comprises a therapeutically effective amount of an anti-5T4 antibody or an antigen-binding fragment thereof (e.g., an anti-5T4 antibody). The term "therapeutically effective amount" as used in the present invention refers to the amount of the active ingredient required to induce a specific biological or pharmacological response in a subject. According to the therapeutic purpose, the therapeutically effective amount can be determined by experience and conventional methods.

[0346] For the CARs of the present invention, a therapeutically effective amount refers to the amount of CAR molecule expression in transduced T cells or NK cells that can modulate the immune response of a subject in need thereof. In addition, for the CARs of the present invention, a therapeutically effective amount refers to the amount of CAR molecule expression in transduced T cells or NK cells that results in the treatment, prevention or delay of the progression of a disease, disorder or condition, or the alleviation or complete remission of the symptoms of a disease, disorder or condition.

[0347] For the CAR-T cells or CAR-NK cells of the present invention, a therapeutically effective amount refers to the dose of CAR-T cells or CAR-NK cells that can modulate the immune response of a subject in need thereof. In addition, for the CAR-T cells or CAR-NK cells of the present invention, a therapeutically effective amount refers to the dose of CAR-T cells or CAR-NK cells that results in the treatment, prevention or delay of the progression of a disease, disorder or condition, or the alleviation or complete remission of the symptoms of a disease, disorder or condition.

[0348] For the anti-5T4 antibody or antigen-binding fragment thereof of the present invention, a therapeutically effective amount refers to the dose of the anti-5T4 antibody or antigen-binding fragment thereof that modulates the immune response of a subject in need thereof. In addition, for the anti-5T4 antibody or antigen-binding fragment thereof of the present invention, a therapeutically effective amount also refers to the dose of the anti-5T4 antibody or antigen-binding fragment thereof that results in the treatment, prevention or delay of the progression of a disease, disorder or condition, or alleviates or completely relieves the symptoms of a disease, disorder or condition.

[0349] According to a specific embodiment of the present invention, the disease, disorder or condition to be treated is cancer, preferably the following cancers: lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors. According to other specific embodiments of the present invention, the disease, disorder or condition to be treated is inflammation and / or autoimmune diseases.

[0350] According to a specific embodiment of the present invention, the therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four or five of the following effects: (i) alleviating or relieving the disease, pathological disorder or clinical condition, or the severity of its accompanying symptoms; (ii) shortening the duration of the disease, pathological disorder or clinical condition, or its accompanying symptoms; (iii) blocking the pathological progression of the disease, pathological disorder or clinical condition, or its accompanying symptoms; (iv) promoting the regression of the disease, pathological disorder or clinical condition, or its accompanying symptoms; (v) preventing the onset and progression of the disease, pathological disorder or clinical condition, or its accompanying symptoms; (vi) reducing the risk of recurrence of the disease, pathological disorder or clinical condition, or its accompanying symptoms; (vii) reducing the number of hospitalizations of a patient due to the disease, disorder or clinical condition, or due to related symptoms; (viii) shortening the length of hospitalization of a patient due to the disease, pathological disorder or clinical condition, or its accompanying symptoms; (ix) prolonging the overall survival of a patient suffering from the disease, pathological disorder or clinical condition, or its accompanying symptoms; (xi) inhibiting the pathological progression of the disease, pathological disorder or clinical condition, or its accompanying symptoms in a subject; or (xii) enhancing or improving the preventive or therapeutic effect of other treatment means.

[0351] The therapeutically effective amount or dose can vary depending on a variety of factors, including, for example, the disease, disorder or condition, the mode of administration, the target site, the physiological state of the subject (including, for example, age, weight, state of health), the species of the subject (whether the subject is human or an animal), other drugs administered, and the purpose of the treatment (whether prophylactic or therapeutic). Preferably, the therapeutic dose should be safe and effective.

[0352] According to specific embodiments of the present invention, the compositions described herein should be formulated into dosage forms suitable for administration to a subject by the intended route of administration. For example, the compositions can be formulated into dosage forms suitable for intravenous injection, subcutaneous injection or intramuscular injection.

[0353] The cells of the present invention can be administered by any suitable route of administration known to those skilled in the art. For example, the cells of the present invention can be administered to a subject by aerosol inhalation, injection, oral administration, infusion, implantation and / or transplantation, etc. The routes of administration of the cells of the present invention include, but are not limited to, intraarterial injection, subcutaneous injection, intradermal injection, intratumoral injection, intranodal injection, intramedullary injection, intramuscular injection, intrapleural injection, intravenous (i.v.) injection or intraperitoneal injection, etc. In certain embodiments, the cells of the present invention can be administered to a subject with or without lymphodepleting chemotherapy.

[0354] The pharmaceutical composition comprises the cells of the present invention expressing the CAR of the present invention, and the pharmaceutical composition can be provided as a sterile liquid preparation, usually an isotonic aqueous solution, emulsion or dispersion containing a cell suspension, and usually a buffer system is used to adjust the pH value to a preset range. The composition comprises a carrier, such as water, physiological saline, phosphate buffer solution, etc., and these carriers can maintain the integrity and activity of the cells and meet the requirements of the above-mentioned routes of administration of the cell composition.

