Anti-5T4 antigen-binding domain, antibody-drug conjugate, and method of use thereof

Abstract

This disclosure provides an anti-5T4 antigen-binding domain that can be incorporated into antibodies and receptors, including a bispecific anti-5T4 antibody, a biparatopic anti-5T4 antibody, and its antibody-drug conjugate, comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antibody-antigen-binding domain that specifically binds to a second 5T4 epitope that is not identical to the first 5T4 epitope (the first antigen-binding domain is operably linked to the second antigen-binding domain), and a chemotherapeutic agent. This disclosure further provides methods for using it for the treatment of cancer.

Description

[Technical Field]

[0001] Related applications This application claims priority and interest in U.S. Provisional Application No. 63 / 453,929, filed on 22 March 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (SBTI-005_001WO_SeqList_St26.xml, size: 163,004 bytes, and creation date: February 29, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] Cancer is one of the leading causes of death in developed countries. In the United States alone, an estimated 1.8 million people are newly diagnosed with cancer, and in 2020, there were more than 600,000 cancer deaths. In cancer, the affected cells proliferate and divide abnormally and spread to surrounding tissues. Each cancer is thought to have a combination of genetic alterations that can differ from one cancer to another, allowing cancer cells to evade the body's natural control over cell proliferation and enabling the cancer to spread. Some cancers are currently treatable, but many are not. In this field of technology, there is a need for compositions and methods for the treatment of cancer.

[0004] Human 5T4 manifests itself in various cancer types, including bladder cancer, breast cancer, cervical cancer, endometrial cancer, lung cancer, esophageal cancer, ovarian cancer, pancreatic cancer, gastric cancer, and testicular cancer. Human 5T4 is generally not found in normal tissues, and when found, is expressed at low levels, making it an ideal therapeutic target for cancer treatment. This disclosure provides antibodies and antibody-drug conjugates comprising chemotherapeutic agents that specifically bind to a first, and in some cases a second, 5T4 epitope, compositions comprising the same, and methods for preparing and using the same for the treatment of diseases such as cancer. [Overview of the project]

[0005] This disclosure provides a 5T4 antigen-binding domain, as well as antibodies, antibody-drug conjugates, and receptors that constitute it.

[0006] This disclosure provides a 5T4 biparatopic antibody-drug conjugate comprising (a) a first antigen-binding domain that specifically binds to a first 5T4 epitope, (b) an antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and (c) a chemotherapeutic agent, wherein the first antigen-binding domain is operably linked to the second antigen-binding domain. In some embodiments of the 5T4 biparatopic antibody and antibody-drug conjugate described herein, the biparatopic antibody or antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, and the second antigen-binding domain contains an scFv.

[0007] In some embodiments of the antibody-drug conjugates of this disclosure, the N-terminus of a second antigen-binding domain is operably linked to the C-terminus of the heavy chain of a first antigen-binding domain. In some embodiments, the C-terminus of a second antigen-binding domain is operably linked to the N-terminus of the heavy chain of a first antigen-binding domain. In some embodiments, the second antigen-binding domain is operably linked to the heavy chain of a first antigen-binding domain using a linker. In some embodiments, the antibody or antibody-drug conjugate comprises a full-length IgG antibody containing a first antigen-binding domain, the second antigen-binding domain containing scFv, and the antibody or antibody-drug conjugate comprises four polypeptides, (a) two polypeptides containing a first antigen-binding domain heavy chain, a linker, and a second antigen-binding domain from N-terminus to C-terminus, and (b) two polypeptides containing a first antigen-binding domain light chain. In some embodiments, the antibody or antibody-drug conjugate comprises a full-length IgG antibody containing a first antigen-binding domain, the second antigen-binding domain containing scFv, and the antibody-drug conjugate comprises four polypeptides, each comprising (a) two polypeptides containing a second antigen-binding domain, a linker, and a first antigen-binding domain heavy chain from the N-terminus to the C-terminus, and (b) two polypeptides containing a first antigen-binding domain light chain.

[0008] In some embodiments, the chemotherapeutic agent is an auristatin selected from the group consisting of auristatins such as auristatin E (AE), monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and synthetic analogs of drastatin.

[0009] This disclosure provides nucleic acid systems and vectors encoding antigen-binding domains, antibodies, and receptors.

[0010] This disclosure provides pharmaceutical compositions comprising immune cells comprising the antigen-binding domain, antibody, antibody-drug conjugate, and receptor of this disclosure.

[0011] This disclosure provides pharmaceutical compositions comprising immune cells, antigen-binding domains, antibodies, antibody-drug conjugates, and receptors, for use in the treatment of cancer in a subject.

[0012] This disclosure provides pharmaceutical compositions comprising immune cells, antigen-binding domains, antibodies, antibody-drug conjugates, and receptors, for use in the manufacture of pharmaceuticals for the treatment of cancer in a subject.

[0013] This disclosure provides a method for treating cancer in a subject in need of cancer treatment, comprising administering to the subject in need a therapeutically effective amount of immune cells comprising an antigen-binding domain, antibody, antibody-drug conjugate, or receptor of this disclosure. In some embodiments, the method comprises administering an antibody-drug conjugate comprising (a) a first antigen-binding domain that specifically binds to a first 5T4 epitope, (b) a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and (c) a chemotherapeutic agent, wherein the first antigen-binding domain is operably linked to the second antigen-binding domain.

[0014] This disclosure provides a method for producing an antibody-drug conjugate, comprising (a) culturing cells containing a nucleic acid system encoding an antibody under conditions that result in antibody expression, (b) recovering the antibody, and (c) conjugating the antibody with a chemotherapeutic agent. [Brief explanation of the drawing]

[0015] [Figure 1] This is a schematic diagram showing the structural configuration of exemplary anti-5T4 biparatopic antibody-drug conjugates. Figure 1A shows the first exemplary antibody-drug conjugate (Bs2 orientation). Figure 1B shows the second exemplary antibody-drug conjugate (Bs3 orientation). VH: variable heavy chain, VL: variable light chain, scFv: single-chain variable fragment.

[0016] [Figure 2]Figure 2A shows graphs and tables illustrating the size exclusion chromatography analysis of four exemplary anti-5T4 biparatopic antibodies after protein A purification. Figure 2A shows two graphs illustrating the monomer peak analysis of two exemplary antibodies in Bs2 orientation, as shown in Figure 1A. The left plot shows the antibody with VL-VH orientation scFv, while the right plot shows the antibody with VH-VL orientation scFv. Figure 2B shows two graphs illustrating the monomer peak analysis of two exemplary antibodies in Bs3 orientation, as shown in Figure 1B. The left plot shows the antibody with VL-VH orientation scFv, while the right plot shows the antibody with VH-VL orientation scFv. Figure 2C is a table showing the quantitative soluble protein yield in mg / L and relative purity by size exclusion chromatography (SEC) peaks for the antibody variations shown in Figures 2A-2B from 10-day culture harvests.

[0017] [Figure 3] These are two graphs and tables showing SPR-based binding assays to confirm that the 5T4 epitope bound by antibodies 1 and 2 is non-competitive. Figure 3A shows an assay in which mouse Ab1 ((m)Ab1) is captured on a sensor chip using an anti-mouse IgG antibody, followed by injection of 5T4, and then humanization of Ab1 or Ab2 using human Fc. Figure 3B shows an assay in which mouse Ab2 ((m)Ab2) is captured on a sensor chip using an anti-mouse IgG antibody, followed by injection of 5T4, and then humanization of Ab1 or Ab2 using human Fc. Figure 3C is a table showing a legend with binding sensorgrams and injection samples for Figures 3A-3B. In Figures 3A and 3B, the y-axis shows the relative response (in resonance units, i.e., RU), and the x-axis shows time (in seconds, i.e., s).

[0018] [Figure 4-1]Figure 4A shows the binding affinity of parental monospecific antibodies and biparatopic antibodies to 5T4, as determined by Biacore analysis. Figure 4A shows the binding of the first exemplary monospecific antibody, humanized antibody Ab1. Figure 4B shows the binding of the second exemplary monospecific antibody, humanized antibody Ab2. Figure 4C shows the binding of an exemplary biparatopic antibody oriented Bs3 to a VH-VL oriented scFv (Bs3-HL). Figure 4D shows the binding of a second exemplary biparatopic antibody (Bs3-HL-FCA, Bs3 with a VH-VL oriented scFv, and Bs3 with L234F, S239C, and N434A mutations). Figure 4E is a table summarizing the equilibrium dissociation constant (KD), dissociation rate constant (Kd), and association rate constant (Ka) of the antibody binding assays from Figures 4A-4D. In Figures 4A to 4D, the y-axis represents the response (in RU), and the x-axis represents time (in seconds). [Figure 4-2] Figure 4A shows the binding affinity of parental monospecific antibodies and biparatopic antibodies to 5T4, as determined by Biacore analysis. Figure 4A shows the binding of the first exemplary monospecific antibody, humanized antibody Ab1. Figure 4B shows the binding of the second exemplary monospecific antibody, humanized antibody Ab2. Figure 4C shows the binding of an exemplary biparatopic antibody oriented Bs3 to a VH-VL oriented scFv (Bs3-HL). Figure 4D shows the binding of a second exemplary biparatopic antibody (Bs3-HL-FCA, Bs3 with a VH-VL oriented scFv, and Bs3 with L234F, S239C, and N434A mutations). Figure 4E is a table summarizing the equilibrium dissociation constant (KD), dissociation rate constant (Kd), and association rate constant (Ka) of the antibody binding assays from Figures 4A-4D. In Figures 4A to 4D, the y-axis represents the response (in RU), and the x-axis represents time (in seconds). [Figure 4-3]Figure 4A shows the binding affinity of parental monospecific antibodies and biparatopic antibodies to 5T4, as determined by Biacore analysis. Figure 4A shows the binding of the first exemplary monospecific antibody, humanized antibody Ab1. Figure 4B shows the binding of the second exemplary monospecific antibody, humanized antibody Ab2. Figure 4C shows the binding of an exemplary biparatopic antibody oriented Bs3 to a VH-VL oriented scFv (Bs3-HL). Figure 4D shows the binding of a second exemplary biparatopic antibody (Bs3-HL-FCA, Bs3 with a VH-VL oriented scFv, and Bs3 with L234F, S239C, and N434A mutations). Figure 4E is a table summarizing the equilibrium dissociation constant (KD), dissociation rate constant (Kd), and association rate constant (Ka) of the antibody binding assays from Figures 4A-4D. In Figures 4A to 4D, the y-axis represents the response (in RU), and the x-axis represents time (in seconds).

[0019] [Figure 5] This table shows the sequence identity percentage of 5T4 proteins derived from rhesus monkeys, cynomolgus monkeys, mice, and rats compared to human 5T4.

[0020] [Figure 6] This graph shows the binding of exemplary Bs3-oriented double paratopic 5T4 antibodies to 5T4 proteins from various species, as measured by an ELISA assay. NHP: Non-human primates.

[0021] [Figure 7-1]This is a series of graphs showing the binding specificity of exemplary biparatopic antibodies with Bs3 orientation to 5T4 proteins from various species, as determined by surface plasmon resonance (SPR). Figure 7A shows binding to human 5T4. Figure 7B shows binding to NHP 5T4. NHP: Non-human primate. Figure 7C shows binding to mouse 5T4. Figure 7D shows binding to rat 5T4. In all of Figures 7A-7D, the y-axis shows the relative response (in RU), and the x-axis shows the time (in seconds). In Figures 7A-7D, the antibody concentrations are as follows: 100 nM (purple), 33.3 nM (yellow), 11.1 nM (pink), 3.7 nM (green), 1.2 nM (orange), and 133 pM (blue). [Figure 7-2] This is a series of graphs showing the binding specificity of exemplary biparatopic antibodies with Bs3 orientation to 5T4 proteins from various species, as determined by surface plasmon resonance (SPR). Figure 7A shows binding to human 5T4. Figure 7B shows binding to NHP 5T4. NHP: Non-human primate. Figure 7C shows binding to mouse 5T4. Figure 7D shows binding to rat 5T4. In all of Figures 7A-7D, the y-axis shows the relative response (in RU), and the x-axis shows the time (in seconds). In Figures 7A-7D, the antibody concentrations are as follows: 100 nM (purple), 33.3 nM (yellow), 11.1 nM (pink), 3.7 nM (green), 1.2 nM (orange), and 133 pM (blue). [Figure 7-3] This is a series of graphs showing the binding specificity of exemplary biparatopic antibodies with Bs3 orientation to 5T4 proteins from various species, as determined by surface plasmon resonance (SPR). Figure 7A shows binding to human 5T4. Figure 7B shows binding to NHP 5T4. NHP: Non-human primate. Figure 7C shows binding to mouse 5T4. Figure 7D shows binding to rat 5T4. In all of Figures 7A-7D, the y-axis shows the relative response (in RU), and the x-axis shows the time (in seconds). In Figures 7A-7D, the antibody concentrations are as follows: 100 nM (purple), 33.3 nM (yellow), 11.1 nM (pink), 3.7 nM (green), 1.2 nM (orange), and 133 pM (blue). [Figure 7-4]This is a series of graphs showing the binding specificity of exemplary biparatopic antibodies with Bs3 orientation to 5T4 proteins from various species, as determined by surface plasmon resonance (SPR). Figure 7A shows binding to human 5T4. Figure 7B shows binding to NHP 5T4. NHP: Non-human primate. Figure 7C shows binding to mouse 5T4. Figure 7D shows binding to rat 5T4. In all of Figures 7A-7D, the y-axis shows the relative response (in RU), and the x-axis shows the time (in seconds). In Figures 7A-7D, the antibody concentrations are as follows: 100 nM (purple), 33.3 nM (yellow), 11.1 nM (pink), 3.7 nM (green), 1.2 nM (orange), and 133 pM (blue).

[0022] [Figure 8] Figure 1A shows a table outlining the equilibrium dissociation constants (KDs) for binding to exemplary Bs3 HL format dual paratopic antibodies with Fc mutations, and parental Ab1 without Fc mutations, for the selection of the Fc gamma receptor (FcγR). FCA: L234F, S239C, and N434A mutations.

[0023] [Figure 9-1] Figure 9A shows a series of plots illustrating flow cytometry analysis of cell surface 5T4 expression in a panel of 5T4-negative and 5T4-positive cell lines. Figure 9A shows AGS cells. Figure 9B shows HepG2 cells. Figure 9C shows LoVo cells. Figure 9D shows PCI-N87 cells. Figure 9E shows A549 cells. Figure 9F shows DU145 cells. Figure 9G shows PANC-1 cells. Figure 9H shows T-47D cells. Figure 9I shows MCF7 cells. Figure 9J shows HEK293-5T4(3G9) cells. Figure 9K shows HEK293-5T4(4F2) cells. Figure 9L shows HEK293-5T4(5C10) cells. [Figure 9-2]Figure 9A shows a series of plots illustrating flow cytometry analysis of cell surface 5T4 expression in a panel of 5T4-negative and 5T4-positive cell lines. Figure 9A shows AGS cells. Figure 9B shows HepG2 cells. Figure 9C shows LoVo cells. Figure 9D shows PCI-N87 cells. Figure 9E shows A549 cells. Figure 9F shows DU145 cells. Figure 9G shows PANC-1 cells. Figure 9H shows T-47D cells. Figure 9I shows MCF7 cells. Figure 9J shows HEK293-5T4(3G9) cells. Figure 9K shows HEK293-5T4(4F2) cells. Figure 9L shows HEK293-5T4(5C10) cells. [Figure 9-3] Figure 9A shows a series of plots illustrating flow cytometry analysis of cell surface 5T4 expression in a panel of 5T4-negative and 5T4-positive cell lines. Figure 9A shows AGS cells. Figure 9B shows HepG2 cells. Figure 9C shows LoVo cells. Figure 9D shows PCI-N87 cells. Figure 9E shows A549 cells. Figure 9F shows DU145 cells. Figure 9G shows PANC-1 cells. Figure 9H shows T-47D cells. Figure 9I shows MCF7 cells. Figure 9J shows HEK293-5T4(3G9) cells. Figure 9K shows HEK293-5T4(4F2) cells. Figure 9L shows HEK293-5T4(5C10) cells. [Figure 9-4] Figure 9A shows a series of plots illustrating flow cytometry analysis of cell surface 5T4 expression in a panel of 5T4-negative and 5T4-positive cell lines. Figure 9A shows AGS cells. Figure 9B shows HepG2 cells. Figure 9C shows LoVo cells. Figure 9D shows PCI-N87 cells. Figure 9E shows A549 cells. Figure 9F shows DU145 cells. Figure 9G shows PANC-1 cells. Figure 9H shows T-47D cells. Figure 9I shows MCF7 cells. Figure 9J shows HEK293-5T4(3G9) cells. Figure 9K shows HEK293-5T4(4F2) cells. Figure 9L shows HEK293-5T4(5C10) cells.

[0024] [Figure 10-1]This is a series of graphs showing the internalization of the 5T4 receptor induced by dual paratopic antibodies in Bs3-HL and Bs2-HL format, with or without FCA (L234F, S239C, and N434A) mutations, compared to parental monospecific antibodies (humanized versions of antibodies Ab1 and Ab2). 5T4 receptor internalization was assayed in multiple 5T4-expressing cell types. Figure 10A shows DU145 cells. Figure 10B shows PANC-1 cells. Figure 10C shows MCF7 cells. Figure 10D shows HEK293-5T4(3G9) cells. Figure 10E shows HEK293-5T4(4F2) cells. Figure 10F shows T-47D cells. [Figure 10-2] This is a series of graphs showing the internalization of the 5T4 receptor induced by dual paratopic antibodies in Bs3-HL and Bs2-HL format, with or without FCA (L234F, S239C, and N434A) mutations, compared to parental monospecific antibodies (humanized versions of antibodies Ab1 and Ab2). 5T4 receptor internalization was assayed in multiple 5T4-expressing cell types. Figure 10A shows DU145 cells. Figure 10B shows PANC-1 cells. Figure 10C shows MCF7 cells. Figure 10D shows HEK293-5T4(3G9) cells. Figure 10E shows HEK293-5T4(4F2) cells. Figure 10F shows T-47D cells. [Figure 10-3] This is a series of graphs showing the internalization of the 5T4 receptor induced by dual paratopic antibodies in Bs3-HL and Bs2-HL format, with or without FCA (L234F, S239C, and N434A) mutations, compared to parental monospecific antibodies (humanized versions of antibodies Ab1 and Ab2). 5T4 receptor internalization was assayed in multiple 5T4-expressing cell types. Figure 10A shows DU145 cells. Figure 10B shows PANC-1 cells. Figure 10C shows MCF7 cells. Figure 10D shows HEK293-5T4(3G9) cells. Figure 10E shows HEK293-5T4(4F2) cells. Figure 10F shows T-47D cells. [Figure 10-4]This is a series of graphs showing the internalization of the 5T4 receptor induced by dual paratopic antibodies in Bs3-HL and Bs2-HL format, with or without FCA (L234F, S239C, and N434A) mutations, compared to parental monospecific antibodies (humanized versions of antibodies Ab1 and Ab2). 5T4 receptor internalization was assayed in multiple 5T4-expressing cell types. Figure 10A shows DU145 cells. Figure 10B shows PANC-1 cells. Figure 10C shows MCF7 cells. Figure 10D shows HEK293-5T4(3G9) cells. Figure 10E shows HEK293-5T4(4F2) cells. Figure 10F shows T-47D cells.

[0025] [Figure 11-1] This is a series of graphs showing the mean percentage survival rates of cancer cell lines incubated with a Bs3-HL format dual paratopic antibody conjugated to MMAE, compared to a 5T4-nonspecific IgG antibody (IgG-Ctrl) conjugated to MMAE and a control with MMAE (no antibody). Survival rates were assayed in multiple 5T4-expressing cell types. Figure 11A shows AGS cells. Figure 11B shows DU145 cells. Figure 11C shows T-47D cells. Figure 11D shows MCF7 cells. Figure 11E shows HEK293-5T4(3G9) cells. Figure 11F shows HEK293-5T4(4F2) cells. cc4 and cc8 show different drug-to-antibody ratios (DAR) of approximately 4 and 8, respectively. The y-axis shows the percentage survival rate, and the x-axis shows the concentration of the antibody-drug conjugate (in pM). MMAE: Monomethyl auristatin E. [Figure 11-2]This is a series of graphs showing the mean percentage survival rates of cancer cell lines incubated with a Bs3-HL format dual paratopic antibody conjugated to MMAE, compared to a 5T4-nonspecific IgG antibody (IgG-Ctrl) conjugated to MMAE and a control with MMAE (no antibody). Survival rates were assayed in multiple 5T4-expressing cell types. Figure 11A shows AGS cells. Figure 11B shows DU145 cells. Figure 11C shows T-47D cells. Figure 11D shows MCF7 cells. Figure 11E shows HEK293-5T4(3G9) cells. Figure 11F shows HEK293-5T4(4F2) cells. cc4 and cc8 show different drug-to-antibody ratios (DAR) of approximately 4 and 8, respectively. The y-axis shows the percentage survival rate, and the x-axis shows the concentration of the antibody-drug conjugate (in pM). MMAE: Monomethyl auristatin E. [Figure 11-3] This is a series of graphs showing the mean percentage survival rates of cancer cell lines incubated with a Bs3-HL format dual paratopic antibody conjugated to MMAE, compared to a 5T4-nonspecific IgG antibody (IgG-Ctrl) conjugated to MMAE and a control with MMAE (no antibody). Survival rates were assayed in multiple 5T4-expressing cell types. Figure 11A shows AGS cells. Figure 11B shows DU145 cells. Figure 11C shows T-47D cells. Figure 11D shows MCF7 cells. Figure 11E shows HEK293-5T4(3G9) cells. Figure 11F shows HEK293-5T4(4F2) cells. cc4 and cc8 show different drug-to-antibody ratios (DAR) of approximately 4 and 8, respectively. The y-axis shows the percentage survival rate, and the x-axis shows the concentration of the antibody-drug conjugate (in pM). MMAE: Monomethyl auristatin E.

[0026] [Figure 12-1]This is a series of graphs showing the mean growth inhibition percentage of cancer cell lines incubated with a dual paratopic 5T4 antibody in Bs3-HL format conjugated to MMAE, compared to a non-specific IgG antibody (IgG-Ctrl) conjugated to MMAE and a control with MMAE (no antibody). Growth inhibition was assayed in multiple 5T4-expressing cell types. Figure 12A shows AGS cells. Figure 12B shows DU145 cells. Figure 12C shows MCF7 cells. Figure 12D shows T-47D cells. Figure 12E shows HEK293-5T4(3G9) cells. Figure 12F shows HEK293-5T4(4F2) cells. cc4 and cc8 show different drug-to-antibody ratios (DAR) of approximately 4 and 8, respectively. The y-axis shows the percentage of growth inhibition, and the x-axis shows the concentration of the antibody-drug conjugate (in pM). MMAE, Monomethyl Auristatin E. [Figure 12-2] This is a series of graphs showing the mean growth inhibition percentage of cancer cell lines incubated with a dual paratopic 5T4 antibody in Bs3-HL format conjugated to MMAE, compared to a non-specific IgG antibody (IgG-Ctrl) conjugated to MMAE and a control with MMAE (no antibody). Growth inhibition was assayed in multiple 5T4-expressing cell types. Figure 12A shows AGS cells. Figure 12B shows DU145 cells. Figure 12C shows MCF7 cells. Figure 12D shows T-47D cells. Figure 12E shows HEK293-5T4(3G9) cells. Figure 12F shows HEK293-5T4(4F2) cells. cc4 and cc8 show different drug-to-antibody ratios (DAR) of approximately 4 and 8, respectively. The y-axis shows the percentage of growth inhibition, and the x-axis shows the concentration of the antibody-drug conjugate (in pM). MMAE, Monomethyl Auristatin E. [Figure 12-3]This is a series of graphs showing the mean growth inhibition percentage of cancer cell lines incubated with a dual paratopic 5T4 antibody in Bs3-HL format conjugated to MMAE, compared to a non-specific IgG antibody (IgG-Ctrl) conjugated to MMAE and a control with MMAE (no antibody). Growth inhibition was assayed in multiple 5T4-expressing cell types. Figure 12A shows AGS cells. Figure 12B shows DU145 cells. Figure 12C shows MCF7 cells. Figure 12D shows T-47D cells. Figure 12E shows HEK293-5T4(3G9) cells. Figure 12F shows HEK293-5T4(4F2) cells. cc4 and cc8 show different drug-to-antibody ratios (DAR) of approximately 4 and 8, respectively. The y-axis shows the percentage of growth inhibition, and the x-axis shows the concentration of the antibody-drug conjugate (in pM). MMAE, Monomethyl Auristatin E.

[0027] [Figure 13] These are two graphs showing tumor growth and mouse survival in immunodeficient mice transplanted with NCI-H1975 lung adenocarcinoma tumors and treated with MMAE-conjugated Bs3-oriented 5T4 biparatopic antibody (two dose concentrations) compared to isotype control antibodies conjugated with MMAE and mock PBS control. Figure 13A shows tumor growth. Figure 13B shows survival probability. ADC: Antibody-drug conjugate, MMAE, monomethyl auristatin E.

[0028] [Figure 14] This is a series of graphs showing tumor growth in immunodeficient mice transplanted with various CDX tumor models and treated with MMAE-conjugated Bs3-oriented 5T4 biparatopic antibody (multiple concentrations), compared to isotype control antibodies conjugated with MMAE or mock PBS controls. Figure 14A shows the MDA-MD-361 breast cancer model. Figure 14B shows the DU145 prostate cancer model. Figure 14C shows the A549 lung cancer model. ADC: Antibody-drug conjugate, MMAE, monomethyl auristatin E.

