Bivalent antibodies comprising Anti-EGFR and Anti-CDCP1 antibody regions

Abstract

The present disclosure relates to bispecific antibodies comprising anti-CDCPl antibody regions and anti-EGFR antibody regions, drug conjugates comprised of one or morecytotoxic agents linked to the bispecific, and methods of making and using these molecules for the treatment of cancer and other diseases. Also disclosed here are monospecific antibodies comprising anti- CDCPl antibody regions. The anti-CDCPl antibody region comprises a single variable domain on a heavy chain (VHH) or a fragment thereof.

Description

[0001] BIVALENT ANTIBODIES COMPRISING ANTI-EGFR AND ANTI-CDCP1 ANTIBODY REGIONS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 734,752 filed December 17, 202 and U.S. Provisional Patent Application No. 63 / 791,484, filed April 19, 2025, each of which is incorporated by reference herein in its entirety.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted in .XML format via EFS-WEB and is hereby incorporated by reference in its entirety. Said .XML copy, created on December 17, 2025, is named 062460-502001 WO. xml and is 1.18 megabytes in size

[0006] BACKGROUND

[0007] Current approaches to target EGFR may provide limited clinical benefit due to off-tumor on-target toxicity and acquired resistance. There is a need for cis-acting bispecific antibodies that leverage the shared biology of inherently proximal proteins on tumor cells for improved therapeutic activity. The present disclosure solves this unmet need.

[0008] SUMMARY

[0009] The present disclosure relates to a bispecific antibody comprising an anti-EGFR antibody region and an anti-CDCPl antibody region capable of binding EGFR and CDCP1 antigens expressed on the surface of a target cell (e.g., a cancer cell). Applicant has demonstrated that the bispecific antibody provided herein is particularly effective in internalizing into cancer cells. The bispecific antibody therefore is contemplated to be effective for treating diseases, including cancers. Additionally, the present disclosure relates to a monospecific antibody comprising an anti- CDCPl antibody region capable of binding CDCP1 antigens expressed on the surface of a target cell (e.g., a cancer cell).

[0010] An aspect of the present disclosure is a bispecific antibody comprising at least one anti- EGFR antibody region and at least one anti-CDCPl antibody region, wherein the anti-CDCPl antibody region comprises a single variable domain on a heavy chain (VHH) or a fragment thereof. In embodiments, the bispecific antibody comprises an antibody region comprising a sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 1 to Table 3 and a sequence that is at least 90% identical (e.g, 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g., SEQ ID NO: 1291.

[0011] In embodiments, an anti-EGFR antibody region is configured as a Fab, a single-chain variable fragment (scFv), and / or a single variable domain on a heavy chain (VHH). In embodiments, an anti-CDCPl antibody region is configured as a single variable domain on a heavy chain (VHH). In embodiments, the bispecific antibody includes one or more Fabs, includes one or more scFvs, and / or includes one or more VHHs.

[0012] In embodiments, the bispecific antibody comprises one or more antibody regions identified by a PROT ID number in at least one of Table 8 to Table 12.

[0013] In embodiments, the bispecific antibody is conjugated with one or more cytotoxic agents, thereby forming an antibody drug conjugate (ADC). In some case, the ADC comprises two (or more copies) of one drug and / or the ADC comprises one or more copies of two different drugs.

[0014] In embodiments, the one or more cytotoxic agents are conjugated to a constant region of the bispecific antibody, e.g., to an Fc region. In some cases, the drug is conjugated to an amino acid position of the bispecific antibody’s Fc region that has been substituted with a cysteine, e.g., a 375C, 360C, 149C, and / or a 140C substituted position. In various cases, the bispecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one SEQ ID NO: 1309-1316.

[0015] In embodiments, the one or more cytotoxic agents include a microtubule-disrupting drug (e.g., an anti-tubulin inhibitor), DNA-modifying drug, RNA-modifying drug, conjugated toxin, a small molecule, radioisotope, or a drug. In some cases, the one or more cytotoxic agents include monomethyl auristatin E (MMAE) or exatecan. In some cases, the MMAE payload is linked to the antibody with a val-cit-PABC linker to form the vcMMAE drug linker. In some cases, the exatecan payload is linked to the antibody with a GGFG linker to form the GGFG-exatecan drug linker. Examples of other conjugatable drugs include monomethyl auristatin F (MMAF) or other auristatins; DM1, DM4, or other maytansinoids, SN-38, Dxd or other camptothecin derivatives; pyrrolobenzodiazepines (PBDs) or other DNA alkylators; duocarmycins, NAMPT inhibitors, TLR agonists, STING agonists, small molecule cell cycle inhibitors such as cell cycle blockers (e.g., Cdk4 / 6 inhibitors including Palbociclib (Ibrance), Ribociclib (Kisqali), and Abemaciclib (Verzenio)) and inhibitors of anti-apoptotic proteins (e.g., Bcl2 family proteins, including Bcl-2, Bcl-XL and Mell inhibitors, Inhibitor of Apoptosis Proteins (IAPS), and Caspase Inhibitors), and / or other common ADC payloads. These payloads may be used in combination on the same antibody.

[0016] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 445 to SEQ ID NO: 450, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0017] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 445 to SEQ ID NO: 450, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g, 95%, 99%, or 100% identical) SEQ ID NO: 1291.

[0018] In embodiments, the bi specific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 630, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g, 95%, 99%, or 100% identical) SEQ ID NO: 1291.

[0019] In embodiments, the bi specific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g, 95%, 99%, or 100% identical) to SEQ ID NO: 630, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0020] Another aspect of the present disclosure is a method of killing a cancer cell, the method comprising contacting the cell with an effective amount of a herein-disclosed bispecific antibody. The method may be in vitro, ex vivo, or in vivo.

[0021] In an aspect, the present disclosure provides a pharmaceutical composition comprising a herein-disclosed bispecific antibody and a pharmaceutically acceptable carrier, diluent, or excipient.

[0022] In an additional aspect, the present disclosure provides a polynucleotide or plurality of polynucleotides encoding a herein-disclosed bispecific antibody. In yet an additional aspect, the present disclosure provides a vector comprising the polynucleotide or plurality of a herein-disclosed polynucleotides.

[0023] In a further aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of any herein-disclosed pharmaceutical composition and administering to the subject an effective amount of a standard of care therapy for the cancer. The standard of care therapy for the cancer may be administered before, simultaneously with, or after the pharmaceutical composition is administered.

[0024] An aspect of the present disclosure is a monospecific antibody comprising at least one anti- CDCP1 antibody region, wherein the anti-CDCPl antibody region comprises a single variable domain on a heavy chain (VHH) or a fragment thereof.

[0025] In embodiments, the monospecific antibody comprises an antibody region comprising a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 4, e.g, SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g., SEQ ID NO: 1291.

[0026] In embodiments, an anti-CDCPl antibody region is configured as a single variable domain on a heavy chain (VHH). In embodiments, the monospecific antibody includes one or more VHHs.

[0027] In embodiments, the monospecific antibody comprises one or more antibody regions identified by a PROT ID number in Table.

[0028] In embodiments, the monospecific antibody is conjugated with one or more cytotoxic agents, thereby forming an antibody drug conjugate (ADC). In some case, the ADC comprises two (or more copies) of one drug and / or the ADC comprises one or more copies of two different drugs.

[0029] In embodiments, the one or more cytotoxic agents are conjugated to a constant region of the monospecific antibody, e.g., to an Fc region. In some cases, the drug is conjugated to an amino acid position of the monospecific antibody’s Fc region that has been substituted with a cysteine, e.g., a 375C, 360C, 149C, and / or a 140C substituted position. In various cases, the monospecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one SEQ ID NO: 1309-1316.

[0030] In embodiments, the one or more cytotoxic agents include a microtubule-disrupting drug (e.g., an anti-tubulin inhibitor), =, DNA-modifying drug, RNA-modifying drug, conjugated toxin, a small molecule, radioisotope, or a drug. In some cases, the one or more cytotoxic agents include monomethyl auristatin E (MMAE) or exatecan. In some cases, the MMAE payload is linked to the antibody with a val-cit-PABC linker to form the vcMMAE drug linker. In some cases, the exatecan payload is linked to the antibody with a GGFG linker to form the GGFG-exatecan drug linker. Examples of other conjugatable drugs include monomethyl auristatin F (MMAF) or other auristatins; DM1, DM4, or other maytansinoids, SN-38, Dxd or other camptothecin derivatives; pyrrolobenzodiazepines (PBDs) or other DNA alkylators; duocarmycins, NAMPT inhibitors, TLR agonists, STING agonists, small molecule cell cycle inhibitors such as cell cycle blockers (e.g., Cdk4 / 6 inhibitors including Palbociclib (Ibrance), Ribociclib (Kisqali), and Abemaciclib (Verzenio)) and inhibitors of anti-apoptotic proteins e.g., Bcl2 family proteins, including Bcl-2, Bcl-XL and Mell inhibitors, Inhibitor of Apoptosis Proteins (IAPS), and Caspase Inhibitors), and / or other common ADC payloads. These payloads may be used in combination on the same antibody.

[0031] Another aspect of the present disclosure is a method of killing a cancer cell, the method comprising contacting the cell with an effective amount of a herein-disclosed monospecific antibody. The method may be in vitro, ex vivo, or in vivo.

[0032] In an aspect, the present disclosure provides a pharmaceutical composition comprising a herein-disclosed monospecific antibody and a pharmaceutically acceptable carrier, diluent, or excipient.

[0033] In an additional aspect, the present disclosure provides a polynucleotide or plurality of polynucleotides encoding a herein-disclosed monospecific antibody.

[0034] In yet an additional aspect, the present disclosure provides a vector comprising the polynucleotide or plurality of a herein-disclosed polynucleotides.

[0035] In a further aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of any herein-disclosed pharmaceutical composition and administering to the subject an effective amount of a standard of care therapy for the cancer. The standard of care therapy for the cancer may be administered before, simultaneously with, or after the pharmaceutical composition is administered.

[0036] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

[0037] BRIEF DESCRIPTION OF THE FIGURES

[0038] FIG. 1 is an alignment of anti-CDCPl antibody variable domain sequences.

[0039] FIG. 2A is an alignment of anti-EGFR antibody heavy chain variable domain sequences.

[0040] FIG. 2B is an alignment of anti-EGFR antibody light chain variable domain sequences. FIG. 3Ato FIG. 3E are diagrams of illustrative formats for EGFR x CDCP 1 bi specific antibodies. The diagrams additionally relate to biparatopic antibodies, where the antibody binds to two distinct epitopes on the same antigens, or in the case for FIG. 3C, when the bispecific in a biparatopic antibody, the antibody binds to two epitopes of EGFR and two epitopes of CDCP1 FIG. 3E shows an illustrative antibody drug conjugate (ADC); here, the drug is conjugated to the bispecific of FIG. 3A; however, a drug can be conjugated to any bispecific antibody format disclosed herein.

[0041] FIG. 4 is a plot of illustrative CDCP1 x EGFR bispecific antibodies analyzed in antibody internalization assays using SKHEP1 cells that either express EGFR and CDCP1 (y-axis) or only express EGFR (x-axis).

[0042] FIG. 5 is a plot of internalization activity of illustrative CDCP1 x Fc antibodies corresponding to distinct CDCP1 epitope bins analyzed in an antibody internalization assay.

[0043] FIG. 6 is a plot of the activity of illustrative CDCP1 x EGFR bispecific antibodies in a cellviability assay of primary keratinocytes (x-axis) compared to the mean of three cancer cell lines (y-axis).

[0044] FIG. 7 shows an isoaffinity plot of CDCP1 monospecific antibodies binding to recombinant human CDCP1 ECD by biolayer interferometry (BLI). CDCP1 binders contained in the selected bispecifics shown in Table 10 are in filled symbols.

[0045] FIG. 8A to FIG. 8D show plots of selected bispecific antibody internalization in WT and CDCP1 knockout SKHEP1 cells for four different EGFR binding arms.

[0046] FIG. 9 A to FIG. 9D show activity in cell viability assays of bispecifics PROT IDs from Table 10 as well as a monovalent EGFR control (open circles) generated using C046 and C065 as the EGFR binding arm coupled to Protein-A MMAE (Monomethyl Auristatin E, is a synthetic antineoplastic agent).

[0047] FIG. 9E to FIG. 9H shows activity in cell viability assays of bispecific PROT IDs from Table 10 as well as a monovalent EGFR control (open circles) generated using C230 and C047 as the EGFR binding arm coupled to Protein-A MMAE.

[0048] FIG.10A to FIG. 10D shows internalization activity of bispecific antibodies (solid and open circles) in WT and CDCP1 knockout SKHEP1 cells with matched monovalent EGFR (solid and open triangles) and CDCP1 (solid and open squares) controls for illustrative bispecific antibodies. FIG. 11A to FIG. 11D shows the internalization activity of illustrative selectively internalizing bispecific antibodies in SW48, HCC827, H1975 and HT29 cell lines, as indicated, with matched monovalent EGFRx Fc (diamond symbol) and CDCP1 x Fc controls (open symbols corresponding to matched bispecific closed symbol).

[0049] FIG. 12 shows the internalization activity of illustrative internalizing monovalent CDCP1 antibodies.

[0050] FIG. 13 shows killing activity in cell viability assays using Protein A-MMAE-labeled monovalent EGFR x Fc binders detuned to have varying affinities, measured by alamarBlue.

[0051] FIG. 14 shows killing activity in cell viability assays using Protein A-MMAE-labeled EGFR x CDCP1 bispecific antibodies consisting of detuned EGFR binders, measured by alamarBlue.

[0052] FIG. 15 shows killing activity in cell viability assays using Protein A-MMAE-labeled EGFR x CDCP1 bispecific antibodies with optimized EGFR and CDCP1 sequences compared to single arm controls, measured by alamarBlue.

[0053] FIG. 16 compares killing activity in cell viability assays using Protein A-MMAE-labeled EGFR x CDCP1 bispecific antibodies with a detuned EGFR arm paired to a CDCP1 arm that targets a different epitope, measured by alamarBlue.

[0054] FIG. 17 compares the killing activity in cell viability assays using Protein A-MMAE-labeled EGFR x CDCP1 bispecific antibodies with a detuned EGFR variant as a monovalent binder or paired with CDCP1, relative to the killing activity of a higher affinity EGFR variant as a monovalent binder or paired with CDCP1, measured by alamarBlue.

[0055] FIG. 18A shows graphs of cell viability assays in indicated cell lines using bispecific EGFR x CDCP1 ADCs, generated using knob-in-hole technology and directly conjugated to vcMMAE, monovalent EGFR controls, and a non-targeting control containing various drug-to-antibody ratios (DARs), measured by alamarBlue.

[0056] FIG. 18B shows graphs of cell viability assays in SW48 and SW900 cells comparing an unconjugated EGFR x CDCP1 bispecific antibody to a directly conjugated vcMMAE EGFR x CDCP1 bispecific antibody as measured by CellTiter-Glo.

[0057] FIG. 19 shows graphs of cell viability assays in indicated primary cells using bispecific EGFR x CDCP1 ADCs, monovalent EGFR controls, and a non-targeting control, as measured by alamarBlue.

[0058] FIG. 20A is a western blot depicting that endogenous full-length (FL) CDCP1 is cleaved in the presence of plasmin in SW756 and SKMES1 cells and exists in a cleaved form in HCC827 cells.

[0059] FIG. 20B shows graphs indicating that an EGFRxCDCPl bispecific (C5192) and an anti-CDCPl monovalent antibody (C9373) that targets the distal region of CDCP1 can bind to full length and plasmin cleaved CDCP1 in SW756 and SKMES1 cells and binds to endogenously cleaved CDCP1 in HCC827 cells.

[0060] FIG. 20C shows graphs depicting that bispecific CDCP1 x EGFR ADCs lead to equivalent killing of SW756 cells in the presence or absence of plasmin treatment in a cell viability assay, as measured by alamarBlue.

[0061] FIG. 20D is a graph depicting that bispecific CDCP1 x EGFR ADCs lead to killing HCC827 cells in a cell viability assay, as measured by alamarBlue.

[0062] FIG. 21 shows graphs depicting the comparison of bispecific EGFR x CDCP1 ADCs with different vcMMAE conjugation strategies to kill cancer cells in a cell viability assay using the indicated cancer cell lines, as measured by alamarBlue.

[0063] FIG. 22 is a graph depicting the ability of EGFR x CDCP1 antibodies labeled with anti-human- Fc-MMAE ADCs with different spacer sequences to kill SW48 cells in a cell viability assay as measured by alamarBlue.

[0064] FIG. 23A shows graphs of cell viability assays in the indicated cancer cell lines comparing bispecific EGFR x CDCP 1 ADCs conjugated to vcMMAE drug linkers, as measured by CellTiter- Glo.

[0065] FIG. 23B shows graphs of cell viability assays in the indicated cancer cell lines comparing bispecific EGFR x CDCP1 ADCs conjugated to GGFG-exatecan drug linkers, as measured by CellTiter-Glo.

[0066] FIG. 24 is a graph depicting the ability of sequence optimized bispecific EGFR x CDCP1 antibodies incubated with anti-human-Fc-MMAE, to kill WT (black symbols) or CDCP1 KO (open symbols) cancer cells compared to EGFR arms alone, in a cell viability assay, as measured by alamarBlue.

[0067] FIG. 25 shows graphs depicting the ability of EGFR x CDCP1 bispecific antibodies incubated with anti-human-Fc-MMAE to kill cancer cells expressing EGFR and CDCP1, or corresponding cells where CDCP1 was knocked out, in a cell viability assay, as measured by alamarBlue.

[0068] FIG. 26A to FIG. 26C are immunohistochemistry images and summary graphs comparing expression of EGFR and CDCP1 in human tumors and primary tissues.

[0069] FIG. 27 shows immunohistochemistry images and a summary graph of EGFR and CDCP1 expression in primary adenocarcinoma tissue and corresponding lymph node metastases.

[0070] FIG. 28 are graphs showing the ability of EGFR x CDCP1 bispecific ADCs to internalize in cancer cell lines as compared to mono-specific EGFR and CDCP1 ADCs. FIG. 29A shows graphs depicting the ability of bispecific EGFR x CDCP1 ADCs to kill cancer cells from the indicated cancer cell lines in a cell viability assay as measured by CellTiter-Glo.

[0071] FIG. 29B is a graph depicting the ability of bispecific EGFR x CDCP1 ADCs to kill primary cells in viability assays as measured by CellTiter-Glo.

[0072] FIG. 30 is a graph depicting EGFR and CDCP1 surface expression levels on the indicated cancer cell lines and primary cells.

[0073] FIG 31A is a diagram of signaling molecules illustrating signal transduction pathways downstream of EGFR, and proteins downstream of EGFR that can become phosphorylated following a stimulus of EGF.

[0074] FIG. 31B are Western blots showing that an EGFR x CDCP1 antibody does not block ERK or AKT phosphorylation following treatment of BxPC3 or SW900 cells with EGF.

[0075] FIG. 31C is a Western blot showing that an EGFR x CDCP1 antibody does not block ERK or AKT phosphorylation following treatment of primary human keratinocytes with EGF.

[0076] FIG. 32A is a graph showing that Osimertinib treatment leads to reduced viability of HCC827 cells (circles) but not an HCC827 cell line that is resistant to Osimertinib (squares) FIG. 32B is a graph showing that parental HCC827 cells (HCC827 pm) and Osimertinib-resistant EGFR mutant NSCLC cells (HCC827 OSR1) respond to a bispecific EGFR x CDCP1 ADC of the present disclosure.

[0077] FIG. 33A to FIG. 33E are diagrams of illustrative formats for CDCP1 monospecific antibodies. FIG. 33E shows an illustrative CDCP1 antibody drug conjugate (ADC); here, the drug is conjugated to the monospecific antibody of FIG. 33B; however, a drug can be conjugated to any monospecific antibody format disclosed herein. The diagrams additionally relate to biparatopic antibodies and multiparatopic antibodies where the antibody binds to two (for bi-) or more (for multi-) distinct epitopes on the same antigen, here, CDCP1. Thus, the monospecific antibodies of the present disclosure may have more than one identical CDCP1 binding domains (which bind the same epitope on CDCP1) or may be comprised of multiple unique CDCP1 binding regions that recognize distinct epitopes, to generate a biparatopic or multiparatopic antibody; any such and similar combination of binders is considered in the present disclosure.

[0078] FIG. 34A is an immunohistochemistry image showing that EGFR and CDCP1 are co-expressed in a xenograft tumor derived from the BxPC3 pancreatic adenocarcinoma cell line.

[0079] FIG. 34B is a graph showing that bispecific EGFR x CDCP1 ADC has antitumor activity in a BxPC3 xenograft model. FIG. 34C is a graph showing that a bispecific EGFR x CDCP1 ADC extends survival in mice bearing BxPC3 xenograft tumors.

[0080] FIG. 35 shows images of EGFR and CDCP1 expression by IHC in tumors from various Cell Line- Derived Xenograft (CDX) models.

[0081] FIG. 36A are graphs showing that a bispecific EGFR x CDCP1 ADC has dose-dependent antitumor activity and extends survival in mice in two distinct CDX models.

[0082] FIG. 36B are graphs showing dose-dependent serum exposure of a bispecific EGFR x CDCP1 ADC as either total antibody or total ADC in a CDX model, and the amount of conjugated antibody over time in a CDX model.

[0083] FIG. 36C are graphs showing that a bi specific EGFR x CDCP1 ADC has dose-dependent antitumor activity and extends overall survival in CDX tumor models and is superior to monovalent controls.

[0084] FIG. 36D and FIG. 36E are graphs showing that bispecific EGFR x CDCP1 ADCs with either topoisomerase 1 inhibitor-DAR4 or MMAE-DAR2 have dose-dependent activity and extend survival in mice in two distinct CDX models.

[0085] FIG. 37 are images and a summary table showing EGFR and CDCP1 mRNA expression or by IHC in patient-derived xenograft (PDX) models of non-small cell lung Cancer (NSCLC).

[0086] FIG. 38 are graphs showing that a bispecific EGFR x CDCP1 ADC has antitumor activity against NSCLC PDX tumors in a mouse clinical trial.

[0087] FIG. 39A and FIG. 39B are graphs showing that modulating EGFR affinity in bispecific EGFR x CDCP1 ADCs can influence their activity in cell viability assays, as measured by CellTiter-Glo.

[0088] FIG. 39C are graphs showing binding of two bi specific EGFR x CDCP1 antibodies with different affinities to parental tumor cells or corresponding tumor cells that lack CDCP1 (CDCP1 KO).

