Anti-5T4 antibody and its use

Abstract

This disclosure relates to anti-5T4 (5T4 tumor embryo antigen) antibodies, antigen-binding fragments thereof, antibody-drug conjugates (ADCs) derived therefrom, and their uses.

Description

Technical Field

[0001] Priority Claim This application claims priority to PCT / CN2023 / 080242, filed on March 8, 2023. The entire contents of the above application are hereby incorporated by reference into this specification.

[0002] Technical Field The present disclosure relates to anti-5T4 (5T4 tumor fetal antigen) antibodies, antigen-binding fragments thereof, antibody-drug conjugates (ADCs) derived therefrom, and their uses.

Background Art

[0003] Trophoblast glycoprotein, also known as 5T4, TPBG, Wnt-activated inhibitory factor 1, or WAIF1, is a human protein encoded by the 5T4 gene. The 5T4 protein is expressed in many different cancers but is rarely expressed in normal adult tissues. 5T4 is an antagonist of the Wnt / β-catenin signaling pathway.

[0004] Considering the important role of 5T4 in tumors, the development of therapeutic agents targeting 5T4 is needed.

Summary of the Invention

[0005] The present disclosure relates to anti-5T4 antibodies, antigen-binding fragments thereof, and their uses.

[0006] In one aspect, the present disclosure is an antibody or an antigen-binding fragment thereof that binds to 5T4 (5T4 tumor fetal antigen), A heavy chain variable region (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence, and a A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, and, comprising a light chain variable region, The amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3 are those of an antibody or an antigen-binding fragment thereof selected from one of the following. (1) The amino acid sequences of the selected VH CDR1, 2, 3 are shown in SEQ ID NOs: 4-6 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are shown in SEQ ID NOs: 1-3 respectively. (2) The amino acid sequences of the selected VH CDR1, 2, 3 are shown in SEQ ID NOs: 7-9 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are shown in SEQ ID NOs: 1-3 respectively. (3) The amino acid sequences of the selected VH CDR1, 2, 3 are shown in SEQ ID NOs: 10-12 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are shown in SEQ ID NOs: 1-3 respectively. (4) The amino acid sequences of the selected VH CDR1, 2, 3 are shown in SEQ ID NOs: 13-15 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are shown in SEQ ID NOs: 1-3 respectively. (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 16-18, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively. (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 19 to 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1 to 3, respectively. (7) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 22 to 24, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1 to 3, respectively. (8) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 25-27, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively. (9) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 28-30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively. (10) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 31 to 33, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1 to 3, respectively. (11) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 34-36, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively, and (12) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 37 to 39, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1 to 3, respectively.

[0007] In some embodiments, based on Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4 to 6, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0008] In some embodiments, based on Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 7 to 9, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0009] In some embodiments, based on Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10 to 12, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0010] In some embodiments, based on Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0011] In some embodiments, based on Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 16-18, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1-3, respectively.

[0012] In some embodiments, based on Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 19 to 21, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0013] In some embodiments, based on Chothia's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 22 to 24, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

[0014] In some embodiments, based on Chothia's definition, the above VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 25 to 27, respectively, and the above VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

[0015] In some embodiments, based on the definition of Chothia, the above VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 28 to 30, respectively, and the above VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

[0016] In some embodiments, based on the definition of Chothia, the above VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 31 to 33, respectively, and the above VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

[0017] In some embodiments, based on the definition of Chothia, the above VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 34 to 36, respectively, and the above VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

[0018] In some embodiments, based on Chothia's definition, the above VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 37 to 39, respectively, and the above VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

[0019] In some embodiments, the antibody or its antigen-binding fragment specifically binds to 5T4 in humans, mice, monkeys, or dogs.

[0020] In some embodiments, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0021] In some embodiments, the antibody or its antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

[0022] In one embodiment, the disclosure relates to nucleic acids comprising polynucleotides that encode a polypeptide including the following: An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 4-6, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 40. An immunoglobulin light chain or fragment thereof comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 1 to 3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 41. An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 7-9, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 40. An immunoglobulin light chain or fragment thereof comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 1 to 3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 42. An immunoglobulin heavy chain or fragment thereof comprising a VH containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 10 to 12, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 1-3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 43, An immunoglobulin heavy chain or fragment thereof comprising a VH containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 13 to 15, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 1-3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 44. An immunoglobulin heavy chain or fragment thereof comprising a VH containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 16-18, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 1-3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 45. An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 1-3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 46, An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 19 to 21, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin light chain or fragment thereof comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 47, An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 22 to 24, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin heavy chain or fragment thereof comprising a VH containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 25-27, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin heavy chain or fragment thereof comprising a VH containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 28-30, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 31-33, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 40. An immunoglobulin heavy chain or fragment thereof comprising a VH containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 34-36, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40, or An immunoglobulin heavy chain or fragment thereof comprising a VH containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 37 to 39, wherein the VH binds to 5T4 when pairing with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40.

[0023] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, which comprises a VL containing an immunoglobulin light chain or fragment thereof, which comprises CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0024] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, which includes an immunoglobulin heavy chain or fragment thereof, and a VH comprising CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 4-6, respectively.

[0025] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, which comprises an immunoglobulin heavy chain or fragment thereof, and includes a VH comprising CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 7 to 9.

[0026] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 10 to 12.

[0027] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 13-15, respectively.

[0028] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, which comprises a VH containing an immunoglobulin heavy chain or fragment thereof, which comprises CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 16-18, respectively.

[0029] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 19-21, respectively.

[0030] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 22-24, respectively.

[0031] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 25-27.

[0032] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 28-30, respectively.

[0033] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 31-33, respectively.

[0034] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 34-36.

[0035] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 37-39, respectively.

[0036] In some embodiments, the VH specifically binds to the 5T4 of humans, mice, monkeys, or dogs when pairing with the VL, or the VL specifically binds to the 5T4 of humans, mice, monkeys, or dogs when pairing with the VH.

[0037] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof or a humanized immunoglobulin heavy chain or fragment thereof (for example, a human IgG1 heavy chain or fragment thereof, or a human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof or a humanized immunoglobulin light chain or fragment thereof.

[0038] In some embodiments, the nucleic acid encodes a single-stranded variable fragment (scFv), a single-arm antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

[0039] In some embodiments, the nucleic acid is cDNA.

[0040] In one embodiment, this disclosure relates to a vector comprising one or more nucleic acids described herein.

[0041] In one embodiment, the present disclosure relates to a vector comprising two of the nucleic acids described herein, wherein the vector encodes the VL region and the VH region that bind together to 5T4.

[0042] In one embodiment, the disclosure relates to a pair of vectors, each comprising one of the nucleic acids described herein, wherein the pair of vectors encode a pair of VL and VH regions that bind together to 5T4.

[0043] In one embodiment, this disclosure relates to a cell comprising a vector described herein, or a pair of vectors described herein.

[0044] In some embodiments, the above cells are CHO cells.

[0045] In one embodiment, this disclosure relates to a cell comprising one or more nucleic acids described herein.

[0046] In one embodiment, the present disclosure relates to a cell comprising two of the nucleic acids described herein.

[0047] In some embodiments, the two nucleic acids encode a pair of VL and VH regions that bind together to 5T4.

[0048] In one embodiment, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing cells described herein under conditions sufficient to enable the cells to produce an antibody or an antigen-binding fragment, and recovering the antibody or antigen-binding fragment produced by the cells.

[0049] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to 5T4, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. The selected VH sequence is sequence number 41, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 42, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 43, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 44, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 45, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 46, the selected VL sequence is sequence number 40, and The selected VH sequence is sequence number 47, and the selected VL sequence is sequence number 40.

[0050] In some embodiments, VH includes the sequence of sequence number 41, and VL includes the sequence of sequence number 40.

[0051] In some embodiments, VH includes the sequence of sequence number 42, and VL includes the sequence of sequence number 40.

[0052] In some embodiments, VH includes the sequence of sequence number 43, and VL includes the sequence of sequence number 40.

[0053] In some embodiments, VH includes the sequence of sequence number 44, and VL includes the sequence of sequence number 40.

[0054] In some embodiments, VH includes the sequence of sequence number 45, and VL includes the sequence of sequence number 40.

[0055] In some embodiments, VH includes the sequence of sequence number 46, and VL includes the sequence of sequence number 40.

[0056] In some embodiments, VH includes the sequence of sequence number 47, and VL includes the sequence of sequence number 40.

[0057] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to 5T4, comprising a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. The selected VH sequence is sequence number 41, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 42, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 43, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 44, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 45, and the selected VL sequence is sequence number 40. The selected VH sequence is sequence number 46, the selected VL sequence is sequence number 40, and The selected VH sequence is sequence number 47, and the selected VL sequence is sequence number 40.

[0058] In some embodiments, the antibody or its antigen-binding fragment specifically binds to 5T4 in humans, mice, monkeys, or dogs.

[0059] In some embodiments, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0060] In some embodiments, the antibody or antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

[0061] In one embodiment, this disclosure relates to an antibody or an antigen-binding fragment that cross-competes with an antibody or an antigen-binding fragment described herein.

[0062] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0063] In some embodiments, the Fc region exhibits increased complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0064] In one embodiment, this disclosure relates to a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof as described herein.

[0065] In one embodiment, the present disclosure relates to an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof, covalently bound to a therapeutic agent, as described herein.

[0066] In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

[0067] In one embodiment, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, a CAR as described herein, or an antibody-drug conjugate as described herein.

[0068] In some embodiments, the subject has a solid tumor.

[0069] In some embodiments, the cancer is colorectal cancer, gastric cancer, ovarian cancer, lung cancer, head and neck cancer, or pancreatic cancer.

[0070] In some embodiments, the subjects are further treated with an effective amount of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-CD40 antibody, or anti-PD-L1 antibody.

[0071] In one aspect, the present disclosure relates to a method for reducing the rate of tumor growth, wherein the method is The present invention relates to a method comprising contacting tumor cells with a composition comprising an effective amount of an antibody or its antigen-binding fragment as described herein, a CAR as described herein, or an antibody-drug conjugate as described herein.

[0072] In one embodiment, the present disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof as described herein, a CAR as described herein, or an antibody-drug conjugate as described herein.

[0073] In one embodiment, the present disclosure relates to a method for increasing an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.

[0074] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier.

[0075] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier.

[0076] In some embodiments, the drug-antibody ratio (DAR) is approximately 4.

[0077] As used herein, the term “cancer” means cells capable of autonomous proliferation. Examples of such cells include cells in an abnormal state or condition characterized by rapid proliferation. The term means cancerous growth, e.g., tumors, oncogenic processes, metastatic tissues, and malignant transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Malignancies of various organ systems, e.g., respiratory, cardiovascular, renal, reproductive, hematologic, nervous, liver, gastrointestinal, and endocrine systems, as well as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, and small intestine cancer. “Spontaneously occurring” cancers include any cancer that is not experimentally induced by transplanting cancer cells into a subject, e.g., spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers resulting from transgenic oncogene insertions or tumor suppressor gene knockouts, and cancers caused by infections, e.g., viral infections. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine glandular tissue. This term also includes carcinosarcoma, which is a malignant tumor composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma of glandular origin, or a carcinoma in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving hematopoietic hyperplasia / neoplastic cells. Hematopoietic neoplastic diseases may originate from the bone marrow, lymphoid system, or erythrocyte lineage, or their progenitor cells.

[0078] As used herein, the term “antibody” means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining regions (CDRs) (e.g., any of three CDRs derived from an immunoglobulin light chain or any of three CDRs derived from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, antibodies may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0079] As used herein, the term “antigen-binding fragment” means a portion of a full-length antibody, the portion of which is specifically capable of binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain in the heavy chain or a variable domain in the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0080] As used herein, the term “human antibody” means an antibody encoded by endogenous nucleic acids present in humans (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, human antibodies are recovered from humans or produced in human cell culture media (e.g., in human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unarranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0081] As used herein, the term “chimeric antibody” means an antibody containing sequences present in at least two different antibodies (e.g., antibodies derived from two different mammalian species, such as human and mouse antibodies). Non-limiting examples of chimeric antibodies include antibodies containing variable domain sequences (e.g., all or part of the light chain and / or heavy chain variable domain sequences) of a non-human (e.g., mouse) antibody, as well as constant domains of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0082] As used herein, the term “humanized antibody” means a non-human antibody that contains minimal sequences derived from non-human (e.g., mouse) immunoglobulin and sequences derived from human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (recipient antibody) in which residues in the hypervariable (e.g., CDR) region of the recipient antibody are replaced by residues in the hypervariable (e.g., CDR) region of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody having desired specificity, affinity, and capability. In some embodiments, Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can further refine the performance of the antibody. In some embodiments, the humanized antibody contains substantially all, at least one, and typically two, variable domains, with all or substantially all of the hypervariable loop (CDR) corresponding to the hypervariable loop of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework region being a human immunoglobulin sequence. The humanized antibody may also contain an immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. The humanized antibody can be produced using molecular biological methods well known in the art. Non-limiting examples of methods for producing the humanized antibody are described herein.

