Claudin 6 bispecific antibody

Abstract

To provide bispecific antigen-binding proteins which bind to Claudin-6 (CLDN6) and a second antigen.SOLUTION: In various aspects, antigen-binding proteins bind to Extracellular Loop 2 (EL2) of the extracellular domain of CLDN6. Related polypeptides, nucleic acids, vectors, host cells, and conjugates are further provided herein. Kits and pharmaceutical compositions comprising such entities are moreover provided. Also provided are methods of making an antigen-binding protein and methods of treating a subject having cancer.SELECTED DRAWING: Figure 61

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 821,399, filed on March 20, 2019, and the entire contents of the said application are incorporated in their entirety by this reference.

[0002] Integration by referencing electronically submitted documents The entirety of which is incorporated by reference is a computer-readable nucleotide / amino acid sequence listing, identified as a 344,064 ASCII (text) file named "54086P2_Seqlisting.txt" created on March 20, 2019, which was submitted simultaneously with the application. [Background technology]

[0003] Antibodies constitute potent therapeutic agents characterized by limited side effects due to their ability to specifically target different antigens on cells, bacteria, viruses, or toxins. In 1986, the first therapeutic monoclonal antibody, Orthoclone OKT3, was introduced to the market. Since then, this class of biopharmaceuticals has grown significantly. In late 2014, 47 monoclonal antibody products were approved in the United States or Europe for the treatment of cancer, as well as a variety of other diseases, including inflammatory diseases, cardiovascular diseases, respiratory diseases, and infections.

[0004] More than 12 monoclonal antibodies are currently approved by the U.S. Food and Drug Administration (FDA) for the treatment of cancer. These include alemtuzumab (Campath®), indicated for chronic lymphocytic leukemia (CLL), and trastuzumab (Herceptin®), used to treat breast cancer. Some antibodies are labeled with chemotherapy drugs, such as brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®). Other antibody products, such as blinatumomab (Blincyto), are designed to recognize and bind to two different antigens. Despite the availability of such antibody products, current cancer incidence and mortality rates remain high. Cancer incidence is reported to be over 450 per 100,000 person-years for both sexes, and cancer mortality is over 170 per 100,000 person-years for both sexes. [Overview of the Initiative]

[0005] This specification provides antigen-binding proteins that bind to claudin 6 (CLDN6) and bispecific forms thereof. In various embodiments, the antigen-binding proteins of this disclosure bind to human CLDN6 and, optionally, to mouse CLDN6. In various embodiments, the antigen-binding proteins bind to the extracellular domain (ECD) of CLDN6. In various embodiments, the antigen-binding proteins bind to the extracellular loop 2 (EL2) of the ECD of CLDN6. In various embodiments, the antigen-binding proteins bind to EL2 and not to the extracellular loop 1 (EL1) of the ECD of CLDN6. In various embodiments, the antigen-binding proteins bind to additional members of the human claudin family, such as claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9), etc. In various embodiments, the antigen-binding proteins bind to CLDN6 and at least one of CLDN4 and CLDN9. In various embodiments, the antigen-binding proteins bind to CLDN6 and not to any other members of the claudin family. In various embodiments, the antigen-binding protein binds to CLDN6 endogenously expressed by human ovarian cancer cells, e.g., OVCA429 cells, exhibiting an IC50 of less than approximately 1200 nM in FACS affinity assays using OVCA429 cells. In various cases, the antigen-binding protein of this disclosure inhibits tumor growth in subjects, e.g., humans, without including any other portion bound to this antigen-binding protein.

[0006] This specification further provides antigen-binding proteins conjugated to heterologous moieties (e.g., any chemotherapeutic agent, drug, or toxic moiety) that inhibit tumor growth in a target, such as in humans. In various cases, the conjugated antigen-binding protein is a monoclonal antibody. In various cases, the antibody is conjugated to a drug that alters microtubule dynamics, such as MMAE. In various cases, the complex includes a cleavable linker, such as MC-VC-PAB. In various embodiments, the complex is a homogeneous or heterogeneous complex. In various embodiments, the heterologous moiety is conjugated at a specific site on the antigen-binding protein. In various cases, the conjugated antigen-binding protein is a bispecific antigen-binding protein.

[0007] In various embodiments, bispecific antigen-binding proteins bind to CLDN6 expressed by human cancer cells. In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and an anti-CLDN6 reference antibody. Without being bound by any particular theory, the inhibitory effect of the antigen-binding proteins provided herein makes such entities useful in methods of suppressing tumor growth and treating tumors or cancerous subjects. As will be further discussed herein, in various embodiments, the antigen-binding protein is an antibody, its antigen-binding antibody fragment, or an antibody protein product.

[0008] This disclosure also provides bispecific antigen-binding proteins comprising at least three, four, five, or all of the amino acid sequences from a specific group of amino acid sequences. In various embodiments, the antigen-binding protein comprises at least three, four, five, or six complementarity-determining region (CDR) amino acid sequences of the CLDN6 antibody disclosed herein.

[0009] Related polypeptides, nucleic acids, vectors, host cells, and complexes are further provided herein. Kits and pharmaceutical compositions containing such entities are further intended.

[0010] Methods for producing bispecific antigen-binding proteins are also provided. In various embodiments, the method comprises culturing host cells containing nucleic acids encoding such bispecific antigen-binding proteins or polypeptides to express such bispecific antigen-binding proteins or polypeptides as described herein.

[0011] Methods for treating subjects having cancer are further provided herein. In various embodiments, the method comprises administering a pharmaceutical composition of the present disclosure to a subject in an amount effective to treat cancer in the subject.

[0012] Furthermore, methods for treating subjects having CLDN6-expressing cancer are also provided, including administering the pharmaceutical compositions described herein to the subject. In addition, methods for inhibiting tumor growth in a subject are intended, including administering the pharmaceutical compositions described herein to the subject.

[0013] A method for reducing tumor size in a subject or preventing cancer recurrence in a subject, comprising administering a pharmaceutical composition described herein to the subject.

[0014] Furthermore, this specification also provides a method for treating cancer in subjects diagnosed with low overexpression of CLDN6, which includes administering the pharmaceutical compositions described herein to the subjects.

[0015] In various embodiments, administration induces apoptosis in tumor cells, such as CLDN6-expressing cells. In various embodiments, administration induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), tumor necrosis and cell death or removal, and / or disruption of tumor cell adhesion, each of which results in tumor regression or slowing of tumor growth. [Brief explanation of the drawing]

[0016] [Figure 1] This graph shows CLDN6 expression in normal (non-cancerous) tissue. [Figure 2] This graph shows CLDN6 expression in cancer cell lines determined by the Agilent44K method. [Figure 3] This graph shows the expression of CLDN6 in cancer cell lines determined by RNASeq. [Figure 4] This image shows a series of fluorescence images illustrating the localization of CLDN6-GFP in different cell models. [Figure 5] This shows the sequence alignments for human CLDN6, human CLDN3, human CLDN4, human CLDN9, and mouse CLDN6. The sequences for EL1 and EL2 are shown. [Figure 6] A is a graph showing the tumor volume (mm3) of endometrial tumor-bearing mice as time (days) after treatment with the control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, AB2, and AB3. B is a graph showing the mean change in tumor volume (mm3) at day 14 of endometrial tumor-bearing mice treated with the control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, AB2, or AB3. [Figure 7] A is a graph showing the tumor volume (mm3) of bladder tumor-bearing mice as time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, and AB3. B is a graph showing the mean change in tumor volume (mm3) at day 35 of bladder tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, reference Ab2, or AB3. [Figure 8] A is a graph showing the tumor volume (mm3) of ovarian tumor-bearing mice as time (days) after treatment with the control IgG2 antibody, AB3, reference Ab1, AB2, and AB3. B is a graph showing the mean change in tumor volume (mm3) at day 20 of ovarian tumor-bearing mice treated with the control IgG2 antibody, AB3, reference Ab1, AB2, or AB3. [Figure 9]A is a graph showing the tumor volume (mm3) of melanoma-bearing mice as time (days) after treatment with the control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, and AB3. B is a graph showing the mean change in tumor volume (mm3) at day 21 of melanoma-bearing mice treated with the control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, or AB3. [Figure 10] A is a graph showing the percentage of tumor growth suppression achieved in tumor-bearing mice treated with AB3 compared to mice treated with a control antibody. B is a Western blot image showing different levels of CLDN6 id in endometrial cancer cell lines (ARK2), bladder cancer cell lines (UMUC4), ovarian cancer cell lines (OV90), melanoma cell lines (M202), and control cells. The levels of α-tubulin were almost the same, indicating that the loaded proteins were equivalent. [Figure 11] This graph shows the percentage change in body weight over time (days) in tumor-bearing mice treated with vehicle control, control antibody, reference Ab1, reference Ab2, reference Ab3, and AB3. [Figure 12A] This graph shows the tumor volume (mm3) of ovarian tumor-bearing mice as time (days) after treatment with the vehicle control, control IgG2 antibody, AB3, reference Ab1, or one of the indicated anti-CLDN6 antibodies. [Figure 12B] This graph shows the mean change in tumor volume (mm3) at day 28 of ovarian tumor-bearing mice treated with a vehicle control, a control IgG2 antibody, AB3, reference Ab1, or one of the indicated anti-CLDN6 antibodies. [Figure 13] This graph shows the percentage change in body weight over time (days) in tumor-bearing mice treated with a vehicle control, control antibody, reference Ab1, and the indicated anti-CLDN6 antibody. [Figure 14] This shows a series of dose-response curves for several anti-CLDN6 antibodies of the present invention, as well as reference Ab1 and reference 2. Mouse IgG was used as a control. [Figure 15]Figure A shows the mean change in tumor volume (mm3) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse type AB3, first humanized form of AB3, and second humanized form of AB3. Figure B shows the change in tumor volume (mm3) for each group in Figure 15A. [Figure 16] Figure A shows the mean change in tumor volume (mm3) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse type AB3, first humanized form of AB3, and second humanized form of AB3. Two control antibodies (one mouse and one chimeric) are also tested in this experiment. Figure B shows the change in tumor volume (mm3) for each group in Figure 16A. [Figure 17] Figure A shows the mean change in tumor volume (mm3) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse type AB1, and humanized type AB1. Figure B shows the change in tumor volume (mm3) for each group in Figure 17A. [Figure 18] Figure A shows the mean change in tumor volume (mm3) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse type AB4, and humanized type AB4. Figure B shows the change in tumor volume (mm3) for each group in Figure 18A. [Figure 19] Figure A shows the mean change in tumor volume (mm3) at day 35 in bladder tumor-bearing mice treated with vehicle control, control IgG antibody, mouse type AB3, chimeric type AB3, first humanized type AB3, second humanized type AB3, mouse type Ab1, humanized type AB1, mouse type AB4, and humanized type AB4. Four control antibodies (one mouse type or chimeric type, and one mouse type or human type) are also tested in this experiment. Figure B shows the graph of the change in tumor volume (mm3) for each group in Figure 19A. [Figure 20] Figure 19A shows a graph of the mean change in tumor volume (mm³) at day 55 in bladder tumor-bearing mice treated as described above. [Figure 21] Figure 19A shows a graph of the percentage change in body weight of treated tumor-bearing mice on day 32 after treatment. [Figure 22] This is a list of the heavy chain and light chain variable region sequences of 12 CLDN6 antibodies (named S1-S12) that were prepared and characterized. S1-S6 are based on the humanized version of AB3 (humanized AB3-7), and S7-S12 are based on the humanized version of AB1 (humanized AB1-11). [Figure 23] This is a schematic diagram of exemplary somatic hypermutations (SHMs) of the CLDN6 antibody of this disclosure, identified by next-generation sequencing (NGS). Examples of NGS-identified SHMs are shown in the AB-3 antibody and heavy chain. Mutations are described using Chothia numbering. [Figure 24] This is a schematic diagram of exemplary somatic hypermutations (SHMs) of the CLDN6 antibody of this disclosure, identified by next-generation sequencing (NGS). Examples of SHMs identified by NGS in the AB-3 antibody and light chain are shown. Mutations are described using Chothia numbering. [Figure 25] This is a schematic diagram of exemplary somatic hypermutations (SHMs) of the CLDN6 antibody of this disclosure, identified by next-generation sequencing (NGS). Examples of SHMs identified by NGS in the AB-1 antibody and heavy chain are shown. Mutations are described using Chothia numbering. [Figure 26] This is a schematic diagram of exemplary somatic hypermutations (SHMs) of the CLDN6 antibody of this disclosure, identified by next-generation sequencing (NGS). Examples of SHMs identified by NGS in the AB-1 antibody and light chain are shown. Mutations are described using Chothia numbering. [Figure 27] This table shows the results of FACS binding assays of S1-S12 antibodies based on humanized AB3-7 (ABS1-S6) or humanized AB1-11 (ABS7-S12). The concentrations tested are shown in column D. [Figure 28] This table shows the results of FACS binding assays of S1-S12 antibodies at different concentrations in various cell lines. [Figure 29] A shows three types of N-glycans (oligomannose, complex, and hybrid) and examples of symbols commonly used for such sugars. B is a diagram of the salvage and de novo pathways of fucose metabolism. In the salvage pathway, free L-fucose is converted to GDP-fucose, while in the de novo pathway, GDP-fucose is synthesized via three reactions catalyzed by GMD and FX. GDP-fucose is then transported from the cytosol to the Golgi lumen by GDP-Fuc transferase and transferred to acceptor oligosaccharides and proteins. Another reaction product, GDP, is converted to guanosine 5-monophosphate (GMP) and inorganic phosphate (Pi) by nucleotide diphosphatases in the lumen. The former is thought to be transported to the cytosol (via a counter-transport system in conjunction with GDP-fucose transport), while the latter is thought to leave the Golgi lumen via the Golgi anion channel GOLAC. For example, see Nordeen et al. 2000; Hirschberg et al. 2001. [Figure 30] This graph shows the tumor volume in mice that underwent the treatment described herein following subcutaneous injection of human cancer cells. [Figure 31] This graph shows the tumor volume in mice that underwent the treatment described herein following subcutaneous injection of human cancer cells. [Figure 32] This graph shows the tumor volume in mice that underwent the treatment described herein following subcutaneous injection of human cancer cells. [Figure 33] This graph shows the tumor volume in mice that underwent the treatment described herein following subcutaneous injection of human cancer cells. [Figure 34A] These are FACS plots of signals from OVCA429 cells incubated with secondary antibody alone (black line) or with full-length antibody (red, blue, and green lines), as described herein. The red, blue, and green lines differ in that they undergo different washing steps before and / or after the secondary antibody. [Figure 34B]This is a FACS plot of the signal of OVCA429 cells incubated with secondary antibody alone (black line) or with Fab (red, blue, and green lines), as described herein. The red, blue, and green lines differ in that they have different washing steps before and / or after the secondary antibody. [Figure 34C] This is a FACS plot of the signal of OVCA429 cells incubated with secondary antibody alone (black line) or with Fab (red, blue, and green lines), as described herein. The red, blue, and green lines differ in that they have different washing steps before and / or after the secondary antibody. [Figure 34D] This is a FACS plot of the signal of OVCA429 cells incubated with secondary antibody alone (black line) or with Fab (red, blue, and green lines), as described herein. The red, blue, and green lines differ in that they have different washing steps before and / or after the secondary antibody. [Figure 34E] These are FACS plots of signals from OVCA429 cells incubated with secondary antibody alone (black line) or with a mixture of full-length antibody and Fab (red, blue, green, and purple lines), as described herein. The red, blue, green, and purple lines differ in that they have different pre- and / or post-secondary antibody washing steps. [Figure 35] This is a table listing the quantified signals from the FACS plot. [Figure 36] Image A shows cells immunostained with only the secondary antibody. Image B shows cells immunostained with only Fab (acting as the primary antibody). Image C shows cells immunostained with both Fab and the secondary antibody. [Figure 37] A is an example of an expression vector used to express scFv for humanized AB3-7 and humanized AB1-11. B shows the sequences encoding the LC variable region and the HC variable region, with a spacer in between. [Figure 38A] This is a FACS plot of signals from ARK2 cells incubated with secondary antibody alone (black) or with AB3-7scFv and secondary antibody (red). [Figure 38B]This is a FACS plot of signals from M202 cells incubated with secondary antibody alone (black) or with AB3-7scFv and secondary antibody (red). [Figure 39A] This bar graph shows signals from 293T cells, M202 cells, or ARK2 cells overexpressing CLDN6 GFP incubated with (A) secondary antibody only (blue bars), (B) AB3-7 scFV followed by a secondary antibody (orange bars), or (C) AB1-11 scFV followed by a secondary antibody (gray bars). [Figure 39B] This bar graph shows the GFP signal from 293T cells, M202 cells, or ARK2 cells overexpressing CLDN6 GFP incubated with (A) secondary antibody alone (blue bars), (B) AB3-7 scFV followed by a secondary antibody (orange bars), or (C) AB1-11 scFV followed by a secondary antibody (gray bars). [Figure 40A] This is a FACS plot of signals generated by CLDN6 GFP-overexpressing 293T cells incubated with secondary antibody alone (black line), AB3-7 scFV followed by secondary antibody (red line), or AB1-11 scFV followed by secondary antibody (blue line). [Figure 40B] This is a FACS plot of signals generated by M202 cells overexpressing CLDN6 GFP, incubated with secondary antibody alone (black line), AB3-7 scFV followed by secondary antibody (red line), or AB1-11 scFV followed by secondary antibody (blue line). [Figure 40C] This is a FACS plot of signals generated by ARK2 cells overexpressing CLDN6 GFP, incubated with secondary antibody alone (black line), AB3-7 scFV followed by secondary antibody (red line), or AB1-11 scFV followed by secondary antibody (blue line). [Figure 40D-F] This is a FACS plot showing the number of cells in each group in Figure 40A. [Figure 41-1]This shows the biochemical characteristics of CLDN6 antibody-drug conjugates (ADCs) containing AB3-7 (also called AB23). A is a table summarizing the biochemical properties of CLDN6 ADCs. [Figure 41-2] This shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) containing AB3-7 (also known as AB23). BH represents the HIC-HPLC chromatogram showing the relative abundance of antibodies conjugated to different numbers of drugs. [Figure 42] This shows the molecular integrity of CLDN6 ADC containing AB3-7, as analyzed by Native PAGE. [Figure 43] This shows the binding activity (flow cytometry) of a CLDN6 antibody-drug conjugate (ADC) containing AB3-7 to naturally occurring CLDN6-positive cells or cells artificially overexpressing CLDN6. [Figure 44] This study demonstrates the binding affinity of CLDN6 ADCs, including AB3-7, to CLDN6-expressing cells. KD (dissociation constant) measurements were performed using HEK293T CLDN6-mGFP A11 cells with KinExA 4000 (Sapidyne Instrument, Boise, Idaho). [Figure 45] This shows the in vitro characterization of CLDN6 ADCs, including AB3-7. The panel shows the intracellular migration of CLDN6 ADCs. H23-7 refers to AB3-7. [Figure 46A] This demonstrates the in vitro anticancer activity of CLDN6 ADC. CLDN6 ADC containing AB-3-7, i.e., MC-VC-PAB-MMAE (conventional), exhibits two-dimensional (2D) growth inhibitory activity against cancer cells. [Figure 46B] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the two-dimensional (2D) growth inhibitory effect of CLDN6 ADC containing AB-3-7, i.e., MC-VC-PAB-MMAE (D4 technology), on cancer cells. [Figure 46C] This demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the two-dimensional (2D) growth inhibitory effect of CLDN6 ADC containing AB-3-7, i.e., MC-GGFG-MMAE (D4 technology), on cancer cells. [Figure 46D] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows that CLDN6 ADC containing AB-3-7, i.e., CL2A-SN38 (conventional type), exhibits two-dimensional (2D) growth inhibition against cancer cells. [Figure 46E] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the two-dimensional (2D) growth inhibitory effect of CLDN6 ADC containing AB-3-7, i.e., CL2A-SN38 (D4 technology), on cancer cells. [Figure 46F] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows that CLDN6 ADC containing AB-3-7, i.e., MC-GGFG-DXD, exhibits two-dimensional (2D) growth inhibitory effects on cancer cells. [Figure 46G] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows that CLDN6 ADC containing AB-3-7, i.e., MC-VC-PAB-DXD, exhibits two-dimensional (2D) growth inhibitory effects on cancer cells. [Figure 46H] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the 2D proliferation inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against the cancer cell line ARK2. [Figure 46I] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the 2D proliferation inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against the cancer cell line OVCA429. [Figure 46J] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against the cancer cell line H841. [Figure 46K] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against the cancer cell line OV90. [Figure 46L]This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the 2D proliferation inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against the cancer cell line H1693. [Figure 46M] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against the cancer cell line M202. [Figure 46N] This study demonstrates the in vitro anticancer activity of CLDN6 ADC. It also shows the 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against the cancer cell line MCF7. [Figure 47] This study demonstrates the in vivo anticancer effect of CLDN6 ADC-11 against xenografts of CLDN6-positive ovarian cancer cell line (OV90). [Figure 48] Figure A shows that CLDN6 ADC-11 has no anticancer activity against xenografts of CLDN6-negative melanoma cancer cell line (M202). Figure B shows that CLDN6 ADC-11 has no anticancer activity against xenografts of CLDN6-negative melanoma cancer cell line (M202). [Figure 49] Image A shows the in vivo anticancer effect of CLDN6 ADC-23 (AB3-7-VC-PAB-MMAE) against cancer cell line xenografts. It shows the anticancer activity of CLDN6 ADC-23 against CLDN6-positive bladder cell line (UMUC4) xenografts. Image B shows the in vivo anticancer effect of CLDN6 ADC-23 (AB3-7-VC-PAB-MMAE) against cancer cell line xenografts. It shows hematoxylin and eosin (H&E) staining of xenograft tissue collected at the indicated time points after treatment with a control antibody or 5 mg / kg of ADC-23. [Figure 50-1]This shows the in vivo anticancer effect of CLDN6 ADC-23 on xenografts (PDX) derived from ovarian cancer patients. A shows a panel of ovarian PDX samples screened for CLDN6 expression by Western blotting. B is a schematic diagram showing the injection of luciferase-transfected PDX ovarian cancer cells into the peritoneal cavity of immunodeficient mice (NSGs). [Figure 50-2] This document demonstrates the in vivo anticancer effect of CLDN6 ADC-23 on xenografts (PDX) derived from ovarian cancer patients. CE represents the survival rate of mice treated with CLDN6 ADC-23 as described herein. [Figure 51] A shows the dose-dependent anticancer effect of CLDN6 ADC-23 against xenografts of CLDN6-positive ovarian cancer cell line (OV90). It shows tumor size reduction at the indicated dose and time. B shows the dose-dependent anticancer effect of CLDN6 ADC-23 against xenografts of CLDN6-positive ovarian cancer cell line (OV90). It shows tumor size reduction at the indicated dose and time. C shows the dose-dependent anticancer effect of CLDN6 ADC-23 against xenografts of CLDN6-positive ovarian cancer cell line (OV90). It shows the change in body weight (%) of mice administered CLDN6 ADC-23. [Figure 52] A shows no off-target activity of CLDN6 ADC-23 in xenografts of CLDN6-negative melanoma cancer cell line (M202). It shows the change in tumor volume. B shows no off-target activity of CLDN6 ADC-23 in xenografts of CLDN6-negative melanoma cancer cell line (M202). It shows the change in tumor volume. Tumor volume was measured on day 21. C shows no off-target activity of CLDN6 ADC-23 in xenografts of CLDN6-negative melanoma cancer cell line (M202). It shows the change in body weight (%) of mice administered CLDN6 ADC-23. [Figure 53]A shows the purification and validation of the CLDN6-CD3 bispecific T cell engager (BiTE). A schematic diagram of the BiTE construct is shown. B shows the purification and validation of the CLDN6-CD3 bispecific T cell engager (BiTE). The purified BiTE protein of BiTE-23 (a fusion of VL and VH of AB3-7 and VH and VL of CD3E) is shown. C shows the purification and validation of the CLDN6-CD3 bispecific T cell engager (BiTE). The purified BiTE protein of BiTE-11 (a fusion of VL and VH of AB1-11 and VH and VL of CD3E) is shown. [Figure 54]A shows the efficient binding of CLDN6-CD3 bispecific protein to native CLDN6-positive cells (ARK2) and cells engineered to overexpress CLDN6 (HEK293T CLDN6-mGFP HIS20 cells). Flow cytometry of cells bound to BiTE-11 (11-1BiTE and 11-2BiTE; independent production lots of BiTE-11) or Bite-23 (23BiTE) is shown. B shows the efficient binding of CLDN6-CD3 bispecific protein to native CLDN6-positive cells (ARK2) and cells engineered to overexpress CLDN6 (HEK293T CLDN6-mGFP HIS20 cells). Flow cytometry of cells bound to BiTE-11 (11-1BiTE and 11-2BiTE; independent production lots of BiTE-11) or Bite-23 (23BiTE) is shown. C shows the efficient binding of CLDN6-CD3 bispecific protein to native CLDN6-positive cells (ARK2) and cells engineered to overexpress CLDN6 (HEK293T CLDN6-mGFP HIS20 cells). Flow cytometry of cells bound to BiTE-11 (11-1BiTE and 11-2BiTE; independent production lots of BiTE-11) or Bite-23 (23BiTE) is shown. D shows the efficient binding of CLDN6-CD3 bispecific protein to native CLDN6-positive cells (ARK2) and cells engineered to overexpress CLDN6 (HEK293T CLDN6-mGFP HIS20 cells). ARK2 cells stained with BiTE-11 are shown. E shows the efficient binding of the CLDN6-CD3 bispecific protein to native CLDN6-positive cells (ARK2) and cells engineered to overexpress CLDN6 (HEK293T CLDN6-mGFP HIS20 cells). BiTE-23 stained ARK2 cells are shown. [Figure 55] Image A shows that the CLDN6-CD3 bispecific protein induces T cell activation (BiTE-11). Image B shows that the CLDN6-CD3 bispecific protein induces T cell activation (BiTE-23). [Figure 56]This study demonstrates that the CLDN6-CD3 bispecific protein induces T cell cluster formation during incubation with CLDN6-positive cells. BiTE-11 (center panel; p11 BiTE). BiTE-23 (right panel (p23 BiTE)). [Figure 57] This study demonstrates that the CLDN6-CD3 bispecific protein induces target-specific and dose-dependent cytotoxicity against cancer cells. BiTE-11 (upper panel; p11-11). BiTE-23 (lower panel; p23-7). Blinatumomab (control). [Figure 58] A schematic diagram of an exemplary CLDN6-CD16A bispecific tandem diabody (TandAb) is shown, which includes CLDN6-binding VL and VH domains fused with CD16A-binding VH and VL domains. [Figure 59]A shows efficient binding of CLDN6-CD16A TandAb, which binds to CLDN6-positive cells (ARK2) but not to CLDN6-negative cells (HUPT4). TandAb-11 (indicated as Tandab-11-his or Tandab 11-CD16A-his). TandAb-23 (indicated as Tandab-23-his or Tandab 23-CD16A-his). B shows efficient binding of CLDN6-CD16A TandAb, which binds to CLDN6-positive cells (ARK2) but not to CLDN6-negative cells (HUPT4). TandAb-11 (indicated as Tandab-11-his or Tandab 11-CD16A-his). TandAb-23 (indicated as Tandab-23-his or Tandab 23-CD16A-his). C shows efficient binding of CLDN6-CD16A TandAb, which binds to CLDN6-positive cells (ARK2) but not to CLDN6-negative cells (HUPT4). TandAb-11 (indicated as Tandab-11-his or Tandab 11-CD16A-his). D shows efficient binding of CLDN6-CD16A TandAb, which binds to CLDN6-positive cells (ARK2) but not to CLDN6-negative cells (HUPT4). TandAb-23 (indicated as Tandab-23-his or Tandab 23-CD16A-his). [Figure 60] This study demonstrates the in vivo anticancer activity of CLDN6-CD3BiTE. The samples included Blincyte (control), AFM13 (non-target-directed CD16 TandAb control), BiTE-23 (labeled as BiTE #23), BiTE-11 (labeled as BiTE #11), AB3-7 (labeled as mAb #23), TandAb-11 (labeled as CD16-TandAb #11), and TandAb-23 (labeled as CD16-TandAb #23). [Figure 61] This study demonstrates the in vivo anticancer activity of CLDN6-CD3BiTE. The sample includes an IgG control, BiTE-23 (labeled as BiTE #23), and TandAb-23 (labeled as CD16-TandAbs #23). [Figure 62A] The amino acid sequence of CLDN6-CD3BiTE is shown. The amino acid sequence and nucleic acid sequence of BiTE-11 are also shown. [Figure 62B] The nucleic acid sequence of CLDN6-CD3BiTE is shown. The amino acid sequence and nucleic acid sequence of BiTE-11 are shown. [Figure 62C] The amino acid sequence of CLDN6-CD3BiTE is shown. The amino acid sequence and nucleic acid sequence of BiTE-23 are also shown. [Figure 62D] The nucleic acid sequence of CLDN6-CD3BiTE is shown. The amino acid sequence and nucleic acid sequence of BiTE-23 are shown. [Figure 63A] The amino acid sequence of CLDN6-CD16A TandAb is shown. The amino acid sequence of TandAb-11 is shown. [Figure 63B] The nucleic acid sequence of CLDN6-CD16A TandAb is shown. The nucleic acid sequence of TandAb-11 is shown. [Figure 63C] The amino acid sequence of CLDN6-CD16A TandAb is shown. The amino acid sequence of TandAb-23 is shown. [Figure 63D] The nucleic acid sequence of CLDN6-CD16A TandAb is shown. The nucleic acid sequence of TandAb-23 is shown. [Modes for carrying out the invention]

[0017] Claudine Family

[0018] Tight junctions, also known as occluding junctions or zonulae occludentes, are vertebrate structures located between two adjacent cells that regulate paracellular permeability and maintain cell polarity in epithelial and endothelial cell sheets. The claudin (CLDN) family of genes encodes membrane proteins that are key components of tight junctions.