[0089] FIG. 40 are graphs showing binding of an EGFR x CDCP1 bispecific antibody by biolayer interferometry (BLI).

[0090] FIG. 41A and FIG. 41B show that engineered cysteine conjugation improves bispecific EGFR x CDCP1 ADC properties.

[0091] DETAILED DESCRIPTION

[0092] Epidermal growth factor receptor “EGFR” is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. EGFR, also known as ErbB 1 or HER1, is a member of the HER family of receptor tyrosine kinases (RTKs). Other HER family members include HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). The HER receptors are Type I transmembrane proteins composed of an extracellular domain (ECD), a transmembrane domain, an intracellular tyrosine kinase domain, and a C-terminal tail. Ligand binding to EGFR leads to homo- or hetero-dimerization, thereby activating the kinase domain via trans-autophosphorylation. This leads to recruitment of adaptor proteins that activate multiple signaling cascades, resulting in enhanced cell proliferation and survival. EGFR is broadly expressed and plays critical roles in various developmental and physiological processes. EGFR is predominantly expressed on cells of epithelial origin, such as those of skin and gastrointestinal tract. Deficient signaling of the EGFR and other receptor tyrosine kinases in humans is associated with diseases such as Alzheimer’s, while over-expression is associated with the development of a wide variety of tumors. Indeed, activating mutations in EGFR or over-expression due to gene amplification have been associated with multiple cancers, leading to the development of multiple EGFR targeting strategies. Interruption of EGFR signaling, either by blocking EGFR binding sites on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can prevent the growth of EGFR-expressing tumors and improve the patient’s condition. Although EGFR is a biologically validated target which is overexpressed and activated in many human cancers and associated with poor prognosis, most current EGFR targeting therapies encompassing multiple modalities have shown limited therapeutic efficacy, frequently due to on-target toxicity in non-malignant tissues where EGFR is expressed. In particular, antibody drug conjugates (ADCs) targeting EGFR exhibit toxicity due to expression on healthy tissue, and EGFR can show heterogeneous tumor expression.

[0093] The CUB domain-containing protein 1 “CDCP1”, also known as CD318 (cluster of differentiation 318), TRASK (Transmembrane and associated with src kinases), and SIMA135, is a transmembrane protein which contains three extracellular CUB domains and acts as a substrate for Src family kinases. CDCP1 is involved in the tyrosine phosphorylation-dependent regulation of cellular events that are involved in tumor invasion and metastasis. Alternative splicing results in multiple transcript variants of this gene. CDCP1 has also been designated as CD318 (cluster of differentiation 318) and Trask (Transmembrane and associated with src kinases). CDCP1 is a 140 kD transmembrane glycoprotein with a large extracellular domain (ECD). CDCP1 is overexpressed in many types of cancer, including breast, lung, colorectal, cervical, urothelical, gastric, ovary, kidney, liver, pancreas, hematopoietic system cancers, and metastatic cancers. High levels of CDCP1 are associated with poorer survival and progressive disease. CDCP1 is a key player in tumorigenesis and metastasis, and is involved in signaling cascades that support cancer cell growth and survival. CDCP1 was identified as a tumor associated proximity antigen (TAPA) which is found in proximity to EGFR. Like EGFR, CDCP1 is over-expressed in multiple cancers and plays key roles in oncogenic signaling and malignant transformation. CDCP1 is upregulated in KRAS transformed cells, and its overexpression plays essential roles associated with loss of adhesion, aggressive metastasis, and poor prognosis. CDCP1 downstream signaling also intersects with multiple RTKs such as EGFR and HER2 to promote tumorigenesis and resistance to RTK therapies. Moreover, EGFR signaling induces upregulation and cell surface localization of CDCP1 as well as inhibition of its degradation. Further exemplifying the inter-dependence of EGFR and CDCP1, in cohorts of EGFR-mutation-positive NSCLC patients treated with EGFR TKI, CDCP1 expression was strongly associated with worsened progression free survival.

[0094] The present disclosure describes the development of a bispecific ADC targeting EGFR and CDCP1. To identify antibodies that display selective activity on cells that express both EGFR and CDCP1, antibodies to both antigens were generated, then combined to form 250+ hlgGl -based 1+1 bispecific antibodies with diverse epitopes and affinities. This bispecific library was screened on internalization and cytotoxicity assays, yielding candidates that bound domain III of EGFR and bound the membrane-distal domain of CDCP1. Further sequence optimization to affinity tune and humanization of these bispecific antibodies, engineering their Fc domains, and conjugating them with drugs was performed. In some cases, antibody drug conjugates (ADCs) with a drug-to- antibody ratio (DAR) of two via engineered cysteines to a vcMMAE drug linker were created.

[0095] The bispecific antibodies described herein can incorporate several design principles that distinguish them from other EGFR ADCs, including possible higher tumor exposure and penetration because of their smaller size and the inherent proximity of the tumor antigen targets, EGFR and CDCP1 in tumors. Due to their formats and conjugation strategy, the bispecific antibodies described herein exhibit excellent biophysical properties and are highly developable. The bispecific antibodies described herein have potent in vitro cytotoxic activity against cancer cell lines with varying target expression levels when engineered as ADCs.

[0096] The bispecific antibodies disclosed herein can be an MMAE-conjugated or exactecan conjugated EGFR-CDCP1 bispecific antibody with a low DAR. These bispecific antibodies display selective internalization and cell killing through co-binding. The bispecific antibodies disclosed herein can be effective in in vitro cell killing assays against a panel of cancer cell lines of varying target expression levels, including osimertinib resistant EGFR mutant NSCLC. The bispecific antibodies disclosed herein can have minimal activity against normal cells expressing both targets. CDCP1 is known to undergo cleavage in some settings, and the present disclosure presents data that confirms that the bispecific antibodies described herein bind both intact and cleaved isoforms of CDCP1. Additionally, the bispecific antibodies disclosed herein demonstrate in vivo efficacy in multiple cell line derived and patient derived xenograft models. Preclinical in vivo assessment of the bispecific antibodies disclosed herein show efficacy against established tumor cell line-derived xenograft (CDX) models representing a range of target expression (measured by flow cytometry and immunohistochemistry) across multiple solid tumor indications. Illustrative bispecific antibodies of the present disclosure and having varying EGFR affinity, or monospecific derivatives thereof, were administered I.V. at a single dose in tumor-bearing mice.

[0097] Unlike other EGFR ADCs, in embodiments, the bispecific antibodies of the present disclosure do not engage immune effector cells. They have a serum half-life of ~16 hours in mice. The bispecific antibodies of the present disclosure are well tolerated in mice at all doses evaluated, with no signs of body weight loss. Robust, dose-dependent anti-tumor activity was observed in tumor models with dual-target expression across a range of target expression levels. At lower doses of bispecific ADC, activity was reduced but not absent in models with low EGFR or CDCP1 expression. Bispecific ADCs were consistently more potent than monospecific monovalent ADC controls, demonstrating improved and more-selective anti-tumor activity through co-binding of EGFR and CDCP1. The activity of the bispecific antibodies of the present disclosure were also evaluated in patient-derived xenograft (PDX) models of squamous lung carcinoma, where a high degree of target co-expression is observed in clinical samples.

[0098] In summary, the present disclosure provides logic-gated targeting and killing of in vitro or in vivo cells, e.g., cancer cells, that express or overexpress both EGFR and CDCP1, with a high degree of selectivity over cells that express one of EGFR and CDCP1, or neither of EGFR nor CDCP1.

[0099] Bispecific Antibodies

[0100] CA-acting bispecific antibodies (cA-bsAbs) represent a rapidly evolving therapeutic modality that can leverage multiple mechanisms of action such as logic-gated cell targeting or signal modulation through induced proximity of transmembrane receptors.

[0101] Provided herein are bispecific antibodies including: (i) an anti-EGFR antibody region capable of recognizing and binding an EGFR epitope, e.g., of an EGFR protein expressed by a cancer cell and (ii) a CDCP1 antibody region capable of recognizing and binding a CDCP1 epitope, e.g., of a CDCP1 protein expressed by the cancer cell. In embodiments, the bispecific antibody is an antibody-drug conjugate (ADC) which comprises one or more cytotoxic agents for killing target cells, e.g., cancer cells that express or overexpress both of EGFR and CDCP1.

[0102] As used herein, the term bispecific antibody is any polypeptide chain, fusion protein, protein, protein complex, and the like that can recognize and bind both an EGFR epitope and a CDCP1 epitope. In some cases, the term “EGFRxCDCPl” is here used to represent every envisioned bispecific antibody of the present disclosure. Illustrative bispecific antibodies are shown in FIG. 3A to FIG. 3G. The diagrams in FIG. 3A to FIG. 3G additionally relate to biparatopic antibodies, where the antibody binds to two distinct epitopes on the same antigens. The biparatopic antibodies may comprise multiple unique binding regions that recognize distinct (which may be non-overlapping or overlapping) epitopes on the antigen. Some illustrative bispecific antibodies comprise Fc regions having either a knob or a hole; see, e.g., FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3G; here, the knob is shown as the circular protrusion and the hole as the indentation. In other cases, bispecific antibodies comprise Fc regions lacking a knob and a hole; see, e.g., the three bispecifics of FIG. 3A, FIG. 3E and FIG. 3F. In embodiments, a bispecific antibody binds domain III of EGFR with a kappa Fab and binds the membrane-distal domain of CDCP1 with an alpaca-derived VHH.

[0103] The term “bispecific antibody” “bispecific antibodies” or “bispecific” or “bispecifics” or “bsAb” or “bsAbs” or “cis-acting bispecific antibodies” or “cis-bsAbs” as provided herein is used according to its conventional meaning well known in the art and refers to a bispecific recombinant protein capable of simultaneously binding to two different antigens. In contrast to traditional monoclonal antibodies, bispecific antibodies include two independently different antibody regions (i.e.., an anti-EGFR antibody region and an anti-CDCPl antibody region).

[0104] An “antibody region” as provided herein refers to a protein moiety that can recognize and bind an antigen (epitope). The antibody region provided herein may include a domain of an antibody or fragment (e.g., Fab, single-chain variable fragment (scFv), and single variable domain on a heavy chain (VHH)) thereof. An antibody region comprises a sufficient number of, e. ., three, complementarity determining regions (CDRs) that permit recognition and binding of the EGFR epitope or the CDCP1 epitope. An antibody region may include a light chain variable domain (VL) and / or a heavy chain variable domain (VH). In embodiments, the antibody region includes a light chain variable (VL) domain. In embodiments, the antibody region includes a heavy chain variable (VH) domain. In embodiments, the antibody region is an HH, also referred to as a nanobody. In embodiments, the antibody region is a Fab, Fab’, or scFv. In embodiments, the antibody region is an Fab. The bispecific antibody may include two Fabs, each of which binds a different antigens. In embodiments, the antibody region is a Fab’. In embodiments, the antibody region is an scFv. The bispecific antibody may include two scFvs, each of which binds a different antigen. In embodiments, the bispecific antibody comprises one or more VHH, one or more scFv, and / or one or more Fab; any combination of VHH, scFv, and VHH is envisioned. In embodiments, the antibody region is a peptibody. A “peptibody” as provided herein refers to a peptide moiety attached (through a covalent or non-covalent linker) to the Fc domain or fragment thereof of an antibody. In short, a bispecific antibody can be any polypeptide chain, fusion protein, protein, protein complex, and the like that comprises a sufficient number of, e.g., three, EGFR-epitope- specific complementarity determining regions (CDRs) that permit recognition and binding of the EGFR epitope and comprise a sufficient number of, e.g., three, CDCP1 -epitope-specific CDRs that permit recognition and binding of the CDCP1 epitope.

[0105] Antibody regions of the bispecific antibodies of the present disclosure may have different affinities for each of the two target antigens. In one embodiment, the anti-EGFR antibody region has low affinity for EGFR whereas the anti-CDCPl antibody region has a high affinity for CDCP1. In another embodiment, the anti-EGFR antibody region has high affinity for EGFR whereas the anti-CDCPl antibody region has a low affinity for CDCP1. Such differential affinities may result in a bispecific antibody that is biased to a specific cancer cell type. Bispecific antibodies with differential affinities may be “tuned” to a specific cancer type. Such differential affinities may not be necessary in some cancer types; here, the anti-EGFR antibody region and the anti-CDCPl antibody region have either low affinities for their respective antigens or high affinities for their respective antigens. Low affinity binding to one or both antigens may contribute to selective bispecific activity, where the bispecific only binds tightly in the presence of both antigens, but binds weakly if only one antigen is present.

[0106] Illustrative bispecific antibodies are shown in FIG. 3A to FIG. 3E. The bispecific of FIG. 3A, comprises an anti-CDCPl VHH and an anti-EGFR Fab; the Fc domains shown here comprise a knob and a hole, Fc domains lacking the knob and hole are envisioned. The bispecific of FIG. 3B, comprises two anti-EGFR Fabs and an anti-CDCPl VHH; the Fc domains shown here comprise a knob and a hole, Fc domains lacking the knob and hole are envisioned. The bispecific of FIG. 3C, comprises two anti-EGFR Fabs and two anti-CDCPl VHHs; the Fc domains shown here lacks a knob and a hole, Fc domains comprising the knob and hole are envisioned. The bispecific of FIG. 3D, comprises one anti-EGFR scFv and one anti-CDCPl VHH; the Fc domains shown here as comprising a knob and a hole, Fc domains lacking the knob and hole are envisioned. The bispecific of FIG. 3H shows an illustrative antibody drug conjugate (ADC); here, the drug is conjugated to the bispecific of FIG. 3A; however, a drug can be conjugated to any bispecific antibody format disclosed herein. Alternately, bispecifics of this general format have two anti- EGFR VHH and one anti-CDCPl VHH; two anti-EGFR VHH and one anti-EGFR Fab, and one anti-CDCPl VHH; two anti-CDCPl VHH, one anti-EGFR Fab, one anti-EGFR scFv; and any other possible configuration, with the anti-CDCPl antibody region comprising a VHH domain. In some embodiments a VHH is attached to the Fc domain. In some embodiments, an scFv is attached to the hinge region of the antibody. In some embodiments a scFv is attached to the Fc domain. The diagrams in FIG. 3A to FIG. 3G additionally relate to biparatopic antibodies, where the antibody binds to two distinct epitopes on the same antigens. Thus, in FIG. 3B for example, the left arm of a biparatopic binds to a first epitope of EGFR whereas the right arm of the biparatopic binds to a second epitope of EGFR. Moreover, the biparatopic may bind to the same molecule of EGFR, albeit at different epitopes, or it may bind to the first epitope on a first molecule of EGFR and to a second epitope on a second molecule of EGFR, where the first and second epitopes are distinct epitopes. The biparatopic antibodies may comprise multiple unique binding regions that recognize distinct (which may be non-overlapping or overlapping) epitopes on the antigen. In FIG. 3C, when the bispecific antibody is a biparatopic antibody, the top left arm of a biparatopic binds to a first epitope of EGFR, the top right arm of the biparatopic binds to a second epitope of EGFR, the bottom left arm of the biparatopic binds to a first epitope of CDCP1, and the bottom right arm of the biparatopic binds to a second epitope of CDCP1; the first and second epitopes may be on the same target molecule or may be on different target molecules. Any possible configuration of VHH, Fab, scFv, knob and hole or absence thereof, and attachment of VHH, Fab, and scFv to the bispecific is envisioned in the present disclosure.

[0107] Binding of the bispecific antibodies provided herein to EGFR and CDCP1 on cancer cells results in cancer cell death when the bispecific antibody is conjugated to one or more cytotoxic payloads (as an antibody drug conjugate). The bispecific antibodies provided herein have demonstrated enhanced internalization upon binding to cancer cells compared to that of monovalent CDCP1 and EGFR antibodies, which may be attributed to the inherent proximity and spatial cell surface localization of EGFR and CDCPlon cancer cells. Without wishing to be bound by scientific theory, binding of a bispecific antibody including an anti-CDCPl antibody region and an anti-EGFR antibody region to both CDCP1 and EGFR expressed on a cancer cell may sequester EGFR and CDCP1 expressed on the same cancer cell. As demonstrated throughout the specification, including in the Examples and Figures, contacting a cancer cell with the bispecific antibody provided herein results in increased internalization of antibody, delivery of conjugated drug and cancer cell death compared to the increased internalization of antibody, delivery of drug conjugate and cancer cell death resulting from contacting a cancer cell with an anti-EGFR or anti- CDCPl antibody (alone or in combination). In embodiments, contacting a cancer cell with the bispecific ADC results in increased cancer cell death compared to contacting a cancer with an anti- EGFR or anti-CDCPl antibody (alone or in combination). Cancer cell death can be quantified, for example, by a decrease in tumor volume or decrease in the number of cancer cells.

[0108] An aspect of the present disclosure is a bispecific antibody comprising at least one anti- EGFR antibody region and at least one anti-CDCPl antibody region.

[0109] In embodiments, the bispecific antibody recognizes and binds to an EGFR antigen and to a CDCP1 antigen on a cancer cell.

[0110] In embodiments, the bispecific antibody includes an antibody region including a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 1 to Table 5, e.g., SEQ ID NO: 1291.

[0111] In embodiments, the bispecific antibody comprises SEQ ID NO: 1291.

[0112] In embodiments, the bispecific antibody includes an antibody region including a sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 1 to Table 3 and a sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g, SEQ ID NO: 1291.

[0113] In embodiments, the bispecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0114] In embodiments, the bispecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1112 to SEQ ID NO: 1123.

[0115] In embodiments, the bispecific antibody includes a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in Table 7.

[0116] In embodiments, an anti-CDCPl antibody region is configured as a single variable domain on a heavy chain (VHH). In embodiments, the bispecific antibody includes at least two anti-CDCPl antibody regions, with the anti-CDCPl antibody region comprising a VHH domain.

[0117] In embodiments, the bispecific antibody includes at least three anti-CDCPl antibody regions, with the anti-CDCPl antibody region comprising a VHH domain.

[0118] In embodiments, an anti-EGFR antibody region is configured as a Fab, a single-chain variable fragment (scFv), and / or a single variable domain on a heavy chain (VHH).

[0119] In embodiments, the bispecific antibody includes at least two anti-EGFR antibody regions.

[0120] In embodiments, the bispecific antibody includes at least three anti-EGFR antibody regions.

[0121] In embodiments, the bispecific antibody includes one or more Fabs, includes one or more scFvs, and / or includes one or more VHHs.

[0122] In embodiments, the bispecific antibody includes at least two Fabs.

[0123] In embodiments, a first Fab includes an anti-EGFR antibody region and a second Fab includes an anti-CDCPl antibody region, with the anti-CDCPl antibody region comprising a VHH domain.

[0124] In embodiments, the bispecific antibody includes at least one Fab and at least one VHH.

[0125] In embodiments, the at least one Fab includes an anti-EGFR antibody region and the at least one VHH includes an anti-CDCPl antibody region, with the anti-CDCPl antibody region comprising a VHH domain.

[0126] In embodiments, the bispecific antibody includes at least one Fab and at least one scFv, with the anti-CDCPl antibody region comprising a VHH domain.

[0127] In embodiments, the bispecific antibody includes at least two Fabs and includes at least one VHH.

[0128] In embodiments, both Fabs include an anti-EGFR antibody region.

[0129] In embodiments, the at least one VHH includes an anti-EGFR antibody region.

[0130] In embodiments, the at least one VHH includes an anti-CDCPl antibody region.

[0131] In embodiments, the bispecific antibody includes at least two Fabs and includes at least one scFv.

[0132] In embodiments, both Fabs include an anti-EGFR antibody region.

[0133] In embodiments, the at least one scFv includes an anti-EGFR antibody region.

[0134] In embodiments, the bispecific antibody includes at least two Fabs and includes at least two VHHs. In embodiments, both Fabs include an anti-EGFR antibody region.

[0135] In embodiments, both VHHs include an anti-EGFR antibody region.

[0136] In embodiments, both VHHs include an anti-CDCPl antibody region.

[0137] In embodiments, one VHH includes an anti-EGFR antibody region and one VHH includes an anti-CDCPl antibody region.

[0138] In embodiments, the bispecific antibody includes at least two Fabs and includes at least two scFvs.

[0139] In embodiments, both Fabs include an anti-EGFR antibody region.

[0140] In embodiments, both scFvs include an anti-EGFR antibody region.

[0141] In any herein disclosed aspect or embodiment comprising a bispecific antibody, the bispecific antibody is an antibody-drug conjugate (ADC) which comprises one or more cytotoxic agents for killing target cells, e.g, cancer cells that express or overexpress both of EGFR and CDCP1. In embodiments, the drug is conjugated, e.g., covalently or non-covalently bonded, to the antibody’s Fc region. In some embodiments the drug is conjugated to an amino acid position of the antibody’s Fc region that has been substituted with a cysteine. As examples, the conjugation site may be a 375C, 360C, 149C, and / or a 140C substituted position; see, e.g., SEQ ID NO: 1309- 1316 which includes S375C, K360C, K149C, and / or A140C substitutions in context. Other positions, or substituted positions, suitable for conjugation may be used. Sequences for Fc regions that may be conjugated with a drug are shown in Table 7.

[0142] Illustrative Antibody Regions, Antibodies, and Bi-Specific Antibodies

[0143] EGFR Antibodies

[0144] The bispecific antibodies of the present disclosure include an antibody region that binds EGFR in vivo, ex vivo, in vitro, and / or in biochemical assays.

[0145] In embodiments, the bispecific antibody includes an antibody region including one to six complementarity determining regions (“CDRs”) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the CDR sequence shown in Table 1.

[0146] In embodiments, the bispecific antibody includes an antibody region including three Heavy Chain CDRs (CDR-H1, CDR-H2, and CDR-H3) sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 447. In embodiments, the bispecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 445 to SEQ ID NO: 447.

[0147] In embodiments, the bispecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 457 to SEQ ID NO: 459.

[0148] In embodiments, the bispecific antibody includes an antibody region including three Light Chain CDRs (CDR-L1, CDR-L2, and CDR-L3) sequences from a single row of Table 1, e.g., SEQ ID NO: 448 to SEQ ID NO: 450.

[0149] In embodiments, the bi specific antibody comprises CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 448 to SEQ ID NO: 450.

[0150] In embodiments, the bispecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 460 to SEQ ID NO: 462.

[0151] In embodiments, the bispecific antibody includes an antibody region including CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 450.

[0152] In embodiments, the bispecific antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR- Ll, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 445 to SEQ ID NO: 450.

[0153] In embodiments, the bispecific antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR- Ll, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 457 to SEQ ID NO: 462.