[0083] As used herein, the term “single-chain antibody” means a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are specifically capable of binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0084] As used herein, the term “multimeric antibody” means an antibody containing four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, a multimeric antibody can crosslink one target molecule (e.g., 5T4) to at least one second target molecule on the surface of a mammalian cell (e.g., human T cell).

[0085] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and refer to animals, humans, or non-humans to which treatment according to the methods of the present invention is provided. Veterinary and non-veterinary uses are conceived by the present invention. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), equids, canids, felines, bovines, and other domestic, livestock, and zoo animals.

[0086] As used herein, when referring to an antibody, the terms "specifically binding" and "specifically binds" mean that the antibody interacts more with its target molecule (e.g., 5T4) than with other molecules, because the interaction depends on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent recognizes and binds to a molecule containing a specific structure, rather than to all general molecules. An antibody that specifically binds to a target molecule may also be called a target-specific antibody. For example, an antibody that specifically binds to the 5T4 molecule may be called a 5T4-specific antibody or an anti-5T4 antibody.

[0087] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and mean polymers of at least two amino acids of any length.

[0088] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and mean, but are not limited to, polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials for use in the present invention are described herein, but other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0090] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0091] [Figure 1] The CDR sequences of the heavy chain variable region and light chain variable region of anti-5T4 antibodies as defined by Kabat are listed below. [Figure 2] The CDR sequences of the heavy chain variable region and light chain variable region of anti-5T4 antibodies as defined by Chothia are listed below. [Figure 3] The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-5T4 antibody are listed below. [Figure 4] The mean tumor volume in different groups of B-NDG mice injected with NUGC-4 cells and treated with 3 mg / kg of 32G1-ADC (G2) or 3 mg / kg of PF06263507-ADC (G3) is shown. PBS was injected as a control (G1). [Figure 5] This shows the mean tumor volume in different groups of B-NDG mice that were transplanted with patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) and treated with phosphate-buffered saline (PBS) or ADC. [Figure 6A] This shows the binding between NCI-H226 cells and the anti-5T4 antibody 32G1, as determined by flow cytometry. [Figure 6B] This shows the binding between NCI-H2030 cells and the anti-5T4 antibody 32G1, as determined by flow cytometry. [Figure 6C] This shows the binding between HCC827 cells and the anti-5T4 antibody 32G1, as determined by flow cytometry. [Figure 7-1] The selected amino acid sequences discussed in this disclosure are listed below. [Figure 7-2] Same as above [Modes for carrying out the invention]

[0092] This disclosure provides examples of antibodies that bind to 5T4, and their antigen-binding fragments.

[0093] 5T4 The 5T4 protein is expressed in many different cancers but is rarely expressed in normal adult tissues. The 5T4 molecule is a 72kD highly N-glycosylated protein containing multiple leucine-rich repeats, often associated with protein-protein interactions. 5T4 expression is associated with epithelial-mesenchymal transition, enhanced CXCL12 / CXCR4 chemotaxis, and directional cell movement mediated by inhibition of standard Wnt / β-catenin, while simultaneously promoting signaling in non-standard pathways. All of these processes help promote cancer cell spread. The selective patterns of 5T4 tumor expression, its association with tumor initiation phenotypes, and its mechanistic involvement in cancer spread will facilitate the clinical development of various immunotherapy strategies, including vaccines, tumor-targeted superantigens, and antibody-drug conjugates.

[0094] The 5T4 trophoblast glycoprotein was discovered in an attempt to identify a shared cell surface molecule that may play a role in enabling fetal survival in the mother as a semi-allogeneic graft, or in tumor survival in the host. The rationale was that such shared expression would reflect a common function related to proliferation, invasion, or changes in immune surveillance in the host. Mouse monoclonal antibodies were generated against purified glycoproteins from trophoblast specimens derived from full-term human placentas and were initially screened against various cancer cell lines and human peripheral blood mononuclear cells. Further screening using 5T4 monoclonal antibodies (mAbs) by immunohistochemistry showed that the antigen is expressed in many different cancers, but its distribution in normal tissues is limited. A series of biochemical and genomic studies revealed that the 5T4 molecule is an N-glycosylated protein with an apparent molecular size of 72 kD and is encoded on chromosomes 6q14-15. The human gene encodes a 42 kD transmembrane protein core containing several leucine-rich repeats (LRRs) associated with protein-protein interactions of a functionally diverse set of molecules. The extracellular portion of the molecule contains multiple LRRs in two domains separated by a short hydrophilic sequence, along with a transmembrane domain and a short cytoplasmic sequence. Importantly, 5T4-specific mAbs recognize a structure-dependent epitope that depends on the integrity of intramolecular disulfide bonds and the indirect presence of complex N-linked glycosylation. Overexpression of human 5T4 in mouse fibroblasts resulted in more spindle-shaped cells and decreased adhesion, while normal epithelial cells showed downregulation of E-cadherin, increased motility, and disruption of the cytoskeleton.

[0095] Immunohistochemistry (IHC) of frozen sections revealed that 5T4-specific monoclonal antibodies detected antigen expression in many different types of carcinoma, although low levels were observed in some normal adult epithelial tissues. Importantly, 5T4 was frequently expressed at high levels in many different primary and metastatic cancers, with stromal expression added in some cases. In relation to the role of 5T4 in tumor development and spread, 5T4 expression in colorectal cancer, gastric cancer, and ovarian cancer has been shown to correlate with poorer clinical outcomes.

[0096] Several "5T4-specific" antibodies are commercially available, formulated against specific peptides or sequences of the 5T4 molecule. However, their specificity may not be identical to the original mAb, as the exact epitope is often not identified, and they may contain parts of the 5T4 molecule that include leucine-rich repeats shared by numerous proteins with diverse functions and expression. However, in antibody-targeted therapy, the individual properties of the reagent in living tissue are the only important relevant properties.

[0097] Overexpression of 5T4 in normal mouse epithelial cells is associated with the downregulation of E-cadherin, a key component of EMT. This develops during embryonic development and is crucial for the metastatic spread of epithelial tumors. 5T4 has been shown to be a marker of early differentiation in mouse and human embryonic stem (ES) cells, a process that involves the switching from E-cadherin to N-cadherin, the upregulation of E-cadherin repressor molecules (Snail and Slug proteins), and increased activity and motility of matrix metalloproteinases (MMP-2 and MMP-9), all of which are typical EMT features. Undifferentiated knockout (KO) E-cadherin ES cells constitutively express the cell surface 5T4 molecule, but antibody-induced downregulation of E-cadherin in ES cells induces 5T4 membrane expression, increased motility, altered actin cytoskeleton arrangement, and a mesenchymal phenotype. These observations are consistent with E-cadherin inhibiting 5T4 cell surface expression in some way, and a possible mechanism is the stabilization of the cortical actin cytoskeleton. Co-expression of 5T4 and factors involved in epithelial-mesenchymal transition was also observed in undifferentiated lung tumor cells, but not in differentiated lung tumor cells.

[0098] The 5T4 molecule has been shown to be involved in the functional expression of CXCR4 on the cell surface of several germ cells and tumor cells. Expression of both CXCL12 and CXCR4 is associated with tumorigenesis in many cancers, and CXCR4 expression is thought to promote diffusion to tissues that highly express CXCL12, including the lungs, liver, lymph nodes, and bone marrow. 5T4 is expressed by putative leukemia-initiating cells in BCP-ALL, and these cells exhibit associated CXCL12 / CXCR4 chemotaxis characteristics. 5T4-positive leukemia-initiating cells appear to be attracted by CXCL12 produced in extramedullary sites, which leads to decreased bioavailability of the treatment after treatment, resulting in disease relapse.

[0099] Intracellular signaling of Wnt proteins is a central element in many aspects of cellular regulation crucial for normal development, homeostasis, and regeneration, but misregulation can lead to diseases, including cancer. There are two pathways, the most characterized being the standard Wnt / β-catenin pathway, but atypical Wnt signaling via cell-autonomous planar cell polarity (PCP) pathways can drive the regulation of actin and the microtubule skeleton, which facilitates cell migration during cancer development. 5T4 has been shown to interfere with Wnt / β-catenin signaling while simultaneously activating the atypical Wnt pathway. 5T4 binds to the Wnt coreceptor LRP6 and inhibits the internalization of Wnt-inducible LRP6 into endocytic vesicles, a process necessary for pathway activation, thereby regulating the intracellular localization of LRP6 and modulating Wnt / β-catenin signaling. Furthermore, 5T4 enhances β-catenin-independent Wnt signaling by promoting the atypical function of Dickkopf1. These results suggest that 5T4 promotes pathway selection in Wnt-receptor cells, inhibiting the standard Wnt / β-catenin pathway while simultaneously activating a non-standard Wnt signaling pathway associated with increased motility. 1.8A resolution crystals of the extracellular domain of 5T4 and related cell biological studies provided a structural basis for the inhibition of Wnt / β-catenin signaling.

[0100] The selective patterns of 5T4 tumor expression, their association with tumor initiation phenotypes, and their mechanistic involvement in cancer spread will facilitate the development of several different immunotherapy strategies.

[0101] Detailed reviews of 5T4 and its function can be found in Stern, Peter L., and Richard Harrop. "5T4 oncofoetal antigen: an attractive target for immune intervention in cancer." Cancer Immunology, Immunotherapy 66.4(2017):415-426, and Harrop, Richard, Eric O'Neill, and Peter L. Stern. "Cancer stem cell mobilization and therapeutic targeting of the 5T4 oncofetal antigen." Therapeutic advances in vaccines and immunotherapy 7 (2019):2515135518821623, with the entirety of each of these references incorporated here.

[0102] This disclosure provides several anti-5T4 antibodies, their antigen-binding fragments, and methods for using these anti-5T4 antibodies and antigen-binding fragments to inhibit tumor growth and treat cancer.

[0103] Antibody and antigen-binding fragments This disclosure provides an anti-5T4 antibody and its antigen-binding fragment. Generally, an antibody (also called an immunoglobulin) consists of two classes of polypeptide chains: a light chain and a heavy chain. The non-limiting antibodies of this disclosure may be an intact four-immunoglobulin chain antibody containing two heavy chains and two light chains. The heavy chain of the antibody may be any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a κ light chain or a λ light chain. The antibody may contain two identical copies of the light chain and two identical copies of the heavy chain, each containing one variable domain (or variable region, V HThe heavy chain, which contains a variable domain (or variable region), binds to each other via disulfide bonds within its constant domains, forming the "stem" of the antibody. Each of these contains one variable domain (or variable region, V L Each light chain containing a constant domain (or constant region) is bonded to a heavy chain via a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bonded. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0104] The hypervariable region, known as the complementarity-determining region (CDR), forms a loop containing the antibody's antigen-binding surface. The four framework regions are largely adapted to the β-sheet structure, and the CDRs form linked loops, sometimes even forming part of the β-sheet structure. The CDRs of each chain are held in close proximity to the framework regions and, together with the CDRs of other chains, contribute to the formation of the antigen-binding region.

[0105] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well-known, and several definitions of CDRs are commonly used. Kabat's definition is based on sequence variability, while Chothia's definition is based on the location of the structural loop region. These methods and definitions are, for example, found in Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody Engineering, Springer Berlin Heidelberg, 2001. 422-439, Abhinandan, et al. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular Immunology 45.14(2008):3832-3839, Wu, T. and Kabat, EA(1970) J. Exp. Med. 132:211-250, Martin et al., Methods Enzymol. 203:121-53(1991), Morea et al., Biophys Chem. 68(1-3):9-16(Oct. 1997), Morea et al., J Mol Biol. 275(2):269-94(Jan. 1998), Chothia This is described in et al., Nature 342(6252):877-83 (Dec. 1989), and Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), the entirety of each of these works is incorporated herein by reference.