[0019] The CLDN protein contains four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2). The extracellular loops of CLDN proteins in adjacent cells interact with each other to tightly bind cell sheets and regulate paracellular transport between the lumen and basolateral space.

[0020] CLDN proteins are involved in various human diseases and pathologies. For example, mutations in the CLDN1 gene have been shown to cause progressive skin scaling along with bile duct obstruction. Mutations in the CLDN16 gene cause magnesium wasting disorders. Mutations in CLDN19 lead to eye diseases such as macular colobomata and myopia, and mutations in CLDN14 can lead to non-symptomatic inferior hearing loss. CLDN3 and CLDN4 are known to be surface receptors for Clostridium perfringens enterotoxin in the intestinal tract, and CLDN1, CLDN6, and CLDN9 are co-receptors associated with hepatitis C virus (HCV) entry. Several CLDN proteins have been shown to be abnormally expressed in cancer. For example, CLDN1 is downregulated in breast and colon cancer, while CLDN3 and CLDN4 are highly upregulated in multiple cancers.

[0021] Claudin 6 (CLDN6) is a member of the CLDN family. The gene encoding the human CLDN6 protein is located at 16p13.3 on the short arm of human chromosome 16 and is conserved in chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, zebrafish, and frogs. In humans, CLDN6 is generally expressed as a precursor protein with 220 amino acids, the first 21 of which constitute a signal peptide. The amino acid sequence of the CLDN6 precursor protein is publicly available on the National Center for Biotechnology Information (NCBI) website as NCBI Reference Sequence NP_067018.2, and is provided herein as Sequence ID No. 1. The amino acid at position 143 of Sequence ID No. 1 is Ile. In some cases, due to a single nucleotide polymorphism (SNP) in the DNA sequence encoding CLDN6, the amino acid at position 143 is Val. The amino acid sequence of human CLDN6 with Val at position 143 is provided herein as Sequence ID No. 178.

[0022] Antigen-binding protein

[0023] This specification provides antigen-binding proteins that bind to claudin 6 (CLDN6). The antigen-binding proteins of this disclosure may take any one of several forms of antigen-binding proteins known in the art. In various embodiments, the antigen-binding proteins of this disclosure may take the form of an antibody, an antigen-binding antibody fragment, or an antibody protein product.

[0024] In various embodiments of this disclosure, the antigen-binding protein includes, essentially consists of, or comprises an antibody. As used herein, the term “antibody” means a protein having a conventional immunoglobulin format, comprising a heavy chain and a light chain, and comprising a variable region and a constant region. For example, an antibody may be an IgG having two identical pairs of polypeptide chains in a “Y-shape,” with each pair having one “light” chain (typically with a molecular weight of about 25 kDa) and one “heavy” chain (typically with a molecular weight of about 50–70 kDa). Antibodies have a variable region and a constant region. In the IgG format, the variable region generally has about 100–110 or more amino acids, includes three complementarity-determining regions (CDRs), and is substantially different from other antibodies that bind to different antigens, primarily involved in antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region consists of the N-terminal regions of the light and heavy chains, while the constant region consists of the C-terminal regions of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0025] The general structure and properties of antibody CDRs have been reported in the art. Simply put, in an antibody scaffold, CDRs are embedded within a framework of variable regions of the heavy and light chains, constituting regions that play a significant role in antigen binding and recognition. The variable region typically includes at least three CDRs of the heavy or light chain (see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md., and Chothia and Lesk, 1987, J.Mol.Biol.196:901-917, and Chothia et al., 1989, Nature 342:877-883) within a framework region (FR1, FR2, FR3, and FR4 of designated framework regions 1-4 by Kabat et al., 1991; see also Chothia and Lesk, 1987).

[0026] Antibodies may include any constant region known in the art. Human light chains are classified into kappa and lambda light chains. Heavy chains are classified into mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Embodiments of this disclosure include all such classes or isotypes of antibodies. The light chain constant region may be, for example, a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or muon heavy chain constant region, such as the human alpha, delta, epsilon, gamma, or muon heavy chain constant region. Thus, in various embodiments, the antibody is an isotype IgA, IgD, IgE, IgG, or IgM antibody, which includes any one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, the antibody includes a constant region with one or more amino acid modifications compared to the naturally occurring amino acids to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In various cases, the antibody includes a constant region in which a C-terminal Lys residue present in the naturally occurring constant region has been removed or clipped.

[0027] The antibody may be a monoclonal antibody. In some embodiments, the antibody contains a sequence substantially similar to naturally occurring antibodies produced by mammals, such as mice, rabbits, goats, horses, chickens, hamsters, and humans. In this regard, the antibody may be considered a mammalian antibody, such as a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, or human antibody. In certain embodiments, the antigen-binding protein is an antibody such as a human antibody. In certain embodiments, the antigen-binding protein is a chimeric antibody or humanized antibody. The term “chimeric antibody” refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more generally, contain a segment of amino acid sequences from at least two species. A chimeric antibody may also contain domains from two or more different antibodies within the same species. The term "humanization," as used in relation to antibodies, refers to antibodies that have at least a CDR region from non-human material and have been manipulated to have a structure and immunological function more similar to a true human antibody than the original source antibody. For example, humanization may involve transplanting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanization may also involve selecting amino acid substitutions to create a non-human sequence that more closely resembles a human sequence. Sequence information, etc., for the constant regions of the heavy and light chains of human antibodies is publicly available from the Uniprot database and other databases well known to those skilled in the art in the field of antibody engineering and production. For example, the IgG2 constant region is available from the Uniprot database as Uniprot number P01859 and is incorporated herein by reference.

[0028] Antibodies can be cleaved into fragments by enzymes, such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody to produce an F(ab')2 fragment and a pFc' fragment. In various aspects of this disclosure, the antigen-binding protein of this disclosure is the antigen-binding fragment of an antibody (also known as an antigen-binding antibody fragment, antigen-binding fragment, or antigen-binding moiety). In various cases, the antigen-binding antibody fragment is either a Fab fragment or an F(ab')2 fragment.

[0029] Antibody structures have been used to create an expanding range of alternative antibody formats, with molecular weights ranging from at least approximately 12 to 150 kDa and binding titers (n) ranging from monomers (n=1), dimers (n=2), trimers (n=3), tetramers (n=4), and potentially even higher. Such alternative antibody formats are referred to herein as “antibody protein products.” Antibody protein products include those based on complete antibody structures, as well as those mimicking antibody fragments that retain complete antigen-binding ability, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists only of a variable (V) region. To stabilize the molecule, a soluble and mobile amino acid peptide linker is used to connect the V region to an scFv (single-chain variable region) fragment, or a constant (C) domain is added to the V region to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be readily produced in host cells, such as prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody formats such as diabodies, triabodies, and tetrabodies, or minibodies (miniAb), which include various formats consisting of disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and scFv linked to an oligomer-forming domain. The smallest fragments are VHH / VH of camel heavy chain Ab and single-domain Ab (sdAb). The most frequently used component for creating novel antibody formats is the single-chain variable (V) domain antibody fragment (scFv), which contains V domains (VH domain and VL domain) from the heavy and light chains linked by a peptide linker of approximately 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of a peptide body consists of a bioactive peptide transplanted into an Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0030] Other antibody protein products include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, and bispecific or trispecific antibodies. Bispecific antibodies are divided into five main classes: BsIgG, IgG with addition, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb complexes. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

[0031] In various embodiments, the antigen-binding proteins of the Disclosure include, essentially consist of, or consist of any one of these antibody protein products. In various embodiments, the antigen-binding proteins of the Disclosure include, essentially consist of, or consist of any one of scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camel heavy chain antibodies, sdAb, diabody; triabody; tetrabody; bispecific or triplicate antibodies, BsIgG, IgG with addition, BsAb fragment, bispecific fusion proteins, and BsAb complexes.

[0032] In various cases, the antigen-binding proteins of this disclosure are antibody-protein products in monomeric form, or in polymeric, oligomeric, or polymeric form. In certain embodiments in which the antibody comprises two or more distinct antigen-binding domain fragments, the antibody may be bispecific, triplicate, or multispecific, or divalent, trivalent, or polyvalent, depending on the number of distinct epitopes that the antibody recognizes and binds to.

[0033] In various embodiments, the anti-CLDN6 antibody or its variant antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, monomeric antibodies, diabodies, triabodies, tetrabodies, Fab fragments, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies.

[0034] In various embodiments, the antigen-binding proteins of this disclosure are linked to therapeutic agents. The therapeutic agents may be any known in the art, including, but not limited to, chemotherapeutic agents, cytokines and growth factors, cytotoxic drugs, etc. See "Conjugates" below.

[0035] Dual Specificity Format

[0036] In exemplary embodiments, the antigen-binding protein is bispecific and can therefore bind to two different distinct antigens. In exemplary embodiments, the antigen-binding protein is bispecific and binds to CLDN6 and a second antigen.

[0037] In exemplary cases, the second antigen is a cell surface protein expressed by T cells. In exemplary embodiments, the cell surface protein is a component of the T cell receptor (TCR), e.g., CD3. In exemplary cases, the second antigen is a costimulatory molecule that assists T cell activation, e.g., CD40 or 4-1BB (CD137). In exemplary embodiments, the second antigen is an Fc receptor. In various embodiments, the Fc receptor is an Fc-gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In exemplary embodiments, the Fc receptor is CD64 (Fc-gamma RI), CD32 (Fc-gamma RIIA), CD16A (Fc-gamma RIIIA), CD16b (Fc-gamma RIIIb), FcεRI, CD23 (Fc-epsilon RII), CD89 (Fc-epsilon RI), Fcα / μR, or FcRn. In exemplary embodiments, the Fc receptor is CD16A. In exemplary cases, the second antigen is an immune checkpoint molecule, e.g., a protein involved in the immune checkpoint pathway. The immune checkpoint pathway and the molecules or proteins that function within it are known in the art. See, for example, Pardoll, Nat Rev Genet 12(4):252-264 (2012). In exemplary cases, the immune checkpoint molecules are A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. Optionally, the immune checkpoint molecules are PD-1, LAG3, TIM3, or CTLA4.

[0038] More than 50 formats of bispecific antigen-binding proteins are known in the art, some of which are described in Kontermann and Brinkmann, Drug Discovery Today 20(7):838-847 (2015), Zhang et al., Exp Hematol Oncol 6:12 (2017), and Spiess et al., Mol Immunol.; 67(2 Pt A):95-106 (2015). In exemplary embodiments, the bispecific antigen-binding proteins of this disclosure are produced by chemical engineering, genetic engineering, or quadroma technology.

[0039] In exemplary embodiments, the bispecific antigen-binding protein is constructed using some or all of the constant domains of an antibody. In exemplary embodiments, the bispecific antigen-binding protein of this disclosure comprises an Fc polypeptide and retains effector function by Fc. In various cases, the bispecific antigen-binding protein is a bispecific monoclonal antibody, formed, for example, by chemical crosslinking of two monoclonal antibodies (mab) or by the knob-and-hold technique. In exemplary embodiments, the bispecific antigen-binding protein is produced by the "knobs-into-holes" technique, in which H chain heterodimerization is forced by introducing different mutations into two CH3 domains to produce an asymmetric antibody. A "knob" mutation is made in one HC and a "hole" mutation in the other HC to promote heterodimerization. In exemplary embodiments, the bispecific antigen-binding protein is a bispecific antibody produced by the quadroma technique, which is based on somatic cell fusion of two different hybridoma cells that produce a monoclonal antibody with desired specificity. See Zhang et al., 2017, above. In exemplary embodiments, bispecific antigen-binding proteins include crossMab, ortho-Fab IgG, DVD-Ig, two-in-one IgG, IgG-scFv, and scFv2-Fc (Kontermann and Brinkmann, 2015, see above). In various embodiments, the bispecific antigen-binding protein is an Ig-scFv fusion, which adds a new antigen-binding moiety to full-length IgG to produce a tetravalent fusion protein for two distinct antigens, such as an IgGC-terminal scFv fusion and an IgGN-terminal scFv fusion. In exemplary embodiments, the bispecific antigen-binding protein is a bivariable domain IgG (DVD-IgG), where the LC and HC variable regions of IgG specific to one antigen are fused via a linker to the N-terminus of the LC and HC variable region of IgG specific to a second antigen to form DVD-IgG. In exemplary embodiments, the bispecific antigen-binding protein is a diabody-Fc fusion involving the replacement of a Fab fragment of IgG in a bispecific diabody.

[0040] In alternative examples, the bispecific antigen-binding proteins of this disclosure do not contain Fc polypeptides. In exemplary embodiments, the bispecific antigen-binding protein comprises the variable domain of each parent monoclonal antibody, and a linker is cloned and ligated to form a single-strand bispecific antibody. In exemplary embodiments, the bispecific antigen-binding protein is a tandem scFv, a diabody format, a single-strand diabody, a tandem diabody (TandAb), a biaffinity retargeting molecule (DART), a dock-and-lock (DNL), and a nanobody (Fan et al., J Hematol Oncol. 2015;8:130). In various embodiments, the bispecific antigen-binding protein is a bispecific F(mab1)2, scFv, bispecific diabody (BsDb), single-stranded bispecific diabody (scBsDb), single-stranded bispecific tandem variable domain (scBsTaFv), dock-and-lock trivalent Fab (DNL-(Fab)3), single-domain antibody (sdAb), or bispecific single-domain antibody (BssdAb). In an exemplary embodiment, the bispecific antigen-binding protein is a tandem scFv containing two scFv fragments linked by an additional peptide linker such as a glycine-serine repeat motif. Optionally, the tandem scFv contains the structure VLA-linker1-VHA-linker2-VHB-linker3-VLB (where VL and VH are derived from the single-stranded antibody fragment, and A and B represent parental monoclonal antibody A and parental monoclonal antibody B). In an exemplary embodiment, the bispecific antigen-binding protein is TandAb, containing two pairs of VL and VH domains linked together to form a single polypeptide chain (Reusch et al., MAbs. 2015;7(3):584-604). The two polypeptide products dimerize in a head-to-tail manner, forming a large (approximately 105 kDa) homodimer upon expression. In an exemplary embodiment, the bispecific antigen-binding protein is prepared using the crossMab technique described in PNAS 108(27):11187-92 (2011).CrossMab is prepared by a method that compels correct light chain association in bispecific heterodimer IgG antibodies, without any chemical linkers or conjugates. In exemplary embodiments, CrossMab is a bispecific crossMab of divalent (1+1), trivalent (2+1), and tetravalent (2+2), or a crossMab using a non-Fc tandem antigen-binding fragment (Fab). In exemplary cases, crossMab is crossMabFab, crossMabVH-VL, or crossMabCH1-CL.

[0041] In exemplary embodiments, the bispecific antigen-binding protein comprises a single-domain antibody or nanobody containing a single monomeric variable antibody domain. Optionally, the variable domain is based on a heavy chain variable domain. In alternative embodiments, the variable domain is based on a light chain variable domain.

[0042] In exemplary embodiments, the bispecific antigen-binding protein is a bispecific T-cell engager or BiTE®. BiTE is a bivalent small molecule containing only the variable region of an antibody, in which the scFv is linked by a mobile peptide linker. In exemplary embodiments, the bispecific antigen-binding protein includes an scFV containing the LC and HC variable regions of the CLDN6 antibody disclosed herein, and the LC and HC variable regions of a second antibody specific to a second antigen. In some embodiments, BiTE includes the LC and HC variable regions of a second antibody specific to CD3. In some embodiments, CD3 is CD3E.

[0043] In an exemplary case, the bispecific antigen-binding protein is a biaffinity retargeting protein (DART), and unlike BiTEs®, the covalent bond between the two strands of DART restricts the freedom of the antigen-binding site. Therefore, DART is structurally small and can form a stable contact between target and effector cells. DART contains two manipulated Fv fragments, one of which has its VH exchanged with the other. The mutually exchanged Fv domains favorably release the mutant fragment due to the steric constraints of the short linked peptide.

[0044] In an exemplary embodiment, the bispecific antigen-binding protein is an HSABody containing two scFvs fused to a modified HSA. The HSABody is described in McDonagh et al., Mol Cancer Ther. 2012;11(3):582-93.

[0045] Accordingly, in exemplary embodiments, the bispecific antigen-binding protein comprises an antigen-binding fragment of any of the CLDN6 antibodies disclosed herein. In exemplary embodiments, the antigen-binding fragment is Fab. In exemplary embodiments, the bispecific antigen-binding protein comprises F(ab)2' of any of the CLDN6 antibodies disclosed herein. In exemplary embodiments, the bispecific antigen-binding protein comprises an scFv containing the LC variable region and the HC variable region of any of the CLDN6 antibodies disclosed herein. In exemplary embodiments, the scFv contains the amino acid sequence of SEQ ID NO: 514 or 515. In various embodiments, the antigen-binding fragment is based on the heavy chain variable region, and in other embodiments, the antigen-binding fragment is based on the light chain variable region. In exemplary embodiments, the antigen-binding fragment comprises at least a portion of both the HC variable region and the LC variable region. In exemplary embodiments, the bispecific antigen-binding protein comprises at least one of the LC variable region and / or HC variable region of the CLDN6 antibody disclosed herein, and at least one of both the LC variable region and / or HC variable region of a second antibody specific to a second antigen. In exemplary cases, the bispecific antigen-binding protein comprises an scFV including the LC variable region and HC variable region of the CLDN6 antibody disclosed herein, and the LC variable region and HC variable region of a second antibody specific to a second antigen.

[0046] CLDN6 and epitopes

[0047] The antigen-binding protein of this disclosure binds to CLDN6. In various embodiments, CLDN6 is human CLDN6 having the following amino acid sequence. TIFF0007856818000001.tif40160 where X is Ile or Val (Sequence ID 202).

[0048] In various embodiments, human CLDN6 contains one of the amino acid sequences of sequence numbers 1, 178, and 200-202.

[0049] In various embodiments, the antigen-binding proteins of this disclosure bind to epitopes within the amino acid sequence of CLDN6. In various embodiments, CLDN6 is human CLDN6, and the antigen-binding proteins of this disclosure bind to epitopes within the amino acid sequence of human CLDN6, for example, within SEQ ID NOs: 1, 178, and 200-202. "Epitope" means a region of CLDN6 or within CLDN6 to which the antigen-binding protein binds. In some embodiments, the epitope is a linear epitope. "Linear epitope" means a region of CLDN6 or within CLDN6 to which the antigen-binding protein binds, and the region consists of consecutive amino acids in the amino acid sequence of CLDN6. The amino acids of a linear epitope are adjacent to each other in the primary structure of CLDN6. Therefore, a linear epitope is an antigen, i.e., a fragment or portion of the amino acid sequence of CLDN6. In various other embodiments, the epitope is a structural epitope or a three-dimensional epitope. A "structural epitope" or "three-dimensional epitope" means an epitope composed of amino acids that are located in close proximity to each other only when CLDN6 is in its properly folded state. Unlike linear epitopes, the amino acids of a structural epitope or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN6. The structural epitope or structural epitope is not made up of consecutive amino acids in the amino acid sequence of the antigen (CLDN6).

[0050] In various embodiments, the epitope is located within the extracellular domain (ECD) of CLDN6, for example, human CLDN6. In various embodiments, the antigen-binding protein binds to extracellular loop 2 (EL2) of the ECD of CLDN6, which has the amino acid sequence WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2). In various embodiments, the epitope to which the antigen-binding protein binds is located within SEQ ID NO: 2. In various embodiments, the antigen-binding protein of this disclosure binds to the N-terminal portion of SEQ ID NO: 2, for example, THAIIRDFYNPL (SEQ ID NO: 3). In various embodiments, the antigen-binding protein of this disclosure binds to the C-terminal portion of SEQ ID NO: 2, for example, LVAEAQKREL (SEQ ID NO: 4). In various cases, the antigen-binding protein of this disclosure binds to EL2 but not to extracellular loop 1 (EL1) of CLDN6. In various embodiments, the epitope(s) to which the antigen-binding protein of this disclosure binds is different from the epitope to which the anti-CLDN6 antibody, which includes a light chain variable region containing the sequence of SEQ ID NO: 185 and a heavy chain variable region containing the sequence of SEQ ID NO: 186, binds. In various embodiments, the epitope(s) to which the antigen-binding protein of this disclosure binds is different from the epitope to which the anti-CLDN6 antibody, which includes a light chain variable region containing the sequence of SEQ ID NO: 181 and a heavy chain variable region containing the sequence of SEQ ID NO: 182, binds.

[0051] In various embodiments, antigen-binding proteins bind to human CLDN6 and non-human CLDN6. In various cases, non-human CLDN6 is CLDN6 from chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, zebrafish, or frogs. In various cases, antigen-binding proteins bind to human CLDN6 and mouse CLDN6.

[0052] Affinity and binding force

[0053] The antigen-binding proteins provided herein bind to CLDN6 in a non-covalent and reversible manner. In various embodiments, the binding strength of the antigen-binding protein to CLDN6 can be represented by its affinity, which is a measure of the strength of the interaction between the binding site of the antigen-binding protein and the epitope. In various aspects, the antigen-binding proteins provided herein have a high affinity for CLDN6 and thus bind to a large amount of CLDN6 in a shorter period than low-affinity antigen-binding proteins. In various aspects, the antigen-binding protein has an equilibrium association constant K A of at least 10 5 mol -1 at least 10 6 mol -1 at least 10 7 mol -1 at least 10 8 mol -1 at least 10 9 mol -1 or at least 10 10 mol -1 or at least 10 10 mol -1 or at least 10 10 mol -1 . As will be understood by those skilled in the art, K A can be affected by factors such as pH, temperature, and buffer composition.

[0054] In various embodiments, the binding strength of the antigen-binding protein to CLDN6 can be represented by its sensitivity. K D is the equilibrium dissociation constant, which is the ratio of k off / k on between the antigen-binding protein and CLDN6. K D and K A are inversely correlated. The K D value is related to the concentration of the antigen-binding protein (the amount of antigen-binding protein required for a specific experiment), so the lower the K D value (lower concentration), the higher the affinity of the antigen-binding protein. In various aspects, the binding strength of the antigen-binding protein to CLDN6 is K DIt can be represented as follows. In various embodiments, the K of the antigen-binding protein provided herein D It is about 10 -1 , about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 or less. In various embodiments, the K of the antigen-binding protein provided herein D This is micromolar, nanomolar, picomolar, or femtomole. In various embodiments, the K of the antigen-binding protein provided herein D It is about 10 -4 ~10 -6 or 10 -7 ~10 -9 or 10 -10 ~10 -12 or 10 -13 ~10 -15 It is within the range. In various embodiments, the K of the antigen-binding protein provided herein D It is approximately 1.0 × 10 -12 M ~ approx. 1.0×10 -8 It is within the range of M. In various embodiments, the K of antigen-binding proteins D It is approximately 1.0 × 10 -11 M ~ approx. 1.0×10 -9 It is within the range of M.

[0055] In various embodiments, the affinity of antigen-binding proteins is measured or ranked using flow cytometry- or fluorescence-activated cell classification (FACS)-based assays. Flow cytometry-based binding assays are known in the art. See, for example, Cedeno-Arias et al., Sci Pharm 79(3):569-581 (2011), Rathanaswami et al., Analytical Biochem 373:52-60 (2008), and Geuijen et al., J Immunol Methods 302(1-2):68-77 (2005). In various embodiments, the affinity of antigen-binding proteins is measured or ranked using Trikha et al., Int J Cancer 110:326-335 (2004) and Tam et al., Circulation 98(11):1085-1091 (1998), as well as the competitive assays described below. See the section titled "Competitive Assays" below. In Trikh et al., antigen-expressing cells were used in a radioassay. 125 The binding of I-labeled antigen-binding proteins (e.g., antibodies) to cell surface antigens is measured using cells in suspension. In various embodiments, the relative affinity of CLDN6 antibodies is determined by a FACS-based assay, in which various concentrations of CLDN6 antibodies conjugated to fluorophores are incubated with CLDN6-expressing cells, and the emitted fluorescence (a direct measure of antibody-antigen binding) is determined. A curve is created plotting each dose or concentration. The maximum value is the lowest concentration at which fluorescence levels off or reaches its maximum, i.e., when binding saturation occurs. Half of the maximum value is considered the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity compared to other antibodies tested in the same manner. Such an assay is described herein in Example 5.

[0056] In various aspects, IC determined by competitive binding inhibition assays 50 The value is the K of the antigen-binding protein. DThis approximates the following. In various cases, as discussed below, the competitive assay is a FACS-based assay performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative embodiments, the cells endogenously express CLDN6. Before performing the FACS-based assay, in some embodiments, the endogenously expressing CLDN6 cells are pre-determined as either CLDN6-low or CLDN6-high expression cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are from cell lines, e.g., ovarian cell lines, endometrial cell lines, bladder cell lines, lung cell lines, upper gastrointestinal (GI) cell lines, hepatocyte cell lines, etc. In various embodiments, cells endogenously expressing CLDN6 are selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper gastrointestinal cells, OV-90 ovarian cells, HUH-7 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 expressed by the cells and a reference antibody, where the reference antibody is known to bind to CLDN6 but is not the antigen-binding protein of this disclosure. In various cases, the antigen-binding protein of this disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human CLDN6 and a reference antibody, and this inhibition is IC 50 Characterized by the following: In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and the reference antibody, resulting in an IC of less than approximately 2500 nM. 50This indicates that, in various embodiments, antigen-binding proteins have an IC of less than approximately 2000 nM, less than approximately 1500 nM, less than approximately 1000 nM, less than approximately 900 nm, less than approximately 800 nm, less than approximately 700 nm, less than approximately 600 nm, less than approximately 500 nm, less than approximately 400 nm, less than approximately 300 nm, less than approximately 200 nm, or less than 100 nm. 50 This indicates that, in various embodiments, the antigen-binding protein has an IC of less than approximately 90 nM, less than approximately 80 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, or less than 10 nM. 50 This demonstrates that, in various cases, the antigen-binding proteins of this disclosure compete for binding to CLDN6 with reference antibodies known to bind to CLDN6 (the reference antibodies being different from any of the antigen-binding proteins of this disclosure). See "Competitive Assays" for details.