[0355] The cells of the present invention are mixed into an appropriate amount of a suitable solvent, and other solvent components are added if necessary to prepare a sterile injection solution. The composition comprises a pharmaceutically acceptable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextran, etc., and such adjuvants are suitable for cell preparations and for administration to a subject, such as a human. Suitable buffer systems for providing cell compositions are well known in the art. Any carrier, diluent or additive used can maintain the structural integrity and viability of the cells of the present invention.

[0356] The cells of the present invention can be administered via any physiologically acceptable carrier. A cell population comprising the cells of the present invention may comprise a purified cell population. Those skilled in the art can easily perform quantitative analysis on the cell population using a variety of well-known technical means. The purity of the cell population comprising the genetically modified cells of the present invention is in the range of about 50% - about 55%, or in the range of about 55% - about 60%, or in the range of about 60% - about 65%, or in the range of about 65% - about 70%, or in the range of about 70% - about 75%, or in the range of about 75% - about 80%, or in the range of about 80% - about 85%, or in the range of about 85% - about 90%, or in the range of about 90% - about 95%, or in the range of about 95% - about 100%. Those skilled in the art can adjust the administration dose accordingly based on the purity of the preparation. For example, when the cell purity decreases, the administration dose needs to be increased accordingly.

[0357] The cells of the present invention are generally administered based on the body weight of the subject to whom the cells are to be administered, in units of cells per kilogram of body weight (cells / kg). Depending on the administration method and site, the cell dose is generally in the range of about 10 4 to about 10 10 cells / kg body weight. For example, about 10 5 to about 10 9 、or about 10 5 to about 10 8 、or about 10 5 to about 10 7 ,or about 10 5 to about 10 6 。Generally, in the case of systemic administration, the dose used is usually higher than the local administration of the immune cells of the present invention in the tumor and / or cancer area. Exemplary dose ranges include, but are not limited to: 1×10 4 -1×10 8 、2×10 4 -1×10 8 、3×10 4 -1×10 8 、4×10 4 -1×10 8 、5×10 4 -6×10 8 、7×10 4 -1×10 8 、8×10 4 -1×10 8 、9×10 4 -1×10 8 、1×10 5 -1×10 8 、1×10 5 -9×10 7 、1×105 -8×10 7 、1×10 5 -7×10 7 、1×10 5 -6×10 7 、1×10 5 -5×10 7 、1×10 5 -4×10 7 、1×10 5- 3×10 7 、1×10 5 -2×10 7 、1×10 5 -1×10 7 、1×10 5 -9×10 6 、1×10 5 -8×10 6 、1×10 5 -7×10 6 、1×10 5 -6×10 6 、1×10 5 -5×10 6 、1×10 5 -4×10 6 、1×10 5 -3×10 6 、1×10 5 -2×10 6 、1×10 5 -1×10 6 、2×10 5 -9×10 7 、2×10 5 -8×10 7 、2×10 5 -7×10 7 、2×10 5 -6×10 7 、2×10 5 -5×10 7 、2×10 5 -4×10 7 、2×10 5 -3×10 7 、2×10 5 -2×10 7 、2×10 5 -1×10 7 、2×10 5 -9×10 6 、2×10 5 -8×10 6, 2×10 5 , -7×10 6 , 2×10 5 , -6×10 6 , 2×10 5 , -5×10 6 , 2×10 5 , -4×10 6 , 2×10 5 , -3×10 6 , 2×10 5 , -2×10 6 , 2×10 5 , -1×10 6 , 3×10 5 , -3×10 6 cells / kg, etc. In addition, the dosage can be adjusted according to the administration method such as single dose or multiple doses. The precise effective dose is determined according to the individual factors of each subject.

[0358] As used herein, the terms "treatment", "therapeutic" and "treatment method" all refer to the improvement or reversal of at least one measurable physiological parameter in cancer, inflammation and / or autoimmune diseases, pathological disorders or clinical conditions, although such parameters may not be directly detectable in the subject. The terms "treatment", "therapeutic" and "treatment method" also refer to the situation where the disease, pathological disorder or clinical condition is promoted to regress, or its further development is prevented, or at least its progression is slowed down to a certain extent. In a specific embodiment, "treatment", "therapeutic" and "treatment method" refer to delaying or preventing the onset and progression of one or more concomitant symptoms of the disease, pathological disorder or clinical condition, or shortening the duration of onset of one or more concomitant symptoms of the disease, pathological disorder or clinical condition. The diseases include, for example, tumors, more preferably cancer. In a specific embodiment, "treatment", "therapeutic" and "treatment method" refer to preventing the recurrence of the disease, pathological disorder or clinical condition. In a specific embodiment, "treatment", "therapeutic" and "treatment method" refer to increasing the survival rate of a subject suffering from the disease, pathological disorder or clinical condition. In a specific embodiment, "treatment", "therapeutic" and "treatment method" refer to curing the disease, pathological disorder or clinical condition in the subject.