[0029] [Figure 15] This is a series of sensorgrams showing the binding affinity of the 5T4 parent antibody compared to the 5T4 biparatopic antibody in the Bs3 configuration. Figure 15A shows the binding affinity of the parent antibody (m)Ab1. Figure 15B shows the binding affinity of the parent antibody (m)Ab2. Figure 15C shows the binding affinity of the bispecific antibody in Bs3-HL orientation. Figure 15D shows the binding affinity of the bispecific antibody in Bs3-HL orientation conjugated to MMAE. Monomethyl auristatin E.

[0030] [Figure 16] This table shows an overview of the equilibrium dissociation constants (KDs) for 5T4 binding of 5T4 antibodies, as shown in Figures 15A-15D, with and without MMAE conjugation. [Modes for carrying out the invention]

[0031] This disclosure provides antigen-binding domains that specifically bind to 5T4, as well as methods for constructing and using them. Examples of 5T4 antigen-binding domains in this disclosure include, but are not limited to, Fab fragments, F(ab')2 fragments, scFv, scab, dAb, single-domain antibodies, and full-length IgG antibodies. Non-limited uses of 5T4 antigen-binding domains intended to be within the scope of this disclosure include use in immunotherapy, as antibody-drug conjugates, and incorporation into chimeric antigen receptors (CARs) used in adoptive cell therapy.

[0032] Accordingly, this disclosure provides antibody-drug conjugates comprising 5T4 antigen-binding domains as described herein. In some embodiments, the antibody-drug conjugate is a dual paratopic, i.e., comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope and a second antigen-binding domain that binds to a second 5T4 epitope, wherein the two 5T4 epitopes are not the same. In some embodiments, the antibody-drug conjugate comprises a first antigen-binding domain that specifically binds to a first 5T4 epitope and a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and comprises a chemotherapeutic agent.

[0033] The dual paratopic antibodies and their antibody-drug conjugates described herein bind to two different epitopes of the same target 5T4 molecule, thereby crosslinking 5T4 on the surface of cells such as cancer cells and inducing the formation of 5T4-antibody immune complexes. This improved crosslinking provides functional benefits over known 5T4-binding proteins known in the art, such as monospecific antibodies or other proteins that specifically bind to a single 5T4 epitope. Such benefits include more robust and rapid internalization of the 5T4 molecule bound to the antibody-drug conjugate, which can result in better cytotoxic death of targeted cancer cells. While we do not wish to be bound by theory, it is thought that increased crosslinking and / or aggregation of antibody-immune complexes leads to more robust internalization, lysosomal transport and degradation of the immune complex within cells, as well as intracellular release of the cytotoxic payload into the host cell. The dual paratopic antibody-drug conjugates described herein demonstrate improved internalization and specific death of 5T4-expressing cancer cells. Therefore, the dual paratopic antibodies and antibody-drug conjugates of this disclosure can provide improved efficacy against cancer cells and more effective treatment for subjects with 5T4-positive cancer.

[0034] In some embodiments, the dual paratopic antibodies and antibody-drug conjugates of this disclosure include an IgG constant (Fc) region domain containing at least one mutation that reduces effector function, extends half-life, or a combination thereof. For example, the Fc mutation in the antibody-drug conjugates provided herein provides additional benefits to 5T4-binding proteins known in the prior art by reducing affinity for at least Fc gamma receptor I (FcγRI) and / or Fc gamma receptor IIIa (FcγRIIIa) compared to antibodies without the Fc mutation, without attenuating the binding affinity of the antibody-drug conjugate to 5T4. Exemplary Fc mutations that minimize antibody effector function and extend the half-life of the antibody-drug conjugate are shown in Table 3.

[0035] In some embodiments, the dual paratopic antibody of this disclosure comprises a first antigen-binding domain operably linked to a second antigen-binding domain. In some embodiments, the first and second antigen-binding domains are independently selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, scab, dAb, single-domain heavy-chain antibodies, single-domain light-chain antibodies, and full-length IgG antibodies. In some embodiments, the dual paratopic antibody comprises a full-length IgG antibody containing the first antigen-binding domain, and the second antigen-binding domain comprises scFv. In some embodiments, the chemotherapeutic agent is conjugated via a linker to at least one of the full-length IgG antibodies containing either the first antigen-binding domain or the second antigen-binding domain. In some embodiments, the chemotherapeutic agent is auristatin, but is not limited to, auristatin E (AE), monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and synthetic analogs of drastatin.

[0036] This specification also provides polynucleotides and vectors encoding antigen-binding domains, antibodies and antibody-drug conjugates containing them, chimeric antigen receptors (CARs) containing the antigen-binding domains of this disclosure, pharmaceutical compositions containing them, and methods for preparing and using them. Antigen-binding domains, antibodies, antibody-drug conjugates and CARs can be used to treat a variety of diseases and disorders, including cancer.

[0037] definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this invention pertains.

[0038] For the purposes of interpreting this Spec., the following definitions apply, and wherever appropriate, a singular term is also included in its plural form, and vice versa. In the event of any conflict between any definition set forth below and any document incorporated herein by reference, the definition set forth below shall prevail.

[0039] As used herein, the term “subject” includes, but is not limited to, mammals including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals, non-mammalian vertebrates including, for example, birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates. In some embodiments, the methods and compositions of the present invention are used to treat non-human animals (both prophylactically and / or therapeutically). The term “subject” may also refer to a patient, i.e., an individual awaiting or receiving medical treatment.

[0040] In this specification, the term "pharmaceutical composition" means a composition suitable for pharmacokinetic use in subjects including animals or humans. A pharmaceutical composition generally comprises an effective amount of an active agent (e.g., an antibody or antibody-drug conjugate as disclosed herein) and a pharmaceutically acceptable carrier, diluent, or excipient (e.g., a buffer, adjuvant, etc.).

[0041] The term "effective dose" means a dosage or quantity sufficient to produce the desired outcome. The desired outcome may include objective or subjective improvement in the recipient of the dosage or quantity (e.g., long-term survival, reduction in the number and / or size of tumors, effective prevention of disease status, etc.).

[0042] "Prophylactic treatment" refers to treatment administered to subjects who do not show signs or symptoms of a disease, pathology, or medical disorder, or who show only early signs or symptoms of a disease, pathology, or medical disorder, and whose purpose is to attenuate, prevent, or reduce the risk of developing the disease, pathology, or medical disorder. Prophylactic treatment functions as a prophylactic treatment for a disease or disorder. "Prophylactic activity" is the activity of a drug, such as the anti-5T4 bispecific antibody-drug conjugate or its composition herein, that, when administered to a subject who does not show signs or symptoms of a pathology, disease, or disorder (or shows only early signs or symptoms of a pathology, disease, or disorder), attenuates, prevents, or reduces the risk of the subject developing a pathology, disease, or disorder. A drug or compound that is "prophylactically useful" (e.g., anti-5T4 bispecific antibody-drug conjugate) refers to a drug or compound that is useful in attenuating, preventing, treating, or reducing the development of a pathology, disease, or disorder.

[0043] "Therapeutic treatment" means a treatment administered to a subject exhibiting symptoms or signs of a pathology, disease, or disorder, and the treatment is administered to the subject with the aim of attenuating or eliminating such signs or symptoms of the pathology, disease, or disorder. "Therapeutic activity" is the activity of a drug, such as an antibody-drug conjugate or composition thereof, that eliminates or attenuates the signs or symptoms of a pathology, disease, or disorder when administered to a subject suffering from such signs or symptoms. "Therapeutably effective" drug or compound (e.g., anti-5T4 bispecific antibody-drug conjugate) indicates that the drug or compound is effective in attenuating, treating, or eliminating such signs or symptoms of a pathology, disease, or disorder.

[0044] As used herein, the term “treat cancer” means, unless otherwise indicated, partially or completely reversing, alleviating, inhibiting the progression of, or preventing tumors, tumor metastases, or other cancers or neoplastic cell growth in a subject. As used herein, the term “treatment” means, unless otherwise indicated, the act of treating.

[0045] In the context of two or more nucleic acid or polypeptide sequences, the terms “identical” or “identity percentage” refer to two or more sequences or subsequences that, when compared and aligned for the greatest correspondence, are identical or have a specific percentage of identical nucleotide or amino acid residues. To determine the identity percentage, the sequences are aligned for the best possible comparison (for example, gaps may be introduced into the sequence of the first amino acid or nucleic acid sequence for the best possible alignment with the second amino acid or nucleic acid sequence). Then, amino acid residues or nucleotides at 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, then the molecules are identical at that position. The identity percentage between two sequences is a function of the number of identical positions shared by the sequences (i.e., identity % is equal to the number of identical positions / the total number of positions (e.g., number of overlapping positions × 100)). In some embodiments, the two sequences are of the same length.

[0046] In the context of two nucleic acids or polypeptides, the term “substantially identical” means two or more sequences or subsequences having at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identity, or at least 99% identity (determined, for example, by using one of the methods described below).

[0047] The determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. A non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs in Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searching is performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid encoding the target protein. BLAST protein searches are performed using the XBLAST program, score=50, and word length=3, to obtain amino acid sequences homologous to the target protein. For gapped alignments for comparison purposes, Gapped BLAST can be used, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches that detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm by Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap length penalty of 4 can be used.Further algorithms for sequence analysis are publicly known in the art and include ADVANCE and ADAM, as described in Torellis and Robotti, 1994, Comput.Appl.Biosci.10:3-5, and FASTA, as described in Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA 85:2444-8. Alternatively, the CLUSTAL W algorithm may be used, as described in Higgins et al., 1996, Methods Enzymol.266:383-402.

[0048] As used herein, “antigen-binding domain” refers to a region on an antibody that binds to an antigen. An exemplary antigen-binding domain includes one constant domain and one variable domain in each of the heavy and light chains of an antibody. However, alternative configurations, such as a single-domain antigen-binding domain, are intended to be within the scope of this disclosure, insofar as such a domain can bind to an antigen. Examples of exemplary antigen-binding domains include, but are not limited to, scFv, Fab fragments, Fab' fragments, and F(ab')2 fragments, which are described in further detail below.

[0049] As used herein, the terms “specifically binding” or “specific” refer to measurable and reproducible interactions, such as binding between a target and an antigen-binding domain, that can determine the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target more readily, with higher affinity, avidity, and / or for a longer period than it would to bind to other targets. In one embodiment, the degree of binding of the antigen-binding domain to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, or <0.01 nM.

[0050] In certain embodiments, the antigen-binding domain specifically binds to an epitope on a protein that is conserved between proteins of different species. In other embodiments, the specific binding may include, but does not have to include, exclusive binding.

[0051] Where used herein, the singular “one” (“a,” “an,” and “the”) also includes multiple references unless the context indicates otherwise. References to “formulations” or “methods” include one or more formulations, methods, and / or processes of the type described herein and / or that would be apparent to those skilled in the art by reading this disclosure.

[0052] The term "polypeptide" refers to polymers of amino acids and their equivalents, and does not refer to products of a specific length; therefore, "peptides" and "proteins" are included within the definition of polypeptide. A protein may have one or more polypeptides. The definition of polypeptide also includes "antibodies" as defined herein. A "polypeptide region" refers to a segment of a polypeptide, which may contain, for example, one or more domains or motifs (for example, the polypeptide region of an antibody may contain, for example, one or more complementarity-determining regions (CDRs)). The term "fragment" refers to a portion of a polypeptide that is smaller than the entire polypeptide, such as those found in nature.

[0053] Unless otherwise indicated by the context, “derivative” refers to a polypeptide or fragment thereof having one or more non-conservative or conservative amino acid substitutions, deletions or insertions, to a second polypeptide (also called a “mutant”), or a polypeptide or fragment thereof modified by, for example, attachment of a heterologous polypeptide, or covalent attachment of a second molecule such as glycosylation, acetylation, or phosphorylation. Furthermore, the definition of “derivative” includes, for example, polypeptides containing one or more analogues of amino acids (e.g., unnatural amino acids), polypeptides having unsubstituted bonds, and other modifications known in the art, both natural and unnatural.

[0054] "Isolated" polypeptides are identified, separated, and / or recovered from components of their natural environment. Contaminating components of their natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes, and may interfere with the polypeptide's diagnostic or therapeutic use. Isolated polypeptides may also include isolated antibodies, or fragments or derivatives thereof.

[0055] As used herein, the term “chimeric antigen receptor (CAR)” refers to an artificial transmembrane protein receptor comprising (i) an extracellular domain capable of binding to at least one predetermined CAR ligand or antigen, such as a 5T4 antigen-binding domain as described herein; (ii) an intracellular segment comprising one or more cytoplasmic domains derived from a signaling protein different from the polypeptide from which the extracellular domain originates; and (iii) a transmembrane domain. In some cases, CARs also comprise a hinge domain. CARs can be used to impart artificial specificity to specific immune effector cells, such as helper T cells (CD4+), cytotoxic T cells (CD8+), or NK cells. CARs can be used to confer specificity of monoclonal antibodies onto T cells, thereby enabling the generation of a large number of specific T cells, for example, for use in adoptive cell therapy. Depending on the specific structure of the CAR and the intracellular signaling domain used, a CAR may be an activator receptor or an inhibitory receptor. Exemplary activator CARs include an intracellular domain of CD3 zeta, one or more intracellular domains for additional co-stimulatory signaling such as ICOS, CD137(4-1BB), CD27, CD28, CD134, CD152(CTLA-4), CD223(LAG4), DAP10, and / or OX-40, and optionally, an extracellular hinge region derived, for example, CD8a or CD28. Many different CARs are known in the art, and it is assumed that all of them fall within the scope of the present invention.

[0056] As used herein, the term “T cell bispecific antibody” refers to an antibody having bispecific binding to cancer-associated antigens such as 5T4, and to one of the CD3 subunits present on the surface of T cells, such as CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta. While we do not wish to be bound by theory, it is thought that this allows T cell bispecific antibodies to bridge and bring T cells and cancer cells closer together, thereby inducing T cell activation and subsequent cancer cell death.

[0057] As used herein, the term “about” means, in quantitative terms, plus or minus 5%, or in another embodiment, plus or minus 10%, or in another embodiment, plus or minus 15%, or in another embodiment, plus or minus 20%.

[0058] All methods described herein may be carried out in any preferred order unless otherwise indicated herein or expressly rejected by the context. Any and all examples or exemplary language provided herein (e.g., "etc.") are intended solely to better illustrate the invention and do not impose limitations on the scope of the invention unless otherwise claimed. No language herein should be construed as indicating any unclaimed element essential to the practice of the invention.

[0059] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.

[0060] 5T4 An example of a cancer-associated antigen is trophotrophoblast glycoprotein (TPBG), also known as human 5T4 antigen (5T4), 5T4 carcinoembryonic antigen, or Wnt activator inhibitor 1 (WAIF1). Human 5T4 antigen is a 72 kDa type I transmembrane glycoprotein expressed in embryonic tissues such as the placenta, as well as in various types of solid tumors and carcinomas, including prostate cancer, gastric cancer, and colorectal cancer. See, for example, U.S. Patent No. 7,074,909 or U.S. Patent No. 7,514,546. However, 5T4 antigen is either expressed at low levels or not expressed at all in most healthy adult epithelial tissues. See Woods et al, Biochem. J. (2002) 366, 353-365.

[0061] Expression or overexpression of the 5T4 antigen in various tumor types, particularly ovarian, gastric, and colorectal tumors, is associated with worse clinical outcomes. Furthermore, overexpression is associated with changes in cell morphology and motility consistent with tumor invasion. Therefore, the 5T4 antigen is thought to play a role in the progression or malignancy of several solid tumors.

[0062] This disclosure provides antigen-binding domains, as well as antibodies, antibody-drug conjugates, and receptors that constitute antigen-binding domains that bind to 5T4.

[0063] This disclosure provides antigen-binding domains useful for targeting cells, such as cancer cells, that express the 5T4 antigen. In some embodiments, the antigen-binding domains are incorporated into antibody-drug conjugates that can be used in 5T4-targeted cancer therapies. Exemplary antibody-drug conjugates include a biparatopic antibody that binds to two different 5T4 epitopes and a 5T4 antibody-drug conjugate that can bind to a single 5T4 epitope. In some embodiments, the 5T4 antibody-drug conjugate comprises a biparatopic 5T4 antibody that binds to two different 5T4 epitopes. The antibody-drug conjugates provided herein may include a 5T4 antigen-binding moiety of an anti-5T4 antibody that has been engineered into a single-stranded form and fused to a chemotherapeutic agent. In some embodiments, the 5T4 antibody-drug conjugate comprises a biparatopic 5T4 antibody containing a full-length IgG antibody linked to an scFv.

[0064] This disclosure provides pharmaceutical compositions comprising antigen-binding domains, antibodies, antibody-drug conjugates, and receptors as described herein. In some embodiments, the pharmaceutical composition comprises a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and a dual paratopic 5T4 antibody-drug conjugate comprising a chemotherapeutic agent.

[0065] Furthermore, the present disclosure provides a method for treating a disease or disorder involving cellular expression of the 5T4 antigen, comprising administering to a subject in need of such treatment a therapeutically effective amount of a pharmaceutical composition as described herein, for example, a pharmaceutical composition comprising a dual paratopic 5T4 antibody-drug conjugate comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and a chemotherapeutic agent.

[0066] This disclosure provides an antigen-binding domain that binds to the human 5T4 antigen. Human 5T4 comprises an amino acid sequence according to the following NCBI reference sequence NP_001363851.1: [Table 1] (Sequence ID 27).

[0067] Any suitable antigen-binding domain or fragment thereof that can specifically bind to 5T4 is envisioned within the scope of this disclosure and includes, but is not limited to, single-domain antibodies (sdAb) such as Fab fragments, F(ab')2 fragments, single-chain variable fragments (scFv), scab, dAb, VHH single-domain antibodies, single-domain heavy-chain antibodies, or single-domain light-chain antibodies, full-length IgG antibodies, antibody fragments, antigen-binding domains, or fragments containing antigen-binding domains as described in more detail below. In addition, full-length antibodies, biparatopic antibodies, T-cell bispecific antibodies, fusion proteins, and receptors such as chimeric antigen receptors described herein are intended to be within the scope of this disclosure.

[0068] This disclosure provides antibodies comprising an antigen-binding domain specific to the 5T4 antigen described herein. For example, the antibody may be a monoclonal antibody, such as a full-length IgG antibody.

[0069] As used herein, “antibody” refers to a protein comprising one or more polypeptides substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit consists of a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one “light” chain (approximately 25 kD) and one “heavy” chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. The term “antibody” includes antibody molecules prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express antibodies. The term “antibody” also includes bispecific antibodies (e.g., T-cell bispecific antibodies) or biparatopic antibodies, which may include heterotetrameric immunoglobulins capable of binding to more than one different epitopes. Bispecific antibodies are generally described in U.S. Patent Application Publication 2010 / 0331527, which is incorporated herein by reference. The term “antibody” as used herein is used in its broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity.

[0070] The term “antibody” also includes one or more fragments of an antibody that possess the ability to specifically bind to an antigen, such as the antibody-binding portion or domain of an antibody, which may be referred to herein as the “antigen-binding domain” or “antigen-binding portion.”

[0071] As used herein, the terms “antigen-binding domain” or “antigen-binding region” refer to the domain of the antigen-binding moiety that is involved in the specific binding between the antigen-binding moiety and the antigen. For example, the antigen-binding region of an antibody or fragment thereof is formed by amino acid residues in the N-terminal variable regions of the heavy chain (abbreviated herein as VH) and the light chain (abbreviated herein as VL). The variable regions of VH and VL each contain three hypervariable regions called complementarity-determining regions (CDRs). The three CDRs of VH and the three CDRs of VL are arranged three-dimensionally relative to each other to form the antigen-binding surface. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH1 domains; (ii) a bivalent fragment containing an F(ab')2 fragment and two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) an isolated CDR; and (vii) scFv consisting of two domains of the Fv fragment, VL and VH, linked by a synthetic linker to form a single protein chain where the VL and VH regions pair up to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also included in the term "antibody" (see, for example, Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1 121-1 123).

[0072] Furthermore, an antibody or its antigen-binding domain may be part of a larger immunoadhesion molecule formed by covalent or non-covalent bonding between the antibody or antigen-binding domain and one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to construct tetrameric scFv molecules (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to construct divalent biotinylated scFv molecules (Kipriyanov et al. (1994) Mol.Immunol.31:1047-1058). Antibody fragments such as Fab and F(ab')2 fragments can be prepared from whole antibodies using conventional techniques, such as through papain or pepsin digestion of the whole antibody. Furthermore, antibodies, antibody fragments, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques commonly known in the art (see Sambrook et al., 1989).

[0073] The terms “human antibody” or “humanized antibody” are intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutagenesis in vivo), for example, in the CDR, particularly CDR3. The “humanized” form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody), which contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient’s complementarity-determining region (CDR) are replaced with residues derived from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and / or capabilities. In some cases, the Fv framework residues of human immunoglobulins are also substituted with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient antibody or in the transferred CDR or framework sequence. These modifications are made to further improve and optimize antibody performance. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR region corresponding to that of a non-human immunoglobulin and all or substantially all of the FR region being a human immunoglobulin sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin.

[0074] As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal transgenic for a human immunoglobulin gene (e.g., mouse), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of the human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic for a human Ig sequence is used, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but are sequences that cannot naturally exist in vivo within the human antibody germline repertoire. The general structure of antibodies is known in the art. In simple terms, an immunoglobulin monomer contains two heavy chains and two light chains linked by disulfide bonds. Each heavy chain pairs with one of the light chains, to which it is directly bound via a disulfide bond. Each heavy chain contains a constant region (which varies depending on the antibody isotype) and a variable region. The variable region, designated CDRH1, CDRH2, and CDRH3, contains three hypervariable regions (or complementarity-determining regions) supported within a framework region. Each light chain contains a constant region and a variable region, and the variable region contains three hypervariable regions (designated CDRL1, CDRL2, and CDRL3) supported by a framework region in a manner similar to that of the variable region of the heavy chain.

[0075] The hypervariable regions of each pair of heavy and light chains cooperate to provide antigen-binding sites that can bind to target antigens. The binding specificity of a pair of heavy and light chains is defined by the sequences of CDR1, CDR2, and CDR3 of the heavy and light chains. Thus, once a set of CDR sequences that produces a particular binding specificity (i.e., the sequences of CDR1, CDR2, and CDR3 for the heavy and light chains) is determined, the set of CDR sequences can, in principle, be inserted at appropriate positions within any other antibody framework region linked to any antibody constant region to provide different antibodies with the same antigen-binding specificity.

[0076] Antibodies or antigen-binding domains exist as intact immunoglobulins or as a multitude of well-characterized fragments produced by digestion with various peptidases. For example, pepsin digests the antibody below the disulfide bond in the hinge region, producing F(ab)'2. This is a dimer of Fab, in which the light chain is linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer to a Fab' monomer. A Fab' monomer is essentially a Fab with a portion of the hinge region. While various antibody fragments are defined with respect to the digestion of intact antibodies, those skilled in the art will understand that such Fab' fragments and the like can be newly synthesized, either chemically or by the use of recombinant DNA methodologies. Therefore, the term antibody, as used herein, also includes either antibody fragments produced by modification of the entire antibody, or antibody fragments newly synthesized using recombinant DNA methodologies.

[0077] The antibody or antigen-binding domain includes single-chain antibodies, such as single-chain Fv (sFv or scFv) antibodies in which a variable heavy chain and a variable light chain are bound together (directly or via a peptide linker) to form a continuous polypeptide.

[0078] The antibody or antigen-binding domain includes a single-domain antibody comprising an antibody fragment consisting of a single monomeric variable antibody domain capable of selectively binding to an antigen domain. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking a light chain, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. A single-domain antibody may be any single-domain antibody of the present art or any future single-domain antibody. A single-domain antibody may be derived from any species, including, but not limited to, mice, humans, camels, llamas, goats, rabbits, and cattle.

[0079] As described above, the antibody or antigen-binding domains used herein may optionally include F(ab)2, F(ab')2, Fab, Fab', scFv, single-domain antibodies, etc., depending on the specific requirements of the embodiment. Some embodiments use antibodies containing an IgG domain. However, other embodiments include alternative immunoglobulins such as IgM, IgA, IgD, and IgE. Furthermore, all possible isotypes of various immunoglobulins are also encompassed in this embodiment. Thus, IgG1, IgG2, IgG3, IgG4, etc., are all possible molecules in the antibody domain used in the present invention. In addition to the selection in the type of immunoglobulin and isotype, different embodiments of the present invention may include various hinge regions (or their functional equivalents). Such hinge regions provide flexibility between different domains of the antibody and, for example, effectors to which the antibody is fused.

[0080] The antibodies and antigen-binding domains of this disclosure include "chimeric" antibodies or antigen-binding domains (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, but the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, insofar as they exhibit desired biological activity.

[0081] In some embodiments, the antibodies and antibody-drug conjugates of this disclosure include a 5T4-specific antigen-binding domain comprising one of the CDRs disclosed in Tables 1-2. [Table 2] [Table 3]

[0082] Those skilled in the art will understand that each row in Table 1 discloses three heavy chain CDR sequences, and each row in Table 2 discloses three light chain CDR sequences, which can bind together to 5T4. Thus, the 5T4-specific antigen-binding domains disclosed herein are understood to have six CDRs in some embodiments, three for variable heavy chain domains and three for variable light chain domains, corresponding to the rows at the same positions in Tables 1 and 2, respectively, and identified by the numbers on the left. Any combination of CDRs, including the columns of CDRs from Table 1, combined with the columns of CDRs from Table 2, is assumed to be within the range of the 5T4 antibodies of this disclosure. Additionally, antibodies, antigen-binding domains, CDRs, and their sequences that specifically bind to the 5T4 epitope can be derived by methods known in the art. For example, the monoclonal antibodies used herein may be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., 1991, Nature 352:624-628 and Marks et al., 1991, J.Mol.Biol.222:581-597, and the antibody sequences and corresponding coding nucleic acids may be determined by methods known in the art.

