Compositions and Methods for Modulating Intracellular Trafficking

Abstract

Provided herein are compositions and methods for modulating the internalization properties of cell surface molecules, for example, for converting non-internalized cell surface antigens into internalized ones, and vice versa. In some embodiments, engineered antibodies are provided, each comprising an antigen-binding portion specific for a 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 cell internalization in a cell or subject, as well as methods for modulating cell-type-selective signaling and / or treating diseases in a subject.

Description

Technical Field

[0001] Description of Research and Development Funded by the Federal Government 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 on January 14, 2019, which is hereby expressly incorporated by reference in its entirety, including all drawings.

[0003] Incorporation of Sequence Listing This application is being filed with an electronic sequence listing. The sequence listing is provided as a file named "Sequence Listing_048536 - 628001WO.txt", created on December 23, 2019, which is approximately 124 KB in size. The information in the electronic sequence listing is hereby incorporated by reference in its entirety into this specification.

[0004] Field Aspects of this application relate to the fields of cell biology and immunology. More specifically, for example, engineered antibodies that convert non - internalizing cell - type - selective surface antigens to internalizing ones and vice versa to modulate and / or amplify cell - type - specific internalization are provided herein. The disclosure also provides compositions and methods useful for producing such engineered antibodies, as well as methods for the treatment of health disorders or diseases related to cancer, including solid tumors and hematological malignancies.

Background Art

[0005] Background The use of biopharmaceuticals or pharmaceutical compositions containing therapeutic proteins for the treatment of diseases, disorders, or health conditions is a core strategy for 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 in the development of targeted therapies. Ideally, by conjugating a potent cytotoxic agent to a cell-type specific antibody, the cytotoxic agent can be directed towards target cells and preferentially accumulated in the target tissue. Another example of targeted therapy includes antibody-drug conjugates (ADCs), which have shown promising efficacy in several clinical studies.

[0007] Conceptually simple, the selection of targets in therapeutic antibodies and ADCs is hampered by the fact that it is rare to discover so-called tumor-specific antigens, and even rarer to discover tumor-specific antigens with the properties desired for therapeutic targeting, i.e., those that are highly and uniformly expressed by cancer cells and efficiently internalize. Additionally, movement across the plasma membrane is a major limiting step in the cellular delivery of macromolecules. Thus, the effectiveness of therapies that rely on intracellular translocation of therapeutic agents depends on both the quality of the target on the surface of the target cell and the rate of intracellular translocation of the surface-bound therapeutic agent complexed with that target. Additionally, although 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 a bystander effect. In some cases, in targeted therapies where intracellular payload delivery is required, many tumor antigens are highly expressed but internalization is not sufficient. In other cases, receptor internalization, which is the receptor-mediated endocytosis process that results in movement of the receptor from the plasma membrane into the cell, is also used to block signaling pathways and cause desensitization. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] Accordingly, new approaches and compositions for the treatment of diseases, disorders, or health conditions, such as inflammatory diseases, immune diseases, and cancer, are currently needed. Specifically, there is a need in the art for more effective compositions and methods for treating diseases, disorders, or health conditions by improving the internalization properties of therapeutic antibodies and ADCs. MEANS FOR SOLVING THE PROBLEMS

[0009] This section provides a general overview of the disclosure and does not purport to cover its full scope or all of its characteristics.

[0010] The present disclosure relates to compositions and methods for engineering cell type-selective antibody internalization by guide-effector bispecific antibody design. Specifically, engineered antibodies are provided herein that can simultaneously bind to an antigen pair referred to as a “guide antigen” and an “effector antigen” expressed on the surface of the same cell. When the engineered antibody binds simultaneously, the guide antigen can affect the cell surface dynamics and / or signaling function of the effector antigen. In some specific 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 the effector function to a desired cell. For example, by using a guide-effector bispecific antibody design that can bind to (i) a non-internalizing effector antigen and (ii) an internalizing guide antigen, the non-internalizing effector antigen can be converted to an internalizing effector antigen. Similarly, by using a guide-effector bispecific antibody design that can bind to (i) an internalizing effector antigen and (ii) a non-internalizing guide antigen, the internalizing effector antigen can be converted to a non-internalizing effector 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 containing the same. The present disclosure also provides compositions and methods useful for modulating cellular internalization in a cell or subject by using such engineered antibodies, as well as methods for modulating cell type-selective signaling in a subject and / or for treating health disorders and diseases associated with cancer, including solid tumors and hematological malignancies.

