Claudin-6 antibodies and drug conjugates

Abstract

To provide antigen-binding proteins which bind to Claudin-6 (CLDN6).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 42A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 821,391, filed March 20, 2019, the entire contents of which are incorporated by this reference in their entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing, identified as a 338,714 ASCII (text) file with the file name "54086P1_Seqlisting.txt," created on March 20, 2019, filed concurrently with the filing of the application. [Background technology]

[0003] Antibodies constitute powerful 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 seen significant growth. As of late 2014, 47 monoclonal antibody products have received approval in the United States or Europe for the treatment of a variety of diseases, including cancer, as well as inflammatory, cardiovascular, respiratory, and infectious diseases.

[0004] More than a dozen 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 conjugated with chemotherapy drugs, such as brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®). Other antibody products, such as blinatumomab (Blincyto), are engineered to recognize and bind to two different antigens. Despite the availability of such antibody products, current cancer incidence and cancer mortality rates remain high. The reported cancer incidence rate is over 450 per 100,000 men and women per year, and the cancer mortality rate is just over 170 per 100,000 men and women per year. Summary of the Invention

[0005] Provided herein are antigen binding proteins that bind to claudin 6 (CLDN6). In various aspects, the antigen binding proteins of the present disclosure bind to human CLDN6, and optionally to murine CLDN6. In various aspects, the antigen binding proteins bind to the extracellular domain (ECD) of CLDN6. In various cases, the antigen binding proteins bind to extracellular loop 2 (EL2) of the ECD of CLDN6. In various aspects, the antigen binding proteins bind to EL2 and not to extracellular loop 1 (EL1) of the ECD of CLDN6. In various cases, 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). In various cases, the antigen binding proteins bind to CLDN6 and at least one of CLDN4 and CLDN9. In various cases, 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, and exhibits an IC50 of less than about 1200 nM in a FACS affinity assay using OVCA429 cells. In various cases, the antigen binding protein of the present disclosure inhibits tumor growth in a subject, e.g., a human, without any other moieties attached to the antigen binding protein.

[0006] In various embodiments, the antigen binding protein has (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 has about 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 has about 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 has about or at least 70% sequence identity; (d) the heavy chain CDR4 amino acid sequence of SEQ ID NO: 44 (e) a light chain CDR2 amino acid sequence of SEQ ID NO: 450 or SEQ ID NO: 477, or a variant thereof, which differs by one or two amino acids or has about or at least 70% sequence identity; (f) a light chain CDR3 amino acid sequence of SEQ ID NO: 451 or SEQ ID NO: 454, or a variant thereof, which differs by one or two amino acids or has about or at least 70% sequence identity; and / or (g) a combination of any two or more of (a) to (f).

[0007] In various aspects, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 490-503, or a heavy chain variable region amino acid sequence designated as S1-S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity; or (b) a light chain variable region amino acid sequence of any one of SEQ ID NOs: 380-383, 388-390, 479, and 481, or a light chain variable region amino acid sequence designated as S1-S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity; or both (a) and (b).

[0008] In various embodiments, the antigen-binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 389 and 490, (b) SEQ ID NO: 389 and 491, (c) SEQ ID NO: 389 and 492, (d) SEQ ID NO: 389 and 493, (e) SEQ ID NO: 389 and 494, (f) SEQ ID NO: 389 and 495, (g) SEQ ID NO: 383 and 496, (h) SEQ ID NO: 383 and 497, (i) SEQ ID NO: 383 and 498, (j) SEQ ID NO: 383 and 499, (k) SEQ ID NO: 383 and 500, (l) SEQ ID NO: 383 and 501, (m) SEQ ID NO: 383 and 503, (n) SEQ ID NO: 389 and 502, (o) the sequence of the heavy chain variable region shown as S1 in FIG. 22 and the sequence of the light chain variable region shown as S1 in FIG. 22, (p) the sequence of the heavy chain variable region shown as S2 in FIG. 22 and the sequence of the light chain variable region shown as S2 in FIG. 22, (q) the sequence of the heavy chain variable region shown as S3 in FIG. 22 and the sequence of the light chain variable region shown as S3 in FIG. 22, (r) the sequence of the heavy chain variable region shown as S4 in FIG. 22 and the sequence of the light chain variable region shown as S4 in FIG. 22, (s) the sequence of the heavy chain variable region shown as S5 in FIG. 22 and the sequence of the light chain variable region shown as S5 in FIG. 22, (t) the sequence of the heavy chain variable region shown as S6 in FIG. 22 and the sequence of the light chain variable region shown as S6 in FIG. 22, (u) the sequence of the heavy chain variable region shown as S7 in FIG. 22 and the sequence of the light chain variable region shown as S7 in FIG. 22, (v) the sequence of the heavy chain variable region shown as S78 in FIG. 22 and the sequence of the light chain variable region shown as S8 in FIG. 22, (w) the sequence of the heavy chain variable region shown as S89 in FIG. 22 and the sequence of the light chain variable region shown as S9 in FIG. 22, (x) the sequence of the heavy chain variable region shown as S910 in FIG. 22 and the sequence of the light chain variable region shown as S10 in FIG. 22, (y) the sequence of the heavy chain variable region shown as S11 in FIG. 22 and the sequence of the light chain variable region shown as S11 in FIG. 22, or (z) the sequence of the heavy chain variable region shown as S12 in FIG. 22 and the sequence of the light chain variable region shown as S12 in FIG. 22.

[0009] In various cases, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence set forth as SEQ ID NO: 510 or 513 or set forth in Figure 23 or Figure 25, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity, or (b) a light chain variable region amino acid sequence set forth as SEQ ID NO: 511 or 512 or set forth in Figure 24 or Figure 26, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity, or (c) both (a) and (b).

[0010] In various instances, the antigen binding protein comprises a pair of amino acid sequences, such pair comprising: (a) a heavy chain variable region amino acid sequence set forth as SEQ ID NO:510 and a light chain variable region amino acid sequence set forth as SEQ ID NO:511, or variants thereof, which differ by only 1-5 amino acids or have about or at least 70% sequence identity, optionally wherein the 1-5 amino acids differ are shown in Figure 23 for the heavy chain or Figure 24 for the light chain; or (b) a heavy chain variable region amino acid sequence set forth as SEQ ID NO:513 and a light chain variable region amino acid sequence set forth as SEQ ID NO:512, or variants thereof, which differ by only 1-5 amino acids or have about or at least 70% sequence identity, optionally wherein the 1-5 amino acids differ are shown in Figure 25 for the heavy chain or Figure 26 for the light chain.

[0011] Further provided herein are antigen binding proteins conjugated to a heterologous moiety (e.g., conjugated to any chemotherapeutic agent, drug, or toxic moiety) that inhibit tumor growth in a subject, e.g., a human. In various cases, the conjugated antigen binding protein is a monoclonal antibody. In various cases, the antibody is conjugated to an agent that alters microtubule dynamics, e.g., MMAE. In various cases, the conjugate includes a cleavable linker, e.g., MC-VC-PAB. In various embodiments, the conjugate is a homogeneous or heterogeneous conjugate. In various embodiments, the heterologous moiety is conjugated at a specific site on the antigen binding protein.

[0012] In various embodiments, the antigen binding protein binds to CLDN6 expressed by human cancer cells. In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN6 and an anti-CLDN6 reference antibody. Without being bound by a particular theory, the inhibitory action of the antigen binding proteins provided herein makes such entities useful in methods of suppressing tumor growth and treating subjects with tumors or cancer. As further discussed herein, in various embodiments, the antigen binding protein is an antibody, an antigen-binding antibody fragment thereof, or an antibody protein product.

[0013] The present disclosure also provides antigen binding proteins comprising at least three, four, five, or all of the amino acid sequences of a particular group of amino acid sequences. In various embodiments, the antigen binding proteins comprise at least three, four, five, or six complementarity determining region (CDR) amino acid sequences of a CLDN6 antibody disclosed herein.

[0014] The present disclosure further provides antigen binding proteins comprising an amino acid sequence as detailed herein. In various embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 490-512, or the amino acid sequence shown in any one of Figures 22-26, or a combination thereof, as detailed herein.

[0015] Related polypeptides, nucleic acids, vectors, host cells, and complexes are further provided herein. Kits and pharmaceutical compositions containing such entities are further contemplated.

[0016] Also provided are methods of making the antigen binding proteins, in various embodiments, the methods comprise culturing a host cell comprising nucleic acid encoding an antigen binding protein or polypeptide as described herein, so as to express such antigen binding protein or polypeptide.

[0017] Further provided herein are methods of treating a subject with cancer. In various embodiments, the methods comprise administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0018] Also provided is a method for treating a subject with a CLDN6-expressing cancer, comprising administering to the subject the pharmaceutical composition described herein.Furthermore, a method for inhibiting tumor growth in a subject, comprising administering to the subject the pharmaceutical composition described herein, is contemplated.

[0019] A method of reducing tumor size in a subject or preventing recurrence of cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein.

[0020] Also provided herein is a method for treating cancer in a subject diagnosed with low overexpression of CLDN6, comprising administering to the subject a pharmaceutical composition described herein.

[0021] In various embodiments, administration induces apoptosis in tumor cells, e.g., CLDN6-expressing cells, hi 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 drawings]

[0022] [Figure 1] 1 shows a graph of CLDN6 expression in normal (non-cancerous) tissues. [Figure 2] 1 depicts a graph of CLDN6 expression in cancer cell lines as determined by the Agilent44K method. [Figure 3] 1 shows a graph of CLDN6 expression in cancer cell lines as determined by RNASeq. [Figure 4] 1 depicts a series of fluorescence images showing the localization of CLDN6-GFP in different cell models. [Figure 5] Figure 1 shows a sequence alignment of human CLDN6, human CLDN3, human CLDN4, human CLDN9, and mouse CLDN6. The sequences of EL1 and EL2 are shown. [Figure 6] A depicts a graph of tumor volume (mm3) of tumors in endometrial tumor-bearing mice as a function of time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, AB2, and AB3. B depicts a graph of the mean change in tumor volume (mm3) at day 14 of tumors in endometrial tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, AB2, or AB3. [Figure 7] A represents a graph of tumor volume (mm3) of tumors in bladder tumor-bearing mice as a function of time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, and AB3. B represents a graph of the mean change in tumor volume (mm3) at day 35 of tumors in bladder tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, reference Ab2, or AB3. [Figure 8] A represents a graph of tumor volume (mm3) of tumors in ovarian tumor-bearing mice as a function of time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, AB2, and AB3. B represents a graph of the mean change in tumor volume (mm3) at day 20 of tumors in ovarian tumor-bearing mice treated with control IgG2 antibody, AB3, reference Ab1, AB2, or AB3. [Figure 9] A represents a graph of tumor volume (mm3) of tumors in mice bearing melanoma tumors as a function of time (days) after treatment with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, and AB3. B represents a graph of the mean change in tumor volume (mm3) at day 21 for tumors in mice bearing melanoma tumors treated with control IgG2 antibody, AB3, reference Ab1, reference Ab2, reference Ab3, or AB3. [Figure 10] (A) Graph of tumor growth inhibition (%) achieved in tumor-bearing mice treated with AB3 compared to mice treated with a control antibody. (B) Western blot images showing different levels of CLDN6 id in endometrial cancer cell lines (ARK2), bladder cancer cell lines (UMUC4), ovarian cancer cell lines (OV90), and melanoma cell lines (M202) and control cells. Alpha-tubulin levels were similar, indicating comparable protein loading. [Figure 11] Graphs of body weight change (%) over time (days) for tumor-bearing mice treated with vehicle control, control antibody, Reference Ab1, Reference Ab2, Reference Ab3, and AB3. [Figure 12A] Graph of tumor volume (mm3) of tumors in ovarian tumor-bearing mice as a function of time (days) after treatment with vehicle control, control IgG2 antibody, AB3, reference Ab1, or one of the indicated anti-CLDN6 antibodies. [Figure 12B] Graphs depict the mean change in tumor volume (mm3) at day 28 for tumors in ovarian tumor-bearing mice treated with vehicle control, control IgG2 antibody, AB3, reference Ab1, or one of the indicated anti-CLDN6 antibodies. [Figure 13] Graphs depicting the percent weight change over time (days) in tumor-bearing mice treated with vehicle control, control antibody, reference Ab1, and the indicated anti-CLDN6 antibodies. [Figure 14] Figure 1 shows a series of dose-response curves for several anti-CLDN6 antibodies of the invention as well as Reference Ab1 and Reference 2. Mouse IgG was used as a control. [Figure 15] A graph shows the mean change in tumor volume (mm) of tumors in bladder tumor-bearing mice treated with a vehicle control, a control IgG antibody, the murine form of AB3, the first humanized form of AB3, and the second humanized form of AB3 at day 35. B graphs the change in tumor volume (mm) for each group in Figure 15A. [Figure 16] (A) Graph of the mean change in tumor volume (mm3) at day 35 for tumors in bladder tumor-bearing mice treated with a vehicle control, a control IgG antibody, the murine form of AB3, the first humanized form of AB3, and the second humanized form of AB3. Two control antibodies (one murine and one chimeric) were also tested in this experiment. (B) Graph of the change in tumor volume (mm3) for each group in Figure 16A. [Figure 17] A graph shows the mean change in tumor volume (mm) of tumors in bladder tumor-bearing mice treated with a vehicle control, a control IgG antibody, the murine form of AB1, and the humanized form of AB1 at day 35. B graphs the change in tumor volume (mm) for each group in Figure 17A. [Figure 18] A graph shows the mean change in tumor volume (mm3) of tumors in bladder tumor-bearing mice treated with a vehicle control, a control IgG antibody, the murine form of AB4, and the humanized form of AB4 at day 35. B graphs the change in tumor volume (mm3) for each group in Figure 18A. [Figure 19] (A) shows a graph of the average change in tumor volume (mm3) at day 35 for bladder tumor-bearing mice treated with a vehicle control, a control IgG antibody, a murine form of AB3, a chimeric form of AB3, a first humanized form of AB3, a second humanized form of AB3, a murine form of Ab1, a humanized form of AB1, a murine form of AB4, and a humanized form of AB4; four control antibodies (one murine or chimeric, one murine or humanized) were also tested in this experiment. (B) shows a graph of the change in tumor volume (mm3) for each group in Figure 19A. [Figure 20]19B depicts a graph of the mean change in tumor volume (mm3) at day 55 for tumors in bladder tumor-bearing mice treated as described in FIG. 19A. [Figure 21] 19B depicts a graph of the weight change (%) of treated tumor-bearing mice on day 32 treated as described in FIG. 19A. [Figure 22] List of heavy and light chain variable region sequences of 12 CLDN6 antibodies (designated S1-S12) that have been generated and characterized. S1-S6 are based on a humanized version of AB3 (humanized AB3-7), and S7-S12 are based on a humanized version of AB1 (humanized AB1-11). [Figure 23] 1 is a schematic diagram of exemplary somatic hypermutations (SHM) in the CLDN6 antibodies of the present disclosure, as identified by next-generation sequencing (NGS). An example of SHM identified by NGS in the AB-3 series antibody and heavy chain. Mutations are listed using Chothia numbering. [Figure 24] 1 is a schematic diagram of exemplary somatic hypermutations (SHM) in the CLDN6 antibodies of the present disclosure, as identified by next-generation sequencing (NGS). An example of SHM identified by NGS in the AB-3 series antibody and light chain. Mutations are listed using Chothia numbering. [Figure 25] 1 is a schematic diagram of exemplary somatic hypermutations (SHM) in the CLDN6 antibodies of the present disclosure, as identified by next-generation sequencing (NGS). An example of SHM identified by NGS in the AB-1 series antibody and heavy chain. Mutations are listed using Chothia numbering. [Figure 26] 1 is a schematic diagram of exemplary somatic hypermutations (SHM) in the CLDN6 antibodies of the present disclosure, as identified by next-generation sequencing (NGS). An example of SHM identified by NGS in the AB-1 series antibody and light chain. Mutations are listed using Chothia numbering. [Figure 27]

[0023] Figure 12 is a table of the results of a FACS binding assay of antibodies S1-S12 based on humanized AB3-7 (ABS1-S6) or humanized AB1-11 (ABS7-S12). The concentrations tested are shown in column D. [Figure 28]1 is a table of the results of a FACS binding assay of antibodies S1-S12 at different concentrations in various cell lines. [Figure 29] A shows examples of three types of N-glycans (oligomannose, complex, and hybrid) and the symbols commonly used for such sugars. B shows 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 through three reactions catalyzed by GMD and FX. GDP-fucose is then transported from the cytosol to the lumen of the Golgi by GDP-Fuc transferase and transferred to acceptor oligosaccharides and proteins. The other reaction product, GDP, is converted to guanosine 5-monophosphate (GMP) and inorganic phosphate (Pi) by luminal nucleotide diphosphatase. The former is exported to the cytosol (via a countertransport system in conjunction with the transport of GDP-fucose), while the latter is thought to leave the Golgi lumen via the Golgi anion channel GOLAC. See, for example, Nordeen et al. 2000; Hirschberg et al. 2001. [Figure 30] 1 is a graph of tumor volume in mice receiving the treatments described herein following subcutaneous injection of human cancer cells. [Figure 31] 1 is a graph of tumor volume in mice receiving the treatments described herein following subcutaneous injection of human cancer cells. [Figure 32] 1 is a graph of tumor volume in mice receiving the treatments described herein following subcutaneous injection of human cancer cells. [Figure 33] 1 is a graph of tumor volume in mice receiving the treatments described herein following subcutaneous injection of human cancer cells. [Figure 34A] 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). FIG. 2 is a table summarizing the biochemical properties of CLDN6 ADCs. [Figure 34B]

[0023] Figure 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). Chromatograms from HIC-HPLC are shown showing the relative abundance of antibody conjugated with different numbers of drugs. [Figure 34C]

[0023] Figure 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). Chromatograms from HIC-HPLC are shown showing the relative abundance of antibody conjugated with different numbers of drugs. [Figure 34D]

[0023] Figure 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). Chromatograms from HIC-HPLC are shown showing the relative abundance of antibody conjugated with different numbers of drugs. [Figure 34E]

[0023] Figure 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). Chromatograms from HIC-HPLC are shown showing the relative abundance of antibody conjugated with different numbers of drugs. [Figure 34F]

[0023] Figure 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). Chromatograms from HIC-HPLC are shown showing the relative abundance of antibody conjugated with different numbers of drugs. [Figure 34G]

[0023] Figure 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). Chromatograms from HIC-HPLC are shown showing the relative abundance of antibody conjugated with different numbers of drugs. [Figure 34H]

[0023] Figure 1 shows the biochemical characterization of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 (also called AB23). Chromatograms from HIC-HPLC are shown showing the relative abundance of antibody conjugated with different numbers of drugs. [Figure 35] 1 shows the molecular integrity of CLDN6 ADCs, including AB3-7, analyzed by native PAGE. [Figure 36] 1 shows the binding activity (flow cytometry) of CLDN6 antibody-drug conjugates (ADCs) including AB3-7 to naturally CLDN6-positive cells or cells in which CLDN6 is artificially overexpressed. [Figure 37]

[0039] Figure 1 shows 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 a KinExA 4000 (Sapidyne Instrument, Boise, Idaho). [Figure 38] 1 shows in vitro characterization of CLDN6 ADCs, including AB3-7. The panels show the cellular internalization of CLDN6 ADCs. H23-7 refers to AB3-7. [Figure 39A] This shows the in vitro anti-cancer activity of CLDN6 ADCs. This shows the two-dimensional (2D) growth inhibitory effect of CLDN6 ADCs, including AB-3-7, namely MC-VC-PAB-MMAE (conventional), on cancer cells. [Figure 39B] This shows the two-dimensional (2D) growth inhibitory effect of CLDN6 ADCs, including AB-3-7, namely MC-VC-PAB-MMAE (D4 technology), on cancer cells. [Figure 39C] This shows the in vitro anti-cancer activity of CLDN6 ADCs. This shows the two-dimensional (2D) growth inhibitory effect of CLDN6 ADCs, including AB-3-7, namely, MC-GGFG-MMAE (D4 technology), on cancer cells. [Figure 39D] This shows the in vitro anti-cancer activity of CLDN6 ADCs. CLDN6 ADCs, including AB-3-7, exhibit two-dimensional (2D) growth inhibitory activity against CL2A-SN38 (conventional) cancer cells. [Figure 39E] This shows the in vitro anti-cancer activity of CLDN6 ADCs. This shows the two-dimensional (2D) growth inhibitory effect of CLDN6 ADCs, including AB-3-7, namely CL2A-SN38 (D4 technology), on cancer cells. [Figure 39F] 1 shows the in vitro anti-cancer activity of CLDN6 ADCs. 2D growth inhibitory effect of CLDN6 ADCs, including AB-3-7, namely, MC-GGFG-DXD, on cancer cells. [Figure 39G]1 shows the in vitro anti-cancer activity of CLDN6 ADCs. 2D growth inhibitory effect of CLDN6 ADCs, including AB-3-7, i.e., MC-VC-PAB-DXD, on cancer cells is shown. [Figure 39H] Figure 1 shows the in vitro anti-cancer activity of CLDN6 ADC. Figure 2 shows the 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) on cancer cell lines, namely, ARK2. [Figure 39I] 1 shows the in vitro anti-cancer activity of CLDN6 ADCs. 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) on cancer cell line, i.e., OVCA429. [Figure 39J] 1 shows the in vitro anti-cancer activity of CLDN6 ADCs. 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) on cancer cell line, i.e., H841. [Figure 39K] Figure 1 shows the in vitro anti-cancer activity of CLDN6 ADC. Figure 2 shows the 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) against a cancer cell line, i.e., OV90. [Figure 39L] 1 shows the in vitro anti-cancer activity of CLDN6 ADCs. 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) on cancer cell line, i.e., H1693. [Figure 39M] 1 shows the in vitro anti-cancer activity of CLDN6 ADCs. 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) on cancer cell line, i.e., M202. [Figure 39N] 1 shows the in vitro anti-cancer activity of CLDN6 ADCs. 2D growth inhibitory effect of CLDN6 ADC-11 (AB1-11 conjugated with VC-PAB-MMAE) on a cancer cell line, i.e., MCF7. [Figure 40]1 shows the in vivo anticancer effect of CLDN6 ADC-11 on CLDN6-positive ovarian cancer cell line (OV90) xenografts. [Figure 41] AB shows that CLDN6 ADC-11 has no anticancer activity against CLDN6-negative melanoma cancer cell line (M202) xenografts. [Figure 42A] Figure 1 shows the in vivo anti-cancer effect of CLDN6 ADC-23 (AB3-7-VC-PAB-MMAE) on cancer cell line xenografts. Figure 2 shows the anti-cancer activity of CLDN6 ADC-23 on CLDN6-positive bladder cell line (UMUC4) xenografts. [Figure 42B] Figure 1 shows the in vivo anti-cancer effect of CLDN6 ADC-23 (AB3-7-VC-PAB-MMAE) on cancer cell line xenografts. Hematoxylin and eosin (H&E) staining of xenograft tissues harvested at the indicated time points after treatment with a control antibody or 5 mg / kg ADC-23 is shown. [Figure 43A] Figure 1 shows the in vivo anti-cancer effect of CLDN6 ADC-23 on ovarian cancer patient-derived xenografts (PDX). A panel of ovarian PDX samples screened for CLDN6 expression by Western blotting is shown. [Figure 43B] 1 shows the in vivo anti-cancer effect of CLDN6 ADC-23 on ovarian cancer patient-derived xenografts (PDX). A schematic diagram showing the injection of luciferase enzyme-transfected PDX ovarian cancer cells into the peritoneal cavity of immunodeficient mice (NSG). [Figure 43C] 1 shows the in vivo anti-cancer effect of CLDN6 ADC-23 on ovarian cancer patient-derived xenografts (PDX). 2 shows the survival rate of mice treated with CLDN6 ADC-23 as described herein. [Figure 43D] 1 shows the in vivo anti-cancer effect of CLDN6 ADC-23 on ovarian cancer patient-derived xenografts (PDX). 2 shows the survival rate of mice treated with CLDN6 ADC-23 as described herein. [Figure 43E]1 shows the in vivo anti-cancer effect of CLDN6 ADC-23 on ovarian cancer patient-derived xenografts (PDX). 2 shows the survival rate of mice treated with CLDN6 ADC-23 as described herein. [Figure 44] Figure 1A shows the dose-dependent anti-cancer effect of CLDN6 ADC-23 on CLDN6-positive ovarian cancer cell line (OV90) xenografts. Tumor size reduction is shown at the doses and times indicated. Figure 1B shows the dose-dependent anti-cancer effect of CLDN6 ADC-23 on CLDN6-positive ovarian cancer cell line (OV90) xenografts. Tumor size reduction is shown at the doses and times indicated. Figure 1C shows the dose-dependent anti-cancer effect of CLDN6 ADC-23 on CLDN6-positive ovarian cancer cell line (OV90) xenografts. The change (%) in body weight of mice administered CLDN6 ADC-23 is shown. [Figure 45] A shows that CLDN6 ADC-23 has no off-target activity in CLDN6-negative melanoma cancer cell line (M202) xenografts. Changes in tumor volume are shown. B shows that CLDN6 ADC-23 has no off-target activity in CLDN6-negative melanoma cancer cell line (M202) xenografts. Changes in tumor volume are shown. Tumor volumes were measured on day 21. C shows that CLDN6 ADC-23 has no off-target activity in CLDN6-negative melanoma cancer cell line (M202) xenografts. Changes in body weight (%) in mice administered CLDN6 ADC-23. DETAILED DESCRIPTION OF THE INVENTION

[0023] Claudine Family

[0024] Tight junctions, also known as occluding junctions or zonulae occludente, are vertebrate structures located between two adjacent cells that regulate paracellular permeability and maintain cell polarity in epithelial and endothelial cell sheets. Claudin (CLDN) family genes encode membrane proteins that are key components of tight junctions. CLDN proteins contain four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2). The extracellular loops of CLDN proteins from adjacent cells interact with each other to tightly seal the cell sheets and regulate paracellular transport between the lumen and the basolateral space.