[0083] A bispecific antibody is an antibody that has binding specificity to at least two different antigens. A biparatopic antibody is an antibody that binds to two different epitopes of the same antigen. Both bispecific antibodies, such as antibodies that bind to 5T4 using the 5T4 antigen-binding domain described herein and also bind to an additional antigen, and biparatopic antibodies that bind to 5T4 are intended to be within the scope of this disclosure.

[0084] This disclosure provides a dual paratopic antibody-drug conjugate having a first antigen-binding domain that binds to a first 5T4 epitope and a second antigen-binding domain that binds to a second 5T4 epitope that is not identical to the first 5T4 epitope, wherein the first antigen-binding domain is operably linked to the second antigen-binding domain. In some embodiments, the first antigen-binding domain is linked to the second antigen-binding domain via a linker.

[0085] Antibodies having more than two binding titers are also intended to be within the scope of this disclosure. For example, triplicate antibodies can be prepared. See Tutt et al., J.Immunol. 147:60 (1991).

[0086] In some embodiments, the first and second antigen-binding domains are independently selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, scab, dAb, single-domain heavy chain antibodies, single-domain light chain antibodies, and full-length IgG antibodies.

[0087] In some embodiments, the dual paratopic antibody of this disclosure comprises a first antibody comprising a full-length IgG antibody containing a first antigen-binding domain. In some embodiments, the second antigen-binding domain comprises an scFv. In some embodiments, the antibody-drug conjugate is tetravalent to bind to the 5T4 antigen.

[0088] In some embodiments, the 5T4 biparatopic antibody described herein comprises an scFv. In some embodiments, the antibody-drug conjugate comprises two scFvs, both specifically bound to the 5T4 epitope. In some embodiments, the scFv comprises a heavy chain and a light chain, wherein the C-terminus of the light chain is operably linked to the N-terminus of the heavy chain via a linker, or the C-terminus of the heavy chain is operably linked to the N-terminus of the light chain via a linker. In some embodiments, the linker comprises the sequence of SEQ ID NO: 153.

[0089] In some embodiments, the biparatopic antibody comprises scFv and a full-length IgG antibody. In some embodiments, the N-terminus of scFv is operably ligated to the C-terminus of the heavy chain of the full-length IgG antibody. In some embodiments, the C-terminus of scFv is operably ligated to the N-terminus of the heavy chain of the full-length IgG antibody.

[0090] In some embodiments, the scFv is operably linked to the heavy chain of a full-length IgG antibody using a linker. In some embodiments, the linker contains or consists of the amino acid sequence of SEQ ID NO: 152.

[0091] In some embodiments of the dual paratopic antibody-drug conjugate described herein, the dual paratopic antibody comprises a full-length IgG antibody having a first antigen-binding domain, the second antigen-binding domain comprising scFv, and the antibody-drug conjugate comprises four polypeptides, each comprising two polypeptides having a full-length IgG antibody heavy chain, a linker, and a second antigen-binding domain from the N-terminus to the C-terminus, and two polypeptides having a full-length IgG antibody light chain.

[0092] In some embodiments of the antibody-drug conjugate, the antibody-drug conjugate comprises a full-length IgG antibody containing a first antigen-binding domain, the second antigen-binding domain containing an scFv, and the antibody-drug conjugate comprises four polypeptides, each containing a second antigen-binding domain, a linker, and a full-length IgG antibody heavy chain from the N-terminus to the C-terminus, and two polypeptides containing a full-length IgG antibody light chain.

[0093] As used herein, “binding affinity” refers to the tendency of one molecule to bind to another molecule (typically non-covalently), e.g., the tendency of one member of a specific binding pair to bind to another member of that specific binding pair. Binding affinity can be measured as a dissociation constant, which is 1 × 10⁻¹⁶ for a specific binding pair (such as an antibody / antigen pair). -5 Less than M, 1 x 10 -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, or 1 × 10 -12 It may be less than M. In one embodiment, binding affinity is calculated by a modified version of the Scatchard method described by Frankel et al., Mol.Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by the binding constant. In some embodiments, binding affinity is measured by the antigen / antibody dissociation rate. In yet another embodiment, high binding affinity is measured by competitive radioimmunoassay.

[0094] As used herein, the term "dissociation constant" or "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. In one aspect, KD is determined, for example, by surface plasmon resonance (SPR) technology on a BIAcore 8000 instrument using an antigen as a ligand and an antibody as an analyte. In some aspects, the antibody binds to a given antigen with an affinity corresponding to a KD that is at least 10-fold lower, e.g., at least 100-fold lower, e.g., at least 1,000-fold lower, e.g., at least 10,000-fold lower, e.g., at least 100,000-fold lower than its affinity for binding to non-specific antigens (e.g., BSA, casein) other than the given antigen or closely related antigens. The lower amount of affinity depends on the KD of the antibody, such that when the KD of the antibody is very low (i.e., the antibody is highly specific), the amount by which the affinity for the antigen is lower than the affinity for non-specific antigens can be at least 10,000-fold.

[0095] In some aspects, the equilibrium dissociation constant (KD) of the antigen-binding domain for 5T4 binding is from about 1×10 -12 to about 1×10 -7 M, from about 1×10 -12 to about 1×10 -8 M, from about 1×10 -12 to about 1×10 -9 M, from about 1×10 -11 to about 1×10 -9 M, or from about 1×10 -11 to about 9×10 -10 M. In some aspects, the KD of the antigen-binding domain for 5T4 binding is from about 7.42×10 -11 to about 7.75×10 -10 M. In some aspects, KD is 7.75×10 -10 M or less, e.g., 3.20×10 -10 M or less, 1.98×10 -10 M or less, or 7.42×10 -11 M or less. In some aspects, the KD for 5T4 binding is from about 3.63×10 -12 to about 1.43×10 -9 M. In some aspects, the KD for 5T4 binding is from about 3.63×10-12 ~Approx. 1.34×10 -9 M. In some embodiments, the KD of the antigen-binding domain for 5T4 binding is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.59×10 -11 M is the value. In some embodiments, the KD for the 5T4 bond is approximately 7.42 × 10⁻⁶. -11 ~Approx. 7.75×10 -10 M is the value. In some embodiments, the KD for the 5T4 bond is approximately 7.42 × 10⁻⁶. -11 ~Approx. 7.75×10 -10 It is M.

[0096] In some embodiments of the dual paratopic antibody or dual paratopic antibody-drug conjugate described herein, the equilibrium dissociation constant (KD) for 5T4 binding of at least one of the first antigen-binding domains or the second antigen-binding domain is approximately 1 × 10⁻¹⁶. -12 ~Approx. 1×10 -7 M, about 1 x 10 -12 ~Approx. 1×10 -8 M, about 1 x 10 -12 ~Approx. 1×10 -9 M, about 1 x 10 -11 ~Approx. 1×10 -9 M, or approximately 1 x 10 -11 ~Approx. 9×10 -10 M. In some embodiments, the KD for 5T4 binding of at least one of the first antigen-binding domains or the second antigen-binding domain is approximately 7.42 × 10⁻⁶. -11 ~Approx. 7.75×10 -10 M. In some embodiments, the KD for 5T4 binding of at least one of the first antigen-binding domains or the second antigen-binding domain is approximately 3.63 × 10⁻⁶. -12 ~Approx. 7.75×10 -10 M is M. In some embodiments, KD is 7.75 × 10 -10 M or less, for example, 3.20 × 10 -10 M or less, 1.98×10 -10 M or less, or 7.42 × 10 -11 It is less than or equal to M. In some embodiments, the KD for 5T4 binding of the first antibody or antigen-binding domain is approximately 7.42 × 10⁻⁶. -11~Approx. 7.75×10 -10 M. In some embodiments, the KD of the first antibody or antigen-binding domain that binds to 5T4 is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.43×10 -9 M. In some embodiments, the KD of the first antibody or antigen-binding domain is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.34×10 -9 M is the value. In some embodiments, the KD of the first antibody or antigen-binding domain is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.59×10 -11 M. In some embodiments, the KD for 5T4 binding of at least the second antibody or antigen-binding domain is approximately 7.42 × 10⁻⁶. -11 ~Approx. 7.75×10 -10 M. In some embodiments, the KD of the second antibody or antigen-binding domain that binds to 5T4 is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.43×10 -9 M. In some embodiments, the KD of the second antibody or antigen-binding domain is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.34×10 -9 M. In some embodiments, the KD of the second antibody or antigen-binding domain is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.59×10 -11 M. In some embodiments, the KD for 5T4 binding of both the first antibody or antigen-binding domain and at least the second antibody or antigen-binding domain is approximately 7.42 × 10⁻⁶. -11 ~Approx. 7.75×10 -10 M is M. In some embodiments, KD is approximately 3.63 × 10⁻⁶. -12 ~Approx. 7.75×10 -10 M is M. In some embodiments, KD is approximately 3.63 × 10⁻⁶. -12 ~Approx. 1.59×10 -11 It is M.

[0097] In some embodiments, the equilibrium dissociation constant (KD) for the binding of an antibody containing an antigen-binding domain to the Fc gamma receptor I is approximately 1.0 × 10⁻⁶. -7 M or less, for example, approximately 5.0 × 10 -8 M or less, approximately 4.0×10 -8Less than about 3.0×10 -8 Less than about 1.0×10 -8 Less than about 5.0×10 -9 Less than or about 1.0×10 -9 Less than. In some embodiments, KD is about 3.96×10 -8 Less than. In some embodiments, KD is 3.73×10 -9 Less than.

[0098] In some embodiments, the KD for the binding of an antibody comprising an antigen-binding domain to Fc gamma receptor IIa is about 3.74×10 -6 Less than, for example, about 5.0×10 -7 Less than, about 1.0×10 -7 Less than, about 9.86×10 -8 Less than, about 9.15×(10 -8 Less than, about 5.0×10 -8 Less than, or about 1.0×10 -8 Less than. In some embodiments, the KD for the binding of an antibody comprising an antigen-binding domain to Fc gamma receptor IIa is about 9.15×10 -8 Less than. In some embodiments, the KD for the binding to Fc gamma receptor IIa is about 9.86×10 -8 Less than.

[0099] In some embodiments, the KD for the binding of an antibody comprising an antigen-binding domain to Fc gamma receptor IIb is about 2.0×10 -7 Less than, for example, about 1.16×10 -7 Less than, about 1.11×10 -7 Less than, about 1.0×10 -7 Less than, about 5.0×10 -8 Less than, about 1.0×10 -8 Less than, or about 5.0×10 -9 Less than. In some embodiments, the KD for the binding to Fc gamma receptor IIb is about 1.16×10 -7 Less than. In some embodiments, the KD for the binding to Fc gamma receptor IIb is about 1.11×10 -7 Less than.

[0100] In some embodiments, the KD for binding of an antibody containing an antigen-binding domain to the Fc gamma receptor IIIa is approximately 6.0 × 10⁻⁶. -7 Less than M, for example, approximately 1.0 × 10 -7 M or less, approximately 7.0×10 -8 M or less, approximately 5.05×10 -8 M or less, approximately 5.0×10 -8 M or less, approximately 1.0×10 -8 M or less, or approximately 5.0 × 10 -9 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIIa is approximately 6.0 × 10⁻⁶. -8 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIIa is approximately 5.05 × 10⁻⁶. -8 The M value is less than or equal to M. In some embodiments, the antibody does not bind to Fc gamma receptor IIIa in a detectable manner.

[0101] In some embodiments, antibodies containing an antigen-binding domain do not bind detectably to Fc gamma receptor IIIb.

[0102] In some embodiments, the equilibrium dissociation constant (KD) for binding an antibody containing at least one of the first and / or second antigen-binding domains to the Fc gamma receptor I is approximately 1.0 × 10⁻⁶ -7 M or less, for example, approximately 5.0 × 10 -8 M or less, approximately 4.0×10 -8 M or less, approximately 3.0×10 -8 M or less, approximately 1.0×10 -8 M or less, approximately 5.0×10 -9 M or less, or approximately 1.0 × 10 -9 It is less than or equal to M. In some embodiments, KD is approximately 3.96 × 10⁻⁶. -8 It is less than or equal to M. In some embodiments, KD is 3.73 × 10 -9 It is less than M.

[0103] In some embodiments, the KD for binding of an antibody containing at least one of the first and / or second antigen-binding domains to the Fc gamma receptor IIa is approximately 3.74 × 10⁻⁶. -6M or less, for example, approximately 5.0 × 10 -7 M or less, approximately 1.0×10 -7 M or less, approximately 9.86×10 -8 M or less, approximately 9.15×10 -8 M or less, approximately 5.0×10 -8 M or less, or approximately 1.0 × 10 -8 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIa is approximately 9.15 × 10⁻⁶. -8 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIa is approximately 9.86 × 10⁻⁶. -8 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIb is approximately 1.16 × 10⁻⁶. -7 It is M or less.

[0104] In some embodiments, the KD for binding of an antibody containing at least one of the first and / or second antigen-binding domains to the Fc gamma receptor IIb is approximately 2.0 × 10⁻⁶. -7 Below M, for example, approximately 1.16 × 10 -7 M or less, approximately 1.11×10 -7 M or less, approximately 1.0×10 -7 M or less, approximately 5.0×10 -8 M or less, approximately 1.0×10 -8 M or less, or approximately 5.0 × 10 -9 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIb is approximately 1.16 × 10⁻⁶. -7 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIb is approximately 1.11 × 10⁻⁶. -7 It is M or less.

[0105] In some embodiments, the KD for binding of an antibody containing at least one of the first and / or second antigen-binding domains to the Fc gamma receptor IIIa is approximately 6.0 × 10⁻¹⁴. -7 Less than M, for example, approximately 1.0 × 10 -7 M or less, approximately 7.0×10 -8 M or less, approximately 5.05×10 -8 M or less, approximately 5.0×10 -8 M or less, approximately 1.0×10 -8M or less, or approximately 5.0 × 10 -9 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIIa is approximately 6.0 × 10⁻⁶. -8 It is less than M. In some embodiments, the KD for binding to Fc gamma receptor IIIa is approximately 5.05 × 10⁻⁶. -8 The M value is less than or equal to M. In some embodiments, the antibody does not bind to Fc gamma receptor IIIa in a detectable manner.

[0106] In some embodiments, an antibody comprising at least one of the first and / or second antigen-binding domains does not bind detectably to the Fc gamma receptor IIIb.

[0107] In some embodiments, the first and second antibodies bind to different 5T4 molecules on the surface of different cancer cells. In some embodiments, the first and second antibodies bind to different 5T4 molecules on the surface of the same cancer cell. In some embodiments, the first and second antibodies bind to the same 5T4 molecules on the surface of the cancer cell. In some embodiments, the first and second 5T4 epitopes are non-overlapping epitopes.

[0108] Fc effect pedal function In some embodiments, site mutations in the Fc region of the antibody-drug conjugate mitigate potential side effects caused by antibody effector function, such as binding affinity to at least the Fc gamma receptor (FcγR). While we do not wish to be bound by theory, binding to the Fc gamma receptor is thought to induce immune system activation or inhibition pathways, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0109] In some embodiments, the antibody-drug conjugate described herein comprises a full-length IgG antibody. In some embodiments, the full-length IgG antibody comprises a constant region (Fc) domain. In some embodiments, for example, in embodiments where the antibody-drug conjugate comprises an IgG Fc domain, the Fc domain is an IgG1 isotype constant region domain. In some embodiments, the IgG1 constant region domain comprises at least one mutation that reduces effector function, extends half-life, or a combination thereof. In some embodiments, the antibody-drug conjugate disclosed herein comprises an Fc mutation that reduces binding affinity to Fc gamma receptors. In some embodiments, the Fc mutation reduces binding affinity to Fc gamma receptors including FcγRI. In some embodiments, the Fc mutation reduces binding affinity to Fc gamma receptors including FcγRII. In some embodiments, the Fc mutation reduces binding affinity to Fc gamma receptors including FcγRIII. In some embodiments, the Fc mutation in the antibody-drug conjugate reduces binding affinity to FcγRI compared to a 5T4 antibody without the Fc mutation. In some embodiments, at least one mutation includes F (L234F) at position 237 of sequence number 100, C or A (S239C / A) at position 242 of sequence number 100, or a combination thereof.

[0110] In some embodiments, the Fc site mutations that reduce binding affinity to FcγRI include L234F and S239C, or S239A.

[0111] In some embodiments, the Fc domain may be the IgG1 isotype constant region domain. In some embodiments, the IgG1 constant region domain includes at least one mutation that reduces effector function, extends half-life, or a combination thereof. In some embodiments, the IgG1 constant region domain mutation extends the half-life of the antibody-drug conjugate. The Fc mutation of the antibody-drug conjugate can extend the half-life of the antibody-drug conjugate. In some embodiments, at least one mutation includes a mutation (N434) at position 437 relative to SEQ ID NO: 100. In some embodiments, at least one mutation includes A (N434A) at position 437 relative to SEQ ID NO: 100. These amino acids are shown in bold and underlined in Table 5 below. In some embodiments, the antibody-drug conjugate includes at least one mutation that extends the half-life of the antibody-drug conjugate, at least one mutation includes a mutation at position 437, and the mutation further includes A (N434A) at position 437 relative to SEQ ID NO: 100. In some embodiments, the antibody-drug conjugate includes a mutation that extends the half-life of the antibody-drug conjugate, the mutation includes a mutation at position 437, and the mutation further includes A(N434A) at position 437 relative to SEQ ID NO: 100. [Table 4]

[0112] In some embodiments, the antibody-drug conjugate disclosed herein comprises a monoclonal antibody. In some embodiments, the monoclonal antibody binds to only a single 5T4 epitope. In some embodiments, the monoclonal antibody comprises only a first antibody, i.e., an antibody that is not a bispecific antibody. In some embodiments, the monoclonal antibody binds to only a single epitope, i.e., a single epitope that is not a biparatopic antibody.

[0113] In some embodiments, the antibodies disclosed herein are bispecific or biparatopic monoclonal antibodies. In some embodiments, the antibody comprises a first antigen-binding domain, e.g., scFv, and a second antigen-binding domain, e.g., a full-length IgG antibody. In some embodiments, the antibody comprises a first antigen-binding domain that binds to a first 5T4 epitope, and a second antigen-binding domain that binds to a second 5T4 epitope.

[0114] In some embodiments, the antibody comprises a first antigen-binding domain that binds to the 5T4 epitope, and a second antigen-binding domain that binds to a second antigen, for example, an antigen expressed by cancer cells. Suitable cancer antigens and antigen-binding domains that bind to these antigens are known to those skilled in the art and include, for example, B cell maturation antigen (BCMA), CD19 molecule (CD19), CD20, CD30, CD33, CD38, CD44, CD123, CD138, cell adhesion molecule (CEA), C-type lectin domain family 12 member A (CLEC12A), chorionic somatomammotropin hormone 1 (CS-1), epidermal growth factor receptor (EGFR), EGFRVIII, epithelial cell adhesion molecule (EPCAM), delta-like molecule Examples include quasi-notch ligand 3 (DLL3), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), mesoserine (MSLN), programmed cell death ligand 1 (PD-L1), folate receptor alpha (FOLR1), folate receptor gamma (FOLR3), erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2), HER3, bone marrow stromal cell antigen 2 (HM1,24), sperm mitochondrial-associated cysteine-rich protein (MCSP), and prostate-specific membrane antigen (PSMA). Alternatively, the bispecific antibodies disclosed herein are T cell bispecific (TCB) antibodies. In these cases, the second antigen is expressed by T cells, e.g., CD3e antigen, and the bispecific antibodies described herein help mobilize and engage T cells in the presence of 5T4-positive cancer cells.

[0115] In some embodiments, the antigen-binding domain of the Disclosure comprises a 5T4-specific antigen-binding domain that includes one of the CDRs disclosed in Tables 1-2. Exemplary variable heavy and light chains of the antigen-binding domain of the Disclosure incorporating the CDRs disclosed in Tables 1-2 are provided in Table 4 below. [Table 5] Exemplary full-length heavy and light chains of the antigen-binding domains of this disclosure are provided in Table 5 below (HC: heavy chain, LC: light chain). In Table 5, the full-sequence heavy chain of the first antibody, the full-length IgG1 antibody, is italicized, and mutations in the constant region are underlined. The first linker is underlined, and the VH and VL sequences of the second antibody, scFv, are shown in bold and linked by the italicized and underlined linker. [Table 6-1] [Table 6-2] Tables 6 and 7 provide nucleotide sequences encoding exemplary CDRs for use in the 5T4 antigen-binding domain of this disclosure. [Table 7] [Table 8] Table 8 provides further humanized variable heavy and light chain amino acid sequences of the 5T4 antigen-binding domain of this disclosure. In Table 8, the CDR sequence is shown in bold. [Table 9] Those skilled in the art will understand that the 5T4 antigen-binding domain may include any combination of the variable heavy chain and variable light chain domains disclosed in Table 8 or 9. Alternatively, the 5T4 antigen-binding domain may include a specific pair of variable heavy chain and variable light chain domains identified by the numbers in the left column of Table 8 or 9. Table 9 provides further mouse variable heavy and light chain amino acid sequences of the 5T4 antigen-binding domain of this disclosure. Those skilled in the art will understand that the mouse variable heavy and light chain domains can be humanized. [Table 10-1] [Table 10-2]

[0116] This disclosure provides antigen-binding domains, antibodies, bispecific antibodies (e.g., T-cell bispecific antibodies), antibody-drug conjugates, and chimeric receptors (CARs), each comprising an antigen-binding domain including a variable heavy chain domain paired with one of the variable light chain domains listed in Tables 8 and 9. In some embodiments, the antibody or antibody-drug conjugate comprises a biparatopic antibody comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope (the first antigen-binding domain is functionally linked to the second antigen-binding domain), and a chemotherapeutic agent. In some embodiments, the first and / or second antigen-binding domains include one of the variable heavy chain domains paired with one of the variable light chain domains listed in Tables 8 and 9.

[0117] This specification provides antibody-drug conjugates that bind to at least one epitope of the 5T4 protein. In some embodiments, the antibody-drug conjugate binds to one epitope of the 5T4 antigen. In some embodiments, the antibody-drug conjugate binds to two epitopes of the 5T4 antigen.

[0118] In some embodiments, the antibody-drug conjugate comprises a first antigen-binding domain that specifically binds to a first 5T4 epitope, and a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, wherein the first antigen-binding domain is functionally linked to the second antigen-binding domain. In some embodiments, the first and second antigen-binding domains are independently selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, scab, dAb, single-domain heavy-chain antibodies, single-domain light-chain antibodies, and full-length IgG antibodies. In some embodiments, the first antigen-binding domain comprises a full-length IgG antibody. In some embodiments, the full-length IgG antibody comprises two heavy chains and two light chains. In some embodiments of the antibody-drug conjugates disclosed herein, the second antigen-binding domain comprises scFv. In some embodiments, the antibody-drug conjugate comprises two second antigen-binding domains scFv, both of which specifically bind to a second 5T4 epitope.

[0119] In some embodiments, scFv includes a heavy chain and a light chain. In some embodiments, the C-terminus of the light chain is operably connected to the N-terminus of the heavy chain via a linker, or the C-terminus of the heavy chain is operably connected to the N-terminus of the light chain via a linker. In some embodiments, the linker connecting the heavy chain and the light chain includes the sequence of SEQ ID NO: 153.

[0120] In some embodiments, the N-terminus of the second antigen-binding domain is operably linked to the C-terminus of the heavy chain of the first antigen-binding domain. In some embodiments, the C-terminus of the second antigen-binding domain is operably linked to the N-terminus of the heavy chain of the first antigen-binding domain. In some embodiments, the second antigen-binding domain is operably linked to the heavy chain of the first antigen-binding domain using a linker. In some embodiments, the linker contains or consists of the amino acid sequence of SEQ ID NO: 152.

[0121] Antibody constant region domain Antibodies including monoclonal antibodies containing full-length IgG antibodies, bispecific antibodies, and biparatopic antibodies are intended to be within the scope of this disclosure. In some embodiments, the full-length IgG antibody comprises two heavy chains and two light chains, each containing a variable region domain and a constant region domain, as described in more detail below. Those skilled in the art will understand that monoclonal antibodies, as well as bispecific antibodies and biparatopic antibodies containing full-length IgG antibodies having the variable and constant domain configurations described below, are intended to be within the scope of this disclosure.

[0122] In some embodiments, the constant region domain is the IgG1 isotype constant region domain. In some embodiments, the constant region domain includes the amino acid sequence of SEQ ID NO: 148.

[0123] In some embodiments, the antibody light chain includes a variable region domain and a constant region domain. In some embodiments, the constant region domain is an IgG1 isotype constant region domain. In some embodiments, the light chain includes an IgG1 isotype constant region domain, and the constant region domain includes the amino acid sequence of SEQ ID NO: 149.

[0124] In some embodiments, for example, in embodiments in which the antibody includes an IgG1 constant region domain, the IgG1 constant region domain includes at least one mutation that reduces effector function, extends half-life, or a combination thereof.

[0125] In some embodiments, at least one mutation includes F (L234F) at position 237 of SEQ ID NO: 100, C or A (S239C / A) at position 242 of SEQ ID NO: 100, A (N434A) at position 437 of SEQ ID NO: 100, or a combination thereof. In some embodiments, the constant region domain includes F (L234F) at position 237 of SEQ ID NO: 100, C or A (S239C / A) at position 242 of SEQ ID NO: 100, and A (N434A) at position 437 of SEQ ID NO: 100. In some embodiments, at least one mutation includes F (L234F) at position 237 of SEQ ID NO: 100. In some embodiments, at least one mutation includes C or A (S239C / A) at position 242 of SEQ ID NO: 100. In some embodiments, at least one mutation includes C (S239C / A) at position 242 of SEQ ID NO: 100. In some embodiments, at least one mutation includes A(S239C / A) at position 242 of sequence number 100. In some embodiments, at least one mutation includes A(N434A) at position 437 of sequence number 100.