[0011] In one embodiment, some embodiments of the present disclosure are engineered antibodies or functional fragments thereof, comprising: a) a first antigen-binding portion capable of binding to a cell surface guide antigen having a first intracellular trafficking rate; and b) a second antigen-binding portion capable of binding to a cell surface effector antigen having a second intracellular trafficking rate, wherein the intracellular trafficking characteristics 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 intracellular trafficking rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate, with respect to the engineered antibody or functional fragment thereof.

[0012] Implementations of engineered antibody embodiments of the present disclosure may include one or more of the following characteristics. 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. In some embodiments, the relative surface density ratio of the guide antigen to the effector antigen is above a threshold. In some embodiments, the relative surface density ratio of the guide antigen to the 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 portion and the second antigen-binding portion are independently selected from the group consisting of antigen-binding fragments (Fab), single-chain variable fragments (scFv), full-length immunoglobulins, nanobodies, single-domain antibodies (sdAb), variable new antigen receptor (VNAR) domains, and VHH domains, multispecific antibodies, diabodies, or functional fragments thereof.In some embodiments, the guide antigen and the 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-bound (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 signal transducer 2 (Trop-2), and tumor-associated glycoprotein 72 (TAG-72).

[0013] In some embodiments, the guide antigen is a cancer-related antigen 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, EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostate acid phosphatase (PAP), ephrin B2, 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 its functional fragment is conjugated or covalently bound to at least one moiety of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a moiety that improves pharmacokinetics. 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 chelating agent, 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 anti-cancer agent is selected from the group consisting of auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, anthracycline, calicheamycin, camptothecin, irinotecan, SN-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 bound to the constant region of an engineered antibody or a functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently bound to the heavy chain constant (e.g., CH1, CH2, or CH3) region of an antibody or a functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently bound to the heavy chain constant (CH1) region of an antibody or a functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently bound to the light chain constant (CL) region of an antibody or a functional fragment thereof. In some embodiments, the average number of MOIs per antibody (e.g., average drug-to-antibody ratio, DAR) ranges from 1 to 20. 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.

[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 is above 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 the VH sequences 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 portion 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 portion comprises a light chain variable (VL) region having at least 80% sequence identity to the VH sequences identified in Table 4. In some embodiments, 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. In some embodiments, the VL region of the first antigen-binding portion comprises three LCDRs identified in the sequence listing. In some embodiments, 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.

[0019] In some embodiments, the second antigen-binding portion comprises a VH region having at least 80% sequence identity to the VH sequences 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 the VH sequences identified in Table 4. In some embodiments, 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. In some embodiments, the VL region of the second antigen-binding portion comprises 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 a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an engineered antibody or a 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 a vector.

[0021] In one aspect, some embodiments of the present disclosure relate to a recombinant cell comprising (a) an engineered antibody or a functional fragment thereof disclosed herein and / or (and / o) (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 and a culture medium disclosed herein.

[0022] In one aspect, some embodiments of the present disclosure relate to a pharmaceutical composition comprising one or more of the following: (a) an engineered antibody or a 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 intracellular trafficking, the method comprising administering to a cell one or more of the following: (a) an engineered antibody or a 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 intracellular trafficking, the method comprising administering an engineered antibody or a functional fragment thereof comprising: (a) a first antigen-binding portion capable of binding to a cell surface guide antigen having a first intracellular trafficking rate, and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen having a second intracellular trafficking rate, wherein the intracellular trafficking characteristics of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two intracellular trafficking 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 are methods for modulating cell-type selective signaling in a subject, the method comprising administering to the cell an engineered antibody or a functional fragment thereof comprising: (a) 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 intracellular trafficking rate; and (b) a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second intracellular trafficking rate, wherein the intracellular trafficking characteristics of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two intracellular trafficking 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 are methods for treating a health condition or a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered antibody or a functional fragment thereof disclosed herein. In some embodiments, the engineered antibody or its functional fragment comprises: (a) a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first intracellular trafficking rate; and (b) a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second intracellular trafficking rate, wherein the intracellular trafficking characteristics of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two intracellular trafficking rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. In some embodiments, the health condition or disease is cancer.