[0154] For the bispecific antibodies provided herein, in embodiments, the anti-EGFR antibody region includes a variant of any one of CDR Hl, CDR H2, CDR H3, CDR LI, CDR L2, and CDR L3, or a combination thereof, i.e., the bispecific antibody comprises a sequence that is less than 100% identical to a sequence shown in Table 1. The bispecific antibody comprises one, two or three amino acid mutations which are independently a substitution mutation, a deletion mutation, an insertion mutation, or a combination thereof.

[0155] CDRs recited throughout the present application are defined based on the Kabat numbering scheme. CDRs from the VH, VL and scFvs of the present application could also be defined by using other conventional antibody numbering schemes, i.e., IMGT or Chothia.

[0156] Table 1 CDRs Corresponding to EGFR antigen binding domains

[0157]

[0158] In embodiments, the bispecific antibody includes an antibody region including one or more Variable Heavy (VH) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VH sequences shown in Table 2.

[0159] In embodiments, the bispecific antibody includes an antibody region including one or more Variable Light (VL) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VL sequences shown in Table 2.

[0160] In embodiments, the bispecific antibody includes an antibody region including one or more VH sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VH sequences shown in Table 2 and one or more VL sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VL sequences shown in Table 2.

[0161] In embodiments, the bispecific antibody includes an antibody region including a VH sequence that is at least 90% identical (e.g. , 95%, 99%, or 100% identical) to a VH sequence shown in a single row of Table 2, e.g., SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VL sequence shown in the row of Table 2, e.g., SEQ ID NO: 630.

[0162] In embodiments, the bispecific antibody comprises an antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 630.

[0163] In embodiments, the bispecific antibody comprises an antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 633, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 634.

[0164] For the bispecific antibodies provided herein, in embodiments, the anti-EGFR antibody region includes a variant of any one of the recited VH or VL, or a combination thereof, i.e., the bispecific antibody comprises a sequence that is less than 100% identical to a sequence shown in Table 2. The bispecific antibody comprises one, two or three amino acid mutations which are independently a substitution mutation, a deletion mutation, an insertion mutation, or a combination thereof.

[0165] Table 2 VHs, VLs, and VHHs corresponding to EGFR antigen binding domains

[0166]

[0167] In some cases, binders C330 or C2004 of Table 2 act alone as VHH and bind to EGFR and without need for a VL component.

[0168] In embodiments, the anti-EGFR antibody region is configured as an scFv and includes an antibody region including a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to an scFv sequence shown in Table 3.

[0169] For the bispecific antibodies provided herein, in embodiments, the anti-EGFR antibody region includes a variant of any one of the recited scFvs, i.e., the bispecific antibody comprises a sequence that is less than 100% identical to a sequence shown in Table 3 comprises one, two or three amino acid mutations which are independently a substitution mutation, a deletion mutation, an insertion mutation, or a combination thereof.

[0170] Table 3 EGFR scFv Binding Domains

[0171] In embodiments, abispecific antibody including a sequence that is less than 100% identical a sequence shown in one or more of Table 1 to Table 3 retains affinity for EGFR that is substantially equivalent to a bispecific antibody including a sequence that is 100% identical to a sequence shown in one or more of Table 1 to Table 3.

[0172] In embodiments, a bispecific antibody including a sequence that is less than 100% identical to a sequence shown in one or more of Table 1 to Table 3 has less affinity for EGFR than a bispecific antibody including a sequence that is 100% identical to a sequence shown in one or more of Table 1 to Table 3.

[0173] CDCP1 Antibodies

[0174] The bispecific antibodies of the present disclosure include an antibody region, with the anti-CDCPl antibody region comprising a VHH domain, that binds CDCP1 in vivo, ex vivo, in vitro, and / or in biochemical assays.

[0175] In embodiments, the bispecific antibody includes an antibody region including one to three complementarity determining regions (“CDRs”) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the CDR sequence shown in Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0176] In embodiments, the bispecific antibody comprises an antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 630.

[0177] In embodiments, the bispecific antibody comprises an antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 633, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 634.

[0178] In embodiments, the bispecific antibody includes an antibody region including three Heavy Chain CDRs (CDR-H1, CDR-H2, and CDR-H3) sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, with the anti-CDCPl antibody region comprising a VHH domain.

[0179] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 445 to SEQ ID NO: 447, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1115 to SEQ ID NO: 1117. In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 457 to SEQ ID NO: 459, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1121 to SEQ ID NO: 1123.

[0180] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 445 to SEQ ID NO: 450, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0181] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 457 to SEQ ID NO: 462, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 1121 to SEQ ID NO: 1123.

[0182] For the bispecific antibodies provided herein, in embodiments, the anti-CDCPl antibody region, comprising a VHH domain, includes a variant of any one of CDR Hl, CDR H2, and CDR H3 or a combination thereof, i.e., the bispecific antibody comprising a sequence that is less than 100% identical to a sequence shown in Table 4 comprises one, two or three amino acid mutations which are independently a substitution mutation, a deletion mutation, an insertion mutation, or a combination thereof.

[0183] In embodiments, the CDR sequences of Table 4 are included in a VHH format antibody or in an anti-CDCPl antibody region comprising a VHH domain.

[0184] Table 4 CDRs Corresponding to CDCP1 antigen binding domains

[0185]

[0186] In embodiments, the bispecific antibody includes an antibody region including one or more VHH sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VHH sequences shown in Table 5, e.g., SEQ ID NO: 1291.

[0187] In embodiments, the bispecific antibody comprises SEQ ID NO: 1291.

[0188] In embodiments, the bispecific antibody comprises SEQ ID NO: 1293.

[0189] For the bispecific antibodies provided herein, in embodiments, the anti-CDCPl antibody region includes a variant of any one of the recited VHH, i.e., the bispecific antibody comprising a sequence that is less than 100% identical to a sequence shown in Table 5, e.g., SEQ ID NO: 1291, comprises one, two or three amino acid mutations which are independently a substitution mutation, a deletion mutation, an insertion mutation, or a combination thereof.

[0190] In embodiments, the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 1291.

[0191] In embodiments, the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 633, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 1291.

[0192] In embodiments, the bi specific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 630, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) SEQ ID NO: 1291.

[0193] In embodiments, the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 633, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 634, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) SEQ ID NO: 1293.

[0194] In embodiments, the sequences of Table 5 are included in a VHH format antibody. 062460-502001W0

[0195] Table 5 VHH corresponding to CDCP1 antigen binding domains

[0196] In embodiments, a bispecific antibody including a sequence that is less than 100% identical to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 11 15 to SEQ ID NO: 11 17, or Table 5, e.g., SEQ ID NO: 1291 retains affinity for CDCP1 that is substantially equivalent to a bispecific antibody including a sequence that is 100% identical to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g., SEQ ID NO: 1291. Table 6 Linker Sequences

[0197] In embodiments, a bispecific antibody includes a sequence that is less than 100% (e.g., 50%, 60%, 70%, 80%, or 90%) identical to a sequence shown in Table 6. Other linkers and especially linkers useful for conjugating a drug to an antibody of the present disclosure can be used herein. Other linkers can be found at the world wide web (www) medchemexpress.com; especially in the sections relating to “ADC Linker” and including the recited cleavable linkers and non- cleavable linkers. The contents of which as of the present filing date is incorporated by references herein in its entirety.

[0198] Illustrative bi-specifics

[0199] In embodiments, the bispecific antibody includes a first antibody region including CDR- Hl, CDR-H2, and CDR-H3 sequences from a single row of Table 1, e.g., NO: 445 to SEQ ID NO: 447, and includes a second antibody region including CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0200] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1 , CDR-H2, and CDR-H3 of SEQ ID NO: 445 to SEQ ID NO: 447, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0201] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 457 to SEQ ID NO: 459, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1115 to SEQ ID NO: 1293. In embodiments, the bispecific antibody includes a third antibody region including CDR- Hl, CDR-H2, and CDR-H3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 447, and / or includes a fourth antibody region including CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0202] In embodiments, the bispecific antibody includes a first antibody region including CDR- Hl, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 450, and includes a second antibody region including CDR- Hl, CDR-H2, and CDR-H3 sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0203] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 445 to SEQ ID NO: 450, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0204] In embodiments, the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 457 to SEQ ID NO: 462, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 1121 to SEQ ID NO: 1123.

[0205] In embodiments, the bispecific antibody includes a third antibody region including CDR- Hl, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 450, and / or includes a fourth antibody region including CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0206] In embodiments, the bispecific antibody includes a first antibody region including a VH sequence that is at least 90% identical (e.g. , 95%, 99%, or 100% identical) to a VH sequence shown in Table 2, e.g., SEQ ID NO: 629, and includes a second antibody region including a VHH sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in Table 5, e.g., SEQ ID NO: 1291.

[0207] In embodiments, the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 1291. In embodiments, the bi specific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e. ., 95%, 99%, or 100% identical) to SEQ ID NO: 633, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 1293.

[0208] In embodiments, the bispecific antibody includes a third antibody region including a VH sequence that is at least 90% identical (e.g. , 95%, 99%, or 100% identical) to a VH sequence shown in Table 2, e.g., SEQ ID NO: 629, and / or includes a fourth antibody region including a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in Table 5, e.g., SEQ ID NO: 1291.

[0209] In embodiments, the bispecific antibody includes a first antibody region including a VH sequence that is at least 90% identical e.g. , 95%, 99%, or 100% identical) to a VH sequence shown in a single row of Table 2, e.g., SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VL sequence shown in the row of Table 2, e.g., SEQ ID NO: 630, and includes a second antibody region including a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in a single row of Table 5, e.g, SEQ ID NO: 1291.

[0210] In embodiments, the bi specific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 630, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) SEQ ID NO: 1291.

[0211] In embodiments, the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 633, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 634, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) SEQ ID NO: 1293.

[0212] In embodiments, the bispecific antibody includes a third antibody region including a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VH sequence shown in a single row of Table 2, e.g., SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VL sequence shown in the row of Table 2, e.g., SEQ ID NO: 630, and / or includes a fourth antibody region including a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in a single row of Table 5, e.g., SEQ ID NO: 1291.

[0213] For the bispecific antibody provided herein, in embodiments, the anti-EGFR antibody region can be an scFv domain, with the anti-CDCPl antibody region comprising a VHH domain. A single chain antibody includes a variable light chain and a variable heavy chain, in contrast to an immunoglobulin antibody, which includes two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. The variable light chain and the variable heavy chain in a single chain antibody may be linked through a linker peptide. In embodiments, the anti-EGFR antibody region includes an scFv domain. In embodiments, the anti-EGFR antibody region is an scFv domain. In embodiments, the anti-EGFR antibody region includes a first single chain variable fragment (scFv). In embodiments, a bispecific comprises an anti-EGFR antibody region that is a first single chain variable fragment (scFv) and an anti-EGFR antibody region that is a second scFv, with an anti-CDCPl antibody region comprising a VHH domain.

[0214] For the bispecific antibody provided herein, in embodiments, the anti-EGFR antibody region is a Fab fragment, with the anti-CDCPl antibody region comprising a VHH domain. Thus, in embodiments the bispecific antibody may include an anti-EGFR antibody region includes a heavy chain (e.g., including a constant and a variable region) and a light chain (e.g., including a constant and a variable region). In embodiments, the Fab fragment includes a humanized heavy chain (e.g., including a constant and a variable region) and a humanized light chain e.g., including a constant and a variable region). In embodiments, the anti-EGFR antibody region includes a Fab fragment. In embodiments, the anti-EGFR antibody region is a Fab fragment. In embodiments, a bispecific comprises an anti-EGFR antibody region that is a first a Fab fragment and an anti-EGFR antibody region that is a second a Fab fragment, with an anti-CDCPl antibody region comprising a VHH domain.

[0215] For the bispecific antibody provided herein, in embodiments, the anti-EGFR antibody region comprises Fab’ fragment. Thus, in embodiments, the anti-EGFR antibody region includes a Fab’ fragment. In embodiments, the anti-EGFR antibody region is a Fab’ fragment. In embodiments, the anti-EGFR antibody region is a first Fab’ fragment. In embodiments, a bispecific comprises a first antibody region is a first anti-EGFR Fab’ fragment and a second antibody region that is a second anti-EGFRFab’ fragment, with an anti-CDCPl antibody region comprising a VHH domain. In embodiments, the anti-CDCPl antibody region includes a variable domain of heavy chain (VHH). In embodiments, the anti-CDCPl antibody region is a variable domain of heavy chain (VHH). In embodiments, the anti-CDCPl antibody region does not include a constant region (e.g. CHI region and / or an Fc domain, or fragment crystallizable region).

[0216] In embodiments, the anti-CDCPl antibody region includes a peptibody. In embodiments, the anti-CDCPl antibody region is a peptibody.

[0217] For the bispecific antibody provided herein, in embodiments, the anti-CDCPl antibody region and the anti-EGFR antibody region are attached by a linker. In embodiments, the linker is a polypeptide linker. In embodiments, the polypeptide linker includes repeats of serine and glycine for flexibility and / or solubility. In one example, an anti-CDCPl antibody region may be attached to an anti-EGFR antibody region by a polypeptide linker, wherein the anti-CDCPl antibody region is a VHH and the anti-EGFR antibody region is an scFv. In another example, an anti-CDCPl antibody region may be attached to an anti-EGFR antibody region by a polypeptide linker, wherein the anti-CDCPl antibody region is an VHH and the anti-EGFR antibody region is a Fab fragment. For the bispecific antibody provided herein, including embodiments thereof, the anti-EGFR antibody region and the anti-CDCPl antibody region may be any VHH, fragment, or variant thereof as described herein attached by a polypeptide linker. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 10 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 15 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 20 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 25 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 30 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 35 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 40 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 45 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 50 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 55 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 60 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 65 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 70 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 75 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 80 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 85 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 90 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 95 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 100 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 105 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 110 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 115 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 120 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 125 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 130 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 135 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 140 amino acid residues to about 150 amino acid residues in length. In embodiments, the polypeptide linker is from about 145 amino acid residues to about 150 amino acid residues in length.

[0218] In embodiments, the polypeptide linker is from about 5 amino acid residues to about 145 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 140 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 135 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 130 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 125 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 120 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 115 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 110 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 105 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 100 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 95 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 90 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 85 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 80 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 75 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 70 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 65 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 60 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 55 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 50 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 45 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 40 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 35 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 30 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 25 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 20 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 15 amino acid residues in length. In embodiments, the polypeptide linker is from about 5 amino acid residues to about 10 amino acid residues in length. In embodiments, the polypeptide linker is about 5 amino acid residues, 10 amino acid residues, 15 amino acid residues, 20 amino acid residues, 25 amino acid residues, 30 amino acid residues, 35 amino acid residues, 40 amino acid residues, 45 amino acid residues, 50 amino acid residues, 55 amino acid residues, 60 amino acid residues, 65 amino acid residues, 70 amino acid residues, 75 amino acid residues, 80 amino acid residues, 85 amino acid residues, 90 amino acid residues, 95 amino acid residues, 100 amino acid residues, 105 amino acid residues, 110 amino acid residues, 115 amino acid residues, 120 amino acid residues, 125 amino acid residues, 130 amino acid residues, 135 amino acid residues, 140 amino acid residues, 145 amino acid residues, or 150 amino acid residues in length.

[0219] For the bispecific antibody provided herein, in embodiments, the anti-CDCPl antibody region, which comprises a VHH domain, and the anti-EGFR antibody region are attached by a first Fc portion and a second Fc portion, wherein the first Fc portion and the second Fc portion dimerize to form an Fc region, thereby attaching the anti-CDCPl antibody region and the anti-EGFR antibody region. Thus, in embodiments, the anti-CDCPl antibody region includes a first Fc portion and the anti-EGFR antibody region includes a second Fc portion. An “Fc portion” as referred to herein is a polypeptide including an antibody CH2 domain or fragment thereof attached (covalently and / or non-covalently) to an antibody CH3 domain or fragment thereof. Upon binding of two Fc portions, an antibody Fc region is formed. Thus, an Fc region may include a first Fc portion non- covalently or covalently bound to a second Fc portion. In embodiments, the CH3 domain of the first Fc portion is non-covalently bound to the CH3 domain of the second Fc portion. In embodiments, the CH2 domain of the first Fc portion is covalently bound to the CH2 domain of the second Fc portion. In embodiments, the CH2 domain of the first Fc portion is bound to the CH2 domain of the second Fc portion through a disulfide linkage. In embodiments, the Fc portion includes a CH2 domain and a CH3 domain. In embodiments, the Fc portion includes from the N- terminus to the C-terminus a CH2 domain and a CH3 domain. Thus, in embodiments, the first Fc portion and the second Fc portion are attached, thereby forming an Fc region.

[0220] The ability of an antibody to bind a specific epitope (e. , CDCP1 or EGFR) can be described by the equilibrium dissociation constant (KD), which is a quantitative measurement of an antibody’s affinity for its antigen. The equilibrium dissociation constant (KD) as defined herein is the ratio of the dissociation rate (K-off) and the association rate (K-on) of the anti-CDCPl antibody region capable of binding a CDCP1 protein or the anti-EGFR antibody region capable of binding an EGFR protein. It is described by the following formula: KD = K-off / K-on. Thus, in embodiments, the anti-CDCPl antibody region is capable of binding a CDCP1 protein with an equilibrium dissociation constant (KD) of about 0.1 nM to about 50 nM.

[0221] In embodiments, the anti-EGFR antibody region is capable of binding said EGFR protein with an equilibrium dissociation constant (KD) from about 0.01 nM to about 500 nM, e.g., 20 nM.

[0222] Antibody regions of the bispecific antibodies of the present disclosure may have different affinities for each of the two target antigens. In one embodiment, the anti-EGFR antibody region has low affinity for EGFR whereas the anti-CDCPl antibody region has a high affinity for CDCP1. In another embodiment, the anti-EGFR antibody region has high affinity for EGFR whereas the anti-CDCPl antibody region has a low affinity for CDCP1. Such differential affinities may result in a bispecific antibody that is biased to a specific cancer cell type. Bispecific antibodies with differential affinities may be “tuned” to a specific cancer type. Such differential affinities may not be necessary in some cancer types; here, the anti-EGFR antibody region and the anti-CDCPl antibody region have either low affinities for their respective antigens or high affinities for their respective antigens.

[0223] In various aspects, a bispecific antibody is in an antibody format comprising a constant region, e.g., an Fc domain, or fragment crystallizable region. In embodiments, the bispecific antibody includes a sequence that is at least 90% identical

[0224] (e.g., 95%, 99%, or 100% identical) to a sequence shown in Table 7.

[0225] Some of the Fc regions comprise “knob” or “hole” motifs, see, e.g., SEQ ID NOs: 1300 to 1314 and SEQ ID NO: 1316 in Table 7. In some cases, the Fc region lacks “knob” and “hole” motifs, see, e.g., SEQ ID NOs: 1297 to 1299 and SEQ ID NO: 1315 in Table 7. Table 7 Constant Regions

[0226]

[0227] SEQ ID 1299 is a light chain (kappa) sequence, not a heavy chain (Fc-containing) sequence; however, for simplicity, in this disclosure, we are refereeing to it as an Fc sequence.

[0228] In some embodiments, theFc region is a silent “LALA” Fc, e.g., comprising L234A / L235 A substitutions, which is a well-known variant of Fc-silenced antibodies that reduces binding to Fc receptor. Illustrative LALAFcs include those of SEQ ID NO: 1302 to SEQ ID NO: 1308, SEQ ID NO: 1311 to SEQ ID NO: 1314, and SEQ ID NO: 1316.

[0229] In an additional aspect, the present disclosure provides a polynucleotide or plurality of polynucleotides encoding a herein-disclosed bispecific antibody.

[0230] In yet an additional aspect, the present disclosure provides a vector comprising the polynucleotide or plurality of a herein-disclosed polynucleotides.

[0231] MonoSpecific CDCP1 antibodies

[0232] The monospecific antibodies of the present disclosure include an antibody region that binds CDCP1 in vivo, ex vivo, in vitro, and / or in biochemical assays. See, e.g., FIG. 33A to FIG. 33E which are diagrams of illustrative formats for CDCP1 monospecific antibodies. FIG. 33E shows an illustrative CDCP1 antibody drug conjugate (ADC); here, the drug is conjugated to the monospecific antibody of FIG. 33B; however, a drug can be conjugated to any monospecific antibody format disclosed herein. The monospecific antibodies of the present disclosure may have more than one identical CDCP1 binding domains (which bind the same epitope on CDCP1) or may be comprised of multiple unique CDCP1 binding regions that recognize distinct (which may be non-overlapping or overlapping) epitopes, to generate a biparatopic or multiparatopic antibody; any such and similar combination of binders is considered in the present disclosure.

[0233] In embodiments, the monospecific antibody includes an antibody region including one to three complementarity determining regions (“CDRs”) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the CDR sequence shown in Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0234] In embodiments, the monospecific antibody includes an antibody region including three Heavy Chain CDRs (CDR-H1, CDR-H2, and CDR-H3) sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

[0235] In embodiments, the monospecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1115 to SEQ ID NO: 1117. In embodiments, the monospecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1121 to SEQ ID NO: 1123.

[0236] For the monospecific antibodies provided herein, i.e., comprising an anti-CDCPl VHH domain, in embodiments, the anti-CDCPl antibody region includes a variant of any one of CDR Hl, CDR H2, and CDR H3 or a combination thereof, i.e., the monospecific antibody comprising a sequence that is less than 100% identical to a sequence shown in Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, comprises one, two or three amino acid mutations which are independently a substitution mutation, a deletion mutation, an insertion mutation, or a combination thereof.

[0237] In embodiments, the CDR sequences of Table 4 are included in a VHH format antibody.

[0238] In embodiments, the monospecific antibody includes an antibody region including one or more VHH sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VHH sequences shown in Table 5, e.g., SEQ ID NO: 1291.

[0239] In embodiments, the monospecific antibody includes an antibody region that is at least 90% identical (e.g., 95%, 99%, or 100% identical) SEQ ID NO: 1291.

[0240] In embodiments, the monospecific antibody includes an antibody region that is at least 90% identical (e.g., 95%, 99%, or 100% identical) SEQ ID NO: 1293.

[0241] For the monospecific antibodies provided herein, in embodiments, the anti-CDCPl antibody region includes a variant of any one of the recited VHH, i.e., the monospecific antibody comprising a sequence that is less than 100% identical to a sequence shown in Table 5, e.g., SEQ ID NO: 1291, comprises one, two or three amino acid mutations which are independently a substitution mutation, a deletion mutation, an insertion mutation, or a combination thereof.

[0242] In embodiments, the VH sequences of Table 5 are included in a VHH format antibody.