[0126] In some embodiments, the antibody heavy chain(s) includes a constant region domain containing the amino acid sequence of SEQ ID NO: 148, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antibody light chain(s) includes a constant region domain containing the amino acid sequence of SEQ ID NO: 149, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antibody heavy chain includes a constant region domain containing the amino acid sequence of SEQ ID NO: 148, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and the first antibody light chain includes a constant region domain containing the amino acid sequence of SEQ ID NO: 149, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0127] In some embodiments, the antibody heavy chain(s) includes a constant region domain containing the amino acid sequence of SEQ ID NO: 148. In some embodiments, the antibody light chain(s) includes a constant region domain containing the amino acid sequence of SEQ ID NO: 149. In some embodiments, the antibody heavy chain(s) includes a constant region domain containing the amino acid sequence of SEQ ID NO: 148, and the first antibody light chain(s) includes a constant region domain containing the amino acid sequence of SEQ ID NO: 149.

[0128] In some embodiments of the antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises a full-length IgG antibody comprising a first antigen-binding domain and a second antigen-binding domain comprising scFv. In some embodiments, the antibody-drug conjugate comprises four polypeptides. In some embodiments, the antibody-drug conjugate comprises two polypeptides comprising an IgG antibody heavy chain, a linker, and a second antigen-binding domain from the N-terminus to the C-terminus. In some embodiments, the antibody-drug conjugate comprises four polypeptides comprising (a) two polypeptides comprising a full-length IgG antibody heavy chain, a linker, and a second antigen-binding domain from the N-terminus to the C-terminus, and (b) two polypeptides comprising a full-length IgG antibody light chain. In some embodiments, the linker connecting the full-length IgG antibody heavy chain and the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 152.

[0129] In some embodiments of the antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises a full-length IgG antibody comprising a first antigen-binding domain and a second antigen-binding domain comprising scFv, and the antibody-drug conjugate comprises two polypeptides comprising a second antigen-binding domain, a linker, and a full-length IgG antibody heavy chain from the N-terminus to the C-terminus. In some embodiments, the first antigen-binding domain comprises a full-length IgG antibody, the second antigen-binding domain comprises scFv, and the antibody-drug conjugate comprises four polypeptides comprising (a) two polypeptides comprising a second antigen-binding domain, a linker, and a first antigen-binding domain heavy chain from the N-terminus to the C-terminus, and (b) two polypeptides comprising a first antigen-binding domain light chain. In some embodiments, the linker connecting the second antigen-binding domain and the first antigen-binding domain heavy chain comprises the amino acid sequence of SEQ ID NO: 152.

[0130] Heavy-chain CDR In some embodiments, the antigen-binding domains described herein, as well as the antibodies, antibody-drug conjugates, and receptors comprising them, include a heavy chain (HC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 13-23, or a sequence having one, two, or three substitutions, insertions, or deletions thereof; an HC CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, and 24-26 and 28-39, or a sequence having one, two, or three substitutions, insertions, or deletions thereof; and an HC CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 6, and 40-52, or a sequence having one, two, or three substitutions, insertions, or deletions thereof.

[0131] In some embodiments, the antigen-binding domain described herein includes a heavy chain (HC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 13-23, an HC CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 24-26, and 28-39, and an HC CDR3 sequence including the amino acid sequence of SEQ ID NO: 3.

[0132] In some embodiments, the antigen-binding domain described herein includes a heavy chain (HC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 13-23, an HC CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 24-26, and 28-39, and an HC CDR3 sequence comprising the amino acid sequences of SEQ ID NOs: 3, 6, and 40-52.

[0133] In some embodiments, the antibodies and antibody-drug conjugates described herein include a first and at least a second antigen-binding domain, the first and / or second antigen-binding domain comprising a heavy chain containing a heavy chain (HC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 13-23, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; an HC CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 24-26, and 28-39, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; and an HC CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 6, and 40-52, or a sequence having one, two, or three substitutions, insertions, or deletions thereto, wherein one or more of the CDR1, CDR2, and CDR3 sequences are not the same between the first and second antigen-binding domains.

[0134] In some embodiments, the antibodies and antibody-drug conjugates described herein include a first and at least a second antigen-binding domain, the first and / or second antigen-binding domain comprising a heavy chain containing a heavy chain (HC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs: 1, 4 and 13-23, an HC CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 24-26 and 28-39, and an HC CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 6 and 40-52, wherein one or more of the CDR1, CDR2, and CDR3 sequences are not the same between the first and second antigen-binding domains.

[0135] In some embodiments, the antibodies and antibody-drug conjugates described herein include an antigen-binding domain comprising a heavy chain (HC) complementarity-determining region (CDR1) sequence selected from any heavy chain CDR1 (CDRH1) sequence listed in Table 1, an HC CDR2 selected from any heavy chain CDR2 (CDRH2) sequence listed in Table 1, and an HC CDR3 selected from any heavy chain CDR3 (CDRH3) sequence listed in Table 1.

[0136] Those skilled in the art will understand that the antigen-binding domains, antibodies, antibody-drug conjugates, and receptors of the present disclosure may include heavy chains containing any CDRH1, CDRH2, or CDRH3 sequences listed in Table 1. In some embodiments, the heavy chain may include a combination of one CDRH1, one CDRH2, and one CDRH3 in a single row in Table 1. In some embodiments, the antibody-drug conjugate may include a heavy chain containing a combination of one CDRH1, one CDRH2, and one CDRH3, where CDRH1, CDRH2, and CDRH3 are not in the same row in Table 1. Those skilled in the art will understand that the antibody-drug conjugates of the present disclosure may include heavy chains containing any one CDRH1 sequence listed in Table 1, combined with any one CDRH2 sequence listed in Table 1, combined with any one CDRH3 sequence listed in Table 1.

[0137] In some embodiments, the heavy chain variable region domains include: (a) HC CDR1 containing SEQ ID NO: 1, HC CDR2 containing SEQ ID NO: 2, and HC CDR3 containing SEQ ID NO: 3; (b) HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 5, and HC CDR3 containing SEQ ID NO: 6; (c) HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 24, and HC CDR3 containing SEQ ID NO: 40; (d) HC CDR1 containing SEQ ID NO: 13, HC CDR2 containing SEQ ID NO: 25, and HC CDR3 containing SEQ ID NO: 41; (e) HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 26, and HC CDR3 containing SEQ ID NO: 42; (f) HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 28, and HC CDR3 containing SEQ ID NO: 43; (g) HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 29, and HC CDR3 containing SEQ ID NO: 44; (h) HC CDR1 containing SEQ ID NO: 16, and HC CDR3 containing SEQ ID NO: 30 (i) HC CDR3 containing CDR2 and SEQ ID NO: 45, (j) HC CDR1 containing SEQ ID NO: 17, HC CDR2 containing SEQ ID NO: 31, and HC CDR3 containing SEQ ID NO: 46, (k) HC CDR1 containing SEQ ID NO: 18, HC CDR2 containing SEQ ID NO: 32, and HC CDR3 containing SEQ ID NO: 47, (l) HC CDR1 containing SEQ ID NO: 19, HC CDR2 containing SEQ ID NO: 33, and HC CDR3 containing SEQ ID NO: 48, (m) HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 35, and HC CDR3 containing SEQ ID NO: 50, (n) HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 36, and HC CDR3 containing SEQ ID NO: 51, (o) HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 37, and HC CDR3 containing SEQ ID NO: 3, (p) HC CDR1 containing SEQ ID NO: 21, and HC CDR3 containing SEQ ID NO: 38 HC CDR3 including CDR2 and SEQ ID NO: 3, (q) HC CDR1 including SEQ ID NO: 22, HC CDR2 including SEQ ID NO: 39, and HC CDR3 including SEQ ID NO: 45, or (s) HC CDR1 including SEQ ID NO: 23, and HC CDR3 including SEQ ID NO: 32Includes CDR2 and HC CDR3, which includes sequence number 52.

[0138] Light Chain CDR In some embodiments, the antigen-binding domains described herein, as well as the antibodies, antibody-drug conjugates, and receptors comprising them, include a light chain (LC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs: 7, 10, and 53-66, or a sequence having one, two, or three substitutions, insertions, or deletions thereof; an LC CDR2 sequence selected from the group consisting of SEQ ID NOs: 8, 11, and 67-75, or a sequence having one, two, or three substitutions, insertions, or deletions thereof; an LC CDR3 sequence selected from the group consisting of SEQ ID NOs: 9, 12, and 76-83, or a sequence having one, two, or three substitutions, insertions, or deletions thereof.

[0139] In some embodiments, the antibody-drug conjugate described herein comprises a first and a second antigen-binding domain, the first and / or second antigen-binding domain comprising a light chain (LC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs. 7, 10, and 53-66, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; an LC CDR2 sequence selected from the group consisting of SEQ ID NOs. 8, 11, and 67-75, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; an LC CDR3 sequence selected from the group consisting of SEQ ID NOs. 9, 12, and 76-83, or a sequence having one, two, or three substitutions, insertions, or deletions thereto, wherein one or more of the CDR1, CDR2, and CDR3 sequences are not the same between the first and second antigen-binding domains.

[0140] In some embodiments, the antibody-drug conjugate described herein comprises a first and a second antigen-binding domain, the first and / or second antigen-binding domain comprising a light chain (LC) complementarity-determining region (CDR1) sequence selected from the group consisting of SEQ ID NOs: 7, 10, and 53-66, an LC CDR2 sequence selected from the group consisting of SEQ ID NOs: 8, 11, and 67-75, and an LC CDR3 sequence selected from the group consisting of SEQ ID NOs: 9, 12, and 76-83, wherein one or more of the CDR1, CDR2, and CDR3 sequences are not the same between the first and second antigen-binding domains.

[0141] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, or a sequence having one, two, or three substitutions, insertions, or deletions thereto.

[0142] In some embodiments, the first antigen-binding domain includes a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, or a sequence having one, two, or three substitutions, insertions, or deletions thereto; and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 9, or a sequence having one, two, or three substitutions, insertions, or deletions thereto.

[0143] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first antigen-binding domain includes a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 9.

[0144] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 9.

[0145] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 10, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 11, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 12.

[0146] In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 4, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 5, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second antigen-binding domain includes a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 10, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 11, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 12.

[0147] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 9, and the second antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 4, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 5, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 6, and a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 10, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 11, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 12.

[0148] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 10, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 11, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 12; the second antigen-binding domain includes a heavy chain variable region domain comprising an HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 4, an HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 5, and an HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 6, and a light chain variable region domain comprising an LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, an LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 9.

[0149] Those skilled in the art will understand that the antigen-binding domains of the present disclosure, the antibodies comprising them, antibody-drug conjugates, and receptors may include a light chain containing any of the CDRL1, CDRL2, or CDRL3 sequences listed in Table 2. In some embodiments, the light chain may contain a combination of one CDRL1, one CDRL2, and one CDRL3 within a single row in Table 2. In some embodiments, the antigen-binding domain may include a light chain containing a combination of one CDRL1, one CDRL2, and one CDRL3, where CDRL1, CDRL2, and CDRL3 are not in the same row in Table 2. Those skilled in the art will understand that the antigen-binding domains of the present disclosure may include a light chain containing any one CDRL1 sequence listed in Table 2 in combination with any one CDRL2 sequence listed in Table 2 in combination with any one CDRL3 sequence listed in Table 2.

[0150] In some embodiments, the antigen-binding domain is (a) LC CDR1 containing SEQ ID NO: 7, LC CDR2 containing SEQ ID NO: 8, and LC CDR3 containing SEQ ID NO: 9; (b) LC CDR1 containing SEQ ID NO: 10, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 12; (c) LC CDR1 containing SEQ ID NO: 53, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76; (d) LC CDR1 containing SEQ ID NO: 54, LC CDR2 containing SEQ ID NO: 67, and LC CDR3 containing SEQ ID NO: 77; (e) LC CDR1 containing SEQ ID NO: 55, LC CDR2 containing SEQ ID NO: 68, and LC CDR3 containing SEQ ID NO: 78; (f) LC CDR1 containing SEQ ID NO: 56, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79; (g) LC CDR1 containing SEQ ID NO: 57, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79; (h) LC CDR1 containing SEQ ID NO: 58, and LC CDR3 containing SEQ ID NO: 11 (i) LC CDR3 including CDR2 and SEQ ID NO: 76, (j) LC CDR1 including SEQ ID NO: 59, LC CDR2 including SEQ ID NO: 70, and LC CDR3 including SEQ ID NO: 80, (k) LC CDR1 including SEQ ID NO: 60, LC CDR2 including SEQ ID NO: 71, and LC CDR3 including SEQ ID NO: 81, (l) LC CDR1 including SEQ ID NO: 62, LC CDR2 including SEQ ID NO: 70, and LC CDR3 including SEQ ID NO: 82, (m) LC CDR1 including SEQ ID NO: 10, LC CDR2 including SEQ ID NO: 73, and LC CDR3 including SEQ ID NO: 12, (n) LC CDR1 including SEQ ID NO: 63, LC CDR2 including SEQ ID NO: 11, and LC CDR3 including SEQ ID NO: 76, (o) LC CDR1 including SEQ ID NO: 64, LC CDR2 including SEQ ID NO: 68, and LC CDR3 including SEQ ID NO: 78, (p) LC CDR3 including SEQ ID NO: 65 LC CDR1, LC CDR2 including SEQ ID NO: 74, and LC CDR3 including SEQ ID NO: 9, (q) LC CDR1 including SEQ ID NO: 66, LC CDR2 including SEQ ID NO: 75, and LC CDR3 including SEQ ID NO: 9, (registered trademark) LC CDR1 including SEQ ID NO: 58The light chain variable region domain includes CDR1, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76, or (s) LC CDR1 containing SEQ ID NO: 60, LC CDR2 containing SEQ ID NO: 71, and LC CDR3 containing SEQ ID NO: 83.

[0151] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 1, HC CDR2 containing SEQ ID NO: 2, and HC CDR3 containing SEQ ID NO: 3, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 7, LC CDR2 containing SEQ ID NO: 8, and LC CDR3 containing SEQ ID NO: 9.

[0152] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 5, and HC CDR3 containing SEQ ID NO: 6, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 10, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 12.

[0153] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 24, and HC CDR3 containing SEQ ID NO: 40, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 53, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76.

[0154] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 13, HC CDR2 containing SEQ ID NO: 25, and HC CDR3 containing SEQ ID NO: 41, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 54, LC CDR2 containing SEQ ID NO: 67, and LC CDR3 containing SEQ ID NO: 77.

[0155] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 26, and HC CDR3 containing SEQ ID NO: 42, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 55, LC CDR2 containing SEQ ID NO: 68, and LC CDR3 containing SEQ ID NO: 78.

[0156] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 28, and HC CDR3 containing SEQ ID NO: 43, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 56, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79.

[0157] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 29, and HC CDR3 containing SEQ ID NO: 44, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 57, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79.

[0158] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 16, HC CDR2 containing SEQ ID NO: 30, and HC CDR3 containing SEQ ID NO: 45, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 58, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76.

[0159] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 17, HC CDR2 containing SEQ ID NO: 31, and HC CDR3 containing SEQ ID NO: 46, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 59, LC CDR2 containing SEQ ID NO: 70, and LC CDR3 containing SEQ ID NO: 80.

[0160] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 18, HC CDR2 containing SEQ ID NO: 32, and HC CDR3 containing SEQ ID NO: 47, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 60, LC CDR2 containing SEQ ID NO: 71, and LC CDR3 containing SEQ ID NO: 81.

[0161] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 19, HC CDR2 containing SEQ ID NO: 33, and HC CDR3 containing SEQ ID NO: 48, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 61, LC CDR2 containing SEQ ID NO: 72, and LC CDR3 containing SEQ ID NO: 77.

[0162] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 20, HC CDR2 containing SEQ ID NO: 34, and HC CDR3 containing SEQ ID NO: 49, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 62, LC CDR2 containing SEQ ID NO: 70, and LC CDR3 containing SEQ ID NO: 82.

[0163] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 35, and HC CDR3 containing SEQ ID NO: 50, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 10, LC CDR2 containing SEQ ID NO: 73, and LC CDR3 containing SEQ ID NO: 12.

[0164] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 5, and HC CDR3 containing SEQ ID NO: 6, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 63, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76.

[0165] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 36, and HC CDR3 containing SEQ ID NO: 51, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 64, LC CDR2 containing SEQ ID NO: 68, and LC CDR3 containing SEQ ID NO: 78.

[0166] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 37, and HC CDR3 containing SEQ ID NO: 3, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 65, LC CDR2 containing SEQ ID NO: 74, and LC CDR3 containing SEQ ID NO: 9.

[0167] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 21, HC CDR2 containing SEQ ID NO: 38, and HC CDR3 containing SEQ ID NO: 3, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 66, LC CDR2 containing SEQ ID NO: 75, and LC CDR3 containing SEQ ID NO: 9.

[0168] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 22, HC CDR2 containing SEQ ID NO: 39, and HC CDR3 containing SEQ ID NO: 45, and the light chain variable region includes LC CDR1 containing SEQ ID NO: 58, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76.

[0169] In some embodiments, the heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 23, HC CDR2 containing SEQ ID NO: 32, and HC CDR3 containing SEQ ID NO: 52, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 60, LC CDR2 containing SEQ ID NO: 71, and LC CDR3 containing SEQ ID NO: 83.

[0170] Heavy chain variable region domain In some embodiments of the antigen-binding domains of the present disclosure, and the antibodies, antibody-drug conjugates, and receptors comprising them, the antigen-binding domain includes at least one heavy chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 169, 170, and 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them. In some embodiments, the antigen-binding domain includes a single heavy chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 169, 170, and 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0171] In some embodiments, the antibody or antibody-drug conjugate includes at least two heavy chain variable region domains, each containing a sequence independently selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 169, 170, and 172, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them. In some embodiments, the antibody or antibody-drug conjugate includes two heavy chain variable region domains, each containing a sequence independently selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 169, 170, and 172, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them. In some embodiments, the two heavy chain variable region domains are not identical. In some embodiments, the antibody or antibody-drug conjugate includes two or more heavy chain variable region domains containing sequences selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 169, 170, and 172, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0172] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0173] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0174] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 102, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0175] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 104, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0176] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 106, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0177] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 108, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0178] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 112, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0179] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 114, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0180] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 116, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0181] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 118, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0182] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 120, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0183] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 122, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0184] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 124, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0185] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 126, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0186] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0187] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 132, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0188] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 134, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0189] In some embodiments, the antibody-drug conjugate includes a heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 136, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0190] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 138, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0191] In some embodiments, the antigen-binding domain comprises a heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 140, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0192] In some embodiments, the antigen-binding domain comprises a heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 156, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0193] In some embodiments, the antigen-binding domain comprises a heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 158, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0194] In some embodiments, the antigen-binding domain comprises a heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 160, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0195] In some embodiments, the antigen-binding domain comprises a heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 162, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0196] In some embodiments, the antigen-binding domain comprises a heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 164, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0197] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 166, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0198] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 168, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0199] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 170, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0200] In some embodiments, the antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0201] In some embodiments, for example, in which the antigen-binding domain is incorporated into a biparatopic antibody or antibody-drug conjugate, the first and / or second antigen-binding domain includes a heavy chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 56, 158, 160, 162, 164, 166, 169, 170, and 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0202] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 102, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 104, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 106, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 108, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 112, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 114, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 116, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 118, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 120, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 122, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 124, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 126, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 132, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 134, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 136, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 138, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 140, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 156, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 158, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 160, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 162, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 164, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 166, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 168, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 170, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0203] In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 102, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 104, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 106, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 108, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 112, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 114, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 116, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 118, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 120, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 122, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 124, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 126, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 132, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 134, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 136, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 138, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 140, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 156, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 158, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 160, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 162, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 164, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 166, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 168, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 170, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0204] Light chain variable region domain In some embodiments of the antigen-binding domains of the present disclosure, and the antibodies, antibody-drug conjugates, and receptors comprising them, the antibody-drug conjugate includes a light chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them. In some embodiments, the antibody or antibody-drug conjugate includes at least one light chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0205] In some embodiments, the antibody or antibody-drug conjugate includes at least two light chain variable region domains, each containing a sequence independently selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them. In some embodiments, the antibody or antibody-drug conjugate includes two light chain variable region domains, each containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them. In some embodiments, the two light chain variable region domains are not identical. In some embodiments of the antibodies or antibody-drug conjugates of the present disclosure, the antibody or antibody-drug conjugate includes two or more light chain variable region domains containing sequences selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0206] In some embodiments, the antigen-binding domain includes a light chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0207] In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 103, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 105, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 107, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 109, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments of the antigen-binding domain of the present disclosure, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 113, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 115, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 117, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 119, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 121, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 123, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 125, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 127, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 129, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 131, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 133, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 135, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 137, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 139, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 141, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 157, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 159, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 161, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 163, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 165, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 167, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 169, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 171, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0208] In some embodiments, the heavy chain variable region domain includes the amino acid sequence of SEQ ID NO: 96, and the light chain variable region domain includes the amino acid sequence of SEQ ID NO: 98.

[0209] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 97, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 99.

[0210] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 102, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 103.

[0211] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 104, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 105.

[0212] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 106, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 107.

[0213] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 108, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 109.

[0214] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 112, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 113.

[0215] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 114, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 115.

[0216] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 116, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 117.

[0217] In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 118, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 119.

[0218] In some embodiments, the heavy chain variable region domain includes the amino acid sequence of SEQ ID NO: 120, and the light chain variable region domain includes the amino acid sequence of SEQ ID NO: 121.

[0219] In some embodiments, for example, in embodiments in which the antigen-binding domain is incorporated into a biparatopic antibody or antibody-drug conjugate, the first and / or second antibody includes a light chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0220] In some embodiments, the first and second antigen-binding domains include light chain variable region domains containing sequences selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

[0221] In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 103, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 105, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 107, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 109, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 113, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 115, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 117, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 119, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 121, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 123, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 125, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 127, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 129, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 131, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 133, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 135, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 137, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 139, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 141, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 157, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 159, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 161, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 163, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 165, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 167, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 169, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 171, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0222] In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 103, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 105, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 107, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 109, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 113, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 115, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 117, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 119, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 121, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 123, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 125, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 127, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 129, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 131, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 133, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 135, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 137, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 139, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 141, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 157, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 159, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 161, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 163, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 165, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 167, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 169, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 171, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0223] In some embodiments of the antibody-antibody-drug conjugates of this disclosure, the first and / or second antigen-binding domain is a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 168, 170, and 172, or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. The invention comprises a heavy chain variable region domain containing a sequence having a specific property, and a light chain variable region domain containing a sequence selected from the group consisting of sequence numbers 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with those sequences.

[0224] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96 or 97. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98 or 99. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96 or 97 and a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98 or 99.

[0225] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0226] In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second antigen-binding domain includes a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0227] In some embodiments, the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0228] In some embodiments, the first antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and the second antigen-binding domain includes a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0229] In some embodiments, the dual paratopic antibody or antibody-drug conjugate comprises a full-length IgG1 antibody containing a first antigen-binding domain, and the antibody or antibody-drug conjugate comprises two polypeptides comprising the first antigen-binding domain heavy chain variable region domain of SEQ ID NO: 96, the first linker of SEQ ID NO: 152, the second antigen-binding domain heavy chain variable region domain of SEQ ID NO: 97, the second linker of SEQ ID NO: 153, and the light chain variable region of SEQ ID NO: 99. In some embodiments, the antibody or antibody-drug conjugate comprises two polypeptides comprising the light chain variable region domain of SEQ ID NO: 98. In some embodiments, the antibody-drug conjugate comprises two polypeptides comprising the first antigen-binding domain heavy chain variable region domain of SEQ ID NO: 96, the first linker of SEQ ID NO: 152, the second antigen-binding domain heavy chain variable region domain of SEQ ID NO: 97, the second linker of SEQ ID NO: 153, and the light chain variable region of SEQ ID NO: 99, and the antibody or antibody-drug conjugate further comprises two polypeptides comprising the antibody light chain variable region domain of SEQ ID NO: 98. In some embodiments, the heavy and light chains of full-length IgG1 include IgG1 isotype constant region domains.

[0230] In some embodiments, the second antigen-binding domain is an scFv comprising a first heavy chain domain containing sequence number 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0231] In some embodiments, the second antigen-binding domain is an scFv comprising a light chain variable region domain containing sequence number 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0232] In some embodiments of the antibody-drug conjugate, the second antigen-binding domain is an scFv comprising a first heavy chain domain containing SEQ ID NO: 97 and a light chain variable region domain containing SEQ ID NO: 99.

[0233] In some embodiments, the antibody or antibody-drug comprises a full-length IgG antibody containing a first antigen-binding domain, the antigen-binding domain antibody contains scFv, and the antibody or antibody-drug conjugate comprises two polypeptides from N-terminus to C-terminus, comprising a first antibody heavy chain variable region domain of SEQ ID NO: 96, an IgG1 isotype constant region domain of SEQ ID NO: 148, a first linker of SEQ ID NO: 152, a second antibody heavy chain variable region domain of SEQ ID NO: 97, a second linker of SEQ ID NO: 153, and a light chain variable region of SEQ ID NO: 99.

[0234] In some embodiments, the antibody or antibody-drug conjugate comprises a full-length IgG antibody containing a first antigen-binding domain, the second antigen-binding domain containing an scFv, and the antibody or antibody-drug conjugate comprises two polypeptides, from the N-terminus to the C-terminus, containing a first antibody variable light chain domain of SEQ ID NO: 98 and a first antibody light chain constant region domain of SEQ ID NO: 149.