[0359] In a specific embodiment, the present invention provides a composition for treating cancer, inflammation, and / or autoimmune diseases, pathological disorders, or clinical conditions. For cancer treatment, the composition can be used in combination with other treatment methods, including but not limited to: chemotherapy, anti-CD20 monoclonal antibody (mAb), anti-TIM-3 monoclonal antibody, anti-LAG-3 monoclonal antibody, anti-EGFR monoclonal antibody, anti-HER-2 monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD33 monoclonal antibody, anti-CD47 monoclonal antibody, anti-CD73 monoclonal antibody, anti-DLL-3 monoclonal antibody, anti-apelin monoclonal antibody, anti-FOLR1 monoclonal antibody, anti-CTLA-4 monoclonal antibody, anti-PD-L1 monoclonal antibody, anti-PD-1 monoclonal antibody, anti-Claudin 18.2 monoclonal antibody, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADCs), targeted therapies, or other anti-cancer drugs. Bispecific antibodies that target 5T4 and are also capable of targeting the following tumor-associated antigens can be constructed, including but not limited to PD-1, PD-L1, LAG3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD73, CD47, CD3, apelin, DLL-3, TIP-1, GPC3, Claudin 18.2, folate receptor alpha (FOLR1), MUC16, mesothelin, PSCA, IL13Ra2, EGFRvIII, p95HER2, and / or any other tumor-associated antigen (TAA) to treat cancers or tumors that co-express the above two antigens. Bispecific antibodies can also be constructed using two antibodies that recognize two different epitopes on 5T4 to treat cancers / tumors that express 5T4.

[0360] In a specific embodiment, a method for treating cancer in a subject includes administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention, in combination with a reagent that enhances the efficacy of cells expressing a chimeric antigen receptor (CAR) molecule. The reagents include but are not limited to: antibody fragments that bind to CD73, CD39, PD1, PD-L1, PD-L2, CTLA4, TIM3, or LAG3, or antagonists of the adenosine A2a receptor.

[0361] In a specific embodiment, a method for treating cancer in a subject includes administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention, in combination with a reagent that alleviates one or more side effects induced by administering cells expressing a CAR molecule. The reagents include but are not limited to: steroids, tumor necrosis factor alpha (TNFα) inhibitors, or interleukin-6 (IL-6) inhibitors.

[0362] In a specific embodiment, a method for treating cancer in a subject includes administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention, while concomitantly using a reagent for treating 5T4-related diseases. The reagent includes, but is not limited to: anti-5T4 monoclonal antibody or bispecific antibody.

[0363] As used herein, the term "concomitantly" means administering two or more therapeutic agents to a subject. The use of the term "concomitantly" does not limit the order of administration of the therapeutic agents. For example, the first therapeutic agent (e.g., the composition described herein) can be administered to the subject before the second therapeutic agent (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks in advance), or concomitantly with the second therapeutic agent, or after the second therapeutic agent (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks later).

[0364] On the other hand, the present invention relates to a method for detecting the level of 5T4 in a subject. The method includes: (a) obtaining a sample from the subject; (b) contacting the sample with the monoclonal antibody of the present invention or its antigen-binding fragment; and (c) detecting the level of 5T4 in the subject.

[0365] As used herein, the term "sample" refers to a biological sample isolated from a subject, including but not limited to whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy samples (e.g., cancer tissues), lymph fluid, ascites, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine or any other secretion, excrement or body fluid. A "blood sample" refers to whole blood or any of its components, including blood cells, serum and plasma.

[0366] In a specific embodiment, the methods for detecting the level of 5T4 in a subject include, but are not limited to: Western blotting, immunohistochemistry (IHC), and ELISA assays. Western blot analysis and immunohistochemistry can be used to detect the relative protein expression level, and ELISA can be used to detect the absolute protein expression level. When detecting the relative expression level of 5T4, it is necessary to detect at least between two samples, for example, between samples at different time points of the same subject, between samples from different tissues of the same subject, and / or between samples of different subjects. Meanwhile, when detecting the absolute expression level of 5T4, for example, when assaying by ELISA, a standard curve can be constructed before detecting the sample, so as to determine the absolute expression level of 5T4 in the sample. Those skilled in the art should know what analytical techniques to use to determine the level of 5T4 in a subject sample using the antibody or its antigen-binding fragment of the present invention.

[0367] The method of measuring the level of 5T4 in a subject sample can be used to diagnose the abnormality (increase, decrease, or deficiency) of the 5T4 level in a disease, and then make a correct treatment strategy. The diseases are selected from, but not limited to, cancer, inflammation, or autoimmune diseases. In addition, by monitoring the level of 5T4 in a subject, the risk of onset of the above diseases can be determined based on the understanding of the 5T4 level in a specific disease and / or during the progression of a specific disease.

[0368] Embodiment

[0369] The present invention also provides the following non-limiting embodiments.