[0243] In embodiments, a monospecific antibody including a sequence that is less than 100% identical to a sequence shown in one or more of Table 4 or Table 5, e.g., SEQ ID NO: 1291, retains affinity for CDCP1 that is substantially equivalent to a monospecific antibody including a sequence that is 100% identical to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g., SEQ ID NO: 1291.

[0244] In embodiments, a monospecific antibody includes a sequence that is less than 100% (e.g., 50%, 60%, 70%, 80%, or 90%) identical to a sequence shown in Table 6. Other linkers and especially linkers useful for conjugating a drug to an antibody of the present disclosure can be used herein. Other linkers can be found at the world wide web (www) medchemexpress.com; especially in the sections relating to “ADC Linker” and including the recited cleavable linkers and non- cleavable linkers. The contents of which as of the present fding date is incorporated by references herein in its entirety.

[0245] The ability of an antibody to bind a specific epitope (e.g., CDCP1) can be described by the equilibrium dissociation constant (KD), which is a quantitative measurement of an antibody’s affinity for its antigen. The equilibrium dissociation constant (KD) as defined herein is the ratio of the dissociation rate (K-off) and the association rate (K-on) of the anti-CDCPl antibody region capable of binding a CDCP1 protein. It is described by the following formula: KD = K-ofl7K-on. Thus, in embodiments, the anti-CDCPl antibody region is capable of binding a CDCP1 protein with an equilibrium dissociation constant (KD) of about 0.1 nM to about 100 nM.

[0246] In various aspects, a monospecific antibody is in an antibody format comprising a constant region, e.g., an Fc domain, or fragment crystallizable region.

[0247] In embodiments, the monospecific antibody includes a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in Table 7.

[0248] Some of the Fc regions comprise “knob” or “hole” motifs, see, e.g., SEQ ID NOs: 1300 to 1314 and SEQ ID NO: 1316 in Table 7. In some cases, the Fc region lacks “knob” and “hole” motifs, see, e.g., SEQ ID NOs: 1297 to 1299 and SEQ ID NO: 1315 in Table 7.

[0249] The diagrams of FIG. 33A to FIG. 33E additionally relate to biparatopic antibodies and multiparatopic antibodies where the antibody binds to two (for bi-) or more (for multi-) distinct epitopes on the same antigen, here, CDCP1. Thus, in FIG. 33A for example, the left arm of a biparatopic binds to a first epitope of CDCP1 whereas the right arm of the biparatopic binds to a second epitope of CDCP1. In FIG. 33C, the antibody may be multiparatopic, in that it binds to more than two epitopes on the same antigen, here CDCP1. For example, the top left arm of a biparatopic binds to a first epitope of CDCP1, the top right arm of the biparatopic binds to a second epitope of CDCP1, the bottom left arm of the biparatopic binds to a third epitope of CDCP1; the bottom right arm may bind to a fourth epitope of CDCP1 or may bind one of the first, second, or third epitopes of CDCP1; any such and similar combination of binders is considered in the present disclosure. Moreover, the biparatopic or multiparatopic may bind to the same molecule of CDCP1, albeit at different epitopes, or it may bind to the first epitope on a first molecule of CDCP1 and to a second epitope on a second molecule of CDCP1, where the first and second epitopes are distinct epitopes, or the first and second epitopes may be on a first molecule of CDCP1 and the third epitope is on a second molecule of CDCP1; any such and similar combination of binders is considered in the present disclosure.

[0250] In an additional aspect, the present disclosure provides a polynucleotide or plurality of polynucleotides encoding a herein-disclosed monospecific antibody.

[0251] In yet an additional aspect, the present disclosure provides a vector comprising the polynucleotide or plurality of a herein-disclosed polynucleotides.

[0252] Pharmaceutical Compositions

[0253] In an aspect, the present disclosure provides a pharmaceutical composition comprising a herein-disclosed bispecific antibody or monospecific antibody and a pharmaceutically acceptable carrier, diluent, or excipient. The bispecific antibody or monospecific antibody provided herein may be provided in a pharmaceutical composition suitable for administration to a subject.

[0254] In various aspects, the pharmaceutical composition is for use as a medicament in the treatment of cancer. In embodiments, the cancer is characterized by expression or overexpression of EGFR and / or CDCP1.

[0255] The pharmaceutical composition may include a combination of a bispecific antibody or monospecific antibody of the present disclosure and an anti-PD-Ll antibody. Non-limiting examples of anti-PD-Ll antibodies includes: BMS-936559, Atezolizumab, Avelumab, Durvalumab or other equivalent antibodies.

[0256] The pharmaceutical composition may include a combination of a bispecific antibody or monospecific antibody of the present disclosure and an anti-PD-1 antibody. Non-limiting examples of anti-PD-1 mono-specific antibodies include: Pembrolizumab, Nivolumab, Cemiplimab, Spartalizumab, Tislelizumab, Dostarlimab, Camrelizumab, Sintilimab, Toripalimab, Penpulimab, and Retifanlimab. Non-limiting examples of anti-PD-1 bispecific antibodies include ivonescimab and cadonilimab.

[0257] The pharmaceutical composition may include a combination of a bispecific antibody or monospecific antibody of the present disclosure and an immunomodulatory drug. Non-limiting examples of immunomodulatory drugs include checkpoint inhibitors, adoptive T cell therapy, monoclonal antibody therapy, cancer vaccines, corticosteroids, tumor necrosis factor inhibitors, interleukin- 1 (IL-1) inhibitors, interleukin-6 (IL-6) inhibitors, T cell inhibitors, B-cell inhibitors and Janus Kinase inhibitors. The pharmaceutical composition may include a combination of a bispecific antibody or monospecific antibody of the present disclosure and a chemotherapy. Non-limiting examples of chemotherapies include alkylating reagents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors and antitumor antibiotics.

[0258] The pharmaceutical composition may include a combination of a bispecific antibody or monospecific antibody of the present disclosure and the standard of care.

[0259] Methods

[0260] Another aspect of the present disclosure is a method of killing a cancer cell, the method comprising contacting the cell with an effective amount of a herein-disclosed bispecific antibody or monospecific antibody of the present disclosure. The method may be in vitro, ex vivo, or in vivo.

[0261] A further aspect of the present disclosure is a method of killing a cancer cell in a subject in need thereof, the method comprising contacting the cell with an effective amount of a herein- disclosed bispecific antibody or monospecific antibody. In embodiments, the cancer cell expresses or overexpresses CDCP1 and / or EGFR.

[0262] An additional aspect of the present disclosure is a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a herein-disclosed bispecific antibody or monospecific antibody. In embodiments, the cancer is characterized by expression or overexpression of CDCP1 and / or EGFR.

[0263] In another aspect, the present disclosure provides a method of killing a cancer cell in a subject in need thereof, the method comprising contacting the cell with an effective amount of herein-disclosed pharmaceutical composition. In embodiments, the cancer cell expresses or overexpresses CDCP1 and / or EGFR.

[0264] In a further aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of herein-disclosed pharmaceutical composition. In embodiments, the cancer is characterized by expression or overexpression of CDCP1 and / or EGFR.

[0265] The bispecific antibodies provided herein are contemplated to be effective for treating diseases (e. cancer). As described throughout the specification, including in the Examples and Figures, the bispecific antibodies provided herein demonstrate superior cancer cell killing compared to mono-specific EGFR or CDCP1 antibodies and other bispecific antibody constructs. In an aspect is provided a method of treating cancer in a subject in need thereof the method including, administering a therapeutically effective amount of the bispecific antibody provided herein including embodiments thereof to the subject.

[0266] A therapeutically effective amount refers to an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend on the condition being treated. When administered in methods to treat a disease, the pharmaceutical compositions described herein will contain an amount of bispecific antibody effective to achieve the desired result, e.g., modulating the activity of a target molecule (e.g., EGFR, CDCP1), and / or reducing, eliminating, or slowing the progression of disease symptoms (e.g., cancer). Determination of a therapeutically effective amount of a bi specific antibody or monospecific antibody provided herein is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.

[0267] For the methods provided herein, in embodiments, the cancer is brain cancer, breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, renal cell cancer, melanoma, uveal melanoma, stomach cancer, pancreatic cancer, prostate cancer, or rectal cancer. In embodiments, the cancer is brain cancer. In embodiments, the cancer is breast cancer. In embodiments, the cancer is bladder cancer. In embodiments, the cancer is cervical cancer. In embodiments, the cancer is colon cancer. In embodiments, the cancer is head and neck cancer. In embodiments, the cancer is Hodgkin lymphoma. In embodiments, the cancer is liver cancer. In embodiments, the cancer is lung cancer. In embodiments, the cancer is renal cell cancer. In embodiments, the cancer is melanoma. In embodiments, the cancer is uveal melanoma. In embodiments, the cancer is stomach cancer. In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is prostate cancer. In embodiments, the cancer is rectal cancer. In embodiments, the cancer is a squamous cancer, e.g., a laryngeal squamous cell carcinoma, skin squamous cell carcinoma, oral cavity squamous cell carcinoma, esophageal squamous cell carcinoma, or lung squamous cancer.

[0268] In embodiments, the method includes administering to the subject a therapeutically effective amount of the bispecific antibody or monospecific antibody. In embodiments, the method includes administering to the subject a therapeutically effective amount of the bispecific antibody or monospecific antibody in the form of an antibody drug conjugate (ADC).

[0269] The cytotoxic payload region of an antibody drug conjugate generally falls into three major categories: microtubule-disrupting, DNA-modifying and RNA-modifying drugs. In embodiments, the drug in an ADC is monomethyl auristatin E (MMAE), which is a synthetic antineoplastic agent which inhibits cell division by blocking the polymerization of tubulin. As known in the art, because of its toxicity, MMAE cannot be used as a drug itself; instead, it must be linked to an antibody to which directs the drug to cancer cells. In other embodiments, the drug in an ADC is exatecan, which is a drug that is a structural analog of camptothecin that is a DNA modifying topoisomerase I inhibitor which is more potent than other clinical TOPI inhibitors. Any anti -neoplastic, chemotherapeutic, cytotoxic, and the like drug that is useful in killing cancer cells and can be conjugated to a bispecific antibody or monospecific antibody of the present disclosure may be used herein in an ADC. Examples of other conjugatable drugs include monomethyl auristatin F (MMAF) or other auristatins; DM1, DM4, or other maytansinoids, SN-38, Dxd or other camptothecin derivatives; pyrrolobenzodiazepines (PBDs) or other DNA alkylators; duocarmycins, NAMPT inhibitors, TLR agonists, STING agonists, small molecule cell cycle inhibitors such as cell cycle blockers (e.g., Cdk4 / 6 inhibitors including Palbociclib (Ibrance), Riboci clib (Kisqali), and Abemaciclib (Verzenio)) and inhibitors of anti-apoptotic proteins (e.g., Bcl2 family proteins, including Bel -2, Bcl-XL and Mcl 1 inhibitors, Inhibitor of Apoptosis Proteins (IAPS), and Caspase Inhibitors), or other common ADC payloads. These payloads may be used in combination on the same antibody.

[0270] In embodiments, the linker conjugating a drug to a bispecific antibody or monospecific antibody of the present disclosure is a cleavable linker that is stable in extracellular fluid, but is cleaved by proteases once the conjugate has entered a cancer cell, thus activating the cytotoxic mechanism of the drug. The cleavable linker may be a val-cit-PABC linker, a GGFG linker, or another linker. In other cases, a non-cleavable linker connects the antibody and cytotoxic payload. Other linkers and especially linkers useful for conjugating a drug to an antibody of the present disclosure can be used herein. Other linkers can be found at the world wide web (www) medchemexpress.com; especially in the sections relating to “ADC Linker” and including the recited cleavable linkers and non-cleavable linkers. The contents of which as of the present filing date is incorporated by references herein in its entirety.

[0271] In embodiments, the method provided herein includes administering to the subject a therapeutically effective amount of ADC wherein the administration of the ADC results in cancer cell death. In embodiments, the therapeutically effective amount of a bispecific ADC results in about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,

[0272] 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

[0273] 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

[0274] 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

[0275] 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% greater decrease in tumor volume or number of cancer cells than the sum of the decrease when the monospecific EGFR and / or CDCP1 antibodies are administered.

[0276] In embodiments, the method includes administering to the subject a combination therapy including a bispecific antibody provided herein including embodiments thereof, with an anti-PD- L1 or an anti-PD-1 antibody. The combination of a bispecific antibody provided herein including embodiments thereof with an anti-PD-Ll or an anti-PD-1 antibody provides potent activity in increasing T cell activation. In embodiments, the combination therapy (e.g. combination of a bispecific antibody provided herein including embodiments thereof with an anti-PD-Ll or an anti- PD-1 antibody) increases T cell activation compared to T cell activation by treatment with the anti- PD-Ll or an anti-PD-1 antibody alone or bispecific antibody alone. In embodiments, T cell activation is measured by one or more markers of T cell activation. In embodiments, T cell activation is measured by a cytometric method. In embodiments, T cell activation is measured by detection of CD8+ T cells. In embodiments, T cell activation is measured by IL -2 expression or IFN-gamma expression. In embodiments, T cell activation is measured by cytotoxic activity e.g. cancer cell death).

[0277] In embodiments, the combination therapy including a bispecific antibody provided herein including embodiments thereof with an anti-PD-Ll or an anti-PD-1 antibody results in about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,

[0278] 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,

[0279] 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,

[0280] 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,

[0281] 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% greater T cell activation compared to T cell activation from treatment with a monotherapy including the bispecific antibody or anti-PD-Ll or an anti-PD-1 antibody alone. In embodiments, the combination therapy including a bispecific antibody provided herein including embodiments thereof with an anti-PD-Ll or an anti -PD-1 antibody results in at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,

[0282] 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,

[0283] 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,

[0284] 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,

[0285] 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% greater T cell activation compared to T cell activation from treatment with a monotherapy including the bispecific antibody or anti-PD-Ll or an anti-PD-1 antibody alone.

[0286] In embodiments, the combination therapy including a bispecific antibody provided herein including embodiments thereof with an anti-PD-Ll or an anti-PD-1 antibody results in about 0.1- fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 1.5-fold, 2- fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold greater T cell activation compared to T cell activation from treatment with a monotherapy including the bispecific antibody or anti-PD- Ll or an anti-PD-1 antibody alone.

[0287] In embodiments, the method provided herein includes administering to the subject a therapeutically effective amount of a bispecific antibody and anti-PD-Ll or an anti -PD-1 antibody, as described above, wherein the combination has synergistic effect. In embodiments, the synergistic effect is more than a sum of effects from individual administration of the bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody.

[0288] In instances, the synergistic effect is cancer cell death. Cancer cell death can be quantified, for example, by a decrease in tumor volume or decrease in the number of cancer cells. In embodiments, the cancer is brain cancer, breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, renal cell cancer, melanoma, uveal melanoma, stomach cancer, pancreatic cancer, prostate cancer, rectal cancer, or a squamous cancer, e.g., a laryngeal squamous cell carcinoma, skin squamous cell carcinoma, oral cavity squamous cell carcinoma, esophageal squamous cell carcinoma, or lung squamous cancer. In embodiments, synergy between the bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody may result in about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,

[0289] 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,

[0290] 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,

[0291] 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15,

[0292] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

[0293] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

[0294] 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

[0295] 94, 95, 96, 97, 98, 99, or 100% greater decrease in tumor volume or number of cancer cells than the sum of the decrease when the bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody are used individually and separately.

[0296] In embodiments, synergy between the bispecific antibody and anti-PD-Ll or an anti-PD- lantibody may result in 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0 9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,

[0297] 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,

[0298] 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,

[0299] 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3,

[0300] 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16,

[0301] 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

[0302] 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,

[0303] 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

[0304] 95, 96, 97, 98, 99, or 100% greater T cell activation than the sum of T cell activation when the bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody are used individually and separately.

[0305] For the method provided herein, in embodiments, the effective amount of a bispecific antibody and anti-PD-Ll or an anti-PD-lantibody are a combined additive amount. In embodiments, the effective amount of a bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody are a combined synergistic amount. In embodiments, the bispecific antibody and anti- PD-Ll or an anti-PD-lantibody are administered sequentially or simultaneously.

[0306] A “synergistic amount” as used herein refers to the sum of a first amount (e.g., a bispecific antibody) and a second amount (e.g, anti-PD-Ll or an anti-PD-lantibody) that results in a synergistic effect (i.e. an effect greater than an additive effect). Therefore, the terms “synergy”, “synergism”, “synergistic”, “combined synergistic amount”, and “synergistic therapeutic effect” which are used herein interchangeably, refer to a measured effect of the compound administered in combination where the measured effect is greater than the sum of the individual effects of each of the compounds provided herein administered alone as a single agent. More specifically, a “combined synergistic amount” is a combined amount of a first agent (e.g. a bispecific antibody) and second agent (e.g. anti-PD-Ll or an anti-PD-1 antibody) effective to provide a synergistic effect (e.g. for treating cancer, including embodiments described herein). In embodiments, the methods herein including administering bispecific antibody and anti-PD-Ll or an anti-PD- 1 antibody, include administering a combined synergistic amount of the bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody. In embodiments, the pharmaceutical compositions herein including a bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody, include a combined synergistic amount of the bispecific antibody and anti-PD-Ll or an anti-PD-1 antibody.

[0307] In embodiments, a synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the amount of the bispecific antibody provided herein when used separately from the anti-PD-Ll or an anti-PD-1 antibody.

[0308] Definitions

[0309] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0310] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure. The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (z.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or one or more elements.

[0311] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0312] The term “and / or” should be understood to mean either one, or both of the alternatives.

[0313] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are present that materially affect the activity or action of the listed elements.

[0314] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in a particular embodiment.

[0315] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0316] In the present description, any value range, e.g.. concentration range, percentage range, or ratio range, or integer range and the like, is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

[0317] The term “increased” or “enhanced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0318] The term “decrease” or “reduced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0319] The term monospecific antibody, and the like, refers to any antibody format disclosed herein that recognizes a single antigen, e.g., EGFR or CDCP1. The terms monospecific antibody and monovalent antibody are synonymous. In contrast, a bispecific antibody, and the like, refers to any antibody format disclosed herein that recognizes at least two antigens, e.g., EGFR and CDCP1. A monospecific antibody may be biparatopic in that it recognizes two epitopes of the same antigen, e.g., CDCP1 or EGFR, or may be multiparatopic in that it recognizes more than two epitopes of the same antigen, e.g., CDCP1 or EGFR. Abispecific antibody may also be biparatopic or multiparatopic in that it recognizes two more epitopes of a first antigen, e.g., CDCP1, and recognizes two more epitopes of a second antigen, e.g., EGFR. Any combination of bispecifics, biparatopics, and multipartopics is considered. For example, a bispecific antibody may have two antibody binding domains each of which recognizes a different CDCP1 antigen and have two antibody binding domains that both recognize the same EGFR epitope.

[0320] The term “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule / composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[0321] “Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0322] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moi eties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non- covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amio acid on a protein or polypeptide through a covalent, non- covalent or other interaction.

[0323] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g, phosphorami date, phosphorodiamidate, phosphor othioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0324] Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0325] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0326] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0327] As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0328] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0329] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0330] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0331] An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5’-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0332] The terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein “corresponds” to a given residue when it occupies the same essential structural position within the protein as the given residue. One skilled in the art will immediately recognize the identity and location of residues corresponding to a specific position in a protein in other proteins with different numbering systems. For example, by performing a simple sequence alignment with a protein the identity and location of residues corresponding to specific positions of the protein are identified in other protein sequences aligning to the protein. For example, a selected residue in a selected protein corresponds to glutamic acid at position 138 when the selected residue occupies the same essential spatial or other structural relationship as a glutamic acid at position 138. In some embodiments, where a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein aligning with glutamic acid 138 is the to correspond to glutamic acid 138. Instead of a primary sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the glutamic acid at position 138, and the overall structures compared. In this case, an amino acid that occupies the same essential position as glutamic acid 138 in the structural model is the to correspond to the glutamic acid 138 residue.

[0333] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0334] The following eight groups each contain amino acids that are conservative substitutions for one another:

[0335] 1) Alanine (A), Glycine (G);

[0336] 2) Aspartic acid (D), Glutamic acid (E);

[0337] 3) Asparagine (N), Glutamine (Q);

[0338] 4) Arginine (R), Lysine (K);

[0339] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0340] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0341] 7) Serine (S), Threonine (T); and

[0342] 8) Cysteine (C), Methionine (M)

[0343] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site at www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0344] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, etal., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873- 5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0345] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g. , Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0346] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0347] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0348] Antibodies are large, complex molecules (molecular weight of -150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region, involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions (also referred to herein as light chain variable (VL) domain and heavy chain variable (VH) domain, respectively) come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3-dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework region, which forms the environment for the CDRs. In embodiments, the framework region is humanized.

[0349] An “antibody variant” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains (e.g., light chain variable domain, heavy chain variable domain) of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgGs, scFv, bispecific antibodies, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A“peptibody” as provided herein refers to a peptide moiety attached (through a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO97 / 49805 and WO 97 / 49805 which are incorporated by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish and the variable regions thereof and methods for their production, isolation, and use may be found in W02005 / 118629, which is incorporated by reference herein in its entirety and for all purposes.

[0350] The terms “CDR LI”, “CDR L2” and “CDR L3” as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N-terminal to C- terminal direction a CDR LI, a CDR L2 and a CDR L3. Likewise, the terms “CDR Hl”, “CDR H2” and “CDR H3” as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a CDR Hl, a CDR H2 and a CDR H3.

[0351] The terms “FR LI”, “FR L2”, “FR L3” and “FR L4” as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N-terminal to C-terminal direction a FR LI, a FR L2, a FR L3 and a FR L4. Likewise, the terms “FR Hl”, “FR H2”, “FR H3” and “FR H4” as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a FR Hl, a FR H2, a FR H3 and a FR H4. In embodiments, the framework region is humanized.

[0352] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL), variable light chain (VL) domain or light chain variable region and variable heavy chain (VH), variable heavy chain (VH) domain or heavy chain variable region refer to these light and heavy chain regions, respectively. The terms variable light chain (VL), variable light chain (VL) domain and light chain variable region as referred to herein may be used interchangeably. The terms variable heavy chain (VH), variable heavy chain (VH) domain and heavy chain variable region as referred to herein may be used interchangeably. The Fc (z.e. fragment crystallizable region) is the “base” or “tail” of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0353] The term “antibody” is used according to its commonly known meaning in the art. Antibodies exist, e.g, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)’2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)’2 dimer into an Fab’ monomer. The Fab’ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)). The term “antibody” as referred to herein further includes antibody variants such as single domain antibodies. Thus, in embodiments an antibody includes a single monomeric variable antibody domain. Thus, in embodiments, the antibody, includes a variable light chain (VL) domain or a variable heavy chain (VH) domain. In embodiments, the antibody is a variable light chain (VL) domain or a variable heavy chain (VH) domain.