[0235] In some embodiments, the antibody or antibody-drug conjugate comprises a full-length IgG antibody containing a first antigen-binding domain, the second antigen-binding domain containing scFv, and the antibody or antibody-drug conjugate comprises four polypeptides, including two polypeptides from N-terminus to C-terminus, each containing the first antigen-binding domain heavy chain variable region domain of SEQ ID NO: 96, the IgG1 isotype constant region domain of SEQ ID NO: 148, the first linker of SEQ ID NO: 152, the second antigen-binding domain heavy chain variable region domain of SEQ ID NO: 97, the second linker of SEQ ID NO: 153, and the light chain variable region of SEQ ID NO: 99, and two polypeptides from N-terminus to C-terminus, each containing the first antigen-binding domain variable light chain domain of SEQ ID NO: 98 and the first antigen-binding domain light chain constant region domain of SEQ ID NO: 149.

[0236] In some embodiments, the antibody or antibody-drug conjugate comprises a full-length IgG antibody containing a first antigen-binding domain, the second antigen-binding domain containing scFv, and the antibody or antibody-drug conjugate comprises four polypeptides, including two polypeptides from N-terminus to C-terminus, each containing the first antigen-binding domain heavy chain variable region domain of SEQ ID NO: 96, the IgG1 isotype constant region domain of SEQ ID NO: 148, the first linker of SEQ ID NO: 152, the second antigen-binding domain light chain variable region of SEQ ID NO: 99, the second linker of SEQ ID NO: 153, and the heavy chain variable region domain of SEQ ID NO: 97, and two polypeptides from N-terminus to C-terminus, each containing the first antigen-binding domain variable light chain domain of SEQ ID NO: 98 and the first antigen-binding domain light chain constant region domain of SEQ ID NO: 149.

[0237] In some embodiments, the antibody or antibody-drug conjugate comprises two polypeptides containing the sequence of SEQ ID NO: 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and two polypeptides containing the sequence of SEQ ID NO: 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0238] In some embodiments, the antibody or antibody-drug conjugate comprises two polypeptides containing the sequence of SEQ ID NO: 100 and two polypeptides containing the sequence of SEQ ID NO: 101.

[0239] This disclosure provides an antibody and an antibody-drug conjugate comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope (the first antigen-binding domain is operably linked to the second antigen-binding domain), and a chemotherapeutic agent, wherein the first antigen-binding domain comprises a full-length IgG antibody, and the second antigen-binding domain comprises an scFv, and the antibody or antibody-drug conjugate comprises two polypeptides comprising (a) two polypeptides comprising at least one heavy chain variable region domain including an HC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 3, further comprising the amino acid sequence of SEQ ID NO: 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and (b) an LC containing the amino acid sequence of SEQ ID NO: 7 The present invention comprises two polypeptides each comprising at least one light chain variable region domain including a CDR1 sequence, an LC CDR2 sequence including the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 sequence including the amino acid sequence of SEQ ID NO: 9, further comprising two polypeptides each comprising the amino acid sequence of SEQ ID NO: 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0240] This disclosure provides an antibody and an antibody-drug conjugate comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope (the first antigen-binding domain is operably linked to the second antigen-binding domain), and a chemotherapeutic agent, wherein the first antigen-binding domain comprises a full-length IgG antibody, and the second antigen-binding domain comprises an scFv, and the antibody or antibody-drug conjugate comprises (a) two polypeptides comprising at least one heavy chain variable region domain comprising the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 6, further comprising the amino acid sequence of SEQ ID NO: 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and (b) an LC comprising the amino acid sequence of SEQ ID NO: 10 The present invention comprises two polypeptides each comprising at least one light chain variable region domain including a CDR1 sequence, an LC CDR2 sequence including the amino acid sequence of SEQ ID NO: 11, and an LC CDR3 sequence including the amino acid sequence of SEQ ID NO: 12, further comprising two polypeptides each comprising the amino acid sequence of SEQ ID NO: 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0241] This disclosure provides an antibody and an antibody-drug conjugate comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope (the first antigen-binding domain is operably linked to the second antigen-binding domain), and a chemotherapeutic agent, wherein the first antigen-binding domain comprises a full-length IgG antibody, and the second antigen-binding domain comprises an scFv, and the antibody or antibody-drug conjugate comprises (a) a first heavy chain variable region domain comprising an HC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 3, and at least a second heavy chain variable region domain comprising an HC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 4, an HC CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 5, and an HC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 6, and an LC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 7 and an LC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 8 (b) Two polypeptides comprising a light chain variable region domain comprising a CDR2 sequence and an LC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 9, further comprising the amino acid sequence of SEQ ID NO: 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto; and two polypeptides comprising a light chain variable region domain comprising an LC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an LC CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 11, and an LC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 12, further comprising the amino acid sequence of SEQ ID NO: 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0242] This disclosure provides an antibody and an antibody-drug conjugate comprising a first antigen-binding domain that specifically binds to a first 5T4 epitope, a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope (the first antigen-binding domain is operably linked to the second antigen-binding domain), and a chemotherapeutic agent, wherein the first antigen-binding domain comprises a full-length IgG antibody, and the second antigen-binding domain comprises an scFv, and the antibody or antibody-drug conjugate comprises (a) a first heavy chain variable region domain comprising an HC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 1, an HC CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 2, and an HC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 3, and at least a second heavy chain variable region domain comprising an HC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 4, an HC CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 5, and an HC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 6, and an LC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 10 and an LC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 11 (b) Two polypeptides comprising a light chain variable region domain comprising a CDR2 sequence and an LC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 12, further comprising the amino acid sequence of SEQ ID NO: 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto; and two polypeptides comprising a light chain variable region domain comprising an LC CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an LC CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 9, further comprising the amino acid sequence of SEQ ID NO: 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0243] Therapeutic drugs The disclosed 5T4-specific antigen-binding domain and antibody may be conjugated to a therapeutic agent or effector molecule (including, but not limited to, a molecule in which the therapeutic agent has a covalent bond to the antigen-binding domain or antibody). The therapeutic agent is a drug having specific biological activity directed towards a specific target molecule or a cell having the target molecule. Those skilled in the art will know that the therapeutic agent may be a variety of drugs such as vinblastine, daunomycin, cytotoxins such as natural or modified Pseudomonas exotoxin or diphtheria toxin, or encapsulating agents (such as liposomes) that themselves contain the pharmacological composition. 125 I, 32 P, 14 C, 3 H and 35 It will be understood that this may include radioactive agents such as S, as well as other labels, target moieties, and ligands.

[0244] In some embodiments, the therapeutic agent is a chemotherapeutic agent.

[0245] The selection of a particular therapeutic agent depends on the specific target molecule or cell and the desired biological effect. For example, a therapeutic agent could be a cytotoxin used to induce the death of a specific target cell (such as a tumor cell). Conversely, if a non-lethal biological response is desired, the therapeutic agent can be a non-lethal pharmacological agent or conjugated into a liposome containing a non-lethal pharmacological agent.

[0246] Effector molecules can be linked to a target antigen-binding domain or antibody using any number of means known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. The procedure for attaching the effector molecule to the antibody varies according to the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid groups (COOH), free amine groups (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on the antibody, resulting in the binding of the effector molecule. Alternatively, the antigen-binding domain or antibody may be derivatized to expose or attach further reactive functional groups. Derivatization may involve the attachment of any of a number of known linker molecules. The linker can be any molecule used to link the antibody to the effector molecule. The linker can form a covalent bond to both the protein and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the protein and effector molecules are polypeptides, linkers can be attached to the constituent amino acids via their side chain groups (e.g., via disulfide bonds to cysteine) or to the alpha-carbon amino group and carboxyl group of the terminal amino acids.

[0247] In some situations, when an immune complex reaches its target site, it is desirable to release an effector molecule from the antigen-binding domain or the antibody. Therefore, in these situations, the immune complex contains a binding that can be cleaved near the target site. Cleavage of the linker to release the effector molecule from the antibody can be facilitated by enzymatic activity or by conditions in which the immune complex is present either within the target cell or near the target site.

[0248] Those skilled in the art can determine a suitable method for attaching a given drug to an antibody or other polypeptide.

[0249] In some embodiments, the chemotherapeutic agent is conjugated via a linker to at least one of the first antigen-binding domain or the second binding domain of a dual paratopic antibody described herein. In some embodiments, the chemotherapeutic agent is auristatin. In some embodiments, the auristatin is selected from the group consisting of auristatin E (AE), monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and synthetic analogs of drastatin. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0250] The anti-5T4 biparatopic antibody-drug conjugate (ADC) of this disclosure comprises a first antigen-binding domain that specifically binds to a first 5T4 epitope, and a second antigen-binding domain that specifically binds to a second 5T4 epitope that is not identical to the first 5T4 epitope, wherein the first antigen-binding domain is operably linked to the second antigen-binding domain and conjugated to a cytotoxic agent or immunosuppressant, the resulting ADC exerts a cytotoxic effect or a cell proliferation inhibitory effect against 5T4-expressing cancer cells. Thus, the anti-5T4 biparatopic antibody-drug conjugate exerts a cytotoxic or cell proliferation inhibitory effect against 5T4-expressing cancer cells. In one embodiment, the anti-5T4 ADC is internalized and accumulates in 5T4-expressing cells, and the ADC exerts a therapeutic effect (e.g., cytotoxicity, cell proliferation inhibition, or immunosuppressive effect).

[0251] Examples of suitable parts for conjugation to antigen-binding domains and antibodies include chemotherapeutic agents, prodrug-converting enzymes, radioisotopes or compounds, or toxins. In exemplary embodiments, an anti-5T4 antibody or its antigen-binding moiety is conjugated to auristatin, e.g., MMAF or MMAE. Any drug that exerts a therapeutic effect against cancer cells or activated immune cells can be used as a therapeutic agent for conjugation to the anti-5T4 antibody or its derivatives (see, for example, International Publication No. 2004 / 010957, “Drug Conjugates and Their Use for Treating Cancer, An Autoimmune, Disease or an Infectious Disease” (above) and U.S. Provisional Application No. 60 / 400,403 (above)). Typically, the therapeutic agent is a cytotoxic agent. In some embodiments, the anti-5T4 antibody-drug conjugate contains more than one therapeutic agent per conjugate, for example, about 1 to about 20 therapeutic agents per conjugate (commonly referred to as the drug-to-antibody ratio, or DAR).

[0252] In some embodiments, an anti-5T4 antibody, or its antigen-binding moiety, is conjugated with auristatin. Auristatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and / or nuclear and cell division, and to possess anticancer and / or antifungal activity.

[0253] The anti-5T4 antibody or antigen-binding domain of this disclosure may be conjugated to at least one auristatin. Auristatins represent a group of drastatin analogs that have generally been shown to have anticancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cell division. For example, auristatin E (described in U.S. Patent No. 5,635,483, incorporated herein by reference) is a synthetic analog of the marine natural product drastatin 10 and is a compound that inhibits tubulin polymerization by binding to the same tubulin site as the anticancer drug vincristine (GRPettit, Prog. Chem. Org. Nat. Prod, 70:1-79 (1997)). Drastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are specific to compounds of the drastatin class. Exemplary embodiments of the auristatin subclass of mitotic inhibitors include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivatives), monomethyl auristatin E (MMAE or auristatin E derivatives), monomethyl auristatin F (MMAF or auristatin F derivatives), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB).The synthesis and structure of auristatin derivatives are described in U.S. Patent Publications 2003-0083263, 2005-0238649, and 2005-0009751, International Publication 04 / 010957, International Publication 02 / 088172, and U.S. Patents 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, and 5,635,48. These are described in No. 3, No. 5,599,902, No. 5,554,725, No. 5,530,097, No. 5,521,284, No. 5,504,191, No. 5,410,024, No. 5,138,036, No. 5,076,973, No. 4,986,988, No. 4,978,744, No. 4,879,278, No. 4,816,444, and No. 4,486,414 (each of these being incorporated by reference).

[0254] In some embodiments, the anti-5T4 antibody or antigen-binding domain is conjugated to at least one MMAF (monomethyl auristatin F) by a linker such as maleimidocaproyl (mc-MMAF), though not limited to this. Anti-5T4 ADCs may have drug-to-antibody ratios (DARs) of 2, 4, 6, or 8. In particular, the DAR of the ADC may range from 0 to 8, but higher loadings, e.g., 10, 12, or 14, are also possible. Monomethyl auristatin F (MMAF) inhibits cell division by blocking tubulin polymerization. It has a charged C-terminal phenylalanine residue that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE. Due to its toxicity, it cannot be used as a drug itself, but it can be conjugated to a monoclonal antibody (mAb) directed at cancer cells. In one embodiment, the linker to the anti-5T4 antibody is stable in the extracellular fluid, but once the conjugate enters tumor cells, it is cleaved by cathepsin, thus activating the anti-mitotic mechanism.

[0255] In some embodiments, the anti-5T4 antibody or antigen-binding domain of the present invention is conjugated to at least one MMAE (monomethyl auristatin E). Monomethyl auristatin E (MMAE, vedotin) inhibits cell division by blocking tubulin polymerization. Due to its toxicity, it is often not usable as a drug itself. In recent cancer treatment developments, it is linked to a monoclonal antibody (mAb) that recognizes the expression of specific markers in cancer cells and directs the MMAE to the cancer cells. In some embodiments, the linker linking the MMAE to the anti-5T4 antibody or antigen-binding domain is stable in extracellular fluid (i.e., culture medium or environment outside the cell), but once the ADC binds to a specific cancer cell antigen and enters the cancer cell, it is cleaved by cathepsin, thus releasing toxic MMAE and activating a potent anti-mitotic mechanism.

[0256] In some embodiments, the anti-5T4 antibody or its antigen-binding moiety is conjugated to auristatin, which is MMAF. In some embodiments, the anti-5T4 ADC is covalently bonded to one or more molecules of monomethyl auristatin F (MMAF). In some embodiments, to produce an anti-5T4 ADC covalently bonded to one or more molecules of MMAF, the interchain disulfide bonds of the ADC are reduced to sulfhydryl groups. The MMAF is then conjugated to the antibody via these sulfhydryl groups. In some embodiments, the anti-5T4 ADC is produced using an uncleavable linker, i.e., an uncleavable maleimidocaproyl (mc) linkage.

[0257] ADCs that can be labeled with detectable or functional labels. Detectable labels include radioactive labels, such as isotopes. 2 H, 3 H, 11 C, 13 C, 14 C, 32 P, 33 S, 34 S, 35 S, 36 S, 36 Cl, 51Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I, 211 At, 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc and 186 Examples include, but are not limited to, Re, which can be attached to the antibodies of the present invention using conventional chemistry known in the art of antibody imaging. Labels also include fluorescent labels and labels conventionally used in the art for MRI-CT imaging. They also include enzymatic labels such as horseradish peroxidase. Labels further include specific homogeneous detectable moieties, such as chemical moieties such as biotin which can be detected via binding to labeled avidin.

[0258] Functional labels may also include substances designed to target tumor sites to cause destruction of tumor tissue. Examples of such functional labels include cytotoxic agents such as 5-fluorouracil or lysine, which can convert prodrugs into active drugs at the tumor site, and enzymes such as bacterial carboxypeptidases or nitroreductases.

[0259] As those skilled in the art will understand, the above-mentioned agents, as well as other suitable agents, may be conjugated or attached to an anti-5T4 antibody, such as the antibody shown in Figures 1A-1B, in any preferred manner for generating the anti-5T4 ADC of this disclosure. For example, in various embodiments of this disclosure, though not limited to these, the anti-5T4 antibody and agent(s) may be covalently attached and / or conjugated using linker, spacer and / or stretcher compounds, which in various embodiments of the present invention result in a therapeutic agent(s) that is cleavable or incleavable and internally transported by target cells.

[0260] In one embodiment, an anti-5T4 antibody or antigen-binding domain is conjugated to an MMAF using an inclementable maleimidocaproyl linkage.

[0261] Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are publicly known in the field (e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For See Cancer Detection and Therapy (Baldwin et al. eds., Academic Press, 1985) and Thorpe et al., 1982, Immunol. Rev. 62: 119-58). See also, for example, PCT International Publication No. 89 / 12624.

[0262] In some embodiments, the ADC includes a linker region between the cytotoxic agent and the antibody or antigen-binding domain. For example, such linker, spacer, and / or stretcher compounds include, but are not limited to, aminobenzoic acid spacers (see, for example, but not limited to, U.S. Patents 7,091,186 and 7,553,816, each of which is incorporated herein by reference in whole), maleimidocaproyl, p-aminobenzylcarbamoyl (PAB), lysosomal enzyme-cleavable linkers (see, for example, but not limited to, U.S. Patent 6,214,345, which is incorporated herein by reference in whole), maleimidocaproyl-polyethylene glycol 20 (MC(PEG)6-OH), N-methyl-valinecitrulline, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (see, for example, but not limited to, Yoshitake et al., which is incorporated herein by reference in whole). See al. (1979) Eur. J. Biochem., 101, 395-399), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, for example, U.S. Patent No. 4,563,304, which is incorporated herein by reference 25, but is not limited to the entirety of the SPDB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), p-citrulline, and other linker, spacer, and / or stretcher compounds (see, for example, U.S. Patent No. 4,563,304, which is incorporated herein by reference 25, but is not limited to). See Patent Nos. 7,090,843, 7,223,837, and 7,659,241, and U.S. Patent Application Publications 2004 / 0018194, 2004 / 0121940, 2006 / 0116422, 2007 / 0258987, 2008 / 0213289, 2008 / 0241128, 2008 / 0311136, 2008 / 0317747, and 2009 / 0010945 (each of which is incorporated herein by reference in whole).Generally speaking, techniques for attaching and / or conjugating the above-mentioned drugs, as well as other drugs, to the specific binding members of the present invention, particularly antibodies and their fragments, are known in the art. For example, but not limited to, see Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, In Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985), each of which is incorporated herein by reference in whole.

[0263] Several different reactions are available for the covalent attachment of drugs to antibody or antigen-binding domains. This is often achieved by the reaction of amino acid residues of antibody molecules, including the amine group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, and various parts of aromatic amino acids. One of the most commonly used methods of nonspecific covalent attachment is the carbodiimide reaction, which links the carboxyl (or amino) group of a compound to the amino (or carboxyl) group of an antibody. Furthermore, bifunctional agents such as dialdehydes or imide esters are used to link the amino group of a compound to the amino group of an antibody molecule. Schiff base reactions are also available for drug attachment to antibodies. This method involves periodic oxidation of a drug containing a glycol or hydroxyl group, thus forming an aldehyde, which is then reacted with the antibody molecule. Attachment occurs via the formation of a Schiff base with the amino group of the antibody molecule. Isothiocyanates can also be used as coupling agents for covalent attachment of drugs to antibodies. Other technologies are known to those skilled in the art and fall within the scope of the present invention. Non-limiting examples of such technologies are described, for example, in U.S. Patents No. 5,665,358, No. 5,643,573 and No. 5,556,623, which are incorporated herein by reference in their entirety.

[0264] In certain embodiments, an intermediate, which is a precursor of the linker, is reacted with a drug under appropriate conditions. In certain embodiments, a reactive group is used on the drug and / or intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with an anti-5T4 antibody under appropriate conditions.

[0265] Other examples of conjugation methods are described in U.S. Patent No. 7,837,980 (Seattle Genetics), Carter and Senter (2008) Cancer J, 14(3):154, as well as in U.S. Patent Publications 2004-0157782 A1 and 2005-0238649, and International Patent Application PCT / US04 / 038392.

[0266] In certain embodiments, anti-5T4 ADCs can be purified to obtain ADCs having a desired drug-to-antibody ratio (DAR). In one embodiment of the present invention, the formulation comprises an anti-5T4 ADC mixture containing anti-5T4 ADCs having a desired average drug-to-antibody ratio (DAR), for example, an average DAR of about 3. In another embodiment of the present invention, the formulation comprises an ADC mixture containing anti-5T4 ADCs having a desired DAR range, for example, about 2 to 4, or about 2 to 8, or about 4 to 8.

[0267] In one embodiment, the formulation contains an ADC mixture in which 70% of the present ADCs have 8 or fewer drug loading species, and the ADCs contain an anti-5T4 antibody and auristatin. Alternatively, 75% of the present ADCs have 8 or fewer drug loading species, 80% of the present ADCs have 8 or fewer drug loading species, 85% of the present ADCs have 4 or fewer drug loading species, 90% of the present ADCs have 8 or fewer drug loading species, or 95% of the present ADCs have 8 or fewer drug loading species.

[0268] Polynucleotides and vectors This disclosure provides polynucleotides comprising an anti-5T4 antibody, an antigen-binding domain, and a receptor as described herein, as well as a polynucleotide system comprising one or more polynucleotides.

[0269] In some embodiments, the polynucleotides or polynucleotide systems described herein include sequences encoding either the light chain CDR sequence and / or the heavy chain CDR sequence of the anti-5T4 biparatopic antibody described herein.

[0270] In some embodiments, the polynucleotides or polynucleotide systems described herein include sequences encoding either the light chain or the heavy chain of the anti-5T4 biparatopic antibody described herein.

[0271] In some embodiments, the polynucleotides or polynucleotide systems described herein include sequences listed in any of Tables 4 to 7.

[0272] This disclosure provides a first polynucleotide encoding a first heavy chain of the anti-5T4 antigen-binding domain of this disclosure, and a second polynucleotide encoding a light chain of the anti-5T4 antigen-binding domain. In some embodiments, the first and second polynucleotides are separate molecules. Alternatively, the first and second polynucleotides may form part of a single continuous polynucleotide molecule.

[0273] In some embodiments, the disclosure provides a first polynucleotide encoding a first anti-5T4 antigen-binding domain heavy chain, a linker, and a second anti-5T4 antigen-binding domain, as well as a second polynucleotide encoding a first anti-5T4 antigen-binding domain light chain. In some embodiments, the disclosure provides a single continuous polynucleotide molecule encoding a first anti-5T4 antigen-binding domain heavy chain, a first anti-5T4 antigen-binding domain light chain, and a second anti-5T4 antigen-binding domain.

[0274] In some embodiments, the first polynucleotide encodes the sequence of a first polypeptide comprising a first anti-5T4 antigen-binding domain heavy chain, a linker, and a second anti-5T4 antigen-binding domain from the N-terminus to the C-terminus. In some embodiments, the first polynucleotide encodes the sequence of a first polypeptide comprising a second anti-5T4 antigen-binding domain, a linker, and a first anti-5T4 antigen-binding domain heavy chain from the N-terminus to the C-terminus. In some embodiments, the first polynucleotide encodes the sequence of a first polypeptide comprising a second anti-5T4 antigen-binding domain, a linker, and a first anti-5T4 antigen-binding domain heavy chain from the N-terminus to the C-terminus. In some embodiments, the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 144 or 145. In some embodiments, the first polynucleotide comprises the nucleotide sequences of SEQ ID NOs: 144 and 150. In some embodiments, the first polynucleotide comprises the nucleotide sequences of SEQ ID NOs: 145 and 150. In some embodiments, the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 142.

[0275] In some embodiments, the second polynucleotide encodes a second polypeptide comprising a first anti-5T4 antigen-binding domain light chain. In some embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 146 or 147. In some embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 146 or 151. In some embodiments, the second polynucleotide comprises the nucleotide sequences of SEQ ID NOs: 147 and 151. In some embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 143.

[0276] In some embodiments, the first polynucleotide encodes a polypeptide comprising the sequence of SEQ ID NO: 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the second polynucleotide encodes a polypeptide comprising the sequence of SEQ ID NO: 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0277] In some embodiments, the polynucleotide sequences encoding the first and second polypeptides are operably ligated to one or more promoters. For example, the polypeptide sequences may be operably ligated to the same promoter (under its control) and separated by one or more elements that generate separate polypeptides (e.g., self-cleaving polypeptides, internal ribosome entry sites, etc.).

[0278] In alternative embodiments, the sequences of the first or second polynucleotide encoding the first or second polypeptide are under the control of separate promoters. For example, each of the first and second polynucleotides may be cloned into separate expression vectors, each vector containing its own promoter and / or regulatory sequence. In some embodiments, the promoters operably linked to each of the first and second polynucleotides are the same. In some embodiments, the promoters operably linked to each of the first and second polynucleotides are not the same.

[0279] In some embodiments, the polynucleotides of the present invention are prepared using PCR techniques employing procedures and methods known to those skilled in the art. In some embodiments, this procedure includes ligation of two different DNA sequences (see, for example, “Current Protocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons, 1992).

[0280] Using the antigen-binding domains, antibodies, and receptors described herein, those skilled in the art can easily construct various clones containing functionally equivalent nucleic acids, such as nucleic acids that encode the same protein sequence but with different sequences. Accordingly, this disclosure provides nucleic acids encoding such antibodies.

[0281] A polynucleotide sequence is "operably linked" when it is in a functional relationship with another polynucleotide sequence. For example, a polynucleotide pre-sequence or secretion leader is operably linked to the nucleic acid encoding a polypeptide when expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it is positioned to facilitate translation. Generally, "operably linked" means that the linked polynucleotide sequences are contiguous, and in the case of a secretion leader, they are contiguous and within a reading frame. However, enhancers are contiguous by choice. Linking can be achieved, for example, by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotide adapters, linkers, or other methods known in the art can be used. In another embodiment, “functionally linked” also refers to a functional pairing of separate amino acid sequences, peptides, or proteins, such as in the combination of a first anti-5T4 antigen-binding domain heavy chain and a second anti-5T4 antigen-binding domain described herein, which are functionally linked via a linker sequence also described herein. For example, the first anti-5T4 antigen-binding domain heavy chain is “operably linked” to the second anti-5T4 antigen-binding domain via a linker sequence described herein.

[0282] This disclosure provides a vector comprising a polynucleotide containing sequences encoding a 5T4 antigen-binding domain, an antibody, and a receptor as described herein.

[0283] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle capable of introducing a DNA or RNA sequence (e.g., a foreign gene) into a host cell, thereby transforming the host and enhancing the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, and the like.

