Compositions and methods for modulating cellular internalization

Abstract

To provide compositions and methods for modulating cellular internalization.SOLUTION: Provided herein are compositions and methods for modulating internalization properties of cell surface molecules, e.g., converting a non-internalizing cell surface antigen into an internalizing one, and vice versa. In some embodiments, provided are engineered antibodies each containing an antigen binding moiety specific for a guide-antigen and another antigen binding moiety specific for an effector antigen, wherein the internalization property of the engineered antibody or functional fragment thereof is determined by a relative surface density ratio of the guide antigen to the effector antigen. Also provided are recombinant cells, recombinant nucleic acids encoding such engineered antibodies, as well as pharmaceutical compositions containing the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant numbers R01 CA118919, R01 CA129491, and R01 CA171315 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 792,359, filed January 14, 2019, which is expressly incorporated by reference in its entirety, including all drawings.

[0003] Incorporation of sequence listings This application has been submitted with an electronic sequence listing. The sequence listing is provided as a file entitled "Sequence Listing_048536-628001WO.txt", created on December 23, 2019, and is approximately 124 KB in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety.

[0004] Field Aspects of the present application relate to the fields of cell biology and immunology. More specifically, engineered antibodies are provided herein that, for example, convert non-internalized cell-type-selective surface antigens into internalized ones, and vice versa, thereby modulating and / or amplifying cell-type-specific internalization. The present disclosure also provides compositions and methods useful for producing such engineered antibodies, as well as methods for treating health disorders or diseases, such as cancer-related diseases, including solid tumors and hematopoietic malignancies. [Background technology]

[0005] background The use of biopharmaceuticals or pharmaceutical compositions containing therapeutic proteins for the treatment of diseases, disorders, or conditions is a core strategy of several pharmaceutical and biotechnology companies. For example, in cancer immunotherapy, the development of antibodies and antibody-drug conjugates (ADCs) that can target specific cancerous cells, prevent their growth, and / or kill them has emerged as a promising therapeutic approach to complement existing treatment strategies.

[0006] In particular, the high specificity of monoclonal antibodies is often utilized to develop targeted therapies. Ideally, potent cytotoxic agents can be conjugated to cell-type-specific antibodies, allowing the cytotoxic agents to be directed to target cells and preferentially accumulate in target tissues. Another example of targeted therapy includes antibody-drug conjugates (ADCs), which have shown promising efficacy in several clinical studies.

[0007] Although conceptually simple, target selection for therapeutic antibodies and ADCs is hampered by the finding that it is rare to find so-called tumor-specific antigens, and even rarer to find tumor-specific antigens with the properties desired for therapeutic targeting, i.e., those that are uniformly expressed at high levels by cancer cells and efficiently internalized. In addition, transport across the plasma membrane is a major limiting step in the cellular delivery of macromolecules. Therefore, the effectiveness of therapies that rely on the internalization of therapeutic agents depends on both the quality of the target on the surface of the target cell and the rate of cellular internalization of the surface-bound therapeutic agent complexed with that target. Additionally, while internalizing therapeutic antibodies are often desired to achieve efficient intracellular payload delivery and tumor killing, this requirement is not absolute for certain drugs, such as monomethyl auristatin E (MMAE), which can diffuse across the cell membrane and cause bystander effects. In some cases, for targeted therapies requiring intracellular payload delivery, many tumor antigens are highly expressed but poorly internalized. In other cases, receptor internalization, a receptor-mediated endocytic process that results in the movement of receptors from the plasma membrane to the interior of the cell, has also been used to block signaling pathways and result in desensitization. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there is an ongoing need for new approaches and compositions for the treatment of diseases, disorders, or conditions, such as inflammatory diseases, immune diseases, and cancer. Specifically, there is a need in the art for more effective compositions and methods for treating diseases, disorders, or conditions by improving the internalization properties of therapeutic antibodies and ADCs. [Means for solving the problem]

[0009] This section provides a general overview of the disclosure and is not intended to encompass its entire scope or all of its characteristics.

[0010] The present disclosure relates to compositions and methods for engineering cell-type-selective antibody internalization through a guide-effector bispecific antibody design. Specifically, engineered antibodies are provided herein that can simultaneously bind to a pair of antigens, termed "guide antigen" and "effector antigen," expressed on the surface of the same cell. When the engineered antibodies simultaneously bind, the guide antigen can affect the cell surface dynamics and / or signaling function of the effector antigen. In certain designs, the effector antigen is an antigen associated with a target signaling pathway, and the guide antigen provides cell-type specificity to redirect and enhance effector function to a cell of interest. For example, a non-internalizing effector antigen can be converted into an internalizing effector antigen by using a guide-effector bispecific antibody design that can bind to (i) a non-internalizing effector antigen and (ii) an internalizing guide antigen. Similarly, internalizing effector antigens can be converted into non-internalizing effector antigens by using a guide-effector bispecific antibody design that can bind (i) an internalizing effector antigen and (ii) a non-internalizing guide antigen. As described in more detail below, modulation of internalization can, in some cases, directly affect intracellular payload delivery and receptor signaling. Also provided are recombinant cells, recombinant nucleic acids encoding such engineered antibodies, and pharmaceutical compositions comprising the same. The present disclosure also provides compositions and methods useful for modulating cell internalization in a cell or subject using such engineered antibodies, as well as methods for modulating cell-type-selective signaling in a subject and / or treating health disorders and diseases, such as cancer-related diseases, including solid tumors and hematopoietic malignancies.

[0011] In one embodiment, some embodiments of the present disclosure relate to an engineered antibody or functional fragment thereof comprising: a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate; and b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate, wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of guide antigen to effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate.

[0012] Implementations of engineered antibody embodiments of the present disclosure may include one or more of the following properties: In some embodiments, the cell surface guide antigen is an internalized cell surface antigen. In some embodiments, the cell surface effector antigen is a non-internalized cell surface antigen. In some embodiments, the relative surface density ratio of guide antigen to effector antigen is above a threshold. In some embodiments, the relative surface density ratio of guide antigen to effector antigen is below a threshold. In some embodiments, the threshold is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are selected from the group consisting of antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), full-length immunoglobulins, nanobodies, single-domain antibodies (sdAbs), variable new antigen receptor (VNAR) domains, and the like. and VHH domains, multispecific antibodies, diabodies, or functional fragments thereof. In some embodiments, the guide antigen and effector antigen are independently selected from the group consisting of activated leukocyte cell adhesion molecule (ALCAM), neural cell adhesion molecule (NCAM), calcium-activated chloride channel 2 (CaCC), carbonic anhydrase IX, carcinoembryonic antigen (CEA), cathepsin G, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD46, CD52, CD71, CD73, CD272, CD276, B-cell maturation antigen (BCMA), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), ephrin type A receptor 3 (EphA3), ephrin type A receptor 4 (EphA4), ephrin B2, Receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PDGFR-beta, SSEA-4, epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 2 (ErbB2), Erb-B2 receptor tyrosine kinase 3 (ErbB3), Erb-B2 receptor tyrosine kinase 4 (ErbB4), folate-binding protein (folate receptor), ganglioside, gangliosides, gp100, gpA33, immature laminin receptor, intercellular adhesion molecule 1 (ICAM-1), Lewis-Y, mesothelin, prostate stem cell antigen (PSCA), mucin 16 (MUC16 or CA-125), mucin 1 cell surface-associated (MUC1), mucin 2 oligomeric mucus gel-forming (MUC2), mucin, prostate membrane-specific antigen (PSMA), TEM1 / CD248, TEM7R, CLDN6, thyroid-stimulating hormone receptor (TSHR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), immunoglobulin lambda-like polypeptide 1 (IGLL1), P-selectin, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), tumor-associated calcium signaling factor 2 (Trop-2), and tumor-associated glycoprotein 72 (TAG-72).

[0013] In some embodiments, the guide antigen is selected from the group consisting of CD19, CD22, HER2 (ErbB2 / neu), mesothelin, PSCA, CD123, CD30, CD71, CD171, CS-1, CLECL1, CD33, EGFRvIII, GD2, GD3, BCMA, PSMA, receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), TAG72, CD38, CD44v6, CD46, CEA, EpCAM, CD272, B7H3 (CD276), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PDGFR-beta, SSEA-4, C The cancer-associated antigen is selected from the group consisting of D20, MUC1, MUC16, EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostatic acid phosphatase (PAP), ephrinB2, fibroblast activation protein (FAP), EphA2, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), GM3, TEM1 / CD248, TEM7R, CLDN6, thyroid-stimulating hormone receptor (TSHR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0014] In some embodiments, the effector antigen is selected from the group consisting of ALCAM, EpCAM, folate binding protein, PSMA, PSCA, mesothelin, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD46, ICAM-1, CD55, CD59, CD70, CD71, CD73, CD97, BCMA, CD272, CD276, MUC1, MUC16, NCAM, CD24, EphA2, EphA3, EphA4, ephrin B2, CEA, c-Met, FGFRs, IGF-1R, VEGFRs, PDGFRs, Trop-2, TAG-72, P-selectin, EGFR, ErbB2, ErbB3, and ErbB4.

[0015] In some embodiments, the antibody or functional fragment thereof is conjugated or covalently linked to at least one moiety of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a pharmacokinetic-improving moiety. In some embodiments, at least one MOI is selected from the group consisting of an anti-cancer agent, an anti-autoimmune disease agent, an anti-inflammatory agent, an anti-bacterial agent, an anti-microbial agent, an antibiotic, an anti-infectious disease agent, and an anti-viral agent. In some embodiments, at least one MOI is selected from the group consisting of a cytotoxic anti-cancer agent, a DNA chelator, a microtubule inhibitor, a topoisomerase inhibitor, a translation initiation inhibitor, a ribosome-inactivating molecule, a nuclear transport inhibitor, an RNA splicing inhibitor, an RNA polymerase inhibitor, and a DNA polymerase inhibitor.

[0016] In some embodiments, the cytotoxic anticancer agent is an auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, anthracycline, calicheamycin, camptothecin, irinotecan, S N-38, combretastatin, duocarmycin, enediyne, epothilone, ethyleneimine, mitomycin, pyrrolobenzodiazepine (PBD), and calicheamicin.

[0017] In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the constant region of the engineered antibody or functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the heavy chain constant (e.g., CH1, CH2, or CH3) region of the antibody or functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the heavy chain constant (CH1) region of the antibody or functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the light chain constant (CL) region of the antibody or functional fragment thereof. In some embodiments, the average MOI number per antibody (e.g., average drug-to-antibody ratio, DAR) ranges from 1 to 20. In some embodiments, the average DAR is from about 1 to about 5, from about 2 to about 6, from about 3 to about 7, from about 3 to about 8, from about 4 to about 9, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 10 to about 20.

[0018] In some embodiments, the engineered antibody or functional fragment disclosed herein comprises a first antigen-binding portion capable of binding to EphA2 expressed on the surface of a cell and a second antigen-binding portion capable of binding to ALCAM expressed on the surface of the same cell. In some embodiments, the surface density ratio of EphA2 to ALCAM exceeds a threshold of about 1:5. In some embodiments, the engineered antibody or functional fragment thereof described herein comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences identified in Table 4. In some embodiments, the first antigen-binding portion comprises a heavy chain variable (VH) region having at least 80% sequence identity to a VH sequence identified in Table 4. In some embodiments, the first antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO:81 or SEQ ID NO:96. In some embodiments, the VH region of the first antigen-binding portion comprises three complementarity-determining regions (HCDRs) identified in the Sequence Listing. In some embodiments, the VH region of the first antigen-binding moiety comprises HCDR1, HCDR2, and HCDR3 comprising SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106, respectively, or SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:110, respectively. In some embodiments, the first antigen-binding moiety comprises a light chain variable (VL) region having at least 80% sequence identity to a VH sequence identified in Table 4. In some embodiments, the first antigen-binding moiety comprises a VL region having at least 80% sequence identity to SEQ ID NO:82 or SEQ ID NO:97. In some embodiments, the VL region of the first antigen-binding moiety comprises three LCDRs identified in the Sequence Listing. In some embodiments, the VL region of the first antigen-binding moiety comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109, respectively.

[0019] In some embodiments, the second antigen-binding moiety comprises a VH region having at least 80% sequence identity to a VH sequence identified in Table 4. In some embodiments, the second antigen-binding moiety comprises a VH region having at least 80% sequence identity to SEQ ID NO:73 or SEQ ID NO:75. In some embodiments, the VH region of the second antigen-binding moiety comprises the three HCDRs identified in the Sequence Listing. In some embodiments, the VH region of the second antigen-binding moiety comprises HCDR1, HCDR2, and HCDR3 comprising SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100, respectively. In some embodiments, the second antigen-binding moiety comprises a VL region having at least 80% sequence identity to a VH sequence identified in Table 4. In some embodiments, the second antigen-binding moiety comprises a VL region having at least 80% sequence identity to SEQ ID NO:74 or SEQ ID NO:76. In some embodiments, the VL region of the second antigen-binding moiety comprises the three LCDRs identified in the Sequence Listing. In some embodiments, the VL region of the second antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively.

[0020] In one aspect, some embodiments of the present disclosure relate to recombinant nucleic acid molecules comprising a nucleic acid sequence encoding an engineered antibody or functional fragment thereof disclosed herein. In some embodiments, the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence. In some embodiments, the recombinant nucleic acid molecule is further defined as an expression cassette or vector.

[0021] In one aspect, some embodiments of the present disclosure comprise (a) an engineered antibody or functional fragment thereof disclosed herein, and / or (b) a nucleic acid molecule disclosed herein. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In a related aspect, some embodiments of the present disclosure relate to a cell culture comprising at least one recombinant cell disclosed herein and a culture medium.

[0022] In one aspect, some embodiments of the present disclosure pertain to pharmaceutical compositions comprising one or more of the following: (a) an engineered antibody or functional fragment thereof disclosed herein, (b) a nucleic acid molecule disclosed herein, and (c) a recombinant cell disclosed herein, and a pharmaceutically acceptable carrier.

[0023] In another aspect, some embodiments of the present disclosure relate to a method for modulating cellular internalization, comprising administering to a cell one or more of the following: (a) an engineered antibody or functional fragment thereof disclosed herein, (b) a nucleic acid molecule disclosed herein, and (c) a pharmaceutical composition disclosed herein.

[0024] In another aspect, some embodiments of the present disclosure relate to a method for modulating cellular internalization, comprising administering an engineered antibody or functional fragment thereof comprising: (a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate; and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate, wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate.

[0025] In yet another aspect, some embodiments of the present disclosure relate to a method for modulating cell-type selective signaling in a subject, the method comprising administering to a cell an engineered antibody or functional fragment thereof comprising: (a) a first antigen-binding portion capable of binding to a cell-surface guide antigen, where the guide antigen is expressed in a cell-type selective manner in the subject and has a first cellular internalization rate; and (b) a second antigen-binding portion capable of binding to a cell-surface effector antigen with a second cellular internalization rate, wherein the internalization property of the engineered antibody or functional fragment thereof is determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate.

[0026] In yet another aspect, some embodiments of the present disclosure relate to a method for treating a condition or disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered antibody or functional fragment thereof disclosed herein. In some embodiments, the engineered antibody or functional fragment thereof comprises (a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate, and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate, wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. In some embodiments, the condition or disease is cancer.

[0027] In yet another aspect, some embodiments of the present disclosure relate to a method for killing cancer cells, comprising administering to said cells an engineered antibody or functional fragment thereof disclosed herein. In some embodiments, the engineered antibody or functional fragment thereof comprises (a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate, and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate.

[0028] In yet another aspect, some embodiments of the present disclosure relate to a method for killing tumor cells, comprising administering to the tumor cells an engineered antibody or functional fragment thereof disclosed herein. In some embodiments of the disclosed method, the engineered antibody or functional fragment thereof comprises a first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of the tumor cells, and a second antigen-binding portion capable of binding to ALCAM expressed on the surface of the same tumor cells. In some embodiments, the surface density ratio of EphA2 to ALCAM is above a threshold of about 1:5.

[0029] In some embodiments of the disclosed methods, the engineered antibody or functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences identified in Table 4. In some embodiments, the first antigen-binding portion comprises a VH region having at least 80% sequence identity to a VH sequence identified in Table 4. In some embodiments, the first antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO:81 or SEQ ID NO:96. In some embodiments, the VH region of the first antigen-binding portion comprises three HCDRs identified in the Sequence Listing. In some embodiments, the VH region of the first antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 comprising (a) SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106, respectively, or (b) SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:110, respectively. In some embodiments, the first antigen-binding portion comprises a VL region having at least 80% sequence identity to a VL sequence identified in Table 4. In some embodiments, the first antigen-binding moiety comprises a VL region having at least 80% sequence identity to SEQ ID NO: 82 or SEQ ID NO: 97. In some embodiments, the VL region of the first antigen-binding moiety comprises the three LCDRs identified in the Sequence Listing. In some embodiments, the VL region of the first antigen-binding moiety comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively.

[0030] In some embodiments of the disclosed methods, the second antigen-binding portion comprises a VH region having at least 80% sequence identity to a VH sequence identified in Table 4. In some embodiments, the second antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO:73 or SEQ ID NO:75. In some embodiments, the VH region of the second antigen-binding portion comprises three HCDRs identified in the Sequence Listing. In some embodiments, the VH region of the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 comprising SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100, respectively. In some embodiments, the second antigen-binding portion comprises a VL region having at least 80% sequence identity to a VL sequence identified in Table 4. In some embodiments, the second antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO:74 (SEC ID NO:74) or SEQ ID NO:76. % sequence identity to the VL region of the second antigen-binding moiety. In some embodiments, the VL region of the second antigen-binding moiety comprises three LCDRs identified in the Sequence Listing. In some embodiments, the VL region of the second antigen-binding moiety comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively.

[0031] In some embodiments, the cancer is pancreatic cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, mesothelioma, breast cancer, urothelial cancer, liver cancer, head and neck cancer, sarcoma, cervical cancer, stomach cancer, gastric cancer, melanoma, uveal melanoma, cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, and glioblastoma.

[0032] In some embodiments, the cell surface guide antigen is an internalized cell surface antigen. In some embodiments, the cell surface effector antigen is a non-internalized cell surface antigen. In some embodiments, the relative surface density ratio of guide antigen to effector antigen is above a threshold. In some embodiments, the relative surface density ratio of guide antigen to effector antigen is below a threshold. In some embodiments, the threshold is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. In some embodiments, the methods disclosed herein further comprise modulating the cell surface density of guide antigen and / or the cell surface density of effector antigen. In some embodiments, the internalization property of the engineered antibodies disclosed herein is converted from internalizing to non-internalizing. In some other embodiments, the internalization property of the engineered antibodies disclosed herein is converted from non-internalizing to internalizing. In some embodiments, the expression of the guide antigen and / or the effector antigen is cell type selective.