[0354] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor etal., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). “Monoclonal” antibodies (mAb) refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0355] A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. The linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.

[0356] A“diabody” is used according to its ordinary meaning in the art and refers to an scFv dimer including the variable heavy (VH) and variable light (VL) regions of an antibody, where the dimers are connected by a linker.

[0357] The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a lx, 5x, lOx, 20x or lOOx excess of one antibody inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans etal., Cancer Res. 50: 1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. For preparation of suitable antibodies of the invention and for use according to the invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93 / 08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121 :210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0358] Methods for humanizing or primatizing non-human antibodies are well known in the art (e.g., U.S. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321 :522; and Verhoyen et al. (1988) Science 239: 1534). Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Morrison etal., PNAS USA, 81:6851-6855 (1984), Jones etal., Nature 321 :522-525 (1986); Riechmann etal., Nature 332:323- 327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988), Verhoeyen et al.. Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. For example, polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells.

[0359] A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. The preferred antibodies of, and for use according to the invention include humanized and / or chimeric monoclonal antibodies.

[0360] The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, ALaboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0361] A “ligand” refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a receptor or antibody, antibody variant, antibody region or fragment thereof.

[0362] Techniques for conjugating therapeutic agents to antibodies are well known (see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery” in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982)). As used herein, the term “antibody-drug conjugate” or “ADC” refers to a therapeutic agent conjugated or otherwise covalently bound to an antibody.

[0363] The term “EGFR” as used herein includes human EGFR (hEGFR), variants, isoforms, and species homologs of EGFR, and analogs having at least one common epitope with hEGFR. Cancers associated with EGFR expression or overexpression include Adenocarcinoma Of The Gastroesophageal Junction, Anal Canal Squamous Cell Carcinoma, Anal Carcinoma, Anaplastic Astrocytoma, Anaplastic Oligoastrocytoma, Anaplastic Oligodendroglioma, Astrocytoma, B-Cell Non-Hodgkin Lymphoma, Bile Duct Carcinoma, Biliary Tract Carcinoma, Bladder Carcinoma, Breast Carcinoma, Bronchogenic Carcinoma, Cancer, Carcinoma, Cervical Carcinoma, Cervical Squamous Cell Carcinoma, Cholangiocarcinoma, Chordoma, Colorectal Adenocarcinoma, Colorectal Carcinoma, Diffuse Astrocytoma, Diffuse Intrinsic Pontine Glioma, Diffuse Midline Glioma, H3 K27M-Mutant, Endometrial Carcinoma, Ependymoma, Esophageal Carcinoma, Esophageal Squamous Cell Carcinoma, Fallopian Tube Carcinoma, Gallbladder Carcinoma, Gastric Adenocarcinoma, Gastric Carcinoma, Gastrointestinal Stromal Tumor, Glioblastoma, IDH-Wildtype, Glioblastoma, Glioma, Gliosarcoma, Head And Neck Carcinoma, Head And Neck Squamous Cell Carcinoma, Hepatobiliary Neoplasm, Hepatocellular Carcinoma, High Grade Ovarian Serous Adenocarcinoma, Hypopharyngeal Squamous Cell Carcinoma, Kidney Carcinoma, Laryngeal Squamous Cell Carcinoma, Lip And Oral Cavity Carcinoma, Low Grade Glioma, Lung Adenocarcinoma, Lung Carcinoma, Lung Neuroendocrine Neoplasm, Lymphoma, Malignant Central Nervous System Neoplasm, Malignant Glioma, Malignant Hepatobiliary Neoplasm, Malignant Laryngeal Neoplasm, Malignant Salivary Gland Neoplasm, Malignant Solid Tumor, Malignant Supratentorial Neoplasm, Malignant Uterine Neoplasm, Medulloblastoma, Melanoma, Meningioma, Mesothelioma, Multiple Myeloma, Nasal Cavity And Paranasal Sinus Carcinoma, Nasopharyngeal Carcinoma, Neuroblastoma, Neuroendocrine Carcinoma, Non-small cell lung cancer (NSCLC), Non-Small Cell Lung Carcinoma, Non-Squamous Non-Small Cell Lung Carcinoma, Oral Cavity Carcinoma, Oral Cavity Squamous Cell Carcinoma, Oropharyngeal Carcinoma, Oropharyngeal Squamous Cell Carcinoma, Ovarian Carcinoma, Ovarian Carcinosarcoma, Ovarian Epithelial Tumor, Pancreatic Adenocarcinoma, Pancreatic Carcinoma, Pancreatic Ductal Adenocarcinoma, Pituitary Gland Carcinoma, Primary Peritoneal Carcinoma, Primitive Neuroectodermal Tumor, Prostate Carcinoma, Renal Cell Carcinoma, Rhabdomyosarcoma, Sarcoma, Skin Squamous Cell Carcinoma, Small Cell Lung Carcinoma, Small Intestinal Carcinoma, Soft Tissue Sarcoma, Squamous Cell Lung Carcinoma, Thymic Carcinoma, Thyroid Gland Undifferentiated (Anaplastic) Carcinoma, Ureter Carcinoma, Urothelial Carcinoma, Uterine Carcinosarcoma, Uveal Melanoma, and WHO Grade III Glioma.

[0364] The term “CDCP1” as used herein includes human CDCP1 (hCDCPl), variants, isoforms, and species homologs of CDCP1, and analogs having at least one common epitope with hCDCPl.

[0365] Cancers associated with CDCP1 expression or overexpression include breast cancer (e.g., triple-negative breast), lung cancer, colorectal cancer, cervical cancer, urothelial cancer, gastric cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, a hematopoietic system cancer, or a metastatic cancer.

[0366] For specific proteins described herein, the named protein includes any of the protein’s naturally occurring forms, variants or homologs that maintain the protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference, homolog or functional fragment thereof.

[0367] The term “gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene.

[0368] The terms “plasmid”, “vector” or “expression vector” refer to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, the gene and the regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0369] The terms “transfection”, “transduction”, “transfecting” or “transducing” can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral -based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8: 1-4 and Prochiantz (2007) Nat. Methods 4: 119-20.

[0370] A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0371] When the label or detectable moiety is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA, TETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups. The chelate is normally linked to the PSMA antibody or functional antibody fragment by a group, which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelates, when complexed with nonradioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223Ra for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an A1-18F complex, to a targeting molecule for use in PET analysis.

[0372] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. antibodies and antigens) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated; however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0373] The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a pharmaceutical composition as provided herein and a cell. In embodiments contacting includes, for example, allowing a pharmaceutical composition as described herein to interact with a cell (e.g. a T cell).

[0374] A “cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0375] The term “recombinant” when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0376] The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0377] The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g, a fusion protein).

[0378] The term “exogenous” refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an “exogenous promoter” as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. Conversely, the term “endogenous” or “endogenous promoter” refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0379] As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasing proliferation) or killing the cell. In some embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein (e.g. EGFR protein, CDCP1 protein, or intracellular proteins bound to EGFR or CDCP1). Similarly, an “inhibitor” is a compound or protein that inhibits a receptor or another protein, e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0380] The term “modulate”, “modulates”, “modulating”, “modulation” and the like relates to a change in a process, e.g., in an amount of the process and the rate of a process. The change may be an increase or may be a decrease.

[0381] The term “activate”, “increase”, “enhance”, “potentiate”, “amplify” and the like relates to an increase in a process, e.g., in an amount of the process and the rate of a process.

[0382] As defined herein, the term “activate”, “increase”, “enhance”, “potentiate”, “amplify” and the like in reference to cell proliferation (e.g., cancer cell proliferation) means positively affecting (e.g., increasing proliferation) or killing the cell. Thus, activation includes, at least in part, partially or totally enhancing stimulation, increasing, potentiating, or accelerating activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein (e.g. CD6 protein, or intracellular proteins bound to CD6). Similarly, an “agonist” is a compound or protein that activates a receptor or another protein, e.g., by binding, increasing, potentiating, accelerating, activating, sensitizing, or up-regulating activity (e.g., a receptor activity or a protein activity).

[0383] The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g, ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0384] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g, serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g, primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0385] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues). One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0386] “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0387] The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. The disease may be a cancer. The cancer may refer to a solid tumor malignancy. Solid tumor malignancies include malignant tumors that may be devoid of fluids or cysts. For example, the solid tumor malignancy may include brain cancer, breast cancer, ovarian cancer, pancreatic cancer, cervical cancer, gastric cancer, renal cancer, head and neck cancer, bone cancer, skin cancer or prostate cancer. In some further instances, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin’s lymphomas (e.g., Burkitt’s, Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including acute myeloid leukemia (AML), ALL, and CML), or multiple myeloma. In embodiments, the cancer is a squamous cancer, e.g., a laryngeal squamous cell carcinoma, skin squamous cell carcinoma, oral cavity squamous cell carcinoma, esophageal squamous cell carcinoma, or lung squamous cancer.

[0388] As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g., humans), including leukemia, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound or method provided herein include brain cancer, breast cancer, colon cancer, kidney cancer, leukemia, lung cancer, melanoma, uveal melanoma, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, liver cancer, gastric cancer or a sarcoma. In embodiments, the cancer is a squamous cancer, e.g., a laryngeal squamous cell carcinoma, skin squamous cell carcinoma, oral cavity squamous cell carcinoma, esophageal squamous cell carcinoma, or lung squamous cancer.

[0389] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adj acent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and / or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.

[0390] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g. a protein associated disease (e.g., cancer, inflammatory disease, autoimmune disease, or infectious disease)) means that the disease (e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease.

[0391] The term “signaling pathway” as used herein refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components. The term “aberrant” as used herein refers to different from normal. When used to describe enzymatic activity, aberrant refers to activity that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non-disease- associated amount (e.g. by using a method as described herein), results in reduction of the disease or one or more disease symptoms.

[0392] A “therapeutic agent” as referred to herein, is a composition useful in treating or preventing a disease such as cancer (e.g., leukemia). In embodiments, the therapeutic agent is an anti-cancer agent. “Anti-cancer agent” is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In embodiments, an anticancer agent is a chemotherapeutic. In embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer.

[0393] As used herein, “treating” or “treatment of’ a condition, disease or disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently. As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

[0394] The terms “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of disease prior to onset or recurrence.

[0395] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

[0396] By “therapeutically effective dose or amount” as used herein is meant a dose that produces effects for which it is administered (e.g. treating or preventing a disease). The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2- fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease. A therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease.

[0397] As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g.. a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be co-administered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0398] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, / .< ., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0399] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0400] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0401] The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0402] The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0403] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0404] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0405] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0406] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0407] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

[0408] EXAMPLES Example 1: Screening for CDCP1 VHH binders.

[0409] Immunizations of a single llama and a single alpaca were performed. The llama was immunized with mRNA encoding the full-length transcript of human CDCP1, and subsequently encapsulated in lipid nanoparticles. The alpaca was immunized with recombinant protein corresponding to the extracellular domain (ECD) of human CDCP1, with a C-terminal His6 tag. At multiple points along the immunization process, bleeds were collected, and serum evaluated by ELISA or flow cytometry for antigen-positive titers. Upon immunization completion, whole blood was drawn, and peripheral blood mononuclear cells (PBMCs) cryopreserved.

[0410] Llama PBMCs were thawed and sorted for CDCP1 -reactive B cells using biotinylated CDCP1 ECD. cDNA was prepared from sorted B cells, and VHH genes amplified for cloning into a mammalian expression vector to yield VHH-human Fc antibodies. These antibodies were then screened for binding to CDCP1.

[0411] Total RNA was purified from alpaca PBMCs, and mRNA converted to cDNA. VHH genes were amplified for cloning into a phagemid expression vector and library construction. Panning was performed on immobilized CDCP1-ECD over several rounds, enriching for high-affinity CDCP1 VHH fragments. After panning completion, individual phage clones were isolated and binding to CDCP1 was confirmed by phage ELISA. A subset of hits was subcloned into a mammalian expression vector to yield VHH-human Fc antibodies. These antibodies were then confirmed to bind CDCP1 by flow cytometry and / or biolayer interferometry (BLI). Table 8 shows the binding affinity and epitope bin of selected CDCP1 binders.

[0412] EXAMPLE 2: Determination of CDCP1 antibody binding using biolayer interferometry (BLI)

[0413] CDCP1 VHH antibodies were immobilized on AHC biosensors and then recombinant human CDCP1 extracellular domain was used as an analyte at three concentrations. Sensorgrams were fit to a 1 : 1 global model and KD, kon, and koir rates were extracted. Antibodies with fitted KDS of under 1 nM are reported as “<1 nM”. To determine the binding epitope, the experiment was repeated using recombinant CDCP1 of only residues 1-343 instead of the full length ECD. Antibodies that still bound are reported as being in epitope bin 1, while antibodies that did not bind to the truncated protein are reported as being in epitope bin 3. “N.b.” indicates there was no or minimal binding in the BLI assay, and “n d ” means there was binding but a good fit could not be achieved. Competition binding assays were performed to further refine epitope bin 1, wherein recombinant his-tagged full-length CDCP1 ECD was immobilized on HIS1K biosensors. In this setup, one antibody was used as analyte, followed by a second antibody; if the second antibody was unable to bind, then the antibodies were determined to bind overlapping epitopes. In this way, two antibodies that bind to a unique epitope in bin 1 were identified, and this is reported as Epitope bin 2.

[0414] Table 8.

[0415] An alignment of CDCP1 antibody variable domain sequences was performed. FIG. 1 shows the range of sequence diversity. Example 3: Screening for EGFR binders using biolayer interferometry (BLI).

[0416] A series of anti-EGFR antibodies were evaluated for binding to EGFR using biolayer interferometry. EGFR antibodies were immobilized on AHC biosensors and then recombinant human EGFR extracellular domain was used as an analyte at three concentrations. Sensorgrams were fit to a 1 : 1 global model and KD, kOn, and koff rates were extracted. Table 9 shows the binding affinities of anti-EGFR antibodies to the recombinant ECD of human EGFR by BLI.

[0417] Table 9.

[0418] An alignment of EGFR antibody variable domain sequences was performed. FIG. 2A and FIG. 2B show the sequence diversity of EGFR heavy and light chain variable domain sequences, respectively. Sequence alterations were engineered to improve conformational stability and eliminate any sequence liabilities that impact expression, purity and stability.

[0419] Example 4: Generation of bispecific CDCP1-EGFR antibodies

[0420] Combinations of the CDCP1 and EGFR binding domains described in the examples above were joined into bispecific molecules capable of binding to both CDCP1 and EGFR. FIG. 3A to FIG. 3E show illustrative formats for EGFR x CDCP1 bispecific antibodies. CDCP1 binding domains corresponding to PROT ID NOs C2019-C5297 (Table 8) and EGFR-binding domains corresponding to PROT ID NOs: C046-C6012 (Table 9) were made and joined into bispecific molecules.

[0421] Example 5: Bispecific CDCP1-EGFR molecules selectively internalize on SKHEP1 cells expressing both CDCP1 and EGFR.

[0422] An important characteristic for the activity of antibody drug conjugates is the ability to be rapidly internalized by target cells and release a cytotoxic payload intracellulary, thereby inducing cell killing. To identify bispecific pairs that were internalized selectively when both CDCP1 and EGFR were expressed, a comparison of internalization assays using WT SKHEP1 cells that express both CDCP1 and EGFR vs. SKHEP1 cells in which CDCP1 was deleted (CDCP1 KO (knockout)), were performed. SKHEP1 cells are endothelial cells isolated from the liver of a male with adenocarcinoma. A Fab pHast Internalization assay was used to evaluate multiple bispecific antibodies simultaneously. WT or CDCP1 KO SKHEP1 cells were plated at 2 x 105cells per well in black plates with clear bottoms and allowed to attach overnight. The following day a 10X mixture of the antibody, at 10X the final concentration indicated, and 500 nM Fab pHast reagent (Advanced Targeting Systems, PH-01) was prepared and allowed to sit for 20 minutes in the dark at room temperature. Then 10 pL of this mixture was added to 90 pL of media in the plate. Cells were then returned to the incubator. 19 hours later the media / antibody / dye mixture was removed and replaced with PBS. Plates were then imaged on a Neo2 plate reader with an excitation of 532 and an emission of 560. This signal was normalized to cells treated with just Fab pHast and no antibody to calculate a relative fluorescence (RFU).

[0423] FIG. 4 is a plot of unique CDCP1 x EGFR bispecific antibodies analyzed in receptor internalization assays using SKHEP1 cells that expressed EGFR and CDCP1 (y-axis) or only expressed EGFR (x-axis). Each symbol represents one bispecific and symbols are coded by their EGFR arm as indicated in the legend.

[0424] The PROT IDs in the legend correspond to the EGFR binder, each paired with different CDCP1 binders. The majority of bispecific antibodies were internalized to a greater degree when both CDCP 1 and EGFR were expressed.

[0425] To initially determine whether CDCP1 binders corresponding to distinct epitope bins could influence internalization, monovalent CDCPlxFc binders were tested for their ability to internalize on WT SKHEP1 cells, which express high levels of CDCP1.

[0426] FIG. 5 shows the internalization activity of CDCP1 x Fc antibodies corresponding to distinct CDCP1 epitope bins (described in Example 2) analyzed in an antibody internalization assay.

[0427] The equilibrium dissociation constant for each anti-CDCPl antibody was plotted against the internalization value of the corresponding monovalent CDCP1 x Fc antibody. This shows that antibodies that bind to the membrane-proximal domain of CDCP1 (bin 3, black circles) tend to be poor internalizers, while some that bind to the membrane-distal domain of CDCPl(bins 1+2, white circles) are stronger internalizers.

[0428] Example 6: CDCP1-EGFR bispecific ADCs induce killing of tumor cells preferentially over non-tumor cells

[0429] A subset of bispecific antibodies that showed selective internalization on WT SKHEP1 cells compared to SKHEP CDCP1 KO cells (shown in FIG. 5) were evaluated in cell cytotoxicity assays. The cancer cell lines SW48, H1975 and HCC827 and primary human keratinocytes all express both CDCP1 and EGFR. To screen the ability of a subset of our bispecific library to selectively and potently kill cancer cells we employed a Protein A-MMAE cell viability assay. Indicated cell lines were plated in 96 well plates at 2.5 to 5.0 x 104cells per well and allowed to attach to the plate overnight. The following day, the concentration of antibody as indicated was added to the well along with 1 nM Protein A-MMAE reagent (Levena biosciences, LEV-100- AME). Plates were returned to the incubator for 72 hours. After 72 hours 10 pL of alamarBlue was added to each well and then plates were incubated for 4 hours at 37° C. Plates were then read out on a Biotek Synergy Neo2 Plate reader at 570 and 600 and the specific absorbance was calculated by subtracting the absorbance of a media only control. These numbers were then normalized to cells treated with just Protein A-MMAE and no antibody to calculate the relative viability. Bispecifics that kill cancer cells at 0.125nM without killing keratinocytes at 0.25nM were potent and selective cancer cell killers (FIG. 6). On the x-axis the relative viability for that bispecific antibody in this assay at 0.25 nM in normal keratinocytes is plotted. On the y-axis the mean viability in this assay in three cancer cell lines (SW48, H1975, and HCC827) at 0.125nM is plotted. Bispecifics are indicated based on which EGFR bin they belong to as indicated in the legend. The PROT IDs in the legend correspond to the EGFR binder, each paired with different CDCP1 binders. A subset of antibodies exhibited the desired profde of potent killing of cancer cells (low viability) with minimal killing activity of keratinocytes (high viability).

[0430] Example 7: Selection of illustrative bispecific antibodies

[0431] Based on the findings from preliminary internalization and cytotoxicity assays, seventeen CDCP1-EGFR bispecific antibodies were selected for further characterization. Table 10 shows the EGFR and CDCP1 PROT IDs of the seventeen illustrative bispecifics selected for further analysis. Table 10.

[0432] The selected CDCP1 IDs represented a range of affinities to CDCP1. FIG. 7 shows an isoaffinity plot of monospecific CDCP1 antibodies binding to recombinant human CDCP1 ECD by BLI. Kinetic rate constants for the anti-CDCPl antibodies in the first screen were determined as described in Example 2. For antibodies that bound, these values were plotted and isoaffinity lines shown in gray with the relevant KD labeled above the plot. The CDCP1 antibodies included in the seventeen bispecifics shown in Table 10 are shown as filled black circles, while those not selected are shown as open white circles.

[0433] The internalization activity of binders against EGFR epitopes in the context of CDCP1- EGFR bispecifics was evaluated in the presence or absence of CDCP1 expression. FIG. 8A to FIG. 8D shows plots of bispecific antibody internalization activity in WT (black circles) and CDCP1 knockout (open triangles) SKHEP1 cells for four different EGFR epitope bins. Relative fluorescence from an antibody internalization assay is plotted on the y-axis, and the concentration of the test article is plotted on the x-axis. EGFR epitope bins corresponding to C230 and C047 paired with CDCP1 binders showed greater internalization and selectivity compared to C046 and C065 in WT SKHEP1 vs CDCP1 KO SKHEP1 cells. These findings highlight that some EGFR epitopes show better bispecific internalization than others.

[0434] Selected CDCP1-EGFR bispecifics were then evaluated in cytotoxicity assays in SW48 and HCC827 cells, both of which express CDCP1 and EGFR. FIG. 9A to FIG. 9D shows activity in cell viability assays of bispecific IDs from Table 10 generated using C046 and C065 as the EGFR binding arm coupled to Protein-A MMAE (as described above). Bispecifics were each compared to EGFR x Fc single arm binders (open circles), e.g., C1259 and C2581. C046 and C065 previously showed limited selectivity on killing of tumor cells and keratinocytes (see FIG. 6).

[0435] FIG. 9E to FIG. 9H shows activity in cell viability assays of bispecific SEQ IDs from Table 10 generated using C230 and C047 as the EGFR binding arm coupled to Protein-A MMAE (as described above). Bispecifics were each compared to EGFR x Fc single arm binders (open circles), e.g., C 1113 and C 1114. C230 and C047 previously showed enhanced selectivity on killing tumor cells and keratinocytes (see FIG. 6).