[0025] CLDN proteins are involved in various human diseases and pathologies. For example, mutations in the CLDN1 gene have been shown to cause progressive scaling of the skin along with bile duct obstruction. Mutations in the CLDN16 gene cause a magnesium-wasting disorder. Mutations in CLDN19 lead to eye diseases such as macular colobomata and myopia, and mutations in CLDN14 can result in nonsyndromic recessive 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 coreceptors for hepatitis C virus (HCV) entry. Several CLDN proteins have been shown to be aberrantly expressed in cancer. For example, CLDN1 is downregulated in breast and colon cancer, while CLDN3 and CLDN4 are highly upregulated in multiple cancers.

[0026] 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, cows, mice, rats, zebrafish, and frogs. CLDN6 is generally expressed in humans as a 220-amino acid precursor protein, of which the first 21 amino acids 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 SEQ ID NO: 1. The amino acid at position 143 of SEQ 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 SEQ ID NO: 178.

[0027] antigen-binding proteins

[0028] Provided herein is an antigen-binding protein that binds to claudin 6 (CLDN6). The antigen-binding protein of the present disclosure may take the form of any one of the many forms of antigen-binding proteins known in the art. In various embodiments, the antigen-binding protein of the present disclosure takes the form of an antibody, or an antigen-binding antibody fragment, or an antibody protein product.

[0029] In various embodiments of the present disclosure, the antigen-binding protein comprises, consists essentially of, or consists of an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, including heavy and light chains, and including variable and constant regions. For example, an antibody may be an IgG, which has two identical pairs of polypeptide chains in a "Y-shaped" configuration, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In the IgG format, the variable region generally has about 100-110 amino acids or more, contains three complementarity-determining regions (CDRs), and is primarily responsible for antigen recognition, substantially differentiating it from other antibodies that bind to different antigens. The constant region enables the antibody to recruit cells and molecules of the immune system. The variable region is made up of the N-terminal regions of the light and heavy chains, respectively, and the constant region is made up of the C-terminal regions of the heavy and light chains, respectively (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes," Immunobiology: The Immune System in Health and Disease, 4 th ed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0030] The general structure and characteristics of antibody CDRs have been reported in the art. Briefly, in antibody scaffolds, CDRs are embedded within the framework of the heavy and light chain variable regions and constitute the regions primarily responsible for antigen binding and recognition. A variable region typically contains at least three CDRs of the heavy or light chain (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883) within framework regions (FR1, FR2, FR3, and FR4, designated framework regions 1 to 4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0031] Antibodies can include any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype 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 the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can 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 can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Thus, in various embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In various aspects, the antibody comprises a constant region containing one or more amino acid modifications relative to naturally occurring amino acids to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In various cases, the antibody comprises a constant region in which the C-terminal Lys residue present in the naturally occurring constant region has been removed or clipped.

[0032] The antibody may be a monoclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. 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, human antibody, etc. In certain aspects, the antigen-binding protein is an antibody such as a human antibody. In certain aspects, the antigen-binding protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody can, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, can contain sections of amino acid sequence from at least two species. A chimeric antibody can also contain domains from two or more different antibodies within the same species. The term "humanized" as used in reference to antibodies refers to antibodies that have at least CDR regions from non-human material and that have been engineered to have a structure and immunological function that more closely resembles that of a true human antibody than the original source antibody. For example, humanization can involve grafting CDRs from a non-human antibody, such as a murine antibody, onto a human antibody. Humanization can also involve selecting amino acid substitutions to make the non-human sequence more similar to a human sequence. Information, such as sequence information for the heavy and light chain constant regions of human antibodies, is publicly available from the Uniprot database and other databases well known to those skilled in the art of antibody engineering and production. For example, the IgG2 constant region is available from the Uniprot database as Uniprot number P01859, incorporated herein by reference.

[0033] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves antibodies to generate two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to generate an F(ab')2 fragment and a pFc' fragment. In various aspects of the present disclosure, the antigen-binding proteins of the present disclosure are antigen-binding fragments of antibodies (also known as antigen-binding antibody fragments, antigen-binding fragments, or antigen-binding portions). In various cases, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0034] The antibody structure has been exploited to generate an ever-expanding range of alternative antibody formats, ranging in molecular weight from at least about 12 to 150 kDa and in valencies (n) from monomers (n=1) to 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 the entire antibody structure 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, consisting of only the variable (V) region. A soluble, flexible amino acid peptide linker is used to stabilize the molecule, connecting the V region to the 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 easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody formats such as disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and diabodies, triabodies, and tetrabodies, including various formats consisting of scFvs linked to oligomerization domains, or minibodies (miniAbs). The smallest fragments are the VHH / VH of camelid heavy chain Abs and single-domain Abs (sdAbs). The most frequently used building blocks for creating novel antibody formats are single-chain variable (V) domain antibody fragments (scFv), which contain V domains (VH and VL domains) from 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 peptibody structure consists of a biologically active peptide grafted onto the Fc domain. Peptibodies are well described in the art, see, e.g., Shimamoto et al., mAbs 4(5):586-591 (2012).

[0035] Other antibody protein products include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, bispecific or triabodies, etc. Bispecific antibodies are divided into five major classes: BsIgG, tagged IgG, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

[0036] In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of these antibody protein products. In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of the following: scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camelid heavy chain antibody, sdAb, diabody; triabody; tetrabody; bispecific or triabody, BsIgG, tagged IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.

[0037] In various cases, the antigen-binding proteins of the present disclosure are antibody protein products in monomeric, or polymeric, oligomeric, or multimeric form. In certain embodiments in which an antibody comprises fragments of two or more distinct antigen-binding regions, the antibody is bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that the antibody recognizes and binds.

[0038] In various embodiments, the anti-CLDN6 antibody or variant thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0039] In various aspects, the antigen binding proteins of the present disclosure are linked to a therapeutic agent. As described below, the therapeutic agent can be any known in the art, including, but not limited to, chemotherapeutic agents, cytokines and growth factors, cytotoxic drugs, and the like. See "Conjugates" below.

[0040] CLDN6 and epitopes

[0041] The antigen binding proteins of the present disclosure bind to CLDN6. In various embodiments, the CLDN6 is human CLDN6 having the following amino acid sequence: TIFF2025122026000002.tif37156 where X is Ile or Val (sequence number 202).

[0042] In various aspects, human CLDN6 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 178, and 200-202.

[0043] In various aspects, the antigen-binding proteins of the present disclosure bind to an epitope within the amino acid sequence of CLDN6. In various aspects, CLDN6 is human CLDN6, and the antigen-binding proteins of the present disclosure bind to an epitope within the amino acid sequence of human CLDN6, for example, SEQ ID NOs: 1, 178, and 200-202. "Epitope" refers to a region of CLDN6 or a region within CLDN6 to which an antigen-binding protein binds. In some embodiments, the epitope is a linear epitope. "Linear epitope" refers to a region of CLDN6 or a region within CLDN6 to which an antigen-binding protein binds, which region is composed 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. Thus, 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 conformational epitope or a structural epitope. "Conformational epitope" or "structural epitope" refers to an epitope composed of amino acids that are located adjacent to each other only when CLDN6 is in its properly folded state. Unlike linear epitopes, the amino acids of a conformational epitope or a structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN6. A conformational epitope or a structural epitope is not made up of consecutive amino acids in the amino acid sequence of the antigen (CLDN6).

[0044] In various embodiments, the epitope is located within the extracellular domain (ECD) of CLDN6, e.g., 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 of WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO:2). In various embodiments, the epitope to which the antigen binding protein binds is within SEQ ID NO:2. In various embodiments, the antigen binding protein of the present disclosure binds to the N-terminal portion of SEQ ID NO:2, e.g., TAHAIIRDFYNPL (SEQ ID NO:3). In various embodiments, the antigen binding protein of the present disclosure binds to the C-terminal portion of SEQ ID NO:2, e.g., LVAEAQKREL (SEQ ID NO:4). In various cases, the antigen binding protein of the present disclosure binds to EL2 but does not bind to extracellular loop 1 (EL1) of CLDN6. In various aspects, the epitope(s) to which an antigen binding protein of the present disclosure binds is different from the epitope to which an anti-CLDN6 antibody comprising a light chain variable region comprising the sequence of SEQ ID NO: 185 and a heavy chain variable region comprising the sequence of SEQ ID NO: 186 is bound. In various aspects, the epitope(s) to which an antigen binding protein of the present disclosure binds is different from the epitope to which an anti-CLDN6 antibody comprising a light chain variable region comprising the sequence of SEQ ID NO: 181 and a heavy chain variable region comprising the sequence of SEQ ID NO: 182 is bound.

[0045] In various aspects, the antigen binding protein binds to human CLDN6 and non-human CLDN6. In various cases, the non-human CLDN6 is CLDN6 from a chimpanzee, a rhesus monkey, a dog, a cow, a mouse, a rat, a zebrafish, or a frog. In various cases, the antigen binding protein binds to human CLDN6 and mouse CLDN6.

[0046] Affinity and avidity

[0047] The antigen binding proteins provided herein bind to CLDN6 in a non-covalent and reversible manner. In various embodiments, the binding strength of an antigen binding protein to CLDN6 can be expressed 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 high affinity for CLDN6 and therefore bind to a larger amount of CLDN6 in a shorter period of time than antigen binding proteins with lower affinity. In various aspects, the antigen binding protein has an equilibrium association constant K A is 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 appreciated by those skilled in the art, K A can be affected by factors such as pH, temperature and buffer composition.

[0048] In various embodiments, the binding strength of an antigen binding protein to CLDN6 can be expressed as its sensitivity. D is the k between the antigen-binding protein and CLDN6 off / k on The equilibrium dissociation constant is the ratio of K D and K. A is inversely correlated. D The K value is related to the concentration of the antigen-binding protein (the amount of antigen-binding protein required for a particular experiment), so D The lower the value (lower concentration), the higher the affinity of the antigen binding protein. In various embodiments, the binding strength of an antigen binding protein to CLDN6 is expressed as K DIn various aspects, the K of the antigen binding proteins provided herein can be expressed as D is about 10 -1 , about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 In various embodiments, the K of the antigen binding proteins provided herein is D is micromolar, nanomolar, picomolar, or femtomolar. In various embodiments, the K of the antigen binding proteins provided herein D is about 10 -4 ~10 -6 or 10 -7 ~10 -9 or 10 -10 ~10 -12 or 10 -13 ~10 -15 In various aspects, the K of the antigen binding proteins provided herein is within the range D is approximately 1.0 x 10 -12 M ~ approx. 1.0×10 -8 In various embodiments, the K of the antigen binding protein is within the range of D is approximately 1.0 x 10 -11 M ~ approx. 1.0×10 -9 It is within the range of M.

[0049] In various embodiments, the affinity of antigen-binding proteins is measured or ranked using flow cytometry- or fluorescence-activated cell sorting (FACS)-based assays. Flow cytometry-based binding assays are known in the art. See, e.g., 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 the competitive assays described in Trikha et al., Int J Cancer 110:326-335 (2004) and Tam et al., Circulation 98(11):1085-1091 (1998), as well as those described below. See below under the heading "Competition Assays." In Trikh et al., cells expressing the antigen were used in a radioassay. 125 The binding of 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 fluorophore-conjugated CLDN6 antibodies are incubated with CLDN6-expressing cells and the fluorescence emitted (a direct measure of antibody-antigen binding) is determined. A curve is generated plotting each dose or concentration. The maximum value is the lowest concentration at which the fluorescence plateaus or reaches a maximum, i.e., 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 in Example 5 herein.

[0050] In various embodiments, the IC determined in a competitive binding inhibition assay 50 The value is the K DIn 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. Prior to performing the FACS-based assay, in some embodiments, the cells endogenously expressing CLDN6 are predetermined as being CLDN6 low-expressing cells or CLDN6 high-expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are cells from a cell line, e.g., an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, an upper gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various aspects, the cells that endogenously express 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 liver cells, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various aspects, the antigen binding protein inhibits the binding interaction of human CLDN6 expressed by the cells with a reference antibody, where the reference antibody is known to bind to CLDN6 but is not an antigen binding protein of the present disclosure. In various cases, the antigen binding protein of the present 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 in an in vitro competitive binding assay. In various aspects, the antigen binding proteins of the present disclosure inhibit the binding interaction between human CLDN6 and a reference antibody, and the inhibition is 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM when inhibiting the binding interaction between human CLDN6 and the reference antibody. 50In various embodiments, the antigen binding protein has an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than 100 nm. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than 10 nM. 50 In various cases, the antigen binding proteins of the disclosure compete for binding to CLDN6 against a reference antibody known to bind to CLDN6 (wherein the reference antibody is different from any of the antigen binding proteins of the disclosure). See under "Competition Assays" for details.

[0051] Avidity provides a measure of the overall strength of the antibody-antigen complex. It depends on three main parameters: the affinity of the antigen-binding protein for the epitope, the valency of both the antigen-binding protein and CLDN6, and the structural arrangement of the interacting moieties. The greater the valency (number of antigen-binding sites) of the antigen-binding protein, the greater the amount of antigen (CLDN6) it can bind. In various embodiments, the antigen-binding protein has a stronger avidity for CLDN6. In various embodiments, the antigen-binding protein is multivalent. In various embodiments, the antigen-binding protein is bivalent. In various cases, the antigen-binding protein is monovalent.

[0052] Cross-reactivity

[0053] In various embodiments, the antigen binding proteins of the present disclosure bind to CLDN6 and do not bind to any other members of the CLDN family, e.g., do not cross-react with any other members of the CLDN family. In various cases, the antigen binding proteins of the present disclosure are specific for CLDN6. In various embodiments, the antigen binding proteins of the present disclosure have selectivity for CLDN6 that is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of the antigen binding protein for CLDN3, CLDN4, CLDN9, or a combination thereof. In various embodiments, the antigen binding proteins of the present disclosure have selectivity for CLDN6 that is at least 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold greater than the selectivity of the antigen binding protein for each of CLDN3, CLDN4, and CLDN9. Selectivity is measured by the K D Based on well, K D can be determined by techniques known in the art, for example, surface plasmon resonance, FACS-based affinity assays.

[0054] In various aspects, the antigen binding proteins of the present disclosure bind to CLDN6 and do not bind to any of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9). In various aspects, the antigen binding proteins do not bind to any of CLDN3, CLDN4, and CLDN9, and have an IC of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) in a FACS-based assay using OVCA429 cells that endogenously express CLDN6. 50In various aspects, the antigen binding protein does not bind to any of CLDN3, CLDN4, and CLDN9, and achieves 50% of binding saturation in OVCA429 cells that endogenously express CLDN6 at a concentration of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various aspects, the antigen binding protein exhibits at least 5-fold greater selectivity for CLDN6 than for CLDN3, CLDN4, and CLDN9, and achieves 50% of binding saturation in OVCA429 cells that endogenously express CLDN6 at a concentration of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM). In various aspects, the antigen binding protein exhibits an IC50 of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) against artificial and endogenous models of CLDN6, and exhibits a ratio of more than about 5-fold that distinguishes the IC50 of CLDN6 from CLDN3, CLDN4, and / or CLDN9. In various cases, the antigen binding protein exhibits an IC50 of less than about 1200 nM (e.g., less than about 1000 nM, less than about 750 nM, less than about 500 nM, less than about 250 nM) against CLDN6, and exhibits an IC50 that is at least 5-fold greater than the IC50 against any one of CLDN3, CLDN4, and CLDN9.

[0055] In various embodiments, the antigen binding proteins of the present disclosure bind to CLDN6 and cross-react with (e.g., bind to) at least one other member of the CLDN family. In various aspects, the antigen binding proteins of the present disclosure bind to CLDN6 and one or more of CLDN3, CLDN4, and CLDN9. In various aspects, the antigen binding proteins of the present disclosure bind to CLDN6 and CLDN4 or CLDN9, but not to CLDN3. In various cases, the antigen binding proteins of the present disclosure bind to CLDN6 and CLDN4, but not to CLDN3 or CLDN9. In various cases, the antigen binding proteins of the present disclosure bind to CLDN6 and CLDN9, but not to either CLDN3 or CLDN4.

[0056] Competition assay

[0057] 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 an antigen binding protein of the present disclosure. In various cases, the antigen binding protein of the present 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 in an in vitro competitive binding assay. In various embodiments, the reference antibody binds to an epitope within the amino acid sequence of the extracellular domain of human CLDN6, optionally within EL2 or EL1. In various aspects, the reference antibody comprises 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 aspects, the reference antibody comprises a light chain variable sequence of SEQ ID NO: 181 and a heavy chain variable sequence of SEQ ID NO: 182. In various aspects, the antigen binding protein of the present disclosure inhibits the binding interaction between human CLDN6 and the reference antibody, and the inhibition is measured by an IC 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM when inhibiting the binding interaction between human CLDN6 and the reference antibody. 50 In various embodiments, the antigen binding protein has an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than 100 nm. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than 10 nM. 50 Shows.

[0058] In various cases, the antigen binding protein of the present disclosure competes with a reference antibody for binding to human CLDN6, thereby reducing the amount of human CLDN6 bound to the reference antibody as determined in an in vitro competitive binding assay. In various embodiments, the in vitro competitive binding assay is a FACS-based assay in which the fluorescence of a fluorophore-conjugated secondary antibody that binds to the Fc of the reference antibody is measured in the absence or presence of a specific amount of an antigen binding protein of the present disclosure. Such FACS-based assays are described herein in the Examples. In various embodiments, the FACS-based assay is performed using the reference antibody, the 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. Prior to performing a FACS-based assay, in some embodiments, cells that endogenously express CLDN6 are predetermined as being CLDN6 low-expressing cells or CLDN6 high-expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are cells from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, an upper gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various embodiments, the cells that endogenously express 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 liver cells, JHOS-4 ovarian cells, H1435 lung cells, and NUGC3 upper gastrointestinal cells. In various cases, the antigen binding proteins of the present disclosure bind with high affinity to CLDN6 endogenously expressed by one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. In various embodiments, the antigen binding proteins exhibit an IC200 activity greater than or equal to 1000 fold as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50In various embodiments, the antigen binding protein exhibits an IC50 of less than about 3000 nM as determined in a FACS-based competitive binding inhibition assay using one or more of ARK2 cells, OVCA429 cells, LS513 cells, or MCF7 cells. 50 In various embodiments, the antigen binding protein exhibits an IC20 of less than about 2500 nM, less than about 2000 nM, less than about 1750 nM, less than about 1500 nM, less than about 1250 nM, less than about 1000 nM, less than about 750 nM, or less than about 500 nM. ... 50 is less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM.

[0059] Other binding assays, such as competitive binding assays or competition assays, that test the ability of an antibody to compete with a second antibody for binding to an antigen or its epitope 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 No. 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), which can be used to determine the binding constant of an antibody and a second antibody, and then the two binding constants can be compared.

[0060] Antibody production methods and related methods

[0061] 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 producing antibodies are described, for example, in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5 th Ed., Garland Publishing, New York, NY (2001)). Various methods for preparing the CLDN6 monoclonal antibodies of the present disclosure are provided in the Examples herein.

[0062] Depending on the host species, various adjuvants can be used to increase the immunological response leading to the production of large amounts of antibodies 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.

[0063] Other antibody production methods are summarized in Table 1. [Table 1]

[0064] Regardless of how the antibody is produced, methods for testing the antibody for its ability to bind to an epitope of CLDN6 are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266, as well as the section above relating to competitive assays).

[0065] Arrays / Structures

[0066] As used herein, a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence is defined as follows: (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence set forth in Table A, 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, and 131, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity with a HC set forth in Table A; (c) an 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 about 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) a CDR3 amino acid sequence, 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 about 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) the amino acid sequence of 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 about 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 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 about or at least 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; Antigen binding proteins are provided that comprise the amino acid sequence of a 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, or a variant thereof, which differs by only one or two amino acids or has about 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 a combination of any two or more of (g)(a)-(f). [Table 2]

[0067] In various aspects, the antigen binding protein comprises an LC CDR1 amino acid sequence, an LC CDR2 amino acid sequence, and an LC CDR3 amino acid sequence set forth in Table A, and at least one or two of the HC CDR amino acid sequences set forth in Table A. In various aspects, the antigen binding protein comprises an HC CDR1 amino acid sequence, an HC CDR2 amino acid sequence, and an HC CDR3 amino acid sequence set forth in Table A, and at least one or two of the LC CDR amino acid sequences set forth in Table A.

[0068] In various embodiments, the antigen binding protein comprises at least three, four, or five of the amino acid sequences specified by SEQ ID NOs in a row of Table A. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A and at least one or two of the HC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A and at least one or two of the LC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A. In various embodiments, the antigen binding protein comprises all six of the CDR amino acid sequences specified by SEQ ID NOs in a row of Table A. In various embodiments, the antigen binding protein comprises 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.

[0069] In various instances, the amino acid sequences of Table A are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). In various instances, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various instances, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various instances, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various cases, there are about 14 to about 16 amino acids between the HC CDR1 and HC CDR2 sequences, and about 30 to about 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0070] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence set forth 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 which has about 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 set forth 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 which has about 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 3]

[0071] In various embodiments, the antigen binding proteins are selected from the group consisting of: (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; and (k) SEQ ID NO: 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, (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.

[0072] In various embodiments, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 179 and 180. In various embodiments, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 181 and 182. In various embodiments, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 183 and 184. In various embodiments, the antigen binding protein does not comprise the pair of amino acid sequences of SEQ ID NOs: 185 and 186.

[0073] In various embodiments, the antigen binding protein comprises an amino acid sequence similar to the amino acid sequences set forth above, but the antigen binding protein still substantially retains its biological function, e.g., its ability to bind to human CLDN6, reduce tumor growth, and treat cancer.