[0106] CDRs are important for recognizing the epitopes of antigens. As used herein, an “epitope” is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, as epitopes can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structures of the antigen.

[0107] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are well known in the art, for example, in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2 (2015):171-182; and Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference.

[0108] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies containing an immunoglobulin-binding domain fused to another polypeptide. The terms “antigen-binding domain” or “antigen-binding fragment” refer to any portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of an antibody that is specifically capable of binding to an epitope on the target molecule of an intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or its antigen-binding fragment may be any polypeptide containing, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and a binding domain that is an antibody-binding domain, or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0109] In some embodiments, antigen-binding fragments can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of single-stranded variable fragments (scFv) described herein, fused to the CD3ζ transmembrane and endodomains. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, for increased potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) expressing the chimeric antigen receptor described herein.

[0110] In some embodiments, the scFv has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains and two light chain variable domains.

[0111] In some embodiments, a bispecific antibody targeting 5T4 and an additional antigen (e.g., OX40, CD3, 4-1BB, CD314, CD47, PD-1, CTLA4, CD40, or PDL1) can be generated using the antibody or antigen-binding fragment sequence described herein (e.g., CDR or VH / VL sequence).

[0112] Anti-5T4 antibody and antigen-binding fragment This disclosure provides antibodies and antigen-binding fragments that specifically bind to 5T4 (e.g., human 5T4). The antibodies and antigen-binding fragments described herein are capable of binding to 5T4. These antibodies may be agonists or antagonists. In some embodiments, these antibodies can increase the immune response. In some embodiments, these antibodies can block the 5T4 pathway.

[0113] This disclosure provides, for example, anti-5T4 antibodies 31G4, 32E3, 32G1, 32G8, 35B12, 35C6, their chimeric antibodies, and their human antibodies or humanized antibodies.

[0114] CDR sequences for 31G4 and antibodies derived from 31G4 (e.g., human antibodies or humanized antibodies) include the heavy chain variable domain CDRs, SEQ ID NOs: 4-6, and the light chain variable domain CDRs, SEQ ID NOs: 1-3, as defined by Kabat. CDRs can also be defined by the Chothia system. Under the Chothia definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 22-24, and the light chain variable domain CDR sequences are shown in SEQ ID NOs: 1-3.

[0115] Similarly, CDR sequences for 32E3 and antibodies derived from 32E3 include the heavy chain variable domain CDRs, SEQ ID NOs. 7-9, and the light chain variable domain CDRs, SEQ ID NOs. 1-3, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 25-27, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 1-3.

[0116] The CDR sequences of 32G1 and antibodies derived from 32G1 include the heavy chain variable domain CDRs, SEQ ID NOs. 10-12, and the light chain variable domain CDRs, SEQ ID NOs. 1-3, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 28-30, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 1-3.

[0117] The CDR sequences of 32G8 and antibodies derived from 32G8 include the heavy chain variable domain CDRs, SEQ ID NOs. 13-15, and the light chain variable domain CDRs, SEQ ID NOs. 1-3, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 31-33, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 1-3.

[0118] The CDR sequences of 35B12 and antibodies derived from 35B12 (e.g., 35B12K, whose VH sequence is shown in SEQ ID NO: 46) include the CDR of the heavy chain variable domain, SEQ ID NOs: 16-18, and the CDR of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 34-36, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 1-3.

[0119] The CDR sequences of 35C6 and antibodies derived from 35C6 include the heavy chain variable domain CDRs, SEQ ID NOs. 19-21, and the light chain variable domain CDRs, SEQ ID NOs. 1-3, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 37-39, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 1-3.

[0120] The amino acid sequence of the heavy chain variable region of the 31G4 antibody is shown in SEQ ID NO: 41. The amino acid sequence of the light chain variable region of the 31G4 antibody is shown in SEQ ID NO: 40.

[0121] The amino acid sequence of the heavy chain variable region of the 32E3 antibody is shown in SEQ ID NO: 42. The amino acid sequence of the light chain variable region of the 32E3 antibody is shown in SEQ ID NO: 40.

[0122] The amino acid sequence of the heavy chain variable region of the 32G1 antibody is shown in SEQ ID NO: 43. The amino acid sequence of the light chain variable region of the 32G1 antibody is shown in SEQ ID NO: 40.

[0123] The amino acid sequence of the heavy chain variable region of the 32G8 antibody is shown in SEQ ID NO: 44. The amino acid sequence of the light chain variable region of the 32G8 antibody is shown in SEQ ID NO: 40.

[0124] The amino acid sequence of the heavy chain variable region of the 35B12 antibody is shown in SEQ ID NO: 45. The amino acid sequence of the light chain variable region of the 35B12 antibody is shown in SEQ ID NO: 40.

[0125] The amino acid sequence of the heavy chain variable region of the 35C6 antibody is shown in SEQ ID NO: 47. The amino acid sequence of the light chain variable region of the 35C6 antibody is shown in SEQ ID NO: 40.

[0126] The amino acid sequences for the heavy chain variable region and light chain variable region of the modified antibody are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs. 41-47. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO. 40. The heavy chain variable region sequence can be paired with a corresponding light chain variable region sequence, which together bind to 5T4.

[0127] Humanization percentage refers to the percentage of identity of a heavy chain or light chain variable region sequence compared to human antibody sequences in the International Immunogenetic Information System (IMGT) database. A top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to other species. For example, a top hit for humans means that the sequence is closer to humans than to other species. A top hit for humans and cynomolgus monkeys means that the sequence has the same percentage of identity to human and cynomolgus monkey sequences, and that these percentages of identity are the highest compared to sequences of other species. In some embodiments, the humanization percentage exceeds 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of methods for determining the humanization ratio and the top hits are well known in the art and are, for example, described in Jones, et al. “The INNs and outs of antibody nonproprietary names.” MAbs. Vol.8. No.1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization ratio often has various advantages, such as being safer and more effective in humans, more likely to be accepted by human subjects, and / or less likely to have side effects. In some embodiments, the variable region is entirely human, for example, derived from human heavy chain immunoglobulin loci (e.g., a combination of human IGHV, human IGHD, and human IGHJ genes) and / or human kappa chain immunoglobulin loci (e.g., a combination of human IGKV and human IGKJ genes).

[0128] Furthermore, in some embodiments, the antibodies or antigen-binding fragments described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, 22-24, 25-27, 28-30, 31-33, 34-36, and 37-39, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1-3.

[0129] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR1; the CDR2 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR2; and the CDR3 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR3. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR1; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR2; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR3. The amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in Figure 1 (Kabat's CDR) and Figure 2 (Chothia's CDR).

[0130] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0131] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9

[0132] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three heavy chain variable domains of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0133] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15

[0134] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18

[0135] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0136] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0137] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0138] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0139] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 33, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0140] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0141] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0142] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3

[0143] Insertions, deletions, and substitutions may be present within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on Kabat's definition. In some embodiments, the CDR is determined based on Chothia's definition. In some embodiments, the CDR is determined based on a combination of Kabat's and Chothia's definitions.

[0144] This disclosure also provides an antibody or antigen-binding fragment thereof that binds to 5T4. The antibody or antigen-binding fragment thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 41 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 42 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 43 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 44 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 45 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 46 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 40. The disclosure also provides an antibody or antigen-binding fragment thereof that can compete with the antibodies described herein. In some embodiments, the antibody or antigen-binding fragment can bind to the same epitope as the antibodies described herein.

[0145] This disclosure also provides an antibody or an antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., “Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.” Journal of virology 70.3(1996):1863-1872, which is incorporated herein by reference in its entirety. In one embodiment, this disclosure also provides an antibody or an antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope-binding assays are well known in the art and are described, for example, in Estep et al., “High throughput solution-based measurement of antibody-antigen affinity and epitope binning.” MAbs. Vol.5. No.2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0146] To measure the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences to ensure optimal alignment for comparison, and non-homologous sequences may be ignored). Subsequently, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and measurement of the percentage of identity between two sequences can be performed using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0147] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides containing immunoglobulin heavy chains or immunoglobulin light chains. The immunoglobulin heavy chain or immunoglobulin light chain comprises a CDR as shown in Figure 1 or Figure 2, or has the sequence shown in Figure 3. When a polypeptide pairs with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to 5T4 (e.g., human 5T4).

[0148] Anti-5T4 antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein include polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0149] Antibody fragments are suitable for use in the provided manner, insofar as they retain the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment that binds to 5T4 retains its ability to bind to 5T4. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are closely associated, which can essentially be covalent, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs, or subsets thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, though usually with lower affinity than the entire binding site.

[0150] A single-stranded Fv or (scFv) antibody fragment contains the VH and VL domains (or regions) of the antibody, and these domains are located within a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a desirable structure for antigen binding.

[0151] The Fab fragment contains variable and constant domains of the light chain, as well as a variable domain and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment contains a pair of Fab fragments, generally commonally linked near the carboxyl terminus by a hinge cysteine ​​between them. Other chemical linkages of antibody fragments are well known in the art.

[0152] A diabody is a small antibody fragment containing two antigen-binding sites, and this fragment contains VH (VH and VL) attached to VL within the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domains can be paired with complementary domains on another chain, thereby generating two antigen-binding sites.

[0153] Linear antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0154] A single-arm antibody may have a heavy chain and a light chain, as well as a heavy chain fragment containing the CH2 and CH3 domains of IgG. In some embodiments, a single-arm antibody is an antibody having only one of the two antigen-binding arms in a typical antibody. In some embodiments, a single-arm antibody includes an antigen-binding arm (e.g., VH+CH1 and VL+CL) and Fc.

[0155] The antibodies and antibody fragments of this disclosure can be modified within the Fc region to provide desired effector function or serum half-life. In some embodiments, the Fc region can be modified to silence or reduce complement-dependent cell injury (CDC) or antibody-dependent cell injury (ADCC). In some embodiments, the Fc region can be modified to enhance or increase complement-dependent cell injury (CDC) or antibody-dependent cell injury (ADCC).

[0156] Antibody multimerization can be achieved by spontaneous aggregation of antibodies or by chemical or recombinant conjugation techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., one purified IgG molecule) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0157] Alternatively, antibody homodimers can be formed by chemical bonding techniques well known in the art. For example, antibody polymers can be formed using heterobifunctional crosslinking agents, including but not limited to SMCC (4-(maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl) and SATA (S-acetylthioacetate N-succinimidyl). Exemplary procedures for forming antibody homodimers are described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is to use the autophilic T15 peptide, as described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0158] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0159] Examples of bispecific antibodies include crosslinked or "heterocomplex" antibodies. For example, one antibody in a heterocomplex can be coupled to avidin and the other to biotin. Heterocomplex antibodies can also be produced using any convenient crosslinking method. Suitable crosslinking agents and techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0160] Methods for generating bispecific antibodies from antibody fragments are also well known in the art. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al. (Science 229:81, 1985) describe a procedure in which an intact antibody is cleaved by proteolysis to generate F(ab')2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then reconverted to a Fab'thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'TNB derivative to form a bispecific antibody.

[0161] Any antibody or antigen-binding fragment described herein can be conjugated with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation with a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment or extend its biological activity, either in vitro (e.g., in tissue culture medium or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0162] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated with a therapeutic agent. The antibody-drug conjugate, comprising an antibody or its antigen-binding fragment, can be covalently or noncovalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), zione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as analogues).

[0163] In some embodiments, the antibodies or antigen-binding fragments described herein recognize endogenous 5T4 or recombinant 5T4. In some embodiments, the antibodies or antigen-binding fragments described herein recognize human 5T4.