[0057] The binding affinity provides a measure of the overall strength of the antibody-antigen complex. This varies depending on three key parameters: the affinity of the antigen-binding protein to the epitope, the binding titer of both the antigen-binding protein and CLDN6, and the structural arrangement of the interacting regions. The higher the binding titer (number of antigen-binding sites) of the antigen-binding protein, the greater the amount of antigen (CLDN6) it can bind to. In various embodiments, antigen-binding proteins have a strong binding affinity to CLDN6. In various embodiments, antigen-binding proteins are polyvalent. In various embodiments, antigen-binding proteins are bivalent. In various cases, antigen-antigen-binding proteins are monovalent.

[0058] Cross-reactivity

[0059] In various embodiments, the antigen-binding proteins of this disclosure bind to CLDN6 and do not bind to any other member of the CLDN family, for example, they do not cross-react with any other member of the CLDN family. In various cases, the antigen-binding proteins of this disclosure are CLDN6-specific. In various embodiments, the antigen-binding proteins of this disclosure have selectivity for CLDN6, which is at least 10, 5, 4, 3, or 2 times greater than the selectivity of the antigen-binding proteins for CLDN3, CLDN4, CLDN9, or any combination thereof. In various embodiments, the antigen-binding proteins of this disclosure have selectivity for CLDN6, which is at least 10, 5, 4, 3, or 2 times greater than the selectivity of the antigen-binding proteins for CLDN3, CLDN4, and CLDN9, respectively. Selectivity is the K value that the antigen-binding protein exhibits towards CLDN6 or a member of the CLDN family. D Based on K D This can be determined by techniques known in the art, such as surface plasmon resonance and FACS-based affinity assays.

[0060] In various embodiments, the antigen-binding protein of this disclosure binds to CLDN6 but not to claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various embodiments, the antigen-binding protein does not bind to CLDN3, CLDN4, and CLDN9 and exhibits IC50 levels of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) in FACS-based assays using OVCA429 cells endogenously expressing CLDN6. 50This shows that, in various embodiments, the antigen-binding protein does not bind to any of CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved in OVCA429 cells endogenously expressing CLDN6 is less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM). In various embodiments, the antigen-binding protein exhibits at least five times greater selectivity for CLDN6 than for CLDN3, CLDN4, and CLDN9, and the concentration at which 50% binding saturation is achieved in OVCA429 cells endogenously expressing CLDN6 is less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM). In various embodiments, the antigen-binding protein exhibits an IC50 of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) relative to artificial and endogenous models of CLDN6, and a ratio of more than approximately 5 times that distinguishes it from the IC50 of CLDN6 to CLDN3, CLDN4, and / or CLDN9. In various cases, the antigen-binding protein exhibits an IC50 of less than approximately 1200 nM (e.g., less than approximately 1000 nM, less than approximately 750 nM, less than approximately 500 nM, less than approximately 250 nM) relative to CLDN6, and an IC50 at least 5 times greater than that for any one of CLDN3, CLDN4, and CLDN9.

[0061] In various embodiments, the antigen-binding protein of the Disclosure binds to CLDN6 and cross-reacts with (e.g., binds to) at least one other member of the CLDN family. In various embodiments, the antigen-binding protein of the Disclosure binds to CLDN6 and one or more of CLDN3, CLDN4, and CLDN9. In various embodiments, the antigen-binding protein of the Disclosure binds to CLDN6 and CLDN4 or CLDN9, but not to CLDN3. In various cases, the antigen-binding protein of the Disclosure binds to CLDN6 and CLDN4, but not to CLDN3 or CLDN9. In various cases, the antigen-binding protein of the Disclosure binds to CLDN6 and CLDN9, but not to either CLDN3 or CLDN4.

[0062] Competitive assay

[0063] In various embodiments, the antigen-binding protein inhibits the binding interaction between human CLDN6 and a reference antibody, where the reference antibody is known to bind to CLDN6 but is not the antigen-binding protein of this disclosure. In various cases, the antigen-binding protein of this disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the reference antibody binds to an epitope in EL2 or EL1, optionally, within the amino acid sequence of the extracellular domain of human CLDN6. In various embodiments, the reference antibody includes a light chain variable sequence encoded by SEQ ID NO: 179 and a heavy chain variable sequence encoded by SEQ ID NO: 180. In various embodiments, the reference antibody includes a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, and this inhibition is reflected in IC 50 Characterized by the following: In various embodiments, antigen-binding proteins inhibit the binding interaction between human CLDN6 and the reference antibody, resulting in an IC of less than approximately 2500 nM. 50 This indicates that, in various embodiments, antigen-binding proteins have an IC of less than approximately 2000 nM, less than approximately 1500 nM, less than approximately 1000 nM, less than approximately 900 nm, less than approximately 800 nm, less than approximately 700 nm, less than approximately 600 nm, less than approximately 500 nm, less than approximately 400 nm, less than approximately 300 nm, less than approximately 200 nm, or less than 100 nm. 50 This indicates that, in various embodiments, the antigen-binding protein has an IC of less than approximately 90 nM, less than approximately 80 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, or less than 10 nM. 50 This indicates.

[0064] In various cases, the antigen-binding protein of this disclosure competes with the reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the in vitro competitive binding assay is a FACS-based assay that measures the fluorescence of a fluorophore-conjugated secondary antibody bound to the Fc of the reference antibody, in the absence or presence of a specific amount of the antigen-binding protein of this disclosure. Such FACS-based assays are described in the examples herein. In various embodiments, the FACS-based assay is performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN6. In various embodiments, the cells are genetically engineered to overexpress CLDN6. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN6. In alternative embodiments, the cells endogenously express CLDN6. Before performing a FACS-based assay, in some embodiments, cells endogenously expressing CLDN6 are pre-determined as either low-CLDN6 or high-CLDN6 expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are cells from cell lines, such as ovarian cell lines, endometrial cell lines, bladder cell lines, lung cell lines, upper gastrointestinal (GI) cell lines, hepatocyte cell lines, etc. In various embodiments, cells endogenously expressing CLDN6 are selected from the group consisting of OVCA429 ovarian cells, ARK2 endometrial cells, OAW28 ovarian cells, UMUC-4 bladder cells, PEO14 ovarian cells, OV177 ovarian cells, H1693 lung cells, MKN7 upper gastrointestinal cells, OV-90 ovarian cells, HUH-7 hepatocytes, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various cases, the antigen-binding proteins of this disclosure bind with high affinity to CLDN6 endogenously expressed in one or more ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various embodiments, the antigen-binding proteins are determined by competitive binding inhibition assays based on FACS using one or more ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50The IC is less than approximately 3000 nM. In various embodiments, the antigen-binding protein is determined by a competitive binding inhibition assay based on FACS using one or more of the following cells: ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 The IC values ​​are approximately less than 2500 nM, less than 2000 nM, less than 1750 nM, less than 1500 nM, less than 1250 nM, less than 1000 nM, less than 750 nM, or less than 500 nM. In various embodiments, the IC values ​​of the antigen-binding protein are determined by a competitive binding inhibition assay based on FACS using one or more ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 This indicates a value of less than approximately 400 nM, less than approximately 300 nM, less than approximately 200 nM, less than approximately 100 nM, less than approximately 75 nM, less than approximately 50 nM, less than approximately 25 nM, or less than approximately 10 nM.

[0065] Other binding assays that test the ability of an antibody to compete with a second antibody for binding to an antigen or its epitope, such as competitive binding assays or competitive assays, are known in the art. See, for example, Trikha et al., Int J Cancer 110:326-335 (2004), Tam et al., Circulation 98(11):1085-1091 (1998), U.S. Patent Application Publication US20140178905, Chand et al., Biologicals 46:168-171 (2017), Liu et al., Anal Biochem 525:89-91 (2017), and Goolia et al., J Vet Diagn Invest 29(2):250-253 (2017). Other methods for comparing two antibodies are also known in the art, including, for example, surface plasmon resonance (SPR). SPR can be used to determine the binding constants of one antibody and a second antibody, and the two binding constants can be compared.

[0066] Antibody production method and related methods

[0067] Suitable methods for producing antigen-binding proteins (e.g., antibodies, antigen-binding antibody fragments, and antibody protein products) are known in the art. For example, standard hybridoma methods for antibody production are described in, for example, Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5. th This is described in Ed., Garland Publishing, New York, NY (2001). Various methods for preparing the CLDN6 monoclonal antibody of this disclosure are provided in the examples herein.

[0068] Depending on the host species, various adjuvants can be used to enhance the immunological response that leads to massive antibody production by the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum may be useful human adjuvants.

[0069] Other antibody production methods are summarized in Table 1. [Table 1] TIFF0007856818000003.tif59161

[0070] Regardless of how the antibody is produced, methods for testing the ability of an antibody to bind to the CLDN6 epitope are known in the art, and these include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blotting, immunoprecipitation, SPR, and competitive inhibition assays (see, for example, Janeway et al., below, and U.S. Patent Application Publication No. 2002 / 0197266, and the above section relating to competitive assays).

[0071] Array / Structure

[0072] In this specification, (a) the amino acid sequences of heavy chain (HC) complementarity-determining region (CDR) 1 listed in Table A, or sequences selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, and 131, or variant sequences thereof that differ by only one or two amino acids, or have approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, and (b) the HC sequences listed in Table A (c) The amino acid sequence of CDR2, or a sequence selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, and 132, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, (c) HC as listed in Table A (d) The amino acid sequence of CDR3, or a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, and 133, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, (d) Light as described in Table A (e) The amino acid sequence of chain (LC)CDR1, or a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, and 128, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, (e) LC as listed in Table A(f) The amino acid sequence of CDR2, or a sequence selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, and 129, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, (f) LC as listed in Table A The following are provided: an antigen-binding protein containing the amino acid sequence of CDR3, or a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, and 130, which differs by only one or two amino acids, or which has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or any combination of two or more of (g)(a) to (f). [Table 2]

[0073] In various embodiments, the antigen-binding protein comprises the LC CDR1 amino acid sequence, the LC CDR2 amino acid sequence, and the LC CDR3 amino acid sequence described in Table A, and at least one or two of the HC CDR amino acid sequences described in Table A.

[0074] In various embodiments, the antigen-binding protein includes at least three, four, or five amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein includes each of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A, and at least one or two of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein includes each of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A, and at least one or two of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein includes all six CDR amino acid sequences specified by the sequence numbers in a row in Table A. In various embodiments, the antigen-binding protein contains six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs. 74-79, (b) SEQ ID NOs. 50-55, (c) SEQ ID NOs. 122-127, (d) SEQ ID NOs. 26-31, (e) SEQ ID NOs. 128-133, (f) SEQ ID NOs. 38-43, (g) SEQ ID NOs. 62-67, (h) SEQ ID NOs. 80-85, (i) SEQ ID NOs. 44-49, (j) SEQ ID NOs. 86-91, (k) SEQ ID NOs. 104-109, (l) SEQ ID NOs. 56-61, (m) SEQ ID NOs. 32-37, (n) SEQ ID NOs. 110-115, (o) SEQ ID NOs. 98-103, (p) SEQ ID NOs. 92-97, (q) SEQ ID NOs. 116-121, (r) SEQ ID NOs. 8-13, (s) SEQ ID NOs. 68-73, (t) SEQ ID NOs. 14-19, and (u) SEQ ID NOs. 20-25.

[0075] In various cases, the amino acid sequences in Table A are separated by at least one (for example, at least two, three, four, five, six, seven, eight, nine, ten, or more) intervening amino acids. In various cases, there are approximately 10 to 20 amino acids between the LC CDR1 and LC CDR2 sequences, and approximately 25 to 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are approximately 14 to 16 amino acids between the LC CDR1 and LC CDR2 sequences, and approximately 30 to 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are approximately 10 to 20 amino acids between the HC CDR1 and HC CDR2 sequences, and approximately 25 to 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various cases, there are approximately 14 to 16 amino acids between the HC CDR1 and HC CDR2 sequences, and approximately 30 to 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0076] In various embodiments, the antigen-binding protein is (a) a heavy chain variable region amino acid sequence as listed in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or (b) Light chain variable region amino acid sequences listed in Table B, or sequences selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176, or variant sequences thereof that differ by only one or two amino acids, or have approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, or (c) including both (a) and (b). [Table 3]

[0077] In various embodiments, the antigen-binding protein is (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 166 It includes a pair of amino acid sequences selected from the group consisting of (l)sequences 150 and 151, (m)sequences 142 and 143, (n)sequences 168 and 169, (o)sequences 164 and 165, (p)sequences 162 and 163, (q)sequences 170 and 171, (r)sequences 134 and 135, (s)sequences 154 and 155, (t)sequences 136 and 137, and (u)sequences 138 and 139.

[0078] In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 179 and 180. In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 181 and 182. In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 183 and 184. In various embodiments, the antigen-binding protein does not contain the pair of amino acid sequences encoded by sequence numbers 185 and 186.

[0079] In various embodiments, antigen-binding proteins contain amino acid sequences similar to those described above, yet they still substantially retain their biological functions, such as their ability to bind to human CLDN6, reduce tumor growth, and treat cancer.

[0080] In various embodiments, the antigen-binding protein contains an amino acid sequence that differs by only one, two, three, four, five, six, or more amino acids from the aforementioned amino acid sequence(s). In various embodiments, the antigen-binding protein contains a variant sequence of the mentioned sequence, the variant sequence differing by only one or two amino acids from the mentioned sequence. In various embodiments, the antigen-binding protein contains one or more amino acid substitutions occurring outside the CDR, for example, one or more amino acid substitutions occurring within the framework region(s) of the heavy or light chain. In various embodiments, the antigen-binding protein contains one or more amino acid substitutions, but the antigen-binding protein still retains the amino acid sequence of the six CDRs. In various embodiments, the antigen-binding protein contains an amino acid sequence that has only one, two, three, four, five, six, or more conserved amino acid substitutions from the aforementioned amino acid sequence(s). As used herein, the term “conservative amino acid substitution” means the substitution of one amino acid with another amino acid having similar properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and this includes substitutions within one of the following five groups: TIFF0007856818000006.tif70161

[0081] In various forms, a conservative amino acid substitution is an exchange within one of the following groups of amino acids: TIFF0007856818000007.tif70161

[0082] In various embodiments, the antigen-binding protein includes an amino acid sequence having approximately 30% or more, approximately 50% or more, or approximately 70% or more sequence identity to the aforementioned amino acid sequence. In various embodiments, the antigen-binding protein includes an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity to the aforementioned amino acid sequence. In various embodiments, the antigen-binding protein includes an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity along the full length of the aforementioned amino acid sequence. In various embodiments, the antigen-binding protein includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity along the full length of the aforementioned amino acid sequence.

[0083] In various embodiments, the antigen-binding protein includes a variant sequence of the sequence mentioned, the variant sequence having approximately or at least 70% sequence identity to the aforementioned sequence. In various embodiments, the antigen-binding protein includes a variant sequence of the sequence mentioned, the variant sequence having approximately or at least 80% sequence identity to the aforementioned sequence. In various embodiments, the antigen-binding protein includes a variant sequence of the sequence mentioned, the variant sequence having approximately or at least 90% sequence identity to the aforementioned sequence. In various embodiments, the antigen-binding protein includes a variant sequence of the sequence mentioned, the variant sequence having approximately or at least 95% sequence identity to the aforementioned sequence.

[0084] In various embodiments, the antigen-binding protein includes at least one mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to one, two, three, four, or five sequences from the sequence numbers in a horizontal row of Table A, and any of sequence numbers 8-133. In various embodiments, the antigen-binding proteins are (a) SEQ ID NOs. 74-79, (b) SEQ ID NOs. 50-55, (c) SEQ ID NOs. 122-127, (d) SEQ ID NOs. 26-31, (e) SEQ ID NOs. 128-133, (f) SEQ ID NOs. 38-43, (g) SEQ ID NOs. 62-67, (h) SEQ ID NOs. 80-85, (i) SEQ ID NOs. 44-49, (j) SEQ ID NOs. 86-91, (k) SEQ ID NOs. 104-109, (l) SEQ ID NOs. 56-61, (m) SEQ ID NOs. 32-37, (n) SEQ ID NOs. 110-115, (o) SEQ ID NOs. 98-103, (p) SEQ ID NOs. 92- 97, comprising one, two, three, four, or five sequences from a set of sequences selected from (q) SEQ ID NOs. 116-121, (r) SEQ ID NOs. 8-13, (s) SEQ ID NOs. 68-73, (t) SEQ ID NOs. 14-19, and (u) SEQ ID NOs. 20-25, wherein such antigen-binding protein further comprises at least one mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to at least one of the sequences in that set. For example, in various embodiments, the antigen-binding protein includes four sequences from sequence numbers 74-79, i.e., sequence numbers 74-77, where the antigen-binding protein includes two mutant sequences: one mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to sequence number 78, and the other mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to sequence number 79.

[0085] In various embodiments, the antigen-binding protein contains a pair of mutant sequences having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175. In various cases, the antigen-binding protein contains (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 166 and 167, (l) SEQ ID NOs: 150 and 151, (m) SEQ ID NOs: 142 The antigen-binding protein includes a pair of mutant sequences having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to (n) SEQ ID NOs: 168 and 169, (o) SEQ ID NOs: 164 and 165, (p) SEQ ID NOs: 162 and 163, (q) SEQ ID NOs: 170 and 171, (r) SEQ ID NOs: 134 and 135, (s) SEQ ID NOs: 154 and 155, (t) SEQ ID NOs: 136 and 137, and (u) SEQ ID NOs: 138 and 139. In various embodiments, the antigen-binding protein includes a pair of sequences, one of which is the sequence in Table B, and the other is a mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 134-175.In various embodiments, the antigen-binding protein comprises a pair of sequences, one of which is (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 166 and 167, (l) SEQ ID NOs: 150 and 151, (m) SEQ ID NOs: 142 and 143, (n (a) Sequence IDs 168 and 169, (o) Sequence IDs 164 and 165, (p) Sequence IDs 162 and 163, (q) Sequence IDs 170 and 171, (r) Sequence IDs 134 and 135, (s) Sequence IDs 154 and 155, (t) Sequence IDs 136 and 137, and (u) Sequence IDs 138 and 139, wherein the other sequence is a variant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to sequences (a) to (u). For example, in various embodiments, an antigen-binding protein contains the sequence of Sequence ID 134, and such antigen-binding protein further contains a variant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to Sequence ID 135.

[0086] In various cases, the antigen-binding protein contains the amino acid sequence described above and has one or more amino acid substitutions to reduce or remove reactive amino acids in order to reduce or prevent undesirable side-chain reactions. For example, the antigen-binding protein contains the amino acid sequence described above and has one or more of the following: (i) a Trp residue is substituted with His, Tyr, or Phe; (ii) an Asn residue is substituted with Gln, Ser, Ala, or Asp; (iii) an Asp residue immediately preceding a Pro residue is substituted with Ala, Ser, or Glu; (iv) an Asn residue is substituted with Gln, Ser, or Ala; and / or (v) a Cys residue is substituted with Tyr, Ser, or Ala. In various embodiments, the antigen-binding protein contains the amino acid sequence described above and has amino acid substitutions that are predicted to have higher binding affinity, higher stability, or other beneficial properties based on SHM events or statistical analysis of many other similar antibody sequences. In some embodiments, the antigen-binding protein is (a) an HC CDR1 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs. 452, 455, 461, 465, 71, and 472, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) an HC CDR2 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs. 475, 456, 462, 466, 468, and 473, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) an HC CDR2 amino acid sequence listed in Table A1(d) CDR3 amino acid sequences, or sequences selected from the group consisting of SEQ ID NOs. 453, 457, 463, 467, 469, and 474, or variant sequences thereof, which differ by only one or two amino acids, or have approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) LC CDR1 amino acid sequences listed in Table A1, or sequences selected from the group consisting of SEQ ID NOs. 449, 476, 458, 464, 68, and 470, or variant sequences thereof, which differ by only one or two amino acids, or have approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) LC CDR1 amino acid sequences listed in Table A1 (f) A CDR2 amino acid sequence, or a sequence selected from the group consisting of SEQ ID NOs. 450, 477, 459, 57, 69, and 471, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (f) An LC CDR3 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs. 451, 454, 460, 58, 70, and 112, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (g) A combination of any two or more of (a) to (f). [Table 4]

[0087] In some embodiments, HC CDR1 contains Gly immediately adjacent to the N-terminus of SEQ ID NO: 452, and optionally, in some embodiments, HC CDR1 contains MX immediately adjacent to the C-terminus of SEQ ID NO: 452, where X is H, N, or S. In various embodiments, HC CDR3 contains Ala immediately adjacent to the N-terminus of SEQ ID NO: 453. In various embodiments, LC CDR1 further contains TAS immediately adjacent to the N-terminus of SEQ ID NO: 449, and optionally, XH immediately adjacent to the C-terminus of SEQ ID NO: 449, where X is H, S, Y, or Q. In some embodiments, the first amino acid of SEQ ID NO: 449 is S or Q, as described below. In some embodiments, the first amino acid of SEQ ID NO: 451 is S or Q, as described below.

[0088] In various embodiments, HC CDR1 includes Gly immediately adjacent to the N-terminus of SEQ ID NO: 455, and optionally includes MX immediately adjacent to the C-terminus of SEQ ID NO: 455, where X is N, S, or H. In some embodiments, HC CDR2 includes Gln immediately adjacent to the N-terminus of SEQ ID NO: 456, and optionally includes H immediately adjacent to the C-terminus of SEQ ID NO: 456. In various embodiments, LC CDR1 includes RIS immediately adjacent to the N-terminus of SEQ ID NO: 476, and optionally includes LA immediately adjacent to the C-terminus of SEQ ID NO: 476. In various embodiments, LC CDR2 includes XLVE immediately adjacent to the C-terminus of SEQ ID NO: 477, where X is I or S.

[0089] In various embodiments, HC CDR1 contains MH immediately adjacent to the C-terminus of SEQ ID NO: 461. In various embodiments, HC CDR2 contains Tyr immediately adjacent to the N-terminus of SEQ ID NO: 462, and optionally contains TH immediately adjacent to the C-terminus of SEQ ID NO: 462. In exemplary embodiments, HC CDR3 does not contain the first two amino acids of SEQ ID NO: 463. In various embodiments, LC CDR1 contains RSS immediately adjacent to the N-terminus of SEQ ID NO: 458, and optionally contains LN immediately adjacent to the C-terminus of SEQ ID NO: 458. In various embodiments, LC CDR2 contains XRFS immediately adjacent to the C-terminus of SEQ ID NO: 459, where X is Q, S, A, or D.

[0090] In various embodiments, HC CDR1 contains MH immediately adjacent to the C-terminus of SEQ ID NO: 465. In various embodiments, HC CDR2 contains YI immediately adjacent to the N-terminus of SEQ ID NO: 466, and optionally contains Xaa immediately adjacent to the C-terminus of SEQ ID NO: 466, where Xaa is N, S, Q, or A. In various embodiments, LC CDR1 contains LAS immediately adjacent to the N-terminus of SEQ ID NO: 464, and optionally contains LA immediately adjacent to the C-terminus of SEQ ID NO: 464. In various embodiments, LC CDR2 contains SLAD immediately adjacent to the C-terminus of SEQ ID NO: 57.

[0091] In various embodiments, HC CDR1 contains MH immediately adjacent to the C-terminus of SEQ ID NO: 71. In various embodiments, HC CDR2 contains Tyr immediately adjacent to the N-terminus of SEQ ID NO: 468, and optionally IY immediately adjacent to the C-terminus of SEQ ID NO: 468. In various embodiments, LC CDR1 contains RAS immediately adjacent to the N-terminus of SEQ ID NO: 68, and optionally SYIH immediately adjacent to the C-terminus of SEQ ID NO: 68. In various embodiments, LC CDR2 contains XLES immediately adjacent to the C-terminus of SEQ ID NO: 69, where X is N, Q, S, A, or D.

[0092] In various embodiments, LC CDR1 includes KSS immediately adjacent to the N-terminus of SEQ ID NO: 470, and optionally YLA immediately adjacent to the C-terminus of SEQ ID NO: 470. In various embodiments, LC CDR2 includes TRES immediately adjacent to the C-terminus of SEQ ID NO: 471. In various embodiments, HC CDR1 includes MN immediately adjacent to the C-terminus of SEQ ID NO: 472. In various embodiments, HC CDR2 includes Xaa immediately adjacent to the N-terminus of SEQ ID NO: 473, where Xaa is N, Q, S, or A, and optionally Thr immediately adjacent to the C-terminus of SEQ ID NO: 473.

[0093] In various embodiments, the antigen-binding protein comprises the LC CDR1 amino acid sequence, the LC CDR2 amino acid sequence, and the LC CDR3 amino acid sequence described in Table A1, and at least one or two of the HC CDR amino acid sequences described in Table A1.

[0094] In various embodiments, the antigen-binding protein includes at least three, four, or five amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein includes each of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A1, and at least one or two of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein includes each of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A1, and at least one or two of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein includes all six CDR amino acid sequences specified by the sequence numbers in a row in Table A1. In various embodiments, the antigen-binding protein contains six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 449-453 and 475, (b) SEQ ID NOs: 476-477 and 454-457, (c) SEQ ID NOs: 458-463, (d) SEQ ID NOs: 57, 58, 464-467, (e) SEQ ID NOs: 68-71 and 468-469, and (f) SEQ ID NOs: 112 and 470-474.

[0095] In various cases, the amino acid sequences in Table A1 are separated by at least one (for example, at least two, three, four, five, six, seven, eight, nine, ten, or more) intervening amino acids. In various cases, there are approximately 10 to 20 amino acids between the LC CDR1 and LC CDR2 sequences, and approximately 25 to 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are approximately 14 to 16 amino acids between the LC CDR1 and LC CDR2 sequences, and approximately 30 to 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various cases, there are approximately 10 to 20 amino acids between the HC CDR1 and HC CDR2 sequences, and approximately 25 to 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various cases, there are approximately 14 to 16 amino acids between the HC CDR1 and HC CDR2 sequences, and approximately 30 to 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0096] In various embodiments, the antigen-binding protein includes (a) a heavy chain variable region amino acid sequence listed in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 478, 480, 482, 484, 486, and 488, or a variant thereof, which differs by only one or two amino acids or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) a light chain variable region amino acid sequence listed in Table B1, or a sequence selected from the group consisting of SEQ ID NOs: 479, 481, 483, 485, 487, and 489, or a variant thereof, which differs by only one or two amino acids or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both (a) and (b). [Table 5]

[0097] In various embodiments, the antigen-binding protein comprises a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 478 and 479, (b) SEQ ID NOs: 480 and 481, (c) SEQ ID NOs: 482 and 483, (d) SEQ ID NOs: 484 and 485, (e) SEQ ID NOs: 486 and 487, and (f) SEQ ID NOs: 488 and 489. In various embodiments, the antigen-binding protein comprises a variant sequence of the sequence having the SEQ ID NOs listed in Table B1, which differs by only one or two amino acids or has approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity, where the differing amino acid(s) are located at the positions described in the "Humanized Antibody" section below.

[0098] Humanized antibodies

[0099] In various embodiments, the antigen-binding protein is a humanized version of the antigen-binding protein described in Table A, Table A1, Table B, or Table B1.