[0074] In various embodiments, the antigen binding protein comprises an amino acid sequence that differs by only one, two, three, four, five, six, or more amino acids compared to the aforementioned amino acid sequence(s). In various embodiments, the antigen binding protein comprises a variant of a reference sequence, which variant sequence differs by only one or two amino acids compared to the reference sequence. In various embodiments, the antigen binding protein comprises one or more amino acid substitutions that occur outside the CDRs, for example, one or more amino acid substitutions that occur within the framework region(s) of the heavy or light chain. In various embodiments, the antigen binding protein comprises 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 comprises an amino acid sequence with only one, two, three, four, five, six, or more conservative amino acid substitutions compared to the aforementioned amino acid sequence(s). As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, including exchanges within one of the following five groups: I. Small non-polar or slightly polar aliphatic residues: Ala, Ser, Thr, Pro, Gly; II. Negatively charged polar residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III. Positively charged polar residues: His, Arg, Lys; Ornithine (Orn) IV. Large aliphatic non-polar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine

[0075] In various aspects, conservative amino acid substitutions are exchanges within one of the following groups of amino acids: TIFF2025122026000006.tif81156

[0076] In various embodiments, the antigen binding protein comprises an amino acid sequence having about 30% or more, about 50% or more, or about 70% or more sequence identity to the aforementioned amino acid sequences. In various embodiments, the antigen binding protein comprises 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 sequences. In various embodiments, the antigen binding protein comprises 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 entire length of the aforementioned amino acid sequences. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity along the entire length of the aforementioned amino acid sequences.

[0077] In various embodiments, the antigen binding proteins comprise variants of the referenced sequences, which variants have about or at least 70% sequence identity to said sequences. In various embodiments, the antigen binding proteins comprise variants of the referenced sequences, which variants have about or at least 80% sequence identity to said sequences. In various embodiments, the antigen binding proteins comprise variants of the referenced sequences, which variants have about or at least 90% sequence identity to said sequences. In various embodiments, the antigen binding proteins comprise variants of the referenced sequences, which variants have about or at least 95% sequence identity to said sequences.

[0078] In various embodiments, the antigen binding protein comprises one, two, three, four, or five of the SEQ ID NOs in a row of Table A, and at least one variant sequence having about 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: 8-133. In various embodiments, the antigen binding proteins are 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: 92-93, 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, wherein the antigen-binding protein further comprises at least one variant sequence having about 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 of the set. For example, in various embodiments, the antigen binding protein comprises four of the sequences set forth in SEQ ID NOs: 74-79, i.e., SEQ ID NOs: 74-77, wherein the antigen binding protein comprises two mutant sequences, one mutant sequence having about 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 SEQ ID NO: 78, and another mutant sequence having about 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 SEQ ID NO: 79.

[0079] In various embodiments, the antigen binding protein comprises a pair of variant sequences having about 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 instances, the antigen binding protein comprises (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 NO: 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. In various embodiments, the antigen binding protein comprises a pair of sequences, one of which is a sequence of Table B, and the other sequence is a variant sequence that has about 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 selected from the group consisting of (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) a sequence selected from 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, and the other sequence is a mutant sequence having about 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 a sequence of (a) through (u). For example, in various embodiments, the antigen binding protein comprises the sequence of SEQ ID NO: 134, and such antigen binding protein further comprises a mutant sequence having about 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 SEQ ID NO: 135.

[0080] In various instances, the antigen binding protein comprises the amino acid sequence of the foregoing amino acid sequences with one or more amino acid substitutions to reduce or eliminate reactive amino acids to reduce or prevent undesired side chain reactions. For example, the antigen binding protein comprises the amino acid sequence of the foregoing amino acid sequences with one or more of: (i) a Trp residue substituted with His, Tyr, or Phe; (ii) an Asn residue substituted with Gln, Ser, Ala, or Asp; (iii) an Asp residue immediately preceding a Pro residue substituted with Ala, Ser, or Glu; (iv) an Asn residue substituted with Gln, Ser, or Ala; and / or (v) a Cys residue substituted with Tyr, Ser, or Ala. In various embodiments, the antigen binding protein comprises the amino acid sequence of the foregoing amino acid sequences with 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 large numbers of similar antibody sequences. In some embodiments, the antigen binding protein comprises: (a) a HC CDR1 amino acid sequence set forth 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 which has about 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 HC CDR2 amino acid sequence set forth 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 which has about 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) a HC CDR2 amino acid sequence set forth 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 which has about 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) a LC CDR1 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 449, 476, 458, 464, 68, and 470, or a variant thereof, which differs by only one or two amino acids or which has about 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) a LC CDR2 amino acid sequence listed in Table A1, or a sequence selected from the group consisting of SEQ ID NOs: 449, 476, 458, 464, 68, and 470, or a variant thereof, which differs by only one or two amino acids or which has about 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) a LC CDR3 amino acid sequence set forth 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 which has about 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) through (f). [Table 4]

[0081] In some embodiments, HC CDR1 comprises a Gly immediately N-terminus of SEQ ID NO:452, and optionally in some embodiments, HC CDR1 comprises an MX immediately C-terminus of SEQ ID NO:452, where X is H, N, or S. In various embodiments, HC CDR3 comprises an Ala immediately N-terminus of SEQ ID NO:453. In various embodiments, LC CDR1 further comprises a TAS immediately N-terminus of SEQ ID NO:449, and optionally comprises an XH immediately 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.

[0082] In various embodiments, HC CDR1 comprises a Gly immediately N-terminus of SEQ ID NO:455, and optionally, in various embodiments, HC CDR1 comprises an MX immediately C-terminus of SEQ ID NO:455, where X is N, S, or H. In some embodiments, HC CDR2 comprises a Gln immediately N-terminus of SEQ ID NO:456, and optionally, an H immediately C-terminus of SEQ ID NO:456. In various embodiments, LC CDR1 comprises a RIS immediately N-terminus of SEQ ID NO:476, and optionally, an LA immediately C-terminus of SEQ ID NO:476. In various embodiments, LC CDR2 comprises an XLVE immediately C-terminus of SEQ ID NO:477, where X is I or S.

[0083] In various embodiments, HC CDR1 comprises an MH immediately C-terminus of SEQ ID NO:461. In various embodiments, HC CDR2 comprises a Tyr immediately N-terminus of SEQ ID NO:462 and, optionally, a TH immediately C-terminus of SEQ ID NO:462. In exemplary embodiments, HC CDR3 does not include the first two amino acids of SEQ ID NO:463. In various embodiments, LC CDR1 comprises an RSS immediately N-terminus of SEQ ID NO:458 and, optionally, an LN immediately C-terminus of SEQ ID NO:458. In various embodiments, LC CDR2 comprises an XRFS immediately C-terminus of SEQ ID NO:459, where X is Q, S, A, or D.

[0084] In various embodiments, HC CDR1 comprises an MH immediately C-terminus of SEQ ID NO: 465. In various embodiments, HC CDR2 comprises a YI immediately N-terminus of SEQ ID NO: 466 and optionally an Xaa immediately C-terminus of SEQ ID NO: 466, where Xaa is N, S, Q, or A. In various embodiments, LC CDR1 comprises an LAS immediately N-terminus of SEQ ID NO: 464 and optionally an LA immediately C-terminus of SEQ ID NO: 464. In various embodiments, LC CDR2 comprises a SLAD immediately C-terminus of SEQ ID NO: 57.

[0085] In various embodiments, HC CDR1 comprises MH immediately C-terminal to SEQ ID NO: 71. In various embodiments, HC CDR2 comprises Tyr immediately N-terminal to SEQ ID NO: 468, and optionally IY immediately C-terminal to SEQ ID NO: 468. In various embodiments, LC CDR1 comprises RAS immediately N-terminal to SEQ ID NO: 68, and optionally SYIH immediately C-terminal to SEQ ID NO: 68. In various embodiments, LC CDR2 comprises XLES immediately C-terminal to SEQ ID NO: 69, where X is N, Q, S, A, or D.

[0086] In various embodiments, the LC CDR1 comprises a KSS immediately N-terminal to SEQ ID NO:470, and optionally a YLA immediately C-terminal to SEQ ID NO:470. In various embodiments, the LC CDR2 comprises a TRES immediately C-terminal to SEQ ID NO:471. In various embodiments, the HC CDR1 comprises a MN immediately C-terminal to SEQ ID NO:472. In various embodiments, the HC CDR2 comprises a Xaa immediately N-terminal to SEQ ID NO:473, where Xaa is N, Q, S, or A, and optionally a Thr immediately C-terminal to SEQ ID NO:473.

[0087] In various aspects, the antigen binding protein comprises an LC CDR1 amino acid sequence, an LC CDR2 amino acid sequence, and an LC CDR3 amino acid sequence set forth in Table A1, and at least one or two of the HC CDR amino acid sequences set forth in Table A1. In various aspects, the antigen binding protein comprises an HC CDR1 amino acid sequence, an HC CDR2 amino acid sequence, and an HC CDR3 amino acid sequence set forth in Table A1, and at least one or two of the LC CDR amino acid sequences set forth in Table A1.

[0088] In various embodiments, the antigen binding protein comprises at least three, four, or five of the amino acid sequences specified by SEQ ID NOs in a row of Table A1. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A1 and at least one or two of the HC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A1. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A1 and at least one or two of the LC CDR amino acid sequences specified by SEQ ID NOs in a row of Table A1. In various embodiments, the antigen binding protein comprises all six of the CDR amino acid sequences specified by SEQ ID NOs in a row of Table A1. In various embodiments, the antigen binding protein comprises 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, 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.

[0089] In various instances, the amino acid sequences of Table A1 are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). In various instances, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences, and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various instances, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences, and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various instances, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences, and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various cases, there are about 14 to about 16 amino acids between the HC CDR1 and HC CDR2 sequences, and about 30 to about 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0090] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence set forth 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 which has about 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) a light chain variable region amino acid sequence set forth 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 which has about 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]

[0091] 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 aspects, the antigen binding protein comprises variants of the sequences having SEQ ID NOs set forth in Table B1 that differ by only one or two amino acids or that share about 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 at the positions described below under "Humanized Antibodies."

[0092] humanized antibodies

[0093] In various embodiments, the antigen binding protein is a humanized version of an antigen binding protein set forth in Table A, Table A1, Table B, or Table B1.

[0094] Humanized AB1

[0095] In various aspects, the antigen binding protein is a humanized version of AB1 as set forth in Table B or B1, with 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) at one or more of the following positions in the heavy chain variable region: 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. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 428. In various embodiments, the antigen binding protein is a humanized version of AB1 as set forth in Table B or B1, with one or more amino acid substitutions at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) within the heavy chain variable region: 20, 31, 35, 48, 50, 59, 67, 70, 74, 79, 98, 101. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 429. In various embodiments, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 6]

[0096] In various embodiments, the antigen binding protein is a humanized version of AB1 as set forth in Table B or B1 and has 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, In various instances, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:430. In various aspects, the antigen binding protein is a humanized version of AB1 as set forth in Table B or B1, with 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 cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 431. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 7]

[0097] Humanized AB3

[0098] In various embodiments, the antigen binding protein is a humanized version of AB3 as set forth in Table B or B1, with one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 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 instances, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 432. In various embodiments, the antigen binding protein is a humanized version of AB3 as set forth in Table B or B1, with one or more amino acid substitutions at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions within the heavy chain variable region: 31, 35, 50, 55, 79, 99, 106. In various instances, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 433. In various embodiments, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 8]

[0099] In various aspects, the antigen binding protein is a humanized version of AB3 as set forth in Table B or B1, with one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 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 comprises the amino acid sequence of SEQ ID NO:434. In various aspects, the antigen binding protein is a humanized version of AB3 as set forth in Table B or B1, with one or more amino acid substitutions in the light chain variable region at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9): 25, 34, 48, 53, 55, 84, 85, 90, and 93. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 435. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 9]

[0100] Humanized AB4

[0101] In various embodiments, the antigen binding protein is a humanized version of AB4 as set forth in Table B or B1 and has 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, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 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, 80, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 2, 84, 87, 91, 97, 101, 117 (e.g., 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 aspects, the antigen binding protein is a humanized version of AB4 as set forth in Table B or B1, with one or more amino acid substitutions at one or more of the following positions within 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 cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 437. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 10]

[0102] In various embodiments, the antigen binding protein is a humanized version of AB4 as set forth in Table B or B1, with one or more amino acid substitutions at one or more of the following positions within the light chain variable region: 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 comprises the amino acid sequence of SEQ ID NO:438. In various aspects, the antigen binding protein is a humanized version of AB4 as set forth in Table B or B1, with one or more amino acid substitutions in the light chain variable region at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions: 33, 39, 55, 57, 81, 95, and 96. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 439. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 11]

[0103] Humanized AB18

[0104] In various aspects, the antigen binding protein is a humanized version of AB18 as described in Table B or B1, with one or more amino acid substitutions at one or more of the following positions in the heavy chain variable region: 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), optionally at one or more of the following positions (e.g., 1, 2, 3, 4, or 5): 20, 48, 68, 70, 79. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 440 or 441. In various embodiments, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 12]

[0105] In various aspects, the antigen binding protein is a humanized version of AB18 as set forth in Table B or B1, with 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), optionally at one or more of the following positions: 19, 21, or 84 (e.g., 1, 2, or 3). In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 442 or 443. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 13]

[0106] Humanized AB9

[0107] In various aspects, the antigen binding protein is a humanized version of AB9 as described in Table B or B1, with one or more amino acid substitutions at one or more of the following positions in 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 comprises the amino acid sequence of SEQ ID NO:444. In various aspects, the amino acids at the positions listed above are selected from the amino acids according to the table below. [Table 14]

[0108] In various aspects, the antigen binding protein is a humanized version of AB9 as set forth in Table B or B1, with one or more amino acid substitutions at one or more of the following positions within 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 cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 445. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 15]

[0109] Humanized AB11

[0110] In various aspects, the antigen binding protein is a humanized version of AB11 as set forth in Table B or B1, with one or more amino acid substitutions in the heavy chain variable region at one or more of the following positions: 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 cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 446. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 16]

[0111] In various aspects, the antigen binding protein is a humanized version of AB11, as set forth in Table B or B1, with 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), optionally at one or more of the following positions: 4, 82, 110. In various cases, the antigen binding protein comprises the amino acid sequence of SEQ ID NO: 447 or 448. In various aspects, the amino acids at the above-listed positions are selected from the amino acids according to the table below. [Table 17]

[0112] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence set forth 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 about or at least 70%, or about 80%, or about 85%, or about 90%, or about 95% sequence identity. or (b) a light chain variable region amino acid sequence set forth in Table C, or a sequence selected from the group consisting of 380-383, 388-390, 397-402, 409-411, 414, 415, 420, and 421, or a variant thereof, which differs by only one or two amino acids or has about or at least 70%, or about 80%, or about 85%, or about 90%, or about 95% sequence identity; or (c) both (a) and (b). [Table 18]

[0113] In various embodiments, the humanized antigen binding protein comprises a pair of amino acid sequences shown in Table D. [Table 19] TIFF2025122026000023.tif100170

[0114] In various embodiments, the antigen binding protein comprises a pair of variant sequences, each having about 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 a SEQ ID NO: set forth in Table C. In various embodiments, the antigen binding protein comprises a pair of sequences, one sequence selected from the SEQ ID NOs set forth in Table C, and the other sequence having a SEQ ID NO: set forth in Table D, a variant sequence having about 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 a SEQ ID NO: set forth in Table C.

[0115] In various embodiments, the antigen binding protein comprises a pair of sequences, one sequence selected from the SEQ ID NOs set forth in Table D, and the other sequence is a variant sequence having about 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 a SEQ ID NO set forth in Table D. For example, in various aspects, the antigen binding protein comprises the sequence of SEQ ID NO: 419, and such antigen binding protein further comprises a variant sequence having about 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 SEQ ID NO: 421.

[0116] In various instances, the antigen binding protein is a humanized antigen binding protein with one or more (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) amino acid substitutions in the heavy chain (HC) variable region or the light chain (LC) variable region, or both, as set forth in Table D. In an exemplary embodiment, the antigen binding protein is a humanized antigen binding protein designated AB1-11 with 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 comprises a HC of SEQ ID NO: 379 with one, two, three, four, or five amino acid substitutions. In exemplary embodiments, the antigen binding protein comprises an HC CDR1 of SEQ ID NO: 504, an HC CDR2 of SEQ ID NO: 505, an HC CDR3 of SEQ ID NO: 506, or a combination thereof. In exemplary cases, the antigen binding protein comprises an HC of SEQ ID NO: 503. In some embodiments, the antigen binding protein comprises an HC of any one of SEQ ID NOs: 496-501. In some embodiments, the antigen binding protein comprises an HC sequence depicted as S7-S12 in Figure 22. In various cases, the light chain variable region comprises an LC CDR1 of SEQ ID NO: 449, an LC CDR2 of SEQ ID NO: 450, an LC CDR3 of SEQ ID NO: 451, or a combination thereof. In some embodiments, the antigen binding protein comprises an LC of any one of SEQ ID NOs: 380-383, and 479. In exemplary cases, the antigen binding protein comprises an LC of SEQ ID NO: 383. In some embodiments, the antigen binding protein comprises an LC sequence depicted as S7-S12 in Figure 22. In an exemplary embodiment, the antigen binding protein is a humanized antigen binding protein designated AB3-7 with 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 comprises an HC of SEQ ID NO: 387 with one, two, three, four, five, or six amino acid substitutions. In an exemplary embodiment, the antigen binding protein comprises an HC CDR1 of SEQ ID NO: 507, an HC CDR2 of SEQ ID NO: 508, an HC CDR3 of SEQ ID NO: 509, or a combination thereof.In an exemplary case, the antigen binding protein comprises an HC of SEQ ID NO: 502. In some embodiments, the antigen binding protein comprises an HC of any one of SEQ ID NOs: 490-495. In some embodiments, the antigen binding protein comprises an HC sequence designated S1-S6 in Figure 22. In various cases, the light chain variable region comprises an LC CDR1 of SEQ ID NO: 476, an LC CDR2 of SEQ ID NO: 477, an LC CDR3 of SEQ ID NO: 454, or a combination thereof. In some embodiments, the antigen binding protein comprises an LC of any one of SEQ ID NOs: 388-390, and 481. In an exemplary case, the antigen binding protein comprises an LC of SEQ ID NO: 389. In some embodiments, the antigen binding protein comprises an LC sequence designated S1-S6 in Figure 22. In exemplary embodiments, the antigen binding protein is a humanized antigen binding protein of AB3, with one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In exemplary embodiments, the antigen binding protein comprises 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 comprises the HC of any one of SEQ ID NO: 510. In some embodiments, the antigen binding protein comprises 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 comprises 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 comprises the HC of any one of SEQ ID NO: 511. In some embodiments, the antigen binding protein comprises 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 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 comprises 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 comprises the HC of any one of SEQ ID NO: 513.In some embodiments, the antigen binding protein comprises 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 comprises the HC 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 comprises the HC of any one of SEQ ID NO: 512. In some embodiments, the antigen binding protein comprises 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.

[0117] Defucosylated antibodies

[0118] Many secreted proteins undergo post-translational glycosylation, a process in which sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of a protein. In eukaryotic cells, two types of glycosylation occur: (1) N-linked glycosylation, in which the glycan is attached to an asparagine in the recognition sequence Asn-X-Thr / Ser, where "X" can be any amino acid except proline, and (2) O-linked glycosylation, in which the glycan is attached to a serine or threonine. Regardless of the type of glycosylation (N-linked [glycosylation] or O-linked [glycosylation]), microheterogeneity exists in protein glycoforms due to the large range of glycan structures associated with each site (O or N).

[0119] All N-glycans share a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr (Man3GlcNAc2Asn) and are classified into one of three types: (A) high-mannose (HM) or oligomannose (OM) types, consisting of two N-acetylglucosamine (GalNAc) moieties and multiple (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 sugar types; or (C) hybrid types, containing a Man residue on one branch and a GlcNAc at the base of a complex branch. Figure 1A (Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2014). nd ed., Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans.

[0120] N-linked glycans typically contain one or more monosaccharides: 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). Commonly used symbols for such sugars are shown in Figure 29A.

[0121] N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex series of reactions results in the attachment of a core glycan structure, essentially composed of two GlcNAc and three Man residues. The glycan complexes formed in the ER are then modified by enzymes in the Golgi apparatus. If the sugars are relatively inaccessible to the enzymes, they typically remain in their original HM form. If the enzymes are able to access the sugars, many of the Man residues are cleaved, and the sugars are further modified to produce complex N-glycan structures. For example, mannosidase-1, located in the cis-Golgi, can cleave or hydrolyze HM glycans, and fucosyltransferase FUT-8, located in the medial Golgi, fucosylates the glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0122] Thus, the sugar composition and structural configuration of the glycan structure will vary depending on, among other factors, the glycosylation machinery in the ER and the Golgi apparatus, the accessibility of the glycan structure to the enzymes of the machinery, the order in which each enzyme acts, and the stage at which the protein is released from the glycosylation machinery.

[0123] In exemplary embodiments of the present disclosure, the antigen binding protein comprises an Fc polypeptide. As used herein, the term "Fc polypeptide" includes native and mutant forms of polypeptides 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 lacks fucose or is defucosylated. In exemplary embodiments, the antigen binding protein comprises a defucosylated glycan. As used herein, the term "defucosylated glycan" or "afucoglycan" or "defucosylated glycoform" or "Afuc" refers to a glycoform that lacks core fucose, e.g., α1,6-linked fucose on the GlcNAc residue involved in the amide bond with Asn at the N-glycosylation site. 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, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015).

[0124] The present disclosure also provides compositions, e.g., pharmaceutical compositions, comprising antigen-binding proteins comprising Fc polypeptides comprising defucosylated glycans. In exemplary embodiments, about or at least 25% of the antigen-binding proteins present in the composition are antigen-binding proteins comprising Fc polypeptides comprising 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 genetically modified to alter the activity of enzymes in the de novo or salvage pathways. These two fucose metabolic pathways are shown in Figure 29B. In exemplary embodiments, the cells are genetically engineered to alter the activity of any one or more of fucosyltransferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In exemplary embodiments, the cells are genetically engineered to knock out the gene encoding FX. See, for example, International Patent Publication No. WO2017 / 079165A1; Kanda et al., J Biotechnol 130, 2007, 300-310; Yamane-Ohunuki et al., Biotechnol Bioeng 87, 2004, 614-622; Malphettes et al., Biotechnol Bioeng 106, 2010, 774-783.