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

[0028] In yet another aspect, some embodiments of the present disclosure relate to a method for killing tumor cells, the method comprising administering to the tumor cells an engineered antibody or a functional fragment thereof disclosed herein. In some embodiments of the disclosed method, the engineered antibody or the 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 method, the engineered antibody or a 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 the VH sequences 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 the 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, respectively, (a) SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, or (b) SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 110. In some embodiments, the first antigen-binding portion comprises a VL region having at least 80% sequence identity to the VL sequences identified in Table 4. In some embodiments, 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. In some embodiments, the VL region of the first antigen-binding portion comprises the three LCDRs identified in the sequence listing. In some embodiments, the VL region of the first antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 comprising, respectively, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109.

[0030] In some embodiments of the disclosed method, the second antigen-binding portion comprises a VH region having at least 80% sequence identity to the VH sequences 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 the VL sequences identified in Table 4. In some embodiments, the second antigen-binding portion comprises a VL region having at least 80% sequence identity to SEQ ID NO: 74 (SEC ID NO: 74) or SEQ ID NO: 76. In some embodiments, the VL region of the second antigen-binding portion comprises 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.

[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 the guide antigen to the effector antigen exceeds a threshold value. In some embodiments, the relative surface density ratio of the guide antigen to the 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. In some embodiments, the methods disclosed herein further comprise modulating the cell surface density of the guide antigen and / or the cell surface density of the effector antigen. In some embodiments, the internalization properties of the engineered antibodies disclosed herein are converted from internalized to non-internalized. In some other embodiments, the internalization properties of the engineered antibodies disclosed herein are converted from non-internalized to internalized. 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) An engineered antibody or a functional fragment thereof, A first antigen-binding portion capable of binding to a cell surface guide antigen having a first intracellular trafficking rate, and A second antigen-binding portion capable of binding to a cell surface effector antigen having a second intracellular trafficking rate comprising, wherein the intracellular trafficking characteristics of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, and wherein one of the two intracellular trafficking rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate, an engineered antibody or a functional fragment thereof. (Item 2) The engineered antibody or a functional fragment thereof according to item 1, wherein the cell surface guide antigen is an intracellular trafficking cell surface antigen. (Item 3) The engineered antibody or a functional fragment thereof according to item 1, wherein the cell surface effector antigen is a non-intracellular trafficking cell surface antigen. (Item 4) The engineered antibody or a functional fragment thereof according to any one of items 1 to 2, wherein the relative surface density ratio of the guide antigen to the effector antigen exceeds a threshold value. (Item 5) The engineered antibody or a functional fragment thereof according to 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) The engineered antibody or a functional fragment thereof according to any one of items 1 to 5, 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 7) The engineered antibody or a functional fragment thereof according to 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 antigen-binding fragments (Fab), single-chain variable fragments (scFv), full-length immunoglobulins, nanobodies, single-domain antibodies (sdAb), VNAR domains, and VHH domains, multispecific antibodies, diabodies, or functional fragments thereof. (Item 8) The guide antigen and the 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-bound (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 signal transducer 2 (Trop-2), and tumor-associated glycoprotein 72 (TAG-72), and the engineered antibody or a functional fragment thereof according to any one of items 1 to 7. (Item 9) The engineered antibody or functional fragment thereof according to any one of items 1 to 8, wherein the guide antigen is a cancer-related antigen 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, EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostate acid phosphatase (PAP), ephrin B2, 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) The engineered antibody or functional fragment thereof according to any one of items 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) An engineered antibody or a functional fragment thereof according to any one of items 1 to 10, conjugated or covalently bound to at least one part of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a moiety that improves pharmacokinetics. (Item 12) The engineered antibody or a