[0100] Humanized AB1

[0101] In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, having one or more amino acid substitutions in the heavy chain variable region at one or more of the following positions: 5, 8, 11, 12, 13, 20, 31, 33, 35, 38, 40, 48, 50, 55, 57, 59, 61, 65, 66, 67, 68, 70, 72, 74, 76, 79, 80, 82, 87, 90, 91, 98, 101, and 116 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 428. In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 20, 31, 35, 48, 50, 59, 67, 70, 74, 79, 98, 101 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 429. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 6]

[0102] In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, with the following positions within the light chain variable region: 1, 3, 4, 9, 10, 11, 15, 17, 21, 24, 27, 29, 32, 34, 35, 43, 44, 48, 51, 52, 53, 54, 55, 56, 61, 67, 71, 72, 73, 79, 80, 81, 84, 90, 92, It has one or more amino acid substitutions in one or more of 93, 94, 95, 96, 101, 107 (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 430. In various embodiments, the antigen-binding protein is a humanized version of AB1 as described in Table B or B1, and has one or more amino acid substitutions in the light chain variable region at one or more of the following positions: 4, 21, 32, 34, 48, 51, 53, 61, 67, 79, 84, 91, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 431. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 7]

[0103] Humanized AB3

[0104] In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, having one or more amino acid substitutions in the heavy chain variable region at one or more of the following positions: 3, 5, 18, 19, 23, 31, 33, 35, 40, 42, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 76, 79, 80, 81, 87, 94, 95, 99, 106, 112, 114 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 432. In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 31, 35, 50, 55, 79, 99, 106 (e.g., 1, 2, 3, 4, 5, 6, or 7). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 433. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below.

[0105] [Table 8]

[0106] In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, and has one or more amino acid substitutions in the light chain variable region at one or more of the following positions: 9, 17, 18, 25, 27, 28, 30, 34, 40, 43, 45, 48, 50, 52, 53, 55, 56, 70, 72, 74, 76, 84, 85, 90, 91, 93, 94, 97, and 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 434. In various embodiments, the antigen-binding protein is a humanized version of AB3 as described in Table B or B1, and has one or more amino acid substitutions in the light chain variable region at one or more of the following positions: 25, 34, 48, 53, 55, 84, 85, 90, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 435. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 9]

[0107] Humanized AB4

[0108] In various embodiments, the antigen-binding protein is a humanized version of AB4 as described in Table B or B1, with the following positions within the heavy chain variable region: 5, 11, 12, 13, 20, 29, 31, 33, 37, 38, 40, 45, 48, 50, 55, 56, 57, 59, 61, 62, 65, 66, 67, 68, 70, 72, 74, 76, 79, 8 It has one or more amino acid substitutions in one or more of the following: 2, 84, 87, 91, 97, 101, 117 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 436. In various embodiments, the antigen-binding protein is a humanized version of AB4 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 20, 29, 31, 37, 45, 48, 56, 59, 61, 62, 65, 66, 68, 70, 74, 79, 84, 97, and 101 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 437. In various embodiments, the amino acids at the positions listed above are selected from the amino acids in the table below. [Table 10]

[0109] In various embodiments, the antigen-binding protein is a humanized version of AB4 as described in Table B or B1, and has one or more amino acid substitutions in the light chain variable region at one or more of the following positions: 7, 14, 17, 18, 31, 33, 39, 41, 42, 44, 50, 51, 55, 57, 60, 81, 88, 92, 94, 95, 96, 99, 100, 105 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 438. In various embodiments, the antigen-binding protein is a humanized version of AB4 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 33, 39, 55, 57, 81, 95, and 96 (e.g., 1, 2, 3, 4, 5, 6, or 7). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 439. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 11]

[0110] Humanized AB18

[0111] In various embodiments, the antigen-binding protein is a humanized version of AB18 as described in Table B or B1, having one or more amino acid substitutions in the heavy chain variable region at one or more of the following positions: 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 65, 67, 68, 70, 72, 74, 76, 79, 82, 84, 87, 91, and 116 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), and optionally, one or more of the following positions: 20, 48, 68, 70, 79 (e.g., 1, 2, 3, 4, or 5). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 440 or 441. In various embodiments, the amino acids at the positions listed above are selected from the amino acids shown in the table below. [Table 12]

[0112] In various embodiments, the antigen-binding protein is a humanized version of AB18 as described in Table B or B1, and has one or more amino acid substitutions in the light chain variable region at one or more of the following positions: 1, 3, 9, 15, 18, 19, 21, 22, 49, 51, 69, 93, 84, 78, 105, and 111 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16), and optionally, one or more of the following positions: 19, 21, or 84 (e.g., 1, 2, or 3). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 442 or 443. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below.

[0113] [Table 13]

[0114] Humanized AB9

[0115] In various embodiments, the antigen-binding protein is a humanized version of AB9 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 1, 5, 9, 11, 12, 20, 38, 40, 41, 43, 44, 48, 61, 63, 65, 67, 69, 70, 72, 73, 74, 76, 79, 84, 87, 91, 93, 112, and 113 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In various cases, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 444. In various embodiments, the amino acids at the positions listed above are selected from the amino acids listed in the table below. [Table 14]

[0116] In various embodiments, the antigen-binding protein is a humanized version of AB9 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions in the light chain variable region: 9, 11, 15, 17, 18, 43, 45, 70, 72, 73, 74, 80, 84, 85, and 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 445. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 15]

[0117] Humanized AB11

[0118] In various embodiments, the antigen-binding protein is a humanized version of AB11 as described in Table B or B1, and has one or more amino acid substitutions at one or more of the following positions within the heavy chain variable region: 1, 15, 18, 19, 42, 49, 63, 75, 76, 78, 80, 84, 88, and 93 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 446. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 16]

[0119] In various embodiments, the antigen-binding protein is a humanized version of AB11 as described in Table B or B1, and has one or more amino acid substitutions in the light chain variable region at one or more of the following positions: 4, 9, 17, 22, 64, 78, 80, 81, 82, 83, 84, 87, 89, 104, and 110 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), and optionally at one or more of the following positions: 4, 82, and 110. In various embodiments, the antigen-binding protein contains the amino acid sequence of SEQ ID NO: 447 or 448. In various embodiments, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 17]

[0120] In various embodiments, the antigen-binding protein is (a) a heavy chain variable region amino acid sequence as described in Table C, or a sequence selected from the group consisting of 376-379, 384-387, 391-396, 403-408, 412, 413, 416-419, and 422-427, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% sequence identity. (b) light chain variable region amino acid sequences listed in Table C, or sequences selected from the group consisting of 380-383, 388-390, 397-402, 409-411, 414, 415, 420, and 421, or variant sequences thereof that differ by only one or two amino acids, or have approximately or at least 70%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% sequence identity, or (c) both (a) and (b).

[0121] [Table 18]

[0122] In various embodiments, the humanized antigen-binding protein comprises a pair of amino acid sequences shown in Table D. [Table 19] TIFF0007856818000024.tif101170

[0123] In various embodiments, the antigen-binding protein comprises a pair of mutant sequences, each having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence numbers listed in Table C. In various embodiments, the antigen-binding protein comprises a pair of sequences, one of which is a sequence selected from the sequence numbers listed in Table C, and the other is a mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence having the sequence number listed in Table D or Table C.

[0124] In various embodiments, the antigen-binding protein comprises a pair of sequences, one of which is a sequence selected from the sequence numbers listed in Table D, and the other sequence is a mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence having the sequence number listed in Table D. For example, in various embodiments, the antigen-binding protein comprises the sequence of sequence number 419, and such antigen-binding protein further comprises a mutant sequence having approximately or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to sequence number 421.

[0125] In various cases, the antigen-binding protein is a humanized antigen-binding protein having one or more amino acid substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) in the heavy chain (HC) variable region, the light chain (LC) variable region, or both, as listed in Table D. In an exemplary embodiment, the antigen-binding protein is a humanized antigen-binding protein named AB1-11, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the antigen-binding protein contains the HC of SEQ ID NO: 379 and has one, two, three, four, or five amino acid substitutions. In exemplary embodiments, the antigen-binding protein includes HC CDR1 of SEQ ID NO: 504, HC CDR2 of SEQ ID NO: 505, HC CDR3 of SEQ ID NO: 506, or a combination thereof. In exemplary cases, the antigen-binding protein includes the HC of SEQ ID NO: 503. In some embodiments, the antigen-binding protein includes one of the HCs from SEQ ID NOs: 496-501. In some embodiments, the antigen-binding protein includes the HC sequences shown as S7-S12 in Figure 22. In various cases, the light chain variable region includes LC CDR1 of SEQ ID NO: 449, LC CDR2 of SEQ ID NO: 450, LC CDR3 of SEQ ID NO: 451, or a combination thereof. In some embodiments, the antigen-binding protein includes one of the LCs from SEQ ID NOs: 380-383 and 479. In exemplary cases, the antigen-binding protein includes the LC of SEQ ID NO: 383. In some embodiments, the antigen-binding protein includes the LC sequences shown as S7-S12 in Figure 22. In exemplary embodiments, the antigen-binding protein is a humanized antigen-binding protein AB3-7 having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the antigen-binding protein contains the HC of SEQ ID NO: 387 and has one, two, three, four, five, or six amino acid substitutions. In exemplary embodiments, the antigen-binding protein includes HC CDR1 of SEQ ID NO: 507, HC CDR2 of SEQ ID NO: 508, HC CDR3 of SEQ ID NO: 509, or a combination thereof.In exemplary cases, the antigen-binding protein contains the HC of SEQ ID NO: 502. In some embodiments, the antigen-binding protein contains one of the HCs of SEQ ID NOs: 490-495. In some embodiments, the antigen-binding protein contains the HC sequences shown as S1-S6 in Figure 22. In various cases, the light chain variable region contains LC CDR1 of SEQ ID NO: 476, LC CDR2 of SEQ ID NO: 477, LC CDR3 of SEQ ID NO: 454, or a combination thereof. In some embodiments, the antigen-binding protein contains one of the LCs of SEQ ID NOs: 388-390 and 481. In exemplary cases, the antigen-binding protein contains the LC of SEQ ID NO: 389. In some embodiments, the antigen-binding protein contains the LC sequences shown as S1-S6 in Figure 22. In exemplary embodiments, the antigen-binding protein is the humanized antigen-binding protein of AB3, and has one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the antigen-binding protein contains the HC of SEQ ID NO: 139 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein contains any one of the HCs of SEQ ID NO: 510. In some embodiments, the antigen-binding protein contains the HC sequence of SEQ ID NO: 510 and has one, two, three, four, or five (or more) amino acid substitutions as shown in Figure 23. In exemplary embodiments, the antigen-binding protein contains the HC of SEQ ID NO: 138 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein contains any one of the HCs of SEQ ID NO: 511. In some embodiments, the antigen-binding protein contains the HC sequence of SEQ ID NO: 511 and has one, two, three, four, or five (or more) amino acid substitutions as shown in Figure 24. In exemplary embodiments, the antigen-binding protein is a humanized antigen-binding protein of AB1, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the antigen-binding protein contains the HC of SEQ ID NO: 135 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein contains one of the HCs of SEQ ID NO: 513.In some embodiments, the antigen-binding protein contains the HC sequence of SEQ ID NO: 513 and has one, two, three, four, or five (or more) amino acid substitutions as shown in Figure 25. In exemplary embodiments, the antigen-binding protein contains the HC sequence of SEQ ID NO: 134 and has one, two, three, four, or five (or more) amino acid substitutions. In some embodiments, the antigen-binding protein contains any one of the HC sequences of SEQ ID NO: 512. In some embodiments, the antigen-binding protein contains the HC sequence of SEQ ID NO: 512 and has one, two, three, four, or five (or more) amino acid substitutions as shown in Figure 26.

[0126] Defucosylated antibodies

[0127] Many secreted proteins undergo post-translational glycosylation, a process in which a sugar moiety (e.g., glycan, sugar) is covalently bonded to a specific amino acid in the protein. In eukaryotic cells, two types of glycosylation occur: (1) N-linked glycosylation, where the glycan is bound to asparagine in the recognition sequence Asn-X-Thr / Ser, where "X" is any amino acid other than proline; and (2) O-linked glycosylation, where the glycan is bound to serine or threonine. Regardless of the type of glycosylation (N-linked [glycosylation] or O-linked [glycosylation]), there is a small heterogeneity in the glycoform of proteins due to the wide range of glycan structures that associate with each site (O or N).

[0128] All N-glycans have a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr(Man3GlcNAc2Asn) and are classified into three types: (A) high-mannose (HM) or oligomannose (OM) types consisting of two N-acetylglucosamine (GalNAc) moieties and numerous (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues; (B) complex types containing more than two GlcNAc moieties and any number of other types of sugars; or (C) hybrid types containing Man residues in one branch and GlcNAc at the base of the complex branch. Figure 1A (Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2 nd (Obtained from Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans.

[0129] N-linked glycans typically contain one or more monosaccharides, including galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (ClcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-doxynononic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (Gal A), and mannuronic acid (Man A). Common symbols used for such sugars are shown in Figure 29A.

[0130] N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex series of reactions leads to the binding of a coreglycan structure essentially composed of two GlcNAc residues and three Man residues. The glycan complex formed in the ER is modified by enzymes in the Golgi apparatus. If the sugar is relatively inaccessible to the enzyme, it typically remains in its original HM form. If the enzyme can access the sugar, many of the Man residues are cleaved, further modifying the sugar to create a complex N-glycan structure. For example, mannosidase-1 located in the cis-Golgi can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8 located in the medial-Golgi fucosylates glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0131] Therefore, the sugar composition and structural configuration of glycan structures differ depending, among other factors, particularly the glycosylation mechanism and Golgi apparatus within the ER, the accessibility of the enzymes of the mechanism to the glycan structure, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation mechanism.

[0132] In exemplary embodiments of this disclosure, the antigen-binding protein comprises an Fc polypeptide. As used herein, the term “Fc polypeptide” includes polypeptides in their native and mutant protein forms derived from the Fc region of an antibody. In exemplary embodiments, the Fc polypeptide of the antigen-binding protein disclosed herein comprises a glycan. In various cases, the glycan is either fucose-deficient or defucosylated. In exemplary embodiments, the antigen-binding protein comprises a defucosylated glycan. As used herein, the terms “defucosylated glycan” or “afucoglycan” or “defucosylated glycoform” or “Afuc” refer to a glycoform lacking core fucose, for example, the α1,6-linked fucose on the GlcNAc residue involved in the amide bond with Asn at the N-glycosylation site. Examples of defucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Additional defucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3]. See, for example, Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015).

[0133] This disclosure also provides compositions comprising antigen-binding proteins comprising Fc polypeptides containing defucosylated glycans, such as pharmaceutical compositions. In exemplary embodiments, about or at least 25% of the antigen-binding proteins present in the composition are antigen-binding proteins comprising Fc polypeptides containing defucosylated glycans. In exemplary embodiments, about or at least 25% of the antigen-binding proteins present in the composition are defucosylated. Optionally, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the antigen-binding proteins present in the composition are defucosylated. Methods for producing compositions comprising antigen-binding proteins with specific glycoprofiles are known in the art. In exemplary embodiments, the antigen-binding proteins are recombinant, produced in cells whose genes have been recombined to alter the activity of enzymes in a de novo or salvage pathway. These two fucose metabolic pathways are shown in Figure 29B. In exemplary embodiments, cells are genetically recombined to alter the activity of one or more of the following: fucosyltransferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinases, GDP-fucospyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase,4-reductase (FX). In exemplary embodiments, cells are genetically recombined to knock out the gene encoding FX. For example, see International Patent Publication WO2017 / 079165A1, Kanda et al., J Biotechnol 130, 2007, 300-310, Yamane-Ohunuki et al., Biotechnol Bioeng 87, 2004, 614-622, and Malphettes et al., Biotechnol Bioeng 106, 2010, 774-783.

[0134] nucleic acid

[0135] This disclosure further provides nucleic acids comprising nucleotide sequences encoding antigen-binding proteins of this disclosure. As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, or a modified form thereof, which may be single-stranded or double-stranded, synthetic, or obtained from naturally occurring materials (e.g., isolated and / or purified), may contain natural, unnatural, or modified nucleotides, and may contain natural, unnatural, or modified nucleotide bonds, such as phosphoramidate bonds or phosphorothioate bonds, instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. A nucleic acid may contain any nucleotide sequence encoding any of the antigen-binding proteins of this disclosure. In various embodiments, nucleic acids are (a) the amino acid sequence of heavy chain (HC) complementarity-determining region (CDR) 1 listed in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a variant thereof, which differs by only one or two amino acids or has approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity, or (b) the HC listed in Table A or A1 (c) CDR2 amino acid sequences, or sequences selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468, and 473, or variant sequences thereof that differ by only one or two amino acids, or have approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity, (c) HC CDR3 amino acid sequences listed in Table A or A1, or SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67,(d) A sequence selected from the group consisting of 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity, (d) a light chain (LC) CDR1 amino acid sequence or sequence number listed in Table A or A1 (e) LCs selected from the group consisting of numbers 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or variant sequences thereof that differ by only one or two amino acids, or have approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity, (e) LCs listed in Table A or A1 (f) LCs listed in Table A, which are CDR2 amino acid sequences, or sequences selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or variant sequences thereof that differ by only one or two amino acids, or have approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity, (f) LCs listed in Table A The amino acid sequence of CDR3, or a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity.Alternatively, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising any two or more combinations of (g)(a) to (f). In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence described in Table A or A1, and at least one or two of the HC CDR amino acid sequences described in Table A or A1. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein comprising the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, and HC CDR3 amino acid sequence described in Table A or A1, and at least one or two of the LC CDR amino acid sequences described in Table A or A1. In various embodiments, the nucleic acid is (a) at least three, four, or five amino acid sequences specified by the sequence numbers in a row in Table A or A1; (b) each of the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A or A1 and at least one or two of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A or A1; (c) each of the HC CDR amino acid sequences specified by the sequence numbers in a row in Table A or A1 and the LC CDR amino acid sequences specified by the sequence numbers in a row in Table A or A1 (d) at least one or two CDR amino acid sequences, all six CDR amino acid sequences specified by the sequence numbers in the horizontal row of Table A, and / or (e) (a) SEQ ID NOs. 74-79, (b) SEQ ID NOs. 50-55, (c) SEQ ID NOs. 122-127, (d) SEQ ID NOs. 26-31, (e) SEQ ID NOs. 128-133, (f) SEQ ID NOs. 38-43, (g) SEQ ID NOs. 62-67, (h) SEQ ID NOs. 80-85, (i) SEQ ID NOs. 44-49, (j) SEQ ID NOs. 86-91 (k) Sequence IDs 104-109, (l) Sequence IDs 56-61, (m) Sequence IDs 32-37, (n) Sequence IDs 110-115, (o) Sequence IDs 98-103, (p) Sequence IDs 92-97, (q) Sequence IDs 116-121, (r) Sequence IDs 8-13, (s) Sequence IDs 68-73, (t) Sequence IDs 14-19, (u) Sequence IDs 20-25, (v) Sequence IDs 449-453 and 475, (w) Sequence IDs 476-477, 454-457, (x) Sequence IDs 458-463,(y) Containing a nucleotide sequence encoding an antigen-binding protein comprising six CDR amino acid sequences selected from the group consisting of SEQ ID NOs. 57, 58, 464-467, (z) SEQ ID NOs. 68-71 and 468-469, and (aa) SEQ ID NOs. 112 and 470-474. In various embodiments, the nucleic acid is (a) a heavy chain variable region amino acid sequence as described in Table B or B1, or a sequence selected from the group consisting of 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 478, 480, 482, 484, 486 and 488, or a variant thereof, which differs by only one or two amino acids or has approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity, or (b) a light chain variable region amino acid sequence as described in Table B or B1. A variant amino acid sequence, or a sequence selected from the group consisting of 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 479, 481, 483, 485, 487, and 489, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., approximately or at least 80%, approximately or at least 85%, approximately or at least 90%, approximately or at least 95%) sequence identity, or a nucleotide sequence encoding an antigen-binding protein including both (a) and (b). In various embodiments, the nucleic acids are (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 166 and 167, (l) SEQ ID NOs: 150 and 151, (m) SEQ ID NOs: 142 and 143, (n) SEQ ID NOs: 168 and 169, (o) SEQ ID NOs: 164 and 165,The nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein containing a pair of amino acid sequences selected from the group consisting of (p) SEQ ID NOs: 162 and 163, (q) SEQ ID NOs: 170 and 171, (r) SEQ ID NOs: 134 and 135, (s) SEQ ID NOs: 154 and 155, (t) SEQ ID NOs: 136 and 137, and (u) SEQ ID NOs: 138 and 139. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein containing a pair of amino acid sequences selected from the group consisting of pairs listed in Table D. In various embodiments, the nucleic acid comprises a nucleotide sequence containing one or more sequences from SEQ ID NOs: 208 to 375. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid contains one or more insertions, deletions, inversions, and / or substitutions.

[0136] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which is a humanized antigen-binding protein having one or more amino acid substitutions in the heavy chain (HC) variable region, the light chain (LC) variable region, or both (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) as listed in Table D. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which is a humanized antigen-binding protein named AB1-11 having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which contains the HC of Sequence ID No. 379 having one, two, three, four, or five amino acid substitutions. In exemplary embodiments, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing HC CDR1 of SEQ ID NO: 504, HC CDR2 of SEQ ID NO: 505, HC CDR3 of SEQ ID NO: 506, or a combination thereof. In exemplary cases, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing HC of SEQ ID NO: 503. In some embodiments, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing any one of SEQ ID NOs: 496-501. In some embodiments, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing HC sequences shown as S7-S12 in Figure 22. In various cases, the nucleic acid includes a nucleotide sequence encoding a light chain variable region containing LC CDR1 of SEQ ID NO: 449, LC CDR2 of SEQ ID NO: 450, LC CDR3 of SEQ ID NO: 451, or a combination thereof. In some embodiments, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing any one of SEQ ID NOs: 380-383 and 479. In exemplary cases, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing LC of SEQ ID NO: 383. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein, which includes the LC sequences shown as S7-S12 in Figure 22.In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which is a humanized antigen-binding protein named AB3-7 having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, five, or six amino acid substitutions, including the HC of SEQ ID NO: 387. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which includes HC CDR1 of SEQ ID NO: 507, HC CDR2 of SEQ ID NO: 508, HC CDR3 of SEQ ID NO: 509, or a combination thereof. In exemplary cases, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which includes the HC of SEQ ID NO: 502. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which includes any one of the HCs from SEQ ID NOs: 490-495. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein which includes the HC sequences shown as S1-S6 in Figure 22. In various cases, the nucleic acid includes a nucleotide sequence encoding a light chain variable region containing LC CDR1 of SEQ ID NO: 476, LC CDR2 of SEQ ID NO: 477, LC CDR3 of SEQ ID NO: 454, or a combination thereof. In some embodiments, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing one of the LCs from SEQ ID NOs: 388-390 and 481. In an exemplary case, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing the LC of SEQ ID NO: 389. In some embodiments, the nucleic acid includes a nucleotide sequence encoding an antigen-binding protein containing the LC sequences shown as S1-S6 in Figure 22.

[0137] In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein, which is a humanized antigen-binding protein of AB3, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, including the HC of SEQ ID NO: 139. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein containing any one of the HCs of SEQ ID NO: 510. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, as shown in Figure 23, including the HC sequence of SEQ ID NO: 510. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, including the HC of SEQ ID NO: 138. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein containing any one of the HCs of SEQ ID NO: 511. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, as shown in Figure 24, including the HC sequence of SEQ ID NO: 511. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein, which is a humanized antigen-binding protein of AB1, having one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, including the HC sequence of SEQ ID NO: 135. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, as shown in Figure 25, including the HC sequence of SEQ ID NO: 513.In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, including the HC of SEQ ID NO: 134. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one of the HCs of SEQ ID NO: 512. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein having one, two, three, four, or five (or more) amino acid substitutions, as shown in Figure 26, including the HC sequence of SEQ ID NO: 512. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid contains one or more insertions, deletions, inversions, and / or substitutions.

[0138] In some embodiments, the nucleic acids of this disclosure are recombinant. As used herein, the term “recombinant” means (i) a molecule constructed by ligating a native or synthetic nucleic acid segment outside a living cell to a nucleic acid molecule that can be replicated within a living cell, or (ii) a molecule obtained from replication of the molecules described in (i) above. For the purposes herein, replication may be either in vitro or in vivo.

[0139] Nucleic acids in some embodiments are constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al. (cited above) and Ausubel et al. (cited above). For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to enhance the biological stability of the molecule or the physical stability of the double helix formed during hybridization. Examples of modified nucleotides that can be used to produce nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosin, inosine, N 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylammomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6Examples of nucleic acids include, but are not limited to, isopentenyl adenine, uracil-5-oxyacetic acid (v), wiebtoxosin, pseudouracil, cueosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of this disclosure may be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0140] vector

[0141] In some embodiments, the nucleic acids of this disclosure are incorporated into vectors. In this regard, this disclosure provides vectors comprising any of the nucleic acids disclosed herein. In various embodiments, the vectors are recombinant expression vectors. For the purposes herein, the term “recombinant expression vector” means a genetically recombinant oligonucleotide or polynucleotide construct that enables the expression of mRNA, protein, polypeptide, or peptide by a host cell, in which case the construct comprises a nucleotide sequence encoding such mRNA, protein, polypeptide, or peptide, and the vector comes into contact with the cell under conditions sufficient to cause the expression of mRNA, protein, polypeptide, or peptide in the cell. The vectors of this disclosure are generally not found in nature; however, portions of the vectors may be found in nature. The vectors disclosed herein may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic, or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The vectors may contain naturally occurring nucleotide-nucleotide bonds, non-natural nucleotide-nucleotide bonds, or both types of bonds. In some embodiments, modified nucleotides or internucleotide bonds not present in nature do not interfere with the transcription or replication of the vector.

[0142] The vectors of this disclosure may be any suitable vector and can be used to transduce, transform, or transfect any suitable host. Suitable vectors include those designed for propagation and / or expansion, or for expression, or both, such as plasmids and viruses. The vector may be an expression vector using a plasmid. In various embodiments, the vector is selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJoIIa, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGTIO, λGTl1, λZapII (Stratagene), λEMBL4, and λNMl149, can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, the vector is a viral vector, such as a retroviral vector. In various embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, a vaccinia virus vector, or a lentivirus vector. See, for example, Howarth et al., Cell Biol. Toxicol. 26(1):1-20 (2010). In various embodiments, the vector is a baculovirus vector that infects arthropods, such as insects. In various forms, the baculovirus vector is either Autographacalifornica multiple nuclear virus (AcMNPV) or Bombyxmorinuclear polyhedrosis (BmNPV).See, for example, Khan, Adv Pharm Bull 3(2):257-263 (2013), Miller, Bioessays 11(4):91-96 (1989), and Atkinson et al., Pestic Sci 28:215-224 (1990).

[0143] The vectors of this disclosure can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al. (cited above) and Ausubel et al. (cited above). The circular or linear expression vector constructs can be prepared to contain a replication system that functions in host prokaryotic or host eukaryotic cells. The replication system may be derived from, for example, CoIEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0144] In some embodiments, the vector includes regulatory sequences such as start and stop codons for transcription and translation that are specific to the type of host to which the vector is introduced (e.g., bacteria, fungi, plants, or animals), and whether the vector is DNA-based or RNA-based is considered as needed.

[0145] The vector may include one or more marker genes that enable selection of the transformed or transfected host. Marker genes include those for biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and complementarity in the nutritionally required host to provide protrophotrophy. Suitable marker genes for the expression vectors disclosed herein include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0146] The vector may include a nucleotide sequence encoding a polypeptide (including its functional portion and functional variants), or a natural or standard promoter functionally linked to a nucleotide sequence complementary to or hybridizing with the polypeptide-encoding nucleotide sequence. The selection of promoters, e.g., strong, weak, inducible, tissue-specific, and developmentally specific, is within the ordinary skill of those skilled in the art. Similarly, matching nucleotide sequences with promoters is also within the skill of those skilled in the art. Promoters may be non-viral promoters or viral promoters, such as cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, and promoters found in long terminal repeats of murine stem cell virus.