[0125] nucleic acid

[0126] The present disclosure further provides nucleic acids comprising nucleotide sequences encoding the antigen-binding proteins of the present disclosure. As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to polymers of DNA or RNA, or modified forms thereof, which may be single-stranded or double-stranded, synthetic, or derived from naturally occurring sources (e.g., isolated and / or purified), and may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides. A nucleic acid may include any nucleotide sequence that encodes any of the antigen-binding proteins of the present disclosure. In various embodiments, the nucleic acid comprises (a) an amino acid sequence of a heavy chain (HC) complementarity determining region (CDR) 1 set forth 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 about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity to a CDR 1 heavy chain (HC) set forth in Table A or A1; (c) a CDR2 amino acid sequence, 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, 132, 475, 456, 462, 466, 468, and 473, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity; (d) a HC CDR3 amino acid sequence set forth in Table A or A1, or SEQ ID NOs: 13, 19, 25, 31, 37, 43, 49, 55, 61, 67;(d) a light chain (LC) CDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 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 about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity; (e) a sequence selected from the group consisting of sequences of 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 about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity with a LC sequence listed in Table A or A1; (f) a CDR2 amino acid sequence, 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, 129, 450, 477, 459, and 471, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity; an 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 about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity;or (g) a combination of any two or more of (a) through (f). In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC CDR1 amino acid sequence, an LC CDR2 amino acid sequence, and an LC CDR3 amino acid sequence set forth in Table A or A1, and at least one or two of the HC CDR amino acid sequences set forth in Table A or A1. In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC CDR1 amino acid sequence, an HC CDR2 amino acid sequence, and an HC CDR3 amino acid sequence set forth in Table A or A1, and at least one or two of the LC CDR amino acid sequences set forth in Table A or A1. In various embodiments, the nucleic acid comprises (a) at least three, four, or five of the amino acid sequences set forth by SEQ ID NOs in a row in Table A or A1, (b) each of the LC CDR amino acid sequences set forth by SEQ ID NOs in a row in Table A or A1 and at least one or two of the HC CDR amino acid sequences set forth by SEQ ID NOs in a row in Table A or A1, or (c) each of the HC CDR amino acid sequences set forth by SEQ ID NOs in a row in Table A or A1 and at least one or two of the LC CDR amino acid sequences set forth by SEQ ID NOs in a row in Table A or A1. (d) all six CDR amino acid sequences designated by the SEQ ID NOs in one row of Table A, and / or (e) (a) SEQ ID NOs: 74 to 79, (b) SEQ ID NOs: 50 to 55, (c) SEQ ID NOs: 122 to 127, (d) SEQ ID NOs: 26 to 31, (e) SEQ ID NOs: 128 to 133, (f) SEQ ID NOs: 38 to 43, (g) SEQ ID NOs: 62 to 67, (h) SEQ ID NOs: 80 to 85, (i) SEQ ID NOs: 44 to 49, and (j) SEQ ID NOs: 86 to 91 , (k) SEQ ID NOs: 104 to 109, (l) SEQ ID NOs: 56 to 61, (m) SEQ ID NOs: 32 to 37, (n) SEQ ID NOs: 110 to 115, (o) SEQ ID NOs: 98 to 103, (p) SEQ ID NOs: 92 to 97, (q) SEQ ID NOs: 116 to 121, (r) SEQ ID NOs: 8 to 13, (s) SEQ ID NOs: 68 to 73, (t) SEQ ID NOs: 14 to 19, (u) SEQ ID NOs: 20 to 25, (v) SEQ ID NOs: 449 to 453 and 475, (w) SEQ ID NOs: 476 to 477, 454 to 457, (x) SEQ ID NOs: 458 to 463,The antigen-binding protein comprises a nucleotide sequence encoding six CDR amino acid sequences selected from the group consisting of (y) SEQ ID NOs: 57, 58, and 464 to 467, (z) SEQ ID NOs: 68 to 71 and 468 to 469, and (aa) SEQ ID NOs: 112, and 470 to 474. In various embodiments, the nucleic acid is selected from the group consisting of (a) a heavy chain variable region amino acid sequence set forth in Table B or B1, or 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 about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity; or (b) a light chain variable region amino acid sequence set forth in Table B or B1. (c) a nucleotide sequence encoding an antigen binding protein comprising a variant region 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 about or at least 70% (e.g., about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%) sequence identity; or (c) both (a) and (b). In various embodiments, the nucleic acids are selected from the group consisting of (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 comprising 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 comprising a pair of amino acid sequences selected from the group consisting of the pairs set forth in Table D. In various aspects, the nucleic acid comprises a nucleotide sequence comprising the sequence of any one or more of SEQ ID NOs: 208-375. 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.

[0127] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein that is a humanized antigen binding protein with one or more (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) amino acid substitutions in the heavy chain (HC) variable region or the light chain (LC) variable region, or both, as set forth in Table D. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein that is a humanized antigen binding protein designated AB1-11 with one or more amino acid substitutions in the HC variable region, the LC variable region, or both. In an exemplary embodiment, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising the HC of SEQ ID NO: 379 with one, two, three, four, or five amino acid substitutions. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC CDR1 of SEQ ID NO: 504, an HC CDR2 of SEQ ID NO: 505, an HC CDR3 of SEQ ID NO: 506, or a combination thereof. In exemplary cases, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC of SEQ ID NO: 503. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC of any one of SEQ ID NOs: 496-501. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC sequence designated as S7-S12 in Figure 22. In various cases, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region comprising an LC CDR1 of SEQ ID NO: 449, an LC CDR2 of SEQ ID NO: 450, an LC CDR3 of SEQ ID NO: 451, or a combination thereof. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of any one of SEQ ID NOs: 380-383, and 479. In exemplary cases, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of SEQ ID NO: 383. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising the LC sequence designated as S7-S12 in FIG.In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein that is a humanized antigen binding protein designated AB3-7 with 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 with one, two, three, four, five, or six amino acid substitutions comprising the HC of SEQ ID NO: 387. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC CDR1 of SEQ ID NO: 507, an HC CDR2 of SEQ ID NO: 508, an 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 comprising an HC of SEQ ID NO: 502. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC of any one of SEQ ID NOs: 490-495. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an HC sequence designated S1-S6 in Figure 22. In various cases, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region comprising an LC CDR1 of SEQ ID NO: 476, an LC CDR2 of SEQ ID NO: 477, an LC CDR3 of SEQ ID NO: 454, or a combination thereof. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of any one of SEQ ID NOs: 388-390, and 481. In an exemplary case, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising an LC of SEQ ID NO: 389. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising the LC sequences designated S1-S6 in Figure 22.

[0128] In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein that is a humanized antigen binding protein of AB3 with 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 with one, two, three, four, or five (or more) amino acid substitutions comprising the HC of SEQ ID NO: 139. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with the HC of any one of SEQ ID NO: 510. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with one, two, three, four, or five (or more) amino acid substitutions as depicted in Figure 23, comprising the HC sequence of SEQ ID NO: 510. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with one, two, three, four, or five (or more) amino acid substitutions comprising the HC of SEQ ID NO: 138. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with the HC of any one of SEQ ID NO: 511. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with one, two, three, four, or five (or more) amino acid substitutions as shown in Figure 24, comprising the HC sequence of SEQ ID NO: 511. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein that is a humanized antigen binding protein of AB1, with 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 with one, two, three, four, or five (or more) amino acid substitutions, comprising the HC of SEQ ID NO: 135. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with the HC of any one of SEQ ID NO: 513. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with one, two, three, four, or five (or more) amino acid substitutions as shown in Figure 25, comprising the HC sequence of SEQ ID NO: 513.In exemplary aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with one, two, three, four, or five (or more) amino acid substitutions comprising the HC of SEQ ID NO: 134. In some aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with the HC of any one of SEQ ID NOs: 512. In some aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein with one, two, three, four, or five (or more) amino acid substitutions as depicted in Figure 26, comprising 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.

[0129] In some embodiments, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by splicing natural or synthetic nucleic acid segments into a nucleic acid molecule that can replicate within the living cell, or (ii) a molecule resulting from replication of the molecule described in (i) above. For purposes herein, replication can be either in vitro or in vivo.

[0130] In some embodiments, nucleic acids are constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al. (supra) and Ausubel et al. (supra). 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 increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate 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-galactosylquefon, inosine, N-acetyl-3-methyl-4-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenines, 7-methylguanine, 5-methylammomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6Examples of nucleic acids include, but are not limited to, -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queosine, 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 the present disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0131] vector

[0132] In some embodiments, the nucleic acids of the present disclosure are incorporated into a vector. In this regard, the present disclosure provides vectors comprising any of the nucleic acids disclosed herein. In various embodiments, the vector is a recombinant expression vector. For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that enables expression of an mRNA, protein, polypeptide, or peptide by a host cell, where the construct includes a nucleotide sequence encoding such mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the present disclosure are generally not naturally occurring, although portions of the vector may be naturally occurring. The vectors disclosed herein can include any type of nucleotide, including, but not limited to, DNA and RNA, and can be single-stranded or double-stranded, synthetic, or derived in part from naturally occurring sources, and can contain natural, non-natural, or modified nucleotides. The vectors can include naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In some embodiments, the modified nucleotides or non-naturally occurring internucleotide linkages do not interfere with the transcription or replication of the vector.

[0133] The vectors of the present disclosure may be any suitable vector and may be used to transduce, transform, or transfect any suitable host. Suitable vectors include those designed for propagation and expansion, expression, or both, such as plasmids and viruses. The vector may be a plasmid-based expression vector. In various embodiments, the vector is selected from the group consisting of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGTIO, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, may also be used. Examples of plant expression vectors include pBI01, 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, e.g., a retroviral vector. In various embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, a vaccinia viral vector, or a lentiviral vector. See, e.g., Howarth et al., Cell Biol. Toxicol. 26(1):1-20 (2010). In various embodiments, the vector is a baculoviral vector that infects arthropods, e.g., insects. In various embodiments, the baculovirus vector is Autographa californica multiple nuclear virus (AcMNPV) or Bombyx mori nuclear polyhedrosis (BmNPV).See, e.g., Khan, Adv Pharm Bull 3(2):257-263 (2013), Miller, Bioessays 11(4):91-96 (1989), Atkinson et al., Pestic Sci 28:215-224 (1990).

[0134] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system that functions in a host prokaryotic or eukaryotic cell. Replication systems can be derived, for example, from CoIE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0135] In some embodiments, the vector includes regulatory sequences, such as transcription and translation initiation and termination codons, specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, taking into account, as appropriate, whether the vector is DNA- or RNA-based.

[0136] Vectors can contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to confer prototrophy, etc. Suitable marker genes for use in 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.

[0137] A vector can include a native or standard promoter operably linked to a nucleotide sequence encoding a polypeptide (including functional portions and functional variants thereof) or a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding the polypeptide. Selection of a promoter, e.g., strong, weak, inducible, tissue-specific, and developmentally specific, is within the ordinary skill of one of ordinary skill in the art. Similarly, matching a nucleotide sequence with a promoter is also within the skill of one of ordinary skill in the art. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long terminal repeat of murine stem cell virus.

[0138] host cell

[0139] Provided herein are host cells comprising the nucleic acids or vectors of the present disclosure. As used herein, the term "host cell" refers to any type of cell that can contain the vectors disclosed herein and produce expression products encoded by the nucleic acids (e.g., mRNA, proteins). In some embodiments, the host cells are adherent cells or suspension cells, i.e., cells that grow in suspension. In various embodiments, the host cells are cultured cells or primary cells, i.e., cells directly isolated from an organism, e.g., a human. The host cells can be of any cell type, can be derived from any type of tissue, and can be at any developmental stage.

[0140] 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, including, but not limited to, a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells have been described 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 are selected from the group consisting of 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 embryonic 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 bone osteosarcoma epithelial cells U2-OS, human alveolar basal adenocarcinoma cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, and mouse embryonic fibroblast cells NIH. 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse myeloma B J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0141] For purposes of amplifying or replicating vectors, the host cells in some embodiments are prokaryotic cells, such as bacterial cells.

[0142] The present disclosure also provides a cell population comprising at least one host cell described herein. In some aspects, the cell population is a heterogeneous population comprising host cells comprising the described vectors, as well as at least one other cell that does not comprise any of the vectors. Alternatively, in some aspects, the cell population is a substantially homogeneous population, wherein the population primarily comprises (e.g., consists essentially of) host cells comprising the vector. In some aspects, the population is a clonal population of cells, wherein all cells in the population are clones of a single host cell comprising the vector, and all cells in the population comprise the vector. In various embodiments of the present disclosure, the cell population is a clonal population comprising host cells comprising the vectors described herein.

[0143] Manufacturing method

[0144] Also provided herein are methods for producing an antigen binding protein that binds to CLDN6. In various embodiments, the methods include culturing host cells comprising 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 cells can be any of the host cells described herein. In various aspects, the host cells are selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, the step of culturing the host cells includes culturing the host cells in a growth medium to support the growth and proliferation of the host cells. In various aspects, the growth medium allows for timely increases in cell density, culture viability, and productivity. In various aspects, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and attachment factors. In various aspects, the growth medium is a synthetic medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to nutrients, growth media also help maintain pH and osmolality. Several growth media are commercially available and have been described in the art. See, e.g., Arora, "Cell Culture Media: A Review" MATER METHODS 3:175 (2013).

[0145] In various embodiments, the method comprises culturing the host cells in a fed-batch medium. In various embodiments, the method comprises culturing the host cells in a fed-batch medium in a fed-batch culture. Methods for recombinant protein production are known in the art. See, e.g., Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5):466-477 (2010).

[0146] Methods for producing antigen-binding proteins can include one or more steps for purifying the protein from a cell culture or its supernatant, and preferably recovering the purified protein. In various embodiments, the methods include one or more chromatography steps, such as affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, or hydrophobic interaction chromatography. In various embodiments, the methods include purifying the protein using a Protein A affinity chromatography resin.

[0147] In various embodiments, the method further includes a step of formulating the purified protein, etc., to thereby obtain a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0148] In various aspects, the antigen binding protein linked to the polypeptide and the antigen binding protein are part of a fusion protein. Accordingly, the present disclosure further provides methods of producing a fusion protein comprising an antigen binding protein that binds to CLDN6. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding the fusion protein in a cell culture medium as described herein, and recovering the fusion protein from the cell culture medium.

[0149] Complex

[0150] The present disclosure also provides antigen binding proteins that are attached, linked, or conjugated to a second moiety (e.g., a heterologous moiety, a conjugate moiety). Accordingly, the present disclosure provides conjugates comprising an antigen binding protein and a heterologous moiety. As used herein, the term "heterologous moiety" is synonymous with "conjugate moiety" and refers to any molecule (chemical or biochemical, naturally occurring, or non-encoded) that is distinct from the antigen binding protein of the present disclosure. Various heterologous moieties 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.

[0151] In some embodiments, the heterologous moiety is a polymer. The polymer can be branched or unbranched. The polymer can be of 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 a preparation of the water-soluble polymer, some molecules may be greater or less than the stated molecular weight). The average molecular weight of the polymer, in some aspects, is about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20 kDa to about 35 kDa.

[0152] 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, to allow for control of the degree of polymerization. In some embodiments, the polymer is water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, the polymer is pharmaceutically acceptable, e.g., when the composition is used for therapeutic purposes. Furthermore, in some aspects, the polymer is a mixture of polymers, e.g., a copolymer, a block copolymer.

[0153] In some embodiments, the polymer is selected from the group consisting of polyamides, polycarbonates, polyalkylenes and their derivatives (including polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates), polymers of acrylic and methacrylic acid esters (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 ethers, polyvinyl esters, halogenated polyvinyls, polyvinyl acrylates, and polyvinyl acrylates). and polyvinylpyrrolidone), polyglycolides, polysiloxanes, polyurethanes and their copolymers, celluloses (including alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and cellulose sulfate sodium salt), polypropylene, polyethylenes (including poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate)), and polystyrene.

[0154] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is intended to encompass any of the forms 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.

[0155] In some embodiments, the heterologous moiety 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), a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0156] In some embodiments, the heterologous moiety is a lipid. In some embodiments, the lipid is a fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acylethanolamine), glycerolipid (e.g., mono-, di-, or tri-substituted glycerol), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol), prenol lipid, glycolipid, or polyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, or phospholipid.

[0157] In some embodiments, the heterologous moiety is a therapeutic agent. The therapeutic agent can be any therapeutic agent known in the art. Examples of therapeutic agents contemplated herein include natural enzymes, proteins derived from naturally occurring substances, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic drugs, immunoglobulins, beta-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmic drugs, cardiac sympathomimetics, angiotensin-converting enzyme (ACE) inhibitors, diuretics, cardiac inotropes, cholesterol and triglyceride lowering drugs, bile acid sequestrants, fibrates, 3-hydroxy-3-methylglucosamine, ... HMG-CoA reductase inhibitors, niacin derivatives, antiadrenergics, alpha-adrenergic blockers, central antiadrenergics, vasodilators, potassium-sparing drugs, thiazides and related drugs, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogs, alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thioazolidinediones, androgens, progestogens, bone metabolism regulators, Anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropin, gonadotropin-releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators, antithyroid drugs, thyroid hormones, bulking agents, laxatives, antikinetic drugs, bacterial flora modifiers, enterosorbents, intestinal anti-infectives, anorexics, anticachexics, antibulimics, appetite suppressants, antiobesity drugs, antacids, upper gastrointestinal drugs, anticholinergics, aminosalicylic acid derivatives , biological response 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 promoters, hematopoietic agents, drugs for anemia, 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, antifungals, 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 derivatives, tetracycline Clinical drugs, anthelmintics, antiamoebic drugs, biguanides, cinchona alkaloids, folate antagonists, quinoline derivatives, drugs for treating Pneumocystis carinii, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, anticholinesterase agents, adjunctive therapy drugs, barbiturates and derivatives, benzodiazepines, gamma-aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigraine preparations, biological response modifiers, carbamic acid eater), tricyclic derivatives, depolarizing agents, non-depolarizing agents, neuromuscular paralytic agents, central nervous system stimulants, dopaminergic agents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, ethylenimines, imidazotetrazines, nitrogen mustard analogues, nitrosoureas, platinum-containing compounds, antimetabolites, purine analogues, pyrimidine analogues, urea derivatives, anthracyclines, actinomycin d, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and analogues, antiandrogens drugs, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitors, anti-cancer drugs, azaspirodecanedione derivatives, anti-anxiety drugs, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole 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, uricosurics, xanthine oxidase inhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetics, halogenated hydrocarbons, esters of aminobenzoic acid, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hypnotics These include, but are not limited to, hypnotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase activators, nicotine replacement therapy, 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.

[0158] The antigen binding proteins of the present disclosure can be conjugated to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis, where the cytokine or growth factor has been shown to have an anti-proliferative effect on at least one cell population. 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 factor, and erythropoietin.Additional growth factors for use herein include angiogenin, 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 alpha, cytokine-inducible neutrophil chemotactic factor 1 , cytokine-induced neutrophil, chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, beta-endothelial 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-related protein, growth-related protein α, growth-related protein β, growth-related 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 Included are nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof.

[0159] In some embodiments, the conjugate 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 present invention, the results obtained are synergistic. In other words, the effectiveness of the combination therapy of the compound and the cytotoxic agent is synergistic, i.e., the effectiveness is greater than that predicted from the additive effects of each agent individually. Thus, the dosage of the cytotoxic agent can be reduced, thereby simultaneously reducing the risk of toxicity and other side effects. 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, such as those described in U.S. Pat. No. 6,630,124, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides.

[0160] 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 selected from the group consisting of 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); ethylenediaminemalonatoplatinum(II); aqua(1,2-diamine)platin. (1,2-diaminocyclohexane)-sulfatoplatinum(II); (1,2-diaminocyclohexane)malonatoplatinum(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.

[0161] In some embodiments, cisplatin is the platinum coordination 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 constitution with water, sterile saline, or other suitable vehicle. Other platinum coordination compounds suitable for use in the present invention are known and can be commercially available and / or prepared by conventional techniques. Cisplatin, or cis-dichlorodiammineplatinum II, has been used successfully as a chemotherapeutic agent in the treatment of various human malignant solid tumors for many years. More recently, other diamino-platinum complexes have also demonstrated 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. Cisplatin and other diamino-platinum complexes have been widely used as chemotherapeutic agents in humans, but have required delivery at high doses that can result in toxicity issues, such as nephrotoxicity.

[0162] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that can alter the DNA topology of eukaryotic cells. They are important for cell function and 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, introduces a transient single-strand break, unwinds (or allows unwinding) the double helix, and then rejoins the break without dissociating 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.

[0163] In some embodiments, the topoisomerase inhibitor is camptothecin or a camptothecin analog. Camptothecin is a water-insoluble cytotoxic alkaloid produced by the Chinese native Camptotheca accuminata tree and the Indian native Nothapodytes foetida tree. Camptothecin exhibits tumor cell growth inhibitory activity against some tumor cells. Camptothecin analog class compounds are typically specific inhibitors of DNA topoisomerase I. The term "topoisomerase inhibitor" refers to any tumor cell growth inhibitor compound structurally related to camptothecin. Camptothecin analog class compounds include, but are not limited to, topotecan, irinotecan, and 9-amino-camptothecin.

[0164] In additional embodiments, the cytotoxic agent is a cytotoxic agent as described in U.S. Patent No. 5,004,758, issued April 2, 1991, and European Patent Application No. 88311366.4, published June 21, 1989, as Publication No. EP 0321122; U.S. Patent No. 4,604,463, issued August 5, 1986, and European Patent Application Publication No. EP 0137145, published April 17, 1985; and European Patent Application Publication No. EP 0074256, published March 16, 1983; U.S. Patent No. 4,545,880, issued October 8, 1985, and European Patent Application Publication No. EP 0074256, published March 16, 1983; European Patent Application Publication No. EP 0088642, published 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 (particularly the compound designated CPT-11). CPT-11 is a camptothecin analogue with a 4-(piperidino)-piperidine side chain linked via a carbamate bond at C-10 of 10-hydroxy-7-ethylcamptothecin. CPT-11 is currently undergoing human clinical trials 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. Pat. No. 4,342,776, issued Aug. 3, 1982; U.S. Pat. Application No. 581,916, filed Sep. 13, 1990, and European Patent Application Publication No. EP 418099, published Mar. 20, 1991; U.S. Pat. No. 4,513,138, issued Apr. 23, 1985, and European Patent Application Publication No. EP 0074770, published Mar. 23, 1983; U.S. Pat. No. 4,399,276, issued Aug. 16, 1983, and European Patent Application Publication No. 0056692, published Jul. 28, 1982; the entire disclosures of each of which are incorporated herein by reference. Any of the camptothecin analog class compounds listed above are commercially available and / or can be prepared by conventional techniques, including those described in the references listed above. The topoisomerase inhibitor may be selected from the group consisting of topotecan, irinotecan, and 9-aminocamptothecin.

[0165] 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 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 Nos. 7,999,083, 8,080,250, 8,759,496, 8,999,344, 10,195,288, and 9,808,537.

[0166] In some embodiments, the camptothecin analog is exatecan methanesulfonate, which exhibits more potent topoisomerase I inhibitory activity and antitumor activity than water-soluble camptothecin (CPT) and other CPT analogs. Furthermore, exatecan is effective against p-glycoprotein (P-gp)-mediated multidrug resistance cells.

[0167] In some embodiments, the camptothecin analog is deruxtecan (Dxd), a potent derivative of exatecan, which has 10-fold greater potency in inhibiting topoisomerase I than SN-38. In some embodiments, Dxd has the following structure: [ka]

[0168] Dxd is described in U.S. Patent Application Nos. 6,407,115, 10,195,288, 9,808,537, and 6,407,115.

[0169] The preparation of numerous camptothecin analog class compounds (including pharmaceutically acceptable salts, hydrates and solvates thereof) and oral and parenteral pharmaceutical compositions comprising such camptothecin analog class compounds and inert pharmaceutically acceptable carriers or diluents are described in detail in U.S. Pat. No. 5,004,758, issued April 2, 1991, and European Patent Application No. 88311366.4, published June 21, 1989, as Publication No. EP 0321122, the teachings of which are incorporated herein by reference.

[0170] In still other embodiments of the present invention, the chemotherapeutic agent is a combination antibiotic. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin.

[0171] In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. Antimitotic alkaloids can generally be extracted from Cantharanthus roseus and have been shown to be effective as anticancer chemotherapeutic agents. Numerous semisynthetic derivatives have been studied chemically and pharmacologically (see O. Van Tellingen et al., Anticancer Research, 12, 1699-1716 (1992)). Antimitotic alkaloids of the present 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. Pat. No. 5,620,985). In one embodiment, the antimitotic alkaloid is vinorelbine.

[0172] In another embodiment of the present invention, the chemotherapeutic agent is a difluoronucleoside. 2'-Deoxy-2',2'-difluoronucleosides are known in the art for their antiviral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication No. 184,365 discloses that these same difluoronucleosides have oncolytic activity. In a particular aspect, 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 is commercially available or can be synthesized in a multi-step process as disclosed and taught in U.S. Patent Nos. 4,526,988, 4,808,614, and 5,223,608, the teachings of which are incorporated herein by reference.

[0173] In various embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking tubulin polymerization, destabilizing microtubules, or altering microtubule dynamics, such as a maytansinoid or a derivative thereof (e.g., DM1 or DM4), or an auristatin or a derivative thereof. In various cases, the chemotherapeutic agent is an auristatin. For example, in some embodiments, 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 embodiments, a conjugate comprises an antibody of the present disclosure in combination with MMAE. Optionally, the conjugate comprises a linker. In some embodiments, 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, the linking group being linked to a spacer linked to MMAE. In embodiments, the linking group is attached to the antibody via a Cys residue in the Fc region of the antibody. In an exemplary embodiment, the linking group comprises the structure of Formula I: [ka] In an exemplary embodiment, the cathepsin-cleavable linker comprises the structure of Formula II: [ka] In an exemplary embodiment, the spacer comprises the structure of Formula III. [ka]

[0174] In some embodiments, MMAE has the following structure: [ka]

[0175] The present disclosure also provides a complex comprising an antigen-binding protein of the present disclosure linked to a polypeptide, such that the complex is a fusion protein. Thus, the present disclosure provides a fusion protein comprising an antigen-binding protein of the present disclosure linked to a polypeptide. In various embodiments, the polypeptide is a diagnostic label, e.g., a fluorescent protein such as green fluorescent protein, or other tag, e.g., a Myc tag. In various aspects, the polypeptide is a cytokine, lymphokine, growth factor, or one of the other hematopoietic factors listed above.