functional fragment thereof according to item 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 antibacterial agent, an antimicrobial agent, an antibiotic, an anti-infectious disease agent, and an antiviral agent. (Item 13) The engineered antibody or a functional fragment thereof according to item 12, wherein the at least one MOI is selected from the group consisting of a cytotoxic anti-cancer agent, a DNA chelating agent, 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) The engineered antibody or a functional fragment thereof according to item 13, wherein the cytotoxic anti-cancer agent is selected from the group consisting of auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, anthracycline, calicheamicin, camptothecin, irinotecan, SN-38, combretastatin, duocarmycin, enediyne, epothilone, ethyleneimine, mitomycin, pyrrolobenzodiazepine (PBD), and calicheamycin. (Item 15) The engineered antibody or a functional fragment thereof according to any one of items 11 to 14, wherein the at least one part of interest (MOI) is conjugated or covalently bound to the constant region of the engineered antibody or a functional fragment thereof. (Item 16) The engineered antibody or a functional fragment thereof according to item 15, wherein the at least one part of interest (MOI) is conjugated or covalently bound to the heavy chain constant (CH1) region of the engineered antibody or a functional fragment thereof. (Item 17) The engineered antibody or a functional fragment thereof according to item 15, wherein the at least one part of interest (MOI) is conjugated or covalently bound to the light chain constant (CL) region of the engineered antibody or a functional fragment thereof. (Item 18) An engineered antibody or a functional fragment thereof according to any one of items 11 to 17, wherein the average number of MOIs per antibody (average DAR) ranges from 1 to 20. (Item 19) The engineered antibody or a 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 portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell, A second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cell An engineered antibody or a functional fragment thereof according to any one of items 1 to 19, comprising (Item 21) The engineered antibody or a functional fragment thereof according to item 20, wherein the surface density ratio of EphA2 to ALCAM exceeds a threshold of about 1:5. (Item 22) An engineered antibody or a functional fragment thereof according to 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 specified in Table 4. (Item 23) The engineered antibody or a functional fragment thereof according to item 22, wherein the first antigen-binding portion comprises a heavy chain variable (VH) region having at least 80% sequence identity to the VH sequence specified in Table 4. (Item 24) The engineered antibody or a functional fragment thereof according to item 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) The engineered antibody or a functional fragment thereof according to 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 specified in the sequence listing. (Item 26) The VH region of the first antigen-binding portion is (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 The engineered antibody or a functional fragment thereof according to item 25, comprising HCDR1, HCDR2, and HCDR3. (Item 27) The engineered antibody or functional fragment thereof according to any one of items 22 to 26, wherein the first antigen-binding portion comprises a variable light (VL) region having at least 80% sequence identity to the VL sequence specified in Table 4. (Item 28) The engineered antibody or functional fragment thereof according to 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) The engineered antibody or functional fragment thereof according to any one of items 22 to 28, wherein the VL region of the first antigen-binding portion comprises the CDRs specified in the Sequence Listing. (Item 30) The engineered antibody or functional fragment thereof according to 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) The engineered antibody or functional fragment thereof according to any one of items 22 to 30, wherein the second antigen-binding portion comprises a VH region having at least 80% sequence identity to the VH sequence specified in Table 4. (Item 32) The engineered antibody or functional fragment thereof according to item 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) The engineered antibody or functional fragment thereof according to any one of items 22 to 32, wherein the VH region of the second antigen-binding portion comprises three HCDRs specified in the Sequence Listing. (Item 34) The engineered antibody or functional fragment thereof according to item 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) The engineered antibody or functional fragment thereof according to any one of items 22 to 34, wherein the second antigen-binding portion comprises a VL region having at least 80% sequence identity to the VL sequence specified in Table 4. (Item 36) The engineered antibody or functional fragment thereof according to item 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) The engineered antibody or a functional fragment thereof according to any one of items 22 to 36, wherein the VL region of the second antigen-binding portion comprises three CDRs specified in the sequence listing. (Item 38) The engineered antibody or a functional fragment thereof according to item 37, wherein the VL region of the second antigen-binding portion comprises LCDR1, LCDR2, and LCDR3, which comprise SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, respectively. (Item 39) A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the