[0164] In some embodiments, the half-life of the antibody or antigen-binding fragment described herein in wild-type mice (e.g., C57BL / 6 mice) is at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In some embodiments, the half-life of the antibody or antigen-binding fragment described herein in 5T4 gene-humanized mice (e.g., h5T4 mice) is at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In some embodiments, the maximum concentration (Cmax) of the antibody or antigen-binding fragment described herein in a 5T4 gene-humanized mouse (e.g., h5T4 mouse) is at least 50 μg / mL, at least 75 μg / mL, at least 100 μg / mL, at least 125 μg / mL, at least 150 μg / mL, at least 175 μg / mL, or at least 200 μg / mL. In some embodiments, the serum clearance rate (CL) of the antibody or antigen-binding fragment described herein in a 5T4 gene-humanized mouse (e.g., h5T4 mouse) is at least 7.5 mL / day / kg, at least 10 mL / day / kg, at least 12.5 mL / day / kg, at least 15 mL / day / kg, at least 17.5 mL / day / kg, at least 20 mL / day / kg, at least 25 mL / day / kg, at least 30 mL / day / kg, at least 35 mL / day / kg, or at least 40 mL / day / kg.

[0165] In some embodiments, the clearance rate (CL) of the antibodies or their antigen-binding fragments described herein in wild-type mice (e.g., C57BL / 6 mice) is less than 7 mL / day / kg, less than 6 mL / day / kg, less than 5 mL / day / kg, or less than 4 mL / day / kg. In some embodiments, the clearance rate (CL) of the antibodies or their antigen-binding fragments described herein in 5T4 gene-humanized mice (e.g., h5T4 mice) is less than 15 mL / day / kg, less than 14 mL / day / kg, less than 13 mL / day / kg, or less than 12 mL / day / kg.

[0166] In some embodiments, the half-life of the antibody or antigen-binding fragment described herein (e.g., in FcRn gene humanized mice) is at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In some embodiments, the clearance rate (CL) of the antibody or antigen-binding fragment described herein (e.g., in FcRn gene humanized mice) is less than 16 mL / day / kg, less than 15 mL / day / kg, less than 14 mL / day / kg, less than 13 mL / day / kg, less than 12 mL / day / kg, less than 11 mL / day / kg, less than 10 mL / day / kg, less than 9 mL / day / kg, less than 8 mL / day / kg, or less than 7 mL / day / kg.

[0167] Antibody-drug conjugates (ADCs) The antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated with therapeutic agents (drugs). The therapeutic agent can be covalently or noncovalently bound to the antibody or antigen-binding fragment, or antigen-binding protein construct (e.g., bispecific antibodies). In some embodiments, the bispecific antibodies have a common light chain.

[0168] In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, meitansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as its analogues). Useful classes of cytotoxic, cell proliferation inhibitor, or immunomodulatory agents include, for example, antitubulins, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0169] In some embodiments, the therapeutic agent may be, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent, an immunotherapeutic agent, etc.), an antiviral agent, or an antibacterial agent. In some embodiments, the conjugateable therapeutic agent may be, but is not limited to, selected from MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0170] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of drastatin-10), or a derivative thereof. The auristatin can be, for example, an ester formed of auristatin E with a keto acid. For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Publication No. 2003-0083263, International Patent Publication No. WO 04 / 010957, and International Patent Publication No. WO U.S. Patent Nos. 02 / 088172, and U.S. Patent Nos. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5, These are described in Patent Nos. 521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, each of which is incorporated herein by reference for all purposes.

[0171] Auristatin has been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and has been shown to possess anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell proliferation inhibitory effects in cancer cells. Numerous well-known assays exist in the art that can be used to measure whether auristatin or the resulting antibody-drug conjugate exerts cell proliferation inhibitory or cytotoxic effects in desired cells.

[0172] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include thiotepa and cyclosphosphamide (CYTOXAN). TMAlkylating agents such as ); alkyl sulfonates such as busulfan, improsulfan, and biposulfan; aziridines such as benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylamelamamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamamine; chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, f Nitrogen mustards such as enesterine, prednimastine, trophosphamide, and uracil mustard; nitrosoureas such as carmastine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; acrasinomycin, actinomycin, anthramycin, azaserine, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophiline, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epi Antibiotics such as rubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; fludarabine, 6-mercaptopri Purine analogs such as thiamiprine and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine, and 5-FU; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrine;Best Love Sil; Bisanthren; Edatrexate; Defofamine; Demecoltin; Diadiquan; Elfomitin; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamin; Mitoguazone; Maitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK 7; Lazoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxanes, e.g., Paclitaxel (TAXOL®, Bristol-Myers Examples include Squibb Oncology (Princeton, New Jersey), doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, platinum analogs such as cisplatin or carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition includes, for example, tamoxifen, raloxifen, aromatase inhibitor 4(5)imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); as well as antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Furthermore, anti-hormonal agents that modulate or inhibit hormonal activity in tumors, such as anti-estrogen agents containing any of the pharmaceutically acceptable salts, acids, or derivatives mentioned above, are also included. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated in its entirety by reference.

[0173] In some embodiments, the antigen-binding construct is coupled to the drug via a cleavable linker, e.g., an SPBD linker, or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is coupled to the drug via an incleavable linker, e.g., an MCC linker formed using SMCC or sulfo-SMCC. The selection of an appropriate linker for a given ADC can be quickly made by a person skilled in the art who has knowledge of the art and takes into account relevant factors such as the binding site to the antigen-binding construct, any structural limitations of the drug, and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). Numerous specific linker-toxin combinations have been described and, in certain embodiments, can be used with the antigen-binding constructs described herein to prepare ADCs. Examples include, but are not limited to, cleavable peptide linkers with auristatins such as MMAE and MMAF, and camptothecines such as SN-38, duocalmycin, and PBD dimer; incleavable MC linkers with auristatin MMAF and MMAE; acid-unstable hydrazone linkers with calicheamycin and doxorubicin; disulfide linkers with meitansinoids such as DM1 and DM4; and bismaleimide trioxyethylene glycol (BMPEO) linkers with meitansinoid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters & Brown, (2015) Biosci.Rep.e00225, Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65, U.S. Patent Publication No. US 2015 / 0374847, and U.S. Patent Publication No. US 20180193477A1, which are incorporated herein by reference in their entirety.

[0174] Depending on the desired drug and the selected linker, those skilled in the art can choose a suitable method for coupling them to each other. For example, several conventional coupling methods, such as amine coupling, can be used to form the desired drug-linker complex, which still contains a reactive group for covalently conjugating the antibody. In some embodiments, a drug-maleimide complex (i.e., a maleimide-bound drug) can be used with the payload having the reactive group in this disclosure. Maleimide is the most common reactive group that can be bound to a thiol group in ADC preparation. Furthermore, organobromids and iodides are also frequently used.

[0175] ADCs can be prepared by one of several well-known routes in the art, using organic chemical reactions, conditions, and reagents familiar to those skilled in the art (see, for example, Bioconjugate Techniques (G. Thermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) forming an antibody-linker intermediate Ab-L by covalent bonding of the nucleophilic or electrophilic group of the antibody with a divalent linker reagent, followed by a reaction with the activated drug site D, or (2) forming a drug-linker intermediate DL by covalent bonding of the nucleophilic or electrophilic group of the drug site with a linker reagent, followed by a reaction with the nucleophilic or electrophilic group of the antibody. Conjugation methods (1) and (2) can be used with various antibodies, drug moieties, and linkers to prepare the ADCs described herein. The various linkers, linker components, and toxins prepared are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley), Toki et al., (2002) J. Org. Chem. 67:1866-1872, Frisch et al., (1997) Bioconj. Chem. 7:180-186, Bioconjugate Techniques (G. Thermanson, 2013, Academic Press), US20210379193A1, and US20180193477A1, which are incorporated herein by reference in their entirety. Furthermore, a number of preformed drug-linkers suitable for reaction with selected antigen-binding constructs are also commercially available. For example, linker-toxins containing DM1, DM4, MMAE, MMAF, or duocalmycin SA are available from Creative BioLabs (Shirley, NY).

[0176] Several specific examples of methods for preparing ADCs are well known in the art and are described in U.S. Patent No. 8,624,003 (Pot Method), U.S. Patent No. 8,163,888 (One-Step Method), U.S. Patent No. 5,208,020 (Two-Step Method), and U.S. 20180193477A1, which are incorporated herein by reference in their entirety. Other methods are well known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0177] The drug load is expressed by the number of drug moieties per antibody in the ADC molecule. For some antibody-drug conjugates, the drug load can be limited by the number of binding sites on the antibody. For example, when the binding is cysteinethiol, as in certain exemplary embodiments described herein, the drug load can range from 0 to 8 drug moieties per antibody. In certain embodiments, a high drug load, e.g., p ≥ 5, may cause aggregation, insolubility, toxicity, or cell permeability of a particular antibody-drug conjugate. In certain embodiments, the average drug load for an antibody-drug conjugate is in the range of 1 to about 8, about 2 to about 6, or about 3 to about 5. In fact, it has been shown that for a particular antibody-drug conjugate, the optimal ratio of drug moieties per antibody can be about 4. In some embodiments, the drug-antibody ratio (DAR) is about, or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0178] Antibody and ADC characteristics The antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, can inhibit the binding between 5T4 and 5T4 ligand.

[0179] The antibody or antigen-binding fragment thereof, or the ADC derived therefrom, described in this specification can be a 5T4 pathway agonist or antagonist. In some embodiments, by binding to 5T4, the antibody can inhibit the 5T4 signaling pathway. In some embodiments, the antibody can upregulate or downregulate the immune response.

[0180] In some embodiments, the antibody (or its antigen-binding fragment) or the ADC derived therefrom specifically binds to 5T4 (e.g., human 5T4, monkey 5T4 (e.g., rhesus monkey, cynomolgus monkey), canine 5T4, mouse 5T4) with a dissociation rate (koff) of less than 2s -1 less than 1s -1 less than 0.1s -1 less than 0.01s -1 less than 0.001s -1 less than 0.0001s -1 less than 0.00001s -1 less than 0.000001s -1 less than, or 0.0000001s -1 less than. In some embodiments, the dissociation rate (koff) is greater than 1s -1 greater than 0.01s -1 greater than 0.001s -1 greater than 0.0001s -1 greater than 0.00001s -1 greater than 0.000001s -1 greater than 0.0000001s -1 greater than, or 0.00000001s -1 greater than.

[0181] In some embodiments, the association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms, greater than 1×10 6 / Ms, greater than 1×10 7 / Ms or greater than 1×10 8 / Ms. In some embodiments, the association rate (kon) is 1×10 5 / Ms less than 1 × 10 6 / Ms less than 1 × 10 7 / Ms less than 1 × 10 8 Less than / Ms, or 1 × 10⁻⁶ 9 It is less than / Ms.

[0182] The affinity can be estimated from the quotient of the velocity constant (KD = koff / kon). In some embodiments, KD is 1 × 10⁻⁶ -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M, 1 x 10 -13 Less than M, or 1 × 10 -14 It is less than M. In some embodiments, KD is 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM. In some embodiments, KD is 1 × 10⁻¹⁶ -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 Super M, 1×10 -12 Super M, 1×10 -13 Super M, 1×10 -14 It is greater than M.

[0183] Common techniques for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human 5T4, monkey 5T4, canine 5T4, and / or mouse 5T4. In some embodiments, the antibody does not bind to human 5T4, monkey 5T4, canine 5T4, and / or mouse 5T4.

[0184] In some embodiments, the antibodies or antigen-binding fragments described herein can bind to 5T4-expressing cells at half-effective concentrations (EC50) of less than 3.5 μg / mL, less than 3 μg / mL, less than 2.5 μg / mL, less than 2 μg / mL, less than 1.5 μg / mL, less than 1 μg / mL, less than 0.75 μg / mL, less than 0.5 μg / mL, less than 0.25 μg / mL, or less than 0.15 μg / mL.

[0185] In some embodiments, the antibodies or antigen-binding fragments described herein can bind to 5T4-expressing cells. In some embodiments, the percentage of positive cells is greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, or greater than 98%.

[0186] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, are added to NCI-H2030 cells (ATCC, catalog number: CRL-5914), CMT cells (ATCC, catalog number: CRL-3456), or D17 cells (ATCC, catalog number: CCL-183) to test the endocytosis rate. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, have an endocytosis ratio greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, or greater than 98%.

[0187] In some embodiments, thermal stability is measured. The antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, may have a Tm of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or greater than 95°C. In some embodiments, Tm is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or less than 95°C.

[0188] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, can bind to the same epitope of 5T4. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, can bind to different epitopes of 5T4.