[0370] Embodiment 1 is an isolated monoclonal antibody or its antigen-binding fragment, comprising a heavy-chain variable region, the heavy-chain variable region comprising heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3; and comprising a light-chain variable region, the light-chain variable region comprising light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences:

[0371] (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8 respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14 respectively;

[0372] (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22 respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28 respectively;

[0373] (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36 respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42 respectively;

[0374] (4) are SEQ ID NOs: 45, 46, 47, 48, 49 and 50, respectively, or are SEQ ID NOs: 51, 52, 53, 54, 55 and 56, respectively;

[0375] (5) are SEQ ID NOs: 59, 60, 61, 62, 63 and 64, respectively, or are SEQ ID NOs: 65, 66, 67, 68, 69 and 70, respectively;

[0376] (6) are SEQ ID NOs: 73, 74, 75, 76, 77 and 78, respectively, or are SEQ ID NOs: 79, 80, 81, 82, 83 and 84, respectively;

[0377] (7) are SEQ ID NOs: 87, 88, 89, 90, 91 and 92, respectively, or are SEQ ID NOs: 93, 94, 95, 96, 97 and 98, respectively;

[0378] (8) are SEQ ID NOs: 101, 102, 103, 104, 105 and 106, respectively, or are SEQ ID NOs: 107, 108, 109, 110, 111 and 112, respectively;

[0379] (9) are SEQ ID NOs: 115, 116, 117, 118, 119 and 120, respectively, or are SEQ ID NOs: 121, 122, 123, 124, 125 and 126, respectively;

[0380] (10) are SEQ ID NOs: 129, 130, 131, 132, 133 and 134, respectively, or are SEQ ID NOs: 135, 136, 137, 138, 139 and 140, respectively; or

[0381] (11) are SEQ ID NOs: 143, 144, 145, 146, 147 and 148, respectively, or are SEQ ID NOs: 149, 150, 151, 152, 153 and 154, respectively;

[0382] Wherein, the antibody or its antigen-binding fragment specifically binds to 5T4, preferably specifically binds to human 5T4.

[0383] Embodiment 2 is a separated monoclonal antibody or its antigen-binding fragment of Embodiment 1, comprising a heavy-chain variable region having a polypeptide sequence with at least 95% sequence identity to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127 or 141, or comprising a light-chain variable region having a polypeptide sequence with at least 95% sequence identity to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128 or 142.

[0384] Embodiment 3 is a separated monoclonal antibody or its antigen-binding fragment of any one of Embodiments 1-2, comprising:

[0385] (1) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 1 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 2;

[0386] (2) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 15 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 16;

[0387] (3) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 29 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 30;

[0388] (4) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 43 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 44;

[0389] (5) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 57 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 58;

[0390] (6) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 71 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 72;

[0391] (7) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 85 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 86;

[0392] (8) A heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 99 and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 100;

[0393] (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114;

[0394] (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128; or

[0395] (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142.

[0396] Embodiment 4 is an isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 1-3, wherein the antibody or its antigen-binding fragment is chimeric and / or human or humanized.

[0397] Embodiment 5 is an isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 4, wherein the humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having a polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs: 155, 156, 159, 160, 161, 165, 166, 169, 170, 173, 174, 177, 178 or 179, or comprises a light chain variable region having a polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs: 157, 158, 162, 163, 164, 167, 168, 171, 172, 175, 176, 180 or 181.

[0398] Embodiment 6 is the isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 5, wherein the humanized monoclonal antibody or its antigen-binding fragment comprises:

[0399] (1) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0400] (2) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0401] (3) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0402] (4) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0403] (5) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0404] (6) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0405] (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0406] (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0407] (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0408] (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0409] (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0410] (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0411] (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0412] (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0413] (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0414] (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0415] (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0416] (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0417] (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0418] (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0419] (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0420] (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0421] (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0422] (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0423] (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0424] (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0425] (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0426] (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0427] (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0428] (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180; or

[0429] (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181.

[0430] Embodiment 7 is an isolated monoclonal antibody or antigen-binding fragment of any one of Embodiments 1-6, wherein the isolated antibody or its antigen-binding fragment can induce effector-mediated tumor cell lysis through antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC); and / or mediate the recruitment of conjugated drugs; and / or form a bispecific antibody with another monoclonal antibody or its antigen-binding fragment having cancer-killing effects.

[0431] Embodiment 8 is an isolated bispecific antibody or its antigen-binding fragment, and the isolated bispecific antibody or its antigen-binding fragment comprises a monoclonal antibody or its antigen-binding fragment of any one of Embodiments 1-7.

[0432] Embodiment 9 is an isolated nucleic acid encoding a monoclonal antibody or its antigen-binding fragment of any one of Embodiments 1-7, or encoding the bispecific antibody or its antigen-binding fragment described in Embodiment 8.

[0433] Embodiment 10 is a vector comprising the isolated nucleic acid described in Embodiment 9.

[0434] Embodiment 11 is a host cell comprising the vector described in Embodiment 10.

[0435] Embodiment 12 is a pharmaceutical composition, which comprises the isolated monoclonal antibody or its antigen-binding fragment according to any one of Embodiments 1-7, or the bispecific antibody or its antigen-binding fragment according to Embodiment 8, and a pharmaceutically acceptable carrier.

[0436] Embodiment 13 is a method for targeting 5T4 on the surface of cancer cells in a subject in need thereof, and / or treating cancer, treating inflammatory diseases and / or treating autoimmune diseases, the method comprising administering the pharmaceutical composition according to Embodiment 12 to the subject. Optionally, the cancer is selected from: lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0437] Embodiment 14 is a method for preparing the monoclonal antibody or its antigen-binding fragment according to any one of Embodiments 1-7, or the bispecific antibody or its antigen-binding fragment according to Embodiment 8, the method comprising culturing cells under conditions for producing the monoclonal antibody or its antigen-binding fragment, or the bispecific antibody or its antigen-binding fragment, the cells comprising a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment, or the bispecific antibody or its antigen-binding fragment, and recovering the monoclonal antibody or its antigen-binding fragment, or the bispecific antibody or its antigen-binding fragment from the cells or the culture.