[0436] Additional internalization assays were performed to compare the ability of bispecific antibodies to induce enhanced internalization as compared to monovalent EGFR or CDCP1 binding.

[0437] FIG.10A to FIG. 10D shows internalization activity of bispecific antibodies (solid and open circles) in WT and CDCP1 knockout SKHEP1 cells with matched monovalent EGFR (solid and open triangles) and CDCP1 (solid and open squares) controls for four selectively internalizing bispecific molecules. In these assays, C2518, C2533, C2523 and C2512 all showed enhanced internalization on WT SKHEP1 cells as compared to CDCP1 KO SKHEP1 cells, and greater internalization than their corresponding monovalent EGFR x Fc or CDCP1 x Fc binders.

[0438] The selected bispecifics were further evaluated for internalization activity in additional cancer cell lines that express both EGFR and CDCP1. FIG. 11 A to FIG. 11D shows the internalization activity of four selectively internalizing bispecific antibodies in SW48, HCC827, Hl 975 and HT29 cell lines, as indicated, with matched monovalent EGFR x Fc and CDCP1 x Fc controls. SW48 and HT29 are human colorectal cancer cell lines, whereas HCC827 and H1975 are non-small cell lung cancer (NSCLC) cell lines. The Fab pHast internalization assay was performed as described above. In each figure four EGFR x CDCP1 bispecifics are graphed in black circle, square, and upside down and upside right triangles. The EGFR single arm control is indicated by a diamond, e.g.. Cl 114. The CDCP1 single arm controls are graphed with open symbols corresponding to the bispecific they are contained in, C2556, C2567, C2562, and C2577. The y-axis indicates the relative fluorescence, and the x-axis indicates the log nM concentration of the test article.

[0439] To better define the relative contribution of the CDCP1 VHH binders to internalization of each bispecific, the internalization activity of selected CDCP1 VHHs was evaluated on WT SKHEP1 cells. FIG. 12 shows that C2556 was especially effective in its ability to internalize when compared to other VHH binders.

[0440] Example 8: Bispecifics with detuned EGFR binders and humanized CDCP1

[0441] To improve selectivity of EGFR - CDCP1 bispecific antibodies to favor dependency on the presence of both CDCP1 and EGFR for enhanced internalization and cell killing, a series of mutant EGFR binder variants derived from PROT ID Cl 114 were generated to reduce affinity to EGFR binding, i.e. , create “detuned” EGFR binders. Table 11 lists the PROT IDs of detuned EGFR x Fc binders and their KD as determined by Octet.

[0442] Table 11.

[0443] The monovalent EGFR x Fc parent (PROT ID Cl 114) and detuned binders were evaluated for their cell killing activity in HCC827 and SW48 cell viability assays using Protein A-MMAE coupling and measured by alamarBlue. FIG. 13 shows that in HCC827 and SW48 cells, the majority of detuned EGFR x Fc binders showed reduced killing activity relative to the parent Cl 114 (black diamond).

[0444] Next, the EGFR detuned variants in FIG. 13 were engineered as bispecifics with CDCP1. The killing activity of these bispecifics with optimized EGFR and CDCP1 sequences was evaluated in viability assays using Protein A-MMAE labeled antibodies and measured by alamarBlue. Unexpectedly, FIG. 14 indicates that the EGFR detuned bispecifics retained potency in the Protein A-MMAE assay despite loss of binding affinity. Both C2512 (Matuzumab with D006) and C4662 (Humanized D006) have the same (not detuned) EGFR arm; C2512 has the original D006 and C4662 has the humanized D006. The other molecules in this graph all have the humanized CDCP1 paired with the various detuned EGFR binders. This finding indicates that cotargeting EGFR and CDCP1 using a bispecific ADC with reduced affinity towards EGFR binding maintains potent cytotoxic activity that could be more selective for tumor cells expressing both targets and provide a greater therapeutic index. One EGFR x CDCP1 bi specific (C4680) with a detuned EGFR binding arm was compared to the activity of monovalent EGFR x Fc or CDCP1 x Fc binders in cell viability assays using Protein A-MMAE labeled antibodies, and measured by alamarBlue. FIG. 15 shows that in both SW48 and HCC827 cell lines, the EGFR x CDCP1 bispecific (black squares, C4680) showed greater killing activity over the monovalent EGFR x Fc (black triangle, C4683) or CDCP1 x Fc (black circle, C4665) controls, showing selectivity for co-expressing cells.

[0445] The detuned EGFR variants were generated as EGFR x CDCP1 bispecifics using another CDCP1 binder with weaker binding and which targets a different epitope. FIG. 16 shows that a reduced binding affinity to CDCP1 resulted in a loss in potency of EGFR x CDCP1 bispecifics that contained a detuned EGFR binding arm (C4663, C4669, C4673, C4677, and C4681) compared to the non-detuned EGFR binder (C2523, open diamond).

[0446] FIG. 17 compares the killing activity of a detuned EGFR variant as a monovalent binder (C4683) or paired with CDCP1 (C4681), relative to the killing activity of a higher affinity EGFR variant as a monovalent binder (Cl 114) or paired with CDCP1 (C4663).

[0447] Example 9: Evaluation of bispecifics generated as knob-in-hole (KiH)

[0448] Selected EGFR and CDCP1 binders were engineered into bispecific antibodies using knobin-hole heterodimerization technology to improve purity and yield and further evaluated. FIG. 18A shows graphs of cell viability assays on indicated cancer cell lines using EGFR x CDCP1 bispecific antibodies with varying EGFR affinity generated using knob-in-hole technology and directly conjugated to vcMMAE to an average DAR of ~2.7. These antibodies were stochastically conjugated on their broken interchain disulfides. The DAR is then measured for each individual antibody after the conjugation. Typically, ADCs conjugated this way will be targeting a DAR of approximately 4; here, a DAR of approximately 3 was sought. Bispecifics (closed circle and square), matching monovalent EGFR controls (corresponding open symbol), and a non-targeting control (open diamond) are shown, and viability was measured by alamarBlue. Two EGFR binders with varying binding affinities were evaluated. Across the majority of cell lines evaluated, the bispecifics showed enhanced killing activity as compared to the corresponding monovalent EGFR x Fc binders (C4992 and C4996) or the Fc x Fc control (C4993).

[0449] To confirm that the cytotoxic activity of bispecific EGFR x CDCP1 antibodies was derived from the conjugated vcMMAE payload and not the antibody itself, viability assays comparing the killing activity of an unconjugated vs. vcMMAE conjugated bispecific EGFR x CDCP1 antibody was compared in cancer cell lines. FIG. 18B shows graphs of cell viability assays indicating that dose dependent killing activity is observed only when the EGFR x CDCP1 bispecific antibody is conjugated with a cytotoxic payload.

[0450] Some primary cells, such as keratinocytes and other cells of epithelial origin, express EGFR and CDCP1 to varying levels. To determine whether the EGFR x CDCP1 bispecific antibodies could selectively kill tumor cells over primary cells, the panel of bispecifics and corresponding monovalent binding controls were evaluated for their killing activity in primary cells. FIG. 19 shows graphs of cell viability assays on indicated primary cells using EGFR x CDCP1 bispecific antibodies (closed circle and square), monovalent EGFR controls (corresponding open symbols), and a non-targeting control (diamond), as measured by alamarBlue. Compared to cancer cell lines, the killing activity of the EGFR x CDCP1 bispecifics on primary cells was reduced. This could be attributed to the differential spatial surface localization of EGFR and CDCP1 on primary vs. tumor cells, which may influence internalization, payload delivery, and subsequent cell killing, highlighting selective killing of tumor cells over primary cells.

[0451] CDCP1 has been reported to be cleaved by extracellular proteases. However, biochemical, biophysical, and structural characterization has shown that the two cleaved fragments of CDCP1 remain tightly associated, with minimal proteolysis-induced conformational change. SW756 and SKMES1 cells are known to contain full-length CDCP1, whereas HCC827 cells contain a cleaved form of CDCP1. SW756 and SKMES1 cells (squamous cell carcinoma cell lines) were treated with 500nM plasmin for 30 minutes, lysed and CDCP1 was evaluated by Western blot. FIG. 20A shows that endogenous full-length (FL) CDCP1 is cleaved in the presence of plasmin in SW756 and SKMES1 cells and is in a cleaved form in HCC827 cells. To determine if an EGFRxCDCPl ADC and a monovalent CDCP1 ADC that binds distal to the CDCP1 cleavage site could bind to both cells with full-length and cleaved forms of CDCP1, CDCP1 binding assays on SW756, SKMES1 and HCC827 either treated with plasmin or not were performed using flow cytometry. FIG. 20B shows that a CDCP1 antibody which targets the distal region of CDCP1 can bind to full length and plasmin cleaved CDCP1 in SW756 and SKMES1 cells and binds to endogenously cleaved CDCP1 in HCC827 cells. EGFR x CDCP1 bispecific antibodies comprised of a CDCP1 binding arm that binds distal to the CDCP1 cleavage site were evaluated for their killing activity in SW756 cells in the presence or absence of plasmin. FIG. 20C shows that CDCP1 x EGFR bispecific antibodies lead to equivalent killing of SW756 cells in the presence or absence of plasmin treatment in a cell viability assay, as measured by alamarBlue. EGFR x CDCP1 bispecific antibodies comprised of a CDCP1 binding arm that binds distal to the CDCP1 cleavage site were evaluated for their killing activity in HCC827 cells, which contain cleaved CDCP1. FIG. 20D shows that EGFR x CDCP1 bispecific antibodies lead to killing of HCC827 cells which contain endogenously cleaved CDCP1.

[0452] Without wishing to be bound by theory, bispecific antibody drug conjugates show activity with a range of drug payloads and conjugation strategies. Two common antibody drug conjugation strategies include interchain disulfide conjugation and engineered cysteine conjugation. EGFR x CDCP1 bispecific antibodies conjugated to vcMMAE via interchain disulfides (C4990, C4991) or by direct conjugation to engineered cysteine residues (C1989, C1991) were compared in cell viability assays using SW48, H1975-T7 (anon-small cell lung cancer cell line), or BxPC3 (human pancreatic cancer cell line) cells. FIG. 21 shows that in all three cell lines, the interchain disulfide MMAE conjugated EGFR x CDCP1 bispecific antibodies had increased killing activity compared to the engineered cysteine conjugated antibodies.

[0453] A spacer peptide sequence is often needed between a VHH and antibody hinge region. Bispecifics herein were made using 3 different linker sequences, including EPKSA (C5204), GGSGG x2 (C5206) and GGSGG (C5208). These linkers, respectively, have SEQ ID NOs: 1319, 1318, and 1317. FIG. 22 shows that EGFR x CDCP1 bispecific antibodies with different spacer sequences show equivalent killing of SW48 cells in a cell viability assay as measured by alamarBlue. An anti-human-Fc Fab conjugated to MMAE was employed for this cell viability assay. Indicated cell lines were plated in 96 well plates at 2.5 to 5.0 x 104cells per well and allowed to attach to the plate overnight. The following day, the concentration of antibody as indicated was added to the well along with 20 nM anti-human-Fc-MMAE reagent (Moardec, Cat # (AH-202AE- 50). Plates were returned to the incubator for 72 hours. After 72 hours 10 pL of alamarBlue was added to each well and then plates were incubated for 4 hours at 37° C. Plates were then read out on a Biotek Synergy Neo2 Plate reader at 570 and 600 and the specific absorbance was calculated by subtracting the absorbance of a media only control. These numbers were then normalized to cells treated with just Protein A-MMAE and no antibody to calculate the relative viability.

[0454] The cytotoxic payload region of an antibody drug conjugate generally falls into three major categories: microtubule-disrupting, DNA-modifying and RNA-modifying drugs. Monomethyl auristatin E (MMAE), a member of the auristatin family, is a synthetic anti neoplastic agent that prevents cell division by blocking tubulin polymerization and acts as a microtubule disruptor. MMAE is frequently used as a cytotoxic payload in ADCs and is a component of several FDA approved ADC-based therapies. MMAE is most commonly used with a val-cit-PABC linker (vcMMAE), but may be used with other linkers. Exatecan is another commonly used payload. Exatecan is a DNA modifying topoisomerase I inhibitor that is more potent than other clinical TOPI inhibitors. EGFR x CDCP1 bispecific antibodies conjugated with a GGFG linker to an exatecan payload (GGFG-exatecan) at either DAR 2 or DAR 8 were generated and evaluated in cell viability assays. For the DAR 2 ADCs, GGFG-exatecan was conjugated to engineered cysteines. For the DAR 8 ADCs, GGFG-exatecan was conjugated to both engineered cysteines and interchain cysteines. EGFR x CDCP1 bispecifics with varying EGFR binding affinities and engineered cysteines were conjugated either to GGFG-exatecan at DAR 2 (C1993, C1995) or conjugated to vcMMAE at DAR 2 (C1989, C1991). These ADCs were subjected to a viability assay wherein they were incubated with the indicated cell lines for six days and then viability was assessed using CellTiter-Glo. FIG. 23A shows that the vcMMAE DAR 2 conjugated EGFR x CDCP1 bispecifics (C1989, C 1991 ) were more potent at killing the majority of cell lines evaluated than the GGFG-exatecan DAR 2 EGFR x CDCP1 bispecifics (C1993, C1995). Both the MMAE and exatecan conjugated DAR 2 bispecifics were more potent than the corresponding single arm EGFR x Fc DAR 2 exatecan binders with varying EGFR affinities (Cl 994, Cl 996).

[0455] EGFR x CDCP1 bispecifics with varying EGFR binding affinities were engineered with DAR 8 GGFG-exatecan (Cl 997, Cl 999) and compared to EGFR x CDCP1 DAR 2 vcMMAE antibodies with varying EGFR affinities (C1989, C 1991 ). FIG. 23B shows that in the majority of cell lines evaluated, the GGFG-exatecan DAR 8 conjugated EGFR x CDCP1 bispecific (C1997) had comparable killing activity to the vcMMAE DAR 2 conjugated EGFR x CDCP1 bispecific (C1989). In some cell lines, the EGFR x CDCP1 bispecifics with reduced EGFR binding affinity showed comparable activity whether as DAR 8 GGFG-exatecan (Cl 999) or DAR 2 vcMMAE (C1991). In some cell lines the EGFR x CDCP1 bispecifics with reduced EGFR binding affinity engineered as GGFG-exatecan DAR 8 (Cl 999) or vcMMAE DAR 2 (Cl 991) showed similar killing activity to the corresponding single arm EGFR x Fc binders (C1998, C2000).

[0456] Binding affinities to human and cynomolgus monkey EGFR and CDCP1 were determined using surface plasmon resonance (SPR) for select EGFR x CDCP1 bispecific antibodies. For SPR binding analysis, antibodies were captured by goat anti-human IgG antibody that was immobilized on the sensor chip. Analytes (recombinant ECD of human and cynomolgus monkey EGFR or CDCP1) were introduced at four concentrations. Sensorgrams were globally fit to a 1 :1 binding model to determine KDs. Table 12 shows the equilibrium dissociation constants of bispecifics for the indicated targets as measured by SPR.

[0457] Table 12:

[0458] Example 10

[0459] Another EGFR binder was combined with a CDCP1 binder into a bispecific antibody using knobin-hole heterodimerization technology to improve purity and yield and further evaluated. FIG. 24 is a graph depicting the ability of sequence optimized EGFR x CDCP1 antibodies incubated with anti-human-Fc-MMAE, to kill WT (black symbols) or CDCP1 KO (open symbols) cancer cells compared to EGFR arms alone, in a cell viability assay, as measured by alamarBlue. FIG. 25 is a graph depicting the ability of EGFR x CDCP1 bispecific antibody with sequence optimizations incubated with anti -Hum an-Fc-MMAE, as indicated, to kill cancer cells, compared to EGFR arms alone, in a cell viability assay, as measured by alamarBlue. Across the cell lines evaluated, the bispecifics showed enhanced killing activity in EGFR / CDCP1 dual positive cells compared to EGFR positive and CDCP1 negative cells.

[0460] Example 11:

[0461] In this example, EGFR and CDCP1 expression and EGFR x CDCP1 bispecific antibodies of the present disclosure are characterized in in vitro assays.

[0462] EGFR and CDCP1 are co-expressed in tumors FIG. 26A includes representative immunohistochemical (IHC) images for HNSCC, NSCLC, cervical, and bladder cancers with high co-expression of EGFR and CDCP1. FIG. 26B includes representative IHC images for selected normal tissues showing limited co-expression of EGFR and CDCP1. FIG. 26C is a graph showing EGFR and CDCP1 co-expression across a pan-cancer tumor microarray and reveals that EGFR and CDCP1 are co-expressed in a number of cancer types but not in normal tissue. FIG. 27 shows representative IHC images and a graph showing coexpression of EGFR and CDCP1 is elevated in metastatic samples.

[0463] EGFR x CDCP1 bispecific antibody internalizes better than EGFR and CDCP1 antibodies

[0464] FIG. 28 are graphs showing that EGFR x CDCP1 bispecific antibodies unexpectedly internalize better than EGFR and CDCP1 mono-specific antibodies. This effect may be due to selective cointernalization over the mono-specific antibodies, specifically dependent upon EGFR and CDCP1 tumor-associated target proximity. To assess the ability of antibodies to internalize an Incucyte internalization assay was used. The plots show the increase in fluorescence intensity over time, with the bispecific antibody showing the greatest internalization at 24 hours. Cells were plated at 2 x 104cells per well in 96 well plates and allowed to adhere overnight. The following day a mix of 20nM antibody and 60nM FabFluor (Sartorius) was prepared in the appropriate cell media and allowed to conjugate for 15 minutes at 37°. 50pL of this mixture was added to the 50pL of media in the wells for a final concentration of lOnM antibody. Plates were then imaged in the Incucyte at the time periods indicated and relative fluorescence intensity per well normalized to phase confluence was calculated on the Incucyte software. These antibody internalization assays reveal that EGFR x CDCP1 bispecific ADC internalizes better than monovalent and bivalent control ADCs in cancer cell lines with varying expression levels of EGFR and CDCP1.

[0465] E TFR x CDCP1 ADC is effective in vitro

[0466] FIG. 29A are graphs showing EGFR x CDCP 1 bispecific antibody-drug conjugates (ADC) display potent cytotoxic activity in vitro. Viability assays were performed in cancer cell lines with a range of EGFR and CDCP1 expression. These demonstrate that EGFR x CDCP1 bispecific ADCs are more effective than EGFR monovalent control ADC (C7828) and CDCP1 monovalent control ADC (C9373), and are at least as effective as EGFR bivalent control ADC (C8066) and CDCP1 bivalent control ADC (C8067). FIG. 29B is a graph showing that the EGFR x CDCP1 bispecific ADCs have substantially reduced cytotoxic activity against primary keratinocytes, bronchial- tracheal epithelial, and cervical epithelial cells. These data show that bispecific EC-conjugated DAR 2 vcMMAE ADCs, and their single- or no-arm control ADCs, display selectivity for tumor vs. primary cells. The cancer cells and primary cells were assessed for EGFR and CDCP1 expression. FIG. 30 is a graph comparing the relative expression of EGFR and CDCP1 in the indicated cancer cell lines and primary cells. The geometric MFI of binding of EGFR x EGFR or CDCP1 x CDCP1 antibodies was divided by the geometric MFI of binding of zero antibody to determine a fold over zero (FOZ) value for the expression of each target. For each cell line, values were obtained from the same plate and analyzed with identical settings on a flow cytometer. The value shown is the average plus / minus standard deviation of two replicate side-by-side wells. Despite comparable co-expression, the differential spatial surface localization of EGFR and CDCP1 on primary cells may influence internalization, payload delivery, and subsequent cell killing, differentiating tumor cells from primary cells.

[0467] EGFR x CDCP1 has minimal impact on ERK andAKT phosphorylation

[0468] The mechanism of action of many EGFR targeted therapies is to block EGF ligand binding to EGFR, which inhibits downstream signaling and the downstream signaling pathways that promote cell growth, proliferation, and survival. Therapies such as small molecule TKIs (tyrosine kinase inhibitors) or antibodies that target EGFR frequently utilize this mechanism of action. EGFR is highly expressed in normal tissues like the skin, so inhibiting its function there leads to common side effects like acneiform rash and dry skin. These on-target side effects of EGFR-targeted therapies, such as skin rash and diarrhea, are caused by inhibition of the EGFR pathway in both cancer cells and healthy tissues. Maintaining intact EGF signaling in a bispecific EGFR x CDCP1 ADC that has enhanced selectivity for tumor vs. normal tissue may serve to diminish some of the adverse events on healthy tissues seen with other EGFR targeting therapies.

[0469] FIG. 31A is a diagram of signaling molecules illustrating signal transduction pathways downstream of EGFR, and proteins downstream of EGFR that can become phosphorylated following a stimulus of EGF, leading to increased cell growth proliferation and survival.

[0470] FIG. 31B is a western blot showing that the EGFR x CDCP1 bispecific antibody has minimal impact on ERK and AKT phosphorylation in the presence of EGF ligand in two cancer cell lines with WT EGFR. Here is shown a western blot of EGFR and downstream signaling pathway activity after treatment with the bispecific EGFR x CDCP1 antibody, a monovalent EGFR antibody, a monovalent CDCP1 antibody, an Fc x Fc control, and two clinical EGFR blocking antibodies (C047 and C230). The bispecific antibodies impair some EGFR phosphorylation but this does not lead to suppression of downstream signaling. The table shows the treatment conditions for the different test articles. These data show that the bispecific antibodies of the present disclosure cause some inhibition of EGFR phosphorylation at high concentrations, but this does not lead to suppression of downstream signaling comparable to other approved EGFR blocking therapies. To assess the ability of the bispecific antibody to impact EGFR signaling lx 106cells per well were plated in six well plates and allowed to adhere overnight. The following day media was replaced with 0.5% serum and the indicated antibody at the indicated concentration. Cells were incubated in antibody for 24 hours, and in the final 15 minutes 10 ng EGF was spiked into the cells were indicated. Cell lysates were collected and a western blot was performed to detect the indicated proteins and phosphorylation. These experiments show that the bispecific antibodies of the present disclosure will have better tolerability over currently available therapies that block EGF signaling. In these data, the bispecific antibodies comprised sequences of C 192 as disclosed herein in Tables 1, 2, 4, and 5. FIG. 31C is a western blot showing that a bispecific EGFR x CDCP1 antibody causes some reduction in EGFR phosphorylation but does not potently suppress downstream signaling in primary human keratinocytes. This is in contrast to clinical EGFR blocking antibody biosimilars (C230 and C047) and a clinical EGFR targeted ADC biosimilar, which more potently suppress EGFR phosphorylation. The table shows the treatment conditions for the different test articles. Most EGFR targeted therapies block EGFR signaling, which can be associated with toxicity in normal tissue such as the skin. These data show that the bispecific antibodies of the present disclosure cause some inhibition of EGFR phosphorylation at high concentrations, but this does not lead to suppression of downstream signaling comparable to EGFR inhibiting antibodies used in the clinic in primary keratinocytes.