[0176] Linker

[0177] In some embodiments, the conjugate is directly linked to the heterologous moiety. In alternative embodiments, the conjugate comprises a linker linking the compound of the present disclosure to the heterologous moiety. In some aspects, the linker comprises a chain length of 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 some embodiments, all of the chain atoms are carbon atoms. In some embodiments, the chain atoms in the linker backbone are selected from the group consisting of C, O, N, and S. The chain atoms and linker can be selected according to their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that undergoes cleavage by an enzyme or other catalyst, or by hydrolysis conditions found in the target tissue, organ, or cell. In some embodiments, the length of the linker is sufficient to reduce the potential for steric hindrance. In some embodiments, the linker is an amino acid or peptidyl linker. Such peptidyl linkers may be of any length. Various linkers are about 1-50 amino acids in length, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length.

[0178] A wide variety of suitable linkers are known in the art. The linker may be cleavable, for example, under physiological conditions, e.g., intracellular conditions (cleavable linker), such that cleavage of the linker releases the drug into the intracellular environment. Alternatively, the linker may be cleavable, such that cleavage of the linker under extracellular conditions, e.g., outside tumor cells or in the vicinity of a tumor mass, releases the drug, which selectively penetrates the tumor cell interior. In other embodiments, the linker is not cleavable (non-cleavable linker), and the drug is released, for example, by degradation of the antibody.

[0179] The linker can be attached to a chemically reactive group on the antibody moiety, such as a free amino group, imino group, hydroxyl group, thiol group, or carboxyl group (e.g., to the N- or C-terminus, the epsilon-amino group of one or more lysine residues, the free carboxylic acid groups of one or more glutamic or aspartic acid residues, the sulfhydryl groups of one or more cysteinyl residues, or the hydroxyl groups of one or more serine or threonine residues). The linker attachment site can be a naturally occurring residue within the amino acid sequence of the antibody moiety, or can be introduced into the antibody moiety by, for example, recombinant DNA techniques (e.g., by introducing a cysteine or protease cleavage site into the amino acid sequence) or protein biochemistry (e.g., reduction, pH adjustment, or proteolysis). The linker attachment site can also be a non-naturally occurring amino acid. The linker attachment site can also be a glycan on the antibody.

[0180] Typically, a linker is substantially inert under the conditions under which the two groups it connects are linked. The terms "bifunctional crosslinker," "bifunctional linker," or "crosslinker" refer to a modifying agent having two reactive groups at each end of the linker, such that one reactive group can first react with a cytotoxic compound to provide a compound bearing a linker moiety, and then the second reactive group can react with an antibody. Alternatively, one end of the bifunctional crosslinker can first react with an antibody to provide an antibody bearing a linker moiety and a second reactive group, which can then react with a cytotoxic compound. The linking moiety can contain a chemical bond that allows for 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-labile bonds, photolabile bonds, protease / peptidase-labile bonds, and esterase-labile bonds. See, e.g., U.S. Patent Nos. 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 linkage is a disulfide bond, a thioether, and / or a protease / peptidase labile bond. Other linkers that can be used in the present invention include non-cleavable linkers such as those detailed in US20050169933, charged linkers or hydrophilic linkers such as those described in US2009 / 0274713, US2010 / 0129314, and WO2009 / 134976, each of which is expressly incorporated herein by reference.

[0181] In some embodiments, the linker is a hydrophilic linker that confers hydrophilicity to the conjugate. In some embodiments, the hydrophilic linker comprises 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. Patent Nos. 8,080,250, 8,759,496, and 10,195,288.

[0182] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E has the following structure: [ka] CL2E is described in U.S. Patent Nos. 8,080,250, 8,759,496, and 10,195,288.

[0183] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., inside a lysosome or endosome or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular or extracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker comprises at least 2 amino acids in length, at least 3 amino acids in length, at least 4 amino acids in length, or at least 5 amino acids in length.

[0184] In some embodiments, the peptide linker is MC-VC-PAB, which comprises valine and citrulline residues. In some embodiments, the MC-VC-PAB linker has the following structure: [ka] MC-VC-PAB is described in U.S. Patent Nos. 7,659,241, 7,829,531, 6,884,869, 6,214,345, and 6,214,345.

[0185] In some embodiments, the peptide linker is maleimidocaproyl glycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure: [ka] MC-GGFG is described in US Patent Nos. 9,808,537 and 10,195,288.

[0186] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. In some embodiments, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, e.g., U.S. Patent Nos. 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 are unstable at pHs below 5.5 or 5.0, which are the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0187] In other embodiments, the linker is cleavable under reducing conditions (e.g., disulfide linker).Bifunctional crosslinkers that allow the antibody to be linked to a cytotoxic compound via a disulfide bond 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, cross-linkers that introduce 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.

[0188] A "heterobifunctional crosslinker" is a bifunctional crosslinker having two different reactive groups. Heterobifunctional crosslinkers containing both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link antibodies to cytotoxic compounds. Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazide terephthalate hydrochloride (SHTH), and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates with acid-labile bonds can also be prepared using the hydrazine-bearing benzodiazepine derivatives of the present invention. Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0189] The linkers described herein may be used in any combination with the heterologous moieties described herein. All of the linkers and heterologous moieties listed herein above are commercially available and / or can be prepared by conventional techniques, including those described in the references listed above.

[0190] complex

[0191] The heterologous moiety-to-antigen-binding protein ratio (HAR) represents the number of heterologous moieties linked per antigen-binding molecule. In some embodiments, the HAR is in the range of 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the HAR is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 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 to about 4. The HAR can be characterized by conventional means, such as mass spectrometry, UV / Vis spectroscopy, ELISA assay, and / or HPLC.

[0192] In some embodiments, the complex is a heterogeneous complex (also called "conventional"), where the antigen binding proteins are conjugated to different numbers of heterologous moieties. In some embodiments, the heterogeneous complex follows a Gaussian or quasi-Gaussian distribution of the complex, where the distribution is centered around the heterologous moiety loading mean, with some antigen binding proteins bound above average and some antigen binding proteins bound below average.

[0193] In some embodiments, the conjugate is a homogeneous conjugate, in which a significant proportion of the antigen binding protein is conjugated to a predetermined number of heterologous moieties. In some embodiments, the homogeneous conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HARs. In some embodiments, the homogeneous conjugate comprises 2, 4, 6, or 8 HARs. In preferred embodiments, the homogeneous conjugate comprises 4 HARs. In other preferred embodiments, the homogeneous conjugate comprises 2 HARs. In some embodiments, the homogeneous conjugate comprises 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 of the conjugates having a defined HAR. In some embodiments, a homogeneous complex comprises about 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 of complexes with a defined HAR. In some embodiments, a homogeneous complex comprises at least 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 of complexes with a defined HAR. In some embodiments, the homogeneous complex comprises an 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 suitable chromatography. In some embodiments, the chromatogram is an HIC chromatogram. The homogeneous complex can be produced by site-specific conjugation.

[0194] In some embodiments, the heterologous moiety is site-specifically attached to an antigen binding protein (eg, an antibody). Various methods of site-specific conjugation are known in the art, such as conjugation at thiomab or 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 cross-linkers (Behrens et al. (2015) Mol. Pharm. 12:3986-98), conjugation at glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15, Dennler et al. (2014) Bioconjug Chem. 25:569-78), conjugation with engineered 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 conjugation (Li et al. (2017) Cell Chem Biol 24:433-442), and glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5), conjugation with galactose or GalNAc analogues (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9, van Geel et al. (2015) Bioconjug Chem 26:2233-42), or glycoengineering (Zhou et al. (2014) Bioconjug Chem 25:510-20, Tang et al.(2017) Nat Protoc 12:1702-1721), by engineering glutamine tags or short peptide tags such as sortase A-mediated transpeptidation (Strop et al. (2013) Chem Biol 20:161-7, Beerli et al. (2015) PLoS One 10:e0131177), and by aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA 106:3000-5).

[0195] Unpredictability of conjugates (e.g., ADCs)

[0196] It is impossible to predict in advance which antibody-drug conjugates will be sufficiently safe and effective for clinical application based solely on antibody or drug payload profiles. For example, a particular drug payload may function perfectly well when conjugated to an antibody directed against one target, but may work poorly when conjugated to an antibody directed against a different target, or even to different antibodies directed against the same target. The reasons why different antibody-drug conjugates exhibit different anti-tumor activities in vivo are not fully understood, making accurate predictions impossible to make when designing new antibody-drug conjugates. It is likely that the 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 ability of the conjugate to penetrate solid tumors, and the circulating half-life required for adequate exposure to tumors without causing toxicity.

[0197] The complexity and unpredictability are fully demonstrated by antibody affinity alone. High-affinity antibodies or antibody-drug conjugates are better internalized into cells, leaving behind a trail of higher levels of cytotoxic payload released inside the cells. High affinity is also known to enhance antibody-dependent cellular cytotoxicity (ADCC). All of these attributes favor the cell-killing properties of antibody-drug conjugates. However, it is also known that high affinity of antibodies or antibody-drug conjugates can hinder efficient tumor penetration due to the "antigen barrier effect," suggesting that to achieve strong anti-tumor activity in vivo, the affinity of an antibody-drug conjugate must be just right, neither too high nor too low. To date, there is no known method for predicting the most efficient or effective level of affinity for a given antibody-drug conjugate.

[0198] Furthermore, in vivo antitumor activity cannot be predicted solely from the linker and payload mechanisms. For example, O. Ab et al., Mol. Cancer Ther. 14(&):1605-1613 (2015) demonstrated that when tested in preclinical cancer models, the same antibody conjugated to the same antitubulin toxin with different linkers exhibited dramatically different antitumor activity. This example is particularly surprising because the chemical structures of the two linkers are very similar. Furthermore, the linker present in the dominant conjugate contained a hydrophilic moiety. Hydrophilic metabolites generally have low membrane permeability and are more slowly released from lysosomes (the site of conjugate degradation), which is thought to result in delayed antitubulin activity of the released payload. While this finding claims an "ideal" kinetics of payload delivery, there is currently no insight into what constitutes such kinetics. Further complicating this, even if ideal kinetics of payload delivery is defined for specific cell types, it remains an open question as to whether it applies to all cell types. Therefore, it is not possible to predict the most effective in vivo antitumor activity simply from the chemical composition of the linker or payload.

[0199] Compositions, pharmaceutical compositions and formulations

[0200] Provided herein are compositions comprising antigen-binding proteins, nucleic acids, vectors, host cells, or complexes as disclosed herein. In some embodiments, the compositions comprise the antigen-binding proteins in isolated and / or purified form. In some embodiments, the compositions comprise a single type (e.g., structure) of antigen-binding protein of the present disclosure, or a combination of two or more antigen-binding proteins of the present disclosure, where such combinations comprise two or more antigen-binding proteins of different types (e.g., structures).

[0201] In some aspects, the composition comprises an agent that enhances the chemical and physical characteristics of the antigen binding protein, e.g., by stabilizing the antigen binding protein at a particular temperature, e.g., room temperature, extending shelf life, reducing degradation, e.g., degradation by oxidative proteases, extending the half-life of the antigen binding protein, etc. In some aspects, the composition comprises any of the agents disclosed herein as a heterologous or conjugated moiety, optionally in admixture with or conjugated to an antigen binding protein of the present disclosure.

[0202] In various aspects of the present disclosure, the composition additionally comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, an antigen-binding protein, nucleic acid, vector, host cell, or complex as disclosed herein (hereinafter referred to as the "active agent") is formulated into a pharmaceutical composition comprising the active agent together with a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, the present disclosure further provides pharmaceutical compositions comprising the active agent intended for administration to a subject, e.g., a mammal.

[0203] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent 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 a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition contains the active agent at a concentration of about 0.001 to about 30.0 mg / ml.

[0204] In various embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil-in-water or water-in-oil emulsions, and various wetting agents. The term also encompasses any drug approved by a regulatory agency of the U.S. federal government or listed in the U.S. Pharmacopeia for use in animals, including humans.

[0205] The pharmaceutical composition can contain any pharmaceutically acceptable ingredient, including, for example, an acidifying agent, an additive, an adsorbent, an aerosol propellant, an air displacing agent, an alkalizing agent, an anticaking agent, an anticoagulant, an antimicrobial preservative, an antioxidant, a disinfectant, a base, a binder, a buffer, a chelating agent, a coating agent, a colorant, a drying agent, a detergent, a diluent, a disinfectant, a disintegrant, a dispersant, a dissolution enhancer, a dye, a softener, an emulsifier, an emulsion stabilizer, and an excipient. Examples of suitable additives include film-forming agents, seasonings, flavoring agents, glidants, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, dyes, plasticizers, glazing agents, preservatives, sequestering agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surface active agents, surfactants, suspending agents, sweeteners, therapeutic agents, thickening agents, isotonicity agents, toxicological agents, thickening agents, water-absorbing agents, water-miscible cosolvents, water softeners, or humectants. See, for example, Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), the entire contents of which are incorporated by reference. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0206] In various aspects, pharmaceutical compositions comprise formulation materials that are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions comprise an active agent and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic agents, tonicity agents, antioxidants, antibiotics, antifungals, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, analgesics, or additional pharmaceutical agents. In various aspects, pharmaceutical compositions comprise one or more polyols and / or one or more surfactants, and optionally one or more other additives, including, but not limited to, pharmaceutically acceptable salts, osmotic agents (isotonic agents), antioxidants, antibiotics, antifungals, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, and analgesics.

[0207] In certain embodiments, pharmaceutical compositions can contain formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); excipients; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorings, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, toriton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability-enhancing substances (such as sucrose or sorbitol); tonicity-enhancing agents (such as 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, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0208] Pharmaceutical compositions can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition can 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 5.5-7.5.

[0209] The present disclosure provides methods of producing a pharmaceutical composition. In various aspects, the methods include combining an antigen binding protein, a conjugate, a fusion protein, a nucleic acid, a vector, a host cell, or a combination thereof with a pharmaceutically acceptable carrier, diluent, or excipient.

[0210] Administration route

[0211] In accordance with the present disclosure, the active substance or pharmaceutical composition comprising the same can be administered to a subject via any suitable route of administration. For example, the active substance can be administered to a subject parenterally, nasally, orally, pulmonary, topically, vaginally, or rectally. The following discussion of routes of administration is provided merely to illustrate various embodiments and should not be construed as limiting the scope in any way.

[0212] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The term "parenteral" means not through the alimentary canal, but through some other route such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agents of the present disclosure can be administered using a pharmaceutical carrier with a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides (with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or detergent), pectin, carbomer, suspending agents such as methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents, and other pharmaceutical adjuvants.

[0213] The oil that can be used in parenteral formulations includes petroleum, animal, vegetable, or synthetic oil.Specific examples of oil include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral oil.The fatty acid suitable for use in parenteral formulations includes oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0214] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, 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 fatty 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.

[0215] In some embodiments, parenteral formulations contain about 0.5% to about 25% by weight of a solution of the active agent of the present disclosure. Preservatives and buffers can be used. To minimize or eliminate injection site irritation, such compositions can 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 typically ranges from 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 a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. In some embodiments, parenteral formulations are packaged in single- or multi-dose hermetically sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid excipient for injection, e.g., water, immediately prior to use. In some embodiments, extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the type described above.

[0216] The injectable formulation is in accordance with the present disclosure. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0217] Dosage

[0218] The disclosed active agents are believed to be useful in other methods as detailed herein, including methods for inhibiting tumor growth and treating or preventing cancer. For purposes of the disclosure, the amount or dose of the active agent 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, a dose of the disclosed active agent should be sufficient to treat the cancers described herein within about 1-4 minutes, 1-4 hours, or 1-4 weeks or longer, e.g., 5-20 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 agent and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0219] Many assays for determining dosage are known in the art.For the purpose herein, any assay that involves comparing the degree of cancer treatment when a given dose of the active substance of the present disclosure is administered to a group of mammals, each group being administered with a different dose of the active substance, can be used to determine the starting dose that should be administered to a mammal.The degree of cancer treatment when a specific dose is administered can be represented, 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.

[0220] The dosage of the active agent of the present disclosure will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular active agent of the present disclosure. Typically, the dosage of the active agent of the present disclosure for treating each individual patient will be determined by the attending physician, taking into account various factors, such as age, body weight, overall health, diet, sex, the active agent of the present disclosure to be administered, the route of administration, and the severity of the condition being treated. By way of example and not intended to be limiting, the dosage of the active agent of the present disclosure may be about 0.0001 to about 1 g / kg body weight (of the subject being treated) per day, about 0.0001 to about 0.001 g / kg body weight per day, or about 0.01 mg to about 1 g / kg body weight per day.

[0221] Controlled-release formulations

[0222] In some embodiments, the active agents described herein can be modified into a depot form, allowing the manner in which the active agent of the present disclosure is released into the body to which it is administered to be controlled with respect to time and location within the body (see, e.g., U.S. Pat. No. 4,450,150). A depot form of an active agent of the present disclosure can be, for example, an implantable composition comprising the active agent and a porous or non-porous material, such as a polymer, where the active agent is encapsulated by or diffuses throughout the material and / or diffuses through the degradation of the non-porous material. The depot is then implanted at a desired location within the subject's body, and the active agent is released from the implant at a predetermined rate.

[0223] In certain embodiments, pharmaceutical compositions containing active substances are modified to have any type of in vivo release profile. In some embodiments, the pharmaceutical compositions are formulations of immediate release, controlled release, sustained release, extended release, delayed release, or biphasic release. Methods for formulating peptides for controlled release are known in the art. For example, see Qian et al., J Pharm 374:46-52 (2009) and International Patent Application Publication Nos. WO2008 / 130158, WO2004 / 033036, WO2000 / 032218, and WO1999 / 040942.

[0224] The compositions may further comprise, for example, micelles or liposomes, or some other encapsulated form, and may also be administered in sustained release form to provide a long-term storage and / or delivery effect.

[0225] use

[0226] The antigen binding proteins of the present disclosure are useful for inhibiting tumor growth. Without being bound by any particular theory, the inhibitory effects of the antigen binding proteins provided herein make such entities useful in methods of treating cancer.

[0227] Thus, provided herein are methods for inhibiting tumor growth in a subject and methods for reducing tumor size in a subject. In various embodiments, the methods comprise administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to inhibit tumor growth or reduce tumor size in the subject. In various aspects, the growth of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is inhibited. In various aspects, the size of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is reduced.

[0228] As used herein, the terms "inhibit" or "reduction" and words derived therefrom may not be 100% or complete inhibition or reduction. Rather, there are various degrees of inhibition or reduction that one of skill in the art will recognize as having a potentially beneficial or therapeutic effect. In this regard, the antigen binding proteins of the disclosure may inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of the 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 a 10% reduction (e.g., about or at least a 20% reduction, about or at least a 30% reduction, about or at least a 40% reduction, about or at least a 50% reduction, about or at least a 60% reduction, about or at least a 70% reduction, about or at least an 80% reduction, about or at least a 90% reduction, about or at least a 95% reduction, about or at least a 98% reduction).

[0229] Further provided herein are methods for treating a subject with cancer, e.g., a CLDN6-expressing cancer. In various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0230] For purposes herein, the cancer of the methods disclosed herein can be any cancer, such as, for example, any malignant growth or tumor caused by abnormal and uncontrolled cell division that can spread to other parts of the body via the lymphatic system or bloodstream. In some embodiments, the cancer is acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid. The cancer is selected from the group consisting of tumors, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, 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, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), and bronchioloalveolar carcinoma. In various embodiments, the cancer is ovarian cancer, melanoma, bladder cancer, lung cancer, liver cancer, or endometrial cancer. In various embodiments, the cancer is any cancer characterized by moderate to high expression of CLDN6. See, e.g., Figures 1-3. In various embodiments, the 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, renal clear cell carcinoma, head and neck cancer, sarcoma, chromophobe carcinoma of the kidney, low-grade glioma, adrenocortical carcinoma, glioblastoma, papillary renal cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, ovarian endometrioid carcinoma, uterine carcinosarcoma, or ovarian cancer. In various aspects, the cancer is selected from ovarian cancer, endometrioid cancer, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), cervical cancer, and bladder cancer.

[0231] As used herein, the term "treat" and related terms do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that those skilled in the art will recognize as having potential benefit or therapeutic effect. In this regard, the disclosed methods of treating cancer can provide any amount or level of treatment. Furthermore, the treatment provided by the disclosed methods can include treating one or more conditions or symptoms or signs of the cancer being treated. The treatment provided by the disclosed methods can also include slowing the progression of cancer. For example, the methods can treat cancer by enhancing T cell activity or immune response to cancer, suppressing tumor or cancer growth, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, etc. In various embodiments, the methods treat 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 more. In various aspects, the methods treat by prolonging survival of the subject.

[0232] The antigen binding proteins of the present disclosure can also be used to detect CLDN6 in a sample or to diagnose CLDN6-positive cancer. Accordingly, the present disclosure provides methods for detecting claudin 6 (CLDN6) in a sample. In various embodiments, the methods include contacting the sample with an antigen binding protein, complex, or fusion protein as described herein, and assessing for an immune complex comprising the antigen binding protein, complex, or fusion protein that binds to CLDN6. The present disclosure also provides methods for diagnosing claudin 6 (CLDN6)-positive cancer in a subject. In various embodiments, the methods include contacting a biological sample comprising cells or tissue obtained from a subject with an antigen binding protein, complex, or fusion protein as described herein, and assessing for an immune complex comprising the antigen binding protein, complex, or fusion protein that binds to CLDN6.

[0233] subject

[0234] In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, mammals from the order Rodentia, such as mice and hamsters, and mammals from the order Lagomorpha, such as rabbits, mammals from the order Carnivora, including Felidae (cats) and Canidae (dogs), mammals from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs), or mammals from the order Perissodactyla, including Equidae (horses). In some aspects, the mammal is a mammal from the order Primates, Superfamily Capuchin, or Simoid (monkey) or Anthropoid (humans and apes). In some aspects, the mammal is a human.

[0235] kit

[0236] In some embodiments, the antigen binding proteins of the present disclosure are provided in a kit. In various aspects, the kit includes the antigen binding protein(s) as a unit dose. For purposes herein, a "unit dose" refers to a discrete amount dispersed in a suitable carrier. In various aspects, a unit dose is an amount sufficient to provide a desired effect in a subject, e.g., inhibiting tumor growth, reducing tumor size, treating cancer. Accordingly, provided herein are kits including the antigen binding proteins of the present disclosure, optionally provided in unit doses. In various aspects, the kit includes several unit doses, e.g., a weekly or monthly supply of unit doses, optionally each individually packaged or otherwise distinguished from the other unit doses. In some embodiments, the components of the kit / unit dose are packaged 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 syringe, etc. In some aspects, the antigen binding protein of the present disclosure, a pharmaceutically acceptable salt thereof, a conjugate comprising the antigen binding protein, or a multimer or dimer comprising the antigen binding protein is pre-packaged in a ready-to-use form, e.g., a syringe, an intravenous bag, etc. In some aspects, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including those described herein. In particular aspects, the kit comprises an antigen binding protein of the present disclosure together with an agent used in chemotherapy or radiation therapy, e.g., a therapeutic agent.