[0438] Embodiment 15 is a method for preparing a pharmaceutical composition comprising the monoclonal antibody or its antigen-binding fragment according to any one of Embodiments 1-7, or the bispecific antibody or its antigen-binding fragment according to Embodiment 8, the method comprising combining the monoclonal antibody or its antigen-binding fragment, or the bispecific antibody or its antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the above-mentioned pharmaceutical composition.

[0439] Embodiment 16 is a method for detecting the level of 5T4 in a subject, the method comprising:

[0440] (a) obtaining a sample from the subject;

[0441] (b) contacting the sample with the isolated monoclonal antibody or its antigen-binding fragment according to any one of Embodiments 1-7; and

[0442] (c) Detect the level of 5T4 in the subject.

[0443] Embodiment 17 is the detection method of Embodiment 16, wherein the sample is a tissue sample or a blood sample. Optionally, the tissue sample is a cancer tissue sample.

[0444] Embodiment 18 is an isolated polynucleotide, which comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises:

[0445] (a) An extracellular domain, which comprises at least one antigen-binding domain that specifically binds to 5T4;

[0446] (b) A hinge region;

[0447] (c) A transmembrane region; and

[0448] (d) An intracellular signaling domain.

[0449] Embodiment 19 is the isolated polynucleotide according to Embodiment 18, wherein the antigen-binding domain comprises a heavy chain variable region, which comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3; and a light chain variable region, which comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. The heavy chain variable region and the light chain variable region have the following polypeptide sequences:

[0450] (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8 respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14 respectively;

[0451] (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22 respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28 respectively;

[0452] (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36 respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42 respectively;

[0453] (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50 respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56 respectively;

[0454] (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64 respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70 respectively;

[0455] (6) are SEQ ID NOs: 73, 74, 75, 76, 77 and 78, or are SEQ ID NOs: 79, 80, 81, 82, 83 and 84, respectively;

[0456] (7) are SEQ ID NOs: 87, 88, 89, 90, 91 and 92, or are SEQ ID NOs: 93, 94, 95, 96, 97 and 98, respectively;

[0457] (8) are SEQ ID NOs: 101, 102, 103, 104, 105 and 106, or are SEQ ID NOs: 107, 108, 109, 110, 111 and 112, respectively;

[0458] (9) are SEQ ID NOs: 115, 116, 117, 118, 119 and 120, or are SEQ ID NOs: 121, 122, 123, 124, 125 and 126, respectively;

[0459] (10) are SEQ ID NOs: 129, 130, 131, 132, 133 and 134, or are SEQ ID NOs: 135, 136, 137, 138, 139 and 140, respectively;

[0460] (11) are SEQ ID NOs: 143, 144, 145, 146, 147 and 148, or are SEQ ID NOs: 149, 150, 151, 152, 153 and 154, respectively;

[0461] Wherein, the antigen-binding domain specifically binds to 5T4, preferably, specifically binds to human 5T4.

[0462] Embodiment 20 is the isolated polynucleotide according to Embodiment 18 or 19, wherein the antigen-binding domain comprises a heavy chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 156, 159, 160, 161, 165, 166, 169, 170, 173, 174, 177, 178 or 179; or comprises a light chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 157, 158, 162, 163, 164, 167, 168, 171, 172, 175, 176, 180 or 181.

[0463] Embodiment 21 is the isolated polynucleotide according to Embodiment 20, wherein the antigen-binding domain comprises:

[0464] (1) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 2;

[0465] (2) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 16;

[0466] (3) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 30;

[0467] (4) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 44;

[0468] (5) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58;

[0469] (6) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72;

[0470] (7) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86;

[0471] (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100;

[0472] (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114;

[0473] (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128;

[0474] (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142;

[0475] (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0476] (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0477] (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157;

[0478] (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158;

[0479] (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0480] (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0481] (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0482] (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0483] (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0484] (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0485] (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162;

[0486] (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163;

[0487] (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164;

[0488] (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0489] (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0490] (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167;

[0491] (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168;

[0492] (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0493] (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0494] (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171;

[0495] (32) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172;

[0496] (33) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0497] (34) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0498] (35) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175;

[0499] (36) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176;

[0500] (37) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0501] (38) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0502] (39) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0503] (40) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181;

[0504] (41) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180;

[0505] Or

[0506] (42) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181.

[0507] Embodiment 22 is the isolated polynucleotide according to any one of Embodiments 18 to 21, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

[0508] Embodiment 23 is the isolated polynucleotide according to Embodiment 22, wherein the single-chain variable fragment (scFv) is humanized.

[0509] Embodiment 24 is the isolated polynucleotide according to any one of Embodiments 18 to 23, wherein the chimeric antigen receptor (CAR) comprises one or more antigen-binding domains, and / or the intracellular signaling domain comprises one or more co-stimulatory domains and one or more activation domains.

[0510] Embodiment 25 is a chimeric antigen receptor (CAR) encoded by the isolated polynucleotide according to any one of Embodiments 18 to 24.

[0511] Embodiment 26 is a vector comprising the isolated polynucleotide according to any one of Embodiments 18 to 24.