[0189] In some embodiments, the antibodies or antigen-binding fragments described herein have a purity greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, or greater than 98%, as determined by size exclusion chromatography (SEC). In some embodiments, the antibodies or antigen-binding fragments described herein have a hydrophobic interaction chromatography (HIC) retention time greater than 2 minutes, greater than 3 minutes, greater than 4 minutes, greater than 5 minutes, greater than 6 minutes, greater than 7 minutes, greater than 8 minutes, greater than 9 minutes, greater than 10 minutes, greater than 11 minutes, greater than 12 minutes, greater than 13 minutes, greater than 14 minutes, greater than 15 minutes, greater than 16 minutes, greater than 17 minutes, greater than 18 minutes, greater than 19 minutes, greater than 20 minutes, greater than 21 minutes, or greater than 22 minutes.

[0190] In some embodiments, the antibodies or antigen-binding fragments described herein have a main peak comprising more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% when determined by capillary isoelectric focusing (cIEF). In some embodiments, the antibodies or antigen-binding fragments described herein have an acidic peak comprising more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% when determined by capillary isoelectric focusing (cIEF). In some embodiments, the antibodies or antigen-binding fragments described herein have an isoelectric point (PI) greater than 2, greater than 2.5, greater than 3, greater than 3.5, greater than 4, greater than 4.5, greater than 5, greater than 5.5, greater than 6, greater than 6.5, greater than 7, greater than 7.5, greater than 8, greater than 8.5, greater than 9, greater than 9.5, greater than 10, greater than 10.5, greater than 11, greater than 11.5, greater than 12, greater than 12.5, greater than 13, greater than 13.5, greater than 14, greater than 14.5, greater than 15, greater than 15.5, greater than 16, greater than 16.5, greater than 17, greater than 17.5, greater than 18, or greater than 18.5, as determined by capillary isoelectric focusing (cIEF). In some embodiments, the antibodies or antigen-binding fragments described herein have half-effective concentrations (EC50) of less than 2.5, less than 2, less than 1.5, less than 1, less than 0.75, less than 0.5, less than 0.25, and less than 0.15 μg / mL.

[0191] In some embodiments, the ADCs described herein have a mean drug-antibody ratio (DAR) greater than 3, greater than 3.2, greater than 3.4, greater than 3.6, greater than 3.8, greater than 4, greater than 4.2, greater than 4.4, or greater than 4.6, as measured by HPLC. In some embodiments, the ADCs described herein have a mean DAR less than 3, less than 3.2, less than 3.4, less than 3.6, less than 3.8, less than 4, less than 4.2, less than 4.4, or less than 4.6, as measured by HPLC.

[0192] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, have a tumor growth inhibition percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, have a tumor growth inhibition percentage less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition rate (TGI%) is calculated using the following formula. TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100 Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0193] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, are 5T4 antagonists. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, reduce 5T4 signaling in target cells expressing 5T4.

[0194] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, can enhance the function of APCs (e.g., DC cells), for example, by inducing the surface expression of costimulatory molecules and MHC molecules, inducing the production of pro-inflammatory cytokines, and / or enhancing T cell triggering function.

[0195] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, can bind to tumor cells expressing 5T4. In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, can induce complement-dependent cell-mediated cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and kill tumor cells.

[0196] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, have a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis.

[0197] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, can induce complement-dependent cell-mediated cytotoxicity (CDC).

[0198] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody optionally having the SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation. In some embodiments, the antibody is a human IgG4 antibody optionally having the SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation.

[0199] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, do not have a functional Fc region. For example, the antibodies or antigen-binding fragments are Fab, Fab', F(ab')2, and Fv fragments. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations according to EU numbering) or LALA-PG mutations (L234A, L235A, and P329G mutations according to EU numbering). In some embodiments, the Fc region has FLAA mutations (F234A and L235A according to EU numbering). In some embodiments, Fc has SI mutations (S239D and I332E mutations according to EU numbering). In some embodiments, Fc has the N297A mutation according to EU numbering. In some embodiments, Fc has YTE mutations (M252Y, S254T, and T256E according to EU numbering).

[0200] Method for producing anti-5T4 antibodies Human 5T4 isolated fragments can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of the antigen peptide or protein. In some embodiments, the antigen peptide or protein is injected with at least one adjuvant. In some embodiments, the antigen peptide or protein can be conjugated with a drug that is immunogenic in the immunized species. Animals may be injected with the antigen peptide or protein two or more times (e.g., two, three, or four times).

[0201] Full-length polypeptides or proteins can be used, or their antigen peptide fragments can be used as immunogens. The protein antigen peptide contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the 5T4 amino acid sequence and includes an epitope of the protein such that the antibody produced against the peptide forms a specific immune complex with the protein. As described above, the full-length sequence of human 5T4 is well known in the art. In some embodiments, Fc-tagged or His-tagged human 5T4 protein is used as an immunogen.

[0202] Immunogens are typically used for antibody preparation by immunizing a suitable target (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., a fragment of human 5T4). The preparation may further contain an adjuvant, such as a Freund complete or incomplete adjuvant, or a similar immunostimulant.

[0203] Polyclonal antibodies can be prepared as described above by immunizing a suitable target with the 5T4 polypeptide or its antigenic peptide (e.g., a portion of 5T4) as an immunogen. The antibody titer in the immunized target can be monitored over time using standard techniques such as enzyme-linked immunosorbent assay (ELISA) with the immobilized 5T4 polypeptide or peptide. If desired, the antibody molecule can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as protein G chromatography or protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the titer of specific antibodies is at its maximum, antibody-producing cells can be obtained from the target organism and used to prepare monoclonal antibodies using standard techniques such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (generally, see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). For example, hybridoma cells that produce monoclonal antibodies can be detected by screening hybridoma culture supernatants for antibodies that bind to a target polypeptide or epitope using a standard ELISA assay.

[0204] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human antibody, a humanized antibody, or a chimeric antibody, or an antibody or antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence that produces the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments exhibit increased affinity for a target protein, such as 5T4. Any combination of deletions, insertions, and / or combinations can be realized in an antibody or antigen-binding fragment with increased binding affinity to the target. Antibodies or antigen-binding fragments can be modified, or novel post-translational modifications can be introduced, by changing the amino acids introduced into the antibody or antigen-binding fragment, such as changing the number of glycosylation sites (e.g., increasing or decreasing them), changing the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are bound by enzymes present in the cell), or introducing novel glycosylation sites.

[0205] The antibodies disclosed herein may originate from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically modified to produce human antibodies.

[0206] Examples of human antibodies and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as) human germline immunoglobulin sequences. Examples of human antibodies include amino acid residues within the CDR that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0207] Humanized antibodies typically have a human framework (FR) into which a non-human CDR has been transplanted. Therefore, humanized antibodies have one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often called “import” residues, and are typically obtained from the “import” variable domain. Humanization can essentially be carried out by substituting, for example, a rodent CDR or CDR sequence with the corresponding sequence of a human antibody. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), and Verhoeyen et al., Science, 239:1534-1536 (1988), each of which is incorporated herein by reference in its entirety. Thus, a “humanized” antibody is a chimeric antibody in which a portion considerably smaller than the intact human V domain is substituted with the corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which several CDR residues and several FR residues are substituted with residues derived from similar sites within the human antibody.

[0208] The selection of human VH and VL domains used in the production of humanized antibodies is crucial for reducing immunogenicity. Following the so-called "best-fit" method, the V domain sequence of a mouse antibody is screened against an entire library of known human domain sequences. The human sequence that most closely matches the mouse sequence is then recognized as the human FR for humanized antibodies (Sims et al., J.Immunol., 151:2296 (1993), Chothia et al., J.Mol.Biol., 196:901 (1987)).

[0209] Furthermore, it is important to humanize antibodies while maintaining high specificity and affinity for antigens, as well as other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by analytical processes of parental sequences and various conceptual humanization products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. By observing these displays, it is possible to analyze the roles that residues can play in the functionalization of candidate immunoglobulin sequences, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0210] Typically, amino acid sequence variants of human anti-5T4 antibodies, humanized anti-5T4 antibodies, or chimeric anti-5T4 antibodies contain amino acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences present in the light or heavy chain of the original antibody.

[0211] In some embodiments, mice having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenMab TMAntibodies are generated using mice. A heavy chain immunoglobulin locus is a chromosomal region containing the gene for the heavy chain of an antibody. Examples of loci include the human IGHV (variable) gene, the human IGHD (variability) gene, the human IGHJ (binding) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a chromosomal region containing the gene encoding the light chain (κ chain) of an antibody. Examples of κ chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (binding) gene, and the mouse light chain constant domain gene. RenMab TM A detailed description of the mouse can be found in PCT / CN2020 / 075698 or US20200390073A1, which are incorporated herein by reference in their entirety.

[0212] In some embodiments, mice having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenLite TM Antibodies are generated using mice. A heavy chain immunoglobulin locus is a chromosomal region containing the gene for the heavy chain of an antibody. Examples of loci include the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (binding) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a chromosomal region containing the gene encoding a common light chain. Examples of κ chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (binding) gene, and the mouse light chain constant domain gene. RenLite TM A detailed description of the mouse can be found in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0213] The antibodies produced by the mouse have a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL are linked to the human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4).

[0214] Identity or homology to the original sequence is typically the percentage of amino acid residues present in a candidate sequence that is identical to a sequence present in a human antibody or fragment, humanized antibody or fragment, or chimeric anti-5T4 antibody or fragment, after aligning the sequences, introducing gaps where necessary, and achieving the maximum percentage of sequence identity, without considering conservative substitutions as part of the sequence identity.

[0215] Further modifications can be made to the anti-5T4 antibody or antigen-binding fragment. For example, a cysteine ​​residue can be introduced into the Fc region to enable the formation of interchain disulfide bonds within this region. The homodimer antibody thus produced may have some kind of extended in vitro and / or in vivo half-life. For example, homodimer antibodies with extended in vitro and / or in vivo half-lives can also be prepared using heterobifunctional crosslinking agents, as described by Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies with a double Fc region can be recombinant (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0216] In some embodiments, covalent modifications can be added to anti-5T4 antibodies or their antigen-binding fragments. These covalent modifications can be added by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues.

[0217] In some embodiments, antibody variants are provided having carbohydrate structures lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans located at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues, or position 314 in Kabat numbering), however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated mutants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further recombined, and asparagine at position 297 can be replaced with alanine (N297A).

[0218] In some embodiments, to enhance production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further recombined, replacing serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is, for example, found in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated in its entirety by reference.

[0219] Recombination vectors This disclosure also provides recombinant vectors (e.g., expression vectors) containing isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such host cells contain polynucleotides and / or the polynucleotide-containing vectors), and the production of recombinant antibody polypeptides or fragments thereof by recombinant technology.

[0220] As used herein, “vector” is any construct that, when introduced into a host cell, can deliver one or more polynucleotides of interest to the host cell. An “expression vector” can deliver and express one or more polynucleotides of interest as encoded polypeptides within the host cell into which the expression vector has been introduced. Thus, within the expression vector, the polynucleotides of interest are positioned for expression within the vector by being operably bound to regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or in the host cell genome, at, near, or adjacent to the integration site of the polynucleotides of interest, so that the polynucleotides of interest are translated within the host cell into which the expression vector has been introduced.

[0221] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (that can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0222] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., a smallpox or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (deficient) replicable virus or a non-replicable virus. In the latter case, viral replication generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317-321, Flexner et al.,1989,Ann.NYAcad Sci.569:86-103, Flexner et al. al., 1990, Vaccine, 8:17-21, U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487, WO 89 / 01973, U.S. Pat. 91 / 02805, Berkner-Biotechniques, 6:616-627, 1988, Rosenfeld et al. Preferred systems are disclosed in al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA may also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating the DNA with biodegradable beads that are efficiently transported into cells.

[0223] For expression, a DNA insert containing a polynucleotide encoding an antibody or polypeptide, as disclosed herein, can be operably ligated to a suitable promoter (e.g., a heterologous promoter), such as, to name a few, the phage λPL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoter of retroviral LTRs. Other suitable promoters are known to those skilled in the art. The expression construct may further contain sites for transcription start and end, and within the transcription region, a ribosome-binding site for translation. The coding portion of the mature transcript expressed by the construct may include a translation start at the beginning and a stop codon (UAA, UGA, or UAG) located approximately at the end of the polypeptide being translated.