[0471] TKI resistant EGFR mutant cells are sensitive to EGFR x CDCP1 bi specific ADC

[0472] TKI (tyrosine kinase inhibitor) resistance occurs when cancer cells develop genetic mutations or activate alternative pathways that bypass the TKI’s effects, leading to treatment failure. This creates a critical need for new and effective second-line and later-line treatments, better strategies to overcome resistance, and improved ways to manage toxicity and quality of life. FIG. 32A is a graph showing an HCC827 cell line, which has an endogenous exon 19 deletion in EGFR that causes EGFR to be overactive, and is resistant to osimertinib, which is a drug that treats non-smallcell lung carcinomas with specific mutations. Osimertinib is a third-generation small molecule TKI that works by inhibiting the epidermal growth factor receptor (EGFR) which is overactive in many types of cancer. FIG. 32B is a graph showing that osimertinib-resistant EGFR mutant NSCLC cells respond similarly to a bispecific EGFR x CDCP1 ADC of the present disclosure. EGFR knockout reduces the response to the ADC confirming this is mediated in part by EGFR expression. The three cell lines used here include the mutant NSCLC cell line HCC827 that was generated to be resistant to the EGFR inhibitor osimertinib (HCC827 osimertinib Resistant), a parental passage matched HCC827 cells that are sensitive to osimertinib (HCC827), and the resistant cell line that had EGFR knocked out using CRISPR (HCC827 OSR1 EGFR KO). These data show that when EGFR is absent in the resistant cell line using CRISPR (HCC827 OSR1 EGFR KO) the cells lose sensitivity, indicating that some of this activity is still due to EGFR expression despite no dependence on EGFR signaling for cell growth. In these data, the bispecific antibodies comprised sequences of C4990 as disclosed herein in Tables 1, 2, 4, and 5.

[0473] These data show that CDCP1 is identified as a tumor associated proximity antigen (TAPA) for EGFR; EGFR and CDCP1 are co-expressed in a variety of tumor types; EGFRx CDCP1 bispecific antibody internalize better than EGFR or CDCP1 monospecific antibodies; EGFR x CDCP1 bispecific ADC show selectivity for cancer cells over normal cell types; EGFR x CDCP1 bispecific antibodies of the present disclosure do not inhibit signaling downstream of EGFR and EGFR x CDCP1 bispecific ADC is effective in TKI resistant and cleaved CDCP1 contexts.

[0474] Example 12: Generation of biparatopic or multiparatopic CDCP1 antibodies

[0475] Combinations of the CDCP1 binding domains described in Table 4 are engineered as biparatopic antibodies or multiparatopic antibodies capable of binding multiple, different epitopes on CDCP1.

[0476] Thus, the diagrams of FIG. 33A to FIG. 33E additionally relate to biparatopic antibodies and multiparatopic antibodies where the antibody binds to two (for bi-) or more (for multi-) distinct epitopes on the same antigen, here, CDCP1. The biparatopic antibodies and multiparatopic antibodies may comprise multiple unique CDCP1 binding regions that recognize distinct (which may be non-overlapping or overlapping) epitopes on CDCP1. Thus, in FIG. 33A for example, the left arm of a biparatopic binds to a first epitope of CDCP1 whereas the right arm of the biparatopic binds to a second epitope of CDCP1. In FIG. 33C, the antibody may be multiparatopic, in that it binds to more than two epitopes on the same antigen, here CDCP1. For example, the top left arm of a biparatopic binds to a first epitope of CDCP1, the top right arm of the biparatopic binds to a second epitope of CDCP1, the bottom left arm of the biparatopic binds to a third epitope of CDCP1; the bottom right arm may bind to a fourth epitope of CDCP1 or may bind one of the first, second, or third epitopes of CDCP1; any such and similar combination of binders is considered in the present disclosure. Moreover, the biparatopic or multiparatopic may bind to the same molecule of CDCP1, albeit at different epitopes, or it may bind to the first epitope on a first molecule of CDCP1 and to a second epitope on a second molecule of CDCP1, where the first and second epitopes are distinct epitopes, or the first and second epitopes may be on a first molecule of CDCP1 and the third epitope is on a second molecule of CDCP1; any such and similar combination of binders is considered in the present disclosure.

[0477] Some illustrative antibodies comprise Fc regions either having a knob or a hole (see FIG. 33B and FIG. 33E). Here, the knob is shown as the circular protrusion and the hole as the indentation. In other cases, the biparatopic antibodies or multiparatopic antibodies comprise Fc regions lacking a knob and a hole fused to an alpaca derived VHH in various formats; see FIG. 33A and FIG. 33C. In other cases antibodies lack Fc regions and are comprised of only an alpaca-derived VHH that binds CDCP1 (see FIG. 33D). CDCP1 binding domains corresponding to PROT ID Nos C2019- C5297 (Table 4) were / can be engineered into bivalent antibodies.

[0478] Combinations of the CDCP1 binding domains described in Table 4 and / or the EGFR binding domains in Table 1 are engineered as biparatopic antibodies capable of binding multiple, different epitopes on CDCP1 and / or multiple, different epitopes on EGFR. Thus, the diagrams in FIG. 3A to FIG. 3G additionally relate to biparatopic antibodies, where the antibody binds to two distinct epitopes on the same antigens. Thus, in FIG. 3B for example, the left arm of a biparatopic binds to a first epitope of EGFR whereas the right arm of the biparatopic binds to a second epitope of EGFR. Moreover, the biparatopic may bind to the same molecule of EGFR, albeit at different epitopes, or it may bind to the first epitope on a first molecule of EGFR and to a second epitope on a second molecule of EGFR, where the first and second epitopes are distinct epitopes. The biparatopic antibodies may comprise multiple unique binding regions that recognize distinct (which may be non-overlapping or overlapping) epitopes on the antigen. In FIG. 3C, when the bi specific antibody is a biparatopic antibody, the top left arm of a biparatopic binds to a first epitope of EGFR, the top right arm of the biparatopic binds to a second epitope of EGFR, the bottom left arm of the biparatopic binds to a first epitope of CDCP1, and the bottom right arm of the biparatopic binds to a second epitope of CDCP1; the first and second epitopes may be on the same target molecule or may be on different target molecules. Example 13:

[0479] In this example, EGFR x CDCP1 bispecific antibodies of the present disclosure are characterized in in vivo tumor models.

[0480] EGFR x CDCP1 bispecific ADC is effective in vivo

[0481] FIG. 34A is an immunohistochemistry image showing co-expression of EGFR and CDCP1 in a pancreatic tumor (BxPC3) xenograft. FIG. 34B shows tumor size changes over time for BxPC3 xenografts formice either treated with an EGFR x CDCP1 bispecific ADC of the present disclosure or an Fc control. Mice were treated on day 10 and then again on day 56 as indicated by the arrows. Tumor growth inhibition (TGI) was assessed at day 45 (d45) post-engraftment. Two-way ANOVA with Tukey’s all groups comparison test; **, p<0.01. FIG. 34C is a Kaplan-Meier curve showing survival for the BxPC3 xenografted mice either treated with a bispecific EGFR x CDCP1 ADC of the present disclosure or an an Fc control. Mice were treated on day 10 and then again on day 56 as indicated by the arrows. Mantel-Cox (survival) test; ****, p<0.0001. In these data, thebispecific antibodies comprised sequences of Cl 989 as disclosed herein in Tables 1, 2, 4, and 5.

[0482] Target expression in CDX tumor models

[0483] FIG. 35 are images showing EGFR and CDCP1 expression by IHC in various Cell Line-Derived Xenograft (CDX) models: colorectal cancer (CRC) and Non-Small Cell Lung Cancer (NSCLC). The tumor cell line-derived xenograft models were selected to represent a range of target expression. Expression level is scored on a scale of 0-4 as shown inset.

[0484] EGFR x CDCP1 has dose-dependent efficacy and exposure in CDX tumor models

[0485] In FIG. 36A, Tumor growth inhibition (TGI) and survival of subcutaneous xenograft tumors in female athymic nude mice is shown. Tumor bearing mice were administered a single intravenous dose of an EGFR x CDCP1-MMAE DAR2 bispecific ADC at 0.3, 1 and 3 mg / kg, or an Fc x Fc control antibody or ADC. TGI vs control (Group 1) is shown (n=8 mice per group). Survival events are recorded when tumors exceed 800mm3. Two-way ANOVA (tumor growth) or Mantel-Cox (survival) test; *, p<0.05, **, p<0.01, ****, p<0.0001. FIG. 36B shows an in vivo mouse PK analysis of an EGFR x CDCP1-MMAE DAR2 bispecific ADC. Samples for analysis were collected at 1, 24, 96 and 168 hour post-dose, (n=2-3 mice per timepoint) and analyzed using an Fc-capture followed by either anti-MMAE or anti-Kappa antibody detection to measure either total MMAE-conjugated antibody or total antibody, respectively. The decreased antibody levels using the anti-MMAE capture method (dashed lines) indicate that some MMAE payload is dissociated from the EGFR x CDCP1 bispecific ADC over time, which is expected. Deconjugation rate (% conjugated) was calculated by dividing the ng / mL concentration of total MMAE-conjugated antibody by the respective ng / mL concentration of total antibody then multiplying by 100 to generate a percentage conjugation for each individual mouse at each timepoint. FIG. 36C shows tumor bearing mice that were treated with a single dose of an EGFR x CDCP1-MMAE DAR2 bispecific ADC or a monovalent control ADC, with the EGFR x CDCP1 bispecific ADC providing dose-dependent efficacy in CDX tumor models. Here, tumor growth inhibition (TGI) of subcutaneous xenograft tumors and survival in female mice was evaluated. Tumor bearing mice were administered a single intravenous dose of EGFR x CDCP1-MMAE DAR2 bispecific ADC at 0.3, 1 and 3 mg / kg, or molar equivalent doses of relevant monovalent control ADCs. TGI vs control (Group 1) is shown (n=8 mice per group). Survival events are recorded when tumors exceed 800mm3. Two-way ANOVA (tumor growth) or Mantel-Cox (survival) test; *, p<0.05, **, p<0.01; ***, p<0.001; ****, p<0.0001. FIG. 36D shows tumor bearing mice that were treated with a single dose of a bispecific EGFR x CDCP1-ADC with either MMAE-DAR2 or topoisomerase 1 inhibitor-DAR4 or an Fc control antibody, with the EGFR x CDCP1 bispecific ADCs providing dose-dependent efficacy in a CDX tumor model. Here, tumor growth inhibition (TGI) of subcutaneous xenograft tumors and survival in female mice was evaluated. Tumor bearing mice were administered a single intravenous dose of EGFR x CDCP1 MMAE-DAR2 or topoisomerase 1 inhibitor-DAR4 bispecific ADC at 1 and 3 mg / kg, or the molar equivalent dose of an Fc control antibody. TGI vs control (Group 1) is shown (n=8 mice per group). Survival events are recorded when tumors exceed 800mm3. Two-way ANOVA (tumor growth) or Mantel-Cox (survival) test; ****, p<0.0001. FIG. 36E shows tumor bearing mice that were treated with a single dose of an EGFR x CDCP1 -bi specific ADC with topoisomerase 1 inhibitor (DAR4) or an Fc control ADC, with the EGFR x CDCP1 bispecific ADC providing dose-dependent efficacy in a CDX tumor model. Here, tumor growth inhibition (TGI) of subcutaneous xenograft tumors and survival in female mice was evaluated. Tumor bearing mice were administered a single intravenous dose of EGFR x CDCP1 topoisomerase 1 inhibitor-DAR4 bispecific ADC at 1 and 3 mg / kg, or the molar equivalent dose of an Fc control ADC. TGI vs control (Group 1) is shown (n=8 mice per group). Survival events are recorded when tumors exceed 800mm3. Two-way ANOVA (tumor growth) or Mantel-Cox (survival) test; *, p<0.05, **, p<0.01. Target expression in PDX tumor models

[0486] FIG. 37 are images showing EGFR and CDCP1 expression by IHC in patient-derived xenograft (PDX) models of Non-Small Cell Lung Cancer (NSCLC). PDX models of NSCLC were selected based on dual target expression by mRNA and retrospectively confirmed by immunohistochemistry. Each numbered “NSCLC” was derived from a separate patient. For IHC, tumors were harvested from Fc control ADC treated tumor bearing mice, fixed in 10% neutral buffered formalin, and stained for EGFR and CDCP1 (n=3). Expression level is scored on a scale of 0-4 as shown inset. Representative images are shown from a subset of PDX models.

[0487] EGFR x CDCP1 has potent activity against NSCLC PDX tumors in a mouse clinical trial

[0488] FIG. 38 shows results from tumor bearing mice treated with a single dose of an EGFR x CDCP1- MMAE DAR2 bispecific ADC or control in a 3+3 mouse clinical trial design. Tumor growth of subcutaneous patient-derived xenograft (PDX) tumors was assayed in female mice. Tumor bearing mice were administered a single intravenous dose of an EGFR x CDCP1-MMAE DAR2 bispecific ADC at 3 mg / kg, or the molar equivalent dose of an Fc control ADC. Tumor volume was assessed twice weekly and reported as mean tumor volume in mm3 + / - SEM (n=3 mice per group). These data show that a bispecific EGFR x CDCP1 ADC of the present disclosure provides broad therapeutic activity and with best-in-class efficacy.

[0489] Example 13:

[0490] In this example, EGFR x CDCP1 bispecific antibodies are further characterized in in vitro biochemical assays.

[0491] Modulating EGFR affinity can influence selectivity of bispecific antibodies

[0492] FIG. 39A is a graph showing a series of EGFR x Fc monovalent antibodies with a range of affinities incubated with proA-MMAE killing cells with various potencies. The weakest-binding EGFR, C4679, and the tightest-binding EGFR, Cl 114, were then reformatted into bispecifics with a CDCP1 binder and conjugated with vcMMAE. FIG. 39B is a graph showing cytotoxic activity of these directly conjugated ADCs with different EGFR affinities as well as their corresponding single arm controls. The less potent EGFR x CDCP1 bispecific (C5193) is selective over the corresponding single arm EGFR control (C5185) and the corresponding single arm CDCP 1 control (C6011), whereas the more potent EGFR x CDCP1 bispecific (C5192) has similar activity to the corresponding single arm EGFR control (C5184). FIG. 39C are graphs showing unconjugated versions of these same bispecific antibodies bound to parental or CDCP1-KO cells and assayed by flow cytometry. The bispecific that bound more weakly to EGFR (C5179) showed greater difference in binding between parental and CDCP1-KO cells, thus indicating greater selectivity on tumor versus normal cells. The more tightly binding bispecific (C5178) has less selectivity and less dependence on CDCP1 -driven avidity. FIG. 40 are graphs showing binding of an EGFR x CDCP1 bispecific antibody by BLI. Here, the avidity of EGFR x CDCP1 bispecifics binding was determined by immobilizing different ratios of recombinantly-expressed target ECDs on a BLI biosensor and binding EGFR x CDCP1 bispecific as an analyte. The bispecific had a fast off-rate when bound to only EGFR, a medium off-rate when bound to only CDCP1, and the slowest off- rate when bound to a mix of both targets.

[0493] Engineered cysteine conjugation improves EGFR x CDCP1 bispecific antibody properties

[0494] Engineered cysteine conjugation involves introducing a single cysteine residue at a specific site for site-specific payload attachment, creating homogeneous antibody-drug conjugates. Interchain disulfide (ICD) conjugation is a traditional method that relies on reducing the antibody’s native interchain disulfide bonds to create multiple reactive cysteine residues, which results in a heterogeneous mixture of ADCs with varying drug-to-antibody ratios (DAR). In FIG. 41A, it is shown that bispecific ADCs conjugated to engineered cysteines (EC) did not aggregate when incubated in formulation buffer, unlike those conjugated on reduced interchain disulfides, and EC ADCs stably retained their drug linker when incubated in rat serum, as analyzed by LC-MS. In FIG. 41B, it is shown that bispecific ADCs conjugated to vcMMAE by ECs retained conjugation longer in vivo and enabled more durable anti-tumor responses than ICD ADCs. A one hour timepoint was not taken for the PK, so an average value of percent conjugation of the other ICD test ADCs in that study was illustratively used instead.

[0495] The EGFR x CDCP1 bispecific antibodies of the present disclosure are expressed at consistently high titer, yield, and purity; they have very high target specificity, they provide low PTM risk; they have excellent thermal, colloidal, and long-term storage stability; and they are amenable to standard manufacturing processes.

[0496] These data show that the EGFR x CDCP1 bispecific ADC of the present disclosure has potent and selective activity against a variety of tumor types and has excellent biophysical properties. REFERENCES

[0497] Adams, Mark N., et al. “EGF inhibits constitutive internalization and palmitoylation-dependent degradation of membrane-spanning procancer CDCP1 promoting its availability on the cell surface.” Oncogene 34.11 (2015): 1375-1383.

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[0499] Khan, Tashbib, et al. “The CDCP1 signaling hub: a target for cancer detection and therapeutic intervention.” Cancer research 81.9 (2021): 2259-2269.

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Claims

1. CLAIMSWhat is claimed is:

1. A bispecific antibody comprising at least one anti-EGFR antibody region and at least one anti-CDCPl antibody region, wherein the anti-CDCPl antibody region comprises a single variable domain on a heavy chain (VHH) or a fragment thereof.

2. The bispecific antibody of claim 1, wherein the bispecific antibody recognizes and binds to an EGFR antigen and to a CDCP1 antigen on a target cell.

3. The bispecific antibody of claim 2, wherein the target cell is a cancer cell that expresses or overexpress EGFR and / or CDCP1.

4. The bispecific antibody of claim 3, wherein the cancer cell expresses or overexpress EGFR and CDCP1.

5. The bispecific antibody of claims 3-4, wherein the cancer cell is a brain cancer, breast cancer (e.g., triple-negative breast) cell, a lung cancer cell, a colorectal cancer cell, an ovarian cancer cell, cervical cancer cell, urothelial cancer cell, gastric cancer cell, a kidney cancer cell, a liver cancer cell, a pancreatic cancer cell, a prostate cancer cell, a squamous cancer, hematopoietic system cancers or metastatic cancers.

6. The bispecific antibody of any one of claims 3 to 5, wherein the cancer cell is associated with non-small cell lung cancer (NSCLC), laryngeal squamous cell carcinoma, malignant salivary gland neoplasm, cholangiocarcinoma, skin squamous cell carcinoma, oral cavity squamous cell carcinoma, esophageal squamous cell carcinoma, lung squamous cancer, bladder carcinoma, malignant glioma, and diffuse astrocytoma.

7. The bispecific antibody of any one of claims 2 to 6, wherein binding of the bispecific antibody to antigens on the target cell promotes internalization of the antibody into the target cell.

8. The bispecific antibody of any one of claims 2 to 7, wherein binding and / or internalization of the bi specific antibody via antigens on the target cell promotes cytotoxicity of the target cell.

9. The bispecific antibody of claim 8, wherein cytotoxicity of the target cell is enhanced when the bispecific antibody is conjugated with one or more cytotoxic agents.

10. The bispecific antibody of any one of claims 7 to 9, wherein internalization of the bispecific antibody to the target cell promotes immunogenic cell death.

11. The bispecific antibody of any one of claims 2 to 10, wherein binding of the bispecific antibody to antigens on the target cell does not block receptor signaling from one or both of EGFR and CDCP1.

12. The bispecific antibody of any one of claims 1 to 11, wherein the bispecific antibody comprises an antibody region comprising a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 1 to Table 7.

13. The bispecific antibody of any one of claims 1 to 12, wherein the bispecific antibody comprises an antibody region comprising a sequence that is at least 90% identical (e.g, 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 1 to Table 3 and a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g, SEQ ID NO: 1291.

14. The bispecific antibody of any one of claims 1 to 13, wherein the bispecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in Table 7.

15. The bispecific antibody of claim 14, wherein the bispecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to one of SEQ ID NO: 1302 to SEQ ID NO: 1308, SEQ ID NO: 1311 to SEQ ID NO: 1314, and SEQ ID NO: 1316.

16. The bispecific antibody of any one of claims 1 to 15, wherein the bispecific antibody comprises an antibody region comprising one to six complementarity determining regions (“CDRs”) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the CDR sequence shown in Table 1 .

17. The bispecific antibody of claim 16, wherein the bispecific antibody comprises an antibody region comprising three Heavy Chain CDRs (CDR-H1, CDR-H2, and CDR-H3) sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 447.

18. The bispecific antibody of claim 17, wherein the bispecific antibody comprises CDR- Hl, CDR-H2, and CDR-H3 of SEQ ID NO: 445 to SEQ ID NO: 447.

19. The bispecific antibody of claims 16-18, wherein the bispecific antibody comprises an antibody region comprising three Light Chain CDRs (CDR-L1, CDR-L2, and CDR-L3) sequences from a single row of Table 1, e.g., SEQ ID NO: 448 to SEQ ID NO: 450.

20. The bispecific antibody of any one of claims 16 to 19, wherein the bispecific antibody comprises an antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR- L2, and CDR-L3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 450.

21. The bispecific antibody of any one of claims 16 to 20, wherein the bispecific antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 445 to SEQ ID NO: 450.

22. The bispecific antibody of any one of claims 1 to 21, wherein the bispecific antibody comprises an antibody region comprising one or more Variable Heavy (VH) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VH sequences shown in Table 2.

23. The bispecific antibody of any one of claims 1 to 22, wherein the bispecific antibody comprises an antibody region comprising one or more Variable Light (VL) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VL sequences shown in Table 2.

24. The bispecific antibody of any one of claims 1 to 23, wherein the bispecific antibody comprises an antibody region comprising one or more VH sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VH sequences shown in Table 2 and one or more VL sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VL sequences shown in Table 2.

25. The bispecific antibody of any one of claims 1 to 24, wherein the bispecific antibody comprises an antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VH sequence shown in a single row of Table 2, e.g., SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VL sequence shown in the row of Table 2, e.g., SEQ ID NO: 630.

26. The bispecific antibody of any one of claims 1 to 24, wherein the bispecific antibody comprises an antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 630.

27. The bispecific antibody of any one of claims 1 to 26, wherein an anti-EGFR antibody region is configured as a Fab, a single-chain variable fragment (scFv), and / or a single variable domain on a heavy chain (VHH).