[0033] In certain embodiments, for example, the following items are provided: (Item 1) 1. An engineered antibody or functional fragment thereof, comprising: a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first cellular internalization rate; a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second rate of cellular internalization; Including, the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen; An engineered antibody or functional fragment thereof, wherein one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. (Item 2) 2. The engineered antibody or functional fragment thereof of item 1, wherein the cell surface guide antigen is an internalizing cell surface antigen. (Item 3) 2. The engineered antibody or functional fragment thereof of item 1, wherein the cell surface effector antigen is a non-internalized cell surface antigen. (Item 4) 3. The engineered antibody or functional fragment thereof of any one of items 1 to 2, wherein the relative surface density ratio of the guide antigen to the effector antigen is above a threshold value. (Item 5) 3. The engineered antibody or functional fragment thereof of any one of items 1 to 2, wherein the relative surface density ratio of the guide antigen to the effector antigen is below a threshold value. (Item 6) 6. The engineered antibody or functional fragment thereof of any one of items 1 to 5, wherein the threshold is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. (Item 7) 7. The engineered antibody or functional fragment thereof of any one of items 1 to 6, wherein the first antigen-binding portion and the second antigen-binding portion are independently selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a full-length immunoglobulin, a nanobody, a single-domain antibody (sdAb), a VNAR domain, and a VHH domain, a multispecific antibody, a diabody, or a functional fragment thereof. (Item 8) The guide antigen and the effector antigen are selected from the group consisting of activated leukocyte cell adhesion molecule (ALCAM), neural cell adhesion molecule (NCAM), calcium-activated chloride channel 2 (CaCC), carbonic anhydrase IX, carcinoembryonic antigen (CEA), cathepsin G, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD46, CD52, CD71, CD73, CD272, CD276, B-cell maturation antigen (BCMA), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), ephrin receptor 3 (EphR3), ephrin receptor 4 (EphR4), ephrin receptor 5 (EphR5), ephrin receptor 6 (EphR6), ephrin receptor 7 (EphR7), ephrin receptor 8 (EphR8), ephrin receptor 9 (EphR9), ephrin receptor 10 (EphR10), ephrin receptor 11 (EphR11), ephrin receptor 12 (EphR12), ephrin receptor 13 (EphR14), ephrin receptor 14 (EphR15), ephrin receptor 15 (EphR16), ephrin receptor 16 (EphR17), ephrin receptor 17 (EphR18), ephrin receptor 18 (EphR19), ephrin receptor 19 ... Ephrin type A receptor 3 (EphA3), ephrin type A receptor 4 (EphA4), ephrin B2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PDGFR-beta, SSEA-4, epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 2 (ErbB2), Erb-B2 receptor tyrosine kinase 3 (ErbB3), Erb-B2 receptor tyrosine kinase ErbB4, folate-binding protein (folate receptor), ganglioside, gangliosides, gp100, gpA33, immature laminin receptor, intercellular adhesion molecule 1 (ICAM-1), Lewis-Y, mesothelin, prostate stem cell antigen (PSCA), mucin 16 (MUC16 or CA-125), mucin 1 cell surface-associated (MUC1), mucin 2 oligomer mucus gel-forming (MUC2), mucin, prostate membrane-specific antigen (PSMA), TEM1 / CD248, TEM7R, CLDN6, thyroid-stimulating hormone receptor (TSR) 8. The engineered antibody or functional fragment thereof of any one of paragraphs 1 to 7, wherein the antibody or functional fragment is independently selected from the group consisting of: HR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), immunoglobulin lambda-like polypeptide 1 (IGLL1), P-selectin, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), tumor-associated calcium signaling factor 2 (Trop-2), and tumor-associated glycoprotein 72 (TAG-72). (Item 9) The guide antigen is CD19, CD22, HER2 (ErbB2 / neu), mesothelin, PSCA, CD123, CD30, CD71, CD171, CS-1, or CLECL1. , CD33, EGFRvIII, GD2, GD3, BCMA, PSMA, receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), TAG72, CD38, CD44v6, CD46, CEA, EpCAM, CD272, B7H3 (CD276), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PDGFR-beta, SSEA-4, CD20, MUC1, MUC16, EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostatic acid phosphatase (PAP), 9. The engineered antibody or functional fragment thereof of any one of paragraphs 1 to 8, wherein the engineered antibody or functional fragment thereof is a cancer associated antigen selected from the group consisting of ephrinB2, fibroblast activation protein (FAP), EphA2, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), GM3, TEM1 / CD248, TEM7R, CLDN6, thyroid-stimulating hormone receptor (TSHR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), and immunoglobulin lambda-like polypeptide 1 (IGLL1). (Item 10) 10. The engineered antibody or functional fragment thereof of any one of paragraphs 1 to 9, wherein the effector antigen is selected from the group consisting of ALCAM, EpCAM, folate binding protein, PSMA, PSCA, mesothelin, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD46, ICAM-1, CD55, CD59, CD70, CD71, CD73, CD97, BCMA, CD272, CD276, MUC1, MUC16, NCAM, CD24, EphA2, EphA3, EphA4, ephrin B2, CEA, c-Met, FGFRs, IGF-1R, VEGFRs, PDGFRs, Trop-2, TAG-72, P-selectin, EGFR, ErbB2, ErbB3, and ErbB4. (Item 11) 11. The engineered antibody or functional fragment thereof of any one of items 1 to 10, conjugated or covalently attached to at least one moiety of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a pharmacokinetic-improving moiety. (Item 12) 12. The engineered antibody or functional fragment thereof of claim 11, wherein the at least one MOI is selected from the group consisting of an anti-cancer agent, an anti-autoimmune disease agent, an anti-inflammatory agent, an anti-bacterial agent, an anti-microbial agent, an antibiotic, an anti-infectious disease agent, and an anti-viral agent. (Item 13) 13. The engineered antibody or functional fragment thereof of claim 12, wherein the at least one MOI is selected from the group consisting of a cytotoxic anticancer drug, a DNA chelator, a microtubule inhibitor, a topoisomerase inhibitor, a translation initiation inhibitor, a ribosome inactivating molecule, a nuclear transport inhibitor, an RNA splicing inhibitor, an RNA polymerase inhibitor, and a DNA polymerase inhibitor. (Item 14) 14. The engineered antibody or functional fragment thereof of item 13, wherein the cytotoxic anticancer agent is selected from the group consisting of an auristatin, a dolastatin, a tubulysin, a maytansinoid, a taxane, a vinca alkaloid, an amatoxin, an anthracycline, a calicheamicin, a camptothecin, an irinotecan, SN-38, a combretastatin, a duocarmycin, an enediyne, an epothilone, an ethyleneimine, a mitomycin, a pyrrolobenzodiazepine (PBD), and a calicheamicin. (Item 15) 15. The engineered antibody or functional fragment thereof of any one of items 11 to 14, wherein the at least one moiety of interest (MOI) is conjugated or covalently attached to a constant region of the engineered antibody or functional fragment thereof. (Item 16) 16. The engineered antibody or functional fragment thereof of claim 15, wherein the at least one moiety of interest (MOI) is conjugated or covalently attached to the heavy chain constant (CH1) region of the engineered antibody or functional fragment thereof. (Item 17) 16. The engineered antibody or functional fragment thereof of claim 15, wherein the at least one moiety of interest (MOI) is conjugated or covalently attached to a light chain constant (CL) region of the engineered antibody or functional fragment thereof. (Item 18) 18. The engineered antibody or functional fragment thereof of any one of items 11 to 17, wherein the average MOI number (average DAR) per antibody ranges from 1 to 20. (Item 19) 19. The engineered antibody or functional fragment thereof according to item 18, wherein the average DAR is about 1 to about 5, about 2 to about 6, about 3 to about 7, about 3 to about 8, about 4 to about 9, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 10 to about 20. (Item 20) a first antigen-binding moiety capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell; a second antigen-binding moiety capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cell; 20. The engineered antibody or functional fragment thereof of any one of items 1 to 19, comprising: (Item 21) 21. The engineered antibody or functional fragment thereof of item 20, wherein the surface density ratio of EphA2 to ALCAM is above a threshold of about 1:5. (Item 22) 22. The engineered antibody or functional fragment thereof of any one of items 1 to 21, comprising an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences identified in Table 4. (Item 23) 23. The engineered antibody or functional fragment thereof of claim 22, wherein the first antigen-binding portion comprises a heavy chain variable (VH) region having at least 80% sequence identity to a VH sequence identified in Table 4. (Item 24) 24. The engineered antibody or functional fragment thereof of claim 23, wherein the first antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO: 81 or SEQ ID NO: 96. (Item 25) 25. The engineered antibody or functional fragment thereof of any one of items 22 to 24, wherein the VH region of the first antigen-binding portion comprises three complementarity determining regions HCDR1, HCDR2, and HCDR3 as identified in the sequence listing. (Item 26) the VH region of the first antigen-binding portion comprises: (a) SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, respectively; or (b) SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 110, respectively 26. The engineered antibody or functional fragment thereof of item 25, comprising HCDR1, HCDR2, and HCDR3 comprising: (Item 27) 27. The engineered antibody or functional fragment thereof of any one of items 22 to 26, wherein the first antigen-binding portion comprises a light chain variable (VL) region having at least 80% sequence identity to a VL sequence identified in Table 4. (Item 28) 28. The engineered antibody or functional fragment thereof of item 27, wherein the first antigen-binding portion comprises a VL region having at least 80% sequence identity to SEQ ID NO: 82 or SEQ ID NO: 97. (Item 29) 29. The engineered antibody or functional fragment thereof of any one of items 22 to 28, wherein the VL region of the first antigen-binding portion comprises the CDRs identified in the sequence listing. (Item 30) 30. The engineered antibody or functional fragment thereof of item 29, wherein the VL region of the first antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively. (Item 31) 31. The engineered antibody or functional fragment thereof of any one of items 22 to 30, wherein the second antigen-binding portion comprises a VH region having at least 80% sequence identity to a VH sequence identified in Table 4. (Item 32) 32. The engineered antibody or functional fragment thereof of claim 31, wherein the second antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO: 73 or SEQ ID NO: 75. (Item 33) 33. The engineered antibody or functional fragment thereof of any one of items 22 to 32, wherein the VH region of the second antigen-binding portion comprises three HCDRs identified in the Sequence Listing. (Item 34) 34. The engineered antibody or functional fragment thereof of claim 33, wherein the VH region of the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 comprising SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively. (Item 35) 35. The engineered antibody or functional fragment thereof of any one of items 22 to 34, wherein the second antigen-binding portion comprises a VL region having at least 80% sequence identity to a VL sequence identified in Table 4. (Item 36) 36. The engineered antibody or functional fragment thereof of claim 35, wherein the second antigen-binding portion comprises a VL region having at least 80% sequence identity to SEQ ID NO: 74 or SEQ ID NO: 76. (Item 37) 37. The engineered antibody or functional fragment thereof of any one of items 22 to 36, wherein the VL region of the second antigen-binding portion comprises the three CDRs identified in the Sequence Listing. (Item 38) 38. The engineered antibody or functional fragment thereof of Item 37, wherein the VL region of the second antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, respectively. (Item 39) 39. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the engineered antibody or functional fragment thereof of any one of items 1 to 38. (Item 40) 40. The recombinant nucleic acid molecule of Item 39, operably linked to a heterologous nucleic acid sequence. (Item 41) 41. The recombinant nucleic acid molecule of any one of items 39 to 40, further defined as an expression cassette or vector. (Item 42) 37. The engineered antibody or functional fragment thereof according to any one of items 1 to 36, and / or 42. The nucleic acid molecule of any one of items 39 to 41. A recombinant cell comprising: (Item 43) 43. The recombinant cell of item 42, which is a prokaryotic or eukaryotic cell. (Item 44) 44. A cell culture comprising at least one recombinant cell according to any one of items 42 to 43 and a culture medium. (Item 45) 39. The engineered antibody or functional fragment thereof according to any one of items 1 to 38. A nucleic acid molecule according to any one of items 39 to 41, and 44. The recombinant cell according to any one of items 42 to 43. and one or more of a pharmaceutically acceptable carrier; 10. A pharmaceutical composition comprising: (Item 46) 1. A method for modulating cell internalization, comprising administering to a cell: 39. The engineered antibody or functional fragment thereof according to any one of items 1 to 38. A nucleic acid molecule according to any one of items 39 to 41, and The pharmaceutical composition according to item 45. The method of claim 1, further comprising administering one or more of: (Item 47) 1. A method for modulating cell internalization, comprising administering to a cell: a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first cellular internalization rate; a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second rate of cellular internalization; administering an engineered antibody or functional fragment thereof comprising: the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen; A method wherein one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. (Item 48) 1. A method for modulating cell type selective signaling in a subject, comprising administering to the subject: a first antigen-binding moiety capable of binding to a cell surface guide antigen, wherein the guide antigen is expressed in a cell-type selective manner in the subject and has a first cellular internalization rate; a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second rate of cellular internalization; administering an engineered antibody or functional fragment thereof comprising: the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen; A method wherein one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. (Item 49) 1. A method for treating a condition or disease in a subject in need thereof, comprising administering to the subject: a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first cellular internalization rate; a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second rate of cellular internalization; wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen; A method wherein one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. (Item 50) 50. The method of claim 49, wherein the condition or disease is cancer. (Item 51) 1. A method for killing cancer cells, comprising administering to said cells: a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first cellular internalization rate; a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second rate of cellular internalization; Administering an engineered antibody or functional fragment thereof comprising: (Item 52) 52. The method of any one of items 50 to 51, wherein the cancer is pancreatic cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, mesothelioma, breast cancer, urothelial cancer, liver cancer, head and neck cancer, sarcoma, cervical cancer, stomach cancer, gastric cancer, melanoma, uveal melanoma, cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, and glioblastoma. (Item 53) 53. The method of any one of items 46 to 52, wherein the cell surface guide antigen is an internalizing cell surface antigen. (Item 54) 53. The method of any one of items 46 to 52, wherein the cell surface effector antigen is a non-internalized cell surface antigen. (Item 55) 55. The method of any one of items 46 to 54, wherein the relative surface density ratio of the guide antigen to the effector antigen is above a threshold value. (Item 56) 55. The method of any one of items 46 to 54, wherein the relative surface density ratio of the guide antigen to the effector antigen is below a threshold value. (Item 57) 57. The method of any one of items 46 to 56, wherein the threshold value is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. (Item 58) 58. The method of any one of items 46 to 57, further comprising modulating the cell surface density of the guide antigen and / or the cell surface density of the effector antigen. (Item 59) 59. The method of any one of items 46 to 58, wherein the internalization property of the engineered antibody or functional fragment thereof is converted from a non-internalizing type to an internalizing type. (Item 60) 59. The method of any one of items 46 to 58, wherein the internalization property of the engineered antibody or functional fragment thereof is converted from an internalizing type to a non-internalizing type. (Item 61) The first antigen-binding portion and the second antigen-binding portion are antigen-binding fragments (Fa b), a single chain variable fragment (scFv), a full length immunoglobulin, a nanobody, a single domain antibody (sdAb), a VNAR domain, and a VHH domain, a multispecific antibody, a diabody, or a functional fragment thereof. (Item 62) 62. The method of any one of items 46 to 61, wherein expression of the guide antigen and / or the effector antigen is cell type selective. (Item 63) The guide antigen and the effector antigen are activated leukocyte cell adhesion molecule (ALCAM), neural cell adhesion molecule (NCAM), calcium-activated chloride channel 2 (CaCC), carbonic anhydrase IX, carcinoembryonic antigen (CEA), cathepsin G, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD46, CD52, CD71, CD73, CD272, CD276, B-cell maturation antigen (BCMA), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), , ephrin type A receptor 3 (EphA3), ephrin type A receptor 4 (EphA4), ephrin B2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PDGFR-beta, SSEA-4, epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 2 (ErbB2), Erb-B2 receptor tyrosine kinase 3 (ErbB3), Erb-B2 receptor Tyrosine kinase 4 (ErbB4), folate-binding protein (folate receptor), ganglioside, gangliosides, gp100, gpA33, immature laminin receptor, intercellular adhesion molecule 1 (ICAM-1), Lewis-Y, mesothelin, prostate stem cell antigen (PSCA), mucin 16 (MUC16 or CA-125), mucin 1 cell surface-associated (MUC1), mucin 2 oligomer mucus gel-forming (MUC2), mucin, prostate membrane-specific antigen (PSMA), TEM1 / CD248, TEM7R, CLDN6, thyroid-stimulating 63. The method of any one of paragraphs 46 to 62, wherein the target polypeptide is independently selected from the group consisting of: steroid hormone receptor (TSHR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), immunoglobulin lambda-like polypeptide 1 (IGLL1), P-selectin, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), tumor-associated calcium signaling factor 2 (Trop-2), and tumor-associated glycoprotein 72 (TAG-72). (Item 64) The guide antigen is selected from the group consisting of CD19, CD22, HER2 (ErbB2 / neu), mesothelin, PSCA, CD123, CD30, CD71, CD171, CS-1, CLECL1, CD33, EGFRvIII, GD2, GD3, BCMA, PSMA, receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), TAG72, CD38, CD44v6, CD46, CEA, EpCAM, CD272, B7H3 (CD276), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PDGFR-beta, SSEA-4, CD20, MUC1, MUC16, 64. The method of any one of paragraphs 46 to 63, wherein the cancer-associated antigen is selected from the group consisting of EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostatic acid phosphatase (PAP), ephrinB2, fibroblast activation protein (FAP), EphA2, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), GM3, TEM1 / CD248, TEM7R, CLDN6, thyroid-stimulating hormone receptor (TSHR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), and immunoglobulin lambda-like polypeptide 1 (IGLL1). (Item 65) The effector antigen is selected from the group consisting of ALCAM, EpCAM, folate-binding protein, PSMA, PSCA, mesothelin, CD19, CD20, CD22, CD30, CD33, and CD3 8, CD44, CD46, ICAM-1, CD55, CD59, CD70, CD71, CD73, CD97, BCMA, CD272, CD276, MUC1, MUC16, NCAM, CD24, EphA2, EphA3, EphA4, ephrin B2, CEA, c-Met, FGFR, IGF-1R, VEGFR, PDGFR, Trop-2, TAG-72, P-selectin, EGFR, ErbB2, ErbB3, and ErbB4. (Item 66) 66. The method of any one of items 46 to 65, wherein the antibody or functional fragment thereof is conjugated or covalently attached to at least one moiety of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a pharmacokinetic-improving moiety. (Item 67) 67. The method of item 66, wherein the at least one MOI is selected from the group consisting of an anti-cancer agent, an anti-autoimmune disease agent, an anti-inflammatory agent, an anti-bacterial agent, an anti-microbial agent, an antibiotic, an anti-infectious disease agent, and an anti-viral agent. (Item 68) 68. The method of item 67, wherein the at least one MOI is selected from the group consisting of cytotoxic anticancer drugs, DNA chelators, microtubule inhibitors, topoisomerase inhibitors, translation initiation inhibitors, ribosome inactivating molecules, nuclear transport inhibitors, RNA splicing inhibitors, RNA polymerase inhibitors, and DNA polymerase inhibitors. (Item 69) 69. The method of claim 68, wherein the cytotoxic anti-drug is selected from the group consisting of an auristatin, a dolastatin, a tubulysin, a maytansinoid, a taxane, a vinca alkaloid, an amatoxin, an anthracycline, a calicheamicin, a camptothecin, an irinotecan, a SN-38, a combretastatin, a duocarmycin, an enediyne, an epothilone, an ethyleneimine, a mitomycin, a pyrrolobenzodiazepine (PBD), and a calicheamicin. (Item 70) 70. The method of any one of items 66 to 69, wherein the at least one moiety of interest (MOI) is conjugated or covalently attached to a constant region of the engineered antibody or functional fragment thereof. (Item 71) 71. The method of claim 70, wherein the at least one moiety of interest (MOI) is conjugated or covalently attached to the CH1 region of the engineered antibody or functional fragment thereof. (Item 72) 71. The method of claim 70, wherein the at least one moiety of interest (MOI) is conjugated or covalently attached to the CL region of the engineered antibody or functional fragment thereof. (Item 73) 73. The method of any one of items 46 to 72, wherein the average MOI (DAR) per antibody ranges from 1 to 20. (Item 74) Item 74. The method of item 73, wherein the average DAR is about 1 to about 5, about 2 to about 6, about 3 to about 7, about 3 to about 8, about 4 to about 9, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 10 to about 20. (Item 75) a first antigen-binding moiety capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell; Binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cells a second antigen-binding moiety capable of: 75. The method according to any one of items 46 to 74, comprising: (Item 76) 76. The method of item 75, wherein the surface density ratio of EphA2 to ALCAM is above a threshold of about 1:5. (Item 77) 77. The method of any one of items 46 to 76, wherein the engineered antibody or functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences identified in Table 4. (Item 78) 1. A method of killing tumor cells in a subject, comprising administering to said tumor cells: a first antigen-binding moiety capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of the tumor cell; a second antigen-binding moiety capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same tumor cells; Administering an engineered antibody or functional fragment thereof comprising: (Item 79) 79. The method of item 78, wherein the surface density ratio of EphA2 to ALCAM is above a threshold of about 1:5. (Item 80) 80. The method of any one of items 46 to 79, wherein the engineered antibody or functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences identified in Table 4. (Item 81) 81. The method of claim 80, wherein the first antigen-binding portion comprises a VH region having at least 80% sequence identity to a VH sequence identified in Table 4. (Item 82) 82. The method of claim 81, wherein the first antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO: 81 or SEQ ID NO: 96. (Item 83) 83. The method of any one of items 80 to 82, wherein the VH region of the first antigen-binding portion comprises the three CDRs identified in the sequence listing. (Item 84) the VH region of the first antigen-binding portion comprises: SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, respectively; or SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 110, respectively 84. The method of item 83, comprising HCDR1, HCDR2, and HCDR3 comprising: (Item 85) 85. The method of any one of items 80 to 84, wherein the first antigen-binding portion comprises a VL region having at least 80% sequence identity to a VL sequence identified in Table 4. (Item 86) 86. The method of claim 85, wherein the first antigen-binding portion comprises a VL region having at least 80% sequence identity to SEQ ID NO: 82 or SEQ ID NO: 97. (Item 87) 87. The method of any one of items 80 to 86, wherein the VL region of the first antigen-binding portion comprises the three CDRs identified in the sequence listing. (Item 88) the VL region of the first antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; Item 87. The method according to item 87. (Item 89) 89. The method of any one of items 80 to 88, wherein the second antigen-binding portion comprises a VH region having at least 80% sequence identity to a VH sequence identified in Table 4. (Item 90) 90. The method of claim 89, wherein the second antigen-binding portion comprises a VH region having at least 80% sequence identity to SEQ ID NO: 73 or SEQ ID NO: 75. (Item 91) 91. The method of any one of items 80 to 90, wherein the VH region of the second antigen-binding portion comprises the three CDRs identified in the sequence listing. (Item 92) 92. The method of claim 91, wherein the VH region of the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 comprising SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively. (Item 93) 93. The method of any one of items 80 to 92, wherein the second antigen-binding portion comprises a VL region having at least 80% sequence identity to a VL sequence identified in Table 4. (Item 94) 94. The method of claim 93, wherein the second antigen-binding portion comprises a VL region having at least 80% sequence identity to SEQ ID NO: 74 or SEQ ID NO: 76. (Item 95) 95. The method of any one of items 80 to 94, wherein the VL region of the second antigen-binding portion comprises the three CDRs identified in the sequence listing. (Item 96) 96. The method of item 95, wherein the VL region of the second antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, respectively. The foregoing summary is illustrative only and is not to be construed as limiting in any way. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings and detailed description, and from the claims. [Brief explanation of the drawings]

[0034] [Figure 1-1]Figures 1A-1G summarize the results obtained from experiments conducted to demonstrate that bispecific antibodies based on a guide-effector design according to some non-limiting embodiments of the present disclosure can significantly affect the internalization kinetics of cell surface antigens. Figure 1A shows a diagram of a tetravalent ALCAM x EphA2 bsIgG. The IgG backbone is based on the non-internalizing anti-ALCAM antibody 3F1. An internalizing anti-EphA2 scFv is fused to the C-terminal end of the light chain. Figure 1B shows a confocal microscopy study of antibody internalization. HEK293 or HEK293-EphA2#2 cells were incubated with the indicated IgG or bsIgG (100 nM) for 2 hours at 37°C. The antibody (red) was detected using an Alexa® 647-labeled anti-human IgG secondary antibody, and cell images were analyzed using a digital laser confocal microscope. Scale bar: 20 μm. Figure 1C shows the kinetics of ALCAM cell surface removal by the bispecific antibody. HEK293-EphA2#2 cells were incubated with the indicated IgG or bsIgG for 1, 4, and 24 hours, and surface ALCAM levels were determined by FACS. Non-internalized ALCAM was removed from the cell surface by a bispecific (3F1 / RYR) antibody but not by a monoclonal antibody. Figure 1D shows the correlation between surface antigen (ALCAM) removal efficiency and the EphA2 / ALCAM (E / A) expression ratio. HEK293 cell models with various EphA2 / ALCAM ratios were incubated with 3F1, 3F1 / RYR, and C10 / RYR (all at 100 nM), and the antigen remaining on the cell surface was determined by an anti-ALCAM antibody that binds to a different epitope than 3F1. Pearson correlation coefficients (r) were calculated (0.3266, -0.7550, and -0.1896 for 3F1, 3F1 / RYR, and C10 / RYR, respectively), and trend lines were drawn according to linear regression analysis. Data represent mean ± standard deviation (duplicates). Figure 1E shows bispecific-induced internalization of ALCAM when the guide-to-effector ratio is above a threshold. CM: cell membrane.Figure 1F shows that EphA2 internalization is significantly delayed by the bispecific 3F1 / RYR when the guide-to-effector ratio is below a threshold. HEK293 cells with a low EphA2 / ALCAM ratio (<0.2) were incubated with the indicated antibodies (100 nM), and surface EphA2 levels were measured by FACS. P values ​​were determined using a two-tailed Student's t-test. *P<0.05, and ***P<0.001. Figure 1G is an illustration of the phenomenon shown in Figure 1F, in which EphA2 internalization is delayed (e.g., reduced) when the EphA2-to-ALCAM (E / A) ratio is below a threshold. [Figure 1-2]Figures 1A-1G summarize the results obtained from experiments conducted to demonstrate that bispecific antibodies based on a guide-effector design according to some non-limiting embodiments of the present disclosure can significantly affect the internalization kinetics of cell surface antigens. Figure 1A shows a diagram of a tetravalent ALCAM x EphA2 bsIgG. The IgG backbone is based on the non-internalizing anti-ALCAM antibody 3F1. An internalizing anti-EphA2 scFv is fused to the C-terminal end of the light chain. Figure 1B shows a confocal microscopy study of antibody internalization. HEK293 or HEK293-EphA2#2 cells were incubated with the indicated IgG or bsIgG (100 nM) for 2 hours at 37°C. The antibody (red) was detected using an Alexa® 647-labeled anti-human IgG secondary antibody, and cell images were analyzed using a digital laser confocal microscope. Scale bar: 20 μm. Figure 1C shows the kinetics of ALCAM cell surface removal by the bispecific antibody. HEK293-EphA2#2 cells were incubated with the indicated IgG or bsIgG for 1, 4, and 24 hours, and surface ALCAM levels were determined by FACS. Non-internalized ALCAM was removed from the cell surface by a bispecific (3F1 / RYR) antibody but not by a monoclonal antibody. Figure 1D shows the correlation between surface antigen (ALCAM) removal efficiency and the EphA2 / ALCAM (E / A) expression ratio. HEK293 cell models with various EphA2 / ALCAM ratios were incubated with 3F1, 3F1 / RYR, and C10 / RYR (all at 100 nM), and the antigen remaining on the cell surface was determined by an anti-ALCAM antibody that binds to a different epitope than 3F1. Pearson correlation coefficients (r) were calculated (0.3266, -0.7550, and -0.1896 for 3F1, 3F1 / RYR, and C10 / RYR, respectively), and trend lines were drawn according to linear regression analysis. Data represent mean ± standard deviation (duplicates). Figure 1E shows bispecific-induced internalization of ALCAM when the guide-to-effector ratio is above a threshold. CM: cell membrane.Figure 1F shows that EphA2 internalization is significantly delayed by the bispecific 3F1 / RYR when the guide-to-effector ratio is below a threshold. HEK293 cells with a low EphA2 / ALCAM ratio (<0.2) were incubated with the indicated antibodies (100 nM), and surface EphA2 levels were measured by FACS. P values ​​were determined using a two-tailed Student's t-test. *P<0.05, and ***P<0.001. Figure 1G is an illustration of the phenomenon shown in Figure 1F, in which EphA2 internalization is delayed (e.g., reduced) when the EphA2-to-ALCAM (E / A) ratio is below a threshold. [Figure 1-3]Figures 1A-1G summarize the results obtained from experiments conducted to demonstrate that bispecific antibodies based on a guide-effector design according to some non-limiting embodiments of the present disclosure can significantly affect the internalization kinetics of cell surface antigens. Figure 1A shows a diagram of a tetravalent ALCAM x EphA2 bsIgG. The IgG backbone is based on the non-internalizing anti-ALCAM antibody 3F1. An internalizing anti-EphA2 scFv is fused to the C-terminal end of the light chain. Figure 1B shows a confocal microscopy study of antibody internalization. HEK293 or HEK293-EphA2#2 cells were incubated with the indicated IgG or bsIgG (100 nM) for 2 hours at 37°C. The antibody (red) was detected using an Alexa® 647-labeled anti-human IgG secondary antibody, and cell images were analyzed using a digital laser confocal microscope. Scale bar: 20 μm. Figure 1C shows the kinetics of ALCAM cell surface removal by the bispecific antibody. HEK293-EphA2#2 cells were incubated with the indicated IgG or bsIgG for 1, 4, and 24 hours, and surface ALCAM levels were determined by FACS. Non-internalized ALCAM was removed from the cell surface by a bispecific (3F1 / RYR) antibody but not by a monoclonal antibody. Figure 1D shows the correlation between surface antigen (ALCAM) removal efficiency and the EphA2 / ALCAM (E / A) expression ratio. HEK293 cell models with various EphA2 / ALCAM ratios were incubated with 3F1, 3F1 / RYR, and C10 / RYR (all at 100 nM), and the antigen remaining on the cell surface was determined by an anti-ALCAM antibody that binds to a different epitope than 3F1. Pearson correlation coefficients (r) were calculated (0.3266, -0.7550, and -0.1896 for 3F1, 3F1 / RYR, and C10 / RYR, respectively), and trend lines were drawn according to linear regression analysis. Data represent mean ± standard deviation (duplicates). Figure 1E shows bispecific-induced internalization of ALCAM when the guide-to-effector ratio is above a threshold. CM: cell membrane.Figure 1F shows that EphA2 internalization is significantly delayed by the bispecific 3F1 / RYR when the guide-to-effector ratio is below a threshold. HEK293 cells with a low EphA2 / ALCAM ratio (<0.2) were incubated with the indicated antibodies (100 nM), and surface EphA2 levels were measured by FACS. P values ​​were determined using a two-tailed Student's t-test. *P<0.05, and ***P<0.001. Figure 1G is an illustration of the phenomenon shown in Figure 1F, in which EphA2 internalization is delayed (e.g., reduced) when the EphA2-to-ALCAM (E / A) ratio is below a threshold.