[0512] Embodiment 27 is a host cell comprising the vector according to Embodiment 26. Optionally, the host cell is a T cell or an NK cell, preferably a human T cell or a human NK cell.

[0513] Embodiment 28 is a method for preparing a host cell expressing a chimeric antigen receptor (CAR), the method comprising transducing a T cell or an NK cell with the vector according to Embodiment 26.

[0514] Embodiment 29 is a method for preparing a chimeric antigen receptor (CAR)-T cell or a chimeric antigen receptor (CAR)-NK cell, the method comprising culturing a T cell or an NK cell under conditions for generating the CAR-T cell or the CAR-NK cell, the T cell or the NK cell comprising the isolated polynucleotide encoding the chimeric antigen receptor (CAR) according to any one of Embodiments 18 to 24, and recovering the CAR-T cell or the CAR-NK cell.

[0515] Embodiment 30 is a method for preparing cells comprising a chimeric antigen receptor (CAR), the method comprising contacting the cells with an isolated polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR) as described in any one of Embodiments 18 to 24, wherein the isolated polynucleotide is in vitro transcribed RNA or synthetic RNA.

[0516] Embodiment 31 is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof the host cell as described in Embodiment 27, optionally, wherein the cancer is selected from: lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, and non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0517] Embodiment 32 is the method as described in Embodiment 31, the treatment method further comprising administering to the subject in need thereof a reagent that enhances the efficacy of cells expressing CAR, or a reagent that alleviates one or more side effects associated with administering cells expressing CAR, or a reagent for treating 5T4-related diseases.

[0518] Example

[0519] Example 1: Identification of anti-5T4 monoclonal antibodies

[0520] Mice were immunized with recombinant 5T4 Ser32-Ser355 (ACROBiosystems, Cat. No. TPG-H52E5), hybridomas were prepared and screened by ELISA and / or FACS. Positive clones were isolated and sequenced.

[0521] The sequences of the heavy chain variable region and the light chain variable region (VH region and VL region respectively) of anti-5T4 monoclonal antibodies are provided in Tables 1 and 2, and the CDR regions of anti-5T4 monoclonal antibodies are provided in Tables 3-6.

[0522] Table 1: Sequence of the heavy chain variable region of anti-5T4 monoclonal antibody.

[0523]

[0524]

[0525] VH: Heavy chain variable region

[0526] Table 2: The light chain variable region sequence of the anti-5T4 monoclonal antibody.

[0527]

[0528]

[0529] VL: Light chain variable region

[0530] Table 3: CDR regions 1-3 of the heavy chain of the anti-5T4 monoclonal antibody.

[0531]

[0532]

[0533] HC: Heavy chain; CDR: Complementary determining region; NO: SEQ ID NO

[0534] The HC CDR of the anti-5T4 monoclonal antibody was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212).

[0535] Table 4: CDR regions 1-3 of the light chain of the anti-5T4 monoclonal antibody.

[0536]

[0537] LC: Light chain; CDR: Complementary determining region; NO: SEQ ID NO

[0538] The LC CDR of the anti-5T4 monoclonal antibody was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212).

[0539] Table 5: CDR regions 1-3 of the heavy chain of the anti-5T4 monoclonal antibody.

[0540]

[0541]

[0542] HC: Heavy chain; CDR: Complementary determining region; NO: SEQ ID NO

[0543] The HC CDR of the anti-5T4 monoclonal antibody was determined using the Kabat method (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. 1991).

[0544] Table 6: CDR regions 1 - 3 of the light chain of the anti-5T4 monoclonal antibody.

[0545]

[0546] LC: light chain; CDR: complementary determining region; NO: SEQ ID NO

[0547] The LC CDR of the anti-5T4 monoclonal antibody was determined using the Kabat method (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. 1991).

[0548] Example 2: The monoclonal antibody was produced and purified from the culture of transfected cells

[0549] To obtain the anti-5T4 chimeric recombinant monoclonal antibody, an expression vector containing the murine variable regions (VH and VL) fused to the human IgG1 heavy chain and κ light chain constant regions respectively was transiently transfected into ExpiCHO-S cells. The recombinant antibody produced in the supernatant of the ExpiCHO-S cell culture was purified by protein A affinity chromatography..

[0550] Example 3: ELISA binding assay of the purified chimeric monoclonal antibody

[0551] The binding ability of the purified chimeric monoclonal antibody to immobilized 5T4 was tested by ELISA. At 4 °C, recombinant human 5T4 (ACROBiosystems, cat. no. TPG-H52E5) prepared in PBS was coated overnight in a 96-well plate (Corning, cat. no. 3361). The next day, the plate was blocked with 5% BSA in TBST solution at room temperature for 1 hour and then washed with TBST. Different concentrations of antibody samples prepared in TBST containing 5% BSA were added to the wells and incubated at room temperature for 1 hour. The plate was washed with TBST, and the bound antibodies were labeled with mouse anti-human IgG Fc HRP (Thermo Fisher Scientific, cat. no. H10007) at room temperature for 1 hour. After washing the plate again with TBST, ELISA color development was performed for 4 minutes using the one-step Ultra TMB detection reagent (Thermo Fisher Scientific, cat. no. 34028), and then the reaction was terminated with ELISA stop solution (Thermo Fisher Scientific, cat. no. SS04). The absorbance at 450 nm was measured with a microplate reader and quantitatively analyzed. The results are as Figure 1A-1C shown.