engineered antibody or a functional fragment thereof according to any one of items 1 to 38. (Item 40) The recombinant nucleic acid molecule according to item 39, which is operably linked to a heterologous nucleic acid sequence. (Item 41) The recombinant nucleic acid molecule according to any one of items 39 to 40, further defined as an expression cassette or a vector. (Item 42) The engineered antibody or a functional fragment thereof according to any one of items 1 to 36, and / or The nucleic acid molecule according to any one of items 39 to 41 Comprising a recombinant cell. (Item 43) The recombinant cell according to item 42, which is a prokaryotic cell or a eukaryotic cell. (Item 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) The engineered antibody or a functional fragment thereof according to any one of items 1 to 38, The nucleic acid molecule according to any one of items 39 to 41, and The recombinant cell according to any one of items 42 to 43 One or more of, A pharmaceutically acceptable carrier Comprising a pharmaceutical composition. (Item 46) A method for modulating intracellular trafficking, comprising administering to a cell The engineered antibody or a functional fragment thereof according to any one of items 1 to 38, The nucleic acid molecule according to any one of items 39 to 41, and The pharmaceutical composition described in item 45 One or more of the steps of administration. (Item 47) A method for modulating intracellular trafficking, comprising administering to a cell A first antigen-binding portion capable of binding to a cell surface guide antigen having a first intracellular trafficking rate, A second antigen-binding portion capable of binding to a cell surface effector antigen having a second intracellular trafficking rate Comprising the step of administering the engineered antibody or a functional fragment thereof. The intracellular trafficking characteristics of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, wherein one of the two intracellular trafficking rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. (Item 48) 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 intracellular trafficking rate, a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second intracellular trafficking rate and an engineered antibody or its functional fragment comprising the same, wherein the intracellular trafficking characteristics of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, wherein one of the two intracellular trafficking rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. (Item 49) A method for performing a treatment of a healthy condition or a 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 intracellular trafficking rate, a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second intracellular trafficking rate and a therapeutically effective amount of an engineered antibody or its functional fragment comprising the same, wherein the intracellular trafficking characteristics of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, wherein one of the two intracellular trafficking rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. (Item 50) The method according to item 49, wherein the healthy condition or the disease is cancer. (Item 51) A method for killing cancer cells, comprising administering to the cells a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first intracellular trafficking rate, a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second intracellular trafficking rate A method comprising the step of administering an engineered antibody or a functional fragment thereof. (Item 52) The method according to 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) The method according to any one of items 46 to 52, wherein the cell surface guide antigen is an internalizing cell surface antigen. (Item 54) The method according to any one of items 46 to 52, wherein the cell surface effector antigen is a non-internalizing cell surface antigen. (Item 55) The method according to any one of items 46 to 54, wherein the relative surface density ratio of the guide antigen to the effector antigen exceeds a threshold value. (Item 56) The method according to 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) The method according to 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) The method according to any one of items 46 to 57, further comprising the step of modulating the cell surface density of the guide antigen and / or the cell surface density of the effector antigen. (Item 59) The method according to any one of items 46 to 58, wherein the internalization property of the engineered antibody or a functional fragment thereof is converted from non-internalizing to internalizing. (Item 60) The method according to any one of items 46 to 58, wherein the internalization property of the engineered antibody or a functional fragment thereof is converted from internalizing to non-internalizing. (Item 61) The method according to any one of items 46 to 60, wherein the first antigen-binding portion and the second antigen-binding portion are independently selected from the group consisting of antigen-binding fragments (Fab), single-chain variable fragments (scFv), full-length immunoglobulins, nanobodies, single-domain antibodies (sdAb), VNAR domains, and VHH domains, bispecific antibodies, diabodies, or functional fragments thereof. (Item 62) The method according to any one of items 46 to 61, wherein the expression of the guide antigen and / or the effector antigen is cell type-selective. (Item 63) The method according to any one of items 46 to 62, wherein the guide antigen and the 