[0035] [Figure 2]Figures 2A-2F summarize the results obtained from experiments conducted to demonstrate that a bispecific antibody (3F1 / RYR) based on a guide-effector design according to certain non-limiting embodiments of the present disclosure effectively removes a non-internalized antigen (ALCAM) from the surface of pancreatic cancer cells. Figure 2A: ALCAM cell surface levels after antibody treatment. Pancreatic cancer cell lines L3.6pl (left bar on the X-axis), Capan-1 (middle bar), and Panc-1 (right bar) were incubated with 3F1, 3F1 / RYR, C10 / RYR, or a mixture of 3F1 and C10 / RYR. After post-treatment washing, cell surface ALCAM levels were determined using Alexa® 647-labeled IgG, which binds to a different epitope on ALCAM than 3F1. MFI values ​​were normalized to cells without antibody treatment. **P<0.01, and ***P<0.001. Duplicate analysis. Figure 2B: Confocal microscopy study of cell-type-selective internalization mediated by bispecific antibodies. L3.6pl (E / A ratio >0.2) and Panc-1 (E / A ratio <0.2) cells were incubated with 3F1, 3F1 / RYR, or C10 / RYR, and internalized antibodies were stained with FITC-labeled anti-human IgG. Scale bar: 20 μm. Figure 2C: Colocalization of antibodies and macropinocytic vesicles. L3.6pl cells were incubated with 100 nM 3F1, 3F1 / RYR, or C10 / RYR and ND70-TR (TR-Dextran, red) for 2 hours. Antibodies were detected with FITC-labeled anti-human IgG (green). Nuclei were labeled with Hoechst 33342 (blue). Scale bar: 10 μm. Figure 2D: Lysosomal transport after internalization. L3.6pl cells were incubated with the indicated antibodies (100 nM) for 2 hours. Internalized antibodies (green) and nuclei (blue) were stained as described in C), and lysosomes were detected using rabbit anti-LAMP1 primary IgG followed by Alexa® 647-labeled anti-rabbit IgG (red). Scale bar: 10 μm. Figure 2E: Delayed EphA2 internalization in Panc-1 cells when targeted by bispecific antibodies. **P<0.01, and ***P<0.001. Duplicate.FIG. 2F: Time course of EphA2 removal from the surface of Panc-1 cells at 0.5, 1, and 4 hours after antibody treatment.

[0036] [Figure 3] Figures 3A-3E summarize the results obtained from experiments conducted to demonstrate that bispecific antibody-induced cell surface ALCAM removal according to some non-limiting embodiments of the present disclosure has an anti-clonogenic effect on pancreatic tumor spheres. Figure 3A: Significant upregulation of ALCAM in L3.6pl sphere cells compared to non-sphere tumor cells. Adherent or sphere-cultured L3.6pl cells were dissociated into single cells, and antigen expression was measured using 3F1 or RYR IgG, followed by Alexa® 647-labeled anti-human IgG. Figure 3B: ALCAM removal from the surface of sphere-forming cells by 3F1 / RYR. Single cell populations of L3.6pl (200 cells / well) were incubated with the indicated antibodies (100 nM) in ultra-low attachment well plates for 2 weeks. Cell surface levels of ALCAM after antibody treatment were determined by FACS. MFI values ​​were normalized to the control (no antibody treatment). **P<0.01. Duplicate. Figure 3C: Antibody internalization into L3.6pl spheres. Tumorspheres incubated with the indicated antibodies were collected by centrifugation, fixed, and permeabilized for analysis by confocal microscopy. Antibodies and nuclei were stained with Alexa® 647-labeled anti-human IgG (red) and Hoechst 33342 (cyan), respectively. Scale bar: 10 μm. Intracellular antibody fluorescence intensity was quantified by Image J and is shown in the right panel. ***P<0.001. Figure 3D: Inhibition of L3.6pl tumorsphere formation by 3F1 / RYR—reduced number. Tumorsphere numbers (>100 μm) were counted 14 days after antibody treatment (left), and images of representative wells are shown (right). Error bars indicate standard deviation of duplicates. *P<0.05. Figure 3E: Inhibition of L3.6pl tumorsphere formation by 3F1 / RYR—reduced size. **P<0.01. Duplicate. Scale bar: 100 μm.

[0037] [Figure 4]Figures 4A-4E show the in vitro potency and selectivity of exemplary antibody-drug conjugates (ADCs) via site-specific conjugation in tumor cell lines with varying EphA2 / ALCAM ratios according to some non-limiting embodiments of the present disclosure. The cytotoxicity of the indicated ADCs or mixtures was investigated in the L3.6pl (Figure 4A) and Capan-1 (Figure 4B) cell lines, which have relatively high EphA2 / ALCAM ratios, and the Panc-1 (Figure 4C) cell line, which has a low EphA2 / ALCAM ratio. The MIA PaCa2 (Figure 4D) and C4-2B (Figure 4E) cell lines were used as ALCAM-low / negative and EphA2-low / negative cancer cell models, respectively. Cell viability (%) was normalized to the control group without ADC treatment.

[0038] [Figure 5] Figures 5A-5B show the anti-tumor efficacy of exemplary bispecific 3F1 / RYR antibody-drug conjugates (ADCs) in a pancreatic cancer xenograft model according to some non-limiting embodiments of the present disclosure. Figure 5A: Effect on tumor growth. Mice were subcutaneously inoculated with 1 x 10 Capan-1 cells and randomly divided into four groups (6 mice / group) with similar mean tumor size. Vehicle (PBS) or ADC (3 mg / kg) was intravenously injected at the indicated time points (arrows). Mean tumor volume ± standard error (mm) was plotted. Figure 5B: Body weight was monitored and plotted to assess toxicity of ADC treatment. None of the groups studied experienced significant weight loss (e.g., greater than 15%).

[0039] [Figure 6]Figures 6A-6D illustrate the selection and characterization of anti-ALCAM scFv from a phage display library. Figure 6A: Enrichment of ALCAM-binding phage through three rounds of selection. Recombinant Fc fusions of the ALCAM-V domain were immobilized on magnetic beads and utilized for scFv phage display library selection. Enrichment was calculated by dividing the phage output titer by the input phage titer (left y-axis). The binding activity of polyclonal phage amplified from each round of output was shown as fold increase over binding of the unselected phage library (right y-axis). Figure 6B: After three rounds of selection, FACS was performed to screen for monoclonal phage binding to the ALCAMhigh DU145 cell line. Figure 6C: Apparent KD of 3F1 IgG against live ALCAM-expressing cells. DU145 cells were incubated overnight at 4°C with various concentrations of 3F1 IgG and analyzed by FACS using Alexa® 647-conjugated anti-human IgG. KD values ​​were estimated by curve fitting using GraphPad Prism (GraphPad Software). Figure 6D: Confocal microscopy study of cellular localization of anti-ALCAM 3F1 IgG. Tumor cell lines were seeded into chamber well slides and incubated with 3F1 IgG for 2 hours at 37°C. The antibody was stained with Alexa® 647-conjugated anti-human antibody (red). Nuclei were stained with Hoechst dye (cyan). Scale bar: 20 μm. ALCAM expression measured using 3F1 IgG is shown below the microscopy images (lower panel).

[0040] [Figure 7]Figures 7A-7B graphically illustrate the characterization of exemplary anti-ALCAM x EphA2 bispecific antibodies according to some embodiments of the present disclosure. Figure 7A: Reducing SDS-PAGE analysis of monoclonal (3F1 and C10) and bispecific (3F1 / RYR and C10 / RYR) antibodies. 3F1 or C10 IgG is composed of a heavy chain (approximately 50 kDa) and a light chain (approximately 25 kDa). 3F1 / RYR or C10 / RYR bsIgG is composed of two similarly sized bands (approximately 50 kDa), a heavy chain and a light chain fused to an scFv. Figure 7A: FACS analysis of binding specificity. Bispecific and monoclonal antibodies were incubated with the HEK293-EphA2#2 cell line stably expressing EphA2 and parental HEK293 (as a specificity control) and analyzed by FACS.

[0041] [Figure 8-1] Figures 8A-8C illustrate surface antigen removal according to some embodiments of the present disclosure. Figure 8A: Insufficient surface ALCAM removal in HEK293 cells lacking expression of the guide antigen EphA2. HEK293 cells were incubated with the indicated IgG or bsIgG for 1, 4, and 24 hours at 37°C, washed, and analyzed by FACS to determine cell surface ALCAM levels after antibody treatment. Figure 8B: EphA2 cell surface removal in pancreatic cancer cell lines with varying EphA2-to-ALCAM ratios. Anti-ALCAM 3F1 IgG did not reduce surface EphA2, as expected, whereas 3F1 / RYR and control C10 / RYR, which bind to EphA2, efficiently removed EphA2 from the cell surface. The ability of the bispecific 3F1 / RYR to remove surface ALCAM is affected by the ratio of EphA2 to ALCAM (guide-to-effector antigen ratio). Figure 8C: EphA2 surface removal from L3.6pl (left) and Capan-1 (right) cells after antibody treatment. E / A ratio: EphA2 to ALCAM ratio. Data represent mean ± standard deviation (duplicates). *P<0.05, **P<0.01, and ***P<0.001. [Figure 8-2]Figures 8A-8C illustrate surface antigen removal according to some embodiments of the present disclosure. Figure 8A: Insufficient surface ALCAM removal in HEK293 cells lacking expression of the guide antigen EphA2. HEK293 cells were incubated with the indicated IgG or bsIgG for 1, 4, and 24 hours at 37°C, washed, and analyzed by FACS to determine cell surface ALCAM levels after antibody treatment. Figure 8B: EphA2 cell surface removal in pancreatic cancer cell lines with varying EphA2-to-ALCAM ratios. Anti-ALCAM 3F1 IgG did not reduce surface EphA2, as expected, whereas 3F1 / RYR and control C10 / RYR, which bind to EphA2, efficiently removed EphA2 from the cell surface. The ability of the bispecific 3F1 / RYR to remove surface ALCAM is affected by the ratio of EphA2 to ALCAM (guide-to-effector antigen ratio). Figure 8C: EphA2 surface removal from L3.6pl (left) and Capan-1 (right) cells after antibody treatment. E / A ratio: EphA2 to ALCAM ratio. Data represent mean ± standard deviation (duplicates). *P<0.05, **P<0.01, and ***P<0.001.

[0042] [Figure 9] Figure 9 graphically illustrates the importance of guide antigens in the cell-selective cytotoxicity of exemplary antibody-drug conjugates (ADCs) according to some embodiments of the present disclosure. In these experiments, various concentrations of the indicated ADCs were incubated with HEK293 cells (ALCAMhighEphA2low, insufficient guide antigen present) at 37°C for 96 hours. Cell viability was determined by calcein-AM and normalized to a control group without ADC treatment.

[0043] [Figure 10A]Figures 10A-10B illustrate the selection and characterization of anti-EphA2 scFvs from a yeast display mutagenesis library. Figure 10A: Apparent KD measurements of the binding affinity of four new EphA2 scFvs to human recombinant EphA2 protein. In this experiment, RYRgerm is the germline version of RYR. The remaining samples were RYRgerm derivatives with high binding affinity. The apparent KD values ​​were estimated by curve fitting of normalized MFI values. Figure 10B: Apparent KD measurements of the binding affinity of four new EphA2 scFvs to mouse recombinant EphA2-Fc fusion protein. The apparent KD values ​​were estimated by curve fitting of normalized MFI values. [Figure 10B] Figures 10A-10B illustrate the selection and characterization of anti-EphA2 scFvs from a yeast display mutagenesis library. Figure 10A: Apparent KD measurements of the binding affinity of four new EphA2 scFvs to human recombinant EphA2 protein. In this experiment, RYRgerm is the germline version of RYR. The remaining samples were RYRgerm derivatives with high binding affinity. The apparent KD values ​​were estimated by curve fitting of normalized MFI values. Figure 10B: Apparent KD measurements of the binding affinity of four new EphA2 scFvs to mouse recombinant EphA2-Fc fusion protein. The apparent KD values ​​were estimated by curve fitting of normalized MFI values.

[0044] [Figure 11] Figure 11 summarizes the results of experiments performed in the human prostate cancer cell line DU145 to evaluate the affinity of recombinant IgG1 between the original RYR and the new improved RYR-binding scFv RYRgerm_102919_15 described in Figures 10A-10B.

[0045] [Figure 12] FIG. 12 summarizes the results of experiments performed to assess the affinity of IgG1 described in FIGS. 10A-10B for recombinant human EphA2. DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description of Disclosure The present disclosure generally relates to the fields of cell biology and immunology. More specifically, compositions and methods are provided herein for modulating the internalization properties of cell surface molecules, for example, for converting a non-internalized cell surface antigen into an internalized one, and vice versa. For example, in some embodiments of the present disclosure, the conversion is achieved through a guide / effector system, in which the internalization properties of the guide antigen are conferred on the effector antigen when a set of conditions are met. In some embodiments of the present disclosure, engineered antibodies are provided, each comprising an antigen-binding portion specific for a cell-type-selective antigen (guide antigen) and another antigen-binding portion specific for an effector antigen, wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen. Also provided are recombinant cells, recombinant nucleic acids encoding such engineered antibodies, and pharmaceutical compositions comprising the same. The present disclosure also provides methods useful for modulating cellular internalization in a cell or a subject, as well as methods for modulating cell-type selective signaling in a subject and / or for treating health disorders and diseases, such as cancer-related diseases, including solid tumors and hematopoietic malignancies.

[0047] Considerable efforts have been made to utilize antibodies to deliver highly toxic payloads to infected or cancerous cells, acting like prodrugs by transporting the drug inside the cell and releasing it. The "antibody-drug conjugate" or "ADC" approach is one such example. In this case, cell-type-selective intracellular payload delivery is desired for the development of antibody-based targeted therapies. However, tumor-specific internalizing antigens are rarely discovered, and even rarer are those expressed at uniformly high levels. The compositions and methods disclosed herein address at least two unmet needs: (1) In targeted therapies requiring intracellular payload delivery, many tumor antigens are highly expressed but poorly internalized. By converting them to internalizing antigens, new targeted therapies can be developed. (2) In some cases, receptor internalization is also used to block signaling pathways and cause desensitization. By converting internalizing receptors to non-internalizing receptors, signaling pathways can be persistently activated.

[0048] As described in more detail below, an exemplary bispecific antibody was constructed using a rapidly internalizing antibody that binds to the tumor-associated antigen EphA2 and a non-internalizing antibody that binds to the highly expressed tumor-associated antigen ALCAM. The overall internalization properties of a bispecific antibody are significantly affected by the relative surface expression levels of EphA2 to ALCAM (antigen density ratio). When the EphA2 to ALCAM ratio exceeds a threshold value (e.g., about 1:5), the amount of bispecific antibody taken up by tumor cells exceeds that achieved by either a monoclonal internalizing antibody or a mixture of the two antibodies, demonstrating a bispecificity-dependent amplification effect in which a small amount of the internalizing antigen EphA2 induces the internalization of a large amount of the non-internalizing antigen ALCAM. When the ratio is below the threshold value, EphA2 may be non-internalized due to the presence of excess ALCAM on the surface of the same cell. In some exemplary experiments described below, bispecific antibody-drug conjugates (ADCs) were constructed based on the bispecific antibody designs described above, and the bispecific ADCs were found to be more potent than monospecific ADCs in terms of tumor cell killing both in vitro and in vivo. Thus, the internalization properties of cell surface antigens can be manipulated in either direction by adjacent antigens, and this phenomenon can be exploited for therapeutic targeting.

[0049] definition Unless otherwise defined, all technical terms, notations, and other scientific terms or terminology used herein are intended to have the meaning that is generally understood by those skilled in the art to which this disclosure pertains.In some cases, terms with generally understood meanings are defined herein for clarity and / or quick reference, and the inclusion of such definitions herein is not necessarily interpreted as representing the substantial difference from what is generally understood in the art.Many of the techniques and procedures described or referred to herein are well understood by those skilled in the art and are commonly used using conventional methods.

[0050] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following alternatives: "A," "B," "A or B," and "A and B." In this disclosure, the use of "or" means "and / or" unless otherwise indicated. Additionally, the use of "including" The words "include", "includes", and other forms thereof are also used. The use of "included" and "included" is not limiting.

[0051] The term "about," as used herein, has its ordinary meaning of approximately. Unless the degree of approximation is otherwise clear from the context, "about" means within 10% above or below the provided value, or rounded to the nearest significant figure, and in all cases includes the provided value. When a range is provided, it includes the boundary values.

[0052] The term "engineered" or "recombinant," when used in reference to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified through human intervention, e.g., by laboratory methods, or is the result of such modification. Thus, for example, recombinant or engineered proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant or engineered proteins may contain amino acid residues not found in the native (non-recombinant or wild-type) form of the protein, or may contain modified, e.g., labeled, amino acid residues. The term may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include: any chemical modification of a peptide, protein, or nucleic acid sequence, including one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; and addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. Thus, an engineered antibody refers to a recombinant polypeptide, including an antibody fragment, that contains at least an antigen-binding site derived from the variable domains of the antibody heavy (VL) and / or light (VH) chains, and may optionally contain all or part of the variable and / or constant domains of an antibody from any of the Ig classes (e.g., IgA, IgD, IgE, IgG, IgM, and IgY). The term "engineered," as used in reference to cells, is not intended to include naturally occurring cells, but rather to encompass cells that have been modified to contain or express polypeptides or nucleic acids that are not present in non-engineered cells.

[0053] As used herein, the term "functional fragment thereof" refers to a molecule that has a qualitative biological activity in common with the wild-type molecule from which the fragment or variant is derived. For example, a functional fragment of an antibody retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment is derived. For example, an antibody capable of binding to an epitope of a cell surface antigen can be truncated at the N-terminus and / or C-terminus, and retention of its epitope-binding activity can be assessed using assays known to those skilled in the art, including exemplary assays provided herein.

[0054] The term "operably linked," as used herein, refers to a physical or functional connection between two or more elements, e.g., polypeptide or polynucleotide sequences, that allows them to function in their intended manner. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that allows expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to a regulatory region and a coding sequence to be transcribed being in a position such that the regulatory region is effective to regulate the transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term "operably linked" refers to a configuration in which a regulatory sequence is appropriately positioned relative to a sequence encoding a polypeptide or functional RNA such that the regulatory sequence directs or regulates the expression or cellular localization of the mRNA, polypeptide, and / or functional RNA encoding the polypeptide. Thus, a promoter is in operable linkage with a nucleic acid sequence if it is capable of mediating transcription of the nucleic acid sequence. Operably linked elements may be contiguous or non-contiguous. Additionally, in the context of polypeptides, "operably linked" refers to a physical connection (direct or indirect) between amino acid sequences (e.g., different fragments, regions, portions, or domains) to provide a desired activity of the polypeptide. In the present disclosure, various segments, regions, or domains of an engineered antibody of the present disclosure may be operably linked to retain proper folding, processing, targeting, expression, binding, and other functional properties of the engineered antibody in a cell. Unless otherwise indicated, the various regions, domains, fragments, and portions of an engineered antibody of the present disclosure are operably linked to one another. Operably linked rregions, domains, fragments, and portions of an engineered antibody of the present disclosure are contiguous. They may be continuous or non-contiguous (eg, connected to each other via a linker).

[0055] The term "percent identity," in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acids (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region when compared and aligned for maximum correspondence over the comparison window or specified region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI website at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be "substantially identical." This definition also refers to or can apply to the complement of a test sequence. This definition includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity typically exists over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0056] If necessary, sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschulet et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be calculated using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group. Measurements can be performed using the ELISA kit at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) using its default parameters.

[0057] As used herein, and unless otherwise indicated, a "therapeutically effective amount" of a therapeutic agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease, such as cancer, or to delay or minimize one or more symptoms associated with the disease. A therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapeutic agents, that provides a therapeutic benefit in the treatment or management of a disease. The term "therapeutically effective amount" can encompass an amount that improves overall treatment of a disease, reduces or avoids symptoms or predisposition to a disease, or enhances the therapeutic effectiveness of another therapeutic agent. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Reducing" a symptom means reducing the severity or frequency of the symptom, or eliminating the symptom. The exact amount of a composition that comprises a "therapeutically effective amount" will depend on the purpose of the treatment, and can be ascertained by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0058] As used herein, a "subject" or "individual" includes animals, e.g., humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient receiving medical care. Thus, a subject may be a human patient or individual who has, is at risk of, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject may also be an individual who is diagnosed as being at risk for a condition of interest, either at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non-mammals, e.g., non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0059] The term "vector" is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. The nucleic acid molecule to be transferred is generally linked, e.g., inserted, into a vector nucleic acid molecule. Generally, a vector is capable of replication when associated with appropriate control elements. The term "vector" includes cloning and expression vectors, as well as viral and integrating vectors. An "expression vector" is a vector containing a regulatory region, thereby allowing expression of DNA sequences and fragments in vitro and / or in vivo. A vector may contain a sequence that induces autonomous replication in a cell, or may contain a sequence sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses. In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle to transfer genes into cells.

[0060] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0061] All ranges disclosed herein also encompass any and all possible subranges and combinations of subranges. All recited ranges can be recognized as fully descriptive and allow for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into lower, middle, and upper thirds, etc. Those skilled in the art will also understand that all terms such as "up to," "at least," "greater than," and "less than" are inclusive of the recited numbers and refer to ranges that can subsequently be broken down into subranges as described above. Finally, those skilled in the art will understand that ranges include each individual member. Thus, for example, a group having 1 to 3 elements refers to groups having 1, 2, or 3 elements. Similarly, a group having 1 to 5 elements refers to groups having 1, 2, 3, 4, or 5 elements, etc.

[0062] Aspects and embodiments of the present disclosure described herein are understood to include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0063] Headings, e.g., (a), (b), (i), etc., are provided solely for ease of reading the specification and claims. The use of headings in the specification or claims does not require that the steps or elements be performed in the alphabetical or numerical order in which they are presented.

[0064] It is understood that certain features of the present disclosure, which are described for clarity in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are described for brevity in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0065] Cellular internalization for targeted therapy The high specificity of monoclonal antibodies is often utilized in the development of targeted therapies. Ideally, potent cytotoxic drugs can be linked to cell-type selective antibodies, allowing the cytotoxic drugs to be directed to target cells and preferentially accumulate in target tissues. Antibody-drug conjugates (ADCs) are a class of targeted therapies that have shown clinical efficacy. Internalizing antibodies are often desired to achieve efficient intracellular payload delivery and tumor killing, but this requirement is not absolute for certain drugs, such as MMAE, which can diffuse across cell membranes and cause bystander effects.

[0066] Although conceptually simple, target selection for ADCs is hampered by the fact that so-called tumor-specific antigens are rarely discovered, and even rarer still are tumor-specific antigens with the desired properties for therapeutic targeting, such as those uniformly expressed at high levels by cancer cells and efficiently internalized. Several approaches have been developed to improve antibody internalization and ADC efficacy. For example, the HER2 antigen has been targeted for improved ADC internalization through a dual paratope design and the resulting cross-linking effect. In another example, bispecific antibodies composed of a moderately internalizing antibody arm (anti-HER2) and an internalization-inducing antibody arm (anti-CD63, anti-PRLR, or anti-APLP2) have been constructed and used to improve ADC uptake. However, despite these efforts, bispecific ADCs have shown only limited improvement over the parent monospecific anti-HER2 ADCs, suggesting that key parameters for this design remain to be elucidated.

[0067] One important question is whether the internalization tendency of a given cell surface antigen can be influenced by its neighboring surface antigen, and if so, what are the parameters that determine the conversion of a non-internalized antigen to an internalized antigen and vice versa. Guide-effector bispecific antibody systems for achieving cell-type-specific signaling modulation have been reported. The key to their design is the guide-to-effector ratio and the threshold of guide antigen expression. We hypothesize that internalization can be manipulated by a guide-effector-based bispecific antibody approach. As described below, we developed an exemplary bispecific antibody targeting the rapidly internalized antigen EphA2 and the non- or slowly internalized antigen ALCAM. We found that this bispecific antibody becomes internalized when the ratio of EphA2 to ALCAM is greater than approximately 1:5. The bispecific effect is due to the single action of two monoclonal antibodies. It has also been shown that, unlike pure mixtures, the number of bispecific molecules delivered to tumor cells is greater than that of antibody mixtures. Thus, the guide-effector design can start with a small number of internalized seed antigens, resulting in an amplification effect in which the internalization effect spreads to more abundant non-internalized antigens. Notably, when the ratio of EphA2 to ALCAM is below a threshold (1:5), internalized EphA2 can be converted to non-internalized or slowly internalized by ALCAM, and this conversion is shown to be reciprocal depending on the ratio of guide to effector antigen. Thus, when targeted by bispecific antibodies, the internalization of cell surface antigens can be easily manipulated by their neighboring antigens, resulting in either amplified endocytosis or significantly delayed internalization depending on the relative abundance of the two antigens, providing an opportunity to therapeutically utilize the guide / effector-based bispecific antibody-induced cell membrane dynamics disclosed herein.

[0068] As shown in the following examples, previously developed guide-effector bispecific antibody designs were employed for cell-type-selective signaling modulation to achieve cell-type-selective modulation of internalization. Specifically, when the guide-to-effector ratio exceeds a threshold (e.g., 1:5 in the example of EphA2 / ALCAM), a non-internalized antigen (ALCAM) can be internalized by the bispecific antibody. When the guide-to-effector ratio falls below the threshold, an internalized antigen (EphA2) can be non-internalized or slowly internalized by the bispecific antibody. Thus, in the context of bispecific targeting, the internalization behavior of a cell surface antigen is significantly influenced by its neighboring antigen and can be easily manipulated in either direction through bispecific targeting of appropriately selected guide / effector pairs.