[0224] As shown, the expression vector may contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, and tetracycline or ampicillin resistance genes for Escherichia coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0225] Non-restrictive vectors for bacterial use include pQE70, pQE60, and pQE-9 from Qiagen, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic cell vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene, and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0226] Suitable non-limiting bacterial promoters for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic cell promoters include the CMV pre-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters such as those for Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0227] In the yeast Saccharomyces cerevisiae, several vectors containing constitutional or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997).

[0228] The construct can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference.

[0229] Transcription of the antibody-encoding DNA in more eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting elements of DNA, approximately 10–300 bp in length, that enhance the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer, located behind the origin of replication at base pairs 100–270, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer.

[0230] Appropriate secretory signals can be incorporated into expressed polypeptides to induce the secretion of translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment. These signals can be endogenous to the polypeptide, or they can be heterologous signals.

[0231] Polypeptides (e.g., antibodies) can be expressed in modified forms such as fusion proteins (e.g., GST fusions) or by histidine tagging, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of a polypeptide to improve stability and endurance in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to induce secretion or excretion, improve stability, and facilitate purification is a well-known and common technique, particularly in the art.

[0232] Treatment method The antibodies or antigen-binding fragments thereof described herein can be used for a variety of therapeutic purposes.

[0233] In one embodiment, the disclosure provides a method for treating cancer in a subject, a method for slowing the rate of increase of tumor volume in a subject over time, a method for reducing the risk of metastasis, or a method for reducing the risk of further metastasis in a subject. In some embodiments, the treatment can interrupt, slow, stop, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction of the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0234] In one embodiment, the Disclosure relates to a method comprising administering a therapeutically effective dose of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need (for example, a subject having, or being identified or diagnosed with, cancer, such as breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, genitourinary cancer, or hematological malignancy). In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the cancer is NSCLC, ovarian cancer, melanoma, colorectal cancer, breast cancer, hematological malignancies, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer. In some embodiments, the subject has solid tumors. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematological malignancies, particularly non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, or progressive solid tumors. In some embodiments, the cancer is colorectal cancer, gastric cancer, ovarian cancer, lung cancer, head and neck cancer, or pancreatic cancer.

[0235] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients at risk of cancer can be identified by various methods well known in the art.

[0236] In one embodiment, the present disclosure provides methods for treating, preventing, or reducing the risk of developing diseases associated with abnormal or undesirable immune responses, such as autoimmune diseases. These autoimmune diseases include alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behçet's disease, pernicious anemia, bullous pemphigoid, polyarteritis nodosa, cardiomyopathy, polychondritis, sprue dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelination, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, and primary biliary tract disease. Examples of conditions that may be included, but are not limited to, sap cirrhosis, pemphigoid scarring, psoriasis, Crest syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, lupus discoid, rheumatoid arthritis, cold globulinemia sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjögren's syndrome, Guillain-Barré syndrome, Stiffman syndrome, Hashimoto's thyroiditis, Takayasu's arteritis, idiopathic alveolar fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (e.g., type 1), vasculitis, lichen planus, and vitiligo. The anti-5T4 antibody or its antigen-binding fragment can also be administered to a subject to treat, prevent, or reduce the risk of progression of abnormal or undesirable immune responses associated with cell, tissue, or organ transplantation, such as kidney, liver, and heart transplantation, or to prevent allograft rejection. In some embodiments, the subject has a skin disease, liver disease (e.g., cirrhosis), hidradenitis, or experimental autoimmune encephalomyelitis. In some embodiments, the subject has a kidney disease, lupus, Sjögren's syndrome, ulcerative colitis, psoriasis, hidradenitis suppurativa, immune thrombocytopenia (ITP), or other inflammatory arthritis. In some embodiments, the subject has multiple sclerosis or myasthenia gravis. In some embodiments, the subject has Crohn's disease, ulcerative colitis, or type 1 diabetes.In some embodiments, the subject has an autoimmune thyroid disease, Graves' disease, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis), rheumatoid arthritis, Sjögren's syndrome, autoimmune nephritis, or systemic lupus erythematosus. In some embodiments, the method comprises administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.

[0237] As used herein, “effective dose” means an amount or dosage sufficient to produce a beneficial or desired outcome, including interrupting, slowing, blocking, or inhibiting the progression of a disease, such as an autoimmune disease or cancer. The effective dose varies depending on the age and weight of the person to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector containing the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and therefore the administration can be determined on an individual basis.

[0238] An effective dose can be administered in one or more doses. For example, an effective dose of antibody or antigen-binding fragment is an amount sufficient to mitigate, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to mitigate, halt, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, an effective dose of antibody or antigen-binding fragment may vary depending on other factors, in particular, the patient's medical history, as well as the type (and / or dose) of antibody used.

[0239] The effective doses and schedules for administering the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein can be determined experimentally, and making such determinations is within the scope of the art. Those skilled in the art will understand that the dose to be administered will vary depending, for example, on the mammal receiving the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein, the route of administration, the specific type of antibody, the polynucleotide encoding the antibody, the antigen-binding fragment, and / or the compositions disclosed herein used, and other agents administered to the mammal. Guidelines for selecting an appropriate dose for an antibody or antigen-binding fragment can be found in literature on the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch.22 and pp.303-357, and Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp.365-389.

[0240] The typical daily dose of an effective amount of antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose may be 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or less than 0.1 mg / kg. In some embodiments, the dose may be 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or greater than 0.01 mg / kg. In some embodiments, the dose is approximately 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0241] In any of the methods described herein, at least one antibody, its antigen-binding fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, can be administered to a target at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0242] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity periods of the one or more additional therapeutic agents and the biological activity periods of at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) overlap within the subject.

[0243] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to a subject over a long period of time (e.g., over a period of at least one week, two weeks, three weeks, one month, two months, three months, four months, twelve months, one year, two years, three years, four years, or five years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein to diagnose or follow up on the effectiveness of the treatment (e.g., to observe at least one symptom of cancer). As described herein, skilled medical professionals may also change (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and may adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to a subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0244] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0245] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of OX40, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of the activated hedgehog signaling pathway, and an agent that selectively degrades estrogen receptor.

[0246] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leflunomide, alimta, dacai’da, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitor, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0247] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF)α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, a HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, a HER2 agonist and an OX40 agonist.

[0248] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0249] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-ICOS antibody, an anti-CD27 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-CD40 antibody, and / or an anti-GITR antibody.

[0250] In one aspect, the present disclosure provides a combination therapy. In some embodiments, an anti-5T4 antibody or an antigen-binding fragment thereof (e.g., any antibody described herein) can be administered together with an anti-OX40 antibody.

[0251] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein. Any two or more (e.g., 2, 3, or 4) of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner well known in the art.

[0252] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antimicrobial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4,522,811). The composition preparations can be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. Where necessary (e.g., in injectable formulations), adequate fluidity can be maintained by coatings such as lecithin or by the use of surfactants. The absorption of antibodies or their antigen-binding fragments can be prolonged by including absorption-delaying agents (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulation delivery systems, including biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, Alza Corporation and Nova Pharmaceutical, Inc.).

[0253] Compositions containing one or more antibodies or antigen-binding fragments described herein can be formulated in unit dosage forms (i.e., physically distinct units containing a predetermined amount of the active compound to facilitate administration and ensure uniformity of dose) for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0254] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be supplied in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. Formulation depends on the chosen route of administration. For injection, antibodies can be formulated in aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain compounding agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form for preparation with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0255] The toxicity and therapeutic effect of a composition can be determined by standard pharmaceutical procedures in cell culture media or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and ED50 (effective dose for 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs exhibiting a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize the potential harm (i.e., mitigate the undesirable side effects). Toxicity and therapeutic effect can be determined by other standard pharmaceutical procedures.

[0256] Data obtained from cell culture assays and animal studies can be used in the formulation of appropriate doses of any given agent for use in subjects (e.g., humans). A therapeutically effective dose of one or more antibodies (e.g., 1, 2, 3, or 4) or their antigen-binding fragments (e.g., any of the antibodies or antibody fragments described herein) is the amount that treats the disease in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who has previously had cancer but is now cured), or the amount that reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a healthcare professional or veterinary professional using methods well known in the art, in addition to observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors may influence the dose and timing required to effectively treat the subject (e.g., severity of the disease or disability, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0257] Exemplary doses include the amount (milligrams or micrograms) of either the antibody or antigen-binding fragment described herein per kilogram of body weight of the subject (e.g., approximately 1 μg / kg to 500 mg / kg, approximately 100 μg / kg to 500 mg / kg, approximately 100 μg / kg to 50 mg / kg, approximately 10 μg / kg to 5 mg / kg, approximately 10 μg / kg to 0.5 mg / kg, or approximately 1 μg / kg to 50 μg / kg). Although these doses cover a wide range, those skilled in the art will understand that the efficacy and effective dose of therapeutic agents containing antibodies and their antigen-binding fragments can be determined by methods well known in the art. Typically, a relatively low dose is administered first, and the dose can be subsequently and gradually increased by the healthcare professional or veterinary professional (in the case of therapeutic use), or the researcher (if still working in the development stage), until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject depends on various factors, including the activity of the specific compound used, the subject's age, weight, overall health, sex, and diet, administration time, route of administration, excretion rate, and the half-life of the antibody or antibody fragment in the body.

[0258] The pharmaceutical composition may be included in a container, pack, or dispenser, along with instructions for administration. This disclosure also provides methods for producing antibodies or their antigen-binding fragments for various applications described herein. [Examples]

[0259] The present invention will be further illustrated by the following embodiments, which are not intended to limit the scope of the invention as described in the claims.

[0260] Example 1. Production of anti-5T4 antibody His-tagged human 5T4 protein (h5T4-His, Sino Biological, catalog number: 19845-H08H) and mouse 5T4 protein (m5T4-His, Novoprotein, catalog number: C00M) are emulsified with an adjuvant, and this is used to develop RenLiteTM Mice were immunized with Biocytogen, a fully human heavy chain variable domain combined with a common light chain substitution in situ. RenLite TM The mice are described, for example, in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety. Postorbital blood was collected as a negative control prior to immunization.

[0261] Freund's complete adjuvant (CFA) was used for the first immunization, and Freund's incomplete adjuvant (IFA) was used for the second, third, and fourth immunizations. A total of four immunizations were performed. The first and second immunizations were spaced two weeks apart, and the remaining immunizations were spaced one week apart. One week after the fourth immunization, post-orbital blood was collected, and serum antibody titers were detected by fluorescence-activated cell sorting (FACS). For impulse immunization, one week later, high-titer mice were further injected with h5T4-His protein and m5T4-His protein by intraperitoneal injection, and with human 5T4-expressing CHO-S cells by tail vein injection.

[0262] Antigen-specific immune cells were isolated from immunized mice, and anti-5T4 antibodies were obtained, or the light and heavy chain variable region sequences of anti-5T4 antibodies were obtained. For example, plasma cells secreting antigen-specific monoclonal antibodies were screened and discovered using single-cell techniques (e.g., Beacon® Optofluidic System, Berkeley Lights Inc.), and then the antibody variable region sequences were obtained using reverse transcription and PCR sequencing. The obtained variable region sequences were cloned into vectors containing sequences encoding the human IgG1 constant region for antibody expression. Binding of the expressed antibodies to 5T4 was confirmed by FACS. Exemplary antibodies obtained by this method include 31G4, 32E3, 32G1, 32G8, 35B12, and 35C6.

[0263] These antibodies contain substantially the same light chain, and their VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3 sequences are shown in Figure 1 or Figure 2. The VH and VL regions of the antibodies and derived antibodies are shown in Figure 3.

[0264] Example 2. Interspecies binding of anti-5T4 antibody CHO-h5T4 cells, CHO-m5T4 cells, CHO-fas5T4 cells, or CHO-d5T4 cells, 2 × 10⁻¹⁴ 5 Cells were seeded at a density of 1 cell / well into each 96-well plate. Anti-5T4 antibody was added to the 96-well plate and incubated at 4°C for 30 minutes. Next, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alex Flour 647(RL1-H) (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170) at 4°C in the dark for 15 minutes, followed by flow cytometry analysis. The test results are shown in the table below.