[0237] Various embodiments

[0238] In various embodiments of the present disclosure, the antigen binding protein binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200), and (a) the antigen binding protein binds to extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6 but not to extracellular loop 1 (EL1) of the ECD of CLDN6, or (b) does not bind to any of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9), and inhibits binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells at less than about 1200 nM, or (c) a combination thereof. In various cases, the antigen binding protein binds to an epitope within the amino acid sequence of WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2), or binds to the amino acid sequence of TAHAIIRDFYNPL (SEQ ID NO: 3) or the amino acid sequence of LVAEAQKREL (SEQ ID NO: 4) of CLDN6. In various aspects, the 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 antigen binding protein does not bind to CLDN3. In various cases, the antigen binding protein binds to CLDN6, CLDN4, and CLDN9, but not to CLDN3. In various cases, the antigen binding protein binds to CLDN6 and CLDN4, but not to CLDN3 or CLDN9. In various aspects, the antigen binding protein binds to CLDN6 and CLDN9, but not to CLDN3 or CLDN4.

[0239] In various cases, antigen binding proteins of the disclosure inhibit binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells by less than about 1200 nM, wherein 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 aspects, antigen binding proteins of the disclosure inhibit binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells by less than 1000 nM or less than 750 nM (e.g., less than 500 nM, less than 250 nM, less than about 100 nM), wherein 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.

[0240] In various embodiments, the antigen binding protein comprises (a) the amino acid sequence of a heavy chain CDR1 set forth 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 about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity. (b) a heavy chain CDR2 amino acid sequence set forth in Table A or A1, 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, 132, 475, 456, 462, 466, 468, and 473, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; c) a heavy chain CDR3 amino acid sequence set forth in Table A or A1, 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, 133, 453, 457, 463, 467, 469, and 474, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (d) a heavy chain CDR3 amino acid sequence set forth in Table A or A1, 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, 133, 453, 457, 463, 467, 469, and 474, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; 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 about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (e) a light chain CDR2 amino acid sequence set forth in Table A or A1;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, 129, 450, 477, 459, and 471, or a variant thereof, which differs by only one or two amino acids or which has about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (f) an amino acid sequence of a light chain CDR3 set forth in Table A or A1, 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 about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (g) a combination of any two or more of (a) to (f).

[0241] In various embodiments, the antigen binding protein comprises a light chain CDR1 amino acid sequence, a light chain CDR2 amino acid sequence, and a light chain CDR3 amino acid sequence set forth in Table A or A1, and one or two of the heavy chain CDR amino acid sequences set forth in Table A or A1. In some cases, the antigen binding protein comprises a heavy chain CDR1 amino acid sequence, a heavy chain CDR2 amino acid sequence, and a heavy chain CDR3 amino acid sequence set forth in Table A or A1, and one or two of the light chain CDR amino acid sequences set forth in Table A or A1. In various aspects, the antigen binding protein is 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: (q) SEQ ID NOs: 92 to 97, (q) SEQ ID NOs: 116 to 121, (r) SEQ ID NOs: 8 to 13, (t) SEQ ID NOs: 68 to 73, (u) SEQ ID NOs: 14 to 19, (v) SEQ ID NOs: 20 to 25, (v) SEQ ID NOs: 449 to 453 and 475, (w) SEQ ID NOs: 476 to 477, 454 to 457, (x) SEQ ID NOs: 458 to 463, (y) SEQ ID NOs: 57, 58, 464 to 467, (z) SEQ ID NOs: 68 to 71 and 468 to 469, and (aa) SEQ ID NOs: 112, and 470 to 474.In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence set forth 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 which shares about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity; (b) a light chain variable region amino acid sequence set forth 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 about or at least 70% (e.g., about or at least 85%, about or at least 90%) sequence identity, or both (a) and (b). In various aspects, the antigen binding proteins are selected from the group consisting of (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, 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.

[0242] In various embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence set forth 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 about or at least 70%, or about 80%, or about 90%, or about 95% sequence identity. or (b) a light chain variable region amino acid sequence set forth 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, which differs by only one or two amino acids or has about or at least 70%, or about 80%, or about 90%, or about 95% sequence identity; or (c) both (a) and (b). In various embodiments, the antigen binding protein comprises a pair of amino acid sequences set forth in Table D.

[0243] The present disclosure provides a HC CDR1 comprising: (A) an HC CDR1 comprising the amino acid sequence of YTFTXYT, where X is T, V, D, or S (SEQ ID NO: 452), optionally comprising the amino acid sequence of YTFTTYT (SEQ ID NO: 11); (B) an HC CDR2 comprising the amino acid sequence of IXPSSGYT, where X is Q, S, A, or N (SEQ ID NO: 475), optionally comprising the amino acid sequence of INPSSGYT (SEQ ID NO: 12); (C) an HC CDR3 comprising the amino acid sequence of AXGDYYVAY, where X is N, Q, H, or D (SEQ ID NO: 453), optionally comprising the amino acid sequence of ANGDYYVAY (SEQ ID NO: 13); (D) an HC CDR4 comprising the amino acid sequence of LC

[0013] Provided is an antigen binding protein comprising: (A) an LC CDR1 comprising the amino acid sequence of SSVSSXY, where X is T, V, F, or D (SEQ ID NO: 449), optionally comprising the amino acid sequence of SSVSSTY (SEQ ID NO: 8); (B) an LC CDR2 comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of STS (SEQ ID NO: 9); and (C) an LC CDR3 comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of HQYHRSPLT (SEQ ID NO: 10).

[0244] (A) HC CDR1 comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of FTFSNYW (SEQ ID NO: 23); (B) HC CDR2 comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of IRLKSDNYAT (SEQ ID NO: 24); (C) HC (D) an HC CDR3 comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of NDGPPSGC (SEQ ID NO: 25); (D) an LC CDR1 comprising the amino acid sequence of EXIYSY, where X is Q, S, A, D, or N (SEQ ID NO: 476), optionally comprising the amino acid sequence of ENIYSY (SEQ ID NO: 20); (E) an LC CDR2 comprising the amino acid sequence of XAK, where X at position 1 is Q, S, A, D, or N (SEQ ID NO: 477), optionally comprising the amino acid sequence of NAK (SEQ ID NO: 21); and (F) an LC CDR2 comprising the amino acid sequence of XAK, where X at position 1 is Q, S, A, D, or N (SEQ ID NO: 477), optionally comprising the amino acid sequence of NAK (SEQ ID NO: 21). An antigen binding protein comprising: a LC CDR3 comprising the amino acid sequence of 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, the amino acid sequence of QHHYTVPWT (SEQ ID NO:22).

[0245] (A) HC CDR1, comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of YTFTSYT (SEQ ID NO: 29); (B) HC CDR2, comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of INPSSTYT (SEQ ID NO: 30); (C) HC CDR3, comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of SRGELGGFAY (SEQ ID NO: 31); CDR3, (D)LC CDR1 comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 26), (E)LC CDR2 comprising the amino acid sequence of 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), optionally comprising the amino acid sequence of KVS (SEQ ID NO: 27), and (F)LC 1. An antigen binding protein comprising: a CDR3 comprising the amino acid sequence of 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, a LC CDR3 comprising the amino acid sequence of SQSTHVPYT (SEQ ID NO: 28).

[0246] In various embodiments, the antigen binding protein comprises: (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 has about or at least 70% sequence identity; (b) a 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 has about or at least 70% sequence identity; (c) a 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 has about 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 has about or at least 70% sequence identity; (e) a 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 has about or at least 70% sequence identity; (f) a 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 has about or at least 70% sequence identity; (g) A combination of two or more of (a) to (f).

[0247] Optionally, the variant sequence has at least about 80%, about or at least 85%, about or at least 90%, or about or at least 95% sequence identity.

[0248] In exemplary embodiments, the antigen binding protein comprises the light chain CDR1 amino acid sequence, the light chain CDR2 amino acid sequence of SEQ ID NO: 449 or SEQ ID NO: 450, and the light chain CDR3 amino acid sequence of SEQ ID NO: 451, and one or two of the heavy chain CDR1 amino acid sequence, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 504 or SEQ ID NO: 505, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 506. In various cases, the antigen binding protein comprises the light chain CDR1 amino acid sequence, the light chain CDR2 amino acid sequence of SEQ ID NO: 476 or SEQ ID NO: 477, and the light chain CDR3 amino acid sequence of SEQ ID NO: 454, and one or two of the heavy chain CDR1 amino acid sequence, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 507 or SEQ ID NO: 508, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 509. Optionally, the antigen binding protein comprises 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.

[0249] In exemplary embodiments, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 490-503, or a heavy chain variable region amino acid sequence designated as S1-S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity, or (b) a light chain variable region amino acid sequence of any one of SEQ ID NOs: 380-383, 388-390, 479, and 481, or a light chain variable region amino acid sequence designated as S1-S12 in Figure 22, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity, or (c) both (a) and (b). In some embodiments, the variant sequence has at least about 80% or at least about 85% sequence identity, or the variant sequence has at least about 90% or at least about 95% sequence identity.

[0250] In an exemplary case, the antigen binding protein comprises the following pair of amino acid sequences: SEQ ID NOs: 389 and 490, SEQ ID NOs: 389 and 491, SEQ ID NOs: 389 and 492, SEQ ID NOs: 389 and 493, SEQ ID NOs: 389 and 494, SEQ ID NOs: 389 and 495, SEQ ID NOs: 383 and 496, SEQ ID NOs: 383 and 497, SEQ ID NOs: 383 and 498, SEQ ID NOs: 383 and 499, SEQ ID NOs: 383 and 500, SEQ ID NOs: 383 and 501, SEQ ID NOs: 383 and 503, SEQ ID NOs: 389 and 502, the sequence of the heavy chain variable region, designated S1 in Figure 22, and the sequence of the light chain variable region, designated S1 in Figure 22; the sequence of the heavy chain variable region, designated S2 in Figure 22, and the sequence of the light chain variable region, designated S2 in Figure 22; the sequence of the heavy chain variable region, designated S3 in Figure 22, and the sequence of the light chain variable region, designated S3 in Figure 22; the sequence of the heavy chain variable region, designated S4 in Figure 22, and the sequence of the light chain variable region, designated S4 in Figure 22; the sequence of the heavy chain variable region, designated S5 in Figure 22, and the sequence of the light chain variable region, designated S5 in Figure 22; the sequence of the heavy chain variable region, designated S6 in Figure 22, and the sequence of the light chain variable region, designated S6 in Figure 22; the sequence of the heavy chain variable region, designated S7 in Figure 22, and the sequence of the light chain variable region, designated S7 in Figure 22; the sequence of the heavy chain variable region designated as S8 in Figure 22 and the sequence of the light chain variable region designated as S8 in Figure 22; the sequence of the heavy chain variable region designated as S9 in Figure 22 and the sequence of the light chain variable region designated as S9 in Figure 22; the sequence of the heavy chain variable region, designated S10 in Figure 22, and the sequence of the light chain variable region, designated S10 in Figure 22; the heavy chain variable region sequence designated as S11 in Figure 22 and the light chain variable region sequence designated as S11 in Figure 22; or The heavy chain variable region sequence is labeled S12 in FIG. 22 and the light chain variable region sequence is labeled S12 in FIG.

[0251] In some embodiments, the antigen-binding protein is an antibody, e.g., a monoclonal antibody. In various embodiments, the antibody is an IgG. Optionally, the antigen-binding protein inhibits at least about 50% 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% tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0252] Thus, in various embodiments, the present disclosure provides an antigen binding protein comprising: (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 has about or at least 70% sequence identity; (b) a 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 has about or at least 70% sequence identity; (c) a 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 has about 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 has about or at least 70% sequence identity; (e) a 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 has about or at least 70% sequence identity; (f) a 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 has about or at least 70% sequence identity; or (g) A combination of two or more of (a) to (f).

[0253] Also provided is an antigen-binding protein comprising six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 449 to 451 and 504 to 506, and SEQ ID NOs: 476, 477, 454 and 507 to 509.

[0254] The present disclosure provides an antigen binding protein comprising: (a) a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 490 to 503, or a heavy chain variable region amino acid sequence designated as S1 to S12 in FIG. 22, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity; or (b) a light chain variable region amino acid sequence of any one of SEQ ID NOs: 380-383, 388-390, 479, and 481, or a light chain variable region amino acid sequence designated as S1-S12 in FIG. 22, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity; or (c) Both (a) and (b).

[0255] In various embodiments, the variant sequence has at least about 85% sequence identity, or about 90% or about 95% sequence identity.

[0256] The present disclosure also provides an antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of: SEQ ID NOs: 389 and 490, SEQ ID NOs: 389 and 491, SEQ ID NOs: 389 and 492, SEQ ID NOs: 389 and 493, SEQ ID NOs: 389 and 494, SEQ ID NOs: 389 and 495, SEQ ID NOs: 383 and 496, SEQ ID NOs: 383 and 497, SEQ ID NOs: 383 and 498, SEQ ID NOs: 383 and 499, SEQ ID NOs: 383 and 500, SEQ ID NOs: 383 and 501, SEQ ID NOs: 383 and 503, SEQ ID NOs: 389 and 502, the sequence of the heavy chain variable region, designated S1 in Figure 22, and the sequence of the light chain variable region, designated S1 in Figure 22; the sequence of the heavy chain variable region, designated S2 in Figure 22, and the sequence of the light chain variable region, designated S2 in Figure 22; the sequence of the heavy chain variable region, designated S3 in Figure 22, and the sequence of the light chain variable region, designated S3 in Figure 22; the sequence of the heavy chain variable region, designated S4 in Figure 22, and the sequence of the light chain variable region, designated S4 in Figure 22; the sequence of the heavy chain variable region, designated S5 in Figure 22, and the sequence of the light chain variable region, designated S5 in Figure 22; the sequence of the heavy chain variable region, designated S6 in Figure 22, and the sequence of the light chain variable region, designated S6 in Figure 22; the sequence of the heavy chain variable region, designated S7 in Figure 22, and the sequence of the light chain variable region, designated S7 in Figure 22; the sequence of the heavy chain variable region designated as S8 in Figure 22 and the sequence of the light chain variable region designated as S8 in Figure 22; the sequence of the heavy chain variable region designated as S9 in Figure 22 and the sequence of the light chain variable region designated as S9 in Figure 22; the sequence of the heavy chain variable region, designated S10 in Figure 22, and the sequence of the light chain variable region, designated S10 in Figure 22; the heavy chain variable region sequence designated as S11 in Figure 22 and the light chain variable region sequence designated as S11 in Figure 22; or The heavy chain variable region sequence is labeled S12 in FIG. 22 and the light chain variable region sequence is labeled S12 in FIG.

[0257] Provided herein are antigen binding proteins comprising: (a) a heavy chain variable region amino acid sequence set forth as SEQ ID NO: 510 or 513, or as set forth in Figure 23 or Figure 25, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity; or (b) a light chain variable region amino acid sequence set forth as SEQ ID NO: 511 or 512, or as set forth in Figure 24 or Figure 26, or a variant thereof, which differs by only one or two amino acids or has about or at least 70% sequence identity; or (c) Both (a) and (b).

[0258] The present disclosure also provides an antigen binding protein comprising a pair of amino acid sequences, wherein the pair comprises: (a) a heavy chain variable region amino acid sequence set forth as SEQ ID NO: 510 and a light chain variable region amino acid sequence set forth as SEQ ID NO: 511, or a variant thereof, which differs by only 1-5 amino acids or has about or at least 70% sequence identity, optionally wherein the 1-5 differing amino acids are set forth in Figure 23 for the heavy chain or Figure 24 for the light chain; or (b) the heavy chain variable region amino acid sequence set forth as SEQ ID NO: 513 and the light chain variable region amino acid sequence set forth as SEQ ID NO: 512, or variants thereof, which differ by only 1 to 5 amino acids or have about or at least 70% sequence identity, or optionally, wherein the 1 to 5 amino acids that differ are set forth in Figure 25 for the heavy chain or Figure 26 for the light chain.

[0259] In various aspects, the antigen binding proteins disclosed herein comprise an Fc polypeptide comprising defucosylated glycans.

[0260] In various embodiments, the antigen binding protein of the present disclosure is an antibody, e.g., a monoclonal antibody. In various cases, the antigen binding protein is an IgG. In various embodiments, the antigen binding protein inhibits at least about 50% colony growth in a soft agar 3D growth assay or inhibits tumor growth in xenograft mice injected with human cancer cells. In various embodiments, the antigen binding protein inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various cases, the antigen binding protein inhibits at least 50% tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0261] The present disclosure provides a conjugate comprising an antigen-binding protein described herein and a heterologous moiety. In exemplary embodiments, the conjugate comprises a cytotoxic 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 an auristatin, and optionally MMAE.

[0262] The present disclosure also provides fusion proteins comprising the antigen-binding proteins described herein. The present disclosure further provides nucleic acids comprising a nucleotide sequence encoding the antigen-binding proteins, complexes, or fusion proteins of the present disclosure. The present disclosure provides vectors comprising nucleic acids comprising a nucleotide sequence encoding the antigen-binding proteins, complexes, or fusion proteins of the present disclosure. The present disclosure additionally provides host cells comprising the nucleic acids or vectors of the present disclosure.

[0263] The present disclosure provides a method for producing an antigen-binding protein that binds to claudin 6 (CLDN6) protein, comprising (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein of any one of the preceding claims, and (ii) recovering the antigen-binding protein from the cell culture medium. Also provided is a method for producing a fusion protein comprising an antigen-binding protein that binds to claudin 6 (CLDN6) protein, comprising (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding the fusion protein of the present disclosure, and (ii) recovering the fusion protein from the cell culture medium.

[0264] The present disclosure further provides methods of producing a pharmaceutical composition comprising combining an antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure, and a pharmaceutically acceptable carrier, diluent, or excipient. Also provided are pharmaceutical compositions comprising an antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0265] Provided herein is a method for treating a subject with a CLDN6-expressing cancer, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to treat the cancer. Also provided is a method for inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to inhibit tumor growth. The present disclosure provides a method for reducing tumor size in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to reduce tumor size. Additionally, provided is a method for preventing cancer recurrence in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to prevent cancer recurrence.

[0266] The present disclosure provides a method for detecting claudin 6 (CLDN6) in a sample, comprising contacting the sample with an antigen-binding protein, complex, or fusion protein of the present disclosure, and evaluating for an immune complex comprising the antigen-binding protein, complex, or fusion protein that binds to CLDN6. Also provided herein is a method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject, comprising contacting a biological sample containing cells or tissue obtained from the subject with an antigen-binding protein, complex, or fusion protein of the present disclosure, and evaluating for an immune complex comprising the antigen-binding protein, complex, or fusion protein that binds to CLDN6.

[0267] The present disclosure also provides methods for treating cancer in a subject diagnosed with low CLDN6 overexpression. In various embodiments, the method comprises administering to the subject a pharmaceutical composition disclosed herein in an amount effective to prevent recurrence of the cancer. In some aspects, the administration induces apoptosis in tumor cells, and optionally, the administration induces apoptosis in CLDN6-expressing cells. In various aspects, the subject has a tumor, and the tumor is semi-quantitatively classified into one of four groups: high expressors, moderate expressors, low expressors, and non-expressors. In various cases, a high expressor is defined as 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, a moderate expressor is defined as a CLDN6 RNA greater than 10 log FPKM, where CLDN6 RNA is measured by RNASeq, or a CLDN6 protein level greater than 2+ when measured by IHC. In various cases, a low expressor is defined as a CLDN6 RNA greater than 6 log FPKM, where CLDN6 RNA is measured by RNASeq, or a CLDN6 protein level greater than 1+ when measured by IHC. In various cases, a non-expressor is defined as a CLDN6 RNA less than 6 log FPKM, where CLDN6 RNA is measured by RNASeq, or a CLDN6 protein level below the IHC detection limit. In various embodiments, subjects with the tumor are similarly described as CLDN6 high expressors, moderate expressors, low expressors, or non-expressors.

[0268] The following examples are provided merely to illustrate the present disclosure and are not intended to limit its scope in any way. [Example]

[0269] Example 1 This example demonstrates the analysis of CLDN6 RNA levels in a variety of cell and tissue sources.

[0270] To establish a baseline for CLDN6 expression in various source materials, we assayed the expression levels of CLDN6 expression in patient samples, normal tissues, and cell lines created by the Translational Oncology Research laboratory (TORL).

[0271] CLDN6 RNA levels in patient samples were measured using information contained in The Cancer Genome Atlas (TCGA) database maintained 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. Analysis of tissues from the GTEX database showed that CLDN6 was detectable in various sites, including the brain, pituitary gland, pancreas, kidney, lung, thyroid, and cervix, among other tissues (Figure 1).

[0272] 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. RNA-Seq was performed by BGI Americas (Cambridge, MA) using their "RNA-Seq for quantification" service. As shown in Figures 2 and 3, ovarian, head and neck, lung, and bladder cancer cells expressed the highest levels of CLDN6, while CLDN6 expression levels were detectable in breast, kidney, colon, sarcoma, and liver cancer cells.

[0273] Example 2 This example demonstrates the production of cells engineered to overexpress CLDN6.

[0274] Models engineered to overexpress CLDN6 were established. These models were used to determine the efficacy of the CLDN6 antibody described in Example 5. Briefly, a nucleotide sequence encoding CLDN6 was engineered into a bicistronic vector containing a CMV promoter and an attenuated internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV). The IRES was positioned between the gene of interest (GOI) cDNA (CLDN6) and the puromycin cDNA. A woodchuck posttranscriptional 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 expression vector containing GFP is provided herein as SEQ ID NO: 189.

[0275] The expression vector was virally transduced into HEK293T cells (for screening) and NIH3T3 cells (for immunization). Positively transduced cells were selected based on their survival in puromycin-containing medium (1 μg / ml). Positively selected cells were subcloned to obtain a stable, homogeneous clonal population of CLDN6-overexpressing cells.

[0276] Expression of the subclone CLDN6 was confirmed by flow cytometry using a reference CLDN6 monoclonal antibody (mAb) on a BD Biosciences Accuri™ flow cytometer (San Jose, CA). Secondary antibodies and conjugates: Goat anti-mouse IgG (minimal cross-reactivity) antibody Alexa Fluor® 647 (Biolegend, San Diego, CA; catalog number 405322) was used to detect binding activity between the reference CLDN6 mAb and the CLDN6 expressed by the subclone.

[0277] The intracellular localization of CLDN6 was determined by fluorescence microscopy using cells expressing CLDN6-green fluorescent protein (GFP) fusion protein with a Cellavista® Imaging System (Synentec, Mountain View, CA). As shown in Figure 4, GFP fluorescence was detected at the plasma membrane, demonstrating that CLDN6 is localized to the plasma membrane.

[0278] Experimental Example 3 This example demonstrates the production of a reference antibody and a control antibody.

[0279] Benchmark (reference) CLDN6-specific and control antibodies were generated by cloning the heavy and light chain variable regions of the antibodies into the ExpiCHO™ Expression System (ThermoFisher Scientific, Waltham, MA) to produce recombinant murine IgG2A chimeric antibodies. These antibodies were tested in parallel with the newly generated CLDN6-specific antibodies described in Example 5.

[0280] Briefly, plasmids containing the control and benchmark antibody sequences were transfected using the ExpiCHO™ Expression System (Cat. No. A29133, ThermoFisher Scientific, USA) according to the manufacturer's instructions. Cells were cultured at 37°C and 8% CO2 on day 1, and then cultured at 32°C and 5% CO2 in the medium provided with the kit after transfection. Antibodies were purified by clarifying the ExpiCHO™ medium by centrifugation at 1,000g for 10 minutes and then at 5,000g for 30 minutes. The supernatant was then filtered using a 0.45µm filter followed by a 0.22µm filter. The supernatant was then subjected to affinity purification using Protein A / G resin (Life Technologies, Carlsbad, CA; Cat. No. 20424) according to the manufacturer's instructions. Prior to ELISA purification, the antibody titer in the medium was roughly determined to ensure that the input medium volume 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, 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 aliquoted and stored at -80°C for long-term storage or kept at 4°C for immediate use.

[0281] Antibody integrity was assessed by SDS-PAGE followed by Coomassie staining under non-reducing versus reducing conditions; under non-reducing conditions, a single dominant band was observed at approximately 150 kDa, whereas under reducing conditions, two bands were observed at 50 kDa and 25 kDa.

[0282] Antibodies specific to other CLDN family members with sequence similarity (Figure 5), i.e., CLDN3, CLDN4, and CLDN9, were generated essentially in the same manner, except that the antibody sequence contained in the plasmid was an antibody sequence specific to CLDN3, CLDN4, or CLDN9.