[0069] This disclosure has relevance to therapeutic drug development. By converting non-internalized antigens into internalized ones, this disclosure has direct relevance to ADC development. ADCs are a class of anticancer drugs that utilize the specificity of antibodies to deliver cytotoxic drugs to tumor cells. While the concept is attractive, clinical development of this class of anticancer drugs has encountered various challenges. To date, only four ADCs have been approved by the FDA for clinical use. While initial issues, such as drug and linker stability, have been addressed, other problems remain. Highly potent drugs, such as DNA chelators, have been used to generate ADCs, but these drugs cause cumulative toxicity and have limited therapeutic windows. Microtubule inhibitors, such as auristatin derivatives, are less potent than DNA chelators, and their toxicity does not accumulate except for peripheral nerve damage. Due to the low potency of auristatins and the limited amount of drug delivered to tumor cells, the therapeutic window remains narrow. Increasing the DAR can improve the efficacy of in It can result in the delivery of more drug molecules to tumor cells in vitro, but in In vivo, ADCs with high DARs are rapidly cleared from the circulation, thus reducing efficacy and increasing toxicity. Site-specific conjugation achieves a nearly uniform DAR (n=2) and improves pharmacokinetics (PK), but the total number of drug molecules delivered to tumor cells remains limited. In principle, measures to improve the therapeutic window of ADCs include (1) increasing cell surface target density and (2) improving target internalization. Both should result in the delivery of a large number of ADCs into tumor cells. While the use of macropinocytotic antibodies in ADC construction to improve internalization has previously been reported, the present disclosure provides an approach to increase target density through a guide-effector bispecific antibody design.

[0070] As a non-limiting example, a rapidly internalizing macropinocytotic anti-EphA2 (guide) antibody and a non- / slowly internalizing anti-ALCAM (effector) antibody are used as a model system to study the bispecific effect. When the antigen density ratio of EphA2 / ALCAM exceeds a threshold value (e.g., 1:5 in the experimental system described herein), the bispecific anti-ALCAM x EphA2 antibody can induce the internalization of both EphA2 and ALCAM. In other words, the bispecific antibody can turn a non-internalizing antigen (ALCAM) into an internalizing antigen. In in vitro cytotoxicity assays, the bispecific ADC is more potent than either of the monospecific ADCs and even the mixture of these ADCs, consistent with the increased amount of ADC delivered and internalized by the bispecific antibody. Thus, there is an amplification effect inherent to bispecific antibodies rather than monospecific antibodies or mixtures thereof, where a small number of internalizing antigens (guide, EphA2) when targeted by a bispecific antibody can induce the internalization of a large number of non-internalizing antigens (effector, ALCAM), resulting in the delivery of greater amounts of ADC and drug molecules to tumor cells compared to monoclonal ADCs and their mixtures.

[0071] In addition to enhancing efficacy through enhanced internalization, the compositions and methods disclosed herein impact the expansion of the range and variety of cell surface targets for ADCs. A major challenge for current ADCs is how to deliver payloads specifically and in high quantities to target cells. In the context of monoclonal antibodies, target antigens must be specifically and uniformly expressed at high levels on the tumor surface. In practice, however, antigens with both absolute specificity and uniformly high levels of expression are rarely found. Therefore, lineage markers expressed by the tissue from which the tumor originates are often used for tumor targeting. These lineage markers have two limitations: (1) they tend to show reduced or heterogeneous expression in late-stage cancers because they are not functionally required for tumor viability. For example, PSMA expression in late-stage prostate cancer is heterogeneous and downregulated in androgen signaling inhibitor-resistant small cell tumors; and (2) they are often expressed in more than one normal tissue type. For example, mesothelin is expressed by some tumors, such as mesothelioma, ovarian cancer, and pancreatic cancer, but also by normal mesothelium. PSMA is expressed by prostate tumors but also by some normal tissues. Similarly, CD19 is expressed by normal tissues other than B cells. With monoclonal antibody approaches, target selection appears to be rather limited or suboptimal. In the context of ADCs, efforts have been made to increase payload efficacy, but the therapeutic window remains narrow, as noted above. An alternative approach is to identify targets that amplify the difference in payload delivered between tumor cells and normal cells. The present disclosure is particularly relevant to this approach because the guide-effector bispecific antibody design described herein allows many non-internalized tumor-associated antigens to be internalized, thus contributing to increased intracellular delivery of ADCs. The amplification effect is specific to tumor cells due to the coexpression of both guide and effector antigens.

[0072] The design of guide-effector bispecific antibodies for cell-type-selective modulation of the Wnt signaling pathway has been reported previously (e.g., Lee NK et al., Sci Rep. 2018 Jan 15;8(1):766. The present disclosure relates to a bispecific antibody approach. This extends the applicability of the bispecific approach to antigen internalization and ADCs. The essence of the guide-effector bispecific antibody system disclosed herein is that the behavior of a given antigen (effector) can be shaped by the adjacent antigen (guide) when the guide to effector ratio exceeds a threshold. In Wnt signaling studies, when the guide / effector ratio exceeds 5-10:1, there is a 1,000-fold increase in the potency of bispecific antibodies compared to monoclonal antibodies, and the enhancement is cell-type selective. In the present disclosure, it has been shown that when the guide / effector ratio exceeds 1:5, a small number of guide antigens (internalized) can convert a large number of effector antigens (non-internalized) into internalized antigens.

[0073] Of note, although some of the experiments described below focus on ADCs and the conversion of non-internalizing antibodies to internalizing antibodies, the converse has also been shown to be true: when the ratio of internalizing antigen to non-internalizing antigen is below a threshold value (e.g., 1:5 in the system described herein), the internalizing antigen EphA2 becomes slowly internalized in the presence of non-internalizing ALCAM. This may be useful in applications where it is desirable to leave the antigen on the cell surface to prevent degradation and prolong signaling function.

[0074] There have been several recent reports of bispecific ADCs with internalization arms that bind either lysosomal proteins or antigens that rapidly traffic to lysosomes. In most cases, the observations are empirical, suggesting that the bispecific effect is rather modest, and important parameters influencing bispecific antibody-induced internalization have not been fully described. For example, it is unclear whether a lysosomal antigen is required for this phenomenon. It is also unclear why bispecific antibodies function in some cells but not others. This disclosure demonstrates that the important variable in bispecific antibody design is the ratio of guide to effector antigen and that no special properties other than internalization are required for the internalization arm. The guide antigen (internalization arm) does not need to be a lysosomal protein to induce internalization and lysosomal trafficking. For example, in this disclosure, micropinocytosis is utilized to select a macropinocytotic antibody against the cell surface antigen EphA2 as a guide to target the bispecific antibody to the lysosomal compartment.

[0075] In summary, this disclosure demonstrates that in the context of bispecific targeting, internalization is no longer an essential property of a given antigen. Instead, antigen internalization is profoundly influenced by its neighboring antigens and can be easily manipulated in either direction in a cell-type-selective manner using appropriately selected guide / effector pairs. This plasticity of bispecific antibody-induced cell surface dynamics can be exploited for therapeutic drug development.

[0076] Compositions of the present disclosure Engineered antibodies As described in more detail below, the present disclosure provides a new class of antibodies that are engineered to modulate the internalization properties of cell surface molecules, e.g., converting non-internalized cell surface antigens into internalized ones, and vice versa. For example, in some embodiments of the present disclosure, this conversion is achieved through a guide / effector system, in which the internalization properties of the guide antigen are conferred on the effector antigen when a set of conditions are met. In some embodiments of the present disclosure, the engineered antibodies disclosed herein can simultaneously bind to a cell-type-selective internalizing antigen (e.g., a guide antigen) and an abundantly expressed receptor (e.g., an effector antigen) on a target cell.

[0077] In one aspect, some embodiments disclosed herein relate to an engineered antibody or functional fragment thereof comprising: a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate; and b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate, wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. The term "internalization" refers to the transport of a moiety from the outside to the inside of a cell. The internalized moiety may be located in an intracellular compartment. An "internalized" or "internalized" antigen or antibody refers to an antigen or antibody that can be transported from the outside to the inside of a target cell.

[0078] In the engineered antibodies disclosed herein, antigens with a high cellular internalization rate are defined as rapidly internalizing antigens, and antigens with a low cellular internalization rate are defined as slowly internalizing antigens. Thus, in some embodiments, the internalization rate of rapidly internalizing antigens is at least 50%, at least 70%, at least 80%, or at least 90% higher than that of slowly internalizing antigens. In certain embodiments, if more than 50% of the surface-bound antibody is internalized within 4 hours at 37°C, the antibody is referred to as rapidly internalizing. In some embodiments, if less than 30% of the surface-bound antibody is internalized after 24 hours at 37°C, the antibody is referred to as slowly internalizing. In some embodiments, if less than 10% of the surface-bound antibody is internalized after 24 hours at 37°C, the antibody is referred to as non-internalizing.

[0079] Those skilled in the art will understand that the process of cellular internalization generally refers to the movement of cell surface molecules across the plasma membrane from the cell surface to the interior of the cell. After internalization, endosomes can transport the molecules to lysosomes for degradation or recycling to the cell surface. The cellular internalization rate of a given cell surface molecule provides a measurement of the kinetics of the movement of the molecule across the plasma membrane from the surface to the interior of the cell. The internalization rate of antigens and antibodies can be experimentally monitored by several techniques known in the art, including acid dissociation (LiN. et al., Methods Mol. Biol., 457:305-17, 2008) and toxin killing assays (Pahara J. et al. ExpCell Res., 316:2237-50, 2010 and Mazor et al., J. Immunol. Methods, 321:41-59, 2007). The internalization kinetics can be assessed and / or measured. Numerous antibody labeling techniques, dyes, and kits for antibody labeling that can be used to quantify and monitor internalization are commercially available (e.g., pHrodo iFL antibody labeling methods, reagents, and kits sold by Thermo Fisher Scientific). For example, the cellular internalization kinetics of the antigens and engineered antibodies of the present disclosure can be assessed and quantified by confocal microscopy or flow cytometry. For example, confocal laser scanning microscopy (CLSM) is widely used to verify cellular internalization. Another suitable technique, imaging flow cytometry (IFC), which provides quantitative FACS data and cellular images, can also be used to quantify cellular internalization kinetics. Additional information on this topic can be found, for example, in Haetal., Mol Cell Proteomics, 13(12):3320-31, 2014 and Vainshtein et al., Pharm. Res. 32:286-299, 2015. In some embodiments, the cellular internalization kinetics of the engineered antibodies of the present disclosure are described previously by Vainshtein et al. (Pharm Res. 2015, 32:286-299), which is incorporated herein by reference. The method described can be used to quantify the internalization rate of fluorescently tagged antibodies in live cells, where confocal microscopy imaging techniques are used to record the internalization kinetics of fluorescently tagged antibodies in live cells, and quantitative image analysis algorithms are used to determine the internalization rate constant (K int In some embodiments, the internalization rate constant, K, of the engineered antibodies disclosed herein is used to determine int is calculated from the internalization time course by curve fitting the data using the following equation: S cyt (t)=S 0,cyt +(1-e- Kint.t ).S max,cyt , where S cyt (t) is the cytoplasmic fluorescence signal at time t, and S 0,cyt and S max,cytare the initial cytoplasmic fluorescence signal and the maximum signal, respectively (see Vainshteinetal. 2015).

[0080] The designation of an antigen-binding moiety capable of binding to a cell surface guide antigen as a "first" antigen-binding moiety and an antigen-binding moiety capable of binding to a cell surface effector antigen as a "second" antigen-binding moiety is not intended to imply a particular structural arrangement of either the "first" or "second" antigen-binding moiety within the engineered antibody. As a non-limiting example, in some embodiments of the present disclosure, an engineered antibody may comprise an N-terminal portion comprising an antigen-binding moiety capable of binding to a cell surface guide antigen and a C-terminal portion comprising an antigen-binding moiety capable of binding to a cell surface effector antigen. In other embodiments, an engineered antibody may comprise an N-terminal portion comprising an antigen-binding moiety capable of binding to a cell surface effector antigen and a C-terminal portion comprising an antigen-binding moiety capable of binding to a cell surface guide antigen.

[0081] As described in more detail below, the first and / or second antigen-binding moieties can be multispecific, e.g., capable of binding more than one antigen, e.g., more than two, more than three, more than four, more than five, or more than six different antigens. For example, in some embodiments, the first antigen-binding moiety can be configured to have bispecificity, i.e., capable of binding to two guide antigens. In some embodiments, the second antigen-binding moiety can be configured to have bispecificity, i.e., capable of binding to two effector antigens. Additional information regarding the design of this dual-functional antibody can be found, for example, in Schaefer G. et al., Cancer Cell. 2011 Oct 18;20(4):472-86 and Lee CV et al., MAbs.2014;6(3):622-627.

[0082] Additionally or alternatively, an engineered antibody may comprise more than one antigen-binding moiety capable of binding to a cell surface guide antigen and / or more than one antigen-binding moiety capable of binding to a cell surface effector antigen. Thus, in some embodiments, an engineered antibody may comprise multiple antigen-binding moieties, each capable of binding to a cell surface guide antigen. In some embodiments, an engineered antibody may comprise multiple antigen-binding moieties, each capable of binding to a cell surface effector antigen. In some embodiments, an engineered antibody comprises multiple antigen-binding moieties, each capable of binding to a cell surface guide antigen, and multiple antigen-binding moieties, each capable of binding to a cell surface effector antigen.

[0083] According to the present disclosure, rapid internalization of a slowly internalizing antigen can be induced by operably linking an antigen-binding moiety specific for a slowly internalizing antigen to another antigen-binding moiety specific for a rapidly internalizing antigen, hi some embodiments, slow internalization of a rapidly internalizing antigen can be induced by operably linking an antigen-binding moiety specific for a rapidly internalizing antigen to another antigen-binding moiety specific for a slowly internalizing antigen.

[0084] In some embodiments, the internalization property of an engineered antibody disclosed herein is converted from internalizing to non-internalizing. In some embodiments, the internalization property of a guide antigen and / or effector antigen is converted from internalizing to non-internalizing. In some embodiments, the internalization property of an internalizing antigen (e.g., a guide antigen or an effector antigen) is converted from internalizing to non-internalizing by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for such an internalizing antigen operably linked to another antigen-binding portion specific for a non-internalizing antigen. For example, in some embodiments, the internalization property of an internalizing guide antigen is converted from internalizing to non-internalizing by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for an internalizing guide antigen operably linked to another antigen-binding portion specific for a non-internalizing effector antigen. In some embodiments, the internalizing property of an internalizing effector antigen is converted from internalizing to non-internalizing by using an engineered antibody disclosed herein that comprises an antigen-binding moiety specific for an internalizing effector antigen operably linked to another antigen-binding moiety specific for a non-internalizing guide antigen.

[0085] In some other embodiments, the internalization property of an engineered antibody disclosed herein is converted from a non-internalizing form to an internalizing form. In some embodiments, the internalization property of a non-internalizing antigen (e.g., a guide antigen or an effector antigen) is converted from a non-internalizing form to an internalizing form. In some other embodiments, the internalization property of a non-internalizing guide antigen is converted from a non-internalizing form to an internalizing form by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for such a non-internalizing guide antigen operably linked to another antigen-binding portion specific for an internalizing effector antigen. In some other embodiments, the internalization property of a non-internalizing effector antigen is converted from a non-internalizing form to an internalizing form by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for such a non-internalizing effector antigen operably linked to another antigen-binding portion specific for an internalizing guide antigen.

[0086] In some embodiments, the guide antigen has a cellular internalization rate that is higher than the cellular internalization rate of the effector antigen, where the guide antigen is a rapidly internalizing antigen and the effector antigen is a slowly internalizing antigen. In some embodiments, the guide antigen has a cellular internalization rate that is at least about 50% higher than the cellular internalization rate of the effector antigen. In some embodiments, the guide antigen has a cellular internalization rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the cellular internalization rate of the effector antigen. In some embodiments, the engineered antibodies of the present disclosure increase the rate of internalization of a slow-internalizing antigen (e.g., an effector antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only a slow-internalizing antigen) by operably linking an antigen-binding portion specific for a slow-internalizing antigen to another antigen-binding portion specific for a rapidly-internalizing antigen (e.g., a guide antigen). In some embodiments, the engineered antibodies of the present disclosure reduce the rate of internalization of a rapidly internalizing antigen (e.g., a guide antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only a rapidly internalizing antigen) by operably linking an antigen-binding portion specific for a rapidly internalizing antigen to another antigen-binding portion specific for a slowly internalizing antigen (e.g., an effector antigen).

[0087] In some embodiments, the effector antigen has a higher cellular internalization rate than the guide antigen, where the effector antigen is a rapidly internalizing antigen and the guide antigen is a slowly internalizing antigen. In some embodiments, the effector antigen has a cellular internalization rate that is at least about 50% higher than the guide antigen. In some embodiments, the effector antigen has a cellular internalization rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the guide antigen. In some embodiments, the engineered antibodies of the present disclosure increase the rate of internalization of a slow-internalizing antigen (e.g., a guide antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only a slow-internalizing antigen) by operably linking an antigen-binding moiety specific for a slow-internalizing antigen to another antigen-binding moiety specific for a rapidly internalizing antigen (e.g., an effector antigen). In some embodiments, the engineered antibodies of the present disclosure reduce the internalization rate of a rapidly internalizing antigen (e.g., an effector antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only the rapidly internalizing antigen) by operably linking an antigen-binding portion specific for a rapidly internalizing antigen to another antigen-binding portion specific for a slowly internalizing antigen (e.g., a guide antigen). In some embodiments, the cell surface guide antigen is an internalizing cell surface antigen. In some embodiments, the cell surface effector antigen is a non-internalizing cell surface antigen.

[0088] The internalization properties of the engineered antibodies or functional fragments thereof disclosed herein are determined by the relative surface density ratio of guide antigen to effector antigen. Those skilled in the art will readily understand that the surface density of a given molecule, e.g., an antigen or polypeptide, refers to the measured and / or estimated number of antigens or polypeptides in a given surface area. For example, the density of an antigen present on a cell surface can be expressed as approximately 10,000 copies per cell, meaning that the measured and / or estimated number of antigen molecules present on the cell surface is approximately 10,000. Numerous techniques, systems, assays, and procedures for determining and / or measuring the density of molecules present on a cell surface are known in the art. Additional information on this can be found in Example 12 below, as well as, for example, Lee NK et al., Sci. Rep. Jan 15;8(1):766, 2018 and Sherbenou, DW et al., J. Clin.Invest. 2016 Nov 14. In some embodiments of the present disclosure, the guide antigen is expressed as approximately 10,000 copies per cell, meaning that the measured and / or estimated number of antigen molecules present on the cell surface is approximately 10,000. The surface densities of the guide antigen and the effector antigen are measured. The results are then compiled and interpreted as a single ratio between the surface density of the guide antigen and the surface density of the effector antigen. A decision rule may indicate that any score above a given threshold indicates internalization of the engineered antibody, while a score below the threshold indicates a lack of internalization, e.g., non-internalization.

[0089] In some embodiments, these scores can be compared to a threshold, and a score above the threshold indicates increased or decreased internalization exhibited by the engineered antibody. The surface density, ratio, and appropriate threshold for each guide / effector pair can be determined by collecting data on a small sample set from both internalized and non-internalized antigens and separating them using a linear model. The linear model can be generated by statistical methods, such as logistic regression or a support vector machine with a first-order kernel function, or the linear model can be generated by testing.

[0090] In some embodiments, the relative surface density ratio of guide antigen to effector antigen is above a threshold value. In some embodiments, the threshold value is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. Thus, in some embodiments, the relative surface density ratio of guide antigen to effector antigen is greater than about 1:1, greater than about 1:2, greater than about 1:3, greater than about 1:4, greater than about 1:5, greater than about 1:10, greater than about 1:20, or greater than about 1:30. In some embodiments, the threshold value is about 1:5. In some embodiments, the relative surface density ratio of guide antigen to effector antigen is below a threshold value. In some embodiments, the threshold value is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. Thus, in some embodiments, the relative surface density ratio of guide antigen to effector antigen is less than about 1:1, less than about 1:2, less than about 1:3, less than about 1:4, less than about 1:5, less than about 1:10, less than about 1:20, or less than about 1:30. In some embodiments, the threshold is about 1:5.

[0091] In some other embodiments, the relative surface density ratio of effector antigen to guide antigen is above a threshold value. In some embodiments, the threshold value is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. Thus, in some embodiments, the relative surface density ratio of effector antigen to guide antigen is greater than about 1:1, greater than about 1:2, greater than about 1:3, greater than about 1:4, greater than about 1:5, greater than about 1:10, greater than about 1:20, or greater than about 1:30. In some embodiments, the threshold value is about 1:5. In some other embodiments, the relative surface density ratio of effector antigen to guide antigen is below a threshold value. In some embodiments, the threshold value is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. Thus, in some embodiments, the relative surface density ratio of effector antigen to guide antigen is less than about 1:1, less than about 1:2, less than about 1:3, less than about 1:4, less than about 1:5, less than about 1:10, less than about 1:20, or less than about 1:30. In some embodiments, the threshold is about 1:5.

[0092] As used herein, the term "antigen-binding moiety" refers to a polypeptide that specifically binds to an antigenic determinant, e.g., an antigen. In some embodiments, an antigen-binding moiety can direct the entity to which it binds (e.g., an engineered antibody comprising a second antigen-binding moiety) to a target site, e.g., a specific cell type, e.g., a type of tumor cell or tumor stroma bearing the antigenic determinant. For example, antibodies, antibody fragments, antibody derivatives, antibody-like scaffolds, and alternative scaffolds comprise at least one antigen-binding moiety. Antigen-binding moieties can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and scFvs. Thus, in some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are independently selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a full-length immunoglobulin, a nanobody, a single-domain antibody (sdAb), a VNAR domain, and a VHH domain, a diabody, or a functional fragment thereof. In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety are monovalent. In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety are multivalent, e.g., comprise more than one antigen-binding site. In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety are monospecific. In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety are multispecific, e.g., comprise antigen-binding sites with specific binding activity for at least two different target antigens, e.g., at least two, at least three, at least four, or at least five target antigens.

[0093] The term "antigen-binding site," as used herein, refers to the portion of an antigen-binding moiety responsible for specific binding between the antigen-binding moiety and an antigenic determinant. The antigen-binding site may be a single domain, e.g., an epitope-binding domain, or it may be a paired VH / VL domain such as can be found in a standard antibody. Thus, in some embodiments, the antigen-binding site of an antibody or fragment thereof described herein is formed by amino acid residues of the N-terminal variable regions of the heavy chain (VH) and light chain (VL). Generally, the variable regions of the VH and VL each contain three hypervariable regions called complementarity-determining regions (CDRs). The three CDRs of the VH (termed HCDR1, HCDR2, and HCDR3) and the three CDRs of the VL (termed LCDR1, LCDR2, and LCDR3) are three-dimensionally arranged relative to each other to form an antigen-binding surface. Unless otherwise indicated, the widely accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD). While any suitable numbering system can be used to designate CDR regions, in the absence of any other designation, the sequences of the CDRs of the engineered antibodies of this disclosure according to the Kabat definition system are summarized in Tables 4 and 5 below.

[0094] Binding of the first and second antigen-binding moieties to their respective targets can be either competitive or non-competitive with the target's natural ligand. Thus, in some embodiments of the present disclosure, binding of the first and / or second antigen-binding moieties to their respective targets can be ligand-blocking. In some other embodiments, binding of the first and / or second antigen-binding moieties to their respective targets does not block binding of the natural ligand. In some embodiments of the present disclosure, the engineered antibody comprises a first amino acid sequence encoding a first antigen-binding moiety linked to a second amino acid sequence encoding a second antigen-binding moiety that is not naturally linked. The amino acid sequences can normally reside in separate proteins that are combined in the fusion polypeptide, or they can normally reside in the same protein but are placed in a new configuration in the fusion polypeptide. The amino acid sequences encoding the first and second antigen-binding moieties can be generated, for example, by chemical synthesis or by generating and translating a polynucleotide that encodes the peptide regions in the desired relationship.

[0095] In some embodiments, the first antigen-binding moiety is directly linked to the second antigen-binding moiety. In some embodiments, the first antigen-binding moiety is directly linked to the second antigen-binding moiety via at least one covalent bond. In some embodiments, the first antigen-binding moiety is directly linked to the second antigen-binding moiety via at least one peptide bond. In some embodiments, the C-terminal amino acid of the first antigen-binding moiety may be operably linked to the N-terminal amino acid of the second antigen-binding moiety. Alternatively, the N-terminal amino acid of the first antigen-binding moiety may be operably linked to the C-terminal amino acid of the second antigen-binding moiety.

[0096] In some embodiments, the first antigen-binding moiety is operably linked to the second antigen-binding moiety via a linker. There are no particular limitations on the linkers that can be used in the engineered antibodies described herein. In some embodiments, the linker is a synthetic compound linker, such as a chemical cross-linking agent. Non-limiting examples of suitable cross-linkers that are commercially available include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0097] In some embodiments, the first antigen-binding moiety is operably linked to the second antigen-binding moiety via a linker peptide sequence. In principle, there are no particular limitations on the length and / or amino acid composition of the linker peptide sequence. In some embodiments, any single-chain peptide containing about 1 to about 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues, etc.) can be used as a peptide linker. In some embodiments, the linker peptide sequence comprises about 5 to about 50, about 10 to about 60, about 20 to about 70, about 30 to about 80, about 40 to about 90, about 50 to about 100, about 60 to about 80, about 70 to about 100, about 30 to about 60, about 20 to about 80, or about 30 to about 90 amino acid residues. In some embodiments, the linker peptide sequence comprises about 1 to about 10, about 5 to about 15, about 10 to about 20, about 15 to about 25, about 20 to about 40, about 30 to about 50, about 40 to about 60, or about 50 to about 70 amino acid residues. In some embodiments, the linker peptide sequence is about 40 to about 70, about 50 to about 80, about 60 to about 80, about 70 to about 90, or or about 80 to about 100 amino acid residues. In some embodiments, the linker peptide sequence comprises about 1 to about 10, about 5 to about 15, about 10 to about 20, or about 15 to about 25 amino acid residues.