[0552] Example 4: FACS binding analysis of the purified monoclonal antibody

[0553] 50,000 SK-OV-3 cells were transferred to a 96-well plate and incubated with different concentrations of the purified chimeric anti-5T4 monoclonal antibody (the variable regions of the mouse monoclonal antibody were fused with the constant regions of human IgG1 heavy chain and κ light chain) on ice for 30 minutes. Subsequently, the cells were washed three times with FACS buffer (HBSS containing 0.1% BSA and 0.05% sodium azide). Then, the cells were stained with propidium iodide and goat anti-human IgG Fc Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. H10120) and incubated in the dark at room temperature for 30 minutes. After washing the cells twice again with FACS buffer, they were resuspended in FACS buffer. The cells were analyzed using an Attune NxT instrument and the data were processed using Attune NxT software. The results are as Figure 2 shown.

[0554] Example 5: Humanization of the anti-5T4 monoclonal antibody

[0555] To reduce the potential immunogenicity of the anti-5T4 monoclonal antibody during its use in human patients, the murine anti-5T4 monoclonal antibody was humanized. The sequences of the variable regions of the heavy and light chains (VH and VL) were aligned with human antibody sequences in the Protein Data Bank (PDB), and a homology model was constructed. The CDRs in the heavy and light chains of the murine monoclonal antibody were grafted onto human frameworks that were most likely to maintain the appropriate structure required for antigen binding. Reverse mutations or other mutations from human residues to murine residues were designed when necessary. The sequences of the humanized VH and VL regions are shown in Table 7. The humanized VH and VL regions were fused with the constant regions of the human IgG1 heavy chain and κ light chain, respectively. L3D5-H1L1 refers to the humanized monoclonal antibody constructed using the L3D5-H1 heavy chain and L3D5-L1 light chain shown in Table 7; other humanized clones follow the same naming rule. FACS was used to detect the binding ability of the humanized monoclonal antibody to SK-OV-3 cells. The results are as Figure 3A-3G shown.

[0556] Table 7: Sequences of the heavy chain variable region and light chain variable region of the humanized anti-5T4 monoclonal antibody

[0557]

[0558]

[0559]

[0560]

[0561] NO:SEQ ID NO

[0562] Example 6: Construct a chimeric antigen receptor construct containing the anti-5T4 antigen-binding domain

[0563] To construct the CAR construct, mAbs were converted into scFv using VH, VL, and (G4S)n linker sequences, and the scFv was fused to the N-terminus of the hinge region and transmembrane domain derived from human CD8α (amino acids 114 - 188; Boursier JP et al., J. Biol. Chem., 1993; 268(3):2013 - 20). The C-terminal intracellular signaling domain of the CAR was constructed by fusing the intracellular co-stimulatory domain of CD28 (amino acids 162 - 202; Aruffo A and Seed B, Proc. Natl. Acad. Sci. USA, 1987; 84(23):8573 - 7), followed by the activation domain of the CD3ζ chain (amino acids 52 - 162; Letourneur F and Klausner RD, Proc. Natl. Acad. Sci. USA, 1991; 88(20):8905 - 9). Using standard molecular biology cloning techniques, the DNA sequence encoding the CAR was assembled and cloned into a suitable expression vector (such as a retroviral vector, lentiviral vector, episomal vector, or integrating vector, etc.) to generate the CAR construct.

[0564] Those skilled in the art should understand that, without departing from the general concept of the present invention, the above-described embodiments can be appropriately modified. Therefore, the present invention is not limited to the specific embodiments disclosed, but is intended to cover all modifications within the scope defined in this specification and consistent with the research concept and scope of the present invention.

Claims

1. An isolated monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof, comprising a heavy chain variable region, the heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, and a light chain variable region, the light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the polypeptide sequences shown below: (1) SEQ ID NOs: 3, 4, 5, 6, 7 and 8 respectively, or SEQ ID NOs: 9, 10, 11, 12, 13 and 14 respectively; (2) SEQ ID NOs: 17, 18, 19, 20, 21 and 22 respectively, or SEQ ID NOs: 23, 24, 25, 26, 27 and 28 respectively; (3) SEQ ID NOs: 31, 32, 33, 34, 35 and 36 respectively, or SEQ ID NOs: 37, 38, 39, 40, 41 and 42 respectively; (4) SEQ ID NOs: 45, 46, 47, 48, 49 and 50 respectively, or SEQ ID NOs: 51, 52, 53, 54, 55 and 56 respectively; (5) SEQ ID NOs: 59, 60, 61, 62, 63 and 64 respectively, or SEQ ID NOs: 65, 66, 67, 68, 69 and 70 respectively; (6) SEQ ID NOs: 73, 74, 75, 76, 77 and 78 respectively, or SEQ ID NOs: 79, 80, 81, 82, 83 and 84 respectively; (7) SEQ ID NOs: 87, 88, 89, 90, 91 and 92 respectively, or SEQ ID NOs: 93, 94, 95, 96, 97 and 98 respectively; (8) SEQ ID NOs: 101, 102, 103, 104, 105 and 106 respectively, or SEQ ID NOs: 107, 108, 109, 110, 111 and 112 respectively; (9) SEQ ID NOs: 115, 116, 117, 118, 119 and 120 respectively, or SEQ ID NOs: 121, 122, 123, 124, 125 and 126 respectively; (10) SEQ ID NOs: 129, 130, 131, 132, 133 and 134 respectively, or SEQ ID NOs: 135, 136, 137, 138, 139 and 140 respectively; or (11) SEQ ID NOs: 143, 144, 145, 146, 147 and 148 respectively, or SEQ ID NOs: 149, 150, 151, 152, 153 and 154 respectively; Among them, The antibody or its antigen-binding fragment specifically binds to 5T4, preferably specifically binds to human 5T4.