[0265] CHO-h5T4 cells, CHO-m5T4 cells, CHO-fas5T4 cells, and CHO-d5T4 cells were obtained by transfecting CHO-S cells with vectors expressing human 5T4 (SEQ ID NO: 48), mouse 5T4 (SEQ ID NO: 49), monkey (cynomolgus monkey) 5T4 (SEQ ID NO: 50), and canine 5T4 (SEQ ID NO: 51).

[0266] [Table 1]

[0267] All anti-5T4 antibodies can bind to human 5T4, monkey 5T4, and canine 5T4 (except 31G4). ​​Furthermore, 32E3, 32G1, 35B12, and 35C6 can also bind to monkey 5T4.

[0268] Example 3. Internalization of anti-5T4 antibody Anti-5T4 antibody (2.5 μg / mL) and pHAb-AffiniPure Fab goat anti-human IgG secondary antibody were added to NCI-H2030 cells (ATCC, catalog number: CRL-5914), CMT cells (ATCC, catalog number: CRL-3456), or D17 cells (ATCC, catalog number: CCL-183) and incubated for 6 - 24 hours. After incubation, the cells were centrifuged and washed in FACS buffer. MFI was detected with a flow cytometer and the endocytosis ratio of the anti-5T4 antibody was calculated. The results are summarized in the following table.

[0269] PF06263507 is a humanized IgG1 monoclonal antibody targeting 5T4, and the VH and VL sequences are shown in SEQ ID NOs: 52 - 53. Human IgG1 protein was used as an isotype control (ISO).

[0270]

Table 2

[0271] According to the data, 31G4, 32E3, 32G1, 32G8, 35B12, and 35C6 showed good endocytosis ratios in NCI-H2030 cells, CMT cells, and D17 cells.

[0272] Example 4. Binding Affinity of Anti-5T4 Antibody The binding affinity of the anti-5T4 antibody to human His-tagged 5T4 protein (h5T4, ACRO Biosystems Inc., catalog number: TPG-H52E5), monkey (fas5T4, ACRO Biosystems Inc., catalog number: TPG-C52H3) or mouse (m5T4, ACRO Biosystems Inc., catalog number: TPG-M52H3) was verified using a Biacore TM (Biacore, Inc., Piscataway, New Jersey) 8K biosensor.

[0273] The purified anti-5T4 antibody was diluted to 2 μg / mL, and then injected into Biacore at a rate of 10 μL / min for approximately 50 seconds. TM The desired protein density (e.g., approximately 50 response units (RU)) was achieved by injecting into an 8K biosensor. Next, His-tagged 5T4 protein at a concentration of 200 nM was injected at 30 μL / min for 180 seconds. Dissociation was monitored for 400 seconds. After the last injection of each titration, the tip was regenerated with glycine (pH 2.0) at 30 μL / min for 30 seconds. For the ISO control, an antibody targeting an unrelated target protein was used.

[0274] Biacore TM Using 8K Evaluation software 3.0, the entire dataset was fitted to a 1:1 Langmuir coupled model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) to simultaneously obtain the motor association velocity (kon) and dissociation velocity (koff). Affinity was estimated from the quotient of the motor velocity constant (KD = koff / kon).

[0275] The same method, with appropriately adjusted parameters (e.g., antibody concentration) as those skilled in the art would understand, was performed for each test antibody. The results for each test antibody are summarized in the table below.

[0276] [Table 3]

[0277] MAB2 is a humanized IgG1 monoclonal antibody targeting 5T4, with its VH and VL sequences shown in SEQ ID NOs. 54-55. Human IgG1 protein was used as an isotype control (ISO).

[0278] The results show that 31G4, 32E3, 32G1, 32G8, 35B12, and 35C6 can all bind to human 5T4 and monkey 5T4. Furthermore, 35B12 and 35C6 also bind to mouse 5T4 with high affinity.

[0279] Example 5. Epitope assay of anti-5T4 antibody Epitope binding assays were performed using the ForteBio Octet system to determine whether two anti-5T4 antibodies targeted the same or overlapping epitopes. A 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4), diluted from 10× HBS-EP+ buffer, was used as the running buffer throughout the experiment. Approximately 50 RU of h5T4-His protein was captured at a flow rate of 10 μL / min, and 200 nM test antibodies were injected at a flow rate of 30 μL / min to bind the ligands. Another comparison antibody was injected under the same conditions to determine whether the binding of different antibodies interfered with each other. Binding times were 180 s per antibody.

[0280] The binding values ​​for each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of one antibody binding to another, the binding ratio was calculated and each pair of antibodies was compared. The binding ratio was defined by dividing the binding value of the second antibody (sample 2) by the binding value of the first antibody (sample 1). The binding ratios for each antibody pair are summarized in the table below. More specifically, when sample 1 showed an inhibitory effect on sample 2, the binding ratio was between -0.02 and 0.5. When sample 1 did not show an inhibitory effect on sample 2, the binding ratio was between 0.5 and 1.05. Generally, antibody pairs that interfere with each other have the same or overlapping epitopes.

[0281] The results showed that 31G4, 32E3, 32G8, and 32G1 recognize different epitopes. 35B12 and 35C6 target the same or overlapping epitopes.

[0282] [Table 4]

[0283] Example 6. Stability analysis of anti-5T4 antibody The biophysical properties and stability of the anti-5T4 antibody were evaluated. The antibody sample processing procedure was as follows:

[0284] 1) The antibody was placed in a buffer solution containing 3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween 80 at pH 6.0 until the final concentration reached 2 mg / mL. The solution was then stored in a sealed Eppendorf tube at 40±2°C and 60%±5% RH (hereinafter referred to as 40°C) for 7 days, and its thermal stability was evaluated.

[0285] 2) The antibody was loaded onto a Protein A column and eluted with a pH 3.5 buffer (0.1 mol / L HAc). Half of the antibody was added to 2 M Tris buffer and the pH was immediately adjusted to 7.5. The remaining half was stored at pH 3.5 for 6 hours, and then the pH was adjusted to 7.5. The diluted antibody was stored in a sealed Eppendorf tube at pH 3.5 ± 0.1, 25 ± 2°C (hereinafter referred to as pH 3.5) for 6 hours to test its stability at low pH.

[0286] The following tests were performed: (1) Detection of antibody purity by size exclusion high-performance liquid chromatography (SEC-HPLC) (expressed as the ratio of the main peak area to the sum of all peak areas (purity, %)), (2) Detection of apparent hydrophobicity of antibody using hydrophobic interaction chromatography-high-performance liquid chromatography (HIC-HPLC) (expressed as the retention time of the main peak (HIC, minutes)), and (3) Detection of antibody pI (isoelectric point) and charge variants by capillary isoelectric focusing (cIEF) (expressed as the ratio of the main component, acidic component, and alkaline component).

[0287] In the SEC-HPLC experiment, the antibody sample was diluted to 1 mg / mL with purified water and used with an Agilent 1290 chromatograph system (connected to an XBridge Protein BEH SEC column (200 Å, Waters Corporation)). The parameters used were as follows: Mobile phase: 0.1 M phosphate buffer (PB) + 10% ACN, pH 7.4, Flow rate: 1.8 mL / min, Column temperature: 25°C, Detection wavelengths: 280 nm, 220 nm, Injection volume: 10 μL, Sample tray temperature: approximately 4°C, and Run time: 7 minutes.

[0288] In the HIC-HPLC experiment, an Agilent 1260 chromatograph system (connected to a ProPac HIC-10 column (4.6 × 250 mm, Thermo Scientific)) was used, and the sample was diluted 10-fold using mobile phase A. The parameters used were as follows: Mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile pH 6.5; Mobile phase B: 0.1 M phosphate buffer (PB), 10% acetonitrile pH 6.5; Flow rate: 0.8 mL / min; Gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, 45 min 100% A; Column temperature: 30°C; Detection wavelengths: 280 nm, 220 nm; Injection volume: 10 μg; Sample tray temperature: approximately 10°C; Run time: 50 minutes.

[0289] For the cIEF experiment, the Maurice cIEF method development kit (Protein Simple, catalog number: PS-MDK01-C) was used for sample preparation. Specifically, 8 μL, 30 μg of protein sample was mixed in the kit with the following reagents: 1 μL of Maurice cIEF pI Marker-7.05, 1 μL of Maurice cIEF pI Marker-10.10, 35 μL of 1% methylcellulose solution, 2 μL of Maurice cIEF 500 mM arginine, 1.33 μL of Ampholytes (Pharmalyte pH range 3-10), and 6.66 μL of Ampholytes (Pharmalyte pH range 8-10.5), and water (added to a final volume of 100 μL). Imaging capillary isoelectric focusing spectra were generated using a Maurice analyzer (Protein Simple, Santa Clara, CA) with a Maurice cIEF cartridge (PS-MC02-C). The sample was focused for a total of 10 minutes.

[0290] The detailed results are shown in the table below. The results demonstrated that these anti-5T4 antibodies possessed excellent stability and physical and chemical properties.

[0291] [Table 5]

[0292] Example 7. Generation of anti-5T4 antibody drug conjugate (ADC) Each purified antibody (31G4, 32E3, 32G1, 32G8, 35B12, and 35C6) was coupled with MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0293] To name an antibody-drug conjugate, add "ADC" immediately after the antibody name. For example, when 31G4 is coupled to MMAE, it is named 31G4-ADC.

[0294] HIC-HPLC was performed to detect the coupling between the antibody and the drug molecule. The results indicate that the drug-antibody ratio (DAR) of the ADC is approximately 4.

[0295] Example 8. Antitumor activity of anti-5T4 ADC in the NUGC-4 model To determine the antitumor activity of anti-5T4 ADCs, B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., catalog number: B-CM-002) were used. Approximately 5 × 10⁻⁶ mice were used. 6 Individual gastric cancer NUGC-4 cells were subcutaneously injected into B-NDG mice, and the tumor volume was approximately 200 mm². 3 Once the tumors had grown to a certain size, the mice were divided into a control group and two treatment groups based on tumor size (5 mice per group). The treatment groups were randomly injected intravenously (iv) with either 3 mg / kg of 32G1-ADC (G2) or 3 mg / kg of PF06263507-ADC (G3). The control group mice were injected with phosphate-buffered saline (PBS) (G1). The administration frequency was once a week (a total of two doses). A PF06263507 analog was coupled with MMAE as a positive control (named PF06263507-ADC). Tumor volume was measured twice a week, and the body weight of the mice was also measured. When the tumor volume of a mouse reached 3000 mm³... 3 Euthanasia was performed when the condition was reached.

[0296] Measure the length of the long axis and short axis of the tumor, and calculate the tumor volume as 0.5 × (long axis) × (short axis). 2The following formula was used for calculation. The tumor growth inhibition rate (TGI%) is calculated using the following formula: (TGI%) = [1 - (Ti - T0) / (Vi - V0)] × 100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0. A t-test was performed for statistical analysis. A TGI% greater than 60% indicates a clear suppression of tumor growth. P < 0.05 is the threshold for showing a statistically significant difference.

[0297] At the end of the experiment, there were no significant differences between the groups, and all mice in the treatment group survived the experiment, demonstrating that 32G01-ADC is well-tolerated and clearly non-toxic to mice.

[0298] The tumor size data for the group treated with ADC are shown in Figure 4. The following table summarizes the results for this experiment and includes tumor volume on the day of group assignment (day 0), 14 days after group assignment (day 14), 28 days after group assignment (day 28), and at the end of the experiment (day 39), as well as mouse survival rate on day 39, tumor growth inhibition (TGI), and the statistical difference (P-value) in tumor volume between the treatment group and the control group.

[0299] [Table 6]

[0300] As shown in Figure 4 and the table above, tumor growth was suppressed to varying degrees in the treatment group compared to the control group. The 32G1-ADC treatment group showed a better tumor inhibitory effect compared to the positive control PF06263507-ADC. In particular, at the end of the experiment (day 39), 32G1-ADC showed a TGI% of 39.1%, demonstrating a stronger antitumor effect than the positive control (TGI% of 3.3%).

[0301] Example 9. Antitumor activity in a xenograft model derived from lung cancer patients. ADCs were tested for their effects on tumor growth in a xenograft model derived from lung cancer patients. Immunofluorescence staining was performed on patient-derived tumor fragments, and the images were analyzed using the HALO® image analysis platform (version 3.2). The results showed that 5T4-positive cells constitute 58.86% of all cells in human lung tumor tissue. Specifically, patient-derived tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. The tumor in the mice was approximately 250-300 mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on their tumor volume. The mice were then injected with either PBS or ADC. Further details are shown in the table below.