[0098] In some embodiments, the length and amino acid composition of the linker peptide sequence can be optimized to vary the orientation and / or proximity of the first and second antigen-binding moieties relative to each other to achieve a desired activity of the engineered antibody. In some embodiments, the orientation and / or proximity of the first and second antigen-binding moieties relative to each other can be varied as a "tuning" tool to achieve a tuning effect that enhances or reduces one or more desired activities of the engineered antibody. For example, in some embodiments, the orientation and / or proximity of the first and second antigen-binding moieties relative to each other can be optimized to create competitive, partially competitive, or non-competitive versions of the engineered antibody. In certain embodiments, the linker contains only glycine and / or serine residues (e.g., a glycine-serine linker).

[0099] antigen In some embodiments, engineered antibodies, e.g., bispecific antibodies, of the present disclosure may have binding specificities for two distinct cell surface antigens, one of which has a more rapid internalization rate than the other. Bispecific antibodies may comprise at least two components, i.e., a first component and a second component, each of which binds to its respective antigen, e.g., a first cell type-associated antigen (guide antigen) and a second antigen (effector antigen) associated with the target signaling pathway, respectively. The first component may comprise a first antigen-binding moiety for the first antigen, and the second component may comprise a second antigen-binding moiety for the second antigen. Such bispecific antibodies allow for increased ability to inhibit the target signaling pathway compared to non-targeting antibodies (e.g., antibodies that do not have binding specificity for effector antigens), and importantly, allow for cell type-specific inhibition.

[0100] Non-limiting examples of cell surface antigens suitable for the engineered antibodies of the present disclosure include activated leukocyte cell adhesion molecule (ALCAM), neural cell adhesion molecule (NCAM), calcium activated chloride channel 2 (CaCC), carbonic anhydrase IX, carcinoembryonic antigen (CEA), cathepsin G, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD46, CD52, CD71, CD73, CD272, CD276, B cell maturation antigen (BCMA), epithelial cell adhesion molecule (EpCAM), epithelial cell adhesion molecule (EpCAM), epithelial cell adhesion molecule (EpCAM), epithelial cell adhesion molecule (E ... Furin type A receptor 2 (EphA2), ephrin type A receptor 3 (EphA3), ephrin type A receptor 4 (EphA4), ephrin B2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PDGFR-beta, SSEA-4, epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 2 (ErbB2), Erb-B2 receptor tyrosine kinase ErbB3, Erb-B2 receptor tyrosine kinase 4 (ErbB4), folate-binding protein (folate receptor), ganglioside, gangliosides, gp100, gpA33, immature laminin receptor, intercellular adhesion molecule 1 (ICAM-1), Lewis-Y, mesothelin, prostate stem cell antigen (PSCA), mucin 16 (MUC16 or CA-125), mucin 1 cell surface-associated (MUC1), mucin 2 oligomer mucus gel-forming (MUC2), mucin, prostate membrane-specific antigen (PSMA), T Examples of surface antigens include EM1 / CD248, TEM7R, CLDN6, thyroid-stimulating hormone receptor (TSHR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), immunoglobulin lambda-like polypeptide 1 (IGLL1), P-selectin, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), tumor-associated calcium signaling factor 2 (Trop-2), and tumor-associated glycoprotein 72 (TAG-72). In some embodiments, the cell surface antigen comprises ICAM-1, EphA2, and ALCAM.

[0101] Guide antigen In some embodiments, the guide antigen recognized by the engineered antibody of the present disclosure is a molecule that functions as a cell-type-associated antigen. A cell-type-associated antigen generally refers to a molecule whose expression level is substantially higher in a particular cell type of interest ("target cell") compared to non-target cells. In some embodiments, the guide antigen can be any cell surface antigen that is overexpressed on target cells. For example, there are molecules that are overexpressed in cancer cells, such as intercellular adhesion molecule 1 (ICAM-1), EphA2, and activated leukocytes. In some embodiments, the guide antigen is intercellular adhesion molecule (ALCAM), and these molecules can be considered cancer-associated antigens or tumor-associated antigens.

[0102] In some embodiments, the guide antigen is a cancer-associated antigen. Non-limiting examples of cancer-associated antigens suitable for the compositions and methods of the present disclosure include CD19, CD22, HER2 (ErbB2 / neu), mesothelin, PSCA, CD123, CD30, CD71, CD171, CS-1, CLECL1, CD33, EGFRvIII, GD2, GD3, BCMA, PSMA, receptor tyrosine kinase-like orphan receptor 1 (ROR1), folate receptor, FLT3 (CD135), TAG72, CD38, CD44v6, CD46, CEA, EpCAM, CD272, B7H3 (CD276), KIT (CD117), CD213A2, IL-1Ra, PRSS21, VEGFR2, CD24, PD These include GFR-beta, SSEA-4, CD20, MUC1, MUC16, EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostatic acid phosphatase (PAP), ephrinB2, fibroblast activation protein (FAP), EphA2, c-Met, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), GM3, TEM1 / CD248, TEM7R, CLDN6, thyroid-stimulating hormone receptor (TSHR), GPRC5D, CD97, CD179a, anaplastic lymphoma kinase (ALK or CD246), and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the engineered antibodies or functional fragments thereof disclosed herein comprise an antigen-binding portion capable of binding to EphA2 expressed on the surface of a cell.

[0103] In some embodiments, by specifically recognizing and binding to a cell-type-associated antigen (e.g., a guide antigen), an engineered antibody of the present disclosure can be recruited to a target cell associated with the guide antigen, resulting in, for example, modulation of a signaling pathway in the target cell. In some embodiments, the guide antigen of an engineered antibody of the present disclosure not only serves as a cell-type selector but also as a potency enhancer, resulting in, for example, potent and selective inhibition of a target signaling pathway. In some embodiments, the guide antigen on the surface of a target cell has a threshold of expression that results in (1) the binding affinity of the engineered antibody to the target cell and (2) enhanced occupancy of the effector antigen by the engineered antibody.

[0104] Effector antigens In some embodiments, the effector antigen recognized by the engineered antibodies described herein is a molecule associated with a cellular activity or function, e.g., a signal transduction pathway. In some embodiments, the effector antigen is expressed on the surface of a cell of interest. In some embodiments, the effector antigen recognized by the engineered antibodies described herein is a molecule associated with a signal transduction pathway of interest (e.g., a target signal transduction pathway). In some cases, the effector antigen comprises a tumor antigen (e.g., a tumor-associated antigen or a tumor-specific antigen). Non-limiting examples of effector antigens suitable for the engineered antibodies of the present disclosure include ALCAM, EpCAM, folate binding protein, PSMA, PSCA, mesothelin, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD46, ICAM-1, CD55, CD59, CD70, CD71, CD73, CD97, BCMA, CD272, CD276, MUC1, MUC16, NCAM, CD24, EphA2, EphA3, EphA4, ephrin B2, CEA, c-Met, FGFR, IGF-1R, VEGFR, PDGFR, Trop-2, TAG-72, and P-selectin. Additional examples of suitable effector antigens are further described below, including EGFR, ErbB2, ErbB3, and ErbB4. In some embodiments, the engineered antibodies or functional fragments thereof disclosed herein comprise an antigen-binding portion capable of binding to ALCAM expressed on the surface of a cell.

[0105] In certain embodiments, the engineered antibody or functional fragment disclosed herein comprises a first antigen-binding portion capable of binding to EphA2 expressed on the surface of a cell and a second antigen-binding portion capable of binding to ALCAM expressed on the surface of the same cell. In some embodiments, the surface density ratio of EphA2 to ALCAM exceeds a threshold value. In some embodiments, the surface density ratio of EphA2 to ALCAM exceeds a threshold value of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. In some embodiments, the surface density ratio of EphA2 to ALCAM exceeds a threshold value of about 1:5.

[0106] In some embodiments, the engineered antibodies or functional fragments thereof described herein comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences disclosed herein. In some embodiments, the engineered antibodies or functional fragments thereof described herein comprise an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to any one of the amino acid sequences disclosed herein. In some embodiments, the engineered antibodies or functional fragments thereof described herein comprise an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to any one of the amino acid sequences identified in Table 4. In some embodiments, the engineered antibodies or functional fragments thereof described herein comprise an amino acid sequence having 100% sequence identity to any one of the amino acid sequences identified in Table 4. In some embodiments, an engineered antibody or functional fragment thereof described herein comprises an amino acid sequence corresponding to any one of the amino acid sequences identified in Table 4, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence are replaced by a different amino acid residue.

[0107] First antigen-binding moiety As outlined above, various embodiments and aspects of the present disclosure include engineered antibodies comprising a first antigen-binding portion capable of binding to a cell surface guide antigen. In some embodiments, the first antigen-binding portion comprises a heavy chain variable (VH) region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to a VH sequence identified in Table 4. In some embodiments, the first antigen-binding portion comprises a VH region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81. In some embodiments, the first antigen-binding portion comprises a VH region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 96. In some embodiments, the first antigen-binding portion comprises a VH region having an amino acid sequence with 100% sequence identity to a VH sequence identified in Table 4. In some embodiments, the first antigen-binding portion comprises a VH region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 81. In some embodiments, the first antigen-binding portion comprises a VH region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 96. In some embodiments, the first antigen-binding portion comprises a VH region having an amino acid sequence corresponding to any one of the VH sequences identified in Table 4, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence are substituted with a different amino acid residue. In some embodiments, the first antigen-binding portion comprises a VH region having an amino acid sequence corresponding to SEQ ID NO: 81, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence of SEQ ID NO: 81 are substituted with a different amino acid residue. In some embodiments, the first antigen-binding portion comprises a VH region having an amino acid sequence corresponding to SEQ ID NO: 96, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence of SEQ ID NO: 96 are replaced by a different amino acid residue.

[0108] In some embodiments, the VH region of the first antigen-binding portion comprises the three CDRs (e.g., HCDR1, HCDR2, and HCDR3) identified in each of the VH sequences disclosed in the Sequence Listing. In some embodiments, HCDR1 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 104. In some embodiments, HCDR2 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 105. In some embodiments, HCDR3 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 106 or SEQ ID NO: 110. In some embodiments, HCDR1, HCDR2, and HCDR3 of the VH region of the first antigen-binding portion comprise the sequences of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 of the VH region of the first antigen-binding portion comprise the sequences of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 110, respectively. In some embodiments, the VH region of the first antigen-binding moiety comprises three HCDRs identified in each of the VH sequences disclosed in the Sequence Listing, wherein one, two, three, four, or five of the amino acid residues in at least one of the HCDRs are substituted with different amino acid residues. In some embodiments, HCDR1 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 104, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 104 are substituted with different amino acid residues. In some embodiments, HCDR2 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 105, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 105 are substituted with different amino acid residues. In some embodiments, HCDR3 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 106, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 106 are substituted with different amino acid residues. In some embodiments, the HCDR3 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 110, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 110 are replaced by a different amino acid residue.

[0109] In some embodiments, the first antigen-binding portion comprises a light chain variable (VL) region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to a VL sequence identified in Table 4. In some embodiments, the first antigen-binding portion comprises a VL region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 82. In some embodiments, the first antigen-binding portion comprises a VL region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 97. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence having 100% sequence identity to a VL sequence identified in Table 4. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 82. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 97. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence corresponding to any one of the VL sequences identified in Table 4, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence are substituted with a different amino acid residue. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 82, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence of SEQ ID NO: 82 are substituted with a different amino acid residue. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 97, wherein one, two, three, four, or five of the amino acid residues in the amino acid sequence of SEQ ID NO: 97 are replaced by a different amino acid residue.

[0110] In some embodiments, the VL region of the first antigen-binding moiety comprises the three CDRs (e.g., LCDR1, LCDR2, and LCDR3) specified in each of the VL sequences disclosed in the Sequence Listing. In some embodiments, LCDR1 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 107. In some embodiments, LCDR2 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 108. In some embodiments, LCDR3 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 109. In some embodiments, LCDR1, LCDR2, and LCDR3 of the VL region of the first antigen-binding moiety comprise the sequences of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively. In some embodiments, the first antigen-binding moiety comprises a VL region having an amino acid sequence corresponding to any one of the VL sequences identified in Table 4, wherein one, two, three, four, or five of the amino acid residues in the amino acid sequence are substituted by a different amino acid residue. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 82, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence of SEQ ID NO: 82 are substituted with a different amino acid residue. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 97, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence of SEQ ID NO: 97 are substituted with a different amino acid residue.

[0111] In some embodiments, the VL region of the first antigen-binding moiety comprises the three CDRs (e.g., LCDR1, LCDR2, and LCDR3) identified in each of the VL sequences disclosed in the Sequence Listing, wherein one, two, three, four, or five of the amino acid residues in at least one of the LCDRs are substituted with different amino acid residues. In some embodiments, LCDR1 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 107, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 107 are substituted with different amino acid residues. In some embodiments, LCDR2 of the first antigen-binding moiety comprises the sequence of SEQ ID NO: 108, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 108 are substituted with different amino acid residues. In some embodiments, the LCDR3 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 109, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 109 are substituted by a different amino acid residue.

[0112] Second antigen-binding moiety As outlined above, various embodiments and aspects of the present disclosure include engineered antibodies comprising a second antigen-binding portion capable of binding to a cell surface effector antigen. In some embodiments, the second antigen-binding portion comprises a VH region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to a VH sequence identified in Table 4. In some embodiments, the second antigen-binding portion comprises a VH region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 73. In some embodiments, the second antigen-binding portion comprises a VH region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 75. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence with 100% sequence identity to a VH sequence identified in Table 4. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 73. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 75. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence corresponding to any one of the VH sequences identified in Table 4, wherein one, two, three, four, or five of the amino acid residues in the amino acid sequence are substituted with a different amino acid residue. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence corresponding to SEQ ID NO: 73, wherein one, two, three, four, or five of the amino acid residues in the amino acid sequence of SEQ ID NO: 73 are substituted with a different amino acid residue. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence corresponding to SEQ ID NO: 75, wherein one, two, three, four, or five of the amino acid residues within the amino acid sequence of SEQ ID NO: 75 are replaced by a different amino acid residue.

[0113] In some embodiments, the VH region of the second antigen-binding moiety comprises three CDRs (e.g., HCDR1, HCDR2, and HCDR3) specified in each of the VH sequences disclosed in the Sequence Listing. In some embodiments, HCDR1 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 98. In some embodiments, HCDR2 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 99. In some embodiments, HCDR3 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 106 or SEQ ID NO: 100. In some embodiments, HCDR1, HCDR2, and HCDR3 of the VH region of the first antigen-binding moiety comprise the sequences of SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively. In some embodiments, the VH region of the second antigen-binding moiety comprises three HCDRs specified in each of the VH sequences disclosed in the Sequence Listing, wherein one, two, three, four, or five of the amino acid residues in at least one of the HCDRs are substituted by a different amino acid residue. In some embodiments, HCDR1 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 98, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 98 are substituted by different amino acid residues. In some embodiments, HCDR2 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 99, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 99 are substituted by different amino acid residues. In some embodiments, HCDR3 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 100, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 100 are substituted by different amino acid residues.

[0114] In some embodiments, the second antigen-binding portion comprises a VL region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to a VL sequence identified in Table 4. In some embodiments, the second antigen-binding portion comprises a VL region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 74. In some embodiments, the second antigen-binding portion comprises a VL region having at least 80%, at least 90%, at least 95%, at least 96%, at least 97, at least 98%, or at least 99% sequence identity to SEQ ID NO: 76. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence with 100% sequence identity to a VL sequence identified in Table 4. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 74. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence with 100% sequence identity to SEQ ID NO: 76. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence corresponding to any one of the VL sequences identified in Table 4, wherein one, two, three, four, or five of the amino acid residues in the amino acid sequence are substituted with a different amino acid residue. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 74, wherein one, two, three, four, or five of the amino acid residues in the amino acid sequence of SEQ ID NO: 74 are substituted with a different amino acid residue. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 76, wherein one, two, three, four, or five of the amino acid residues in the amino acid sequence of SEQ ID NO: 76 are replaced by a different amino acid residue.

[0115] In some embodiments, the VL region of the second antigen-binding moiety is as identified in each of the VL sequences disclosed in the sequence listing. In some embodiments, the VL region of the second antigen-binding moiety comprises three CDRs (e.g., LCDR1, LCDR2, and LCDR3). In some embodiments, LCDR1 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 101. In some embodiments, LCDR2 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 102. In some embodiments, LCDR3 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 103. In some embodiments, LCDR1, LCDR2, and LCDR3 of the VL region of the second antigen-binding moiety comprise the sequences of SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, respectively. In some embodiments, the VL region of the second antigen-binding moiety comprises three CDRs identified in the Sequence Listing, wherein one, two, three, four, or five of the amino acid residues in at least one of the CDRs are substituted by a different amino acid residue. In some embodiments, LCDR1 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 101, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 101 are substituted with a different amino acid residue. In some embodiments, LCDR2 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 102, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 102 are substituted with a different amino acid residue. In some embodiments, LCDR3 of the second antigen-binding moiety comprises the sequence of SEQ ID NO: 103, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 103 are substituted with a different amino acid residue.

[0116] Purpose In some embodiments of the present disclosure, the antibody or functional fragment thereof is conjugated or covalently linked to at least one moiety of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a pharmacokinetic-improving moiety. In some embodiments, the at least one MOI is selected from the group consisting of an anti-cancer agent, an anti-autoimmune disease agent, an anti-inflammatory agent, an anti-bacterial agent, an anti-microbial agent, an antibiotic, an anti-infectious disease agent, and an anti-viral agent. In some embodiments, the at least one MOI is selected from the group consisting of a cytotoxic anti-cancer agent, a DNA chelator, a microtubule inhibitor, a topoisomerase inhibitor, a translation initiation inhibitor, a ribosome-inactivating molecule, a nuclear transport inhibitor, an RNA splicing inhibitor, an RNA polymerase inhibitor, and a DNA polymerase inhibitor.

[0117] In some embodiments, the cytotoxic anticancer agent is an auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, or the like. The compound is selected from the group consisting of benzodiazepines, anthracyclines, calicheamicins, camptothecins, irinotecans, SN-38, combretastatins, duocarmycins, enediynes, epothilones, ethyleneimines, mitomycins, pyrrolobenzodiazepines (PBDs), and calicheamicins.

[0118] In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the constant region of the engineered antibody or functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the heavy chain constant (e.g., CH1, CH2, or CH3) region of the antibody or functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the CH1 region of the antibody or functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the light chain constant (CL) region of the antibody or functional fragment thereof. In principle, there is no particular limit to the number of MOIs that can be conjugated or covalently attached to the engineered antibodies of the present disclosure. In some embodiments, the engineered antibodies of the present disclosure have an average MOI number per antibody (i.e., average drug-to-antibody ratio, DAR) ranging from 1 to 20. In some embodiments, engineered antibodies of the present disclosure have an average MOI per antibody ranging from about 1 to about 10. In some embodiments, the average DAR is about 1 to about 5, about 2 to about 6, about 3 to about 7, about 3 to about 8, about 4 to about 9, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 10 to about 20.

[0119] Those skilled in the art will understand that the complete amino acid sequence of the engineered antibody disclosed herein can be used to construct a reverse-translated gene.For example, a DNA oligomer containing a nucleotide sequence encoding a given antibody can be synthesized.For example, multiple small oligonucleotides encoding desired antibody portions can be synthesized and then ligated together.Each oligonucleotide typically contains a 5' or 3' overhang for complementary assembly.

[0120] In addition to producing engineered antibodies through the expression of nucleic acid molecules that have been altered by recombinant molecular biology techniques, the subject engineered antibodies or functional fragments thereof according to the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by those of skill in the art.

[0121] Once assembled (by synthesis, site-directed mutagenesis, or another method), the DNA sequence encoding the engineered antibody or functional fragment thereof disclosed herein is inserted into an expression vector and operably linked to expression control sequences appropriate for expression of the engineered antibody or functional fragment thereof in a desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is known in the art, to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0122] The binding activity of an engineered antibody or functional fragment thereof of the present disclosure can be assayed by any suitable method known in the art. Antibodies or polypeptides that "preferentially bind" or "specifically bind" (used interchangeably herein) to a target antigen or target epitope are terms well understood in the art, and methods for determining such specific or preferential binding are also known in the art. An antibody or polypeptide is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular antigen or epitope more frequently, more rapidly, for a longer duration, and / or with a higher affinity than with alternative antigens or epitopes. An antibody or polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. An antibody or polypeptide also "specifically binds" or "preferentially binds" to a target in a sample if it binds with greater affinity, avidity, more readily, and / or for a longer duration to the target than it binds to other substances present in the sample. For example, an antibody or polypeptide that specifically or preferentially binds to an EphA2 epitope is an antibody or polypeptide that binds to this epitope with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other EphA2 epitopes or non-EphA2 epitopes. It is understood by reading this definition that, for example, an antibody or polypeptide (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding.

[0123] Various assay formats can be used to select antibodies or polypeptides that specifically bind to a molecule of interest. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA), and Western blot analysis are among the many assays that can be used to identify antibodies that specifically react with an antigen or its ligand-binding portion and specifically bind to a cognate ligand or binding partner. Generally, a specific or selective response will be at least twice the background signal or noise, more typically greater than 10 times the background, even more typically greater than 50 times the background, more typically greater than 100 times the background, even more typically greater than 500 times the background, even more typically greater than 1000 times the background, and even more typically greater than 10,000 times the background. Also, in some embodiments, antibodies may be selected that have an equilibrium dissociation constant (K D ) is less than 43 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, or less than 7 nM.

[0124] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, for example, an antibody or portion thereof, and an antigen. The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity). The binding affinity between two molecules is determined by the dissociation constant (K D ) can be quantified by determining K D can be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) methods (Biacore). The rate constants corresponding to the association and dissociation of the monovalent complex are the binding rate constant k, a (or k on ) and the dissociation rate constant kd (or k off ) is called K D is the equation K D =k d / k a By k a and k d The value of the dissociation constant can be determined directly by well-known methods, and can also be calculated for mixtures of complexes by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K D can be established using a double-filter nitrocellulose filter binding assay, such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for evaluating the binding ability of the engineered antibodies of the present disclosure to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody can also be assessed by standard assays known in the art, such as surface plasmon resonance (SPR), for example, using a Biacore™ system or KinExA.

[0125] nucleic acid molecule In another aspect, various recombinant nucleic acid molecules encoding the engineered antibodies of the present disclosure are provided herein, including expression cassettes and expression vectors comprising these nucleic acid molecules operably linked to heterologous nucleic acid sequences, such as, for example, regulatory sequences, that allow for expression of the engineered antibodies in a host cell or an ex vivo cell-free expression system.

[0126] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules comprising DNA or RNA molecules, including cDNA, genomic DNA, synthetic DNA, and nucleic acid analogs. Nucleic acid molecules may be double-stranded or single-stranded (e.g., sense or antisense strands). Nucleic acid molecules may contain unconventional or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence," when used interchangeably herein, refer to the sequence of a polynucleotide molecule. The nucleotide base nomenclature set forth in 37 CFR §1.822 is used herein.

[0127] Nucleic acid molecules of the present disclosure can be of any length, and generally include nucleic acid molecules that are about 0.5 Kb to about 20 Kb, e.g., about 0.5 Kb to about 20 Kb, about 1 Kb to about 15 Kb, about 2 Kb to about 10 Kb, or about 5 Kb to about 25 Kb, e.g., about 10 Kb to 15 Kb, about 15 Kb to about 20 Kb, about 5 Kb to about 20 Kb, about 5 Kb to about 10 Kb, or about 10 Kb to about 25 Kb.

[0128] The term "recombinant" nucleic acid molecule, as used herein, refers to a nucleic acid molecule that has been altered through human intervention. As a non-limiting example, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule produced by in vitro polymerase reaction(s), or to which a linker has been attached, or which has been incorporated into a vector, e.g., a cloning vector or an expression vector. As non-limiting examples, a recombinant nucleic acid molecule is 1) synthesized or modified in vitro, e.g., using chemical or enzymatic techniques or recombination of nucleic acid molecules; 2) contains nucleotide sequences that are not naturally linked; 3) has been genetically engineered using molecular cloning techniques to lack one or more nucleotides compared to the naturally occurring nucleic acid molecule sequence; and / or 4) has been engineered using molecular cloning techniques to have one or more sequence changes or rearrangements compared to the naturally occurring nucleic acid sequence.

[0129] In some embodiments disclosed herein, a nucleic acid molecule of the disclosure comprises a nucleotide sequence encoding an engineered antibody having an amino acid sequence with at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to the amino acid sequence of an engineered antibody disclosed herein. In some embodiments, a nucleic acid molecule of the disclosure comprises a nucleotide sequence encoding an engineered antibody having an amino acid sequence with at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of the amino acid sequences identified in Table 4. In some embodiments, a nucleic acid molecule of the disclosure comprises a nucleotide sequence encoding an engineered antibody having an amino acid sequence with at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of the VH amino acid sequences identified in Table 3. In some embodiments, a nucleic acid molecule of the disclosure comprises a nucleotide sequence encoding an engineered antibody having an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of the VL amino acid sequences identified in Table 4.