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-bound type (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 signal transducer 2 (Trop-2), and tumor-associated glycoprotein 72 (TAG-72). (Item 64) The method according to any one of items 46 to 63, wherein the guide antigen is a cancer-related antigen 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, EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostate acid phosphatase (PAP), ephrin B2, 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 method according to any one of items 46 to 64, 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, FGFR, IGF-1R, VEGFR, PDGFR, Trop-2, TAG-72, P-selectin, EGFR, ErbB2, ErbB3, and ErbB4. (Item 66) The method according to any one of items 46 to 65, wherein the antibody or a functional fragment thereof is conjugated or covalently bound to at least one part of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a moiety that improves pharmacokinetics. (Item 67) The method according to 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 antibacterial agent, an antimicrobial agent, an antibiotic, an anti-infectious disease agent, and an antiviral agent. (Item 68) The method according to item 67, wherein the at least one MOI is selected from the group consisting of a cytotoxic anti-cancer agent, a DNA chelating agent, 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 69) The method according to item 68, wherein the cytotoxic anti-agent is selected from the group consisting of auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, anthracycline, calicheamicin, camptothecin, irinotecan, SN-38, combretastatin, duocarmycin, enediyne, epothilone, ethyleneimine, mitomycin, pyrrolobenzodiazepine (PBD), and calicheamicin. (Item 70) The method according to any one of items 66 to 69, wherein the at least one part of interest (MOI) is conjugated or covalently bound to the constant region of the engineered antibody or a functional fragment thereof. (Item 71) The method according to item 70, wherein the at least one part of interest (MOI) is conjugated or covalently bound to the CH1 region of the engineered antibody or a functional fragment thereof. (Item 72) The method according to item 70, wherein the at least one part of interest (MOI) is conjugated or covalently bound to the CL region of the engineered antibody or a functional fragment thereof. (Item 73) The method according to any one of items 46 to 72, wherein the average number of MOIs per antibody (DAR) ranges from 1 to 20. (Item 74) The method according to item 73, wherein 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. (Item 75) A first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell, A second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cell, The method according to any one of items 46 to 74, comprising: (Item 76) The method according to item 75, wherein the surface density ratio of EphA2 to ALCAM exceeds a threshold value of about 1:5. (Item 77) The method according to any one of items 46 to 76, wherein the engineered antibody or a functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences specified in Table 4. (Item 78) A method for killing tumor cells in a subject, the method comprising administering to the tumor cells A first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of the tumor cells, A second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same tumor cells An engineered antibody or a functional fragment thereof comprising: (Item 79) The method according to item 78, wherein the surface density ratio of EphA2 to ALCAM exceeds a threshold value of about 1:5. (Item 80) The method according to any one of items 46 to 79, wherein the engineered antibody or a functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences specified in Table 4. (Item 81) The method according to item 80, wherein the first antigen-binding portion comprises a VH region having at least 80% sequence identity to the VH sequence specified in Table 4. (Item 82) The method according to item 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) The method according to any one of items 80 to 82, wherein the VH region of the first antigen-binding portion comprises three CDRs specified in the sequence listing. (Item 84) The VH region of the first antigen-binding portion is each of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, or each of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 110 The method according to item 83, comprising HCDR1, HCDR2, and HCDR3 each containing the above. (Item 85) The method according to any one of items 80 to 84, wherein the first antigen-binding portion comprises a VL region having at least 80% sequence identity to the VL sequence specified in Table 4. (Item 86) The method according to item 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) The method according to any one of items 80 to 86, wherein the VL region of the first antigen-binding portion comprises three CDRs specified in the sequence listing. (Item 88) The method according to item 87, wherein the VL region of the first antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 each containing SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively. (Item 89) The method according to any one of items 80 to 88, wherein the second antigen-binding portion comprises a VH region having at least 80% sequence identity to the VH sequence specified in Table 4. (Item 90) The method according to item 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) The method according to any one of items 80 to 90, wherein the VH region of the second antigen-binding portion comprises three CDRs specified in the sequence listing. (Item 92) The method according to