[0147] host cell

[0148] This specification provides host cells containing the nucleic acids or vectors of this disclosure. As used herein, the term “host cell” means any type of cell that may contain the vectors disclosed herein and may produce expression products encoded by nucleic acids (e.g., mRNA, proteins). In some embodiments, the host cell is an adherent cell or a suspension cell, i.e., a cell that grows in a suspension state. In various embodiments, the host cell is a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, e.g., a human. The host cell may be any cell type, may originate from any type of tissue, and may be at any developmental stage.

[0149] In various embodiments, the antigen-binding protein is a glycosylated protein, and the host cell is a cell capable of glycosylation. In various embodiments, the cell capable of glycosylation is a eukaryotic cell, which includes, but is not limited to, yeast cells, filamentous fungal cells, protozoan cells, algal cells, insect cells, or mammalian cells. Such host cells have been reported in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In various embodiments, the eukaryotic cell is a mammalian cell. In various embodiments, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells include Chinese hamster ovary (CHO) cells and cells derived therefrom (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to be deficient in dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human fetal kidney 293 (HEK293) cells or cells derived therefrom (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, human alveolar basal epithelial adenocarcinoma cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, and mouse embryonic fibroblasts NIH. These include 3T3, mouse fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human hepatocellular carcinoma cells Hep G2, mouse myeloma B cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0150] In some cases, the host cell used for the purpose of amplifying or replicating a vector is a prokaryotic cell, such as a bacterial cell.

[0151] The Disclosure also provides a cell population comprising at least one host cell as described herein. In some embodiments, the cell population is a heterogeneous population comprising host cells containing the described vectors, in addition to at least one other cell that does not contain any of the vectors. Alternatively, in some embodiments, the cell population is a substantially homogeneous population, and the population comprises (e.g., essentially consists of) host cells containing vectors. In some embodiments, the population is a clonal population of cells, where all cells in the population are clones of a single host cell containing a vector, and all cells in the population contain the vector. In various embodiments of the Disclosure, the cell population is a clonal population comprising host cells containing the vectors described herein.

[0152] Manufacturing method

[0153] Furthermore, this specification also provides a method for producing an antigen-binding protein that binds to CLDN6. In various embodiments, the method comprises culturing a host cell containing a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein in a cell culture medium as described herein, and recovering the antigen-binding protein from the cell culture medium. The host cell may be any of the host cells described herein. In various embodiments, the host cell is selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various embodiments, the step of culturing the host cell comprises culturing the host cell in a growth medium to support the growth and proliferation of the host cell. In various embodiments, the growth medium increases the cell density, culture viability, and productivity at appropriate times. In various embodiments, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and adhesion factors. In various embodiments, the growth medium is a fully synthetic medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factor. In addition to providing nutrients, growth media also help maintain pH and osmotic pressure. Several growth media are commercially available and have been reported in the field. See, for example, Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).

[0154] In various embodiments, the method includes culturing host cells in fed-batch medium. In various embodiments, the method includes culturing in fed-batch medium using a fed-batch culture method. Methods for recombinant protein production are known in the art. See, for example, Li et al., “Cell culture processes for monoclonal antibody production” MAbs 2(5):466-477(2010).

[0155] A method for producing antigen-binding proteins may include one or more steps for purifying proteins from cell cultures or their supernatants, and preferably for recovering the purified proteins. In various embodiments, the method may include one or more chromatographic steps, such as affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, or hydrophobic interaction chromatography. In various embodiments, the method may include purifying proteins using protein A affinity chromatography resin.

[0156] In various embodiments, the method further includes a step of formulating a purified protein or the like to obtain a formulation containing the purified protein. Such a step is described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0157] In various embodiments, antigen-binding proteins linked to polypeptides and antigen-binding proteins are part of a fusion protein. Therefore, this disclosure further provides a method for producing a fusion protein comprising an antigen-binding protein that binds to CLDN6. In various embodiments, the method comprises culturing a host cell containing a nucleic acid comprising a nucleotide sequence encoding a fusion protein in a cell culture medium as described herein, and recovering the fusion protein from the cell culture medium.

[0158] complex

[0159] This disclosure also provides antigen-binding proteins that are attached, linked, or conjugated to a second portion (e.g., a heterologous portion, a complex portion). Thus, this disclosure provides complexes comprising an antigen-binding protein and a heterologous portion. As used herein, the term “heterologous portion” is synonymous with “complex portion” and refers to any molecule different from the antigen-binding protein of this disclosure (a chemical or biochemical molecule, a naturally occurring molecule, or a non-encoding molecule). Various heterologous portions include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic drugs, cytokines), or diagnostic agents.

[0160] In some embodiments, the heterogeneous portion is a polymer. The polymer may be branched or unbranched. The polymer can have any molecular weight. In some embodiments, the polymer has an average molecular weight of about 2 kDa to about 100 kDa (the term "about" indicates that in preparations of water-soluble polymers, there may be molecules with higher or lower molecular weights than those described). In some embodiments, the average molecular weight of the polymer is about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20 kDa to about 35 kDa.

[0161] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. In some embodiments, the polymer is water-soluble so that the protein it binds to does not precipitate in an aqueous environment such as a physiological environment. In some embodiments, the polymer is pharmaceutically acceptable, for example, when the composition is used therapeutically. Furthermore, in some embodiments, the polymer is a mixture of polymers, such as a copolymer or a block copolymer.

[0162] In some embodiments, the polymer includes polyamides, polycarbonates, polyalkylenes and their derivatives (including polyalkylene glycols, polyalkylene oxides, and polyalkylene terephthalates), acrylic acid esters and methacrylic acid ester polymers (including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate, and poly(octadecyl acrylate)), polyvinyl polymers (polyvinyl alcohol, polyvinyl ether, polyvinyl ester, halogenated polyvinyl, poly The material is selected from the group consisting of poly(vinyl acetate and polyvinylpyrrolidone), polyglycolides, polysiloxanes, polyurethanes and their copolymers, cellulose (including alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, nitrocellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethylcellulose, cellulose triacetate, and sodium cellulose sulfate), polypropylene, polyethylene (including poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate)), and polystyrene.

[0163] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). Where used herein, polyethylene glycol is intended to encompass any form of PEG that can be used to derivatize other proteins, such as mono-(C1-C10)alkoxy- or aryloxy- polyethylene glycol. PEG is a linear or branched neutral polyether available in a wide range of molecular weights and is soluble in water and most organic solvents.

[0164] In some embodiments, the heterogeneous portion is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0165] In some embodiments, the heterogeneous portion is a lipid. In some embodiments, the lipid is a fatty acid (eicosanoid, prostaglandin, leukotriene, thromboxane, N-acylethanolamine), glycerolipid (e.g., monosubstituted, disubstituted, trisubstituted glycerol), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenolipid, glycolipid, or polyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, or phospholipid.

[0166] In some embodiments, the heterogeneous portion is a therapeutic agent. The therapeutic agent may be any therapeutic agent known in the art. Examples of therapeutic agents intended herein include natural enzymes, proteins derived from naturally occurring substances, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic drugs, immunoglobins, beta-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathogenetics, angiotensin-converting enzyme (ACE) inhibitors, diuretics, cardiotonic agents, cholesterol and triglyceride lowering agents, bile acid scavengers, fibrates, 3-hydroxy-3-methylglycerides Lutaril (HMG)-CoA reductase inhibitors, niacin derivatives, antiadrenergic agonists, alpha-adrenergic blockers, central antiadrenergic agonists, vasodilators, potassium-sparing agents, thiazide diuretics and related drugs, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and its analogues, alpha-glucosidase inhibitors, biguanides, meglitinide, sulfonylurea, thioazolidinedione, androgens, progestogens, bone metabolism modifiers, Anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators, antithyroid drugs, thyroid hormones, bulk-forming agents, laxatives, peristalsis inhibitors, bacterial flora modifiers, intestinal adsorbents, intestinal anti-infective agents, drugs for anorexia (antianorexics), drugs for cachexia (anticachexics), drugs for bulimia (antibulimics), appetite suppressants, anti-obesity drugs, antacids, drugs for the upper gastrointestinal tract, anticholinergics, aminosalicylic acid derivatives Bioresponse modifiers, corticosteroids, antispasmodics, 5-HT4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotective agents, histamine H2 receptor antagonists, mucosal protective agents, proton pump inhibitors, H. pylori eradication therapy, erythropoiesis-promoting agents, hematopoietic agents, anemia medications, heparin, antifibrinolytic agents, hemostatic agents, blood coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane-A2 inhibitors, plasminogen activators, antithrombotic agents,Glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal agents, drugs associated with prophylactic treatment, AIDS-related infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma (idiopathic multiple hemorrhagic sarcoma), aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and its derivatives, tetracycline Culinarian drugs, anthelmintics, anti-amebic drugs, biguanides, quinine alkaloids, folic acid antagonists, quinoline derivatives, Pneumocystis carinii treatments, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, anticholinesterase drugs, adjuvant therapies, barbiturates and derivatives, benzodiazepines, gamma-aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, anti-migraine preparations, bioresponse modifiers, carbamic acid eaters Eater), tricyclic derivatives, depolarizers, non-depolarizers, neuromuscular paralytics, central nervous system stimulants, dopamine agonists, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, ethyleneimine, imidazotetrazine, nitrogen mustard analogs, nitrosourea, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, anthracycline drugs, actinomycin d, camptothecin derivatives, epipodophyllotoxin, taxanes, vinca alkaloids and analogs, antiandrogens Anti-estrogen drugs, non-steroidal aromatase inhibitors, protein kinase inhibitors, antineoplastic drugs, azaspirodecandione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzoisoxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperazine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergen extracts, non-steroidal drugs,Leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytics, antimitotics, uric acid excretion agents, xanthine oxidase inhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuran derivatives, quinolone drugs, smooth muscle relaxants, parasympathomimetic agents, halogenated hydrocarbons, aminobenzoic acid esters, amides (e.g., lidocaine, alticaine hydrochloride, bupivacaine hydrochloride), antipyretics, Examples include, but are not limited to, sleeping pills and sedatives, cyclopyrrolone, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapy drugs, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, and vitamin K analogs and antagonists.

[0167] The antigen-binding proteins of this disclosure can be conjugated to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis, wherein the cytokine or growth factor has been shown to have antiproliferative activity against at least one cell population. Examples of such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factors, and erythropoietin.Additional growth factors for use in this specification include angiotensin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophil chemoattractant 2α, cytokine-induced neutrophil chemoattractant 2β, β-endothelial cell growth factor, endothelin 1, epithelial-derived neutrophil attractant, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-associated protein, growth-associated protein α, growth-associated protein β, growth-associated protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor-binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth-stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β-binding protein I, transforming growth factor β-binding protein II, transforming growth factor β-binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and their biological or immunological active fragments are included.

[0168] In some embodiments, the complex comprises a compound described herein and a cytotoxic agent. A cytotoxic agent is any molecule (chemical or biochemical) that is toxic to cells. In some aspects, when a cytotoxic agent is conjugated to a compound of the invention, the resulting effect is synergistic. In other words, the efficacy of the combination therapy of the compound and the cytotoxic agent is synergistic, i.e., the efficacy is greater than the efficacy predicted from the individual additive effects of each. Thus, the dosage of the cytotoxic agent can be reduced, and therefore the risks of toxicity problems and other side effects are simultaneously reduced. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. Chemotherapeutic agents are known in the art and include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides such as those described in U.S. Patent No. 6,630,124.

[0169] In some embodiments, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any platinum coordination compound that provides platinum in ionic form and inhibits tumor cell growth. In some embodiments, the platinum coordination compound is cis-diamminediaquoplatinum(II)-ion; chloro(diethylenetriamine)-platinum(II) chloride; dichloro(ethylenediamine)-platinum(II), diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin); spiroplatin; iproplatin; diammine(2-ethylmalonato)-platinum(II); ethylenediaminemalonato platinum(II); aqua(1,2-diaminocyclohexane)-sulfatoplatinum(II); (1,2-diaminocyclohexane)malonato platinum(II); (4-carboxyphthalato)(1,2-diaminocyclohexane)platinum(II); (1,2-diaminocyclohexane)-(isocitrato)platinum(II); (1,2-diaminocyclohexane)cis(pyruvato)platinum(II); (1,2-diaminocyclohexane)oxalatoplatinum(II); ormaplatin; and tetraplatin.

[0170] In some embodiments, cisplatin is the platinum-coordinate compound used in the compositions and methods of the present invention. Cisplatin is commercially available from Bristol Myers-Squibb Corporation under the generic name PLATINOL® and is available as a powder for composition with water, sterile saline, or other suitable vehicle. Other platinum-coordinate compounds suitable for use in the present invention are known and can be used commercially and / or prepared by the prior art. Cisplatin, or cis-dichlorodiammineplatinum II, has been used for many years as a chemotherapeutic agent in the treatment of various human malignant solid tumors with good results. More recently, other diamino-platinum complexes have also shown efficacy as chemotherapeutic agents in the treatment of various human malignant solid tumors. Such diamino-platinum complexes include, but are not limited to, spiroplatinum and carboplatinum. Although cisplatin and other diamino-platinum complexes are widely used as chemotherapeutic agents in humans, they require delivery at high doses, which can lead to toxicity problems such as renal impairment.

[0171] In some embodiments, the chemotherapy drug is a topoisomerase inhibitor. Topoisomerases are enzymes that can alter the DNA topology of eukaryotic cells. They are important for cell function and cell proliferation. Generally, there are two classes of topoisomerases in eukaryotic cells: type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of approximately 100,000. This enzyme binds to DNA and introduces transient single-strand breaks to unwind (or enable unwinding) the double helix, and then rejoins the break site before it dissociates from the DNA strand. Various topoisomerase inhibitors have recently shown clinical efficacy in treating humans suffering from ovarian cancer, esophageal cancer, or non-small cell lung cancer.

[0172] In some embodiments, topoisomerase inhibitors are camptothecin or camptothecin analogs. Camptothecin is a water-insoluble cytotoxic alkaloid produced by the Chinese native tree Camptotheca accuminata and the Indian native tree Nothapodytes foetida. Camptothecin exhibits tumor cell growth inhibitory activity against several tumor cells. Camptothecin analog class compounds are typically specific inhibitors of DNA topoisomerase I. The term “topoisomerase inhibitor” refers to any tumor cell growth inhibitory compound structurally related to camptothecin. Examples of camptothecin analog class compounds include, but are not limited to, topotecan, irinotecan, and 9-aminocamptothecin.

[0173] In additional embodiments, the cytotoxic agent is: U.S. Patent No. 5,004,758 issued on April 2, 1991 and European Patent Application No. 88311366.4 published on June 21, 1989 as EP0 321 122; U.S. Patent No. 4,604,463 issued on August 5, 1986 and European Patent Application No. EP0 137 145 published on April 17, 1985; U.S. Patent No. 4,473,692 issued on September 25, 1984 and European Patent Application No. EP0 074 256 published on March 16, 1983; U.S. Patent No. 4,545,880 issued on October 8, 1985 and European Patent Application No. EP0 074 published on March 16, 1983 No. 256; European Patent Application Publication No. EP0 088 642, published on September 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363 (1986); Nitta et al., Proc. 14th International Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section 1, pp. 28-30 (in particular the compound called CPT-11), which is any tumor cell growth inhibitory camptothecin analog. CPT-11 is a camptothecin analog having a 4-(piperidino)-piperidine side chain linked via a carbamate bond at C-10 of 10-hydroxy-7-ethylcamptothecin. CPT-11 is currently undergoing clinical trials in humans and is also known as irinotecan.Wani et al, J.Med.Chem., 23,554 (1980), Wani et al., J.Med.Chem., 30,1774 (1987), U.S. Patent No. 4,342,776 issued on August 3, 1982; U.S. Patent Application No. 581,916 filed on September 13, 1990 and European Patent Application Publication No. EP418099 published on March 20, 1991; U.S. Patent No. 4,513,138 issued on April 23, 1985 and European Patent Application Publication No. EP0074770 published on March 23, 1983; U.S. Patent No. 4,399,276 issued on August 16, 1983 and European Patent Application Publication No. 0056 published on July 28, 1982 Patent No. 692; the full disclosure of each thereof is incorporated herein by reference. Any of the camptothecin analog class compounds listed above can be commercially available and / or prepared by the prior art, including those described in the references listed above. The topoisomerase inhibitors may be selected from the group consisting of topotecan, irinotecan, and 9-aminocamptothecin.

[0174] In some embodiments, the camptothecin analog is an active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is formed by the hydrolysis of irinotecan by carboxylesterase. In some embodiments, SN-38 has one of the following structures. [ka] SN-38 is described in U.S. Patent Application No. 7,999,083, U.S. Patent Application No. 8,080,250, U.S. Patent Application No. 8,759,496, U.S. Patent Application No. 8,999,344, U.S. Patent Application No. 10,195,288, and U.S. Patent Application No. 9,808,537.

[0175] In some embodiments, the camptothecin analog is exatecan methanesulfonic acid. Exatecan methanesulfonic acid exhibits more potent topoisomerase I inhibitory and antitumor activity than water-soluble camptothecin (CPT) and other CPT analogs. Furthermore, exatecan is effective against p-glycoprotein (P-gp)-mediated multidrug-resistant cells.

[0176] In some embodiments, the camptothecin analog is deruxtecan (Dxd), a potent derivative of exatecan, which has 10 times higher topoisomerase I inhibitory activity than SN-38. In some embodiments, Dxd has the following structure. [ka]

[0177] Dxd is described in U.S. Patent Application No. 6,407,115, U.S. Patent Application No. 10,195,288, U.S. Patent Application No. 9,808,537, and U.S. Patent Application No. 6,407,115.

[0178] Numerous preparations of camptothecin analog class compounds (including pharmaceutically acceptable salts, hydrates, and solvates thereof), as well as preparations of oral and parenteral pharmaceutical compositions comprising such camptothecin analog class compounds with inert, pharmaceutically acceptable carriers or diluents, are described in detail in U.S. Patent No. 5,004,758, granted on 2 April 1991, and European Patent Application No. 88311366.4, published on 21 June 1989 as EP0 321 122, the teachings of which are incorporated herein by reference.

[0179] In yet another embodiment of the present invention, the chemotherapeutic agent is a combination antibiotic. Preferred antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin.

[0180] In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. Generally, antimitotic alkaloids can be extracted from Cantharanthus roseus and have been shown to be effective as anticancer chemotherapeutic agents. Numerous semi-synthetic derivatives have been studied chemically and pharmacologically (see O. Van Tellingen et al, Anticancer Research, 12, 1699-1716 (1992)). Examples of antimitotic alkaloids in this invention include, but are not limited to, vinblastine, vincristine, vindesine, taxol, and vinorelbine. The latter two antimitotic alkaloids are commercially available from Eli Lilly and Company and Pierre Fabre Laboratories, respectively (see U.S. Patent No. 5,620,985). In one embodiment, the antimitotic alkaloid is vinorelbine.

[0181] In other embodiments of the present invention, the chemotherapeutic agent is a difluoronucleoside. 2'-deoxy-2',2'-difluoronucleosides are known in the art as having antiviral activity. Such compounds are disclosed and taught in U.S. Patents 4,526,988 and 4,808,614. European Patent Application Publication 184,365 discloses that these same difluoronucleosides have oncolytic activity. In certain embodiments, the 2'-deoxy-2',2'-difluoronucleoside used in the compositions and methods of the present invention is 2'-deoxy-2',2'-difluorocytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine can be made available commercially or synthesized in a multi-step process as disclosed and taught in U.S. Patents 4,526,988, 4,808,614 and 5,223,608, whose teachings are incorporated herein by reference.

[0182] In various aspects, the chemotherapeutic agent is an anti-mitotic agent that inhibits cell division by blocking tubulin polymerization, destabilizing microtubules, or altering microtubule dynamics, such as a maytansinoid or its derivative (e.g., DM1 or DM4), an auristatin or its derivative. In various cases, the chemotherapeutic agent is an auristatin. For example, in some aspects, the auristatin is dolastatin, monomethyl auristatin E (MMAE), monomethyl auristatin E (MMAE), or PF-06380101. Auristatins have been reported in the art. See, for example, Maderna, A.; et al., Mol Pharmaceutics 12(6):1798-1812 (2015). In various aspects, the conjugate comprises an antibody of the present disclosure in combination with MMAE. Optionally, the conjugate comprises a linker. In some aspects, the linker comprises a cleavable linking moiety. In various cases, the conjugate comprises an antibody of the present disclosure linked to a linking group linked to a cathepsin-cleavable linker and linked to a spacer linked to MMAE. In an aspect, the linking group is linked to the antibody via a Cys residue in the Fc region of the antibody. In an exemplary aspect, the linking group comprises the structure of Formula I.

Chemical formula

[0183] In an exemplary aspect, the cathepsin-cleavable linker comprises the structure of Formula II.

Chemical formula

[0184] In an exemplary aspect, the spacer comprises the structure of Formula III.

Chemical formula

[0185] In some embodiments, MMAE has the following structure. [ka]

[0186] The disclosure also provides a complex comprising an antigen-binding protein of the disclosure linked to a polypeptide, such that the complex is a fusion protein. Thus, the disclosure provides a fusion protein comprising an antigen-binding protein of the disclosure linked to a polypeptide. In various embodiments, the polypeptide is a diagnostic label, such as a fluorescent protein such as green fluorescent protein, or another tag, such as a Myc tag. In various embodiments, the polypeptide is a cytokine, lymphokine, growth factor, or one of the other hematopoietic factors listed above.

[0187] Linker

[0188] In some embodiments, the complex is directly linked to the heterogeneous moiety. In alternative embodiments, the complex includes a linker that links the compound of the disclosure to the heterogeneous moiety. In some embodiments, the linker includes a chain with 1 to about 60 atoms, or 1 to 30 atoms or more, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. In some embodiments, all chain atoms are carbon atoms. In some embodiments, the chain atoms in the linker skeleton are selected from the group consisting of C, O, N, and S. The chain atoms and linker can be selected according to their predicted solubility (hydrophilicity) to provide a more soluble complex. In some embodiments, the linker provides a functional group that is subject to cleavage by enzymes or other catalysts, or by hydrolytic conditions found in the target tissue, organ, or cell. In some embodiments, the length of the linker is long enough to reduce the possibility of steric hindrance. In some embodiments, the linker is an amino acid or a peptidyl linker. Such a peptidyl linker may be of any length. Various linkers have amino acid lengths of approximately 1 to 50, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30.

[0189] A wide variety of suitable linkers are known in the art. The linker may be cleavable, for example, under physiological conditions, such as intracellular conditions (cleavable linker), allowing a drug to be released into the intracellular environment upon cleavage. Alternatively, the linker may be cleavable under extracellular conditions, such as outside tumor cells or near the tumor, allowing a drug to be released upon cleavage that selectively permeates into the tumor cells. In other embodiments, the linker is not cleavable (non-cleavable linker), and the drug is released, for example, by antibody degradation.

[0190] Linkers can be attached to chemically reactive groups on the antibody moiety, such as free amino groups, imino groups, hydroxyl groups, thiol groups, or carboxyl groups (e.g., the N-terminus or C-terminus, the epsilon-amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, the sulfhydryl group of one or more cysteinyl residues, or the hydroxyl group of one or more serine or threonine residues). The linker attachment site may be a native residue in the amino acid sequence of the antibody moiety, or it may be introduced into the antibody moiety by, for example, DNA recombination (e.g., by introducing a cysteine ​​or protease cleavage site into the amino acid sequence) or by protein biochemistry (e.g., reduction, pH adjustment, or proteolysis). The linker attachment site may also be a non-native amino acid. The linker attachment site may also be a glycan on the antibody.

[0191] Typically, a linker is substantially inactive under conditions in which the two groups it connects are linked. The terms “bifunctional crosslinker,” “bifunctional linker,” or “crosslinker” refer to a modifier having two reactive groups at each end of a linker, wherein one reactive group can be reacted first with a cytotoxic compound to provide a linker-supported compound, and then the second reactive group can react with an antibody. Alternatively, one end of a bifunctional crosslinker can be reacted first with an antibody to provide an antibody supporting the linker moiety and the second reactive group, which can then react with a cytotoxic compound. The linkage moiety may contain a chemical bond that allows for the release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-unstable bonds, photo-unstable bonds, protease / peptidase-unstable bonds, and esterase-unstable bonds. See, for example, U.S. Patents 5,208,020, 5,475,092, 6,441,163, 6,716,821, 6,913,748, 7,276,497, 7,276,499, 7,368,565, 7,388,026, and 7,414,073. In some embodiments, the bond is a disulfide bond, a thioether, and / or a protease / peptidase unstable bond. Other linkers that can be used in the present invention include non-cuttable linkers such as those detailed in US20050169933, each of which is explicitly incorporated herein by reference, and charged or hydrophilic linkers such as those described in US2009 / 0274713, US2010 / 0129314, and WO2009 / 134976.

[0192] In some embodiments, the linker is a hydrophilic linker that imparts hydrophilicity to the composite. In some embodiments, the hydrophilic linker contains polyethylene glycol (PEG). In some embodiments, the hydrophilic linker is CLA2. In some embodiments, the CLA2 linker has the following structure. [ka] CLA2 is described in U.S. Patents No. 8,080,250, No. 8,759,496, and No. 10,195,288.

[0193] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E has the following structure. [ka] CL2E is described in U.S. Patents No. 8,080,250, No. 8,759,496, and No. 10,195,288.

[0194] In some embodiments, the linker can be cleaved by cleavage substances present in the intracellular environment (e.g., inside lysosomes or endosomes or caveolae). The linker may be a peptide linker that is cleaved by intracellular or extracellular peptidase or protease enzymes, including but not limited to lysosome or endosome proteases. In some embodiments, the peptide linker comprises at least 2 amino acid lengths, at least 3 amino acid lengths, at least 4 amino acid lengths, or at least 5 amino acid lengths.

[0195] In some embodiments, the peptide linker is an MC-VC-PAB containing a valine residue and a citrulline residue. In some embodiments, the MC-VC-PAB linker has the following structure. [ka] MC-VC-PAB is described in U.S. Patents No. 7,659,241, 7,829,531, 6,884,869, 6,214,345, and 6,214,345.

[0196] In some embodiments, the peptide linker is maleimidocaproylglycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure. [ka] MC-GGFG is described in U.S. Patents 9,808,537 and 10,195,288.

[0197] In other embodiments, the cleavable linker is pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. In some embodiments, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, hydrolyzable, acid-unstable linkers (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitamide, orthoester, acetal, ketal, etc.) can be used in lysosomes (see, for example, U.S. Patents 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al, 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but unstable at pH levels below 5.5 or 5.0, which are approximate lysosomal pH values. In certain embodiments, the hydrolyzable linker is a thioether linker (for example, a thioether bonded to the therapeutic agent via an acylhydrazone linkage (see, for example, U.S. Patent No. 5,622,929)).

[0198] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Examples of bifunctional crosslinking agents that enable the binding of antibodies to cytotoxic compounds via disulfide bonds include, but are not limited to, N-succinimidyl-4-(4-nitropyridyl-2-dithio)butanoate, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Sulfo-SPDB is described, for example, in U.S. Patent No. 8,236,319, which is incorporated herein by reference. Alternatively, a crosslinking agent that introduces thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, can be used. In other embodiments, the linker may contain a combination of one or more of the aforementioned peptide linkers, pH-sensitive linkers, or disulfide linkers.

[0199] A "hetero-bifunctional crosslinking agent" is a bifunctional crosslinking agent having two different reactive groups. Hetero-bifunctional crosslinking agents containing both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link cytotoxic compounds with antibodies. Examples of such commercially available hetero-bifunctional crosslinking agents include succinimidyl 6-hydrazinonicotinamideacetonehydrazone (SANH), succinimidyl 4-hydrazide terephthalate hydrochloride (SHTH), and succinimidylhydrazinium nicotinate hydrochloride (SHNH). Complexes with acid-unstable bonds can also be prepared using the hydrazine-supported benzodiazepine derivatives of the present invention. Examples of bifunctional crosslinking agents that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0200] The linkers described herein may be used in any combination with the heterogeneous components described herein. All of the linkers and heterogeneous components described herein above can be commercially available and / or prepared by prior art, including those described in the references cited above.