[0130] Some embodiments disclosed herein relate to vectors or expression cassettes comprising recombinant nucleic acid molecules encoding the engineered antibodies disclosed herein. As used herein, the term "expression cassette" refers to a construct of genetic material containing a coding sequence and sufficient regulatory information to direct the proper transcription and / or translation of the coding sequence in a recipient cell in vivo and / or ex vivo. The expression cassette can be inserted into a vector for targeting to a desired host cell and / or subject. As such, the term expression cassette can be used interchangeably with the term "expression construct." As used herein, the term "construct" is intended to mean any recombinant nucleic acid molecule, e.g., an expression cassette, plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, and comprising one or more nucleic acid sequences linked in a functionally operable manner, e.g., operably linked nucleic acid molecules.

[0131] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any of the engineered antibodies disclosed herein. The above-mentioned nucleic acid molecules can be contained in a vector that can induce its expression in cells transformed / transduced with the vector, for example. Vectors suitable for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or easily prepared by those skilled in the art. Additional vectors can also be found, for example, in Ausubel, FM, et al., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., "Molecular Cloning: A Laboratory Manual," 2nd Ed. (1989).

[0132] It should be understood that not all vectors and expression control sequences function equally well to express the DNA sequences described herein. Also, not all hosts function equally well in the same expression system. However, one skilled in the art can make a selection from among these vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host must be considered, since the vector must replicate in the host. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow DNA encoding the engineered antibodies of the present disclosure to be amplified by multiple copies. Such amplifiable vectors are known in the art.

[0133] Thus, in some embodiments, the engineered antibodies described herein can be expressed from a vector, e.g., an expression vector. Vectors are useful for autonomous replication in a host cell or can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included. Exemplary recombinant expression vectors are selected based on the host cell intended to be used for expression and can include one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed.

[0134] DNA vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989, supra) and other standard molecular biology laboratory manuals.

[0135] The nucleic acid sequence encoding the engineered antibody of the present disclosure can be optimized for expression in a target host cell. For example, the GC content of the sequence can be adjusted to the average level for a given cellular host, calculated with reference to known genes expressed in the host cell. Methods for codon optimization are known in the art. The codon usage within the coding sequence of the engineered antibody disclosed herein can be optimized to enhance expression in a host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence are optimized for expression in a specific host cell.

[0136] Suitable vectors for use include T7-based vectors for use in bacteria, pMSXND expression vectors for use in mammalian cells, and baculovirus-derived vectors for use in insect cells. In some embodiments, the nucleic acid insert encoding an engineered antibody in such a vector may be operably linked to a promoter selected, for example, based on the cell type in which expression is desired.

[0137] When selecting an expression control sequence, various factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject polypeptide, particularly with respect to potential secondary structure. Hosts should be selected taking into consideration their compatibility with the selected vector, the toxicity of the products encoded by the DNA sequences of the present disclosure, their secretion characteristics, their ability to correctly fold the polypeptide, their fermentation or cultivation requirements, and the ease of purification of the products encoded by the DNA sequences.

[0138] Within these parameters, one skilled in the art can select a variety of vector / expression control sequence / host combinations that will express the desired DNA sequence in fermentation or large scale animal culture using, for example, CHO or COS 7 cells.

[0139] The choice of expression control sequences and expression vectors depends, in some embodiments, on the host cell of choice. A wide variety of expression host / vector combinations are available. Non-limiting examples of expression vectors useful for eukaryotic hosts include vectors with expression control sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Non-limiting examples of expression vectors useful for bacterial hosts include known bacterial plasmids, e.g., E. coli-derived plasmids, including colEl, pCRI, pER32z, pMB9, and their derivatives; broad-host-range plasmids, e.g., RP4; phage DNA, e.g., phage lambda, e.g., NM989; and many derivatives of other DNA phages, e.g., M13 and filamentous single-stranded DNA phages. Non-limiting examples of expression vectors useful for yeast cells include the 2μ plasmid and its derivatives. Non-limiting examples of vectors useful for insect cells include pVL941 and pFastBac™1.

[0140] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors may contain origins of replication and other genes encoding selectable markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells in which it is expressed, thereby allowing phenotypic selection of transfected cells. Those skilled in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental situation.

[0141] Viral vectors that can be used in the present disclosure include, for example, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, herpes viruses, simian virus 40 (SV40), and bovine papillomavirus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY). In some embodiments, the vector is a lentiviral vector, adenoviral vector, adeno-associated viral vector, or retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0142] Recombinant prokaryotic or eukaryotic cells comprising an engineered antibody or functional fragment thereof disclosed herein and / or comprising and expressing a nucleic acid molecule encoding any one of the engineered antibodies or functional fragments thereof disclosed herein are also a feature of the disclosure. In some embodiments, a recombinant cell of the disclosure is a transfected cell, e.g., a cell into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding an engineered antibody disclosed herein, has been introduced using recombinant methods and techniques. Progeny of such cells are also considered within the scope of the disclosure. Cell cultures comprising at least one recombinant cell disclosed herein are also within the scope of the disclosure. The terms "cell," "cell culture," "cell line," "recombinant cell," "recipient cell," and "host cell," as used herein, include the primary subject cell and any progeny thereof, regardless of the number of transfers. Not all progeny will be exactly identical to the parent cell (due to intentional or unintended mutations or differences in environment); however, such altered progeny are included within these terms so long as the progeny retain the same functionality as that of the originally transformed cell.

[0143] The exact components of the expression system are not critical. For example, the engineered antibodies disclosed herein can be produced in prokaryotic hosts, such as the bacterium E. coli, or in eukaryotic hosts, such as insect cells (e.g., Sf21 cells), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from a number of suppliers, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, it is important only that the components are compatible with each other. Such determinations are within the skill of the art. Furthermore, if guidance is needed in selecting an expression system, the skilled artisan may refer to Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0144] The expressed antibodies can be purified from the expression system using routine biochemical techniques and can be used, for example, as therapeutic agents, as described herein.

[0145] In some embodiments, the resulting engineered antibody is glycosylated or non-glycosylated, depending on the host organism used to produce the engineered antibody. When bacteria are selected as the host, the engineered antibody produced is non-glycosylated. Eukaryotic cells, on the other hand, will glycosylate the engineered antibody, although perhaps not in the same manner as the native polypeptide. Recombinant antibodies produced by the transformed host can be purified according to any suitable method known in the art. The recombinant antibodies produced can be isolated from inclusion bodies produced in bacteria, e.g., E. coli, or from the conditioned medium of either mammalian or yeast cultures producing the engineered antibodies of the present disclosure using cation exchange, gel filtration, and / or reverse-phase liquid chromatography.

[0146] Additionally or alternatively, another exemplary method for constructing a DNA sequence encoding an engineered antibody of the present disclosure is by chemical synthesis. This includes directly synthesizing a peptide by chemical means of an amino acid sequence encoding an engineered antibody exhibiting the described properties. This method can incorporate both natural and unnatural amino acids at positions that affect the binding affinity of the engineered antibody to its target protein. Alternatively, a gene encoding a desired engineered antibody can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired engineered antibody, generally selecting codons preferred by the host cell in which the engineered antibody of the present disclosure will be produced. In this regard, it is well recognized in the art that the genetic code is degenerate, such that an amino acid can be encoded by more than one codon. For example, Phe (F) is encoded by two codons, TIC or TTT, Tyr (Y) is encoded by TAC or TAT, and his (H) is encoded by CAC or CAT. Trp (W) is encoded by a single codon, TGG. Therefore, those skilled in the art will understand that for a given DNA sequence encoding a particular engineered antibody, there are many degenerate DNA sequences that encode that engineered antibody. For example, in addition to the DNA sequences of the engineered antibodies provided herein, it will be understood that there are many degenerate DNA sequences that encode the engineered antibodies disclosed herein. These degenerate DNA sequences are considered to be within the scope of the present disclosure. Therefore, in the context of the present disclosure, "degenerate variants thereof" refers to all DNA sequences that encode a particular engineered antibody and thereby enable its expression.

[0147] The DNA sequence encoding the subject engineered antibody, whether prepared by site-directed mutagenesis, chemical synthesis, or other methods, may also include a DNA sequence encoding a signal sequence. If present, such a signal sequence must be one that is recognized by the cell selected for expression of the engineered antibody. It may be prokaryotic, eukaryotic, or a combination of the two. The inclusion of a signal sequence generally depends on whether it is desired to secrete the engineered antibody disclosed herein from the recombinant cell in which it is produced. If the selected cell is prokaryotic, the DNA sequence generally does not encode a signal sequence. If the selected cell is eukaryotic, a signal sequence is generally included.

[0148] The provided nucleic acid molecules can include naturally occurring sequences or can include sequences that differ from naturally occurring sequences but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g., an antibody. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. In addition, nucleic acid molecules can be double-stranded or single-stranded (e.g., either the sense or antisense strand).

[0149] Nucleic acid molecules are not limited to sequences encoding polypeptides (e.g., antibodies), but may also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence of an engineered antibody). Those skilled in the art of molecular biology are familiar with routine techniques for isolating nucleic acid molecules. They can be produced, for example, by treating genomic DNA with restriction nucleases or by performing polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0150] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found in nature. Thus, the present disclosure encompasses recombinant molecules, e.g., those in which a nucleic acid sequence (e.g., a sequence encoding an engineered antibody disclosed herein) has been incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a location different from its natural chromosomal location).

[0151] Pharmaceutical Composition In some embodiments, the engineered antibodies, nucleic acids, and / or recombinant cells of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally comprise the engineered antibodies, nucleic acids, and / or recombinant cells of the present disclosure and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., anticancer agents) can also be incorporated into the compositions.

[0152] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, for example, sodium dodecyl sulfate. Protection against the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, one or more isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, are included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0153] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount with one or a combination of the above-mentioned ingredients in a suitable solvent, and then, if necessary, sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients other than those mentioned above. In the case of sterile powders for preparing sterile injectable solutions, an exemplary method of preparation is to obtain a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof by vacuum drying and freeze-drying.

[0154] Oral compositions, if used, generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound (e.g., the engineered antibody and / or nucleic acid molecule of the present disclosure) can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders, e.g., microcrystalline cellulose, gum tragacanth, or gelatin; excipients, e.g., starch or lactose; disintegrants, e.g., alginic acid, Primogel™, or corn starch; lubricants, e.g., magnesium stearate or Sterotes™; glidants, ), such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring.

[0155] For administration by inhalation, the engineered antibodies of the presently disclosed subject matter are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer, methods including those described in U.S. Patent No. 6,468,798.

[0156] The systemic administration of the engineered antibody of the present disclosure can also be carried out by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams, as is generally known in the art.

[0157] In some embodiments, the engineered antibodies of the disclosure can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0158] In some embodiments, the engineered antibodies of the present disclosure can also be administered by transfection or infection using methods known in the art, including, but not limited to, those described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (NatureBiotechnol. 20: 1006-1010,2002), or Putnam (Am. J. Health Syst. Pharm. 53:151-160, 1996, erratum at Am. J.Health Syst. Pharm. 53:325, 1996).

[0159] In some embodiments, the engineered antibodies of the presently disclosed subject matter are prepared with carriers that will protect the engineered antibodies against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. Materials are also available from Alza Corporation and Nova. Pharmaceuticals, Inc. Liposomal suspensions (containing liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0160] In some embodiments, the engineered antibodies of the present disclosure can be used in vivo and / or ex vivo. They may also be further modified to extend their half-life in vivo. Non-limiting examples of known strategies and techniques suitable for modifying the engineered antibodies of the present disclosure include (1) chemical modification of the engineered antibodies with highly soluble macromolecules, such as polyethylene glycol ("PEG"), which prevent the engineered antibodies described herein from contacting proteases, and (2) covalently linking or conjugating the engineered antibodies described herein to stable proteins, such as albumin. Thus, in some embodiments, the engineered antibodies of the present disclosure may be fused to stable proteins, such as albumin. For example, human albumin is known to be one of the most effective proteins for enhancing the stability of polypeptides fused to it, and many such fusion proteins have been reported.

[0161] In some embodiments, engineered antibodies of the present disclosure are chemically modified with one or more polyethylene glycol moieties, e.g., PEGylated, or similarly modified, e.g., PASylated. In some embodiments, a PEG or PAS molecule is conjugated to one or more amino acid side chains of an interferon. In some embodiments, a PEGylated or PASylated antibody contains a PEG or PAS moiety at only one amino acid. In other embodiments, a PEGylated or PASylated antibody contains a PEG or PAS moiety at two or more amino acids, e.g., attached to two or more, five or more, fifteen or more, or twenty or more different amino acid residues. In some embodiments, the PEG or PAS chain is 2000 Da, greater than 2000 Da, 5000 Da, greater than 5,000 Da, 10,000 Da, greater than 10,000 Da, greater than 10,000 Da, 20,000 Da, greater than 20,000 Da, and 30,000 Da. The engineered antibody can be linked directly (e.g., without a linking group) to the PEG or PAS through an amino, sulfhydryl, hydroxyl, or carboxyl group.

[0162] In some embodiments, pharmaceutical compositions of the present disclosure comprise one or more PEGylation reagents. As used herein, the term "PEGylation" means and refers to modifying a protein by covalently attaching polyethylene glycol (PEG) to the protein, and "PEGylated" refers to a protein to which PEG has been attached. PEG or PEG derivatives of a range of sizes, with an optimal range of about 10,000 daltons to about 40,000 daltons, can be attached to the engineered antibodies of the present disclosure using a variety of chemistries. In some embodiments, the PEGylation reagent is selected from methoxypolyethyleneglycol-succinimidylpropionate (mPEG-SPA), mPEG-succinimidylbutyrate (mPEG-SBA), mPEG-succinimidylsuccinate (mPEG-SS), mPEG-succinimidylcarbonate (mPEG-SC), mPEG-succinimidylglutarate (mPEG-SG), mPEG-N-hydroxyl-succinimide (mPEG-NHS), mPEG-tresylate, and mPEG-aldehyde. In some embodiments, the PEGylation reagent is methoxypolyethyleneglycol-succinimidylpropionate, e.g., methoxypolyethyleneglycol-succinimidylpropionate 5000, having an average molecular weight of 5,000 daltons.

[0163] Methods of the present disclosure Methods for modulating cell internalization and cell type-selective signal transduction In various aspects of the present disclosure, the engineered antibodies and functional fragments thereof disclosed herein, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions comprising same can be used to modulate cellular internalization of cell surface molecules. The term "modulate" refers to decreasing, reducing, inhibiting, increasing, inducing, activating, or otherwise affecting cellular internalization of cell surface molecules.

[0164] In one aspect, some embodiments of the present disclosure relate to a method for modulating cellular internalization, comprising administering to a cell one or more of the following: (a) an engineered antibody or functional fragment thereof disclosed herein, (b) a nucleic acid molecule disclosed herein, and (c) a pharmaceutical composition disclosed herein.

[0165] In another aspect, some embodiments of the present disclosure relate to a method for modulating cellular internalization, comprising administering an engineered antibody or functional fragment thereof comprising: (a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate; and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate, wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate.

[0166] In yet another aspect, as discussed in more detail below, some embodiments of the present disclosure relate to methods for treating a condition or disease (e.g., cancer) in a subject using the engineered antibodies or conjugates thereof disclosed herein.

[0167] According to the methods disclosed herein, it is possible to induce rapid internalization of a slowly internalizing antigen by operably linking an antigen-binding portion specific for a slowly internalizing antigen to another antigen-binding portion specific for a rapidly internalizing antigen. In some embodiments, it is possible to induce slow internalization of a rapidly internalizing antigen by operably linking an antigen-binding portion specific for a rapidly internalizing antigen to another antigen-binding portion specific for a slowly internalizing antigen. In some embodiments of the methods described herein, the internalization property of the engineered antibody is converted from internalizing to non-internalizing. In some embodiments, the internalization property of the guide antigen and / or effector antigen is converted from internalizing to non-internalizing. In some embodiments, the internalization property of an internalizing antigen (e.g., a guide antigen or effector antigen) is converted from internalizing to non-internalizing by using an engineered antibody disclosed herein, including an antigen-binding portion specific for such an internalizing antigen operably linked to another antigen-binding portion specific for a non-internalizing antigen. For example, in some embodiments of the disclosed methods, the internalization property of an internalizing guide antigen is converted from internalizing to non-internalizing by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for the internalizing guide antigen operably linked to another antigen-binding portion specific for a non-internalizing effector antigen. In some embodiments, the internalization property of an internalizing effector antigen is converted from internalizing to non-internalizing by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for the internalizing effector antigen operably linked to another antigen-binding portion specific for the non-internalizing guide antigen. In some embodiments of the disclosed methods, the internalization property of the engineered antibody is converted from non-internalizing to internalizing. In some embodiments, the internalization property of a non-internalizing antigen (e.g., guide antigen or effector antigen) is converted from non-internalizing to internalizing.In some other embodiments, the internalization property of a non-internalizing guide antigen is converted from non-internalizing to internalizing by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for such a non-internalizing guide antigen operably linked to another antigen-binding portion specific for an internalizing effector antigen. In some other embodiments, the internalization property of a non-internalizing effector antigen is converted from non-internalizing to internalizing by using an engineered antibody disclosed herein that comprises an antigen-binding portion specific for such a non-internalizing effector antigen operably linked to another antigen-binding portion specific for an internalizing guide antigen.

[0168] In some embodiments, the guide antigen has a cellular internalization rate that is higher than the cellular internalization rate of the effector antigen, where the guide antigen is a rapidly internalizing antigen and the effector antigen is a slowly internalizing antigen. In some embodiments, the guide antigen has a cellular internalization rate that is at least about 50% higher than the cellular internalization rate of the effector antigen. In some embodiments, the guide antigen has a cellular internalization rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the cellular internalization rate of the effector antigen. In some embodiments, the engineered antibodies of the present disclosure increase the rate of internalization of a slow-internalizing antigen (e.g., an effector antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only a slow-internalizing antigen) by operably linking an antigen-binding portion specific for a slow-internalizing antigen to another antigen-binding portion specific for a rapidly-internalizing antigen (e.g., a guide antigen). In some embodiments, the engineered antibodies of the present disclosure reduce the rate of internalization of a rapidly internalizing antigen (e.g., a guide antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only a rapidly internalizing antigen) by operably linking an antigen-binding portion specific for a rapidly internalizing antigen to another antigen-binding portion specific for a slowly internalizing antigen (e.g., an effector antigen).

[0169] In some embodiments, the effector antigen has a higher cellular internalization rate than the guide antigen, where the effector antigen is a rapidly internalizing antigen and the guide antigen is a slowly internalizing antigen. In some embodiments, the effector antigen has a cellular internalization rate that is at least about 50% higher than the guide antigen. In some embodiments, the effector antigen has a cellular internalization rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the guide antigen. In some embodiments, the engineered antibodies of the present disclosure increase the rate of internalization of a slow-internalizing antigen (e.g., a guide antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only a slow-internalizing antigen) by operably linking an antigen-binding portion specific for a slow-internalizing antigen to another antigen-binding portion specific for a rapidly internalizing antigen (e.g., an effector antigen). In some embodiments, the engineered antibodies of the present disclosure reduce the rate of internalization of a rapidly internalizing antigen (e.g., an effector antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., a monospecific antibody comprising only a rapidly internalizing antigen) by operably linking an antigen-binding portion specific for a rapidly internalizing antigen to another antigen-binding portion specific for a slowly internalizing antigen (e.g., a guide antigen).

[0170] In some embodiments, the cell surface guide antigen is an internalized cell surface antigen. In some embodiments, the cell surface effector antigen is a non-internalized cell surface antigen. In some embodiments, the relative surface density ratio of guide antigen to effector antigen is above a threshold. In some embodiments, the threshold is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. Thus, in some embodiments, the relative surface density ratio of guide antigen to effector antigen is greater than about 1:1, greater than about 1:2, greater than about 1:3, greater than about 1:4, greater than about 1:5, greater than about 1:10, greater than about 1:20, or greater than about 1:30. In some specific embodiments, the relative surface density ratio of guide antigen to effector antigen is greater than about 1:5. In some embodiments, the relative surface density ratio of guide antigen to effector antigen is below a threshold. In some embodiments, the threshold is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. Thus, in some embodiments, the relative surface density ratio of guide antigen to effector antigen is less than about 1:1, less than about 1:2, less than about 1:3, less than about 1:4, less than about 1:5, less than about 1:10, less than about 1:20, or less than about 1:30. In certain embodiments, the relative surface density ratio of guide antigen to effector antigen is less than about 1:5.

[0171] In some other embodiments, the relative surface density ratio of effector antigen to guide antigen is above a threshold value. In some embodiments, the threshold value is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, or about 1:30. Thus, in some embodiments, the relative surface density ratio of effector antigen to guide antigen is greater than about 1:1, greater than about 1:2, greater than about 1:3, greater than about 1:4, greater than about 1:5, greater than about 1:10, greater than about 1:20, or greater than about 1:30.

[0172] In some embodiments, the method of the present disclosure further comprises modulating the cell surface density of guide antigen and / or the cell surface density of effector antigen.Those skilled in the art will easily understand that the ratio of guide to effector can be modulated by using techniques known in the art for modulating the expression and / or function of target gene or target protein.Non-limiting examples of such techniques include gene suppression, small interfering RNA, partial gene knockout, small molecules or proteins / peptides with signal transduction functions that change cell metabolism, proliferation, migration, death, aging, differentiation and immune regulation.

[0173] In yet another aspect, some embodiments of the present disclosure relate to a method for modulating cell-type selective signal transduction in a subject, the method comprising administering to a cell an engineered antibody or functional fragment thereof comprising: (a) a first antigen-binding portion capable of binding to a cell-surface guide antigen, where the guide antigen is expressed in a cell-type selective manner in the subject and has a first cellular internalization rate; and (b) a second antigen-binding portion capable of binding to a cell-surface effector antigen with a second cellular internalization rate, wherein the internalization property of the engineered antibody or functional fragment thereof is determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. In some embodiments, the engineered antibodies described herein modulate a signal transduction pathway, which may be an upregulation or downregulation of such a signal transduction pathway. In some embodiments, the engineered antibodies of the present disclosure may function as agonists, upregulating (enhancing, stimulating, promoting, activating, or increasing) a signaling pathway of interest, i.e., a target pathway. In some embodiments, the engineered antibodies described herein increase the activity of a target pathway by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., no antibody or a monospecific antibody). In some other embodiments, upregulating a target signaling pathway includes turning on or initiating a pathway that was shut off or substantially inactive. In another example, the engineered antibodies described herein may function as antagonists, downregulating (suppressing, inhibiting, reducing, diminishing, or attenuating) a target pathway. In some embodiments, the engineered antibodies disclosed herein reduce the activity of a target pathway by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% compared to a control (e.g., no antibody or a monospecific antibody).In some embodiments, downregulating a target signaling pathway includes stopping or substantially blocking a pathway that was on or substantially active.

[0174] Treatment method As discussed above, the experimental results presented herein demonstrate that the guide-effector bispecific antibody designs disclosed herein can be used to develop new tools for manipulating the internalization properties of cell surface antigens. Specifically, the experimental results presented herein demonstrate that the internalization propensity of a given cell surface antigen is manipulable and significantly influenced by its neighboring antigen(s) and can be readily manipulated in either direction through bispecific targeting of appropriately selected guide / effector pairs; this phenomenon can be exploited for therapeutic targeting.

[0175] Some embodiments of the present disclosure relate to methods for treating a condition or disease (e.g., cancer) in a subject using an engineered antibody or conjugate thereof disclosed herein. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an engineered antibody disclosed herein, a conjugate thereof, or a pharmaceutical composition comprising same, alone (e.g., as monotherapy) or in combination with one or more additional agents, e.g., pharmaceutically acceptable excipients (e.g., as combination therapy). In certain aspects, the engineered antibody or pharmaceutical composition administered to the subject specifically targets cells in which a signaling pathway is modulated as a result of the treatment.

[0176] In one aspect, some embodiments of the present disclosure relate to a method for treating a condition or disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered antibody or functional fragment thereof comprising: (a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate; and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate, wherein the internalization properties of the engineered antibody or functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two cellular internalization rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate.

[0177] In another aspect, some embodiments of the present disclosure relate to a method for killing cancer cells, comprising administering to said cells an engineered antibody or functional fragment thereof disclosed herein. In some embodiments, the engineered antibody or functional fragment thereof comprises (a) a first antigen-binding portion capable of binding to a cell surface guide antigen with a first cellular internalization rate, and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen with a second cellular internalization rate.

[0178] In yet another aspect, some embodiments of the present disclosure relate to a method for killing tumor cells, comprising administering to the tumor cells an engineered antibody or functional fragment thereof disclosed herein. In some embodiments of the disclosed method, the engineered antibody or functional fragment thereof comprises a first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of the tumor cells, and a second antigen-binding portion capable of binding to ALCAM expressed on the surface of the same tumor cells. In some embodiments, the surface density ratio of EphA2 to ALCAM is above a threshold of about 1:5.