[0284] The terms “expression” and “expression” mean enabling or making available information in a gene or DNA sequence, for example, producing a protein by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form “expression products,” such as proteins. The expression product itself, for example, the resulting protein, can also be said to be “expressed” by a cell. Expression products can be characterized as intracellular, extracellular, or transmembrane. The term “intracellular” means something inside the cell. The term “extracellular” means something outside the cell. The term transmembrane means something that has an extracellular domain, a portion embedded in the cell membrane, and an intracellular domain.

[0285] In some embodiments, the polynucleotides of this disclosure are inserted into an expression vector (i.e., a nucleic acid construct) to enable the expression of the polypeptides described herein.

[0286] In some embodiments, the expression vectors of the Disclosure include additional sequences that make the vectors suitable for replication and integration in prokaryotes. In some embodiments, the expression vectors of the Disclosure include additional sequences that make the vectors suitable for replication and integration in eukaryotes. In some embodiments, the expression vectors of the Disclosure include a shuttle vector that makes the vectors suitable for replication and integration in both prokaryotes and eukaryotes. For example, such vectors may include a selection marker suitable for both eukaryotic and prokaryotic cells. Suitable markers will be apparent to those skilled in the art.

[0287] In some embodiments, the cloning vector comprises transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals) to enhance the expression of polypeptides expressed therefrom. Suitable translation terminators include, but are not limited to, the bovine growth hormone polyadenylation signal (BGH polyA). Suitable promoters will be obvious to those skilled in the art and include the CMV promoter and the actin promoter.

[0288] In some embodiments, the expression vectors of the present disclosure may further include additional polynucleotide sequences that enable the translation of several proteins from a single mRNA, such as an internal ribosome entry site (IRES), and sequences for genomic integration of promoter-chimeric polypeptides.

[0289] In some embodiments, the expression vector of the Disclosure includes elements that increase the expression of the antibody of the Disclosure. Such features include, but are not limited to, promoter selection and polyadenylation. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence. In some embodiments, the promoter includes a constitutively active promoter. In some embodiments, the promoter includes a cytomegalovirus promoter (pCMV). In some embodiments, the promoter can be combined with additional elements to enhance the expression of the recombinant protein of the Disclosure, such as introns (e.g., rabbit betaglobin intron, EF1a intron, etc.) and enhancer elements (CMV immediate early enhancer, SV40 enhancer, EF1a enhancer, adenovirus major late protein enhancer, etc.).

[0290] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (these are available from Invitrogen), pCI (this is available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (these are available from Strategene), pTRES (this is available from Clontech), and their derivatives.

[0291] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used by the present invention. The SV40 vector includes pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1 MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vectors that enable protein expression under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary cancer virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that have been shown to be effective for expression in eukaryotic cells.

[0292] In some embodiments, for example, in a bacterial system used to express the polypeptide of the present invention, a number of expression vectors can be advantageously selected depending on the intended use of the protein to be expressed. In some embodiments, a vector directed to high levels of protein product expression (directing the expressed product to the bacterial periplasm or a culture medium where the protein product is easily purified) is desired, perhaps as a fusion with a hydrophobic signal sequence. In one embodiment, vectors suitable for such operations include, but are not limited to, the E. coli expression vectors of the pET series (see Studier et al., Methods in Enzymol. 185:60-89 (1990)).

[0293] In some embodiments, a yeast expression system is used to express the polypeptides of this disclosure. In one embodiment, a number of vectors containing constitutive or inducible promoters may be used in yeast as disclosed in U.S. Patent No. 5,932,447. In another embodiment, a vector that enhances the incorporation of foreign DNA sequences into yeast chromosomes is used.

[0294] In some embodiments, recombinant viral vectors are useful for in vivo expression of polypeptides of the present invention because they offer advantages such as lateral infection and target specificity. In one embodiment, lateral infection is a process inherent in the life cycle of retroviruses, for example, in which a single infected cell produces many progeny virions that budding and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, the majority of which was not initially infected by the original viral particle. In one embodiment, a viral vector is produced that cannot spread laterally. In one embodiment, this feature may be useful when the desired objective is to introduce a specific gene into only a localized number of target cells.

[0295] In some embodiments, a mammalian cell expression system is used to express the polypeptides of this disclosure. The mammalian cells may be, for example, Chinese hamster ovary (CHO) cells or derivatives thereof, and the vector is a vector suitable for polypeptide expression in CHO cells. In some embodiments, the mammalian cells may be ExpiCHO-S® cells.

[0296] Aside from containing elements necessary for the transcription and translation of the inserted coding sequence (encoding the polypeptide), it is understood that the expression construct of the present invention may also contain sequences manipulated to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0297] Manufacturing method This disclosure provides a method for producing an anti-5T4 antibody as described herein, comprising: (a) contacting a plurality of cells with a polynucleotide, polynucleotide system, or vector encoding the antibody; (b) culturing the plurality of cells under conditions in which the antibody is expressed by at least one of the plurality of cells; and (c) purifying the antibody.

[0298] This disclosure provides a method for preparing an anti-5T4 antibody-drug conjugate as described herein, comprising: (a) contacting a plurality of cells with a polynucleotide, polynucleotide system, or vector encoding an antibody; (b) culturing the plurality of cells under conditions in which a bispecific antibody is expressed by at least one of the plurality of cells; (c) purifying the antibody; and (d) conjugating the antibody with a chemotherapeutic agent. In some embodiments, the antibody is bispecific or biparatopic as described herein.

[0299] This disclosure provides a method for producing an antibody-drug conjugate as described herein, comprising: (a) culturing cells containing a nucleic acid construct encoding a bispecific or biparatopic antibody under conditions that result in the expression of a bispecific or biparatopic antibody; (b) recovering the bispecific or biparatopic antibody; and (c) conjugating the bispecific or biparatopic antibody to a chemotherapeutic agent.

[0300] This disclosure provides a method for producing an antibody as described herein, comprising (a) culturing cells containing a nucleic acid construct encoding the antibody under conditions that result in antibody expression, wherein the antibody comprises: i) a first antibody that specifically binds to a first 5T4 epitope; and ii) a second antibody that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope (the first antibody is operably linked to the second antibody); and (b) recovering the bispecific antibody.

[0301] Various prokaryotic or eukaryotic cells can be used as host expression systems for expressing the antibodies of the present invention. In some embodiments, these include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing polypeptide coding sequences, and yeast transformed with recombinant yeast expression vectors containing polypeptide coding sequences.

[0302] In some embodiments, the cells include eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. Suitable mammalian cells for antibody-drug conjugate expression include CHO cells, PER.C6 cells, mouse NS0 cells, and HEK293 cells. The selection of suitable cell lines will be apparent to those skilled in the art.

[0303] In some embodiments, the group of cells includes prokaryotic cells, such as E. coli cells.

[0304] Various methods can be used to introduce the expression vector encoding the antibody of this disclosure into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988), and Gilboa et al. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. For further information on positive-negative selection methods, please refer to U.S. Patents No. 5,464,764 and No. 5,487,992.

[0305] In some embodiments, contacting a plurality of cells with a polynucleotide or vector encoding an antibody of the present disclosure includes transfection.

[0306] The term “transfection” refers to the introduction of foreign nucleic acids into cells using recombinant DNA technology. The term “transformation” refers to the introduction of “foreign” (i.e., exogenous or extracellular) genes, DNA, or RNA sequences into host cells, resulting in the host cells expressing the introduced genes or sequences and producing a desired substance, typically a protein or enzyme encoded by the introduced genes or sequences. The introduced genes or sequences are also called “cloned” or “foreign” genes or sequences and may include regulatory or control sequences such as start, stop, promoter, signal, secretion, or other sequences used by the cell’s genetic mechanism. The genes or sequences may include non-functional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA is “transformed,” “transformed,” or “clone.” The DNA or RNA introduced into host cells may originate from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

[0307] In some embodiments, contacting multiple cells with a polynucleotide or vector encoding an antibody of the present disclosure includes transduction. The term "transduction" means the introduction of foreign nucleic acid into cells using a viral vector, such as a lentiviral vector.

[0308] In some embodiments, a non-bacterial expression system (e.g., a mammalian expression system such as CHO cells) is used to express the antibody polypeptide. In some embodiments, the expression vector includes a CMV promoter and a neomycin resistance gene. In alternative embodiments, the expression vector includes a glutamine synthase marker (GS) under the control of the SV40 promoter.

[0309] In some embodiments, the introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, as it can achieve higher transfection efficiency due to the infectivity of the virus.

[0310] In some embodiments, transformed cells are cultured under effective conditions that allow for the expression of large amounts of antibodies or polypeptides. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactors, temperature, pH, and oxygen conditions that allow for protein production. The media typically comprises an aqueous solution having assimilated carbon, nitrogen, and phosphate sources, as well as other nutrients such as appropriate salts, minerals, metals, and vitamins. The cells of the present invention can be cultured in conventional fermentation bioreactors, shaking flasks, test tubes, microtiter dishes, and Petri dishes. In some embodiments, the culture is carried out at a temperature, pH, and oxygen content appropriate for recombinant cells. The culture conditions are within the scope of the expertise of those skilled in the art.

[0311] For example, suitable culture media for eukaryotic cells include, but are not limited to, Iskov-modified Dulbecco's medium, RPMI 1640, minimal essential medium-alpha (MEM-α), Dulbecco's modified Eagle medium (DMEM), Grace's complete insect medium, Ham F-10 or F-12 containing L-glutamine, Schneider's insect medium, or any other medium known to those skilled in the art. Furthermore, culture media described herein include, but are not limited to, chemically defined media, hydrolyzate-containing media, and simple media. The selection of appropriate media and cell culture conditions for a particular cell type will be obvious to those skilled in the art.

[0312] In some embodiments, depending on the vector and host system used for production, the resulting polypeptide of the present invention may remain in recombinant cells, be secreted into fermentation media, be secreted into the space between two cell membranes, for example, into the perimembranous space of Escherichia coli (E. coli), or be retained on the outer surface of a cell or viral membrane.

[0313] In some embodiments, the antibody or polypeptide is recovered after a predetermined incubation period.

[0314] The polypeptides of the present invention are purified using a variety of standard protein purification techniques (e.g., affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed-phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubilization).

[0315] In some embodiments, to facilitate recovery, the expressed coding sequence may be manipulated to encode the polypeptide and the fused cleavable moiety of the present invention. For example, the polypeptide may be designed so that it can be easily isolated by affinity chromatography, e.g., immobilization on a column specific to the cleavable moiety. In one embodiment, the cleavage site is manipulated between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with a suitable enzyme or agent that specifically cleaves the polypeptide at this site [see, for example, Booth et al., Immunol. Lett. 19:65-70 (1988) and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0316] In some embodiments, the polypeptides of the present invention are recovered in a “substantially pure” form. The phrase “substantially pure” refers to a purity that enables the effective use of the protein in the applications described herein.

[0317] In some embodiments, the polypeptides of the present invention may also be synthesized using an in vitro expression system. In one embodiment, the in vitro synthesis method is well known in the art, and the components of the system are commercially available.

[0318] In some embodiments, polypeptides are synthesized, purified, and their therapeutic effects are assayed in vivo or in vitro.

[0319] Pharmaceutical composition This disclosure provides pharmaceutical compositions comprising anti-5T4 antigen-binding domains, antibodies, and bispecific or biparatopic antibody-drug conjugates as described herein, as well as pharmaceutically acceptable carriers, diluents, or excipients. Pharmaceutical compositions comprising immune cells containing CARs comprising antigen-binding domains as described herein are also intended to be within the scope of this disclosure.

[0320] As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent and absorption retarder, etc. The carrier material is non-toxic and does not interfere with the efficacy of the biological activity of the active ingredient. Such preparations may conventionally contain salts, buffers, preservatives, a compatible carrier, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may routinely contain a compatible solid or liquid filler, diluent, or encapsulant suitable for administration to humans. The term “carrier” means a natural or synthetic organic or inorganic component that is combined with the active ingredient to facilitate application. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., injection or infusion).

[0321] Pharmaceutically acceptable diluents include physiological saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the rapid preparation of sterile injection solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0322] The pharmaceutical composition may be known in the art and may exist in a form acceptable for therapeutic use. In some embodiments, the pharmaceutical composition of the present invention is a liquid formulation. In other embodiments, the pharmaceutical composition of the present invention is lyophilized. In further embodiments, the pharmaceutical composition of the present invention is a reconstituted liquid formulation. In some embodiments, the liquid formulation of the present invention is an aqueous formulation. In some embodiments, the liquid formulation is non-aqueous.

[0323] The compositions comprising the anti-5T4 antigen-binding domain, antibody, and bispecific antibody-drug conjugate of the present disclosure can be formulated for administration by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. To administer the compositions of the present disclosure by a particular route of administration, it may be necessary to administer the compositions co-administer them with a material to prevent their inactivation. For example, the antibody-drug conjugate may be administered to a subject in a suitable carrier, e.g., liposomes or diluents.

[0324] In some embodiments, preparations for administration to a subject include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Some embodiments include non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), organic esters (e.g., ethyl oleate), and other solvents known to those skilled in the art. Physiologically acceptable carriers (or excipients) are optionally used in certain embodiments of the present invention. Examples of such are, for example, physiological saline, PBS, Ringer's solution, and lactated Ringer's solution. Furthermore, preservatives and additives are optionally added to the composition to help ensure stability and sterility. For example, antibiotics and other bactericides, antioxidants, chelating agents, etc., are all optionally present in various embodiments of the compositions herein.

[0325] Regardless of the selected route of administration, the compositions of this disclosure, and / or the pharmaceutical compositions of the present invention, which can be used in a preferred hydrated form, are formulated in a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0326] The pharmaceutical composition is optionally administered to subjects requiring treatment (either therapeutic or prophylactic) in any suitable sterile pharmaceutical carrier. Such a pharmaceutical carrier acts to maintain the solubility and action of the anti-5T4 antibody or antibody-drug conjugate.

[0327] In some embodiments, the compositions for use in the manner disclosed herein comprise a solution or emulsion, which in some embodiments comprises a safe and effective amount of the compounds disclosed herein, and optionally other compounds intended for various routes of administration.

[0328] The composition must be sterile and fluid enough to be delivered by syringe. In addition to water, the carrier is preferably isotonic buffered saline. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride.

[0329] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dose level depends on various pharmacokinetic factors, including the activity of the particular compound of the present invention used, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and prior medical history of the subject being treated, as well as similar factors well known in the medical field.

[0330] Treatment method This disclosure provides a method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising administering a therapeutically effective amount of an anti-5T4 bispecific antibody-drug conjugate or a pharmaceutical composition comprising a bispecific antibody-drug conjugate as disclosed herein. For example, a method comprising administering an anti-5T4 antigen-binding domain as part of an immunotherapy is also intended to be within the scope of this disclosure. The methods of this disclosure also include adoptive cell therapy comprising administering immune cells expressing a receptor comprising the 5T4 antigen-binding domain described herein, such as T cells or NK cells.

[0331] In some embodiments, the disease or disorder is cancer. In some embodiments, cancer includes solid tumors.

[0332] In some embodiments, cancer includes solid tumors. In some embodiments, cancer is selected from the group consisting of melanoma, renal cell carcinoma, mesothelioma, small cell lung cancer, uveal melanoma, bladder cancer, gastric cancer, squamous cell carcinoma of the head and neck, skin cancer, non-small cell lung cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer, pancreatic cancer, urothelial carcinoma, esophageal cancer, hepatocellular carcinoma, glioblastoma, glioma, or sarcoma.

[0333] In some embodiments, cancers include adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancers, anorectal cancers, cancers of the anal canal, appendiceal cancers, pediatric cerebellar astrocytomas, pediatric cerebral astrocytomas, basal cell carcinomas, skin cancers (non-melanomas), cholangiocarcinomas, extrahepatic cholangiocarcinomas, intrahepatic cholangiocarcinomas, bladder cancers, supratentorial and joint cancers, osteosarcomas and malignant fibrous histiocytomas, brain cancers, brainstem gliomas, cerebellar astrocytomas, cerebral astrocytomas / malignant gliomas, ependymomas, medulloblastomas, skeletal ectodermal tumors, visual pathway and hypothalamic gliomas, breast cancers, bronchial adenomas / carcinoids, carcinoid tumors, gastrointestinal cancers, nervous system cancers, and nerve cancers. Lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid tumors, mycosis fungoides, Cediary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumors, non-gonadal germ cell tumors, extrahepatic cholangiocarcinoma, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, ovarian germ cell tumors, gestational trophoblastoma, glioblastoma, head and neck cancer, hepatocellular carcinoma (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer Cancer, intraocular melanoma, eye cancer, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, kidney cancer, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-associated lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenström macroglobulinemia, medulloblastoma, melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous cell carcinoma of the neck, oral cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, Myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial carcinoma, low-grade ovarian tumor, pancreatic cancer, islet cell carcinoma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal blastoma and primitive neuroectodermal tumors of the upper cerebral hemispheres, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing's sarcomatoid tumor, Kaposi's sarcoma,The following group of cancers is selected: soft tissue sarcoma, epithelioid sarcoma, synovial sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, primordial neuroectodermal tumors of the cerebral hemispheres, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis, ureter and other urinary tract organs, gestational trophoblastic tumor, urethral cancer, endometrial cancer, uterine sarcoma, endometrial cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0334] Cancers treated with the antigen-binding domains, antibodies, immune cells, or antibody-drug conjugates of this disclosure, or pharmaceutical compositions containing them, may be staged as stage I, stage IIA, stage IIB, stage IIIA, stage IIIB, stage IIIC, or stage IV according to the American Joint Committee on Cancer (AJCC) classification. Cancers treated may be graded as grade GX (e.g., grade cannot be assessed), grade 1, grade 2, grade 3, or grade 4 according to the AJCC classification. Cancers treated may be staged as pNX, pN0, pN0(I-), pN0(I+), pN0(mol-), pN0(mol+), PN1, PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c according to the AJCC pathological classification (pN). Alternatively, or additionally, cancer can be staged according to the TNM staging system, which divides most types of cancer into four stages. Stage 1 usually means the cancer is relatively small and contained within the organ of origin. Stage 2 cancer is usually larger than Stage 1, but has not yet begun to spread into surrounding tissues. In some embodiments, Stage 2 means the cancer has spread to lymph nodes close to the tumor. Stage 3 cancer is usually larger and has begun to spread into surrounding tissues and lymph nodes. Stage 4, or metastatic cancer, is typically cancer that has spread from its place of origin to other organs in the body.

[0335] As used herein, “normal cells” are cells that cannot be classified as part of “proliferative disorders.” Normal cells lack uncontrolled proliferation or abnormal growth, or both, which can lead to the development of undesirable conditions or diseases. Preferably, normal cells have a normally functioning cell cycle checkpoint control mechanism.

[0336] As used herein, “contacting cells” means that the antibody-drug conjugate or other composition is in direct contact with cells or is in a state sufficiently close to inducing a desired biological effect in the cells.

[0337] As used herein, “monotherapy” refers to the administration of a single active or therapeutic compound to a subject requiring it. Preferably, monotherapy involves the administration of a therapeutically effective amount of the active compound. Monotherapy can be contrasted with combination therapy, in which a combination of multiple active compounds is administered, preferably with each component of the combination present in therapeutically effective amounts.

[0338] As used herein, “to treat” or “to treat” describes the management and care of a subject for the purpose of combating a disease, condition, or disorder, and includes the administration of the anti-5T4 antigen-binding domains, antibodies, antibody-drug conjugates, immune cells expressing anti-5T4 receptors, or pharmaceutical compositions comprising the same, for the purpose of alleviating the symptoms or complications of cancer or eliminating cancer.

[0339] As used herein, the term “alleviate” means to describe a process by which the severity of a sign or symptom of cancer is reduced. Importantly, the sign or symptom may be alleviated without being eliminated. In preferred embodiments, administration of a recombinant anti-5T4 bispecific antibody-drug conjugate or pharmaceutical composition of the present disclosure results in the elimination of the sign or symptom, but elimination is not required. An effective dosage is expected to reduce the severity of the sign or symptom. For example, the sign or symptom of a disorder such as cancer, which may occur in multiple locations, is alleviated when the severity of the cancer is reduced in at least one of the multiple locations.

[0340] As used herein, the term “severity” means the likelihood of cancer progressing from a precancerous or benign state to a malignant state. Alternatively, or additionally, severity means describing the stage of cancer, for example, according to the TNM system (approved by the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)) or by other methods recognized in the art. Cancer staging refers to the extent or severity of cancer based on factors such as the location of the primary tumor, the size of the tumor, the number of tumors, and lymph node metastasis (spread of cancer to lymph nodes). Alternatively, or additionally, severity means describing tumor grade by methods recognized in the art (see National Cancer Institute, www.cancer.gov). Tumor grading is a system used to classify cancer cells in terms of how abnormally the cancer cells appear under a microscope and how rapidly the tumor is likely to grow and spread. Many factors are considered when determining tumor grade, including cell structure and growth patterns. The specific factors used to determine tumor grade differ for each type of cancer. Severity also describes the histological grade, also known as differentiation, which indicates how similar tumor cells are to normal cells of the same tissue type (see National Cancer Institute, www.cancer.gov). Furthermore, severity describes the nuclear grade, which refers to the size and shape of the nuclei in tumor cells, as well as the percentage of dividing tumor cells (see National Cancer Institute, www.cancer.gov).

[0341] As used herein, the term “invasive” refers to cancer that can grow, form, or spread rapidly. Cancers referred to as invasive may be susceptible to treatment or may be resistant to treatment. Invasive cancers may include any type of cancer. Alternatively or additionally, the term “invasive” may describe cancers that require more severe or intense treatment than the usual form of treatment for that cancer.

[0342] As used herein, the term “refractory” refers to cancer that does not respond to the forms of treatment attempted. Refractory cancer may also be called resistant cancer.

[0343] In another aspect of this disclosure, severity represents the extent to which a tumor has secreted growth factors, degraded the extracellular matrix, become vascularized, lost adhesion to juxtaposed tissues, or metastasized. Furthermore, severity represents the number of sites to which the primary tumor has metastasized. Finally, severity includes the difficulty of treating tumors of various types and locations. For example, inoperable tumors, cancers with greater access to multiple bodily systems (hematological and immunological tumors), and cancers that are most resistant to conventional treatments are considered the most severe. In these situations, extending the life expectancy of the subject and / or reducing pain, decreasing the percentage of cancerous cells, or limiting cells to one system, as well as improving the stage / tumor grade / histological malignancy / nuclear grade of the cancer, are considered to alleviate the signs or symptoms of cancer.

[0344] As used herein, the term “symptom” is defined as an indicator of a disease, illness, injury, or something being wrong in the body. A symptom is felt or noticed by the individual experiencing it, but may not be readily noticed by others. Others are defined as non-healthcare professionals.

[0345] As used herein, the term “sign” is also defined as an indicator that something is wrong in the body. However, a sign is defined as something that can be seen by a physician, nurse, or other healthcare professional.

[0346] Cancer is a group of diseases that can cause almost any sign or symptom. The signs and symptoms depend on where the cancer is located, its size, and how much it affects nearby organs or structures. If the cancer spreads (metastasizes), symptoms may appear in different parts of the body.

[0347] As cancer grows, it begins to press on nearby organs, blood vessels, and nerves. This pressure can cause some of the signs and symptoms of cancer. Cancer can form in places where it doesn't cause any symptoms until it grows quite large.

[0348] Cancer may also cause symptoms such as fever, fatigue, or weight loss. This may be because cancer cells deplete much of the body's energy supply or release substances that alter the body's metabolism. Alternatively, cancer may trigger the immune system to react in a way that produces these symptoms. The signs and symptoms listed above are more common than those seen in cancer, but there are many others that are less common and not listed herein. However, all signs and symptoms of cancer recognized in the art are intended and encompassed by this disclosure.

[0349] Cancer treatment can result in a reduction in tumor size. This reduction in tumor size may also be referred to as "tumor regression." Preferably, after treatment by the method of this disclosure, the tumor size is reduced by 5% or more compared to its size before treatment; more preferably by 10% or more; more preferably by 20% or more; more preferably by 30% or more; more preferably by 40% or more; even more preferably by 50% or more; and most preferably by 75% or more. Tumor size can be measured by any reproducible measuring means. Tumor size can be measured as the diameter of the tumor.

[0350] Cancer treatment can result in a reduction in tumor volume. Preferably, after treatment by the method of this disclosure, the tumor volume is reduced by 5% or more compared to its size before treatment; more preferably, by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by 75% or more. The tumor volume can be measured by any reproducible measuring means.

[0351] Cancer treatment can result in a reduction in the number of tumors. Preferably, after treatment, the number of tumors is reduced by 5% or more compared to the number before treatment; more preferably, by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by more than 75%. The number of tumors can be measured by any reproducible measuring means. The number of tumors can be measured by counting tumors that can be seen with the naked eye or at a specific magnification. Preferably, the specific magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0352] Cancer treatment can result in a reduction in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment by the method of this disclosure, the number of metastatic lesions is reduced by 5% or more compared to the number before treatment; more preferably, by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by more than 75%. The number of metastatic lesions can be measured by any reproducible measuring means. The number of metastatic lesions can be measured by counting metastatic lesions that can be seen with the naked eye or at a specific magnification. Preferably, the specific magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0353] Cancer treatment may result in an extension of the mean survival time of the treated population compared to a population that has not received the anti-5T4 bispecific antibody-drug conjugate or a pharmaceutical composition comprising the same. Preferably, the mean survival time increases by more than 30 days, more preferably more than 60 days, more preferably more than 90 days, and most preferably more than 120 days. The increase in mean survival time of the population may be measured by any reproducible means. The increase in mean survival time of the population may be measured, for example, by calculating the mean length of survival for the population after the initiation of treatment with the active compound. The increase in mean survival time of the population may also be measured, for example, by calculating the mean length of survival for the population after the completion of the first round of treatment with the active compound.