[0201] complex

[0202] The heterologous moiety-to-antigen-binding protein ratio (HAR) represents the number of linked heterologous moieties per antigen-binding molecule. In some embodiments, the HAR is in the range of 1–15, 1–10, 1–9, 1–8, 1–7, 1–6, 1–5, 1–4, 1–3, or 1–2. In some embodiments, the HAR is in the range of 2–10, 2–9, 2–8, 2–7, 2–6, 2–5, 2–4, or 2–3. In other embodiments, the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6. In some embodiments, the HAR is in the range of about 2–4. The HAR can be characterized by conventional means such as mass spectrometry, UV / Vis spectroscopy, ELISA assay, and / or HPLC.

[0203] In some embodiments, the complex is a heterogeneous complex (also called a "conventional" complex), where antigen-binding proteins bind to different numbers of heterologous regions. In some embodiments, the heterogeneous complex follows a Gaussian or quasi-Gaussian distribution of the complex, in which case the distribution is centered at the mean value of heterologous region binding, with some antigen-binding proteins binding at above-average values ​​and some at below-average values.

[0204] In some embodiments, the complex is a homogeneous complex, in which a significant proportion of antigen-binding proteins are bound to a predetermined number of heterologous moieties. In some embodiments, the homogeneous complex contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HARs. In some embodiments, the homogeneous complex contains 2, 4, 6, or 8 HARs. In a preferred embodiment, the homogeneous complex contains 4 HARs. In another preferred embodiment, the homogeneous complex contains 2 HARs. In some embodiments, the homogeneous complex contains a complex having defined HARs of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent or more. In some embodiments, the homogeneous composite includes a composite having a defined HAR of approximately 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent. In some embodiments, the homogeneous complex includes a HAR distribution that is neither Gaussian nor quasi-Gaussian. In some embodiments, the homogeneity of the homogeneous complex is determined by a chromatogram, e.g., HPLC or any preferred chromatography. In some embodiments, the chromatogram is an HIC chromatogram. The homogeneous complex may be produced by site-specific complexization.

[0205] In some embodiments, a heterologous portion is site-specifically bound to an antigen-binding protein (e.g., an antibody). Various methods of site-specific complexation are known in the art, for example, complexation with thiomab, TDC, or unpaired cysteine ​​residues (Junutula et al. (2008) Nat. Biotechnol. 26:925-932, Dimasi et al. (2017) Mol. Pharm. 14:1501-1516, Shen et al. (2012) Nat. Biotechnol. 30:184-9), thiol crosslinking linkers (Behrens et al. (2015) Mol. Pharm. 12:3986-98), and complexation with glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15, Dennler et al. (2014) Bioconjug Complexation with manipulated non-natural amino acid residues (Chem. 25:569-78), complexation with manipulated non-natural amino acid residues (Axup et al. (2012) Proc Natl Acad Sci USA 104-16101-6, Tian et al. (2014) Proc Natl Acad Sci USA 111:1766-71, VanBrunt et al. (2015) Bioconjug Chem 26:2249-60, Zimmerman et al. (2014) Bioconjug Chem 25:351-61), selenocysteine ​​complexation (Li et al. (2017) Cell Chem Biol 24:433-442), glycan-mediated complexation (Okeley et al. (2013) Bioconjug Chem 24:1650-5), complexation with galactose or GalNAc analogs (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9, van Geel et al. (2015) Bioconjug Chem 26:2233-42), glycotechnology (Zhou et al. (2014) Bioconjug Chem 25:510-20, Tang et al.)These methods include (2017) Nat Protoc 12:1702-1721), manipulation of glutamine tags or short-chain peptide tags such as saltase A-mediated peptide transfer (Strop et al. (2013) Chem Biol 20:161-7, Beerli et al. (2015) PLoS One 10:e0131177), and aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA 106:3000-5).

[0206] Unpredictability of complexes (e.g., ADCs)

[0207] It is impossible to predict in advance which antibody-drug conjugates will be sufficiently safe and effective for clinical application based solely on antibody profiles or drug payload profiles. For example, a particular drug payload may function perfectly well when bound to an antibody directed to a single target, but may be largely ineffective when bound to antibodies directed to different targets, or to different antibodies directed to the same target. The reasons why different antibody-drug conjugates exhibit different antitumor activities in vivo are not fully understood, making accurate predictions difficult in the design of new antibody-drug conjugates. It is suspected that unpredictable interactions of many factors are at play. These factors may include, for example, the binding affinity of the antibody-drug conjugate to the target antigen, the solid tumor penetration ability of the conjugate, and the circulating half-life for appropriate exposure to the tumor without causing toxicity.

[0208] The complexity and unpredictability are clearly demonstrated by antibody affinity alone. High-affinity antibodies or antibody-drug conjugates are better taken up into cells, leaving a trail of higher levels of cytotoxic payloads released intracellularly. High affinity is also known to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). All of these attributes are favorable to the cytotoxic properties of antibody-drug conjugates. However, high affinity of antibodies or antibody-drug conjugates is also known to hinder efficient tumor penetration through an "antigen barrier effect," suggesting that to achieve strong antitumor activity in vivo, the affinity of the antibody-drug conjugate must be just right—neither too high nor too low. To date, there is no known way to predict what the most efficient or effective level of affinity is for a given antibody-drug conjugate.

[0209] Furthermore, in vivo antitumor activity cannot be predicted from the linker and payload mechanisms alone. For example, O. Ab et al, Mol. Cancer Ther. 14(&):1605-1613 (2015), demonstrated that when the same antibody was conjugated to the same antitubulin toxin with different linkers in a preclinical cancer model, dramatically different antitumor activities were observed. This example is particularly surprising because the chemical structures of the two linkers are very similar. Moreover, the linker present in the dominant complex contained a hydrophilic moiety. Hydrophilic metabolites generally have low membrane permeability and are thought to leach more slowly from lysosomes (the sites where the complex is degraded), resulting in a delay in the antitubulin activity of the released payload. While this finding asserts an "ideal" dynamic for payload delivery, there is currently no insight into what constitutes such dynamics. Further complicating matters is the unresolved question of whether the ideal dynamics of payload delivery, while defined for specific cell types, apply to all cell types. Therefore, it is impossible to predict the most effective in vivo antitumor activity simply from the chemical composition of the linker or payload.

[0210] Compositions, pharmaceutical compositions and formulations

[0211] Compositions comprising antigen-binding proteins, nucleic acids, vectors, host cells, or complexes as disclosed herein are provided herein. In some embodiments, compositions comprise antigen-binding proteins in isolated and / or purified forms. In some embodiments, compositions comprise a single type (e.g., structure) of the antigen-binding proteins of the disclosure, or a combination of two or more antigen-binding proteins of the disclosure, such combination comprising two or more antigen-binding proteins of different types (e.g., structures).

[0212] In some embodiments, the composition comprises an agent that enhances the chemophysical properties of an antigen-binding protein, for example, by stabilizing the antigen-binding protein at a specific temperature, e.g., room temperature, extending its shelf life, reducing degradation, e.g., degradation by oxidative proteases, or extending the half-life of the antigen-binding protein. In some embodiments, the composition comprises any of the agents disclosed herein as a heterogeneous or complex moiety, optionally in the form of a mixture with the antigen-binding protein of this disclosure, or conjugated to such antigen-binding protein.

[0213] In various aspects of this disclosure, the composition is a pharmaceutically acceptable carrier, diluent, or additive. (Excipients) The disclosure further includes the following. In some embodiments, antigen-binding proteins, nucleic acids, vectors, host cells, or complexes as disclosed herein (hereinafter referred to as "active substances") are formulated into pharmaceutical compositions comprising the active substances together with pharmaceutically acceptable carriers, diluents, or additives. In this regard, the disclosure further provides pharmaceutical compositions comprising active substances intended for administration to subjects, e.g., mammals.

[0214] In some embodiments, the active substance is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active substance has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and has a pharmaceutically acceptable diluent, carrier, or additive. In some embodiments, the composition contains the active substance at a concentration of about 0.001 to about 30.0 mg / ml.

[0215] In various embodiments, pharmaceutical compositions include pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various wetting agents. The term also includes any drug approved by a U.S. federal government regulatory authority or a drug listed in the United States Pharmacopeia for use in animals, including humans.

[0216] Pharmaceutical compositions may contain any pharmaceutically acceptable components, such as acidifiers, additives, adsorbents, aerosol sprays, air replacement agents, alkalizing agents, anticoagulants, antibacterial preservatives, antioxidants, disinfectants, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, cleaning agents, diluents, bactericides, disintegrants, dispersants, dissolution accelerators, pigments, softeners, emulsifiers, emulsion stabilizers, and excipients. Examples include film-forming agents, seasonings, flavoring agents, flow promoters, gelling agents, granulating agents, moisturizing agents, lubricants, mucosal adhesives, ointment bases, ointments, oily vehicles, organic bases, tablet bases, pigments, plasticizers, glossing agents, preservatives, chelating agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, thickening agents, isotonic agents, toxic agents, thickeners, water-absorbent agents, water-miscible cosolvents, water softeners, or wetting agents. For example, see Handbook of Pharmaceutical Excipients, Third Edition, AHKibbe (Pharmaceutical Press, London, UK, 2000), which incorporates the entire list by reference. The entire text is incorporated by reference in Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980).

[0217] In various embodiments, a pharmaceutical composition comprises formulation materials that are nontoxic to the recipient at the dosage and concentration used. In specific embodiments, a pharmaceutical composition comprises an active substance and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic balancers, isotonic agents, antioxidants, antibiotics, antifungal agents, bulking agents, lyophilization protectants, defoamers, chelating agents, preservatives, colorants, analgesics, or additional pharmaceutical agents. In various embodiments, a pharmaceutical composition comprises one or more polyols and / or one or more surfactants, and optionally one or more other additives, which include, but are not limited to, pharmaceutically acceptable salts, osmotic balancers (isotonic agents), antioxidants, antibiotics, antifungal agents, bulking agents, lyophilization protectants, defoamers, chelating agents, preservatives, colorants, and analgesics.

[0218] In certain embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving the composition's properties, such as pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of elution or release, adsorption, or osmosis. In such embodiments, suitable formulation materials include amino acids (glycine, glutamine, asparagine, arginine, or lysine, etc.); antibacterial agents; antioxidants (ascorbic acid, sodium sulfite, or sodium bisulfite, etc.); buffering agents (borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids, etc.); bulking agents (mannitol or glycine, etc.); chelating agents (ethylenediaminetetraacetic acid (EDTA), etc.); complexing agents (caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin, etc.); excipients; monosaccharides; disaccharides; and other carbohydrates (glucose, mannose, or dextrin, etc.); proteins (serum albumin, gelatin, or immunoglobulin, etc.); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (polyvinylpyrrolidone, etc.); low molecular weight polypeptides; salt-forming counterions (sodium Examples of additives include, but are not limited to, iodine, sorbate, and / or pharmaceutical adjuvants; preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (mannitol or sorbitol); suspending agents; surfactants or wetting agents (pluronic acid, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers (sucrose or sorbitol, etc.); isotonic enhancers (alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; additives and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (ARGenrmo, ed.), 1990, Mack Publishing Company.

[0219] Pharmaceutical compositions can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, between about 4 or about 5 and about 8.0, or between about 4.5 and about 7.5, or between about 5.0 and about 7.5. In various embodiments, the pH of the pharmaceutical composition is between 5.5 and 7.5.

[0220] This disclosure provides a method for producing a pharmaceutical composition. In various embodiments, the method includes combining an antigen-binding protein, complex, fusion protein, nucleic acid, vector, host cell, or combination thereof with a pharmaceutically acceptable carrier, diluent, or additive.

[0221] Route of administration

[0222] With respect to this disclosure, the active substance, or a pharmaceutical composition containing the same, may be administered to a subject via any preferred route of administration. For example, the active substance may be administered to a subject by parenteral administration, nasal administration, oral administration, pulmonary administration, topical administration, vaginal administration, or rectal administration. The following considerations regarding routes of administration are provided solely to illustrate various embodiments and should not be construed as limiting the scope in any way.

[0223] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injections that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the intended recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspensions, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means administration via any other route, such as subcutaneous, intramuscular, intraspinal, or intravenous, rather than through the gastrointestinal tract. The active substances of this disclosure can be administered using a pharmaceutical carrier containing a physiologically acceptable diluent such as a sterile liquid or liquid mixture, which includes water, physiological saline, aqueous dextrose and related sugar solution, alcohol such as ethanol or hexadecyl alcohol, glycol such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketal such as 2,2-dimethyl-153-dioxolane-4-methanol, ether, poly(ethylene glycol) 400, oil, fatty acid, fatty acid ester or glyceride, or acetylated fatty acid glyceride (with or without the addition of a pharmaceutically acceptable surfactant such as soap or detergent), suspending agents such as pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose, or carboxymethylcellulose, or emulsifiers, and other pharmaceutical adjuvants.

[0224] Oils that can be used in parenteral formulations include petroleum, animal, plant, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and minerals. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0225] Suitable soaps for use in parenteral formulations include salts of aliphatic alkali metals, ammonium, and triethanolamine, and suitable detergents include (a) cationic detergents, such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents, such as alkyl sulfonates, aryl sulfonates, olefin sulfonates, alkyl sulfates, olefin sulfates, ether sulfates, monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents, such as aliphatic amine oxides, fatty acid alkanolamides, and copolymers of polyoxyethylene and polypropylene; (d) amphoteric detergents, such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0226] Parenteral formulations in some embodiments contain about 0.5% to about 25% by weight of the active substance solution of the present disclosure. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is typically in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide and propylene glycol. Parenteral formulations in some embodiments are contained in single-dose or multi-dose sealed containers such as ampoules and vials and can be stored in a freeze-dried state, requiring only the addition of a sterile liquid additive for injection, such as water, immediately before use. Solutions and suspensions for immediate injection in some embodiments are prepared from sterile powders, granules, and tablets of the types described above.

[0227] The injectable formulations conform to the disclosures herein. Requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceuticals and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0228] Dosage

[0229] The disclosed active substances are considered useful in other methods, as detailed herein, including methods for inhibiting tumor growth and methods for treating or preventing cancer. For the purposes of the disclosure, the amount or dose of the active substance administered should be sufficient for an effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the active substance disclosed herein should be sufficient to treat the cancer described herein over a period of approximately 1 to 4 minutes, 1 to 4 hours, 1 to 4 weeks, or longer, e.g., 5 to 20 weeks, or longer, from the time of administration. In certain embodiments, the time period may be longer. The dose is determined by the efficacy of the particular active substance and the condition of the animal (e.g., human), as well as the body weight of the animal to be treated (e.g., human).

[0230] Many assays for determining dosages are known in the art. For the purposes herein, any assay that includes comparing the extent to which cancer is treated at a given dose of the active substance in mammals among groups of mammals administered different doses of the active substance in each group is considered usable for determining the starting dose to be administered to mammals. The extent to which cancer is treated at a particular dose can be expressed, for example, by the degree of tumor regression achieved by the active substance in a mouse xenograft model. Methods for evaluating tumor regression are known in the art and are described in the examples herein.

[0231] The dosage of the active substance of this disclosure will also be determined by the presence, nature, and extent of any adverse side effects that may occur with the administration of the particular active substance of this disclosure. Typically, the attending physician will determine the dosage of the active substance of this disclosure for each individual patient, taking into account various factors such as age, weight, overall health, diet, sex, the active substance of this disclosure to be administered, the route of administration, and the severity of the condition being treated. Without intending to limit this disclosure, for example, the dosage of the active substance of this disclosure may be about 0.0001 to about 1 g / kg body weight (body weight of the person being treated) / day, about 0.0001 to about 0.001 g / kg body weight / day, or about 0.01 mg to about 1 g / kg body weight / day.

[0232] Controlled-release formulations

[0233] In some embodiments, the active substances described herein can be modified into a depot form so that the manner in which the active substance of the Disclosure is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). The depot form of the active substance of the Disclosure may be, for example, an implantable composition comprising the active substance and a porous or non-porous material such as a polymer, wherein the active substance is encapsulated by the material, diffused throughout the material, and / or diffused through the decomposition of the non-porous material. The depot is then implanted at a desired location within the body of the subject, and the active substance is released from the implantable tablet at a predetermined rate.

[0234] In certain embodiments, a pharmaceutical composition containing an active substance is modified to have any type of in vivo release profile. In some embodiments, the pharmaceutical composition is a rapid-release, controlled-release, sustained-release, prolonged-release, delayed-release, or biphasic-release formulation. Methods for formulating release-controlled peptides are known in the art. See, for example, Qian et al., J Pharm 374:46-52 (2009) and International Patent Application Publications WO2008 / 130158, WO2004 / 033036, WO2000 / 032218, and WO1999 / 040942.

[0235] The composition may further include, for example, micelles or liposomes, or some other encapsulation form, and may also be administered in a sustained-release form to obtain long-term storage and / or delivery effects.

[0236] use

[0237] The antigen-binding proteins of this disclosure are useful for inhibiting tumor growth. Without being bound by any particular theory, the inhibitory effect of the antigen-binding proteins provided herein makes such entities useful in methods of treating cancer.

[0238] Accordingly, this specification provides methods for inhibiting tumor growth in a subject and methods for reducing tumor size in a subject. In various embodiments, the methods include administering the pharmaceutical composition of this disclosure to a subject in an amount effective to inhibit tumor growth or reduce tumor size in the subject. In various embodiments, the growth of ovarian tumors, melanomas, bladder tumors, or endometrial tumors is inhibited. In various embodiments, the size of ovarian tumors, melanomas, bladder tumors, or endometrial tumors is reduced.

[0239] As used herein, the terms “inhibit” or “reduce” and the words derived therefrom may not mean 100% or complete inhibition or reduction. Rather, the degree of inhibition or reduction that a person skilled in the art would recognize as having potential benefit or therapeutic effect varies. In this regard, the antigen-binding proteins of this disclosure may inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of this disclosure is about or at least 10% inhibition (e.g., about or at least 20% inhibition, about or at least 30% inhibition, about or at least 40% inhibition, about or at least 50% inhibition, about or at least 60% inhibition, about or at least 70% inhibition, about or at least 80% inhibition, about or at least 90% inhibition, about or at least 95% inhibition, about or at least 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is about or at least 10% reduction (e.g., about or at least 20% reduction, about or at least 30% reduction, about or at least 40% reduction, about or at least 50% reduction, about or at least 60% reduction, about or at least 70% reduction, about or at least 80% reduction, about or at least 90% reduction, about or at least 95% reduction, about or at least 98% reduction).

[0240] This specification further provides a method for treating a subject having cancer, for example, CLDN6-expressing cancer. In various embodiments, the method comprises administering the pharmaceutical composition of this disclosure to the subject in an amount effective to treat the cancer in the subject.

[0241] For the purposes herein, cancers of the methods disclosed herein may be any cancer, such as any malignant proliferation or tumor resulting from abnormal and uncontrolled cell division that can spread to other parts of the body via the lymphatic system or bloodstream. In some embodiments, cancers may include acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain tumor, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid Cancers selected from the group consisting of tumors, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, cancers of the peritoneum, omentum, and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer. In certain embodiments, cancers are selected from the group consisting of head and neck cancers, ovarian cancers, cervical cancers, bladder cancers, and esophageal cancers, pancreatic cancers, gastrointestinal cancers, gastric cancers, breast cancers, endometrial cancers, and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancers (e.g., non-small cell lung cancer (NSCLC)), and bronchioloalveolar carcinoma. In various aspects, cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, or endometrial cancer. In various aspects, cancer is any cancer characterized by moderate to high expression of CLDN6. See, for example, Figures 1-3. In various aspects, cancer is acute myeloid leukemia, large B-cell lymphoma, gastric cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, clear cell carcinoma of the kidney, head and neck cancer, sarcoma, chromophobic carcinoma of the kidney, low-grade glioma, adrenocortical carcinoma, glioblastoma, papillary renal cell carcinoma, squamous cell carcinoma of the lung, thyroid cancer, lung adenocarcinoma, pancreatic cancer, ovarian endometrioid carcinoma, uterine carcinosarcoma, or ovarian cancer. In various embodiments, cancer is selected from ovarian cancer, endometrioid carcinoma, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), cervical cancer, and bladder cancer.

[0242] As used herein, the term “to treat” and related terms do not necessarily mean 100% or complete treatment. Rather, the degree of treatment that a person skilled in the art would recognize as having potential benefits or therapeutic effects varies. In this regard, the methods for treating cancer of this disclosure may provide any amount or any level of treatment. Furthermore, the treatment provided by the methods of this disclosure may include treatment of one or more conditions, symptoms or signs of the cancer being treated. The treatment provided by the methods of this disclosure may also include slowing the progression of cancer. For example, the methods may treat cancer by enhancing T-cell activity or the immune response against cancer, suppressing the growth of tumors or cancer cells, reducing the metastasis of tumor cells, or increasing cell death of tumor or cancer cells. In various embodiments, the methods treat cancer by delaying the onset or recurrence of cancer by at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years, or longer. In various forms, the treatment involves extending the patient's survival.

[0243] The antigen-binding proteins of this disclosure may also be used to detect CLDN6 in a sample or to diagnose CLDN6-positive cancer. Accordingly, this disclosure provides a method for detecting claudin 6 (CLDN6) in a sample. In various embodiments, the method includes contacting a sample with an antigen-binding protein, complex, or fusion protein as described herein, and evaluating an immune complex containing the antigen-binding protein, complex, or fusion protein bound to CLDN6. This disclosure also provides a method for diagnosing a claudin 6 (CLDN6)-positive cancer of a subject. In various embodiments, the method includes contacting a biological sample, including cells or tissue obtained from a subject, with an antigen-binding protein, complex, or fusion protein as described herein, and evaluating an immune complex containing the antigen-binding protein, complex, or fusion protein bound to CLDN6.

[0244] subject

[0245] In some embodiments of this disclosure, the subject matter is mammals, which include, but are not limited to, rodent mammals such as mice and hamsters, as well as lagomorph mammals such as rabbits, mammals from the order Carnivora including Felidae (cats) and Canidae (dogs), mammals from the order Artiodactyla including Bovidae (female cats) and Suidae (pigs), or perissodactyla mammals including Equidae (horses). In some embodiments, the mammals are primates, Cebidae, or Simoids or Haplorhini (humans and apes). In some embodiments, the mammals are humans.

[0246] kit

[0247] In some embodiments, the antigen-binding proteins of this disclosure are provided in a kit. In various embodiments, the kit contains antigen-binding proteins as unit doses. For the purposes herein, “unit dose” means an individual amount dispersed in a suitable carrier. In various embodiments, the unit dose is an amount sufficient to provide a desired effect to a subject, e.g., inhibition of tumor growth, reduction of tumor size, or treatment of cancer. Accordingly, this specification provides kits comprising antigen-binding proteins of this disclosure, provided in unit doses, at the option of. In various embodiments, the kit includes several unit doses, e.g., a week's supply or a month's supply of unit doses, each optionally individually packaged or otherwise distinguished from other unit doses. In some embodiments, the components of the kit / unit dose are packaged together with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, e.g., a needle and a syringe, etc. In some embodiments, the antigen-binding protein of this disclosure, its pharmaceutically acceptable salt, a complex containing the antigen-binding protein, or a polymer or dimer containing the antigen-binding protein is packaged in an immediate-use form, such as a syringe or an intravenous bag. In some embodiments, the kit further includes other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including those described herein. In certain embodiments, the kit includes the antigen-binding protein of this disclosure together with an agent used in chemotherapy or radiotherapy, such as a therapeutic agent.

[0248] Various embodiments

[0249] In various embodiments of this disclosure, the bispecific antigen-binding protein binds to the human claudin 6 (CLDN6) protein (SEQ ID NO: 200) and a second antigen, in which (a) the antigen-binding protein binds to the extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6 but not to the extracellular loop 1 (EL1) of the ECD of CLDN6, or (b) it does not bind to any of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9), inhibiting the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than approximately 1200 nM, or (c) a combination thereof. In various cases, the bispecific antigen-binding protein binds to an epitope in the amino acid sequence of WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2), or to the amino acid sequence of TAHAIIRDFYNPL (SEQ ID NO: 3) or LVAEAQKREL (SEQ ID NO: 4) of CLDN6. In various embodiments, the bispecific antigen-binding protein does not bind to one or more of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various cases, the bispecific antigen-binding protein does not bind to CLDN3. In various cases, the bispecific antigen-binding protein binds to CLDN6, CLDN4, and CLDN9, but not to CLDN3. In various cases, the bispecific antigen-binding protein binds to CLDN6 and CLDN4, but not to CLDN3 or CLDN9. In various embodiments, the bispecific antigen-binding protein binds to CLDN6 and CLDN9, but not to CLDN3 or CLDN4.

[0250] In various embodiments, the bispecific antigen-binding protein of this disclosure inhibits the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than approximately 1200 nM, and the reference antibody comprises the light chain variable sequence of SEQ ID NO: 181 and the heavy chain variable sequence of SEQ ID NO: 182 or the light chain variable sequence of SEQ ID NO: 185 and the heavy chain variable sequence of SEQ ID NO: 186. In various embodiments, the bispecific antigen-binding protein of this disclosure inhibits the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than 1000 nM or less than 750 nM (e.g., less than 500 nM, less than 250 nM, or less than approximately 100 nM), and the reference antibody comprises the light chain variable sequence of SEQ ID NO: 181 and the heavy chain variable sequence of SEQ ID NO: 182 or the light chain variable sequence of SEQ ID NO: 185 and the heavy chain variable sequence of SEQ ID NO: 186.

[0251] In various embodiments, the bispecific antigen-binding protein is (a) the amino acid sequence of heavy chain CDR1 listed in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 452, 455, 461, 465, and 472, or a variant thereof, which differs by only one or two amino acids, or by about or at least 70% (e.g., about or at least 85%, about or less) (b) Sequences having sequence identity of 90% or more, (b) amino acid sequences of heavy chain CDR2 listed in Table A or A1, or sequences selected from the group consisting of SEQ ID NOs: 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 86, 102, 108, 114, 120, 126, 132, 475, 456, 462, 466, 468, and 473, or variant sequences thereof that differ by only one or two amino acids, or by approximately or at least 70% (e.g., approximately or at least 85%, approximately or at least 9%) (c) Sequences having 0% sequence identity, (c) amino acid sequences of heavy chain CDR3 listed in Table A or A1, or sequences selected from the group consisting of SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 453, 457, 463, 467, 469, and 474, or variant sequences thereof that differ by only one or two amino acids, or by approximately or at least 70% (e.g., approximately or at least 85%, approximately or at least 90%) (d) Sequences having the same sequence identity as (A) the amino acid sequence of the light chain CDR1 listed in Table A or A1, or a sequence selected from the group consisting of SEQ ID NOs: 8, 14, 20, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 449, 476, 458, 464, and 470, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., approximately or at least 85%, approximately or at least 90%) sequence identity.(e) The amino acid sequences of light chain CDR2 listed in Table A or A1, or sequences selected from the group consisting of SEQ ID NOs: 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 450, 477, 459, and 471, or variant sequences thereof that differ by only one or two amino acids, or have approximately or at least 70% (e.g., approximately or at least 85%, approximately or at least 90%) sequence identity, (f) The light chain CDR2 sequences listed in Table A or A1 The amino acid sequence of chain CDR3, or a sequence selected from the group consisting of SEQ ID NOs: 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 451, 454, and 460, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., approximately or at least 85%, approximately or at least 90%) sequence identity, including any two or more combinations of (g)(a) to (f).