[0302] [Table 7]

[0303] Body weight was measured twice a week. During the experiment, there were no significant differences in body weight between the groups, indicating that the test ADC was well-tolerated and clearly non-toxic to mice.

[0304] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and statistical differences (P-values) in tumor volume and body weight between the treatment group and the control group on the day of group assignment (day 0), 14 days after group assignment (day 14), and 28 days after group assignment (day 28).

[0305] [Table 8]

[0306] The tumor volumes of mice from different groups are shown in Figure 5. The treatment groups (G3-G4) showed better tumor inhibition compared to the PBS group (G1). Furthermore, 32G1-ADC (G3) showed a higher TGI% on day 28 than the positive controls PF06263507-ADC (G4) and ISO-ADC (G2).

[0307] Example 10. Verification of the binding activity of anti-5T4 antibody to NCI-H226 cells, NCI-H2030 cells, or HCC827 cells. The binding activity of anti--5T4 antibody 32G1 to NCI-H226 cells (ATCC, catalog number: CRL-5826), NCI-H2030 cells, or HCC827 cells (ATCC, catalog number: CRL-2868) was verified by flow cytometry.

[0308] NCI-H226 cells, NCI-H2030 cells, or HCC827 cells, 2 × 10⁻⁶ 5 Cells were seeded in 96-well plates at a density of 10 cells / well. Serially diluted purified anti-5T4 antibody 32G1 or positive control PF06263507 analog-SI (maximum concentration: 20 μg / mL, 2-fold dilution, 12 gradients) was added to each well, and incubated at 4°C for 30 minutes. After a single wash with PBS, the cells were incubated with the secondary antibody Alexa Fluor® 647 anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170) at 4°C for 15 minutes, and flow cytometry analysis was performed.

[0309] The SI mutations (EU numbering: S239D and I332E mutations) are introduced into the Fc region of the PF06263507 analog, and the resulting antibody is named PF06263507 analog-SI.

[0310] Cells were harvested and the mean fluorescence intensity (MFI) was determined. The results are shown in Figures 6A-6C, where 32G1 showed higher binding activity to NCI-H226, NCI-H2030, and HCC827 cells than the positive control PF06263507 analog-SI.

[0311] Other Embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody that binds to 5T4 (5T4 tumor fetal antigen) or an antigen-binding fragment thereof, A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR1, the VH CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR2, and the VH CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR3, and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR1, the VL CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR2, and the VL CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR3. An antibody or antigen-binding fragment thereof, wherein the amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3, are one of the following: (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (7) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (8) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 25 to 27, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1 to 3, respectively. (9) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (10) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (11) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 34 to 36, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively, and (12) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

2. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4 to 6, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

3. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 7 to 9, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

4. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10 to 12, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

5. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 13 to 15, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3.

6. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 16 to 18, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

7. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 19 to 21, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

8. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 22 to 24, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

9. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 25 to 27, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

10. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 28 to 30, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3.

11. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 31 to 33, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

12. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 34 to 36, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

13. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 37 to 39, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof specifically binds to 5T4 in humans, mice, monkeys, or dogs.

15. The antibody or antigen-binding fragment according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

16. The antibody or antigen-binding fragment according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof is a human IgG1 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

17. A nucleic acid containing a polynucleotide that codes for a polypeptide including the following: (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 4 to 6, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 40, (2) An immunoglobulin light chain or fragment thereof comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which each contain the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 41, (3) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 7 to 9, wherein the VH binds to 5T4 when pairing with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 40, (4) An immunoglobulin light chain or fragment thereof comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which each contain the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 42, (5) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 10 to 12, wherein the VH binds to 5T4 when pairing with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.

40. (6) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when pairing with a VH containing the amino acid sequence shown in SEQ ID NO: 43, (7) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 13 to 15, wherein the VH binds to 5T4 when pairing with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.

40. (8) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when pairing with a VH containing the amino acid sequence shown in SEQ ID NO:

44. (9) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 16 to 18, wherein the VH binds to 5T4 when pairing with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.

40. (10) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when pairing with a VH containing the amino acid sequence shown in SEQ ID NO: 45, (11) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when pairing with a VH containing the amino acid sequence shown in SEQ ID NO: 46, (12) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 19 to 21, wherein the VH binds to 5T4 when pairing with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.

40. (13) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein the VL binds to 5T4 when pairing with a VH containing the amino acid sequence shown in SEQ ID NO: 47, (14) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 22 to 24, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO.

40. (15) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 25 to 27, wherein the VH binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO.

40. (16) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 28 to 30, wherein the VH forms a pair with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 40, and the immunoglobulin heavy chain or fragment thereof binds to 5T4. (17) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 31 to 33, wherein the VH forms a pair with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 40, and the immunoglobulin heavy chain or fragment thereof binds to 5T4. (18) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 34 to 36, wherein the VH is an immunoglobulin heavy chain or fragment thereof that binds to 5T4 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 40, or (19) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which comprise the amino acid sequences shown in SEQ ID NOs. 37 to 39, wherein the VH binds to 5T4 when pairing with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.

40.

18. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1 to 3.

19. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 4 to 6.

20. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH including CDR1, 2, and 3, which respectively contain the amino acid sequences shown in SEQ ID NOs. 7 to 9.

21. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof containing VH including CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 10 to 12.

22. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof containing VH including CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 13 to 15.

23. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 16 to 18.

24. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof containing VH including CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 19 to 21.

25. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 22 to 24.

26. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH including CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 25 to 27.

27. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 28 to 30.

28. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 31 to 33.

29. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 34 to 36.

30. The nucleic acid according to claim 17, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 37 to 39.

31. The nucleic acid according to any one of claims 17 to 30, wherein the VH specifically binds to 5T4 of humans, mice, monkeys, or dogs when pairing with VL, or the VL specifically binds to 5T4 of humans, mice, monkeys, or dogs when pairing with VH.

32. The nucleic acid according to any one of claims 17 to 31, wherein the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof or a humanized immunoglobulin heavy chain or fragment thereof (for example, a human IgG1 heavy chain or fragment thereof, or a human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof or a humanized immunoglobulin light chain or fragment thereof.

33. The nucleic acid according to any one of claims 17 to 32, wherein the nucleic acid encodes a single-stranded variable fragment (scFv), a single-arm antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

34. The nucleic acid according to any one of claims 17 to 33, wherein the nucleic acid is cDNA.

35. A vector comprising one or more nucleic acids as described in any one of claims 17 to 34.

36. A vector comprising two nucleic acids according to any one of claims 17 to 34, wherein the vector encodes the VL region and the VH region that bind together to 5T4.

37. A pair of vectors, each vector comprising one of the nucleic acids described in any one of claims 17 to 34, wherein the pair of vectors encode the VL region and the VH region together which bind to 5T4.

38. A cell comprising the vector according to claim 35 or 36, or the pair of vectors according to claim 38.

39. The cell according to claim 38, wherein the cell is a CHO cell.

40. A cell comprising one or more nucleic acids as described in any one of claims 17 to 34.

41. A cell comprising two nucleic acids according to any one of claims 17 to 34.

42. The cell according to claim 41, wherein the two nucleic acids encode a pair of VL and VH regions that bind together to 5T4.

43. A method for producing an antibody or an antigen-binding fragment thereof, wherein the method is: (a) Culturing the cells according to any one of claims 39 to 43 under conditions sufficient for the cells to produce the antibody or the antigen-binding fragment, (b) recovering the antibody or antigen-binding fragment produced by the cells, method.

44. An antibody that binds to 5T4 or an antigen-binding fragment thereof, An antibody or antigen-binding fragment comprising a heavy chain variable region (VH) containing an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 41, and the selected VL sequence is sequence number 40. (2) The selected VH sequence is sequence number 42, and the selected VL sequence is sequence number 40. (3) The selected VH sequence is sequence number 43, and the selected VL sequence is sequence number 40. (4) The selected VH sequence is sequence number 44, and the selected VL sequence is sequence number 40. (5) The selected VH sequence is sequence number 45, and the selected VL sequence is sequence number 40. (6) The selected VH sequence is sequence number 46, and the selected VL sequence is sequence number 40, and (7) The selected VH sequence is sequence number 47, and the selected VL sequence is sequence number 40.

45. The antibody or antigen-binding fragment thereof according to claim 44, wherein VH comprises the sequence of SEQ ID NO: 41 and VL comprises the sequence of SEQ ID NO:

40.

46. The antibody or antigen-binding fragment thereof according to claim 44, wherein VH comprises the sequence of SEQ ID NO: 42 and VL comprises the sequence of SEQ ID NO:

40.

47. The antibody or antigen-binding fragment thereof according to claim 44, wherein VH comprises the sequence of SEQ ID NO: 43 and VL comprises the sequence of SEQ ID NO:

40.

48. The antibody or antigen-binding fragment thereof according to claim 44, wherein VH comprises the sequence of SEQ ID NO: 44 and VL comprises the sequence of SEQ ID NO:

40.

49. The antibody or antigen-binding fragment thereof according to claim 44, wherein VH comprises the sequence of SEQ ID NO: 45 and VL comprises the sequence of SEQ ID NO:

40.

50. The antibody or antigen-binding fragment thereof according to claim 44, wherein VH comprises the sequence of SEQ ID NO: 46 and VL comprises the sequence of SEQ ID NO:

40.

51. The antibody or antigen-binding fragment thereof according to claim 44, wherein VH comprises the sequence of SEQ ID NO: 47 and VL comprises the sequence of SEQ ID NO:

40.

52. An antibody that binds to 5T4 or an antigen-binding fragment thereof, An antibody or antigen-binding fragment comprising a heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 41, and the selected VL sequence is sequence number 40. (2) The selected VH sequence is sequence number 42, and the selected VL sequence is sequence number 40. (3) The selected VH sequence is sequence number 43, and the selected VL sequence is sequence number 40. (4) The selected VH sequence is sequence number 44, and the selected VL sequence is sequence number 40. (5) The selected VH sequence is sequence number 45, and the selected VL sequence is sequence number 40. (6) The selected VH sequence is sequence number 46, and the selected VL sequence is sequence number 40, and (7) The selected VH sequence is sequence number 47, and the selected VL sequence is sequence number 40.

53. The antibody or antigen-binding fragment thereof according to any one of claims 44 to 52, wherein the antibody or antigen-binding fragment thereof specifically binds to 5T4 in humans, mice, monkeys, or dogs.

54. The antibody or antigen-binding fragment according to any one of claims 44 to 53, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

55. The antibody or antigen-binding fragment according to any one of claims 44 to 54, wherein the antibody or antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

56. An antibody or an antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment described in any one of claims 1 to 16 and 44 to 55.

57. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 56, wherein the antibody or antigen-binding fragment thereof includes a fragment crystallizable region (Fc region).

58. The antibody or antigen-binding fragment thereof according to claim 57, wherein complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) in the Fc region is increased.

59. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 56.

60. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 58, covalently bound to a therapeutic agent.

61. The antibody-drug conjugate according to claim 60, wherein the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

62. A method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 58, a CAR according to claim 59, or an antibody-drug conjugate according to claim 60 or 61.

63. The method according to claim 62, wherein the subject has a solid tumor.

64. The method according to claim 62, wherein the cancer is colorectal cancer, gastric cancer, ovarian cancer, lung cancer, head and neck cancer, or pancreatic cancer.

65. The method according to claim 62, wherein the subject is further treated with an effective amount of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-CD40 antibody, or anti-PD-L1 antibody.

66. A method for reducing the rate of tumor growth, wherein the method is A method comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 58, a CAR according to claim 59, or an antibody-drug conjugate according to claim 60 or 61.

67. A method for killing tumor cells, wherein the method is A method comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 58, a CAR according to claim 59, or an antibody-drug conjugate according to claim 60 or 61.

68. A method for increasing the immune response in a target, wherein the method is A method comprising administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 58, a CAR according to claim 59, or an antibody-drug conjugate according to claim 60 or 61.

69. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 44 to 58, and a pharmaceutically acceptable carrier.

70. A pharmaceutical composition comprising an antibody-drug conjugate according to claim 60 or 61 and a pharmaceutically acceptable carrier.

71. The antibody-drug conjugate according to claim 60 or 61, wherein the drug-antibody ratio (DAR) is approximately 4.

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