[0354] Cancer treatment may result in a reduction in mortality in the treated population compared to a population that has not received the anti-5T4 bispecific antibody-drug conjugate or a pharmaceutical composition containing the same. Cancer treatment may also result in a reduction in mortality in the treated population compared to an untreated population. Cancer treatment may result in a reduction in mortality in the treated population compared to a population that has received monotherapy with a drug other than the anti-5T4 bispecific antibody-drug conjugate or a pharmaceutical composition containing the same. The reduction in mortality in the treated population may be measured by any reproducible means. The reduction in population mortality may also be measured, for example, by calculating the mean number of disease-related deaths per unit time after the start of treatment with the active compound for the population. The reduction in population mortality may also be measured, for example, by calculating the mean number of disease-related deaths per unit time after the completion of the first round of treatment with the antibody-drug conjugate for the population.

[0355] Cancer treatment can result in a reduction in tumor growth rate. Preferably, after treatment, the tumor growth rate is reduced by at least 5% compared to the pre-treatment rate; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. Tumor growth rate can be measured by any reproducible measuring means. Tumor growth rate can be measured according to the change in tumor diameter per unit time.

[0356] Cancer treatment can result in a reduction in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%, more preferably less than 10%, more preferably less than 20%, more preferably less than 30%, more preferably less than 40%, more preferably less than 50%, even more preferably less than 50%, and most preferably less than 75%. Tumor regrowth can be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring the increase in tumor diameter after the reduction of the previous tumor after treatment. A reduction in tumor regrowth is indicated by the absence of tumor recurrence after discontinuation of treatment.

[0357] Cancer treatment can lead to a decrease in the rate of cell proliferation. Preferably, after treatment, the rate of cell proliferation is reduced by at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 50%, and most preferably at least 75%. The rate of cell proliferation can be measured by any reproducible measuring means. The rate of cell proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0358] Cancer treatment can result in a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 50%, and most preferably at least 75%. The proportion of proliferating cells can be measured by any reproducible measurement means. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of non-dividing cells in a tissue sample. The proportion of proliferating cells may be equivalent to the mitotic index.

[0359] Cancer treatment can result in a reduction in the size of areas or zones of cell proliferation. Preferably, after treatment, the size of the areas or zones of cell proliferation is reduced by at least 5%, more preferably by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, even more preferably by at least 50%, and most preferably by at least 75% compared to its size before treatment. The size of areas or zones of cell proliferation can be measured by any reproducible measuring means. The size of areas or zones of cell proliferation can be measured as the diameter or width of the areas or zones of cell proliferation.

[0360] Cancer treatment can result in a reduction in the number or percentage of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5%, more preferably by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, even more preferably by at least 50%, and most preferably by at least 75% compared to their size before treatment. The appearance or morphology of abnormal cells can be measured by any reproducible measurement means. Abnormal cell morphology can be measured by microscopy, for example, using an inverted tissue culture microscope. Abnormal cell morphology can take the form of nuclear pleomorphism.

[0361] Cancer treatment can induce cell death, preferably resulting in a reduction of at least 10% in the number of cells in the population. More preferably, the cell death means a reduction of at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, and most preferably at least 75%. The number of cells in the population can be measured by any reproducible means. The number of cells in the population can be measured by fluorescence-activated cell sorting (FACS), immunofluorescence microscopy, and light microscopy. A method for measuring cell death is described in Li et al., Proc Natl Acad Sci US A.100(5):2674-8, 2003. In one embodiment, cell death occurs by apoptosis.

[0362] Combination therapy In some embodiments, it may be desirable to administer additional cancer treatments in combination with antigen-binding domains, antibodies, antibody-drug conjugates, adoptive cell therapy, or pharmaceutical compositions containing them. For example, in some treatment regimens, additional formulations including chemotherapeutic agents, antibiotics, antibody-drug conjugates of the Disclosure and one or more standard therapeutic agents are all optionally included with the compositions of the Invention. In some embodiments, antibody-drug conjugates are administered in combination with one or more of the following: chemotherapy, small molecule inhibitors, radiation, surgery, immunotherapy, or adoptive cell therapy.

[0363] As used herein, the terms “combination therapy,” “combination treatment,” and “co-therapy” are interchangeable and generally refer to modes of treatment characterized by antibody-drug conjugates or pharmaceutical compositions containing them, and additional therapeutic agents or methods provided herein. Typically, a mode of combination therapy is part of a particular treatment regimen intended to provide beneficial effects from the simultaneous action of a combination of therapeutic agents. Beneficial effects of combination may include, but are not limited to, pharmacokinetic or pharmacodynamic interactions resulting from the combination of therapeutic agents. Administration of these therapeutic agent combinations is typically carried out over a specified period (usually minutes, hours, days, or weeks, depending on the combination selected). In some embodiments, combination therapy includes administering two or more therapeutic agents sequentially, each therapeutic agent being administered at a different time, and also including administering these therapeutic agents, or at least two of the therapeutic agents, substantially simultaneously. Substantially simultaneous administration can be achieved, for example, by administering a single dosage form having a fixed ratio of each therapeutic agent, or multiple distinct dosage forms for the therapeutic agents. The sequential or substantially simultaneous administration of each therapeutic agent can be carried out by any suitable route (including, but not limited to, oral, intravenous, intramuscular, and direct absorption via mucosal tissue). The therapeutic agents can be administered by the same or different routes. The therapeutic agents can be administered according to the same or different dosing intervals. For example, the first therapeutic agent of a selected combination may be administered by intravenous injection, while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally, or all therapeutic agents may be administered by intravenous injection.

[0364] In some embodiments, combination therapy also includes the administration of the therapeutic agent described above, further combined with other bioactive components and non-pharmacological therapies (e.g., surgery or radiation therapy). Where combination therapy further includes non-pharmacological therapy, the non-pharmacological therapy may be performed at any suitable time, insofar as beneficial effects from the synergistic effects of the combination of the therapeutic agent and the non-pharmacological therapy are achieved. For example, where appropriate, beneficial effects can still be achieved even if the non-pharmacological therapy is temporarily removed from the administration of the therapeutic agent, perhaps for several days or even weeks.

[0365] In some embodiments, additional therapeutic agents are chemotherapeutic agents (also referred to as antineoplastic agents or antiproliferative agents), such as alkylating agents, antibiotics, antimetabolites, antidotes, interferons, polyclonal or monoclonal antibodies, EGFR inhibitors, HER2 inhibitors, histone deacetylase inhibitors, hormones, mitotic inhibitors, MTOR inhibitors, multikinase inhibitors, serine / threonine kinase inhibitors, tyrosine kinase inhibitors, VEGF / VEGFR inhibitors, taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, microtubule-targeting agents, topoisomerase poisons, inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases), cytidine analogs or any chemotherapeutic agents, immune checkpoint inhibitors, platinum-based antitumor agents, CDK inhibitors, PARP inhibitors, or any antineoplastic or antiproliferative agents known to those skilled in the art.

[0366] Examples of alkylating agents suitable for use according to the combination therapy modalities provided herein include, but are not limited to, cyclophosphamide (Cytoxan, Neosar), chlorambucil (Leukeran), melphalan (Alkeran), carmustine (BiCNU), busulfen (Busulfex), lomustine (CeeNU), dacarbazine (DTIC-Dome), oxaliplatin (Eloxatin), carmustine (Gliadel), ifosfamide (Ifex), mechloretamine (Mustargen), busulfen (Myleran), carboplatin (Paraplatin), cisplatin (CDDP, Platinol), temozolomide (Temodar), thiotepa (Thioplex), bendamustine (Treanda), or streptozocin (Zanosar).

[0367] Examples of suitable anthracyclines include, but are not limited to, doxorubicin (Adriamycin), doxorubicin liposome (Doxil), mitoxantrone (Novantrone), bleomycin (Blenoxane), daunorubicin (Cerubidine), daunorubicin liposome (DaunoXome), dactinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), plicamycin (Mithracin), mitomycin (Mutamycin), pentostatin (Nipent), or valrubicin (Valstar).

[0368] Examples of antimetabolites include fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fludara), nelarabine (Arranon), and cladribine (Cladribine). Examples include, but are not limited to, Novaplus, clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposome (DepoCyt), hydroxyurea (Droxia), pralatrexate (Folotyn), phloxuridine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX, Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1, or cytarabine (Tarabine PFS).

[0369] Examples of antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0370] Examples of interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon A).

[0371] Examples of polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin), ofatumumab (Arzerra), bevacizumab (Avastin), rituximab (Rituxan), cetuximab (Erbitux), panitumumab (Vectibix), tositumomab / iodine-131 tositumomab (Bexxar), alemtuzumab (Campath), ibritumomab (Zevalin, In-111, Y-90Zevalin), gemtuzumab (Mylotarg), eculizumab (Soliris), or denosumab.

[0372] Examples of EGFR inhibitors include, but are not limited to, gefitinib (Iressa), lapatinib (Tykerb), cetuximab (Erbitux), erlotinib (Tarceva), panitumumab (Vectibix), PKI-166, canertinib (CI-1033), matsuzumab (EMD72000), or EKB-569.

[0373] Examples of HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb), or AC-480.

[0374] Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0375] Examples of hormones include tamoxifen (Soltamox, Nolvadex), raloxifene (Evista), megestrol (Megace), leuprolide (Lupron, Lupron Depot, Eligard, Viadur), fulvestrant (Faslodex), letrozole (Femara), and triptorelin (Trelstar LA, Trelstar LA). Examples include, but are not limited to, Depot, exemestane (Aromasin), goserelin (Zoladex), bicalutamide (Casodex), anastrozole (Arimidex), fluoxymesterone (Androxy, Halotestin), medroxyprogesterone (Provera, Depo-Provera), estramustine (Emcyt), flutamide (Eulexin), toremifene (Fareston), degarelix (Firmagon), nilutamide (Nilandron), abarelix (Plenaxis), or testolactone (Teslac).

[0376] Examples of mitotic inhibitors include, but are not limited to, paclitaxel (Taxol, Onxol, Abraxane), docetaxel (Taxotere), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etoposide (Toposar, Etopophos, VePesid), teniposide (Vumon), ixabepyrone (Ixempra), nocodazole, epothirone, vinorelbine (Navelbine), camptothecin (CPT), irinotecan (Camptosar), topotecan (Hycamtin), amsacrin, or lamelalin D (LAM-D).

[0377] Examples of MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel), rapamune, ridafololimus, or AP23573.

[0378] Examples of multikinase inhibitors include, but are not limited to, sorafenib (Nexavar), sunitinib (Sutent), BIBW2992, E7080, Zd6474, PKC-412, motesanib, or AP24534.

[0379] Examples of serine / threonine kinase inhibitors include, but are not limited to, ruboxystaurin, eril / fasudil hydrochloride, flavopyridol, sericiclib (CYC202, Roscovitine), SNS-032 (BMS-387032), Pkc412, briostatin, KAI-9803, SF1126, VX-680, Azd1152, Arry-142886 (AZD-6244), SCIO-469, GW681323, CC-401, CEP-1347, or PD332991.

[0380] Examples of tyrosine kinase inhibitors include erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), trastuzumab (Herceptin), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), and eve Examples include, but are not limited to, rolimus (Afinitor), alemtuzumab (Campath), gemtuzumab (Mylotarg), temsirolimus (Torisel), pazopanib (Votrient), dasatinib (Sprycel), nilotinib (Tasigna), batalanib (Ptk787, ZK222584), CEP-701, SU5614, MLN518, XL999, VX-322, Azd0530, BMS-354825, SKI-606, CP-690, AG-490, WHI-P154, WHI-P131, AC-220, or AMG888.

[0381] Examples of VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetinib.

[0382] Examples of microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epotilon, and navelbine.

[0383] Examples of topoisomerase-toxic drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrin, epirubicin, and idarubicin.

[0384] Examples of taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0385] Exemplary immune checkpoint inhibitors include programmed cell death 1 (PD-1) and CD274 molecule (PD-L1) inhibitors. Exemplary PD-1 inhibitors include pembrolizumab, nivolumab, and semiprimab. Further examples of PD-1 inhibitors include retifanlimab, spartalizumab, karelizumab, tislerizumab, tripalimab, and dostarlimab. Exemplary PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab. Further examples of PD-L1 inhibitors include emfavorimab.

[0386] Exemplary platinum-based antineoplastic agents include cisplatin and carboplatin.

[0387] Examples of cyclin-dependent kinase (CDK) inhibitors include abemaciclib, palbociclib, and ribociclib.

[0388] Examples of poly(ADP-ribose) polymerase (PARP) inhibitors include talazoparib, olaparib, lucaparib, niraparib, and veliparib.

[0389] Examples of common chemotherapy agents, antineoplastic agents, and antiproliferative agents include altretamine (Hexalen), isotretinoin (Accutane, Amnesteem, Claravis, Soret), tretinoin (Vesanoid), azacitidine (Vidaza), bortezomib (Velcade), asparaginase (Elspar), rebamisole (Ergamidisol), mitotane (Lysodren), procarbazine (Matulane), pegaspar gauze (Oncaspar), and denileukin difutitex (O Examples include, but are not limited to, ntak, porfimer (Photofrin), aldesleukin (Proleukin), lenalidomide (Revlimid), bexarotene (Targretin), thalidomide (Thalomid), temsirolimus (Torisel), arsenic trioxide (Trisenox), verteporfin (Visudyne), mimosine (Leucenol), (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxonate) and lovastatin.

[0390] Small molecule inhibitors refer to drugs that, due to their small size, can be used to target both extracellular and intracellular proteins expressed by cancer cells. Small molecule inhibitors target serine / threonine / tyrosine kinases, matrix metalloproteinases (MMPs), heat shock proteins (HSPs), proteasomes, and other proteins that play a role in signaling pathways. Examples of small molecular weight inhibitors include atitinib, erlotinib, imatinib, gefitinib, sunitinib, lapatinib, nolitinib, cabozantinib, crizotinib, sorafenib, vemurafenib, trametinib, everolimus, temsirolimus, ruxolitinib, bortezomib, pazopanib, ruzolitinib, vandetanib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, perifosine, batimistat, neovastat, prinomast, levimast, ganetespib, marimast, ovatocrax, navitocrax, and carfilzomib.

[0391] In some embodiments, a combination therapy modality is provided in which the additional therapeutic agent is a cytokine, such as G-CSF (granulocyte colony-stimulating factor).

[0392] In some embodiments, the pharmaceutical compositions provided herein may be administered in combination with radiotherapy. Radiotherapy may also be administered as part of a multi-drug therapy in combination with the pharmaceutical compositions provided herein and other chemotherapeutic agents described herein. In yet another embodiment, the pharmaceutical compositions provided herein may include CMF (cyclophosphamide, methotrexate and 5-fluorouracil), CAF (cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide and paclitaxel), rituximab, Xeloda (capecitabine), cisplatin (CDDP), carboplatin, TS-1 (tegafur, gimesus in a molar ratio of 1:0.4:1). These may be administered in combination with standard chemotherapy regimens, including but not limited to, Tatt and Otastat potassium, Camptothecin-11 (CPT-11, irinotecan, or Camptosar®), CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisone or prednisolone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, Oncovin, prednisone, or prednisolone), or CMMFP (cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone).

[0393] In some preferred embodiments, the pharmaceutical compositions provided herein may be administered together with inhibitors of enzymes such as receptor or non-receptor kinases. Receptor and non-receptor kinases are, for example, tyrosine kinases or serine / threonine kinases. The kinase inhibitors described herein are small molecules, polynucleic acids, polypeptides, or antibodies.

[0394] Examples of kinase inhibitors include bevacizumab (targets VEGF), BIBW2992 (targets EGFR and Erb2), cetuximab / Erbitux (targets Erb1), imatinib / Gleevec (targets Bcr-Abl), trastuzumab (targets Erb2), gefitinib / Iressa (targets EGFR), ranibizumab (targets VEGF), pegaptanib (targets VEGF), erlotinib / Tarceva (targets Erb1), and nilotinib (targets Bcr-Abl). ), lapatinib (targets Erb1 and Erb2 / Her2), GW-572016 / lapatinib ditosylate (targets HER2 / Erb2), panitumumab / Vectibix (targets EGFR), vandetinib (targets RET / VEGFR), E7080 (targets multiple receptors including RET and VEGFR), Herceptin (targets HER2 / Erb2), PKI-166 (targets EGFR), canertinib / CI-1033 (targets EGFR), sunitinib / SU-11464 / S utent (targets EGFR and FLT3), matsuzumab / Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3), batalanib / Ptk787 / ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3) (Targeted as), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targets JAK), sorafenib / Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3, and RET), dasatinib / Sprycel (targets BCR / ABL and Src),Examples include, but are not limited to, AC-220 (targeting Flt3), AC-480 (targeting all HER proteins, including "panHER"), motesanib diphosphate (targeting VEGF1-3, PDGFRβ, and c-kit), denosumab (targeting RANKL and inhibiting SRC), AMG888 (targeting HER3), and AP24534 (targeting multiple proteins, including Flt3).

[0395] In some embodiments, an antibody-drug conjugate or a pharmaceutical composition containing the same is administered in combination with adoptive cell therapy. In some embodiments, the adoptive cell therapy is chimeric antigen receptor T cell (CAR T) or CAR NK cell therapy.

[0396] Medication and administration In some embodiments, the method involves administering a double paratopic 5T4 antibody-drug conjugate. In some embodiments, the anti-5T4 antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 2 months, every 3 months, or every 4 months.

[0397] In some embodiments, the anti-5T4 antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject daily, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, every ten days, every two weeks, every three weeks, or monthly.

[0398] In some embodiments, the anti-5T4 antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject once daily. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject every two days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every three days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every four days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every five days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every six days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every seven days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every eight days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every nine days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every ten days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every 11 days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every 12 days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every 13 days. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every 2 weeks. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every 3 weeks. In some embodiments, the antibody-drug conjugate or a composition containing the same is administered to the subject every month. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject at least twice a year. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject at least twice every 2 years. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject at least twice every 2 years or more.

[0399] In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject once every 7 to 14 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject once every 10 to 20 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject once every 5 to 15 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject once every 15 to 30 days.

[0400] In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 36 hours. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 48 hours. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 60 hours. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 72 hours. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 84 hours. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 96 hours. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 5 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 6 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 7 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 8 to 10 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 10 to 12 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 12 to 15 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 15 to 25 days. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every 20 to 30 days.

[0401] In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered to the subject at least once every month, at least once every two months, at least once every three months, at least once every four months, or at least once every six months. In one embodiment, the dose of the antibody-drug conjugate or a pharmaceutical composition containing the same is administered at least once every six to twelve months. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same is administered quarterly. In some embodiments, the antibody-drug conjugate or a pharmaceutical composit...

Claims

1. Antibody-drug conjugate, a. A first antigen-binding domain that specifically binds to the first 5T4 epitope, b. A second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and c. Contains chemotherapeutic agents, An antibody-drug conjugate in which the first antigen-binding domain is operably linked to the second antigen-binding domain.

2. The antibody-drug conjugate according to claim 1, wherein the first and second antigen-binding domains are independently selected from the group consisting of a Fab fragment, an F(ab')2 fragment, scFv, scab, dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody.

3. The antibody-drug conjugate according to claim 1, comprising a full-length IgG antibody containing the first antigen-binding domain.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the second antigen-binding domain comprises scFv.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein the full-length IgG antigen-binding domain comprises two heavy chains and two light chains.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the second antigen-binding domain comprises an scFv, and the antibody-drug conjugate comprises two second antigen-binding domains scFv, both of which specifically bind to the second 5T4 epitope.

7. The scFv includes a heavy chain and a light chain, (a) The C-terminus of the light chain is operably connected to the N-terminus of the heavy chain via a linker, or (b) The antibody-drug conjugate according to any one of claims 2 to 6, wherein the C-terminus of the heavy chain is operably linked to the N-terminus of the light chain via a linker.

8. The antibody-drug conjugate according to claim 7, wherein the linker comprises the sequence of Sequence ID No.

153.

9. The antibody-drug conjugate according to any one of claims 6 to 8, wherein the N-terminus of the second antigen-binding domain is operably ligated to the C-terminus of the heavy chain of the full-length IgG antibody comprising the first antigen-binding domain.

10. The antibody-drug conjugate according to any one of claims 6 to 8, wherein the C-terminus of the second antigen-binding domain is operably ligated to the N-terminus of the heavy chain of the full-length IgG antibody.

11. The antibody-drug conjugate according to claim 9 or 10, wherein the second antigen-binding domain is operably linked to the heavy chain of the full-length IgG antibody using a linker.

12. The antibody-drug conjugate according to any one of claims 9 to 11, wherein the linker comprises or consists of the amino acid sequence of SEQ ID NO:

152.

13. The antibody-drug conjugate according to any one of claims 5 to 12, wherein the full-length IgG antibody heavy chain comprises a heavy chain variable region domain and a heavy chain constant region domain.

14. The antibody-drug conjugate according to claim 13, wherein the heavy chain constant region domain is an IgG1 isotype constant region domain.

15. The antibody-drug conjugate according to any one of claims 5 to 14, wherein the full-length IgG antibody light chain comprises a light chain variable region domain and a light chain constant region domain.

16. The antibody-drug conjugate according to claim 15, wherein the light chain constant region domain is an IgG1 isotype constant region domain.

17. The antibody-drug conjugate according to any one of claims 13 to 16, wherein the heavy chain constant region domain includes at least one mutation that reduces effector function, extends half-life, or a combination thereof.

18. The antibody-drug conjugate according to claim 17, wherein the at least one mutation includes F at position 237 of SEQ ID NO: 100 (L234F), C or A at position 242 of SEQ ID NO: 100 (S239C / A), A at position 437 of SEQ ID NO: 100 (N434A), or a combination thereof.

19. The antibody-drug conjugate according to claim 17, wherein the at least one mutation comprises F at position 237 of SEQ ID NO: 100 (L234F), C or A at position 242 of SEQ ID NO: 100 (S239C / A), and A at position 437 of SEQ ID NO: 100 (N434A).

20. The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, the second antigen-binding domain containing an scFv, and the antibody-drug conjugate (a) Two polypeptides comprising the full-length IgG antibody heavy chain, a linker, and the second antigen-binding domain from the N-terminus to the C-terminus, (b) The antibody-drug conjugate according to any one of claims 1 to 19, comprising two polypeptides containing the full-length IgG antibody light chain and four polypeptides containing the full-length IgG antibody light chain.

21. The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, the second antigen-binding domain containing an scFv, and the antibody-drug conjugate (a) Two polypeptides comprising the second antigen-binding domain, a linker, and the full-length IgG antibody from the N-terminus to the C-terminus, (b) The antibody-drug conjugate according to any one of claims 1 to 19, comprising two polypeptides containing the full-length IgG antibody light chain and four polypeptides containing the full-length IgG antibody light chain.

22. The antibody-drug conjugate according to claim 20 or 21, wherein the linker comprises the amino acid sequence of SEQ ID NO:

152.

23. Both the first and second antigen-binding domains described above are a. Heavy chain (HC) complementarity determination region (CDR1) sequences selected from the group consisting of sequence numbers 1, 4, and 13-23, b. HC CDR2 sequences selected from the group consisting of sequence numbers 2, 5, and 24-26 and 28-39, and c. Containing an HC CDR3 sequence selected from the group consisting of sequence numbers 3, 6, and 40-52, The antibody-drug conjugate according to any one of claims 1 to 22, wherein one or more of the CDR1, CDR2, and CDR3 sequences are not the same between the first and second antigen-binding domains.

24. Both the first and second antigen-binding domains described above are a. A light chain (LC) complementarity determination region (CDR1) sequence selected from the group consisting of sequence numbers 7, 10, and 53-66. b. LC CDR2 sequences selected from the group consisting of sequence numbers 8, 11, and 67-75, c. Containing an LC CDR3 sequence selected from the group consisting of sequence numbers 9, 12, and 76-83, The antibody-drug conjugate according to any one of claims 1 to 23, wherein one or more of the CDR1, CDR2, and CDR3 sequences are not the same between the first and second antigen-binding domains.

25. The first antigen-binding domain described above is a. An HC CDR1 sequence containing the amino acid sequence of Sequence ID No. 1, or a sequence having one, two, or three substitutions, insertions, or deletions thereto. b. An HC CDR2 sequence containing the amino acid sequence of Sequence ID No. 2, or a sequence having one, two, or three substitutions, insertions, or deletions thereto. c. A heavy chain variable region domain comprising the HC CDR3 sequence, which includes the amino acid sequence of SEQ ID NO: 3, or a sequence having one, two, or three substitutions, insertions, or deletions thereto, The first antigen-binding domain described above is a. An LC CDR1 sequence containing the amino acid sequence of Sequence ID No. 7, or a sequence having one, two, or three substitutions, insertions, or deletions thereto. b. An LC CDR2 sequence containing the amino acid sequence of Sequence ID No. 8, or a sequence having one, two, or three substitutions, insertions, or deletions thereto, and c. The antibody-drug conjugate according to any one of claims 1 to 24, comprising a light chain variable region domain including an LC CDR3 sequence having the amino acid sequence of SEQ ID NO: 9, or a sequence having one, two, or three substitutions, insertions, or deletions thereto.