[0283] Example 4 This example demonstrates the characterization of cell lines with high endogenous CLDN6 expression.

[0284] A panel of cancer cell lines was analyzed for their endogenous CLDN6 expression by FACS and Western blot. Briefly, antibody binding to the target was assessed by FACS using cells overexpressing CLDN6 (e.g., CLDN6-overexpressing HEK293T cells as described in Example 2) and cell lines endogenously expressing high or low levels of CLDN6 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).

[0285] Western blots were performed on nitrocellulose using reference and control antibodies. Briefly, samples from cell lysates were boiled to denature the protein content. The 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 using a 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 recognized the reference or control antibody, and the secondary antibody was detected by chemiluminescence.

[0286] Control and reference antibodies were evaluated using overexpressing lines, and at the time of evaluation, the control and reference antibodies were used to characterize the endogenous cell lines. Cells overexpressing CLDN6 were included in these assays as a positive control.

[0287] FACS assays showed that endometrial cancer cell lines, as well as four ovarian cancer cell lines, a bladder cancer cell line, a lung cancer cell line, and an upper gastrointestinal cancer cell line, expressed high levels of CLDN6 on their surface. High levels of CLDN6 expression were also detected by Western blot. Two additional ovarian cancer cell lines, an additional liver cancer cell line, an additional lung cancer cell line, and an additional upper gastrointestinal cancer cell line, were shown to express moderate levels of CLDN6 on their surface as detected by Western blot. Endometrial and bladder tumor cells also expressed high levels of CLDN6 as xenografts in vivo. The endogenous expression levels of CLDN6 by the cancer cell lines tested are summarized in Table 2. [Table 20] TIFF2025122026000035.tif222167

[0288] Example 5 This example demonstrates the immunization of mice to produce CLDN6-specific antibodies.

[0289] CLDN6-specific antibodies were produced by immunizing Balb / c and CD1 mice with a mixture of three different peptide immunogens according to the procedures of the Fred Hutchinson Cancer Research Center. The three peptides spanned the second loop of the CLDN6 extracellular domain (i.e., EL2). The peptides included the full length of EL2, a peptide extending to the first (N-terminal) half of EL2, and a peptide extending to the remaining (C-terminal) half of EL2. The sequences of the three peptides are listed in Table 3. [Table 21]

[0290] Mice were also immunized with 3T3 cells overexpressing full-length CLDN6 using a plasmid containing the human CLDN6-myc-DDK expression vector.

[0291] Splenocytes were harvested from immunized mice and fused with myeloma lines using BTX Electrofusion (BTX, Holliston, MA) to generate hybridomas. 7,680 primary hybridoma cultures were generated and cultured in 384-well plates. The ability of antibodies to bind to peptides was assessed by bead array using beads expressing three different peptide targets. 1,920 potential antibodies were again arrayed in 96-well plates and further screened by flow cytometry against endogenous and artificial cell line models.

[0292] Positive hybridoma supernatants were then counterscreened by flow cytometry against endogenous and artificial models of proteins with sequence similarity to the target region (e.g., other CLDN6 proteins). From the secondary and counterscreening, approximately 20 CLDN6-specific antibodies were selected for further testing. These antibodies were subcloned, and the variable heavy and light chain sequences were determined. See Table B and the Sequence Listing.

[0293] The CLDN6 antibody was formatted as a full-length IgG antibody using ExpiCHO™ expression. The variable regions of the antibody heavy and light chains were cloned into an antibody expression vector engineered in-house based on the pcDNA™ 3.4-TOPO® vector (catalog number: A14697, ThermoFisher Scientific, USA) and transfected into CHO cells according to the instructions provided with the kit (ExpiCHO™ Expression System, catalog number: A29133, ThermoFisher Scientific, USA). The antibody was purified, and the cell surface binding of the antibody to CLDN6 and the IC50 of the antibody 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 instructions. CLDN6 antibodies were tested from 0.32 nM to 1000 nM (serial dilution 1:5, 6 steps) in a volume of 50 μl using a system of 150,000 cells.

[0294] To determine the ability of CLDN6 antibodies to bind to cell surface CLDN6 and cross-react with other CLDN6 family members, CLDN6-expressing cells were used in FACS assays. HEK293T cells engineered to express human CLDN6 fused to GFP, mouse CLDN6 fused to GFP, CLDN9-GFP, CLDN4-GFP, or CLDN3-GFP, or GFP alone (without CLDN6), were used as artificial models of CLDN6 expression. ARK2, OVCA429, LS513, and MCF7 cells were used as endogenous models of CLDN6 expression and models of CLDN3 / 4 expression.

[0295] 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 mAb at preset concentrations on ice for 30 minutes in the dark. CLDN6 mAb was directly labeled with Alexa Fluor® 647 NHS ester (succinimidyl ester). After washing, cells were read on a BD Accuri™ Flow Cytometer C6 to detect antibody-antigen protein binding in channel FL4H. Each antibody was tested at various concentrations, and dose-fluorescence curves were generated. The antibody EC50 / IC50 (antibody concentration at half-maximum) was calculated based on the FL4H values (gated on live singlet cells) using the Very Simple IC50 Toolkit, available online, which allows for plotting biological dose-response data and fitting curve types to obtain EC50 / IC50 values. The maximum was defined as the lowest antibody concentration at which fluorescence reached its maximum. 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 panel of antibodies tested. Cross-reactivity data were obtained using similar methods, but using cells with different expression profiles for CLDN6, CLDN3, CLDN4, and CLDN9.

[0296] The relative affinity and cross-reactivity data thus determined are set forth in Tables 4 and 5. [Table 22] TIFF2025122026000038.tif89162AB numbers correspond to the AB numbers listed in Tables A and B. [Table 23] The AB numbers correspond to those listed in Tables A and B.

[0297] Example 6 This example demonstrates the characterization of IgG mAbs from chimeric mice.

[0298] To further characterize the mAbs described in Example 5, soft agar 3D growth assays and xenograft binding assays were performed. Briefly, for 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 a 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 a mouse IgG2a control, starting at 150 ng / mL (1 μM) and ending at 1.5 ng / mL (1:10 dilution). Each test condition was performed in duplicate. Cells were allowed to form colonies for 3 weeks, then stained with 0.05% neutral red and imaged using an EVOS XL inverted light microscope. Cell lines were considered sensitive if they showed a 20% or greater reduction in colony numbers between treated and control.

[0299] As shown in Table 6, many of the cell lines showed a decrease in colony numbers when treated with the indicated antibodies. [Table 24]

[0300] In vivo binding studies were performed in xenograft mice injected with human cancer cell lines. Briefly, xenograft models of human cancer cell lines were established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories). Subcutaneous injection of each cell line was performed as follows: ARK2 0.75 x 10 7 cells, UMUC4 1.0×10 7 Cells, OV90 1.0×10 7 Cells and M202 0.5 x 10 7 Both were injected in 50% Matrigel (BD Biosciences). Enough mice were injected to ensure there were eight mice per treatment group. Tumors were 150–300 mm 3 Mice were randomized into treatment groups when tumors reached an average size of 1 mg / ml. For treatment, each therapeutic antibody (AB3, AB2, ref. Ab1, ref. Ab2, ref. Ab3 (trastuzumab)) and a non-targeting IgG2 control were 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) on a 5-day on, 2-day off schedule in a weekly cycle, then tapered to 0.5 mg / kg for the remaining 2 weeks. Tumor xenografts were measured with calipers three times weekly and tumor volume was calculated in mm by multiplying height x width x length. 3The tumor volume was measured in units of 0.01 mg / kg. Mice were treated for 2 to 7 weeks. At the end of the study, animals were euthanized, and tumor tissue was excised and sectioned for storage as snap-frozen or formalin-fixed, paraffin-embedded (FFPE) tissue for biomarker analysis. All animal work was performed under 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 expressed as the mean volume for each group. Error bars represent the standard error of the mean (SE).

[0301] 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 each caused 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 caused 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 each caused a substantial mean change in tumor volume compared to the control IgG2 antibody at day 20. Figures 9A and 9B demonstrate that AB3 functions specifically in a CLDN-specific manner, as the model used in Figures 9A and 9B does not express CLDN6, CLDN3, CLDN4, or CLDN9 and was therefore used as a negative control. 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 of Figures 6 to 9. As shown in Figure 10A, AB3 significantly suppressed tumor growth in mice bearing endometrial tumors, bladder tumors, and ovarian tumors, each of which express CLDN6, but did not suppress tumor growth in mice bearing melanoma tumors, which did not express CLDN6 (Figure 10B). As shown in Figure 11, there was no significant change in the average body weight of mice during treatment, suggesting the safety of the treatment.

[0302] A second set of experiments was performed 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 (murine IgG2a antibody, reference CLDN6 mAb), or a PBS vehicle control. Eight mice per group received intravenous injections of 10 mg / kg of antibody every four days. As shown in Figures 12A and 12B, several of the antibodies described in Example 5 reduced tumor volume in ovarian tumor-bearing mice. While AB3, AB4, AB7, and AB10 performed best, all tested antibodies reduced tumor volume compared to the vehicle control. As shown in Figure 13, there was no significant change in the body weight of animals treated with AB3, AB4, AB7, or AB10, suggesting their safety.

[0303] Example 7 This example presents further characterization of chimeric mouse IgG mAbs.

[0304] An internalization quantification assay was performed. Briefly, we examined the internalization of CLDN6 protein induced by binding of reference Ab1, AB3, or AB4, using the ubiquitously expressed transferrin receptor (TfR) as a positive control, a cell surface receptor known to be internalized after antibody binding.

[0305] 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 into μ-Slide 8-well chambers (catalog number 80826, ibidi Cells In Focus Inc.) to allow cells to adhere and proliferate. Cells were incubated with the labeled antibodies for 30 minutes on ice in the dark. The chambers containing CLDN6- or TfR-labeled cells were then read using an Echo Lab fluorescence microscope, and images were collected before internalization. The chambers were then incubated at 37°C for 40 minutes to allow internalization to proceed, and images were again collected using the Echo Lab fluorescence microscope. With AB3 and AB4, the extent of CLDN6 internalization was greater than that achieved with reference Ab1 (data not shown).

[0306] Example 8 This example presents further characterization of chimeric mouse IgG mAbs.

[0307] Two-dimensional (2D) proliferation assays using the selected antibodies described in Example 5 were performed as follows: cells were seeded in duplicate at 5,000–20,000 cells per well in 24-well plates. The following day, cells were treated with six dilutions of mAb (starting with either 100 nM trastuzumab, Cldn6 mAb, or mouse IgG2A control) from 1 to 5, followed by a fixed concentration of 1 ng / μL monomethyl auristatin E (MMAE)-conjugated anti-mouse secondary antibody (Moradec, LLC) to generate dose-response curves. To determine the range of cell proliferation, cells in untreated wells were quantified on day 1, the day of antibody treatment, and then on day 6. mAb-treated wells were quantified on day 6, and proliferation in each treatment condition was determined as a normalized percentage of proliferation in untreated cells. Quantification was performed using a Z1 Particle Counter (Beckman Coulter, Inc.).

[0308] The results are shown in Figure 14. AB2, AB3, AB4, and AB5 demonstrated the most potency in growth inhibition. The IC50 for each of these antibodies ranged from 0.1 nM to 1 nM. AB7, AB10, AB11, and AB15 also each demonstrated growth inhibition in this assay, although to a lesser extent than AB2, AB3, AB4, and AB5.

[0309] Example 9 This example demonstrates the humanization of an antibody of the present disclosure.

[0310] 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 antibodies AB1, AB3, AB4, AB9, AB11, and AB18 were compared to a library of known human germline sequences from human VH genes and human VL kappa genes (IMGT®, the international ImMunoGeneTics information system®, www.imgt.org; founder and curator: Marie-Paule Lefranc, Montpellier, France). (The databases used were IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT human VL kappa genes (F+ORF, 74 germline sequences).) Acceptor human germline sequences were selected from those closest in sequence to the parent antibodies.

[0311] Table 7 provides information on the human germline sequence selected as the acceptor sequence and the selected human heavy chain joining region (J gene) for each antibody VH and VL, respectively. The joining regions (J genes) were selected from human joining region sequences deposited in IMGT®, the international ImMunoGeneTics information system®, www.imgt.org (founded and curated by Marie-Paule Lefranc, Montpellier, France). [Table 25]

[0312] CDRs were defined according to AbM definitions (see CDR definitions reference table on Dr. Andrew CMartin's website www.bioinf.org.uk / abs / ).

[0313] Changes in human germline framework positions (i.e., non-CDR residues of VH and VL) relative to the corresponding murine parental sequences may be necessary to optimize binding of the humanized antibody. Sequences of versions of the humanized antibodies are provided as SEQ ID NOS: 376-421.

[0314] In the case of AB1, Asn52 (consecutive numbering) of CDR2 of HC and Asn54 of CDR2 of LC were each determined to have low potential for deamidation based on sequence and conformation.

[0315] In the case of AB3, Asn31 (consecutively numbered) in CDR1 of HC, Asn57 in CDR2 of HC, Asn28 in CDR1 of LC, and Asn50 in CDR2 of LC were each determined to have low potential for deamidation based on their sequence and conformation. Trp33 in CDR1 of HC was determined to be potentially solvent-exposed and potentially susceptible to oxidation, especially under stress conditions. CDR3 of HC was determined to have a free Cys106 within the CDR, which could be problematic during antibody manufacturing due to its potential for solvent exposure. Modification of this Cys residue to Tyr, Ser, or Ala was recommended. The binding retention of these modified antibodies was tested. Ile53 in CDR2 of LC was determined to be potentially solvent-exposed and could potentially cause nonspecific binding. Modification of this Ile residue to Ser was proposed. The binding retention of these modified antibodies was tested.

[0316] In the case of AB4, Asn52 (consecutively numbered) in CDR2 of the HC and Asn58 in CDR2 of the LC were each determined to have low potential for deamidation based on sequence and conformation. The sequence DGNT within CDR1 of the LC was determined to be problematic because it was determined to have a high potential for isoaspartate formation (sequence DG) and potential for deamidation (sequence NT). This sequence was recommended for modification.

[0317] In the case of AB9, Asn33 (consecutively numbered) in CDR1 of HC and Asn52 and Asn59 in CDR2 of HC were determined to have a low potential for deamidation based on their sequence and conformation. Asn54 was determined to have a moderate potential for deamidation based on their sequence and conformation. The NGG sequence in CDR2 of HC was determined to have a high / moderate potential for deamidation following isoaspartate formation. Therefore, it was recommended that this amino acid sequence be modified. The free Cys106 in CDR3 of HC was determined to be potentially solvent-exposed and thus could be problematic during antibody manufacturing. Modification of this Cys residue to Tyr, Ser, or Ala is proposed. The binding retention of these modified antibodies will be tested. Arg28 in CDR1 of HC is not commonly found in human antibodies. This residue will be modified to Thr and the binding retention will be tested. In the case of AB9, Trp32 (sequential numbering) within CDR1 of the LC was determined to be solvent-exposed and susceptible to oxidation, particularly under stress conditions. Leu24 in the same CDR is less commonly found in human antibodies. This residue was modified to Arg and tested for retention of binding.

[0318] For AB11, Asp54-Ser55 (consecutive numbering) within CDR2 of the HC was determined to have a low probability of isoaspartate formation, and Asn57 within CDR2 of the LC was determined to have a low probability of deamidation based on sequence and conformation.

[0319] In the case of AB18, Asn33 in CDR1 of the HC and CDR-H2 Asn50 (consecutive numbering) were determined to have low potential for deamidation based on sequence and conformation. In CDR2 of the HC, the Asp-Pro (DP) sequence was determined to be susceptible to fragmentation under acidic conditions. In the VL domain, Asn34 and Asn37 in CDR1 of the LC were determined to have low potential for deamidation based on sequence and conformation. In CDR3 of the LC, Trp56 was determined to be solvent-exposed and susceptible to oxidation, particularly under stress conditions.

[0320] Table 8 shows a scheme for matching humanized VH and VL when neither humanized version is equivalent to the chimeric mAb. Preferred pairs are shown in underlined bold text. [Table 26] TIFF2025122026000043.tif108162

[0321] The humanized antibodies listed in Table 8 were constructed and expressed essentially as described in Example 5. To determine the relative antigen-binding strength of the humanized antibodies, FACS assays were performed essentially as described in Example 5. Two doses of humanized antibodies (1.5 μg or 0.3 μg) were tested for binding to human CLDN6 or mouse CLDN6 proteins expressed by the engineered 293T clone. The results of the assay are listed in Table 9. [Table 27] TIFF2025122026000045.tif250156

[0322] FACS assays were also performed to determine the relative antigen-binding strength of the humanized antibodies (1.5 μg or 0.3 μg) for binding to CLDN6 expressed by the indicated cancer cell lines. The results of the assays are listed 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 parent antibodies (antibodies before humanization) were used as controls and are also labeled "chim." [Table 28] TIFF2025122026000047.tif250158TIFF2025122026000048.tif38162

[0323] Based on the in vitro antigen binding data, three humanized antibodies were selected for further testing and development. The antibodies were derived from AB1, AB3, and AB4.

[0324] In vivo binding studies of the humanized versions of AB1, AB3, and AB4 were performed in xenograft mice injected with the bladder cancer cell line UMUC4 essentially as described in Example 6. Briefly, UMUC4 xenograft models were established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories). Tumors were 150-300 mm 3 After reaching an average size of 1 mg / ml, mice were randomized 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 weekly with calipers and tumor volume was calculated in mm by multiplying height x width x length. 3 The tumor mass was measured in units of malignant tumor tissue. Mice were treated for 2 to 7 weeks. At the end of the study, animals were euthanized, and tumor tissue was excised and sectioned for storage as snap-frozen or formalin-fixed, paraffin-embedded (FFPE) tissue for biomarker analysis.

[0325] The results of the xenograft assay are shown in Figures 15-21. Figure 15 shows the results of the xenograft assay of two versions of humanized AB3 (AB3-2 and AB3-4), where treatment involved administration of 10 mg / kg every four days. Controls included a vehicle control (PBS), human IgG (10 mg / kg every four days), and the murine version of AB3 (10 mg / kg every four days). As shown in this figure, humanized AB3-4 demonstrated a reduction in tumor volume over the 35-day treatment period. Figure 16 shows the results of the xenograft assay with the same treatments as in the experiment shown in Figure 15, except that two additional controls were administered: 64A MSE and its chimeric version (64A-CHIM). As in Figure 15, Figure 16 demonstrates a significant reduction in tumor volume upon treatment with humanized AB3-4.

[0326] Figure 17 shows the results of a xenograft assay of humanized AB1-5. Controls included vehicle control (PBS), human IgG (10 mg / kg every four days), and the mouse version of AB1 (10 mg / kg every four days). As shown in Figure 17, mice treated with humanized AB1-5 showed a significant reduction in tumor volume.

[0327] Figure 18 shows the results of a xenograft assay of humanized AB4-3. Controls included vehicle control (PBS), human IgG (10 mg / kg every 4 days), and the mouse version of AB4 (10 mg / kg every 4 days). Mice treated with humanized AB4-3 did not show a significant reduction in tumor volume.

[0328] Figures 19-21 show the results of xenograft assays testing all four humanized antibodies from Figures 15-18. Mouse and chimeric versions of the reference CLDN6 antibody were used as controls. As shown in Figure 19, mice treated with humanized AB3-4 and AB1-5 showed a reduction in tumor volume. Figure 20 shows tumor volume over time up to day 55. Mouse weights at the assay are shown in Figure 21.

[0329] Example 10 An in silico analysis was performed using the different sequences of AB1, AB3, and AB4. Specifically, for each antibody, (a) the sequence of the original parent clone, (b) the closest mouse germline sequence, (c) the closest human germline sequence, and (d) the humanized sequence were aligned. Amino acids that are likely to have undergone affinity maturation are marked with an asterisk, and amino acids that differ from the amino acid at that position according to antibody database information are marked with a hashtag. The CDRs of each sequence are boxed. Based on this analysis, several humanized antibodies were generated containing the sequences listed in Table 10. [Table 29]

[0330] Antibodies having sequences defined by these consensus sequences were generated in large numbers essentially as described in Example 5 and tested for antigen binding in vitro by FACS (essentially as described in Example 5) and for the ability to reduce tumor volume in mice in vivo (essentially as described in Example 6).

[0331] Example 11 This example describes next-generation sequencing (NGS) analysis of the CLDN6 antibodies of the present disclosure.

[0332] The sequences of AB1 and AB3 were subjected to NGS analysis to identify somatic hypermutation (SHM)-associated mutations in the heavy and light chains of each antibody. NGS analysis identified SHM sites in both the heavy and light chain sequences of each antibody. The analysis revealed 1,452 distinct heavy chain sequences and 326 distinct light chain sequences for AB3, and 372 distinct heavy chain sequences and 3,081 distinct light chain sequences for AB1. Exemplary results are shown in Figures 23-26. Figures 23 and 24 show the identified alterations in the heavy and light chains of mutant AB3, respectively, and Figures 25 and 26 show the identified alterations in the heavy and light chains of mutant AB1, respectively. The mutations are listed at the bottom of each figure using Chothia numbering.

[0333] Antibodies with SHMs in their heavy chains identified by NGS were selected based on their potential binding involvement. Six antibodies were produced based on humanized AB3-7 (AB S1-S6) and six based on humanized AB1-11 (AB S7-S12), and then phenotypes were evaluated. Figure 22 lists the parent heavy chain sequences and 12 mutant heavy chain sequences paired with the light chain sequences. FACS binding assays of the 12 antibodies (AB S1-S12) were performed essentially as described herein, and the results are shown in Figure 27.

[0334] A FACS binding assay was also performed using different amounts of ABs S1-S12. The antibody concentrations tested in this limiting dilution assay were 0.32 nM, 1.6 nM, 8 nM, 40 nM, 200 nM, and 1000 nM, using different cell lines expressing CLDN6. The results are shown in Figure 28.

[0335] These data support that the newly identified antibodies (S1-S12) exhibit increased binding relative to the corresponding parental humanized antibodies.

[0336] Example 12 This example demonstrates in vivo analysis of the humanized antibodies described herein.

[0337] In vivo studies were performed in xenograft mice injected with human cancer cell lines essentially as described in Example 6. In this study, cells from the UMUC4 cell line, a bladder cancer cell line that strongly expresses CLDN6, and cells from the OV-90 cell line, an ovarian cancer cell line that expresses CLDN6, were used. Both cell lines are tumorigenic when administered subcutaneously to mice. Briefly, xenograft models of human cancer cell lines were established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories). The following conditions were observed for subcutaneous injection of each cell line: UMUC4 1.0 × 10 7 and OV90 1.0×10 7In both cases, 50% Matrigel (BD Biosciences) was used. CD-1 nude mice (8 mice per group) were subcutaneously injected with UMUC4 or OV-90 cell lines in the right flank. Tumors were 150–300 mm 3 Once the tumors reached an average size of 100 mm, mice were randomized into treatment groups. For treatment, each therapeutic antibody (humanized AB1-11, humanized AB3-7) and a human IgG control antibody were diluted in sterile saline and administered intravenously via tail vein (IV) injection at 10 mg / kg every four days (Q4D - Figure 30) or once a week (QW - Figure 31). Tumor xenografts were measured with calipers three times a week and tumor volume was calculated in mm by multiplying height x width x length. 3 The tumor volume was measured in units of 1000 mg / kg. Mice were treated for 21 to 36 days. At the end of the study, animals were euthanized, and tumor tissue was excised and sectioned for storage as snap-frozen or formalin-fixed, paraffin-embedded (FFPE) tissue for biomarker analysis. All animal work was performed under 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 expressed as the mean volume for each group. Error bars represent the standard error of the mean (SE).

[0338] The results of the xenograft assay are shown in Figures 30 and 31. As shown in Figure 30, treatment with AB1-11 or AB3-7 every four days resulted in a reduction in tumor volume or UMUC4 tumors compared to control-treated mice. Both humanized antibodies achieved a 60% reduction in tumor volume compared to control mice by the end of the study period.