28. The bispecific antibody of claim 27, wherein the anti-EGFR antibody region is configured as an scFv and comprises an antibody region comprising a sequence that is at least 90% identical (e.g, 95%, 99%, or 100% identical) to an scFv sequence shown in Table 3.

29. The bispecific antibody of any one of claims 1 to 28, wherein the bispecific antibody comprises at least two anti-EGFR antibody regions.

30. The bispecific antibody of any one of claims 1 to 29, wherein the bispecific antibody comprises at least three anti-EGFR antibody regions.

31. The bispecific antibody of any one of claims 12 to 30, wherein a bispecific antibody comprising a sequence that is less than 100% identical to a sequence shown in one or more of Table 1 to Table 3 retains affinity for EGFR that is substantially equivalent to a bispecific antibody comprising a sequence that is 100% identical to a sequence shown in one or more of Table 1 to Table 3.

32. The bispecific antibody of any one of claims 12 to 30, wherein a bi specific antibody comprising a sequence that is less than 100% identical to a sequence shown in one or more of Table 1 to Table 3 has less affinity for EGFR than a bispecific antibody comprising a sequence that is 100% identical to a sequence shown in one or more of Table 1 to Table 3.

33. The bispecific antibody of any one of claims 1 to 32, wherein the bispecific antibody comprises an antibody region identified by a PROT ID number in Table 9 or Table 11.

34. The bispecific antibody of any one of claims 1 to 33, wherein the bispecific antibody comprises an antibody region comprising one to three complementarity determining regions (“CDRs”) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the CDR sequence shown in Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

35. The bispecific antibody of claim 34, wherein the bispecific antibody comprises an antibody region comprising three Heavy Chain CDRs (CDR-H1, CDR-H2, and CDR-H3) sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

36. The bispecific antibody of claim 34 or claim 33, wherein the bispecific antibody comprises CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1115 to SEQ ID NO: 1117.

37. The bispecific antibody of any one of claims 1 to 36, wherein the bispecific antibody comprises an antibody region comprising one or more VHH sequences that are at least 90% identical (e.g, 95%, 99%, or 100% identical) to the VHH sequences shown in Table 5, e.g., SEQ ID NO: 1291.

38. The bispecific antibody of any one of claims 1 to 37, wherein the bispecific antibody comprises a sequence that is 90% identical (e.g, 95%, 99%, or 100% identical) to SEQ ID NO: 1291.

39. The bispecific antibody of any one of claims 1 to 38, wherein an anti-CDCPl antibody region is configured as a single variable domain on a heavy chain (VHH) or a fragment thereof.

40. The bispecific antibody of any one of claims 1 to 39, wherein the bispecific antibody comprises at least two anti-CDCPl antibody regions.

41. The bispecific antibody of any one of claims 1 to 40, wherein the bispecific antibody comprises at least three anti-CDCPl antibody regions.

42. The bispecific antibody of any one of claims 34 to 41, wherein a bispecific antibody comprising a sequence that is less than 100% identical to a sequence shown in Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g, SEQ ID NO: 1291, retains affinity for CDCP1 that is substantially equivalent to a bispecific antibody comprising a sequence that is 100% identical to a sequence shown in Table 4 or Table 5.

43. The bispecific antibody of any one of claims 34 to 41, wherein a bispecific antibody comprising a sequence that is less than 100% identical to a sequence shown in Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g, SEQ ID NO: 1291 has less affinity for CDCP1 than a bispecific antibody comprising a sequence that is 100% identical to a sequence shown in Table 4 or Table 5.

44. The bispecific antibody of any one of claims 1 to 43, wherein the bispecific antibody comprises an antibody region identified by a PROT ID number in Table 8.

45. The bispecific antibody of any one of claims 1 to 44, wherein the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 447, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 4, e.g, SEQ ID NO: 1115 to SEQ ID NO: 1117.

46. The bispecific antibody of any one of claims 1 to 45, wherein the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 445 to SEQ ID NO: 447, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 1115 to SEQ ID NO: 1117.

47. The bispecific antibody of any one of claims 1 to 46, wherein the bispecific antibody comprises a third antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 447, and / or comprises a fourth antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 4, e.g, SEQ ID NO: 1115 to SEQ ID NO: 1117.

48. The bispecific antibody of any one of claims 1 to 47, wherein the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ ID NO: 450, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CORED sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

49. The bispecific antibody of any one of claims 1 to 48, wherein the bispecific antibody comprises a first antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 445 to SEQ ID NO: 450, and comprises a second antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 1115 to SEQ ID NO: 1117.

50. The bispecific antibody of any one of claims 1 to 48, wherein the bispecific antibody comprises a third antibody region comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from a single row of Table 1, e.g., SEQ ID NO: 445 to SEQ IDNO: 450, and / or comprises a fourth antibody region comprising CDR-H1, CDR-H2, and CDR-H3 sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

51. The bispecific antibody of any one of claims 1 to 50, wherein the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VH sequence shown in Table 2, e.g., SEQ ID NO: 629, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in Table 5, e.g., SEQ ID NO: 1291.

52. The bispecific antibody of any one of claims 1 to 51, wherein the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 1291.

53. The bispecific antibody of any one of claims 1 to 52, wherein the bispecific antibody comprises a third antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VH sequence shown in Table 2, e.g., SEQ ID NO: 629, and / or comprises a fourth antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in Table 5, e.g., SEQ ID NO: 1291.

54. The bispecific antibody of any one of claims 1 to 53, wherein the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VH sequence shown in a single row of Table 2, e.g., SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VL sequence shown in the row of Table 2, e.g., SEQ ID NO: 630, and comprises a second antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in a single row of Table 5, e.g., SEQ ID NO: 1291.

55. The bispecific antibody of any one of claims 1 to 53, wherein the bispecific antibody comprises a first antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 629, and one VL sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to SEQ ID NO: 630, and comprises a second antibodyregion comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) SEQ ID NO: 1291.

56. The bispecific antibody of any one of claims 1 to 55, wherein the bispecific antibody comprises a third antibody region comprising a VH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VH sequence shown in a single row of Table 2, e.g., SEQ ID NO: 629, and one VL sequence that is at least 90% identical e.g., 95%, 99%, or 100% identical) to a VL sequence shown in the row of Table 2, e.g., SEQ ID NO: 630, and / or comprises a fourth antibody region comprising a VHH sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a VHH sequence shown in a single row of Table 5, e.g., SEQ ID NO: 1291.

57. The bispecific antibody of any one of claims 1 to 32, wherein the bispecific antibody comprises antibody regions identified by a PROT ID number in Table 10 or Table 12.

58. The bispecific antibody of any one of claims 1 to 57, wherein the bispecific antibody comprises one or more Fabs, comprises one or more scFvs, and / or comprises one or more VHHs.

59. The bispecific antibody of any one of claims 1 to 58, wherein the bispecific antibody comprises at least two Fabs.

60. The bispecific antibody of claim 59, wherein both Fabs comprises an anti-EGFR antibody region.

61. The bispecific antibody of any one of claims 1 to 58, wherein the bispecific antibody comprises at least one Fab and at least one VHH.

62. The bispecific antibody of claim 61, wherein the at least one Fab comprises an anti- EGFR antibody region and the at least one VHH comprises an anti-CDCPl antibody region.

63. The bispecific antibody of any one of claims 1 to 58, wherein the bispecific antibody comprises at least one Fab and at least one scFv.

64. The bispecific antibody of claim 63, wherein the at least one Fab comprises an anti- EGFR antibody region and the at least one scFv comprises an anti-EGFR antibody region.

65. The bispecific antibody of any one of claims 1 to 58, wherein the bispecific antibody comprises at least two Fabs and comprises at least one VHH.

66. The bispecific antibody of claim 65, wherein both Fabs comprise an anti-EGFR antibody region.

67. The bispecific antibody of any one of claims 65 to 66, wherein the at least one VHH comprises an anti-EGFR antibody region.

68. The bispecific antibody of any one of claims 65 to 66, wherein the at least one VHH comprises an anti-CDCPl antibody region.

69. The bispecific antibody of any one of claims 1 to 58, wherein the bispecific antibody comprises at least two Fabs and comprises at least one scFv.

70. The bispecific antibody of claim 69, wherein both Fabs comprise an anti-EGFR antibody region.

71. The bispecific antibody of any one of claims 69 to 70, wherein the at least one scFv comprises an anti-EGFR antibody region.

72. The bispecific antibody of any one of claims 1 to 58, wherein the bispecific antibody comprises at least two Fabs and comprises at least two VHHs.

73. The bispecific antibody of claim 72, wherein both Fabs comprise an anti-EGFR antibody region.

74. The bispecific antibody of any one of claims 72 to 73, wherein both VHHs comprise an anti-CDCPl antibody region.

75. The bispecific antibody of any one of claims 72 to 73, wherein one VHH comprises an anti-EGFR antibody region and one VHH comprises an anti-CDCPl antibody region.

76. The bispecific antibody of any one of claims 9 to 75, wherein the bispecific antibody is conjugated with one or more cytotoxic agents, thereby forming an antibody drug conjugate (ADC).

77. The bispecific antibody of claim 76, wherein the one or more cytotoxic agents are conjugated to a constant region of the bispecific antibody.

78. The bispecific antibody of claim 77, wherein the constant region of the bispecific antibody is an Fc region.

79. The bispecific antibody of claim 78, wherein the drug is conjugated to an amino acid position of the bispecific antibody’s Fc region that has been substituted with a cysteine.

80. The bispecific antibody of claim 79, wherein a conjugation site is at 375C, 360C, 149C, and / or a 140C substituted position.

81. The bispecific antibody of any one of claims 78 to 80, wherein the bispecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one SEQ ID NO: 1309-1316.

82. The bispecific antibody of any one of claims 9 to 81, wherein the one or more cytotoxic agents comprise a microtubule-disrupting drug, DNA-modifying drug, RNA-modifying drug, conjugated toxin, a small molecule, radioisotope, and / or a drug.

83. The bispecific antibody of any one of claims 9 to 82, wherein the one or more cytotoxic agents comprise monomethyl auristatin E (MMAE) and / or exatecan.

84. The bispecific antibody of claim 83, wherein the MMAE is conjugated via a val- cit-PABC linker or another linker or the exatecan is conjugated via a GGFG linker or other linker.

85. The bispecific antibody of any one of claims 9 to 84, wherein the one or more cytotoxic agents comprise monomethyl auristatin F (MMAF) or other auri statins; DM1, DM4, or other maytansinoids, SN-38, Dxd or other camptothecin derivatives; pyrrolobenzodiazepines (PBDs) or other DNA alkylators; duocarmycins, NAMPT inhibitors, TLR agonists, STING agonists, small molecule cell cycle inhibitors such as cell cycle blockers and inhibitors of anti-apoptotic proteins, and / or other common ADC payloads.

86. The bispecific antibody of any one of claims 1 to 84, wherein the bispecific is a biparatopic antibody or a multiparatopic antibody.

87. The bispecific antibody of claim 86, wherein the biparatopic antibody or the multiparatopic antibody binds one, two, or more epitopes from CDCP1 and one, two, or more epitopes from EGFR.

88. The bispecific antibody of claim 87, wherein the biparatopic antibody or the multiparatopic antibody binds one or two epitopes from CDCP1 and one or two epitopes from EGFR.

89. The bispecific antibody of claim 87, wherein the biparatopic antibody or the multiparatopic antibody binds one epitope from CDCP1 and one or two epitopes from EGFR.

90. The bispecific antibody of claim 87, wherein the biparatopic antibody or the multiparatopic antibody binds two or more epitopes from CDCP1 and one epitope from EGFR.

91. A method of killing a cancer cell, the method comprising contacting the cancer cell with an effective amount of the bispecific antibody of any one of claims 1 to 90.

92. The method of claim 91, wherein the cancer cell is in vitro, ex vivo, or in vivo.

93. A method of killing a cancer cell in a subj ect in need thereof, the method comprising contacting the cancer cell with an effective amount of the bi specific antibody of any one of claims 1 to 90.

94. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the bispecific antibody of any one of claims 1 to 90.

95. The method of claims 93 and 81, wherein the bispecific antibody provides dosedependent antitumor activity.

96. The method of any one of claims 93 to 95, wherein the bispecific antibody extends survival of a subject relative to a subject that is not administered the bispecific.

97. The method of any one of claims 93 to 95, wherein the bispecific antibody, when conjugated with one or more cytotoxic agents, displays potent cytotoxic activity against cancer cells.

98. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 90 and a pharmaceutically acceptable carrier, diluent, or excipient.

99. The pharmaceutical composition of claim 98, for use as a medicament in the treatment of cancer.

100. A method of killing a cancer cell in a subject in need thereof, the method comprising contacting the cancer cell with an effective amount of the pharmaceutical composition of any one of claims 98-99.

101. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 98-99.

102. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 98-99 and administering to the subject an effective amount of a standard of care therapy for the cancer.

103. The method of claim 102, wherein the pharmaceutical composition is administered before the standard of care therapy for the cancer.

104. The method of claim 102, wherein the pharmaceutical composition is administered simultaneous with the standard of care therapy for the cancer.

105. The method of claim 102, wherein the pharmaceutical composition is administered after the standard of care therapy for the cancer.

106. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 98-99, wherein the subject has previously been treated with the standard of care therapy for the cancer.

107. A polynucleotide or plurality of polynucleotides encoding the bispecific antibody of any one of claims 1 to 90.

108. A vector comprising the polynucleotide or plurality of polynucleotides of claim 107.

109. A monospecific antibody comprising at least one anti-CDCPl antibody region configured as a single variable domain on a heavy chain (VHH).

110. The monospecific antibody of claim 109, wherein the monospecific antibody recognizes and binds to a CDCP1 antigen on a target cell.

111. The monospecific antibody of claim 110, wherein the target cell is a cancer cell that expresses or overexpress CDCP1.

112. The monospecific antibody of claim 111, wherein the cancer cell is a brain cancer, breast cancer (e.g, triple-negative breast) cell, a lung cancer cell, a colorectal cancer cell, an ovarian cancer cell, cervical cancer cell, urothelial cancer cell, gastric cancer cell, a kidney cancer cell, a liver cancer cell, a pancreatic cancer cell, a prostate cancer cell, a squamous cancer, hematopoietic system cancers, or metastatic cancers.

113. The monospecific antibody of any one of claims 110 to 112, wherein binding of the monospecific antibody to antigens on the target cell promotes internalization of the antibody into the target cell.

114. The monospecific antibody of any one of claims 110 to 113, wherein binding of the monospecific antibody to antigens on the target cell promotes cytotoxicity of the target cell.

115. The monospecific antibody of claim 114, wherein cytotoxicity of the target cell is enhanced when the monospecific antibody is conjugated with one or more cytotoxic agents.

116. The monospecific antibody of any one of claims 110 to 115, wherein internalization of the monospecific antibody to the target cell promotes immunogenic cell death.

117. The monospecific antibody of any one of claims 110 to 116, wherein binding of the monospecific antibody to antigens on the target cellblocks receptor signaling from CDCP1.

118. The monospecific antibody of any one of claims 109 to 117, wherein the monospecific antibody comprises an antibody region comprising a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 4 to Table 7.

119. The monospecific antibody of any one of claims 109 to 118, wherein the monospecific antibody comprises an antibody region comprising a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g, SEQ ID NO: 1291.

120. The monospecific antibody of any one of claims 109 to 119, wherein the monospecific antibody comprises an antibody region comprising a sequence that is at least 90% identical (e.g, 95%, 99%, or 100% identical) to SEQ ID NO: 1115 to SEQ ID NO: 1117, or to SEQ ID NO: 1291.

121. The monospecific antibody of any one of claims 109 to 119, wherein the monospecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in Table 7.

122. The monospecific antibody of claim 121, wherein the monospecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to one ofSEQ ID NO: 1302 to SEQ ID NO: 1308, SEQ ID NO: 1311 to SEQ ID NO: 1314, and SEQ ID NO: 1316.

123. The monospecific antibody of any one of claims 109 to 121, wherein the monospecific antibody lacks a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in Table 7.

124. The monospecific antibody of any one of claims 109 to 123, wherein the monospecific antibody comprises an antibody region comprising one to three complementarity determining regions (“CDRs”) sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the CDR sequence shown in Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

125. The monospecific antibody of claim 124, wherein the monospecific antibody comprises an antibody region comprising three Heavy Chain CDRs (CDR-H1, CDR-H2, and CDR-H3) sequences from a single row of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117.

126. The monospecific antibody of any one of claims 109 to 125, wherein the monospecific antibody comprises an antibody region comprising one or more VHH sequences that are at least 90% identical (e.g., 95%, 99%, or 100% identical) to the VHH sequences shown in Table 5, e.g., SEQ ID NO: 1291.

127. The monospecific antibody of any one of claims 109 to 126, wherein the monospecific antibody comprises at least two anti-CDCPl antibody regions.

128. The monospecific antibody of any one of claims 109 to 126, wherein the monospecific antibody comprises at least three anti-CDCPl antibody regions.

129. The monospecific antibody of any one of claims 109 to 128, wherein the monospecific antibody comprises four anti-CDCPl antibody regions.

130. The monospecific antibody of any one of claims 124 to 129, wherein a monospecific antibody comprising a sequence that is less than 100% identical to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g., SEQ ID NO: 1291, retains affinity for CDCP1 that is substantially equivalent to a monospecific antibody comprising a sequence that is 100% identical to a sequence shown in one or more of Table 4 or Table 5.

131. The monospecific antibody of any one of claims 124 to 129, wherein a monospecific antibody comprising a sequence that is less than 100% identical to a sequence shown in one or more of Table 4, e.g., SEQ ID NO: 1115 to SEQ ID NO: 1117, or Table 5, e.g, SEQ ID NO: 1291, has less affinity for CDCP1 than a monospecific antibody comprising a sequence that is 100% identical to a sequence shown in one or more of Table 4 or Table 5.

132. The monospecific antibody of any one of claims 109 to 131, wherein the monospecific antibody comprises an antibody region identified by a PROT ID number in Table 8.

133. The monospecific antibody of any one of claims 115 to 132, wherein the monospecific antibody is conjugated with one or more cytotoxic agents, thereby forming an antibody drug conjugate (ADC).

134. The monospecific antibody of claim 133, wherein the one or more cytotoxic agents are conjugated to a constant region of the monospecific antibody.

135. The monospecific antibody of claim 134, wherein the constant region of the monospecific antibody is an Fc region.

136. The monospecific antibody of claim 135, wherein the drug is conjugated to an amino acid position of the monospecific antibody’s Fc region that has been substituted with a cysteine.

137. The monospecific antibody of claim 136, wherein a conjugation site is a 375C, 360C, 149C, and / or a 140C substituted position.

138. The monospecific antibody of any one of claims 135 to 137, wherein the monospecific antibody comprises a sequence that is at least 90% identical (e.g., 95%, 99%, or 100% identical) to a sequence shown in one SEQ ID NO: 1309-1316.

139. The monospecific antibody of any one of claims 115 to 138, wherein the one or more cytotoxic agents comprise a microtubule-disrupting drug, DNA-modifying drug, RNA- modifying drug, conjugated toxin, a small molecule, radioisotope, and / or a drug.

140. The monospecific antibody of any one of claims 115 to 139, wherein the one or more cytotoxic agents comprise monomethyl auristatin E (MMAE) and / or exatecan.

141. The monospecific antibody of claim 140, wherein the MMAE is conjugated via a val-cit-PABC linker or another linker or the exatecan is conjugated via a GGFG linker or other linker.

142. The monospecific antibody of any one of claims 115 to 141, wherein the one or more cytotoxic agents comprise monomethyl auristatin F (MMAF) or other auristatins; DM1, DM4, or other maytansinoids, SN-38, Dxd or other camptothecin derivatives; pyrrol obenzodi azepines (PBDs) or other DNA alkylators; duocarmycins, NAMPT inhibitors, TLR agonists, STING agonists, small molecule cell cycle inhibitors such as cell cycle blockers and inhibitors of anti- apoptotic proteins, and / or other common ADC payloads.

143. The monospecific antibody of any one of claims 109 to 142, wherein the monospecific is a biparatopic antibody or a multiparatopic antibody.

144. The bispecific antibody of claim 143, wherein the biparatopic antibody or the multiparatopic antibody binds two, three, or more epitopes from CDCP1.

145. The monospecific antibody of claim 143 or 144, wherein the biparatopic antibody or the multiparatopic antibody comprises two first binding domains that each bind to a first epitope from CDCP1 and at least one second binding domain that binds to second epitope from CDCP1.

146. The monospecific antibody of claim 143 or 144, wherein the biparatopic antibody or the multiparatopic antibody comprises a first binding domain that bind to a first epitope from CDCP1, a second binding domain that bind to a second epitope from CDCP1, and a third binding domain that bind to a third epitope from CDCP1.

147. The monospecific antibody of claim 146, wherein the biparatopic antibody or the multiparatopic antibody comprises a fourth binding domain that bind to a fourth epitope from CDCP1.

148. A method of killing a cancer cell, the method comprising contacting the cancer cell with an effective amount of the monospecific antibody of any one of claims 109 to 147.

149. The method of claim 148, wherein the cancer cell is in vitro, ex vivo, or in vivo.

150. A method of killing a cancer cell in a subject in need thereof, the method comprising contacting the cancer cell with an effective amount of the monospecific antibody of any one of claims 109 to 140.

151. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the monospecific antibody of any one of claims 109 to 147.

152. A pharmaceutical composition comprising the monospecific antibody of any one of claims 109 to 147 and a pharmaceutically acceptable carrier, diluent, or excipient.

153. The pharmaceutical composition of claim 152, for use as a medicament in the treatment of cancer.

154. A method of killing a cancer cell in a subject in need thereof, the method comprising contacting the cancer cell with an effective amount of the pharmaceutical composition of any one of claims 152-153.

155. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 152-153.

156. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 152-153 and administering to the subject an effective amount of a standard of care therapy for the cancer.

157. The method of claim 156, wherein the pharmaceutical composition is administered before the standard of care therapy for the cancer.

158. The method of claim 156, wherein the pharmaceutical composition is administered simultaneous with the standard of care therapy for the cancer.

159. The method of claim 156, wherein the pharmaceutical composition is administered after the standard of care therapy for the cancer.

160. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 152-153, wherein the subject has previously been treated with the standard of care therapy for the cancer.

161. A polynucleotide or plurality of polynucleotides encoding the monospecific antibody of any one of claims 109 to 147.

162. A vector comprising the polynucleotide or plurality of polynucleotides of claim 161.

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