26. The second antigen-binding domain described above is a. An HC CDR1 sequence containing the amino acid sequence of Sequence ID No. 4, or a sequence having one, two, or three substitutions, insertions, or deletions thereto. b. An HC CDR2 sequence containing the amino acid sequence of Sequence ID No. 5, or a sequence having one, two, or three substitutions, insertions, or deletions thereto. c. A heavy chain variable region domain comprising the HC CDR3 sequence, which includes the amino acid sequence of SEQ ID NO: 6, or a sequence having one, two, or three substitutions, insertions, or deletions thereto, The second antigen-binding domain described above is a. An LC CDR1 sequence containing the amino acid sequence of Sequence ID No. 10, or a sequence having one, two, or three substitutions, insertions, or deletions thereto. b. An LC CDR2 sequence containing the amino acid sequence of Sequence ID No. 11, or a sequence having one, two, or three substitutions, insertions, or deletions thereto, and c. The antibody-drug conjugate according to any one of claims 1 to 25, comprising a light chain variable region domain including an LC CDR3 sequence having the amino acid sequence of SEQ ID NO: 12, or a sequence having one, two, or three substitutions, insertions, or deletions thereto.

27. The first and / or second antigen-binding domain includes a heavy chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 168, 170, and 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them, The antibody-drug conjugate according to any one of claims 1 to 26, wherein the first and / or second antigen-binding domain includes a light chain variable region domain containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171, and 173, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with them.

28. The antibody-drug conjugate according to any one of claims 1 to 27, wherein the first and / or second antigen-binding domain comprises a heavy chain variable region domain containing the amino acid sequence of SEQ ID NO: 96 or 97 and a light chain variable region domain containing the amino acid sequence of SEQ ID NO: 98 or 99.

29. The first antigen-binding domain described above is a. A heavy chain variable region domain comprising the amino acid sequence of Sequence ID No. 96, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, b. A light chain variable region domain comprising the amino acid sequence of Sequence ID No. 98, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, The second antigen-binding domain described above is a. A heavy chain variable region domain comprising the amino acid sequence of Sequence ID No. 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, b. An antibody-drug conjugate according to any one of claims 1 to 28, comprising a light chain variable region domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

30. The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, An antibody-drug conjugate according to any one of claims 1 to 29, comprising two polypeptides: a first antigen-binding domain heavy chain variable region domain containing the sequence of SEQ ID NO: 96, a first linker containing the sequence of SEQ ID NO: 152, a second antigen-binding domain heavy chain variable region domain containing the sequence of SEQ ID NO: 97, a second linker containing the sequence of SEQ ID NO: 153, and a second antigen-binding domain light chain variable region containing the sequence of SEQ ID NO:

99.

31. The antibody-drug conjugate according to claim 30, comprising two polypeptides, each comprising a second antigen-binding domain light chain variable region domain containing the sequence of SEQ ID NO:

98.

32. The antibody-drug conjugate according to claim 31, wherein the full-length IgG antibody comprises an IgG1 isotype constant region domain.

33. The antibody-drug conjugate according to any one of claims 1 to 32, wherein the second antigen-binding domain is an scFv comprising a heavy chain domain containing the sequence of SEQ ID NO: 97, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a light chain variable region domain containing the sequence of SEQ ID NO: 99, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

34. The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, the second antigen-binding domain containing an scFv, and the antibody-drug conjugate (a) From the N-terminus to the C-terminus, Two polypeptides comprising: a first antigen-binding domain heavy chain variable region domain containing the sequence of SEQ ID NO: 96; an IgG1 isotype constant region domain containing the sequence of SEQ ID NO: 148; a first linker containing the sequence of SEQ ID NO: 152; a second antigen-binding domain heavy chain variable region domain containing the sequence of SEQ ID NO: 97; a second linker containing the sequence of SEQ ID NO: 153; and a second antigen-binding domain light chain variable region containing the sequence of SEQ ID NO:

99. (b) A first antigen-binding domain variable light chain domain containing the sequence of Sequence ID No. 98 from the N-terminus to the C-terminus, and An antibody-drug conjugate according to any one of claims 1 to 33, comprising four polypeptides: two polypeptides each comprising a light chain constant region domain including the sequence of sequence number 149.

35. The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, the second antigen-binding domain containing an scFv, and the antibody-drug conjugate (a) Two polypeptides comprising the sequence of Sequence ID No. 100, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, (b) The antibody-drug conjugate according to any one of claims 1 to 34, comprising four polypeptides, each comprising two polypeptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto with the sequence of SEQ ID NO:

101.

36. The antibody-drug conjugate according to any one of claims 1 to 35, wherein the chemotherapeutic agent is conjugated via a linker to at least one full-length IgG antibody containing the first antigen-binding domain or the second antigen-binding domain.

37. The antibody-drug conjugate according to any one of claims 1 to 36, wherein the chemotherapeutic agent is auristatin.

38. The antibody-drug conjugate according to claim 37, wherein the auristatin is selected from the group consisting of auristatin E (AE), monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and synthetic analogs of drastatin.

39. The antibody-drug conjugate according to any one of claims 36 to 38, wherein the linker is a cleavable linker.

40. The antibody-drug conjugate according to any one of claims 36 to 38, wherein the linker is an inseparable linker.

41. The antibody-drug conjugate according to any one of claims 1 to 40, wherein the antibody-drug conjugate is tetravalent in order to bind to the 5T4 antigen.

42. The equilibrium dissociation constant (KD) for 5T4 binding of at least one of the first antigen-binding domains or the second antigen-binding domain is 3.63 × 10⁻¹⁴. －１２ ~7.75 x 10 －１０ The antibody-drug conjugate according to any one of claims 1 to 41, wherein M.

43. KD is 7.75 x 10 －１０ The antibody-drug conjugate according to claim 42, wherein the m is less than or equal to M.

44. The equilibrium dissociation constant (KD) for 5T4 binding of the first antigen-binding domain is 3.63 × 10⁻⁶. －１２ ~1.43 x 10 －９ The antibody-drug conjugate according to any one of claims 1 to 43, wherein M.

45. The equilibrium dissociation constant (KD) for 5T4 binding of the second antigen-binding domain is 3.63 × 10⁻⁶. －１２ ~1.34 x 10 －９ The antibody-drug conjugate according to any one of claims 1 to 44, wherein M.

46. The equilibrium dissociation constant (KD) for the binding of both the first antigen-binding domain or the second antigen-binding domain to 5T4 is 3.63×10 －１２ to 7.75×10 －１０ M, and the antibody-drug conjugate according to any one of claims 1 to 45.

47. The equilibrium dissociation constant (KD) of Fc gamma receptor IIa for binding to an IgG antibody containing the first and / or second antigen-binding domain is 3.74 × 10⁻¹⁴. －６ An antibody-drug conjugate according to any one of claims 1 to 46, wherein the m is less than or equal to M.

48. The equilibrium dissociation constant (KD) of Fc gamma receptor IIb for binding to an IgG antibody containing the first and / or second antigen-binding domain is 1.16 × 10⁻¹⁶. －７ An antibody-drug conjugate according to any one of claims 1 to 47, wherein the m is less than or equal to M.

49. The equilibrium dissociation constant (KD) of Fc gamma receptor IIIa for binding to an IgG antibody containing the first and / or second antigen-binding domain is 5.05 × 10⁻⁶. ８ An antibody-drug conjugate according to any one of claims 1 to 48, wherein the m is less than or equal to M.

50. A nucleic acid system encoding a bispecific antibody according to any one of claims 1 to 49.

51. One or more vectors comprising the nucleic acid system described in claim 50.

52. A cell comprising the nucleic acid system described in claim 50 or the vector described in claim 51.

53. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent, or excipient.

54. A method for producing the aforementioned antibody-drug conjugate, a. Contacting multiple cells with the nucleic acid system described in claim 50 or the vector described in claim 51, b. Culturing the plurality of cells under conditions in which the bispecific antibody is expressed by at least one of the plurality of cells, c. Purifying the bispecific antibody, d. A method comprising conjugating the bispecific antibody with a chemotherapeutic agent.

55. An antibody-drug conjugate according to any one of claims 1 to 49 or a pharmaceutical composition according to claim 52, for use in a method of treating cancer in a subject.

56. An antibody-drug conjugate according to any one of claims 1 to 49, for use in the manufacture of a pharmaceutical product for treating cancer in a subject.

57. A method for treating cancer in a subject, comprising administering a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 49.

58. A method for treating cancer in a subject requiring cancer treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to the subject requiring such treatment, wherein the antibody-drug conjugate is a. A first antigen-binding domain that specifically binds to the first 5T4 epitope, b. A second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and c. Contains chemotherapeutic agents, A method wherein the first antigen-binding domain is operably linked to the second antigen-binding domain.

59. The antibody-drug conjugate comprises the amino acid sequence of SEQ ID NO: 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or two polypeptides containing such a sequence. The method according to claim 58, comprising two polypeptides having the amino acid sequence of Sequence ID No. 101, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

60. The method according to any one of claims 57 to 59, wherein the cancer cells express 5T4.

61. The method according to any one of claims 57 to 60, wherein the cancer is selected from the group consisting of lung cancer, stomach cancer, ovarian cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, and kidney cancer.

62. The method according to claim 61, wherein the lung cancer is non-small cell lung cancer.

63. The method according to any one of claims 57 to 62, wherein the first antibody and the second antibody bind to different 5T4 molecules on the surface of at least one cancer cell.

64. The method according to any one of claims 57 to 62, wherein the first antibody and the second antibody bind to the same 5T4 molecule on the surface of a cancer cell.

65. The method according to any one of claims 57 to 64, wherein the method reduces the signs or symptoms of the cancer.

66. The method according to any one of claims 57 to 65, wherein the method reduces tumor volume, the number of tumors, slows the tumor growth rate, or a combination thereof.

67. The method according to any one of claims 57 to 66, wherein the method increases the survival rate of the subject.

68. A method for producing a bispecific antibody-drug conjugate, (a) Culturing cells containing a nucleic acid system encoding the bispecific antibody under conditions that result in the expression of the bispecific antibody, wherein the bispecific antibody has the following structure: a. A first antigen-binding domain that specifically binds to the first 5T4 epitope, b. A second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and c. Contains chemotherapeutic agents, The first antigen-binding domain is operably linked to the second antigen-binding domain, and the culture is performed. (b) Recovering the bispecific antibody, (c) A method comprising conjugating the bispecific antibody to a chemotherapeutic agent.

69. A kit comprising an antibody-drug conjugate according to any one of claims 1 to 49, a nucleic acid system according to claim 50, a vector according to claim 51, cells according to claim 52, or a pharmaceutical composition according to claim 53.

70. An antigen-binding domain comprising a heavy chain variable region domain and a light chain variable region domain, The heavy chain variable region domain, a. A heavy chain (HC) complementarity determination region (CDR1) sequence selected from the group consisting of sequence numbers 1, 4, and 13-23, or a sequence having one, two, or three substitutions, insertions, or deletions thereof. b. HC CDR2 sequences selected from the group consisting of sequence numbers 2, 5, and 24-39, or sequences having one, two, or three substitutions, insertions, or deletions thereof, and c. An HC CDR3 sequence selected from the group consisting of sequence numbers 3, 6, and 40-52, or a sequence having one, two, or three substitutions, insertions, or deletions thereof, The light chain variable region domain, a. A light chain (LC) complementarity-determining region (CDR1) sequence selected from the group consisting of sequence numbers 7, 10, and 53-66, or a sequence having one, two, or three substitutions, insertions, or deletions thereof. b. LC CDR2 sequences selected from the group consisting of sequence numbers 8, 11, and 67-75, or sequences having one, two, or three substitutions, insertions, or deletions thereof, and c. An antigen-binding domain comprising an LC CDR3 sequence selected from the group consisting of Sequence IDs 9, 12, and 76-83, or a sequence having one, two, or three substitutions, insertions, or deletions thereof.

71. The heavy chain variable region domain, a. HC CDR1 containing SEQ ID NO: 1, HC CDR2 containing SEQ ID NO: 2, and HC CDR3 containing SEQ ID NO: 3 b. HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 5, and HC CDR3 containing SEQ ID NO: 6 c. HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 24, and HC CDR3 containing SEQ ID NO: 40 d. HC CDR1 containing SEQ ID NO: 13, HC CDR2 containing SEQ ID NO: 25, and HC CDR3 containing SEQ ID NO: 41 e. HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 26, and HC CDR3 containing SEQ ID NO: 42 f. HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 28, and HC CDR3 containing SEQ ID NO: 43 g. HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 29, and HC CDR3 containing SEQ ID NO: 44 h. HC CDR1 containing SEQ ID NO: 16, HC CDR2 containing SEQ ID NO: 30, and HC CDR3 containing SEQ ID NO: 45 i. HC CDR1 containing SEQ ID NO: 17, HC CDR2 containing SEQ ID NO: 31, and HC CDR3 containing SEQ ID NO: 46 j. HC CDR1 containing SEQ ID NO: 18, HC CDR2 containing SEQ ID NO: 32, and HC CDR3 containing SEQ ID NO: 47 k. HC CDR1 containing SEQ ID NO: 19, HC CDR2 containing SEQ ID NO: 33, and HC CDR3 containing SEQ ID NO: 48 l. HC CDR1 containing SEQ ID NO: 20, HC CDR2 containing SEQ ID NO: 34, and HC CDR3 containing SEQ ID NO: 49 m. HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 35, and HC CDR3 containing SEQ ID NO:

50. n. HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 36, and HC CDR3 containing SEQ ID NO:

51. o. HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 37, and HC CDR3 containing SEQ ID NO:

3. p. HC CDR1 containing SEQ ID NO: 21, HC CDR2 containing SEQ ID NO: 38, and HC CDR3 containing SEQ ID NO:

3. q. HC CDR1 containing SEQ ID NO: 22, HC CDR2 containing SEQ ID NO: 39, and HC CDR3 containing SEQ ID NO: 45, or r. The antigen-binding domain according to claim 70, comprising HC CDR1 containing SEQ ID NO: 23, HC CDR2 containing SEQ ID NO: 32, and HC CDR3 containing SEQ ID NO:

52.

72. The light chain variable region domain, a. LC CDR1 containing SEQ ID NO: 7, LC CDR2 containing SEQ ID NO: 8, and LC CDR3 containing SEQ ID NO: 9 b. LC CDR1 containing SEQ ID NO: 10, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 12 c. LC CDR1 containing SEQ ID NO: 53, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76 d. LC CDR1 containing SEQ ID NO: 54, LC CDR2 containing SEQ ID NO: 67, and LC CDR3 containing SEQ ID NO:

77. e. LC CDR1 containing SEQ ID NO: 55, LC CDR2 containing SEQ ID NO: 68, and LC CDR3 containing SEQ ID NO:

78. f. LC CDR1 containing SEQ ID NO: 56, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79 g. LC CDR1 containing SEQ ID NO: 57, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79 h. LC CDR1 containing SEQ ID NO: 58, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76 i. LC CDR1 containing SEQ ID NO: 59, LC CDR2 containing SEQ ID NO: 70, and LC CDR3 containing SEQ ID NO: 80 j. LC CDR1 containing SEQ ID NO: 60, LC CDR2 containing SEQ ID NO: 71, and LC CDR3 containing SEQ ID NO: 81, k. LC CDR1 containing SEQ ID NO: 61, LC CDR2 containing SEQ ID NO: 72, and LC CDR3 containing SEQ ID NO: 77 l. LC CDR1 containing SEQ ID NO: 62, LC CDR2 containing SEQ ID NO: 70, and LC CDR3 containing SEQ ID NO: 82 m. LC CDR1 containing SEQ ID NO: 10, LC CDR2 containing SEQ ID NO: 73, and LC CDR3 containing SEQ ID NO:

12. n. LC CDR1 containing SEQ ID NO: 63, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76 o. LC CDR1 containing SEQ ID NO: 64, LC CDR2 containing SEQ ID NO: 68, and LC CDR3 containing SEQ ID NO:

78. p. LC CDR1 containing SEQ ID NO: 65, LC CDR2 containing SEQ ID NO: 74, and LC CDR3 containing SEQ ID NO: 9 q. LC CDR1 containing SEQ ID NO: 66, LC CDR2 containing SEQ ID NO: 75, and LC CDR3 containing SEQ ID NO: 9 r. LC CDR1 containing SEQ ID NO: 58, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76, or s. The antigen-binding domain according to claim 70 or 71, comprising LC CDR1 containing SEQ ID NO: 60, LC CDR2 containing SEQ ID NO: 71, and LC CDR3 containing SEQ ID NO:

83.

73. a. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 1, HC CDR2 containing SEQ ID NO: 2, and HC CDR3 containing SEQ ID NO: 3, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 7, LC CDR2 containing SEQ ID NO: 8, and LC CDR3 containing SEQ ID NO: 9, b. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 5, and HC CDR3 containing SEQ ID NO: 6, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 10, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 12, c. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 24, and HC CDR3 containing SEQ ID NO: 40, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 53, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76, d. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 13, HC CDR2 containing SEQ ID NO: 25, and HC CDR3 containing SEQ ID NO: 41, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 54, LC CDR2 containing SEQ ID NO: 67, and LC CDR3 containing SEQ ID NO: 77, e. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 26, and HC CDR3 containing SEQ ID NO: 42, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 55, LC CDR2 containing SEQ ID NO: 68, and LC CDR3 containing SEQ ID NO: 78, f. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 28, and HC CDR3 containing SEQ ID NO: 43, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 56, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79, g. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 29, and HC CDR3 containing SEQ ID NO: 44, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 57, LC CDR2 containing SEQ ID NO: 69, and LC CDR3 containing SEQ ID NO: 79, h. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 16, HC CDR2 containing SEQ ID NO: 30, and HC CDR3 containing SEQ ID NO: 45, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 58, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76, i. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 17, HC CDR2 containing SEQ ID NO: 31, and HC CDR3 containing SEQ ID NO: 46, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 59, LC CDR2 containing SEQ ID NO: 70, and LC CDR3 containing SEQ ID NO: 80, j. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 18, HC CDR2 containing SEQ ID NO: 32, and HC CDR3 containing SEQ ID NO: 47, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 60, LC CDR2 containing SEQ ID NO: 71, and LC CDR3 containing SEQ ID NO: 81, k. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 19, HC CDR2 containing SEQ ID NO: 33, and HC CDR3 containing SEQ ID NO: 48, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 61, LC CDR2 containing SEQ ID NO: 72, and LC CDR3 containing SEQ ID NO: 77, l. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 20, HC CDR2 containing SEQ ID NO: 34, and HC CDR3 containing SEQ ID NO: 49, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 62, LC CDR2 containing SEQ ID NO: 70, and LC CDR3 containing SEQ ID NO: 82, m. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 35, and HC CDR3 containing SEQ ID NO: 50, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 10, LC CDR2 containing SEQ ID NO: 73, and LC CDR3 containing SEQ ID NO: 12, n. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 4, HC CDR2 containing SEQ ID NO: 5, and HC CDR3 containing SEQ ID NO: 6, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 63, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76, o. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 14, HC CDR2 containing SEQ ID NO: 36, and HC CDR3 containing SEQ ID NO: 51, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 64, LC CDR2 containing SEQ ID NO: 68, and LC CDR3 containing SEQ ID NO: 78, p. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 15, HC CDR2 containing SEQ ID NO: 37, and HC CDR3 containing SEQ ID NO: 3, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 65, LC CDR2 containing SEQ ID NO: 74, and LC CDR3 containing SEQ ID NO: 9, q. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 21, HC CDR2 containing SEQ ID NO: 38, and HC CDR3 containing SEQ ID NO: 3, and the light chain variable region domain includes LC CDR1 containing SEQ ID NO: 66, LC CDR2 containing SEQ ID NO: 75, and LC CDR3 containing SEQ ID NO: 9, r. The heavy chain variable region domain includes HC CDR1 containing SEQ ID NO: 22, HC CDR2 containing SEQ ID NO: 39, and HC CDR3 containing SEQ ID NO: 45, and the light chain variable region includes LC CDR1 containing SEQ ID NO: 58, LC CDR2 containing SEQ ID NO: 11, and LC CDR3 containing SEQ ID NO: 76, or s. The antigen-binding domain according to any one of claims 70 to 72, wherein the heavy chain variable region domain comprises HC CDR1 containing SEQ ID NO: 23, HC CDR2 containing SEQ ID NO: 32, and HC CDR3 containing SEQ ID NO: 52, and the light chain variable region domain comprises LC CDR1 containing SEQ ID NO: 60, LC CDR2 containing SEQ ID NO: 71, and LC CDR3 containing SEQ ID NO:

83.

74. The antigen-binding domain according to any one of claims 70 to 73, wherein the heavy chain variable region domain includes a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 156, 158, 160, 162, 164, 166, 168, 170, and 172, or a sequence having at least 80%, at least 90%, or at least 95% identity with them.

75. The antigen-binding domain according to any one of claims 70 to 74, wherein the light chain variable region domain includes a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 103, 105, 107, 109, 113, 115, 117, 119, 121, 122, 125, 127, 129, 131, 133, 135, 137, 139, 141, 157, 159, 161, 163, 165, 167, 169, 171 and 173, or a sequence having at least 80%, at least 90%, or at least 95% identity with them.

76. a. The heavy chain variable region domain includes the amino acid sequence of SEQ ID NO: 96, and the light chain variable region domain includes the amino acid sequence of SEQ ID NO:

98. b. The heavy chain variable region domain includes the amino acid sequence of SEQ ID NO: 97, and the light chain variable region domain includes the amino acid sequence of SEQ ID NO:

99. c. The heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 102, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

103. d. The heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 104, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

105. e. The heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 106, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

107. f. The heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 108, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

109. g. The heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 112, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

113. h. The heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 114, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

115. i. The heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 116, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

117. j. The heavy chain variable region domain contains the amino acid sequence of SEQ ID NO: 118, and the light chain variable region domain contains the amino acid sequence of SEQ ID NO: 119, or k. The antigen-binding domain according to any one of claims 70 to 75, wherein the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 120, and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

121.

77. The antigen-binding domain is the Fab fragment, F(ab') ２ An antigen-binding domain according to any one of claims 70 to 76, comprising a fragment, scFv, scab, dAb, a single-domain heavy chain antibody, or a single-domain light chain antibody.

78. An antibody comprising an antigen-binding domain as described in any one of claims 70 to 76.

79. A chimeric antigen receptor (CAR) comprising the antigen-binding domain described in any one of claims 70 to 76.

80. An immune cell comprising the CAR described in claim 79.

81. The immune cell according to claim 80, wherein the immune cell is a T cell or a natural killer (NK) cell.

82. Antibody-drug conjugate, (a) A first antigen-binding domain that specifically binds to the first 5T4 epitope, (b) A second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and (c) Chemotherapy agents, The first antigen-binding domain is operably linked to the second antigen-binding domain, The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, and the second antigen-binding domain comprises an scFv. The antibody-drug conjugate is (a) i. HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, ii. The HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and iii. Two polypeptides comprising a heavy chain variable region domain comprising the HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, Two polypeptides comprising the amino acid sequence of Sequence ID No. 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, (b) i. LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, ii. The LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, and iii. Two polypeptides comprising a light chain variable region domain containing the LC CDR3 sequence containing the amino acid sequence of Sequence ID No. 9, An antibody-drug conjugate comprising two polypeptides, each containing the amino acid sequence of Sequence ID No. 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

83. Antibody-drug conjugate, (a) A first antigen-binding domain that specifically binds to the first 5T4 epitope, (b) A second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and (c) Chemotherapy agents, The first antigen-binding domain is operably linked to the second antigen-binding domain, The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, and the second antigen-binding domain comprises an scFv. The antibody-drug conjugate is (a) i. HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 4, ii. The HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 5, and iii. Two polypeptides comprising a heavy chain variable region domain comprising the HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 6, Two polypeptides comprising the amino acid sequence of Sequence ID No. 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, (b) i. LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 10, ii. The LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 11, and iii. Two polypeptides comprising a light chain variable region domain containing the LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 12, An antibody-drug conjugate comprising two polypeptides, each containing the amino acid sequence of Sequence ID No. 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

84. Antibody-drug conjugate, (a) A first antigen-binding domain that specifically binds to the first 5T4 epitope, (b) A second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and (c) Chemotherapy agents, The first antigen-binding domain is operably linked to the second antibody, The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, and the second antigen-binding domain comprises an scFv. The antibody-drug conjugate is (a) i. HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, ii. The HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and iii. A first heavy chain variable region domain containing the HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, i. HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 4, ii. The HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 5, and iii. A second heavy chain variable region domain containing the HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 6, i. LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, ii. The LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, and iii. Two polypeptides comprising a light chain variable region domain containing the LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 9, Two polypeptides comprising the amino acid sequence of Sequence ID No. 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, (b) i. LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 10, ii. The LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 11, and iii. Two polypeptides comprising a light chain variable region domain containing the LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 12, An antibody-drug conjugate comprising two polypeptides, each containing the amino acid sequence of Sequence ID No. 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

85. Antibody-drug conjugate, (a) A first antigen-binding domain that specifically binds to the first 5T4 epitope, (b) A second antigen-binding domain that specifically binds to a second 5T4 epitope that is not the same as the first 5T4 epitope, and (c) Chemotherapy agents, The first antigen-binding domain is operably linked to the second antigen-binding domain, The antibody-drug conjugate comprises a full-length IgG antibody containing the first antigen-binding domain, and the second antigen-binding domain comprises an scFv. The antibody-drug conjugate is (a) i. HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 1, ii. The HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 2, and iii. A first heavy chain variable region domain containing the HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 3, i. HC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 4, ii. The HC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 5, and iii. A second heavy chain variable region domain containing the HC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 6, i. LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 10, ii. The LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 11, and iii. Two polypeptides comprising a light chain variable region domain containing the LC CDR3 sequence containing the amino acid sequence of SEQ ID NO: 12, Two polypeptides comprising the amino acid sequence of Sequence ID No. 100, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, (b) i. LC CDR1 sequence containing the amino acid sequence of SEQ ID NO: 7, ii. The LC CDR2 sequence containing the amino acid sequence of SEQ ID NO: 8, and iii. Two polypeptides comprising a light chain variable region domain containing the LC CDR3 sequence containing the amino acid sequence of Sequence ID No. 9, An antibody-drug conjugate comprising two polypeptides, each containing the amino acid sequence of Sequence ID No. 101, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

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