[0179] The terms "administration" and "administering," as used herein, refer to the delivery of a bioactive composition or formulation by a route of administration, including, but not limited to, oral, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or a combination thereof. This term includes, but is not limited to, administration by a healthcare professional and self-administration.

[0180] The effectiveness of treatment can be determined by a skilled clinician. However, those skilled in the art will understand that treatment is considered effective if any one or all of the signs or symptoms or markers of the disease are improved or alleviated. Efficacy can also be measured by the individual not getting worse, as assessed by a reduction in the need for hospitalization or medical intervention (e.g., the progression of the disease is stopped or at least slowed). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals), including (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms, or (2) alleviating the disease, e.g., causing regression of symptoms, and (3) preventing or reducing the likelihood of the onset of symptoms.

[0181] As noted above, a therapeutically effective amount of a composition disclosed herein includes an amount sufficient to promote a particular beneficial effect when administered to an individual, e.g., one having, suspected of having, or at risk for a disease. In some embodiments, an effective amount also includes an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, delaying the progression of disease symptoms), or reverse disease symptoms. It will be understood that for any given case, the appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0182] In some embodiments, the condition or disease is cancer. In some embodiments, the engineered antibodies, conjugates thereof, and functional fragments thereof disclosed herein, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions comprising the same are administered to an individual (e.g., a human patient), e.g., to reduce the viability and / or invasiveness of cancerous cells, e.g., to reduce tumor size or metastasis, reduce tumor burden, and / or improve the patient's clinical prognosis. In certain aspects, antibody compositions can be used to disrupt the cell cycle of cancer cells, e.g., by inducing cancerous cells to enter the early GO phase of the cell cycle, thereby promoting the cells' entry into apoptosis. Cancer-related methods contemplated herein include, for example, the use of antibodies alone or in combination with anti-cancer vaccines or therapies, and the use of antibodies generated using effector and / or guide antigens in anti-cancer vaccines (e.g., by passive immunization) or therapies. The methods are useful in the context of treating or preventing a wide range of cancers. In one aspect, cancer refers to a general term encompassing primary and metastatic cancers. In some embodiments, a primary cancer may refer to a group of tumor cells that have acquired at least one characteristic property of cancer cells but have not invaded adjacent tissues and are grouped together in a tumor localized at the site of primary origin. In some other embodiments, a metastatic cancer may refer to a group of tumor cells that originate from cells of a primary cancer, invade tissues surrounding the primary cancer, disseminate throughout the body, and attach to new distant locations, resulting in the growth of a new tumor. Examples of cancers include, but are not limited to, pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, lung cancer, mesothelioma, breast cancer, urothelial cancer, liver cancer, head and neck cancer, sarcoma, cervical cancer, stomach cancer, gastric cancer, melanoma, uveal melanoma, cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, and glioblastoma.

[0183] In some embodiments, the engineered antibodies, conjugates thereof, and functional fragments thereof disclosed herein, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions comprising the same are used in anti-cancer therapy, where cancerous cells exhibit cell-specific markers that can serve as guide antigens for the bispecific antibodies of the present disclosure on the extracellularly accessible cell surface. Cancers particularly suitable for treatment using the bispecific antibodies of the present disclosure include those targeted by the antibody through binding to the guide antigen. In some embodiments, the presence or expression level of such guide antigens in normal human tissues or cells may be transient and low in abundance compared to cancer cells that overexpress the guide antigen. The guide antigen may be predominantly abundant in abnormal cells, e.g., cancer cells. Because high levels of guide antigen expression may be predominantly present in cancer cells, treatment with the bispecific antibodies of the present disclosure, or compositions comprising the antibodies, can be used to treat cancer cells with high specificity or selectivity, minimizing nonspecific cytotoxicity to non-cancerous or healthy cells.

[0184] In some embodiments, the treatment modality is to use the engineered antibody of the present disclosure to modulate signal transduction pathways.Dysregulation of signal transduction pathways is often associated with the occurrence and / or progression of a disease or condition, in that modulation of such signal transduction pathways can result in effective treatment of the disease or condition.In some cases, a disease or condition may be associated with the dysregulation of one or more signal transduction pathways, and such dysregulation can be alleviated or attenuated by modulation of another signal transduction pathway.In such situations, up-regulation or down-regulation of a signal transduction pathway using the engineered antibody of the present disclosure, which can counteract or reduce the activity of the dysregulated signal transduction pathway, can provide an effective means of treatment.

[0185] In some embodiments, cells subjected to treatment with the engineered antibodies of the present disclosure or compositions comprising the antibodies are not limited to cancer cells, but include all cells in which modulation of cell internalization and signaling may be desired, including, but not limited to, immune effector cells, such as natural killer cells, T cells, dendritic cells, and macrophages.

[0186] Dosage The dosage, toxicity, and therapeutic efficacy of the engineered antibodies of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. For example, compounds that exhibit high therapeutic indices are generally preferred. Compounds that exhibit toxic side effects can be used, but care must be taken to design delivery systems that target such compounds to the site of affected tissues in order to minimize the potential for damage to uninfected cells, thereby reducing side effects.

[0187] In the methods of the present disclosure, an effective amount of an engineered antibody or composition comprising the antibody of the present disclosure is administered to an individual in need thereof. For example, in some embodiments, the engineered antibody or composition thereof inhibits the growth, metastasis, and / or invasiveness of cancer cell(s) in the individual when the antibody or composition thereof is administered in an effective amount. The amount administered will vary depending on the purpose of administration, the health and physical condition of the individual being treated, their age, the taxonomic group of the individual being treated (e.g., human, non-human primate, primate, etc.), the degree of resolution desired, the formulation of the engineered antibody or composition, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall within a relatively broad range that can be determined by routine testing. For example, the amount of an engineered antibody or composition thereof used to inhibit cancer cell growth, metastasis, and / or invasiveness will be below about an amount that would otherwise be irreversibly toxic to the subject (i.e., the maximum tolerated dose). In other cases, the amount will be near or well below the toxicity threshold, but still within the immunoeffective concentration range, or even as low as the threshold dose.

[0188] The individual dose will generally be no less than the amount required to produce a measurable effect in the individual and can be determined based on the pharmacokinetics and pharmacology of the antibody's absorption, distribution, metabolism, and excretion ("ADME"), and thus the disposition of the composition within the individual. This includes consideration of the route of administration, as well as the dosage, which can be adjusted, for example, for parenteral applications (applied by a route other than the digestive tract for systemic or local effect). For example, administration of engineered antibodies or compositions thereof is generally by injection, often intravenously, intramuscularly, intratumorally, or a combination thereof.

[0189] The engineered antibody or composition thereof can be administered by infusion or local injection at a rate of, for example, about 10 mg / hour to about 200 mg / hour, about 50 mg / hour to about 400 mg / hour, including about 75 mg / hour to about 375 mg / hour, about 100 mg / hour to about 350 mg / hour, about 150 mg / hour to about 350 mg / hour, about 200 mg / hour to about 300 mg / hour, and about 225 mg / hour to about 275 mg / hour. Exemplary infusion rates include, for example, about 1 mg / m 2 / day ~ approx. 9mg / m2 / day, approx. 2mg / m 2 / day~about 8mg / m 2 / day, about 3mg / m 2 / day~about 7mg / m 2 / day, approximately 4mg / m 2 / day~about 6mg / m 2 / day, approximately 4.5mg / m 2 / day~about 5.5mg / m 2 / day, approximately 0.5 mg / m 2 / day~about 10mg / m 2 A desired therapeutic dose of 100 mg / day can be achieved. Administration (e.g., by infusion) can be repeated for a desired period, e.g., for a period of about 1 day to about 5 days, or for several days, e.g., about once every 5 days, for about 1 month, for about 2 months. It can also be administered before, at the time of, or after other therapeutic interventions, e.g., surgical intervention to remove cancerous cells. The engineered antibody or composition thereof can also be administered as part of a combination therapy, in which at least one of immunotherapy, cancer chemotherapy, or radiation therapy is administered to the subject.

[0190] Route of administration In some embodiments of the present disclosure, the engineered antibodies, conjugates thereof, and functional fragments thereof, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions comprising the same disclosed herein may be formulated to be compatible with their intended route of administration. For example, while the engineered antibodies, conjugates thereof, and functional fragments thereof, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions comprising the same disclosed herein may be provided orally or by inhalation, they are likely to be administered via parenteral routes. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral administration may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting isotonicity such as sodium chloride or dextrose. The pH can be adjusted (e.g., to about 7.2 to 7.8, e.g., 7.5) with an acid or base such as mono- and / or di-sodium phosphate, hydrochloric acid, or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0191] In practicing the present methods, the route of administration (the route by which the engineered antibodies disclosed herein, conjugates thereof, and functional fragments thereof, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions comprising same are administered to an individual or subject) can be varied. The engineered antibodies or compositions thereof can be administered systemically (e.g., parenterally, e.g., by intravenous route) or locally (e.g., to a local tumor site, e.g., by intratumoral administration (e.g., into a solid tumor, into an associated lymph node in the case of a lymphoma or leukemia), or by administration to a blood vessel supplying a solid tumor).

[0192] In some embodiments, the engineered antibodies described herein are formulated for parenteral administration. In some cases, the engineered antibodies are formulated for intravenous, subcutaneous, intramuscular, intraarterial, intracranial, intracerebral, intraventricular, or intrathecal administration. In some cases, the engineered antibodies are administered to a subject as an injection. In other cases, the engineered antibodies are administered to a subject as an infusion.

[0193] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The preparations may be presented in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid vehicle for injection, such as water, immediately prior to use. Extemporaneous injection solutions and suspensions The formulations can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0194] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of a compound of the present disclosure calculated to be sufficient to produce a desired effect, together with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the novel unit dosage forms depend on the particular compounds employed and the effect to be achieved, as well as the pharmacokinetics associated with each compound in an individual, and the target disease or condition and its stage in the individual.

[0195] Systems and Kits Also provided herein are systems and kits that include the engineered antibodies, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein, as well as written instructions for making and using them.For example, in some embodiments, systems and / or kits are provided herein that include one or more of the engineered antibodies described herein, the recombinant nucleic acid molecules described herein, the recombinant cells described herein, or the pharmaceutical compositions described herein.In some embodiments, the systems and / or kits of the present disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) that are used to administer any one of the engineered antibodies, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided to an individual.In some embodiments, the kit may have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, such as to modulate cell internalization, to modulate cell type-selective signaling in a subject, or to treat a disease in a subject in need thereof.

[0196] Any of the above-described systems and kits may further comprise one or more additional reagents, where such additional reagents may be selected from a dilution buffer, a reconstitution solution, a wash buffer, a control reagent, a control expression vector, a negative control antibody, a positive control antibody, a reagent for in vitro production of an engineered antibody.

[0197] In some embodiments, the system or kit may further include instructions for practicing the method using the components of the kit. The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic. The instructions may be present in the kit as a package insert, on a label on the container (i.e., associated with the packaging or sub-packaging) of the kit or its components, or the like. The instructions may be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, or the like. In some cases, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

[0198] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0199] No admission is made that any reference cited herein constitutes prior art. The discussion of references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. Although several sources of information, including scientific journal articles, patent documents, and textbooks, have been referenced herein, it is expressly understood that these references do not constitute an admission that any of these documents form part of the general general knowledge in the art.

[0200] The discussion of general methods provided herein is intended for illustrative purposes only. Other alternative methods and substitutes will be apparent to those skilled in the art upon consideration of this disclosure, and are within the spirit and scope of this application. [Example]

[0201] Further embodiments are disclosed in more detail in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the disclosure or the claims in any way.

[0202] Example 1 Identification of a high-affinity ALCAM antibody and generation of an ALCAM x EphA2 bispecific antibody To identify human antibodies against ALCAM, scFv phage display library selection was performed against the N-terminal Ig-like V1-V2 domain of ALCAM (Figure 6A). High A panel of binding phages was identified by FACS screening against the DU145 prostate cancer cell line (Figure 6B), further identifying antibody 3F1, which binds with high affinity as an IgG1 to DU145 cells (apparent K D =20.6 pM) (Figure 6C). We studied the internalization of 3F1 IgG in a panel of tumor cell lines by confocal microscopy and found that this antibody was non-internalizing or slowly internalizing (Figure 6D).

[0203] A tetravalent bispecific IgG-scFv (bsIgG) was constructed, consisting of a non-internalizing anti-ALCAM 3F1 IgG backbone and an internalizing anti-EphA2 scFv (RYR) fused to the C-terminus of the 3F1 light chain (Figure 1A). The anti-EphA2 scFv (RYR) was identified from previous studies, where macropinocytotic antibodies were identified using high-content analysis. For control, a control C10 / RYR bsIgG (binding only to EphA2) was constructed using non-binding C10 IgG. SDS-PAGE analysis showed the expected electrophoretic patterns of the monoclonal and bispecific antibodies (Figure 7A). The binding specificity of the bsIgG was then investigated using a HEK293 cell line expressing ALCAM and an engineered HEK293 cell line (HEK293-EphA2#2) stably expressing high levels of EphA2. As shown in Figure 7B, anti-ALCAM 3F1 IgG bound to both HEK293 and HEK293-EphA2#2 cells, as expected. high EphA2 low ), and HEK293-EphA2#2 (ALCAM high EphA2 high) and bound to HEK293-EphA2#2 cells. The control C10 / RYR bsIgG, which binds to EphA2, only showed specific binding to HEK293-EphA2#2 cells, but not to HEK293 cells. Using these two cell line models, the internalization activity of 3F1 / RYR bsIgG was studied by confocal microscopy. As shown in Figure 1B, 3F1 / RYR bsIgG acquired effective internalization ability in an EphA2-dependent manner, and was internalized by HEK293-EphA2#2 but not by HEK293 cell line. The control C10 / RYR bsIgG was internalized by HEK293-EphA2#2 but not by HEK293. The results show that in the guide-effector bispecific antibody design described herein, the internalizing arm (EphA2, guide) can confer internalizing properties to the non-internalizing arm (ALCAM, effector) and to the bispecific antibody as a whole.

[0204] Example 2 Non-internalized antigens can be internalized by bispecific antibodies in a time- and guide-to-effector ratio-dependent manner To quantitatively examine the removal of cell surface antigens by antibody-induced antigen internalization, quantitative FACS analysis was performed to measure the copy numbers of ALCAM and EphA2 on the cell surface (referred to herein as antigen density). As shown in Figure 1C, the level of ALCAM on HEK293-EphA2#2 cells incubated with the bispecific 3F1 / RYR was reduced by approximately 90% within the first 4 hours of incubation. There was no significant change in surface ALCAM levels after treatment with monoclonal anti-ALCAM 3F1, control monoclonal C10, or control bispecific C10 / RYR (Figure 1C). This confirms that efficient removal of ALCAM from the cell surface by 3F1 / RYR is consistent with the ALCAM removal from HEK293-EphA2#2 (ALCAM high EphA2 high ) and HEK293 (ALCAM high EphA2 low) was not observed (Figure 8A). We further explored whether antigen removal efficiency was affected by the guide-to-effector ratio (EphA2 / ALCAM). To generate HEK293-based cell models with various EphA2 / ALCAM ratios, the levels of EphA2 and / or ALCAM were manipulated in three ways: 1) transient transfection of an EphA2-expressing plasmid, 2) transient cotransfection of an EphA2-expressing plasmid and ALCAM-siRNA, and 3) lentiviral transfection of the EphA2 gene to achieve stable EphA2 expression. These cells exhibited various EphA2 / ALCAM ratios and various patterns of surface antigen removal after bispecific 3F1 / RYR treatment. For example, monoclonal anti-ALCAM 3F1 IgG or control C10 / RYR bsIgG did not remove ALCAM from the cell surface, whereas 3F1 / RYR bsIgG efficiently removed surface ALCAM (Figure 1D). Pearson's correlation coefficient analysis showed that the effect significantly increased with increasing EphA2 / ALCAM ratio (Figure 1D). Regarding EphA2, anti-ALCAM 3F1 IgG did not reduce surface EphA2, as expected, whereas EphA2-binding 3F1 / RYR and control C10 / RYR efficiently removed EphA2 from the cell surface (Figure 8B). The ability of the bispecific 3F1 / RYR to remove surface ALCAM was affected by the EphA2 to ALCAM ratio (guide-to-effector antigen ratio, outlined in Figure 1E). As summarized in Table 1, when the ratio was below 1:5 (0.2), only a small percentage of ALCAM was removed (20-35%). When the ratio was 0.9-3.5, 45-65% of surface ALCAM was removed. When the ratio was above 3.5, more than 70% of surface ALCAM was removed.

[0205] Example 3 Internalizing and non-internalizing are interchangeable properties in bispecific antibody design Although it has been shown that a non-internalizing antigen (ALCAM) can be induced to internalize by anti-EphA2 / ALCAM bispecific antibodies when the ratio of EphA2 to ALCAM exceeds a threshold, this experiment examines whether rapidly internalizing EphA2 can be made slower or non-internalizing by the presence of ALCAM at a specific EphA2 to ALCAM ratio. high EphA2 low ) as a model, we found that EphA2 internalization was significantly delayed when the EphA2-to-ALCAM ratio was below 0.2, resulting in a high proportion of surface-bound EphA2 when targeted by 3F1 / RYR bsIgG, but not by the control C10 / RYR (Figure 1F), suggesting that internalization and non-internalization are interconvertible properties and that the relative abundance of internalized versus non-internalized antigen significantly affects cell surface antigen turnover when targeted by bispecific antibodies (outlined in Figure 1G). [Table 1]

[0206] Example 4 Expanding beyond model cell lines: Modulating internalization kinetics in tumor cells with guide / effector-based bispecific antibodies This example demonstrates the dynamics of bispecific antibody-induced surface antigen expression in a panel of pancreatic cancer cell lines with various guide-to-effector ratios. The cell surface antigen densities of ALCAM and EphA2 were first quantified by quantitative FACS (Table 2). ALCAM was found to be highly expressed by these cells, and the guide-to-effector (EphA2 to ALCAM) ratios of L3.6pl, Capan-1, and Panc-1 were estimated to be 0.31, 0.23, and 0.08, respectively. Next, two sets of experiments were performed to determine: (1) how non-internalized ALCAM is converted into an internalized antigen by bispecific antibodies at EphA2 to ALCAM ratios above a threshold (above 0.2), and (2) how rapidly internalized EphA2 is converted into a slowly internalized antigen by bispecific antibodies at EphA2 to ALCAM ratios below a threshold (below 0.2). The internalization kinetics of EphA2 and ALCAM were studied by measuring surface antigen levels by FACS after antibody treatment. For ALCAM, the bispecific 3F1 / RYR was effective in removing approximately 60% of cell surface ALCAM in both L3.6pl and Capan-1 cells, where the guide-to-effector ratio was greater than 0.2, but not in Panc-1 cells, where the ratio was 0.08, suggesting cell type selectivity based on the guide-to-effector ratio (Figure 2A).

[0207] The non-internalizing monoclonal anti-ALCAM antibody 3F1 did not remove any ALCAM antigen from the cell surface. The control C10 / RYR or the antibody mixture of 3F1 and C10 / RYR removed approximately 85% of surface EphA2 (Figure 8C), but failed to remove ALCAM (Figure 2A), suggesting that ALCAM removal is a bispecific-dependent phenomenon that cannot be achieved with oligoclonal antibody mixtures. The above-described bispecific effect on antigen internalization was also studied by confocal microscopy. As shown in Figure 2B, in L3.6pl cells with an EphA2 / ALCAM ratio greater than 0.2 (approximately 0.31), anti-ALCAM 3F1 was most frequently detected on the cell surface, whereas the bispecific 3F1 / RYR was primarily detected in the cytoplasm, with some plasma membrane staining. The control C10 / RYR, which binds EphA2, was primarily detected in the cytoplasm, consistent with its ability to induce rapid EphA2 internalization. In contrast, for the Panc-1 cell line, which has an EphA2 / ALCAM ratio below 0.2 (approximately 0.08), bispecific 3F1 / RYR was detected primarily at the cell surface ( FIG. 2B ), suggesting that bispecific antibody internalization is again dependent on the EphA2 / ALCAM ratio. These data confirm that the ability of bispecific antibodies to convert non-internalized to internalized effector antigens in the guide-effector bispecific antibody design described herein is dependent on the guide-to-effector ratio. [Table 2]

[0208] To assess the internalization pathway and trafficking to lysosomes, confocal microscopy studies were performed using L3.6pl cells. As shown in Figure 2C, the bispecific 3F1 / RYR colocalized with the macropinocytosis marker 70 kDa neutral dextran (ND70), suggesting that the mode of internalization was macropinocytosis. After internalization, 3F1 / RYR and the control C10 / RYR colocalized with the lysosomal marker lysosome-associated membrane protein 1 (LAMP1) (Figure 2D), suggesting that the anti-EphA2 antibody-guided bispecific antibody was trafficked to lysosomes.

[0209] To investigate the opposite direction of the interconversion between internalization and non-internalization, i.e., from a rapidly internalizing antibody to a slow or non-internalizing antibody, we studied the surface removal of EphA2 by the bispecific antibody in the presence of the adjacent non-internalizing antigen, ALCAM. As shown in Figure 2E, in Panc-1 cells where the EphA2-to-ALCAM ratio was below 0.2 (approximately 0.08), EphA2 remained primarily on the cell surface when targeted by the bispecific 3F1 / RYR. The control C10 / RYR removed EphA2 from the cell surface. A mixture of C10 / RYR and 3F1 did not slow EphA2 internalization, suggesting that this phenomenon is bispecific antibody-dependent. Time-course internalization studies showed that 3F1 / RYR significantly delayed EphA2 internalization kinetics, while the control C10 / RYR induced rapid EphA2 internalization (Figure 2F). These studies demonstrate that EphA2 internalization can be significantly delayed by bispecific antibodies when ALCAM is present on the same cells in amounts above a threshold EphA2 / ALCAM ratio.

[0210] Example 5 Bispecific 3F1 / RYR inhibits pancreatic tumorsphere formation The effect of anti-EphA2-guided bsIgG (3F1 / RYR) on the survival and expansion of pancreatic tumorspheres was investigated to assess the functional consequences of surface antigen removal. Previous studies have shown that cancer cells overexpressing ALCAM actively form tumorspheres, suggesting that ALCAM plays a role in tumor clonogenicity. Therefore, in this example, experiments were performed to investigate whether ALCAM removal by tetravalent ALCAM × EphA2 bsIgG could inhibit pancreatic tumorsphere formation. First, antigen expression was assessed in L3.6pl tumorspheres, and ALCAM was found to be significantly upregulated on the surface of tumor cells forming spheres (Figure 3A). There was no difference in EphA2 surface levels between monolayer and sphere-forming L3.6pl cells (Figure 3A). After two weeks of incubation of L3.6pl tumorspheres with the antibody, the bispecific 3F1 / RYR reduced ALCAM surface density by 70%, whereas the anti-ALCAM mAb 3F1 or the control C10 / RYR, which binds only to EphA2, showed no effect on ALCAM surface levels (Figure 3B). Confocal microscopy studies were then performed to confirm antibody internalization. As shown in Figure 3C, 3F1 / RYR was efficiently internalized in L3.6pl sphere-forming cells. In contrast, the monoclonal anti-ALCAM antibody 3F1 showed primarily surface staining (Figure 3C). The control bispecific C10 / RYR was internalized, consistent with its monospecific binding to EphA2 (Figure 3C). Notably, based on the fluorescence signal intensity per cell, a greater amount of 3F1 / RYR was taken up by tumorsphere-forming cells compared with the control bispecific C10 / RYR (Figure 3C, right panel), suggesting an amplification effect inherent to the bispecific antibody. With regard to functional effects on tumor clonogenic activity, the number (Fig. 3D) and size (Fig. 3E) of L3.6pl spheres were significantly reduced by treatment with 3F1 / RYR, but not with 3F1 or C10 / RYR, consistent with previous studies of the role of ALCAM in tumorsphere formation and growth.Thus, a bispecific antibody with one arm that binds to the internalized antigen EphA2 can effectively remove the non-internalized antigen ALCAM from the tumor cell surface, resulting in inhibition of pancreatic tumorsphere growth.