[0252] In various embodiments, the bispecific antigen-binding protein comprises the light chain CDR1 amino acid sequence, the light chain CDR2 amino acid sequence, and the light chain CDR3 amino acid sequence described in Table A or A1, and one or two of the heavy chain CDR amino acid sequences described in Table A or A1. In some cases, the bispecific antigen-binding protein comprises the heavy chain CDR1 amino acid sequence, the heavy chain CDR2 amino acid sequence, and the heavy chain CDR3 amino acid sequence described in Table A or A1, and one or two of the light chain CDR amino acid sequences described in Table A or A1. In various aspects, bispecific antigen-binding proteins include (a) SEQ ID NOs. 74-79, (b) SEQ ID NOs. 50-55, (c) SEQ ID NOs. 122-127, (d) SEQ ID NOs. 26-31, (e) SEQ ID NOs. 128-133, (f) SEQ ID NOs. 38-43, (g) SEQ ID NOs. 62-67, (h) SEQ ID NOs. 80-85, (i) SEQ ID NOs. 44-49, (j) SEQ ID NOs. 86-91, (k) SEQ ID NOs. 104-109, (l) SEQ ID NOs. 56-61, (m) SEQ ID NOs. 32-37, (n) SEQ ID NOs. 110-115, (o) SEQ ID NOs. 98-103, (p (aa) The sequence contains six CDR amino acid sequences selected from the group consisting of (q)Sequences 92-97, (r)Sequences 116-121, (t)Sequences 8-13, (u)Sequences 68-73, (u)Sequences 14-19, (v)Sequences 20-25, (v)Sequences 449-453 and 475, (w)Sequences 476-477, 454-457, (x)Sequences 458-463, (y)Sequences 57, 58, 464-467, (z)Sequences 68-71 and 468-469, and (aa)Sequences 112 and 470-474.In various embodiments, the bispecific antigen-binding protein is (a) a heavy chain variable region amino acid sequence as listed in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., approximately or at least 85%, approximately or at least 90%) sequence identity. Or (b) a light chain variable region amino acid sequence listed in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70% (e.g., approximately or at least 85%, approximately or at least 90%) sequence identity, or both (a) and (b). In various embodiments, the bispecific antigen-binding proteins include (a) SEQ ID NOs: 156 and 157, (b) SEQ ID NOs: 148 and 149, (c) SEQ ID NOs: 172 and 173, (d) SEQ ID NOs: 140 and 141, (e) SEQ ID NOs: 174 and 175, (f) SEQ ID NOs: 144 and 145, (g) SEQ ID NOs: 152 and 153, (h) SEQ ID NOs: 158 and 159, (i) SEQ ID NOs: 146 and 147, (j) SEQ ID NOs: 160 and 161, (k) SEQ ID NOs: 1 It contains a pair of amino acid sequences selected from the group consisting of 66 and 167, (l) SEQ ID NOs: 150 and 151, (m) SEQ ID NOs: 142 and 143, (n) SEQ ID NOs: 168 and 169, (o) SEQ ID NOs: 164 and 165, (p) SEQ ID NOs: 162 and 163, (q) SEQ ID NOs: 170 and 171, (r) SEQ ID NOs: 134 and 135, (s) SEQ ID NOs: 154 and 155, (t) SEQ ID NOs: 136 and 137, and (u) SEQ ID NOs: 138 and 139.

[0253] In various embodiments, the bispecific antigen-binding protein is (a) a heavy chain variable region amino acid sequence as described in Table B1 or C, or a sequence selected from the group consisting of SEQ ID NOs: 376-379, 384-387, 391-396, 403-408, 412, 413, 416-419, 422-427, 478, 480, 482, 484, 486, and 488, or a variant thereof, which differs by only one or two amino acids, or has approximately or at least 70%, approximately 80%, approximately 90%, or approximately 95% sequence identity. (b) a light chain variable region amino acid sequence as described in Table B1 or C, or a sequence selected from the group consisting of SEQ ID NOs: 380-383, 388-390, 397-402, 409-411, 414, 415, 420, 421, and 479, 481, 483, 485, 487, and 489, or a variant thereof that differs by only one or two amino acids, or has approximately or at least 70%, approximately 80%, approximately 90%, or approximately 95% sequence identity, or (c) both (a) and (b). In various embodiments, the bispecific antigen-binding protein contains a pair of amino acid sequences as described in Table D.

[0254] This disclosure includes (A) HC CDR1 comprising the amino acid sequence YTFTXYT, where X is T, V, D, or S (SEQ ID NO: 452), and optionally comprising the amino acid sequence YTFTTYT (SEQ ID NO: 11); (B) HC CDR2 comprising the amino acid sequence IXPSSGYT, where X is Q, S, A, or N (SEQ ID NO: 475), and optionally comprising the amino acid sequence INPSSGYT (SEQ ID NO: 12); (C) HC CDR3 comprising the amino acid sequence AXGDYYVAY, where X is N, Q, H, or D (SEQ ID NO: 453), and optionally comprising the amino acid sequence ANGDYYVAY (SEQ ID NO: 13); and (D) HC CDR3 The present invention provides a bispecific antigen-binding protein comprising: (E)LC CDR1, which comprises the amino acid sequence SSVSSXY, where X is T, V, F, or D (SEQ ID NO: 449), and optionally comprises the amino acid sequence SSVSSTY (SEQ ID NO: 8); (F)LC CDR2, which comprises the amino acid sequence XTX, where X at position 1 is S, T, Q, or A, and X at position 3 is S, T, D, or Q (SEQ ID NO: 450), and optionally comprises the amino acid sequence STS (SEQ ID NO: 9); and (F)LC CDR3, which comprises the amino acid sequence HXYXRSPLT, where X at position 2 is Q, H, or S, and X at position 4 is H, Y, Q, or S (SEQ ID NO: 451), and optionally comprises the amino acid sequence HQYHRSPLT (SEQ ID NO: 10).

[0255] A bispecific antigen-binding protein is provided, which is (A) HC CDR1 containing the amino acid sequence FTFSXYX, where X at position 5 is N, S, R, Q, or A, and X at position 7 is W, H, Y, or F (SEQ ID NO: 455), and optionally containing the amino acid sequence FTFSNYW (SEQ ID NO: 23), (B) HC CDR2 containing the amino acid sequence IRLKXDXYAT, where X at position 5 is S, N, A, or T, and X at position 7 is Q, S, A, or N (SEQ ID NO: 456), and optionally containing the amino acid sequence IRLKSDNYAT (SEQ ID NO: 24), (C) HC (D)LC CDR3, which contains the amino acid sequence XDGPPSGX, where X at position 1 is N, D, or T, and X at position 8 is S, T, A, C, or Y (SEQ ID NO: 457), and optionally contains the amino acid sequence NDGPPSGC (SEQ ID NO: 25), (D)LC CDR1, which contains the amino acid sequence EXIYSY, where X is Q, S, A, D, or N (SEQ ID NO: 476), and optionally contains the amino acid sequence ENIYSY (SEQ ID NO: 20), (E)LC CDR2, which contains the amino acid sequence XAK, where X at position 1 is Q, S, A, D, or N (SEQ ID NO: 477), and optionally contains the amino acid sequence NAK (SEQ ID NO: 21), and (F)LC CDR3 comprising the amino acid sequence QXHYXVPWT, where X at position 2 is H, Q, S, or T, and X at position 5 is T, S, N, or G (SEQ ID NO: 454), and optionally comprising the amino acid sequence QHHYTVPWT (SEQ ID NO: 22), including LC CDR3.

[0256] A bispecific antigen-binding protein is provided, which is (A) HC CDR1 containing the amino acid sequence YTXTXYT, where X at position 3 is F, Y, S, or T, and X at position 5 is S, T, Y, or D (SEQ ID NO: 461), and optionally containing the amino acid sequence YTFTSYT (SEQ ID NO: 29), (B) HC CDR2 containing the amino acid sequence IXPSSXYT, where X at position 2 is Q, S, A, or N, and X at position 6 is T, S, V, D, or G (SEQ ID NO: 462), and optionally containing the amino acid sequence INPSSTYT (SEQ ID NO: 30), (C) HC (D) LC CDR3, which contains the amino acid sequence XRGEXGGFAY, where X at position 1 is S, A, T, or V, and X at position 5 is L, V, or F (SEQ ID NO: 463), and optionally contains the amino acid sequence SRGELGGFAY (SEQ ID NO: 31), (E) LC CDR1, which contains the amino acid sequence QSLVHSXGXTY, where X at position 7 is D, N, E, Q, S, or A, and X at position 9 is Q, S, A, D, or N (SEQ ID NO: 458), and optionally contains the amino acid sequence QSLVHSDGNTY (SEQ ID NO: 26), (E) LC CDR2, which contains the amino acid sequence XVX, where X at position 1 is K, Q, or R, and X at position 3 is S, T, or V (SEQ ID NO: 459), and optionally contains the amino acid sequence KVS (SEQ ID NO: 27), LC CDR2 and (F)LC CDR3, comprising the amino acid sequence SXXTHVPYT, where X at position 2 is Q, H, or T, and X at position 3 is S, G, T, or D (SEQ ID NO: 460), and optionally comprising the amino acid sequence SQSTHVPYT (SEQ ID NO: 28), wherein LC CDR3 is also included.

[0257] In various embodiments, the bispecific antigen-binding protein includes the following: (a) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 504 or SEQ ID NO: 507, or a variant thereof, which differs by only one or two amino acids, or which have approximately or at least 70% sequence identity. (b) The heavy chain CDR2 amino acid sequence of SEQ ID NO: 505 or SEQ ID NO: 508, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (c) The heavy chain CDR3 amino acid sequence of SEQ ID NO: 506 or SEQ ID NO: 509, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (d) The light chain CDR1 amino acid sequence of SEQ ID NO: 449 or SEQ ID NO: 476, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (e) The light chain CDR2 amino acid sequence of SEQ ID NO: 450 or SEQ ID NO: 477, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (f) The light chain CDR3 amino acid sequence of SEQ ID NO: 451 or SEQ ID NO: 454, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (g) Any combination of two or more of (a) to (f).

[0258] With arbitrary selection, the variant sequence has sequence identity of at least approximately 80%, approximately or at least 85%, approximately or at least 90%, or approximately or at least 95%.

[0259] In exemplary embodiments, the bispecific antigen-binding protein comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 449, the light chain CDR2 amino acid sequence or SEQ ID NO: 450, and the light chain CDR3 amino acid sequence or SEQ ID NO: 451, and one or two of the heavy chain CDR1 amino acid sequence of SEQ ID NO: 504, the heavy chain CDR2 amino acid sequence or SEQ ID NO: 505, and the heavy chain CDR3 amino acid sequence or SEQ ID NO: 506. In various cases, the antigen-binding protein comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 476, the light chain CDR2 amino acid sequence or SEQ ID NO: 477, and the light chain CDR3 amino acid sequence or SEQ ID NO: 454, and one or two of the heavy chain CDR1 amino acid sequence of SEQ ID NO: 507, the heavy chain CDR2 amino acid sequence or SEQ ID NO: 508, and the heavy chain CDR3 amino acid sequence or SEQ ID NO: 509. Optionally, the antigen-binding protein contains six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 449-451 and 504-506, and SEQ ID NOs: 476, 477, 454, and 507-509.

[0260] In exemplary embodiments, the bispecific antigen-binding protein includes (a) any one heavy chain variable region amino acid sequence from sequence numbers 490-503, or the heavy chain variable region amino acid sequence shown as S1-S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids or has approximately or at least 70% sequence identity; (b) any one light chain variable region amino acid sequence from sequence numbers 380-383, 388-390, 479, and 481, or the light chain variable region amino acid sequence shown as S1-S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids or has approximately or at least 70% sequence identity; or (c) both (a) and (b). In some embodiments, the variant sequence has at least approximately 80% or at least approximately 85% sequence identity, or the variant sequence has at least approximately 90% or at least approximately 95% sequence identity.

[0261] In exemplary cases, a bispecific antigen-binding protein contains the following pair of amino acid sequences: Sequence IDs 389 and 490, Sequence IDs 389 and 491, Sequence IDs 389 and 492, Sequence numbers 389 and 493, Sequence numbers 389 and 494, Sequence numbers 389 and 495, Sequence numbers 383 and 496, Sequence numbers 383 and 497, Sequence numbers 383 and 498, Sequence numbers 383 and 499, Sequence numbers 383 and 500, Sequence numbers 383 and 501, Sequence numbers 383 and 503, Sequence IDs 389 and 502, The arrangement of the heavy chain variable region shown as S1 in Figure 22 and the arrangement of the light chain variable region shown as S1 in Figure 22, The arrangement of the heavy chain variable region shown as S2 in Figure 22 and the arrangement of the light chain variable region shown as S2 in Figure 22, The arrangement of the heavy chain variable region shown as S3 in Figure 22 and the arrangement of the light chain variable region shown as S3 in Figure 22, The arrangement of the heavy chain variable region shown as S4 in Figure 22 and the arrangement of the light chain variable region shown as S4 in Figure 22, The arrangement of the heavy chain variable region shown as S5 in Figure 22 and the arrangement of the light chain variable region shown as S5 in Figure 22, The arrangement of the heavy chain variable region shown as S6 in Figure 22 and the arrangement of the light chain variable region shown as S6 in Figure 22, The arrangement of the heavy chain variable region shown as S7 in Figure 22 and the arrangement of the light chain variable region shown as S7 in Figure 22, The arrangement of the heavy chain variable region shown as S8 in Figure 22 and the arrangement of the light chain variable region shown as S8 in Figure 22, The arrangement of the heavy chain variable region shown as S9 in Figure 22 and the arrangement of the light chain variable region shown as S9 in Figure 22, The arrangement of the heavy chain variable region shown as S10 in Figure 22 and the arrangement of the light chain variable region shown as S10 in Figure 22, The arrangement of the heavy chain variable region shown as S11 in Figure 22 and the arrangement of the light chain variable region shown as S11 in Figure 22, or The arrangement of the heavy chain variable region, shown as S12 in Figure 22, and the arrangement of the light chain variable region, also shown as S12 in Figure 22.

[0262] In some embodiments, the bispecific antigen-binding protein is an antibody, such as a monoclonal antibody. In various embodiments, the antibody is IgG. Optionally, the antigen-binding protein inhibits at least approximately 50% of colony growth in a soft agar 3D growth assay, inhibits tumor growth in xenograft mice injected with human cancer cells, inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells, or inhibits at least 50% of tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0263] Therefore, in various embodiments, the present disclosure provides bispecific antigen-binding proteins including the following: (a) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 504 or SEQ ID NO: 507, or a variant thereof, which differs by only one or two amino acids, or which have approximately or at least 70% sequence identity. (b) The heavy chain CDR2 amino acid sequence of SEQ ID NO: 505 or SEQ ID NO: 508, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (c) The heavy chain CDR3 amino acid sequence of SEQ ID NO: 506 or SEQ ID NO: 509, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (d) The light chain CDR1 amino acid sequence of SEQ ID NO: 449 or SEQ ID NO: 476, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (e) The light chain CDR2 amino acid sequence of SEQ ID NO: 450 or SEQ ID NO: 477, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity. (f) The light chain CDR3 amino acid sequence of SEQ ID NO: 451 or SEQ ID NO: 454, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity, (g) Any combination of two or more of (a) to (f).

[0264] Also provided are bispecific antigen-binding proteins containing six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 449-451 and 504-506, and SEQ ID NOs: 476, 477, 454, and 507-509.

[0265] This disclosure provides a bispecific antigen-binding protein comprising the following: (a) Any one of the heavy chain variable region amino acid sequences from sequence numbers 490 to 503, or the heavy chain variable region amino acid sequences shown as S1 to S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids, or which have approximately or at least 70% sequence identity, (b) Any one of the light chain variable region amino acid sequences among sequence numbers 380-383, 388-390, 479, and 481, or the light chain variable region amino acid sequences shown as S1-S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids, or which have approximately or at least 70% sequence identity, (c) Both (a) and (b).

[0266] In various embodiments, the variant sequence has at least approximately 85% sequence identity, or approximately 90% or 95% sequence identity.

[0267] This disclosure also provides a bispecific antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of the following: Sequence IDs 389 and 490, Sequence IDs 389 and 491, Sequence IDs 389 and 492, Sequence numbers 389 and 493, Sequence numbers 389 and 494, Sequence numbers 389 and 495, Sequence numbers 383 and 496, Sequence numbers 383 and 497, Sequence numbers 383 and 498, Sequence numbers 383 and 499, Sequence numbers 383 and 500, Sequence numbers 383 and 501, Sequence numbers 383 and 503, Sequence IDs 389 and 502, The arrangement of the heavy chain variable region shown as S1 in Figure 22 and the arrangement of the light chain variable region shown as S1 in Figure 22, The arrangement of the heavy chain variable region shown as S2 in Figure 22 and the arrangement of the light chain variable region shown as S2 in Figure 22, The arrangement of the heavy chain variable region shown as S3 in Figure 22 and the arrangement of the light chain variable region shown as S3 in Figure 22, The arrangement of the heavy chain variable region shown as S4 in Figure 22 and the arrangement of the light chain variable region shown as S4 in Figure 22, The arrangement of the heavy chain variable region shown as S5 in Figure 22 and the arrangement of the light chain variable region shown as S5 in Figure 22, The arrangement of the heavy chain variable region shown as S6 in Figure 22 and the arrangement of the light chain variable region shown as S6 in Figure 22, The arrangement of the heavy chain variable region shown as S7 in Figure 22 and the arrangement of the light chain variable region shown as S7 in Figure 22, The arrangement of the heavy chain variable region shown as S8 in Figure 22 and the arrangement of the light chain variable region shown as S8 in Figure 22, The arrangement of the heavy chain variable region shown as S9 in Figure 22 and the arrangement of the light chain variable region shown as S9 in Figure 22, The arrangement of the heavy chain variable region shown as S10 in Figure 22 and the arrangement of the light chain variable region shown as S10 in Figure 22, The arrangement of the heavy chain variable region shown as S11 in Figure 22 and the arrangement of the light chain variable region shown as S11 in Figure 22, or The arrangement of the heavy chain variable region, shown as S12 in Figure 22, and the arrangement of the light chain variable region, also shown as S12 in Figure 22.

[0268] This specification provides bispecific antigen-binding proteins, including the following: (a) A heavy chain variable region amino acid sequence described as Sequence ID No. 510 or 513 or shown in Figure 23 or Figure 25, or a variant thereof, which differs by only one or two amino acids, or which has approximately or at least 70% sequence identity, (b) Light chain variable region amino acid sequences described as sequence number 511 or 512 or as shown in Figure 24 or Figure 26, or variant sequences thereof, which differ by only one or two amino acids, or which have approximately or at least 70% sequence identity, (c) Both (a) and (b).

[0269] This disclosure also provides antigen-binding proteins comprising a pair of amino acid sequences, such pair including: (a) The heavy chain variable region amino acid sequence described as SEQ ID NO: 510 and the light chain variable region amino acid sequence described as SEQ ID NO: 511, or a variant thereof, which differs by only 1 to 5 amino acids, or has approximately or at least 70% sequence identity, and which, at any discretion, differs by 1 to 5 amino acids as shown in Figure 23 in the case of the heavy chain, or as shown in Figure 24 in the case of the light chain, or (b) The heavy chain variable region amino acid sequence described as Sequence ID No. 513 and the light chain variable region amino acid sequence described as Sequence ID No. 512, or a variant thereof, wherein only 1 to 5 amino acids are different, or there is approximately or at least 70% sequence identity, or optionally, the 1 to 5 different amino acids are shown in Figure 25 in the case of the heavy chain, or in Figure 26 in the case of the light chain.

[0270] In various embodiments, the antigen-binding proteins disclosed herein include an Fc polypeptide containing a defucosylated glycan.

[0271] In various embodiments, the bispecific antigen-binding proteins of this disclosure are based on antibodies, e.g., monoclonal antibodies, e.g., IgG. In various embodiments, the bispecific antigen-binding proteins inhibit at least about 50% of colony growth in a soft agar 3D growth assay or inhibit tumor growth in xenograft mice injected with human cancer cells. In various embodiments, the bispecific antigen-binding proteins inhibit tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various cases, the bispecific antigen-binding proteins inhibit at least 50% of tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0272] This disclosure provides a complex comprising a bispecific antigen-binding protein and a heterologous moiety as described herein. In exemplary embodiments, the complex comprises a cytotoxic agent or chemotherapeutic agent, such as any of those described herein. In various embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking tubulin polymerization. In some cases, the antimitotic agent is auristatin, and optionally MMAE.

[0273] This disclosure also provides fusion proteins comprising the bispecific antigen-binding proteins described herein. This disclosure further provides nucleic acids comprising nucleotide sequences encoding the antigen-binding proteins, complexes, or fusion proteins of this disclosure. This disclosure provides vectors comprising nucleic acids comprising nucleotide sequences encoding the antigen-binding proteins, complexes, or fusion proteins of this disclosure. This disclosure further provides host cells comprising the nucleic acids or vectors of this disclosure.

[0274] The present disclosure provides a method for producing a bispecific antigen-binding protein that binds to the claudin 6 (CLDN6) protein, comprising (i) culturing host cells of the present disclosure in a cell culture medium, wherein the host cells contain nucleic acids comprising a nucleotide sequence encoding the antigen-binding protein described in any one of the prior claims, and (ii) collecting the antigen-binding protein from the cell culture medium. The present disclosure also provides a method for producing a fusion protein comprising a bispecific antigen-binding protein that binds to the claudin 6 (CLDN6) protein, comprising (i) culturing host cells of the present disclosure in a cell culture medium, wherein the host cells contain nucleic acids comprising a nucleotide sequence encoding the fusion protein of the present disclosure, and (ii) collecting the fusion protein from the cell culture medium.

[0275] The Disclosure further provides a method for producing a pharmaceutical composition comprising combining a bispecific antigen-binding protein, complex, fusion protein, nucleic acid, vector, host cell, or combination thereof, with a pharmaceutically acceptable carrier, diluent, or additive. The Disclosure also provides a pharmaceutical composition comprising a bispecific antigen-binding protein, complex, fusion protein, nucleic acid, vector, host cell, or combination thereof, with a pharmaceutically acceptable carrier, diluent, or additive.

[0276] This specification provides a method for treating a subject having CLDN6-expressing cancer, comprising administering the subject a pharmaceutical composition described herein in an amount effective for treating cancer. It also provides a method for inhibiting tumor growth in a subject, comprising administering the subject a pharmaceutical composition described herein in an amount effective for inhibiting tumor growth. This disclosure also provides a method for reducing tumor size in a subject, comprising administering the subject a pharmaceutical composition described herein in an amount effective for reducing tumor size. Furthermore, it provides a method for preventing cancer recurrence in a subject, comprising administering the subject a pharmaceutical composition described herein in an amount effective for preventing cancer recurrence.

[0277] This disclosure provides a method for detecting claudin 6 (CLDN6) in a sample, which includes contacting the sample with an antigen-binding protein, complex, or fusion protein of this disclosure, and evaluating an immune complex containing the antigen-binding protein, complex, or fusion protein bound to CLDN6. This specification also provides a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject, which includes contacting a biological sample containing cells or tissue obtained from the subject with an antigen-binding protein, complex, or fusion protein of this disclosure, and evaluating an immune complex containing the antigen-binding protein, complex, or fusion protein bound to CLDN6.

[0278] This disclosure also provides a method for treating cancer in subjects diagnosed with low overexpression of CLDN6. In various embodiments, the method involves administering the pharmaceutical composition disclosed herein to a subject in an amount effective to prevent cancer recurrence. In some embodiments, the administration induces apoptosis in tumor cells, and optionally, the administration induces apoptosis in CLDN6-expressing cells. In various embodiments, the subjects have tumors, which are semi-quantitatively classified into one of four groups: high-expression individuals, moderate-expression individuals, low-expression individuals, and non-expression individuals. In various cases, high-expression individuals are defined as having CLDN6 RNA greater than 12 log Fragments Per Kilobase Million (FPKM), where CLDN6 RNA is measured by RNASeq or CLDN6 protein levels greater than 3+ as measured by immunohistochemistry (IHC). In various cases, moderate expression individuals are defined as having CLDN6 RNA greater than 10log FPKM, where CLDN6 RNA is measured by RNASeq or CLDN6 protein levels are greater than 2+ when measured by IHC. In various cases, low expression individuals are defined as having CLDN6 RNA greater than 6log FPKM, where CLDN6 RNA is measured by RNASeq or CLDN6 protein levels are greater than 1+ when measured by IHC. In various cases, non-expressing individuals are defined as having CLDN6 RNA less than 6log FPKM, where CLDN6 RNA is measured by RNASeq or CLDN6 protein levels are below the IHC detection limit. In various embodiments, subjects having the tumor are similarly described as high-expressing, moderate-expressing, low-expressing, or non-expressing individuals of CLDN6.

[0279] The following embodiments are provided solely to illustrate the present disclosure and are not intended to limit its scope in any way. [Examples]

[0280] Example 1 This example demonstrates the analysis of CLDN6 RNA levels in various cell and tissue sources.

[0281] To establish a baseline for CLDN6 expression in various source materials, CLDN6 expression levels were assayed in patient samples, normal tissues, and cell lines created by the Translational Oncology Research Laboratory (TORL).

[0282] CLDN6 RNA levels in patient samples were measured using information from The Cancer Genome Atlas (TCGA) database, managed by the National Cancer Institute (NCI). CLDN6 levels in normal tissues were measured using information from the Genotype-Tissue Expression (GTEX) database, maintained by the Common Fund. Tissue analysis from the GTEX database showed that CLDN6 is detectable in various tissues, particularly in the brain, pituitary gland, pancreas, kidneys, lungs, thyroid gland, and neck (Figure 1).

[0283] CLDN6 expression levels were measured in TORL cancer cell lines using an Agilent 44K microarray (4x44K array chip, Agilent Technologies, Santa Clara, CA) and RNA sequencing (RNA-Seq) assay. RNASeq was performed by BGI Americas (Cambridge, MA) using their "RNASeq for quantification" service. As shown in Figures 2 and 3, ovarian cancer, head and neck cancer, lung cancer, and bladder cancer cells expressed the highest levels of CLDN6, but CLDN6 expression levels were detectable in breast cancer, kidney cancer, colon cancer, sarcoma, and liver cancer cells.

[0284] Example 2 This example demonstrates the production of cells engineered to overexpress CLDN6.

[0285] Models were established that were engineered to overexpress CLDN6. These models were used to determine the efficacy of the CLDN6 antibody described in Example 5. Briefly, the nucleotide sequence encoding CLDN6 was engineered within a bicistronic vector having a CMV promoter and an attenuated intra-ribosome entry site (IRES) of encephalomyocarditis virus (EMCV). The IRES was positioned between the target gene (GOI) cDNA (CLDN6) and the puromycin cDNA. A Woodchuck post-transcriptional regulatory element (WPRE) was positioned downstream of the puromycin cDNA. The vector also expressed a GFP marker sequence or a MycDDK tag. The sequence of the GFP-containing expression vector is provided herein as Sequence ID No. 189.

[0286] The expression vector was transduced into HEK293T cells (for screening) and NIH3T3 cells (for immunization) using a virus. Positively transduced cells were selected based on their survival in puromycin-containing medium (1 μg / ml). The positively selected cells were subcloned to obtain a stable, homogeneous CLDN6-overexpressing cell clonal population.

[0287] Subclone CLDN6 expression was confirmed by flow cytometry using a reference CLDN6 monoclonal antibody (mAb) on a BD Biosciences Accuri® flow cytometer (San Jose, CA). Secondary antibody and conjugate: The binding activity between the reference CLDN6 mAb and the CLDN6 expressed by the subclone was detected using the goat anti-mouse IgG (minimal cross-reactivity) antibody Alexa Fluor® 647 (Biolegend, San Diego, CA; catalog number 405322).

[0288] The intracellular localization of CLDN6 was determined by fluorescence microscopy using the Cellavista® imaging system (Synentec (Mountain View, CA)) with cells expressing the CLDN6-green fluorescent protein (GFP) fusion protein. As shown in Figure 4, GFP fluorescence was detected at the cell membrane, demonstrating that CLDN6 is localized to the cell membrane.

[0289] Experimental Example 3 This example demonstrates the production of a reference antibody and a control antibody.