2. The isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to claim 1, comprising: A heavy chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 156, 159, 160, 161, 165, 166, 169, 170, 173, 174, 177, 178 or 179, or A light chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 157, 158, 162, 163, 164, 167, 168, 171, 172, 175, 176, 180 or 181.

3. The isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to claim 1 or 2, comprising: (1) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 2; (2) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 16; (3) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 30; (4) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 44; (5) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58; (6) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72; (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86; (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100; (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114; (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128; (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142; (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157; (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158; (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 157; (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 158; (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162; (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163; (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164; (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162; (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163; (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164; (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 162; (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 163; (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 164; (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167; (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 165 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168; (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 167; (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 168; (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171; (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172; (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 171; (32) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 172; (33) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175; (34) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 173 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176; (35) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 175; (36) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 174 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 176; (37) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180; (38) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 177 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181; (39) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180; (40) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 178 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 181; (41) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 180; or (42) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 179 and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:

181.

4. The isolated monoclonal antibody or its antigen-binding fragment or antigen-binding domain according to any one of claims 1-3, wherein the antibody or its antigen-binding fragment or antigen-binding domain is chimeric or human or humanized.

5. The isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1-4, wherein, The antibody or its antigen-binding fragment or antigen-binding domain induces effector-mediated tumor cell lysis through antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC); and / or mediates the recruitment of conjugated drugs; and / or forms a bispecific antibody with another monoclonal antibody or its antigen-binding fragment having an anti-cancer effect.

6. A bispecific antibody or its antigen-binding fragment or bispecific antigen-binding domain, comprising the monoclonal antibody or its antigen-binding fragment or antigen-binding domain according to any one of claims 1-5.

7. An isolated nucleic acid encoding the monoclonal antibody or its antigen-binding fragment or antigen-binding domain according to any one of claims 1-5; or encoding the bispecific antibody or its antigen-binding fragment or bispecific antigen-binding domain according to claim 6.

8. A vector comprising the isolated nucleic acid according to claim 7.

9. A host cell comprising the vector according to claim 8.

10. A pharmaceutical composition comprising the isolated monoclonal antibody or its antigen-binding fragment or antigen-binding domain according to any one of claims 1-5, or comprising the bispecific antibody or its antigen-binding fragment or bispecific antigen-binding domain according to claim 6, and a pharmaceutically acceptable carrier.

11. A method of targeting 5T4 on the surface of cancer cells of a subject in need thereof, and / or treating cancer of a subject in need thereof, and / or treating an inflammatory disease of a subject in need thereof and / or treating an autoimmune disease of a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to claim 10, optionally, the cancer is selected from: lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

12. A method for preparing a monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1-5, or a bispecific antibody or an antigen-binding fragment or bispecific antigen-binding domain thereof according to claim 6, comprising culturing cells under conditions for producing said monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof or said bispecific antibody or an antigen-binding fragment or bispecific antigen-binding domain thereof, said cells comprising a nucleic acid encoding said monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof or said bispecific antibody or an antigen-binding fragment or bispecific antigen-binding domain thereof, and recovering said monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof or said bispecific antibody or an antigen-binding fragment or bispecific antigen-binding domain thereof from said cells or the culture.

13. A method for preparing a pharmaceutical composition, said pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1-5, or a bispecific antibody or an antigen-binding fragment or bispecific antigen-binding domain thereof according to claim 6, said method comprising combining said monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof or bispecific antibody or an antigen-binding fragment or bispecific antigen-binding domain thereof with a pharmaceutically acceptable carrier, thereby obtaining said pharmaceutical composition.

14. A method for detecting the level of 5T4 in a subject, wherein said method comprises: (a) obtaining a sample from said subject; (b) contacting said sample with an isolated monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1-5; and (c) detecting the expression level of 5T4 in said subject; Optionally, wherein said sample is a tissue sample or a blood sample, and optionally, said tissue sample is a cancer tissue sample.

15. An isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said CAR comprises: (a) an extracellular domain comprising at least one antigen-binding domain according to any one of claims 1-5, or comprising a bispecific antigen-binding domain according to claim 6; (b) a hinge region; (c) a transmembrane region; and (d) an intracellular signal transduction domain.

16. A chimeric antigen receptor (CAR) encoded by the isolated polynucleotide according to claim 15.

17. A host cell comprising a vector, wherein said vector comprises the isolated polynucleotide according to claim 15, optionally, said host cell is a T cell or an NK cell, preferably, a human T cell or a human NK cell.

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