[0339] As shown in Figure 31, OV-90 tumor volumes in mice treated with either humanized antibody were smaller than those in control-treated mice. By the end of the study, AB1-11 achieved a 33% reduction in tumor volume compared to control mice, and AB3-7 achieved a 16% reduction.

[0340] These data support that the humanized antibodies maintain the therapeutic efficacy initially observed with the corresponding murine antibodies.

[0341] Example 13 This example demonstrates antibody drug conjugates tested in vivo.

[0342] Conjugates containing MMAE and humanized AB1-11 were prepared and tested in vivo essentially as described in Example 6 using xenograft mice injected with the OV-90 ovarian cancer cell line expressing CLDN6. In this experiment, CD-1 nude mice (8 mice per group) received subcutaneous injections of OV-90 cells into the right flank. Tumors were 150-300 mm 3 Once tumors reached an average size of 1000 mg / kg, mice were randomized into treatment groups. For treatment, mice received weekly tail vein injections of (1) 10 mg / kg of humanized AB1-11 antibody, (2) 10 mg / kg of human IgG control antibody, (3) 5 mg / kg of a non-targeted conjugate containing MMAE without AB1-11, and (4) 10 mg / kg of a targeted antibody-drug conjugate (ADC) containing MMAE and AB1-11. The non-targeted conjugate contained MMAE conjugated to a non-targeted human IgG control antibody. Tumor xenografts were measured with calipers three times weekly and tumor volume was calculated in mm by multiplying height x width x length. 3 The tumor volume was measured in units of 0.01 mg / kg. Mice were treated for 20 to 36 days. At the end of the study, animals were euthanized, and tumor tissue was excised and sectioned for storage as snap-frozen or formalin-fixed, paraffin-embedded (FFPE) tissue for biomarker analysis. All animal work was performed under 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 expressed as the mean volume for each group. Error bars represent the standard error of the mean (SE).

[0343] As shown in Figure 32, administration of the targeted ADC resulted in a substantial reduction in tumor volume. By the end of the study, mice treated with the targeted ADC showed a greater than 70% reduction in tumor volume compared to control-treated mice.

[0344] The same in vivo analysis was performed using mice subcutaneously injected with UMUC4 bladder cancer cells instead of OV-90 cells. The following treatments were administered weekly via tail vein injection: (1) 10 mg / kg humanized AB1-11 antibody, (2) 10 mg / kg human IgG control antibody, (3) 5 mg / kg non-targeted conjugate containing MMAE without AB1-11, and (4) 10 mg / kg targeted antibody-drug conjugate (ADC) containing MMAE and AB1-11. The non-targeted conjugate contained MMAE conjugated to a non-targeted human IgG control antibody. The targeted conjugate was administered three times in total, and the other treatments were administered weekly for a total of five times. Tumor volume measurements were performed as described above, except that measurements were taken in the targeted ADC-treated mice for up to 80 days.

[0345] As shown in Figure 33, administration of targeted ADCs increased the 3 From almost 0 mm 3 The results demonstrate a reduction in tumor volume up to day 80 of the study, or approximately 60 days after the final ADC treatment.

[0346] These data support that antibody drug conjugates comprising the antibodies of the present disclosure are effective in reducing tumor size and treating cancer.

[0347] Example 14 This example demonstrates the in vitro characterization of CLDN6 ADCs.

[0348] The humanized AB3-7 antibody (also referred to as AB23; see, e.g., Table 8) was used to generate ADCs containing various linker-drug combinations: (a) VC-PAB-MMAE, (b) GGFG-MMAE, (c) CL2A-SN38, (d) GGFG-Dxd, and (e) VC-PAB-Dxd. The ADCs were prepared at WuXi Biologics (Shanghai, China).

[0349] Figures 34A-34H show biochemical characterization of CLDN6 ADCs, including AB3-7. Figure 34A is a table summarizing the biochemical properties of seven CLDN6 ADCs, including AB3-7. The ADC labeled D4 refers to a substantially homogeneous conjugate containing a significant number of antibodies, with four drugs conjugated per antibody (D4 Technology, a WuXi Biologics technology). The ADC labeled CTRL refers to a conventional heterogeneous conjugate (conventional) containing a Gaussian distribution of drug numbers per antibody. The ADCs contained low proportions of unconjugated antibody, high molecular weight (HMW) species, and unconjugated free drug. The ADCs also contained low levels of endotoxin, making them suitable for in vivo testing. Figures 34B-34H show HIC-HPLC chromatograms showing the relative abundance of antibodies conjugated with different numbers of drugs for each ADC. For example, the peak area under D0 indicates the relative abundance of unconjugated AB3-7. Similarly, the peak areas under D1, D2, D3...D8 indicate the relative abundance of AB3-7 antibody conjugated to 1, 2, 3...8 drugs per antibody.

[0350] Figure 35 shows that the molecular integrity of CLDN6 ADCs, including AB3-7, was not altered by conjugation. The molecular integrity of the CLDN6 ADCs and unconjugated AB3-7 antibody was assessed by NativePAGE (NativePAGE 4-16% Bis-Tris protein gels (catalog no. BN1002BOX, ThermoFisher)). Native PAGE separates proteins according to the net charge, size, and shape of their native conformation. Under native conditions, all seven CLDN6 ADCs remained structurally intact and showed no signs of degradation compared to the unconjugated AB3-7 antibody. Therefore, conjugation does not alter the molecular integrity of the AB3-7 antibody.

[0351] Figure 36 shows that conjugation does not affect the binding affinity of the AB3-7 antibody. Flow cytometry demonstrates that the CLDN6 ADC containing AB3-7 retains specific and high binding affinity for CLDN6. ADC binding activity was analyzed by flow cytometry in three cell lines: (a) the UMUC4 bladder cancer cell line (naturally expressing CLDN6), (b) HEK293T CLDN6-mGFP A11 (engineered to artificially overexpress (OE) CLDN6), and (c) the M202 melanoma cell line (CLDN6-negative cells). Specifically, the ADC was incubated with approximately 150,000 cells in 50 μl of 2% FBS / PBS at 4°C for 30 minutes, followed by washing with 2% FBS / PBS. The ADC was then incubated with Alexa Fluor® 647 anti-human IgG Fc antibody (Biolegend, 409320) at 4°C for 30 minutes. Binding was measured on a BD Accuri™ C6 flow cytometer. Seven different CLDN6 ADCs showed similar binding affinities by flow cytometry compared to their unconjugated antibody AB3-7 and CLDN6-positive cell lines (UMUC4 cells and HEK293T CLDN6-m...

Claims

1. a. a 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 has about or at least 70% sequence identity; b. a 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 has about or at least 70% sequence identity; c. A 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 has about or at least 70% sequence identity; d. a 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 has about or at least 70% sequence identity; e. a 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 has about or at least 70% sequence identity; f. a 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 about or at least 70% sequence identity; and / or g. A combination of two or more of (a) to (f) 1. An antigen-binding protein comprising:

2. 2. The antigen binding protein of claim 1, wherein the variant sequence has at least about 80% or about or at least 85% sequence identity.

3. 3. The antigen binding protein of claim 2, wherein the variant sequence has at least about 90% sequence identity or about or at least 95% sequence identity.

4. a. the antigen binding protein binds to human claudin 6 (CLDN6) protein (SEQ ID NO: 200); b. The antigen binding protein binds to extracellular loop 2 (EL2) of the extracellular domain (ECD) of CLDN6, but does not bind to extracellular loop 1 (EL1) of the ECD of CLDN6; c. does not bind to claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9), and inhibits the binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells by less than about 1200 nM; or d. A combination of these 4. The antigen-binding protein of any one of claims 1 to 3, wherein

5. 5. The antigen-binding protein of any one of claims 1 to 4, which binds to an epitope within the amino acid sequence WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2).

6. 6. The antigen-binding protein of claim 5, which binds to the amino acid sequence of TAHAIIRDFYNPL (SEQ ID NO: 3) or LVAEAQKREL (SEQ ID NO: 4) of CLDN6.

7. 7. The antigen-binding protein of any one of claims 1 to 6, which does not bind to one or more of claudin 3 (CLDN3), claudin 4 (CLDN4), and claudin 9 (CLDN9).

8. 8. The antigen-binding protein of claim 7, which does not bind to CLDN3.

9. 9. The antigen-binding protein of claim 8, which binds to CLDN6, CLDN4, and CLDN9.

10. 9. The antigen-binding protein of claim 8, which does not bind to CLDN9.

11. 11. The antigen-binding protein of claim 10, which binds to CLDN6 and CLDN4.

12. 9. The antigen-binding protein of claim 8, which does not bind to CLDN4.

13. 13. The antigen-binding protein of claim 12, which binds to CLDN6 and CLDN9.

14. a light chain CDR1 amino acid sequence of SEQ ID NO: 449, a light chain CDR2 amino acid sequence or SEQ ID NO: 450, and a light chain CDR3 amino acid sequence or SEQ ID NO: 451; 10. An antigen-binding protein according to any one of the preceding claims, comprising 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.

15. a light chain CDR1 amino acid sequence of SEQ ID NO: 476, a light chain CDR2 amino acid sequence or SEQ ID NO: 477, and a light chain CDR3 amino acid sequence or SEQ ID NO: 454; 10. An antigen binding protein according to any one of the preceding claims, comprising 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.

16. a. SEQ ID NOs: 449-451 and 504-506, and b. six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 476, 477, 454, and 507-509 16. The antigen-binding protein of any one of claims 11 to 15, comprising:

17. a. a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 490-503, or a heavy chain variable region amino acid sequence designated as S1-S12 in FIG. 22, or a variant thereof, which differs by only one or two amino acids or which shares about or at least 70% sequence identity; or b. A light chain variable region amino acid sequence of any one of SEQ ID NOs: 380-383, 388-390, 479, and 481, or a light chain variable region amino acid sequence designated as S1-S12 in FIG. 22, or a variant thereof, which differs by only one or two amino acids or which shares about or at least 70% sequence identity; or c. Both (a) and (b) 17. The antigen-binding protein of any one of claims 11 to 16, comprising:

18. 18. The antigen binding protein of claim 17, wherein the variant sequence has at least about 80% or at least about 85% sequence identity.

19. 19. The antigen binding protein of claim 18, wherein the variant sequence has at least about 90% or at least about 95% sequence identity.

20. A pair of amino acid sequences, i.e. a. SEQ ID NOs: 389 and 490; b. SEQ ID NOs: 389 and 491; c. SEQ ID NOs: 389 and 492; d. SEQ ID NOs: 389 and 493; e. SEQ ID NOs: 389 and 494; f. SEQ ID NOs: 389 and 495; g. SEQ ID NOs: 383 and 496; h. SEQ ID NOs: 383 and 497; i. SEQ ID NOs: 383 and 498; j. SEQ ID NOs: 383 and 499; k. SEQ ID NOs: 383 and 500; l. SEQ ID NOs: 383 and 501; m. SEQ ID NOs: 383 and 503; n. SEQ ID NOs: 389 and 502; o. the sequence of the heavy chain variable region designated as S1 in FIG. 22 and the sequence of the light chain variable region designated as S1 in FIG. 22; p. the sequence of the heavy chain variable region shown as S2 in FIG. 22 and the sequence of the light chain variable region shown as S2 in FIG. 22; q. the sequence of the heavy chain variable region designated as S3 in FIG. 22 and the sequence of the light chain variable region designated as S3 in FIG. 22; r. the sequence of the heavy chain variable region designated as S4 in FIG. 22 and the sequence of the light chain variable region designated as S4 in FIG. 22; s. the sequence of the heavy chain variable region designated as S5 in FIG. 22 and the sequence of the light chain variable region designated as S5 in FIG. 22; t. the sequence of the heavy chain variable region shown as S6 in Figure 22 and the sequence of the light chain variable region shown as S6 in Figure 22; u. the sequence of the heavy chain variable region designated as S7 in FIG. 22 and the sequence of the light chain variable region designated as S7 in FIG. 22; v. the heavy chain variable region sequence designated as S78 in FIG. 22 and the light chain variable region sequence designated as S8 in FIG. 22; w. the sequence of the heavy chain variable region designated as S89 in FIG. 22 and the sequence of the light chain variable region designated as S9 in FIG. 22; x. the sequence of the heavy chain variable region designated as S910 in FIG. 22 and the sequence of the light chain variable region designated as S10 in FIG. 22; y. the heavy chain variable region sequence designated as S11 in FIG. 22 and the light chain variable region sequence designated as S11 in FIG. 22; or z. The sequence of the heavy chain variable region shown as S12 in Figure 22 and the sequence of the light chain variable region shown as S12 in Figure 22 20. The antigen-binding protein of any one of claims 17 to 19, comprising:

21. 10. An antigen-binding protein according to any one of the preceding claims which is an antibody.

22. 22. The antigen-binding protein of claim 21, which is a monoclonal antibody.

23. 23. The antigen-binding protein of claim 21 or 22, which is an IgG.

24. 10. The antigen binding protein of any one of the preceding claims, which inhibits colony growth by at least about 50% in a soft agar 3D growth assay.

25. 10. An antigen-binding protein according to any one of the preceding claims which inhibits tumour growth in xenograft mice injected with human cancer cells.

26. 26. The antigen binding protein of claim 25, which inhibits tumor growth in xenograft mice injected with ovarian, melanoma, bladder, or endometrial cancer cells.

27. 27. The antigen binding protein of claim 26, which inhibits tumor growth by at least 50% in xenograft mice injected with ovarian, bladder, or endometrial cancer cells.

28. a. a 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 has about or at least 70% sequence identity; b. a 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 has about or at least 70% sequence identity; c. A 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 has about or at least 70% sequence identity; d. a 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 has about or at least 70% sequence identity; e. a 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 has about or at least 70% sequence identity; f. a 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 about or at least 70% sequence identity; or g. A combination of two or more of (a) to (f) 1. An antigen-binding protein comprising:

29. a. SEQ ID NOs: 449-451 and 504-506, and b. six CDR amino acid sequences selected from the group consisting of SEQ ID NOs: 476, 477, 454, and 507-509 1. An antigen-binding protein comprising:

30. a. a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 490-503, or a heavy chain variable region amino acid sequence designated as S1-S12 in FIG. 22, or a variant thereof, which differs by only one or two amino acids or which shares about or at least 70% sequence identity; or b. A light chain variable region amino acid sequence of any one of SEQ ID NOs: 380-383, 388-390, 479, and 481, or a light chain variable region amino acid sequence designated as S1-S12 in FIG. 22, or a variant thereof, which differs by only one or two amino acids or which shares about or at least 70% sequence identity; or c. Both (a) and (b) 1. An antigen-binding protein comprising:

31. 31. The antigen binding protein of claim 30, wherein the variant sequence has at least about 85% sequence identity, or about 90% or about 95% sequence identity.

32. a. SEQ ID NOs: 389 and 490; b. SEQ ID NOs: 389 and 491; c. SEQ ID NOs: 389 and 492; d. SEQ ID NOs: 389 and 493; e. SEQ ID NOs: 389 and 494; f. SEQ ID NOs: 389 and 495; g. SEQ ID NOs: 383 and 496; h. SEQ ID NOs: 383 and 497; i. SEQ ID NOs: 383 and 498; j. SEQ ID NOs: 383 and 499; k. SEQ ID NOs: 383 and 500; l. SEQ ID NOs: 383 and 501; m. SEQ ID NOs: 383 and 503; n. SEQ ID NOs: 389 and 502; o. the sequence of the heavy chain variable region designated as S1 in FIG. 22 and the sequence of the light chain variable region designated as S1 in FIG. 22; p. the sequence of the heavy chain variable region shown as S2 in FIG. 22 and the sequence of the light chain variable region shown as S2 in FIG. 22; q. the sequence of the heavy chain variable region designated as S3 in FIG. 22 and the sequence of the light chain variable region designated as S3 in FIG. 22; r. the sequence of the heavy chain variable region designated as S4 in FIG. 22 and the sequence of the light chain variable region designated as S4 in FIG. 22; s. the sequence of the heavy chain variable region designated as S5 in FIG. 22 and the sequence of the light chain variable region designated as S5 in FIG. 22; t. the sequence of the heavy chain variable region shown as S6 in Figure 22 and the sequence of the light chain variable region shown as S6 in Figure 22; u. the sequence of the heavy chain variable region designated as S7 in FIG. 22 and the sequence of the light chain variable region designated as S7 in FIG. 22; v. the heavy chain variable region sequence designated as S78 in FIG. 22 and the light chain variable region sequence designated as S8 in FIG. 22; w. the sequence of the heavy chain variable region designated as S89 in FIG. 22 and the sequence of the light chain variable region designated as S9 in FIG. 22; x. the sequence of the heavy chain variable region designated as S910 in FIG. 22 and the sequence of the light chain variable region designated as S10 in FIG. 22; y. the heavy chain variable region sequence designated as S11 in FIG. 22 and the light chain variable region sequence designated as S11 in FIG. 22; or z. The sequence of the heavy chain variable region shown as S12 in Figure 22 and the sequence of the light chain variable region shown as S12 in Figure 22 An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of:

33. a. a heavy chain variable region amino acid sequence set forth as SEQ ID NO: 510 or 513, or as set forth in Figure 23 or Figure 25, or a variant thereof, which differs by only one or two amino acids or which has about or at least 70% sequence identity; or b. a light chain variable region amino acid sequence set forth as SEQ ID NO: 511 or 512, or as set forth in Figure 24 or Figure 26, or a variant thereof, which differs by only one or two amino acids or which shares about or at least 70% sequence identity; or c. Both (a) and (b) 1. An antigen-binding protein comprising:

34. 34. The antigen binding protein of claim 33, wherein the variant sequence has at least about 85% sequence identity.

35. 35. The antigen binding protein of claim 34, wherein the variant sequence has at least about 90% or about 95% sequence identity.

36. The pair is a. a heavy chain variable region amino acid sequence set forth as SEQ ID NO:510 and a light chain variable region amino acid sequence set forth as SEQ ID NO:511, or a variant thereof, which differs by only 1-5 amino acids or has about or at least 70% sequence identity, optionally wherein the 1-5 differing amino acids are as set forth in Figure 23 for the heavy chain or Figure 24 for the light chain; b. a heavy chain variable region amino acid sequence set forth as SEQ ID NO:513 and a light chain variable region amino acid sequence set forth as SEQ ID NO:512, or a variant thereof, which differs by only 1 to 5 amino acids or which has about or at least 70% sequence identity, or optionally, wherein the 1 to 5 amino acids that differ are those shown in Figure 25 for the heavy chain or those shown in Figure 26 for the light chain. An antigen-binding protein comprising a pair of amino acid sequences comprising:

37. 10. The antigen-binding protein of any one of the preceding claims, comprising an Fc polypeptide comprising defucosylated glycans.

38. 10. A conjugate comprising an antigen-binding protein as defined in any one of the preceding claims or as described herein.

39. 39. The conjugate of claim 38, wherein the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 387 and SEQ ID NO:

389.

40. 39. The conjugate of claim 38, wherein the antigen binding protein comprises the amino acid sequences set forth in SEQ ID NO: 379 and SEQ ID NO:

383.

41. The conjugate of any one of claims 38 to 40, comprising a cytotoxic or chemotherapeutic drug.

42. 42. The conjugate of claim 41, wherein the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking tubulin polymerization.

43. 43. The conjugate of claim 42, wherein the antimitotic agent is an auristatin.

44. 44. The conjugate of claim 43, wherein the auristatin is MMAE.

45. 45. The conjugate of any one of claims 38 to 44, wherein the agent is attached to the antigen-binding protein via a cleavable linker.

46. 46. The conjugate of claim 45, wherein the cleavable linker is VC-PAB-MMAE.

47. The conjugate of any one of claims 38 to 46, wherein the antigen-binding protein is an antibody.

48. 48. The conjugate of claim 47, wherein the antibody is a monoclonal antibody, and optionally the monoclonal antibody is an IgG antibody.

49. 49. The conjugate of claim 47 or 48, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

50. 50. The conjugate of any one of claims 38 to 49, wherein the average number of drug units bound per antibody is in the range of 1 to 8, preferably the average number of drug units bound per antibody is in the range of 3 to 8.

51. The complex of any one of claims 38 to 50, wherein the complex is a heterogeneous complex.

52. The complex of any one of claims 38 to 50, wherein the complex is a homogeneous complex.

53. 53. The conjugate of any one of claims 38 to 50 and 52, wherein the agent is attached at a specific site on the antigen-binding protein.

54. 54. The conjugate of claim 53, wherein the specific site is an unpaired cysteine residue.

55. The complex of any one of claims 47 to 54, wherein the complex comprises a polypeptide comprising the amino acid sequences set forth in SEQ ID NO: 387 and SEQ ID NO: 389 bound to MC-VC-PAB-MMAE.

56. The complex of any one of claims 47 to 54, wherein the complex comprises a polypeptide comprising the amino acid sequences set forth in SEQ ID NO: 379 and SEQ ID NO: 383 bound to MC-VC-PAB-MMAE.

57. A fusion protein comprising an antigen-binding protein according to any one of the preceding claims.

58. 58. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to any one of the preceding claims, a conjugate according to claims 38 to 56, or a fusion protein according to claim 57.

59. A vector comprising the nucleic acid of claim 58.

60. 60. A host cell comprising the nucleic acid of claim 58 or the vector of claim 59.

61. 1. A method for producing an antigen-binding protein that binds to claudin 6 (CLDN6) protein, comprising: (i) culturing a host cell according to claim 60 in a cell culture medium, said host cell comprising a nucleic acid comprising a nucleotide sequence encoding the antigen binding protein of any one of the preceding claims; and (ii) recovering the antigen-binding protein from the cell culture medium. The method comprising:

62. 1. A method for producing a fusion protein comprising an antigen binding protein that binds to claudin 6 (CLDN6) protein, comprising: (i) culturing a host cell of claim 60 in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding the fusion protein of claim 43; and (ii) recovering the fusion protein from the cell culture medium. The method comprising:

63. 60. A method of producing a pharmaceutical composition comprising combining an antigen binding protein of any one of claims 1 to 37, a conjugate of any one of claims 38 to 56, a fusion protein of claim 57, a nucleic acid of claim 58, a vector of claim 59, a host cell of claim 60, or a combination thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

64. 61. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 37, a conjugate according to any one of claims 38 to 56, a fusion protein according to claim 57, a nucleic acid according to claim 58, a vector according to claim 59, a host cell according to claim 60, and a pharmaceutically acceptable carrier, diluent or excipient.

65. 65. A method for treating a subject having a CLDN6-expressing cancer, comprising administering to the subject a pharmaceutical composition of claim 64 in an amount effective to treat the cancer.

66. 65. A method of inhibiting tumor growth in a subject, comprising administering to the subject the pharmaceutical composition of claim 64 in an amount effective to inhibit tumor growth.

67. 65. A method of reducing tumor size in a subject, comprising administering to the subject the pharmaceutical composition of claim 64 in an amount effective to reduce tumor size.

68. 65. A method of preventing recurrence of cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 64 in an amount effective to prevent recurrence of cancer.

69. A method for treating cancer in a subject diagnosed as having low overexpression of CLDN6, comprising administering to the subject the pharmaceutical composition of claim 64 in an amount effective to prevent recurrence of the cancer.

70. 70. The method of any one of claims 65 to 69, wherein said administering induces apoptosis in tumor cells.

71. The method of any one of claims 65 to 69, wherein the administration induces apoptosis in CLDN6-expressing cells.

72. A method for detecting claudin 6 (CLDN6) in a sample, comprising: contacting the sample with an antigen-binding protein according to any one of claims 1 to 37, a complex according to any one of claims 38 to 56, or a fusion protein according to claim 57; and Evaluating an immune complex comprising an antigen-binding protein, complex, or fusion protein that binds to the CLDN6. The method comprising:

73. 1. A method for diagnosing claudin 6 (CLDN6)-positive cancer in a subject, comprising: contacting a biological sample comprising cells or tissue obtained from said subject with an antigen-binding protein of any one of claims 1 to 37, a conjugate of any one of claims 38 to 56, or a fusion protein of claim 57; and Evaluating an immune complex comprising an antigen-binding protein, complex, or fusion protein that binds to the CLDN6. The method comprising:

Images