[0290] The benchmark CLDN6-specific antibody and control antibody were prepared by cloning the heavy chain and light chain variable regions of the antibody into the ExpiCHO® expression system (ThermoFisher Scientific, Waltham, MA) to produce recombinant mouse IgG2A chimeric antibody. These antibodies were tested in parallel with the newly prepared CLDN6-specific antibody described in Example 5.

[0291] In short, plasmids containing the sequences of the control antibody and the benchmark antibody were transfected using the ExpiCHO® expression system (catalog number: A29133, ThermoFisher Scientific, USA) according to the manufacturer's instructions. Cells were cultured at 37°C and 8% CO2 for day 1, and then, after transfection, cultured in the kit-provided medium at 32°C and 5% CO2. The antibodies were purified by centrifugation at 1,000 g for 10 minutes, followed by 5,000 g for 30 minutes, to clarify the ExpiCHO® medium. The supernatant was then filtered using a 0.45 μm filter, followed by a 0.22 μm filter. Subsequently, the supernatant was subjected to affinity purification using Protein A / G resin (Life Technologies, Carlsbad, CA; catalog number 20424) according to the manufacturer's instructions. Prior to ELISA purification, the antibody titer in the culture medium was roughly determined to ensure that the volume of culture medium occupied less than 80% of the resin's binding capacity. After incubation, the resin was washed with PBS and eluted with elution buffer (Life Technologies, catalog no. 21004). The eluted fraction was immediately adjusted to physiological pH by adding Tris buffer at pH 8.0. The purified antibody was then subjected to buffer exchange and protein concentration using an Amicon Ultra-15 centrifugal filter unit (Life Technologies, catalog no. UFC900324) in PBS buffer. The antibody concentration was determined by BCA protein assay. SDS-PAGE and Coomassie staining were performed to test antibody purity. The purified protein was divided into equal volumes and stored at -80°C for long-term storage or maintained at 4°C for immediate use.

[0292] The integrity of the antibody was evaluated by comparing non-reducing and reducing conditions using SDS-PAGE followed by Coomassie staining. Under non-reducing conditions, one dominant band was observed around 150 kDa, while under reducing conditions, two bands were observed at 50 kDa and 25 kDa.

[0293] Antibodies specific to other CLDN family members with sequence similarity (Figure 5), namely CLDN3, CLDN4, and CLDN9, were constructed in essentially the same manner, except that the antibody sequences contained in the plasmids were specific to CLDN3, CLDN4, or CLDN9.

[0294] Example 4 This example demonstrates the characterization of cell lines with high endogenous CLDN6 expression.

[0295] Cancer cell line panels were analyzed for their endogenous CLDN6 expression by FACS and Western blotting. In short, antibody binding to targets was evaluated by FACS using cells overexpressing CLDN6 (e.g., HEK293T cells overexpressing CLDN6 as described in Example 2) and cell lines endogenously expressing CLDN6 at high or low levels, as determined in Example 1. CLDN6-expressing cells were incubated with a reference antibody or control antibody (described in Example 3) on ice for 30 minutes, washed, and then incubated with Alexa Fluor® 647-conjugated goat anti-mouse IgG (minimal cross-reactivity) antibody (Biolegend catalog number 405322) on ice for 30 minutes. Fluorescence was read using a BD Biosciences Accuri® flow cytometer (San Jose, CA).

[0296] Western blotting was performed using nitrocellulose with reference and control antibodies. Simply put, samples from cell lysates were boiled to denature protein-containing substances. Denatured proteins were separated by polypeptide length using SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were then transferred from the acrylamide gel to a nitrocellulose membrane. The membrane was blocked with 2% bovine serum albumin (BSA) solution to minimize nonspecific antibody binding. The membrane was incubated with the reference or control antibody. The membrane was stained with a horseradish peroxidase (HRP)-conjugated secondary antibody that recognizes the reference or control antibody, and the secondary antibody was detected by chemiluminescence.

[0297] Overexpression cells were used to evaluate control and reference antibodies, and at the time of evaluation, these control and reference antibodies were used to characterize endogenous cell lines. Cells overexpressing CLDN6 were included in these assays as positive controls.

[0298] FACS assays showed that, in addition to endometrial cancer cell lines, four ovarian cancer cell lines, bladder cancer cell lines, lung cancer cell lines, and upper gastrointestinal cancer cell lines expressed CLDN6 at high levels on their surface. High levels of CLDN6 expression were also detected by Western blotting. Two additional ovarian cancer cell lines, additional hepatic cancer cell lines, additional lung cancer cell lines, and additional upper gastrointestinal cancer cell lines showed moderate levels of CLDN6 expression on their surface when detected by Western blotting. Endometrial tumor cells and bladder tumor cells also expressed high levels of CLDN6 in in vivo xenografts. The endogenous expression levels of CLDN6 in the tested cancer cell lines are summarized in Table 2. [Table 20] TIFF0007856818000036.tif71169

[0299] Example 5 This example demonstrates the immunization of mice to produce CLDN6-specific antibodies.

[0300] CLDN6-specific antibodies were produced by immunizing Balb / c and CD1 mice with a mixture of three different peptide immunogens, following the methodology of the Fred Hutchinson Cancer Research Center. The three peptides extended to the second loop (i.e., EL2) of the CLDN6 extracellular domain. The peptides included the full length of EL2, the peptide up to the first (N-terminal) half of EL2, and the peptide up to the remaining (C-terminal) half of EL2. The sequences of the three peptides are listed in Table 3. [Table 21]

[0301] Mice were also immunized with 3T3 cells overexpressing full-length CLDN6 using a plasmid containing a human CLDN6-myc-DDK expression vector.

[0302] Splenocytes were isolated from immunized mice and fused with myeloma cells using BTX electrofusion (BTX, Holliston, MA) to create hybridomas. 7680 primary hybridoma cultures were prepared and cultured in 384-well plates. The ability of antibodies to bind peptides was evaluated using a bead array with beads expressing three different peptide targets. 1920 potential antibodies were again aligned in 96-well plates and further screened against endogenous and artificial cell line models by flow cytometry.

[0303] Subsequently, the supernatant of positive hybridomas was counter-screened by flow cytometry against endogenous and artificial models of proteins with sequence similarity to the target region (e.g., other CLDN proteins). From the secondary and counter-screenings, approximately 20 CLDN6-specific antibodies were selected for further testing. These antibodies were subcloned, and their variable heavy chain and variable light chain sequences were determined. See Table B and the sequence listing.

[0304] CLDN6 antibody was formatted as a full-length IgG antibody using ExpiCHO® expression. The variable regions of the antibody's heavy and light chains were cloned into a laboratory-engineered antibody expression vector based on the pcDNA® 3.4-TOPO® vector (catalog number: A14697, ThermoFisher Scientific, USA) and transfected into CHO cells (following the operating procedure provided in the kit (ExpiCHO® Expression System, catalog number: A29133, ThermoFisher Scientific, USA)). The antibody was purified, and its cell surface binding to CLDN6 and its IC50 were determined by FACS. The CLDN6 antibody was directly conjugated to Alexa Fluor® 647 NHS ester (succinimidyl ester), catalog number A20106 (ThermoFisher Scientific) according to the manufacturer's operating procedure. The CLDN6 antibody was tested in 50 μl volumes using a 150,000-cell system from 0.32 nM to 1000 nM (series dilution 1:5, 6 steps).

[0305] CLDN6-expressing cells were used in a FACS assay to determine the ability of CLDN6 antibodies to bind to CLDN6 on the cell surface and to cross-react with other CLDN family members. HEK293T cells engineered to express GFP-fused human CLDN6, GFP-fused mouse CLDN6, CLDN9-GFP, CLDN4-GFP, or CLDN3-GFP, or GFP alone (without CLDN6), were used as an artificial model for CLDN6 expression. ARK2, OVCA429, LS513, and MCF7 cells were used as endogenous models for CLDN6 expression, as well as models for CLDN3 / 4 expression.

[0306] For each cell type and each mAb tested, cells were detached from the culture flask surface with EDTA (instead of trypsin) to protect cell surface proteins. The detached cells were then incubated with Alexa Fluor®-labeled CLDN6 mAbs at a pre-set concentration in the dark on ice for 30 minutes. CLDN6 mAbs were directly labeled with Alexa Fluor® 647 NHS ester (succinimidyl ester). After washing, cells were read using a BD Accuri® flow cytometer C6 to detect antibody-antigen protein binding at channel FL4H. Each antibody was tested at various concentrations, and dose-fluorescence curves were created. The EC50 / IC50 of the antibody (half the maximum antibody concentration) was calculated based on the FL4H value (gated in singlet live cells) using the online-available Very Simple IC50 Tool kit, which allows plotting biological dose-response data, fitting curve types, and obtaining EC50 / IC50. The maximum value was defined as the lowest antibody concentration at which fluorescence was maximized. The antibodies were also screened for their ability to cross-react with other CLDN proteins, such as CLDN9, CLDN3, and CLDN4. These values ​​were used to determine the relative affinity of each antibody in the series tested. Cross-reactivity data were obtained using similar methods, but cells with different expression profiles for CLDN6, CLDN3, CLDN4, and CLDN9 were used.

[0307] The relative affinity and cross-reactivity data determined in this manner are shown in Tables 4 and 5. [Table 22] The TIFF0007856818000039.tif89160AB number corresponds to the AB numbers listed in Tables A and B. [Table 23] The AB numbers correspond to the AB numbers listed in Tables A and B.

[0308] Example 6 This example demonstrates the characterization of IgG mAbs from chimeric mice.

[0309] To further clarify the characteristics of the mAb described in Example 5, a soft agar 3D proliferation assay and a xenograft binding assay were performed. Briefly, in each well of a 48-well plate, 250 μL of a top layer mixture containing 10,000 cells in 1×RPMI medium containing 0.6% SeaPlaque agarose was plated on top of the 250 μL bottom layer of solidified 1×RPMI medium containing 0.6% SeaPlaque agarose. A 250 μL liquid feeder layer containing 1×RPMI medium was placed on top of the solidified top layer. All three layers of the soft agar assay were prepared with or without trastuzumab, Cldn6 mAb, or mouse IgG2a control, starting at 150 ng / mL (1 μM) and ending at 1.5 ng / mL (diluted 1:10). Each test condition was performed in double rows. Cells were allowed to colonize for three weeks, then stained with 0.05% neutral red and imaged using an EVOS XL inverted optical microscope. Cell lines showing a reduction in colony number of 20% or more compared to the control were considered sensitive.

[0310] As shown in Table 6, many cell lines showed a decrease in colony count when treated with the antibodies indicated. [Table 24]

[0311] In vivo binding studies were performed in xenograft mice injected with human cancer cell lines. Simply put, a xenograft model of human cancer cell lines was established in 6-week-old CD-1 thymus-deficient nude mice (Charles River Laboratories). Subcutaneous injection of each cell line followed the following conditions: ARK2 0.75 × 10⁻¹⁶ 7 cells, UMUC4 1.0×10 7 Cells, OV90 1.0×10 7 Cells and M202 0.5×10 7 Cellular samples were used, and 50% Matrigel (BD Biosciences) was used in all cases. A sufficient number of mice were injected, with 8 mice per treatment group. Tumors were 150-300 mm in size. 3 When the average size was reached, the mice were randomly divided into treatment groups. For treatment, each therapeutic antibody (AB3, AB2, reference Ab1, reference Ab2, reference Ab3 (trastuzumab) and non-targeted IgG2 control) was diluted to a working concentration of 1 mg / ml in sterile saline for intravenous (IV) injection into the tail vein. In the M202 study, trametinib (DMSO solvate, MedChem Express) was administered orally at 1.0 mg / kg (10% Cremaphor, 10% PEG400) in the first week of a weekly cycle with a 5-day administration, 2-day rest period schedule, and then reduced to 0.5 mg / kg for the remaining two weeks. Tumor xenografts were measured three times a week with calipers, and the tumor volume was calculated by multiplying the height × width × length in mm². 3The results were calculated in units of 10. Mice were treated for 2–7 weeks. At the end of the study, the animals were euthanized, the tumor tissue was excised, and the tissue was divided for preservation as flash-frozen or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis. All animal work was performed according to operating procedures approved by the IACUC and the University of California at Los Angeles Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). Results are presented as the mean volume for each group. Error bars represent the standard error (SE) of that mean.

[0312] The results of the xenograft assay are shown in Figures 6-10. As shown in Figures 6A and 6B, in endometrial tumor-bearing mice, AB2 and AB3, respectively, resulted in a substantial mean change in tumor volume compared to the control IgG2 antibody at day 14. As shown in Figures 7A and 7B, in bladder tumor-bearing mice, AB3 resulted in a substantial mean change in tumor volume compared to the control IgG2 antibody at day 35. Figures 8A and 8B show that in ovarian tumor-bearing mice, AB2 and AB3, respectively, resulted in a substantial mean change in tumor volume compared to the control IgG2 antibody at day 20. Figures 9A and 9B show that AB3 functions specifically for CLDN, as the models used in Figures 9A and 9B did not express any of CLDN6, CLDN3, CLDN4, and CLDN9, and were therefore used as negative controls. The data in Figures 9A and 9B also suggest that AB3 has less off-target activity than reference Ab1 and reference Ab2. Figure 10A summarizes the results from Figures 6 to 9. As shown in Figure 10A, AB3 significantly suppressed tumor growth in endometrial tumor, bladder tumor, and ovarian tumor-bearing mice expressing CLDN6, but did not suppress tumor growth in melanoma tumor-bearing mice that did not express CLDN6 (Figure 10B). As shown in Figure 11, there was no significant change in the average body weight of the treated mice, suggesting the safety of the treatment.

[0313] A second set of experiments was conducted in a xenograft model of the human ovarian cancer cell line OV90. Mice were injected with one of the 10 mAbs described in Example 5, a control antibody (mouse IgG2a antibody, reference CLDN6 ab), or a PBS vehicle control. There were 8 mice per group, and each animal received 10 mg / kg of antibody intravenously every 4 days. As shown in Figures 12A and 12B, several of the antibodies described in Example 5 reduced the tumor volume of ovarian tumor-bearing mice. The best results were obtained with AB3, AB4, AB7, and AB10, but all antibodies tested reduced tumor volume compared to the vehicle control. As shown in Figure 13, there was no significant change in body weight of animals treated with AB3, AB4, AB7, or AB10, suggesting their safety.

[0314] Example 7 This example demonstrates further characterization of IgG mAbs from chimeric mice.

[0315] A quantitative assay for internal translocation was performed. In short, the internal translocation of the CLDN6 protein induced by binding to reference Ab1, AB3, or AB4 was tested, using the transferrin receptor (TfR), a known cell surface receptor that translocates internally after antibody binding and is expressed everywhere, as a positive control.

[0316] TfR and CLDN6 antibodies were labeled with Texas Red®-X, succinimidyl ester, mixed isomers, catalog number T6134 (ThermoFisher Scientific). The day before antibody treatment, cells were seeded in an 8-well μ-slide chamber (catalog number 80826, ibidi Cells In Focus Inc.), and the cells were allowed to adhere and grow. The cells were incubated with the labeled antibodies in the dark on ice for 30 minutes. Subsequently, the chambers containing cells labeled with CLDN6 or TfR were read using an Echo Lab fluorescence microscope to collect images before internal migration. The chambers were then incubated at 37°C for 40 minutes to allow internal migration to proceed, and images were collected again using an Echo Lab fluorescence microscope. In AB3 and AB4, the degree of CLDN6 internal migration was greater than that obtained in reference Ab1 (data not shown).

[0317] Example 8 This example demonstrates further characterization of IgG mAbs from chimeric mice.

[0318] A two-dimensional (2D) proliferation assay was performed using the selected antibody described in Example 5 as follows: Cells were seeded in double rows of 5,000 to 20,000 cells per well in a 24-well plate. The following day, cells were treated with six dilutions of mAbs (starting with either 100 nM trastuzumab, Cldn6 mAb, or mouse IgG2A control) from 1 to 5, and a fixed concentration of 1 ng / μL of monomethyl auristatin E (MMAE)-conjugated anti-mouse secondary antibody (Moradec, LLC) was used to create dose-response curves. To determine the range of cell proliferation, cells in the untreated wells were quantified on day 1 (the day of antibody treatment) and on day 6 thereafter. Wells treated with mAbs were quantified on day 6, and proliferation under each treatment condition was determined as a normalized percentage ratio to the proliferation of untreated cells. Quantification was performed using a Z1 Particle Counter (Beckman Coulter, Inc.).

[0319] The results are shown in Figure 14. AB2, AB3, AB4, and AB5 showed the highest efficacy in inhibiting growth. The IC50 of each of these antibodies was 0.1 nM to 1 nM. AB7, AB10, AB11, and AB15 also showed growth inhibitory activity in this assay, albeit to a lesser degree than AB2, AB3, AB4, and AB5.

[0320] Example 9 This example demonstrates the humanization of the antibody of this disclosure.

[0321] A subset of antibodies listed in Table A was selected for humanization analysis. The heavy chain variable (VH) and light chain variable (VL) sequences of AB1, AB3, AB4, AB9, AB11, and AB18 antibodies were compared with libraries of known human germline sequences from the human VH gene and human VL kappa gene (IMGT®, the international ImMunoGeneTics information system®, www.imgt.org; founder and manager: Marie-Paule Lefranc, Montpellier, France). (The databases used were the IMGT human VH gene (F+ORF, 273 germline sequences) and the IMGT human VL kappa gene (F+ORF, 74 germline sequences)). Acceptor human germline sequences were selected from those that most closely matched the parental antibodies.

[0322] Table 7 provides information on the human germline sequence selected as the acceptor sequence and the selected human heavy chain linkage region (J gene) for both the VH and VL versions of each antibody. The linkage region (J gene) was selected from human linkage region sequences stored in IMGT® (the international ImMunoGeneTics information system) www.imgt.org (founder and administrator: Marie-Paule Lefranc, Montpellier, France). [Table 25]

[0323] CDR is defined according to the AbM definition (see the CDR definition comparison table on Dr. Andrew CRMartin's website www.bioinf.org.uk / abs / ).

[0324] Alterations in the position of the human germline framework (i.e., non-CDR residues in VH and VL) relative to the corresponding mouse parent sequence may be necessary to optimize the binding of the humanized antibody. Sequences of humanized antibody versions are provided as SEQ ID NOs. 376–421.

[0325] In the case of AB1, Asn52 (sequentially numbered) of the HC CDR2 and Asn54 of the LC CDR2 were determined to have a low probability of deamidation based on their sequence and conformation.

[0326] In the case of AB3, Asn31 (sequentially numbered) of HC's CDR1, Asn57 of HC's CDR2, Asn28 of LC's CDR1, and Asn50 of LC's CDR2 were determined to have a low probability of deamidation based on their sequence and conformation. Trp33 of HC's CDR1 was determined to be potentially exposed to solvents and potentially oxidized, especially under stress conditions. In HC's CDR3, it was determined that there is a free Cys106 inside the CDR that could be problematic during antibody production due to the possibility of solvent exposure. Modification of this Cys residue to Tyr, Ser, or Ala was recommended. The binding maintenance of these modified antibodies was tested. Ile53 of LC's CDR2 was determined to be potentially exposed to solvents and could cause nonspecific binding. Modification of this Ile residue to Ser was proposed. The binding maintenance of this modified antibody will be tested.

[0327] In the case of AB4, Asn52 (sequential numbering) in CDR2 of HC and Asn58 in CDR2 of LC were determined to have a low probability of deamidation based on their sequence and conformation. The sequence DGNT within CDR1 of LC was determined to be problematic because it was determined to have a high probability of isoalpartate formation (sequence DG) and a high probability of deamidation (sequence NT). Modification of this sequence was recommended.

[0328] In the case of AB9, Asn33 (sequentially numbered) in HC's CDR1, and Asn52 and Asn59 in HC's CDR2 were judged to have a low probability of deamidation based on their sequence and conformation. Asn54 was determined to have a moderate probability of deamidation based on its sequence and conformation. The NGG sequence in HC's CDR2 was determined to have a high / moderate probability of deamidation following isoalpartate formation. Therefore, modification of this amino acid sequence was recommended. Free Cys106 in HC's CDR3 was determined to be potentially solvent-exposed and could pose a problem during antibody production. Modification of this Cys residue to Tyr, Ser, or Ala is proposed. The binding maintenance of these modified antibodies will be tested. Arg28 in HC's CDR1 is rarely found in human antibodies. This residue will be modified to Thr and the binding maintenance will be tested. In the case of AB9, Trp32 (sequentially numbered) within the LC CDR1 was determined to be susceptible to solvent exposure and, especially under stress conditions, to oxidation. Leu24 in the same CDR is rarely found in human antibodies. This residue will be modified to Arg, and binding maintenance will be tested.

[0329] In the case of AB11, Asp54-Ser55 (sequentially numbered) within the CDR2 of HC was determined to have a low probability of isoalpartate formation. Asn57 within the CDR2 of LC was determined to have a low probability of deamidation based on its sequence and conformation.

[0330] In the case of AB18, Asn33 in HC's CDR1 and Asn50 (sequentially numbered) in CDR-H2 were determined to have a low probability of deamidation based on their sequence and conformation. In HC's CDR2, the Asp-Pro(DP) sequence was determined to be susceptible to fragmentation under acidic conditions. In the VL domain, Asn34 and Asn37 in LC's CDR1 were determined to have a low probability of deamidation based on their sequence and conformation. In LC's CDR3, Trp56 was determined to be susceptible to solvent exposure and may undergo oxidation, particularly under stress conditions.

[0331] Table 8 shows a scheme for matching humanized VH and VL when neither humanized version is equivalent to the chimeric mAb. Preferred pairs are indicated in underlined bold text. [Table 26] TIFF0007856818000044.tif248157

[0332] Humanized antibodies were constructed as described in Table 8 and essentially represented as described in Example 5. A FACS assay was performed essentially as described in Example 5 to determine the relative antigen-binding strength of the humanized antibodies. Two doses of the humanized antibodies (1.5 μg or 0.3 μg) were tested for binding of the protein expressed by the engineered 293T clone to human or mouse CLDN6. The assay results are shown in Table 9. [Table 27] TIFF0007856818000046.tif163160

[0333] Furthermore, FACS assays were performed (1.5 μg or 0.3 μg) to determine the relative antigen-binding strength of humanized antibodies to CLDN6 expressed by the indicated cancer cell lines. The assay results are shown in Table 10. "Second Ab only" was used as a negative control. 64A-chim, h64A, and SC27-108-chim were used as reference antibodies. The corresponding parental antibodies (antibodies before humanization) were used as controls and are indicated with "chim".

[0334] [Table 28] TIFF0007856818000048.tif249160TIFF0007856818000049.tif39160

[0335] Based on in vitro antigen-binding data, three humanized antibodies were selected for further testing and development. The antibodies were derived from AB1, AB3, and AB4.

[0336] Essentially, as described in Example 6, in vivo binding studies of humanized versions of AB1, AB3, and AB4 were performed in xenograft mice injected with the bladder cancer cell line UMUC4. In short, a xenograft model of UMUC4 was established in 6-week-old CD-1 thymus-deficient nude mice (Charles River Laboratories). The tumor size was 150-300 mm. 3 After reaching an average size, the mice were randomly divided into treatment groups. Humanized antibodies were diluted to a working concentration of 1 mg / ml in sterile saline for intravenous (IV) injection into the tail vein. Tumor xenografts were measured three times a week with calipe...

Claims

1. A bispecific antigen-binding protein that binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200) and a second antigen which is CD3 or CD16A, Antigen-binding proteins, (i) HC CDR1 containing the amino acid sequence GTFFSNYW (SEQ ID NO: 23), (ii) HC CDR2 containing the amino acid sequence IRLKSDNYAT (SEQ ID NO: 24), (iii) HC CDR3 containing the amino acid sequence XDGPPSGX (Sequence ID: 457), wherein the X at position 1 is N and the X at position 8 is S, T, A, C, or Y. (iv) LC CDR1 containing amino acid sequence ENIYSY (SEQ ID NO: 20), (v) LC CDR2 containing the amino acid sequence NAK (SEQ ID NO: 21), (vi) f. LC CDR3 containing the amino acid sequence QHHYTVPWT (SEQ ID NO: 22) It contains and binds to the human claudin 6 (CLDN6) protein (SEQ ID NO: 200). The aforementioned bispecific antigen-binding protein.

2. The bispecific antigen-binding protein according to claim 1, wherein the X at position 8 in formula (iii) is S.

3. The bispecific antigen-binding protein according to claim 1, wherein the X at position 8 in formula (iii) is T.

4. The bispecific antigen-binding protein according to claim 1, wherein the X at position 8 in formula (iii) is A.

5. The bispecific antigen-binding protein according to claim 1, wherein the X at position 8 in formula (iii) is C.

6. The bispecific antigen-binding protein according to claim 1, wherein the X at position 8 in formula (iii) is Y.

7. The antigen-binding protein is scFv, F(ab') ２ A bispecific antigen-binding protein according to any one of claims 1 to 6, which is an antigen-binding fragment of an antibody selected from the group consisting of Fab and Fv.

8. The bispecific antigen-binding protein according to any one of claims 1 to 6, wherein the antigen-binding protein is an antibody.

9. Antibodies, (i) Monoclonal antibodies, and (ii) Human antibodies, humanized antibodies, or chimeric antibodies, The bispecific antigen-binding protein according to claim 8, which is any one of the following.

10. The bispecific antigen-binding protein according to claim 8, wherein the antibody is an IgG antibody.

11. The bispecific antigen-binding protein according to claim 10, wherein IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

12. The bispecific antigen-binding protein according to claim 11, wherein IgG is IgG1.

13. The bispecific antigen-binding protein according to claim 2, wherein the bispecific antigen-binding protein that binds to the human claudin 6 (CLDN6) protein has the heavy chain variable domain sequence of SEQ ID NO: 387 and the light chain variable domain sequence of SEQ ID NO:

389.

14. The bispecific antigen-binding protein according to any one of claims 1 to 6, wherein the bispecific antigen-binding protein is a bispecific antibody or a bispecific antigen-binding antibody fragment.

15. The bispecific antigen-binding protein according to claim 14, wherein the bispecific antigen-binding protein is a bispecific monoclonal antibody.

16. The bispecific antigen-binding protein according to claim 14 or 15, wherein the bispecific antigen-binding protein is a chimeric or humanized bispecific antibody.

17. The bispecific antigen-binding protein according to any one of claims 1 to 6, wherein the bispecific antigen-binding protein is a bispecific T cell engager or a tandem diabody.

18. (a) A bispecific antigen-binding protein according to any one of claims 1 to 17, and (b) pharmaceutically acceptable carriers, diluents and / or excipients A pharmaceutical composition containing the following:

19. A bispecific antigen-binding protein according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, for use in the treatment of cancer, inhibition of tumor growth, reduction of tumor size, or prevention of cancer recurrence.

20. A method for manufacturing a pharmaceutical composition, pharmaceutically acceptable carriers, diluents and / or excipients, a. The bispecific antigen-binding protein according to claim 1, b. A complex comprising the bispecific antigen-binding protein described in claim 1, c. A fusion protein comprising the bispecific antigen-binding protein described in claim 1, d. A nucleic acid encoding a bispecific antigen-binding protein as described in claim 1, e. A vector containing the nucleic acid of (d) above, f. A host cell containing the nucleic acid of (d) or the vector of (e), or g. A combination of a. to f. above, The method, which includes combining the following.

21. A method for producing a bispecific antigen-binding protein or a bispecific fusion protein thereof as described in claim 1, a. Culturing host cells in a cell culture medium, wherein the host cells contain nucleic acids comprising a nucleotide sequence encoding the bispecific antigen-binding protein or bispecific fusion protein described in claim 1, and b. Recovering the bispecific antigen-binding protein or the bispecific fusion protein thereof from the cell culture medium. The method, including the method described above.

22. Use of a bispecific antigen-binding protein according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, in the manufacture of a pharmaceutical for use in the treatment of cancer, inhibition of tumor growth, reduction of tumor size, or prevention of cancer recurrence.

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