[0211] Example 6 Potent and cell-type-selective in vitro tumor cell killing by bispecific ADCs To explore the therapeutic potential of amplifying bispecific antibody-induced cellular uptake, several monospecific and bispecific ADCs were generated by site-specific conjugation of MC-VC-pab-MMAF. The conjugation products were analyzed by HIC-HPLC to determine the drug-to-antibody ratio (approximately 1.9). In these experiments, the ADCs were tested in a panel of cancer cell lines exhibiting different levels of cell surface EphA2 and ALCAM, as well as EphA2-to-ALCAM ratios (see, for example, Table 2). The 3F1 / RYR ADC exhibited potent cytotoxicity, with an EC50 of 23 pM in L3.6pl cells and 22 pM in Capan-1 cells (Figures 4A and 4B and Table 3). These two cell lines have EphA2-to-ALCAM ratios above the threshold (0.2, Table 2), resulting in more efficient internalization. In contrast, 3F1 and C10 / RYR Both ADCs showed low potency. The EC50 values ​​for the 3F1 and C10 / RYR ADCs in L3.6pl were 2.37 nM and 0.35 nM, respectively, and for Capan-1 they were 0.87 nM and 0.18 nM, respectively (Figures 4A and 4B). More notably, the bispecific ADC was more potent than the mixture of monoclonal ADCs (C10 / RYR ADC + 3F1 ADC, Figures 4A and 4B), again suggesting that this enhanced potency is inherent to the bispecific antibodies. The cytotoxicity of the ADCs against Panc-1 cells, which have a low guide-to-effector (EphA2 vs. ALCAM) ratio, and MIA PaCa2 cells, which lack expression of the effector antigen (ALCAM), was also studied. In Panc-1 cells, which have a low EphA2 / ALCAM ratio (0.08), the bispecific 3F1 / RYR ADC showed reduced potency (EC50 = 0.46 nM), but was still more potent than the 3F1 (EC50 = 9.3 nM) and C10 / RYR ADC (EC50 > 100 nM) (Figure 4C and Table 3). Again, the cytotoxic potency of the 3F1 / RYR ADC was higher than that of the mixture of 3F1 and C10 / RYR ADC (EC50 = 0.46 nM vs. 7.14 nM for the mixture) (Table 3). In the ALCAM-negative MIA PaCa2 cell line, the 3F1 ADC, as expected, showed little cytotoxicity (Figure 4D). The 3F1 / RYR and C10 / RYR ADCs exhibited similarly low cytotoxicity due to the lack of ALCAM expression and low expression levels of EphA2 (Figure 4D). To further evaluate cell-type selectivity, we investigated the LNCaP-C4-2B and HEK293 cell lines, which express very low levels of EphA2. In LNCaP-C4-2B, as shown in Figure 4E, the bispecific 3F1 / RYR ADC did not exhibit enhanced cytotoxicity compared to the monoclonal 3F1 ADC due to the lack of expression of the guide antigen EphA2 (EC50 = 1.25 nM vs. 1.64 nM, Table 3), demonstrating guide-antigen-dependent cell-type selectivity. Similar results were obtained from studies using HEK293 cells, which lack expression of the guide antigen (Figure 9).Collectively, these data indicate that bispecific ADCs are more potent than monospecific ADCs or their mixtures and exhibit cell-type-selective enhanced potency depending on the guide-to-effector antigen ratio. [Table 3]

[0212] Example 7 In vivo antitumor efficacy of ALCAM×EphA2 bispecific ADC This example summarizes experiments conducted to study the in vivo efficacy of the bispecific 3F1 / RYR ADC, along with a control ADC, in pancreatic cancer xenografts. Capan-1 cells were implanted subcutaneously into NSG mice. When tumors reached an average volume of 110 mm, 3F1 / RYR, 3F1, or C10 / RYR ADCs were injected every four days at 3 mg / kg for a total of four doses. Tumor status was monitored by caliper measurement. Overt toxicity was monitored by weight loss. As shown in Figure 5A, the bispecific 3F1 / RYR ADC significantly inhibited tumor growth, while the monoclonal 3F1 ADC or the control bispecific C10 / RYR ADC had only a moderate effect on reducing tumor size. There was no significant change in body weight over the course of the study for any of the ADCs studied (Figure 5B). These data demonstrate that in the guide-effector bispecific antibody design described herein, the rapidly internalizing anti-guide (EphA2) scFv can induce internalization of an otherwise non-internalizing effector antigen (ALCAM), resulting in greater tumor cell uptake of the bispecific ADC compared to the monospecific ADC, and therefore, enhanced anti-tumor efficacy in vivo.

[0213] Example 8 Cell lines and plasmids Human embryonic kidney (HEK) lines HEK293 and HEK293A, prostate cancer cell lines DU145 and PC3, and pancreatic cancer cell lines Capan-1, Panc-1, and MIA PaCa2 were obtained from the American Type Culture Collection (ATCC). The L3.6pl line was obtained from Dr. Isaiah Fidler (MD Anderson Cancer Center, Houston, TX). LNCap-C4-2B was originally obtained from UroCor Inc. and maintained in the laboratory. Cells were cultured in 10% FBS (Fisher Scientific), 100 μg / ml penicillin / streptomycin (Axenia The cells were maintained in DMEM or RPMI 1640 supplemented with 100% ethanol (BioLogix) at 37°C and 5% CO. Full-length human EphA2 cDNA cloned into pCMV-Entry (Origene) or pLV202 (Origene) was used for transient or stable expression of EphA2, respectively.

[0214] Example 9 Generation of anti-ALCAM scFv antibodies A naive scFv-phagemid display library was used for antibody selection. Recombinant human IgG-like V1-V2 domains of ALCAM fused with human IgG2 Fc (AV-Fc) were produced in HEK293A cells and used as antigen. AV-Fc was coated onto SPHERO™ polystyrene magnetic particles (Spherotech) overnight at 4°C. The phage library was depleted with uncoated beads in PBS / 2% milk, and unbound phages were allowed to bind to AV-Fc-coated beads. The beads were then washed, eluted, and expanded as described above. Individual phage binders were screened by FACS using the ALCAM-expressing DU145 cell line, and the DNA sequences of the scFvs were analyzed using the IgAT tool.

[0215] Example 10 Generation of anti-EphA2 scFv antibodies This example describes experiments performed to identify new versions of EphA2-binding scFv antibodies with improved binding affinity. The original EphA2-binding scFv RYR was previously described in International Application No. PCT / US2015 / 039741, where the EphA2-binding scFv RYR was designated HCA-F1, and the germline version of RYR was designated RYRgerm. To identify new versions of EphA2-binding scFv antibodies with improved binding affinity to EphA2, a yeast display mutagenesis library based on RYRgerm was generated, and higher affinity binders were selected by FACS. Four new EphA2 scFvs with high binding affinity to EphA2 were identified and designated RYRgerm_102019_14, RYRgerm_102919_15, RYRgerm_102919_22, and RYRgerm_102919_33, respectively. The amino acid sequences of the VH and VL regions and CDRs of these newly identified EphA2 scFvs are listed in Tables 4-5 and the Sequence Listing. In these experiments, both human and mouse recombinant EphA2 proteins were used in the selection to maintain cross-species binding. Apparent affinities for binding to both human and mouse EphA2 were measured by flow cytometry. As shown in Figure 10A, the new versions of the EphA2-binding scFv antibodies identified in the yeast display mutagenesis library exhibited enhanced binding affinity to human EphA2 compared to the original EphA2-binding scFv RYR, with increases in binding affinity ranging from approximately 8-fold to approximately 70-fold. The apparent K of human EphA2 binding affinity was approximately 8-fold to approximately 70-fold. D The value was 354.9 nM for RYRgerm (original EphA2 scFv), RYRgerm_102019_14 was 21.27 nM, RYRgerm_102919_15 was 5.58 nM, RYRgerm_102919_22 was 28.13 nM, and RYRgerm_102919_33 was 42.49 nM. Similarly, as shown in Figure 10B, the new versions of EphA2-binding scFv antibodies identified in the yeast display mutagenesis library showed improved binding affinity to mouse recombinant EphA2-Fc compared to the original EphA2-binding scFv RYR, with an increase in binding affinity of approximately 40- to approximately 80-fold. The apparent K of the binding affinity of the mouse recombinant EphA2-Fc fusions was D The values ​​were 114.7 nM for RYRgerm (original EphA2 scFv), 2.12 nM for RYRgerm_102019_14, 2.01 nM for RYRgerm_102919_15, 1.34 nM for RYRgerm_102919_22, and 2.87 nM for RYRgerm_102919_33.

[0216] Subsequently, to go beyond evaluating scFv binding activity in yeast cells, additional recombinant human IgG1s were designed and constructed using the original EphA2 scFv (RYR) and the new, improved RYRgerm_102919_15, and their binding affinities in live cells and recombinant antigens were studied. Figure 11 summarizes the results of experiments performed in the human prostate cancer cell line DU145 to compare the affinity of recombinant IgG1s between the original RYR and the new, improved RYR-binding scFv RYRgerm_102919_15 described in Figures 10A-10B. In these experiments, the apparent binding affinity in DU145 cells of RYR IgG1 compared with RYRgerm_102919_15 IgG1 was assessed. In these experiments, DU145 cells were incubated with RYR or RYRgerm_102919_15 at concentrations ranging from 40 pM to 125 nM for 1 hour at 25 °C, washed, and binding was detected using anti-human Alexa Fluor 647. MFI values ​​were calculated by curve fitting to obtain the apparent K D Generated value: RYR K of IgG1 and RYRgerm_102919_15 D The values ​​were 23.7 nM and 0.23 nM, respectively, which represents an approximately 100-fold increase in binding affinity.

[0217] Additional experiments were also performed to evaluate the binding affinity of the new EphA2 scFv RYRgerm_102919_15 on recombinant human EphA2 (see, e.g., Figure 12). In these experiments, label-free Biolayer interferometry (BLI) analysis was performed using a Probe Life Gator instrument. An anti-human Fc probe was loaded onto RYR or RYRgerm_102919_15 IgG1, and 100 nM recombinant human EphA2 (R&D System) was used in the binding assay at 25°C. The apparent affinity K of RYR IgG1 was D is approximately 28 nM (K off / K on =1.45E-02 / 5.18E+05), while the apparent affinity K of RYRgerm_102919_15 IgG1 was improved. D is approximately 5.0 nM (K off / K on =1.32E-03 / 2.62E+05), indicating an approximately 5-fold increase in binding affinity.

[0218] Example 11 Recombinant antibody production VH and VL antibody genes were amplified by PCR from candidate scFv phagemids and subcloned into Abvec Ig-γ and -λ expression vectors, respectively. To generate bispecific IgG-scFvs, anti-ALCAM 3F1 or nonbinding control C10 was used as the IgG scaffold, and the internalizing scFv was introduced at the C-terminus of the λ light chain constant region (CL) via fusion with a (Gly4Ser)3 linker. HEK293A cells were transfected with antibody expression plasmids mixed with polyethylenimine (Sigma-Aldrich) in Opti-MEM (Life Technologies) for 24 hours. The transfection medium was replaced with Freestyle™ 293 (Gibco), and the cells were further cultured for up to 8 days. Secreted antibodies were purified from the culture supernatant using protein A agarose (Thermo Scientific) and analyzed on SDS-PAGE gradient gels (4-20%).

[0219] Example 12 Generation of stable HEK293-EphA2 cell lines HEK293 cells were transduced with a lentiviral vector expressing EpAh2 and maintained in normal growth medium containing G418 (Sigma). Stable EphA2-expressing clones were identified by FACS using a human anti-EphA2 antibody followed by Alexa Fluor® 647-labeled goat anti-human IgG (Jackson ImmunoResearch). Stable clones were further screened by FACS to obtain clones expressing various levels of EphA2.

[0220] Example 13 Cell surface antigen copy number measurement Cell surface antigen copy number (or antigen density) was measured as previously described. Briefly, cells were dissociated by 0.25% trypsin digestion, washed and resuspended in FACS assay buffer (PBS, 1% FBS, pH 7.4), and incubated with anti-EphA2 or ALCAM antibodies conjugated with Alexa Fluor® 647 using a monoclonal antibody labeling kit (Molecular Probes) to detect EphA2 or ALCAM, respectively, and analyzed by a BD Accuri C6 (BD Biosciences). Mean fluorescence intensity (MFI) was converted to antibody binding capacity (ABC) using Quantum™ Alexa Fluor® 647 MESF and Quantum™ Simple Cellular® anti-human IgG (Bang's Laboratory) according to the manufacturer's recommendations. For each cell model studied, EphA2 was measured using Alexa Fluor® 647 MESF and Quantum™ Simple Cellular® anti-human IgG (Bang's Laboratory) according to the manufacturer's recommendations. The E / A (EphA2 / ALCAM) ratio was calculated by dividing the copy number of A2 by the copy number of ALCAM.

[0221] Example 14 Apparent K D Decision Dissociated cells (approximately 2 × 10 5 Cells (800 cells / ml) were incubated with various concentrations of human IgG for 16 hours at 4°C. After washing three times with ice-cold PBS, cell-bound IgG was detected with Alexa Fluor® 647-labeled goat anti-human IgG (Jackson ImmunoResearch) and analyzed by FACS. Apparent K D Values ​​were calculated by curve fitting methods using GraphPad Prism software.

[0222] Example 15 Cell surface antigen depletion Monospecific or bispecific antibodies (100 nM) were incubated with cells cultured in 24-well plates (approximately 80% confluence) for 24 hours, and residual EphA2 or ALCAM on the cell surface was determined using Alexa Fluor® 647-labeled L1A1 anti-EphA2 human IgG or L50 anti-ALCAM mouse IgG (Fisher Scientific), respectively. Cell surface copy numbers were calculated using the method described above and normalized to the no-antibody control group.

[0223] Example 16 Immunofluorescence confocal microscopy Antibodies were incubated with cells seeded in 8-well culture chamber slides (Fisher Scientific) for the indicated times. To assess the internalization pathway (macropinocytosis), cells were co-incubated with Texas Red-conjugated 70 kDa neutral dextran (ND70-TR, Life Technologies), a marker for macropinocytosis. After incubation, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS / 1% FBS / 0.2% Triton-X100. Cell-associated antibodies were stained with Alexa Fluor® 488- or 647-conjugated goat anti-human IgG (Jackson ImmunoResearch) for 1 hour at room temperature. Lysosomes were detected with rabbit anti-lysosome-associated membrane protein 1 (LAMP1) antibody (Cell Signaling) followed by incubation with Alexa Fluor® 647-labeled goat anti-rabbit IgG (Jackson ImmunoResearch). To analyze antibody localization in tumor spheres, spheres were collected by centrifugation at 500 × g for 5 minutes, washed, fixed, permeabilized, and immunolabeled using the above-mentioned antibodies. CyGEL™ (Abcam) was used to immobilize spheres on 8-well chamber slides for microscopic analysis. For imaging, cells or spheres were counterstained with Hoechst 33342 (Thermo Scientific) and imaged using a FluoView® FV10i laser confocal microscope (Olympus) equipped with an Olympus 60x phase-contrast water-immersion objective.

[0224] Example 17 Tumorsphere formation Tumorspheres were formed by culturing suspension tumor cells obtained from monolayer cultures in serum-free medium (SFM) containing DMEM / F12 (Gibco), 20 ng / ml EGF, 10 ng / ml bFGF, 10 ng / ml IGF, and 2% B27 supplement (Gibco) in ultra-low attachment 24-well plates (Corning) at 37°C / 5% CO2. For sphere growth assays, first-generation spheres were trypsinized and sieved through a 40 μm nylon mesh cell strainer (Fisher Scientific) to obtain a single cell population. 200 cells per well were resuspended in 500 μl of SFM and seeded into ultra-low attachment 24-well plates (Corning) at 37°C / 5% CO2 for 24 hours and treated with the indicated antibodies for 2 weeks. Cells were fed with 100 μl of SFM every 3–4 days. Each well was scanned in cross section using a BIOREVO digital microscope (BZ-9000, Keyence), and the images were merged to show an image of the entire well. Spheres with a diameter greater than 100 μm were counted.

[0225] Example 18 Site-specific ADC generation A cysteine ​​residue was introduced into position 116 (T116C) of the heavy chain of IgG or bsIgG, and site-specific ADCs bearing the modification were generated as described previously. Briefly, the antibody in PBS was reduced by incubation with a 10-fold molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Thermo Scientific) at 37°C for 2 hours, purified using a Zeba spin desalting column (Thermo Scientific), and buffer-exchanged into PBS / 5mM EDTA. To reoxidize the interchain disulfide bonds, the reduced antibody was incubated with a 20-fold molar excess of dehydroascorbic acid (dhAA) (Sigma) at 25°C for 3 hours. After buffer exchange with PBS / 5 mM EDTA, the antibody was incubated with a 3-fold molar excess of maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl monomethyl auristatin F (MC-vc-PAB-MMAF), synthesized as described above, for 1 hour at 25°C. The final conjugated product was purified by two runs on a Zeba™ spin desalting column (Fisher Scientific) and analyzed by hydrophobic interaction chromatography (HIC)-HPLC using an Infinity 1220 LC system (Agilent). The drug-to-antibody ratio (DAR) was estimated by area integration using OpenLab CDS software (Agilent).

[0226] Example 19 ADC cytotoxicity 2 x 10 cells 3 Cells were seeded at 1000 cells / well into 96-well cell culture plates overnight and incubated with various concentrations of ADC for 96 hours. Cell viability was determined using a Calcein-AM Cell Viability Assay Kit (Biotium Inc.).

[0227] Example 20 In vivo xenograft studies All animal studies were approved by the UCSF Animal Care and Use Committee (AN092211) and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. - / - (NSG) female mice, 1 × 10 6 Capan-1 cells were engrafted and randomly divided into four groups on day 5 (n=6 for each group). Mice were treated intravenously with vehicle PBS or monospecific or bispecific ADCs at 3 mg / kg every four days for a total of four injections. Tumor size was measured by caliper and tumor volume was calculated using the formula V=(width) / (width). 2 × length) / 2. Body weight was monitored during the course of the study. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 5]

[0228] While certain alternatives of the present disclosure are disclosed, it should be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended patent application. Accordingly, there is no limitation to the precise abstract and disclosure presented herein. References 1. de Goeij BE,Lambert JM. New developments for antibody-drug conjugate-basedtherapeuticapproaches. Curr Opin Immunol 2016;40:14-23 doi10.1016 / j.coi.2016.02.008. 2. Polakis P.Antibody Drug Conjugates for Cancer Therapy. Pharmacol Rev2016;68(1):3-19 doi10.1124 / pr.114.009373. 3. Gebleux R,Stringhini M, Casanova R, Soltermann A, Neri D. Non-internalizingantibody-drugconjugates display potent anti-cancer activity upon proteolyticrelease ofmonomethyl auristatin E in the subendothelial extracellular matrix.Int JCancer 2017;140(7):1670-9 doi 10.1002 / ijc.30569. 4. Li JY, PerrySR, Muniz-Medina V, Wang X, Wetzel LK, Rebelatto MC, et al. ABiparatopicHER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression inPrimaryModels Refractory to or Ineligible for HER2-Targeted Therapy. CancerCell2016;29(1):117-29 doi 10.1016 / j.ccell.2015.12.008. 5. de Goeij BE,Vink T, Ten Napel H, Breij EC, Satijn D, Wubbolts R, et al.Efficient PayloadDelivery by a Bispecific Antibody-Drug Conjugate TargetingHER2 and CD63. MolCancer Ther 2016;15(11):2688-97 doi 10.1158 / 1535-7163.MCT-16-0364. 6. Andreev J,Thambi N, Perez Bay AE, Delfino F, Martin J, Kelly MP, et al.BispecificAntibodies and Antibody-Drug Conjugates (ADCs) Bridging HER2 andProlactin ReceptorImprove Efficacy of HER2 ADCs. Mol Cancer Ther 2017;16(4):681-93doi10.1158 / 1535-7163.MCT-16-0658. 7. DeVay RM,Delaria K, Zhu G, Holz C, Foletti D, Sutton J, et al. ImprovedLysosomalTrafficking Can Modulate the Potency of Antibody Drug Conjugates.BioconjugChem 2017;28(4):1102-14 doi 10.1021 / acs.bioconjchem.7b00013. 8. Lee NK,Zhang Y, Su Y, Bidlingmaier S, Sherbenou DW, Ha KD, et al. Cell-typespecificpotent Wnt signaling blockade by bispecific antibody. Sci Rep2018;8(1):766 doi10.1038 / s41598-017-17539-z. 9. Bruns CJ,Harbison MT, Kuniyasu H, Eue I, Fidler IJ. In vivo selectionandcharacterization of metastatic variants from human pancreatic adenocarcinomabyusing orthotopic implantation in nude mice. Neoplasia 1999;1(1):50-62. 10. Su Y, Liu Y,Behrens CR, Bidlingmaier S, Lee NK, Aggarwal R, et al.Targeting CD46 for bothadenocarcinoma and neuroendocrine prostate cancer. JCIInsight 2018;3(17):pii:121497 doi 10.1172 / jci.insight.121497. 11. Lee NK,Bidlingmaier S, Su Y, Liu B. Modular Construction of LargeNon-Immune HumanAntibody Phage-Display Libraries from Variable Heavy and LightChain GeneCassettes. Methods Mol Biol 2018;1701:61-82 doi10.1007 / 978-1-4939-7447-4_4. 12. Rogosch T,Kerzel S, Hoi KH, Zhang Z, Maier RF, Ippolito GC, et al.Immunoglobulinanalysis tool: a novel tool for the analysis of human and mouseheavy and lightchain transcripts. Front Immunol 2012;3:176 doi10.3389 / fimmu.2012.00176. 13. Smith K,Garman L, Wrammert J, Zheng NY, Capra JD, Ahmed R, et al. Rapidgeneration offully human monoclonal antibodies specific to a vaccinatingantigen. Nat Protoc2009;4(3):372-84 doi 10.1038 / nprot.2009.3. 14. SherbenouDW, Aftab BT, Su Y, Behrens CR, Wiita A, Logan AC, et al.Antibody-drugconjugate targeting CD46 eliminates multiple myeloma cells. J ClinInvest2016;126(12):4640-53 doi 10.1172 / JCI85856. 15. Orcutt KD,Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL, Frangioni JV, et al. Amodular IgG-scFv bispecific antibody topology. Protein Eng DesSel2010;23(4):221-8 doi 10.1093 / protein / gzp077. 16. Ha KD,Bidlingmaier SM, Zhang Y, Su Y, Liu B. High-content analysis ofantibodyphage-display library selection outputs identifies tumorselectivemacropinocytosis-dependent rapidly internalizing antibodies. MolCellProteomics 2014;13(12):3320-31 doi 10.1074 / mcp.M114.039768. 17. JunutulaJR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, et al.Site-specificconjugation of a cytotoxic drug to an antibody improves thetherapeutic index.Nat Biotechnol 2008;26(8):925-32 doi 10.1038 / nbt.1480. 18. Jiao J,Hindoyan A, Wang S, Tran LM, Goldstein AS, Lawson D, et al.Identification ofCD166 as a surface marker for enriching prostatestem / progenitor and cancerinitiating cells. PLoS One 2012;7(8):e42564 doi10.1371 / journal.pone.0042564. 19. Yan M,YangX, Wang L, Clark D, Zuo H, Ye D, et al. Plasma membrane proteomics oftumorspheres identify CD166 as a novel marker for cancer stem-like cells inhead andneck squamous cell carcinoma. Mol Cell Proteomics 2013;12(11):3271-84doi10.1074 / mcp.M112.025460. 20. Moon BS,Jeong WJ, Park J, Kim TI, Min do S, Choi KY. Role of oncogenicK-Ras in cancerstem cell activation by aberrant Wnt / beta-catenin signaling. JNatl Cancer Inst2014;106(2):djt373 doi 10.1093 / jnci / djt373. 21. Behrens CR,Liu B. Methods for site-specific drug conjugation to antibodies. MAbs2014;6(1):46-53 doi 10.4161 / mabs.26632. 22. Fu Y, Ho M.DNA damaging agent-based antibody-drug conjugates for cancertherapy. AntibTher 2018;1(2):33-43 doi 10.1093 / abt / tby007. 23. Panowski S,Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drugconjugates for cancer therapy. MAbs 2014;6(1):34-45 doi10.4161 / mabs.27022.24. Ha KD,Bidlingmaier SM, Su Y, Lee NK, Liu B.Identification of Novel MacropinocytosingHuman Antibodies by Phage Display andHigh-Content Analysis. Methods Enzymol2017;585:91-110 doi10.1016 / bs.mie.2016.10.004. 25. Chang K,Pastan I. Molecular cloning of mesothelin, a differentiationantigen present onmesothelium, mesotheliomas, and ovarian cancers. Proc NatlAcad Sci U S A1996;93(1):136-40. 26. Silver DA,Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membraneantigen expression in normal and malignant human tissues. Clin CancerRes1997;3(1):81-5. 27. KinoshitaY, Kuratsukuri K, Landas S, Imaida K, Rovito PM, Jr., Wang CY, etal.Expression of prostate-specific membrane antigen in normal and malignanthumantissues. World J Surg 2006;30(4):628-36 doi 10.1007 / s00268-005-0544-5. 28. Wang K, WeiG, Liu D. CD19: a biomarker for B cell development, lymphomadiagnosis andtherapy. Exp Hematol Oncol 2012;1(1):36 doi10.1186 / 2162-3619-1-36. 29. VainshteinI, Roskos LK, Cheng J, Sleeman MA, Wang B, Liang M. Quantitativemeasurement ofthe target-mediated internalization kinetics ofbiopharmaceuticals. Pharm Res.2015 Jan;32(1):286-99. 30. Li N, HillKS, Elferink LA. Analysis of receptor tyrosine kinase internalizationusingflow cytometry. Methods Mol Biol. 2008;457:305-17. 31. Pahara J,Shi H, Chen X, Wang Z. Dimerization drives PDGF receptorendocytosis through aC-terminal hydrophobicmotif shared by EGF receptor. ExpCell Res.2010;316:2237-50. 32. Mazor Y,Barnea I, Keydar I, Benhar I. Antibody internalization studiedusing a novelIgG binding toxin fusion. J Immunol Methods. 2007;321:41-59. 33. Schaefer Get al. A two-in-one antibody against HER3 and EGFR has superiorinhibitoryactivity compared with monospecific antibodies. Cancer Cell. 2011Oct18;20(4):472-86. doi: 10.1016 / j.ccr.2011.09.003 34. Lee CV,Koenig P, Fuh G. A two-in-one antibody engineered from a humanizedinterleukin4 antibody through mutation in heavy chaincomplementarity-determining regions.MAbs. 2014;6(3):622-627. doi:10.4161 / mabs.28483)

Claims

[Claim 1] The invention described in the specification.

Images