item 91, wherein the VH region of the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 each containing SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively. (Item 93) The method according to any one of items 80 to 92, wherein the second antigen-binding portion comprises a VL region having at least 80% sequence identity to the VL sequence specified in Table 4. (Item 94) The method according to item 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) The method according to any one of items 80 to 94, wherein the VL region of the second antigen-binding portion comprises three CDRs specified in the sequence listing. (Item 96) The method according to item 95, wherein the VL regions of the second antigen-binding portions each comprise 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 fully apparent from the drawings and detailed description, as well as from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] [Figure 1-1]Figures 1A - 1G summarize the results obtained from experiments conducted to show that bispecific antibodies based on the guide - effector design according to some non - limiting embodiments of the present disclosure can significantly affect the internal trafficking dynamics of cell - surface antigens. Figure 1A is a diagram of tetravalent ALCAM×EphA2 bsIgG. The IgG backbone is based on the non - internalizing anti - ALCAM antibody 3F1. The internalizing anti - EphA2 scFv is fused to the end of the light - chain C - terminus. Figure 1B shows a study of antibody internalization by confocal microscopy. 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 (R) 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 the surface ALCAM levels were determined by FACS. Non - internalizing ALCAM is removed from the cell surface by the bispecific (3F1 / RYR) antibody but not by the 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 diverse EphA2 / ALCAM ratios were incubated with 3F1, 3F1 / RYR, and C10 / RYR (all 100 nM), and the antigen remaining on the cell surface was determined by an anti - ALCAM antibody that binds to an epitope different from 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 (duplicate). Figure 1E is a diagram showing bispecific - induced internalization of ALCAM when the guide - to - effector ratio exceeds a threshold. CM: cell membrane.Figure 1F shows a significant delay in the internalization of EphA2 by bispecific 3F1 / RYR when the guide-to-effector ratio is below the threshold. HEK293 cells with a low EphA2 / ALCAM ratio (less than 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-sided Student's t-test. *P < 0.05, and ***P < 0.001. Figure 1G is a diagram of the phenomenon shown in Figure 1F, where EphA2 internalization is delayed (e.g., reduced) when the ratio of EphA2 to ALCAM (E / A) is below the threshold. [Figure 1-2]Figures 1A-1G summarize the results obtained from experiments conducted to show 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 is a diagram of tetravalent ALCAM×EphA2 bsIgG. The IgG backbone is based on the non-internalizing anti-ALCAM antibody 3F1. The internalizing anti-EphA2 scFv is fused to the end of the C-terminus of the light chain. Figure 1B shows a study of antibody internalization by confocal microscopy. 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 (registered trademark) 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-internalizing ALCAM is removed from the cell surface by the bispecific (3F1 / RYR) antibody, but not by the monoclonal antibody. Figure 1D shows the correlation between surface antigen (ALCAM) removal efficiency and EphA2 / ALCAM (E / A) expression ratio. HEK293 cell models with various EphA2 / ALCAM ratios were incubated with 3F1, 3F1 / RYR, and C10 / RYR (all 100 nM), and the antigen remaining on the cell surface was determined by an anti-ALCAM antibody that binds to an epitope different from 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 are represented as mean ± standard deviation (duplicate). Figure 1E is a diagram showing bispecific-induced internalization of ALCAM when the guide-to-effector ratio exceeds a threshold. CM: cell membrane.Figure 1F shows a significant delay in the internalization of EphA2 by bispecific 3F1 / RYR when the guide-to-effector ratio is below the threshold. HEK293 cells with a low EphA2 / ALCAM ratio (less than 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-sided Student's t-test. *P < 0.05, and ***P < 0.001. Figure 1G is a diagram of the phenomenon shown in Figure 1F, where EphA2 internalization is delayed (e.g., reduced) when the ratio of EphA2 to ALCAM (E / A) is below the threshold. [Figure 1-3]Figures 1A - 1G summarize the results obtained from experiments conducted to show that bispecific antibodies based on the 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 is a diagram of tetravalent ALCAM×EphA2 bsIgG. The IgG backbone is based on the non - internalizing anti - ALCAM antibody 3F1. The internalizing anti - EphA2 scFv is fused to the end of the light - chain C - terminus. Figure 1B shows a study of antibody internalization by confocal microscopy. HEK293 or HEK293 - EphA2#2 cells were incubated with the indicated IgG or bsIgG (100 nM) at 37°C for 2 hours. The antibody (red) was detected using an Alexa (R) 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 the surface ALCAM levels were determined by FACS. Non - internalizing ALCAM is removed from the cell surface by the bispecific (3F1 / RYR) antibody, but not by the monoclonal antibody. Figure 1D shows the correlation between the 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 100 nM), and the antigen remaining on the cell surface was determined by an anti - ALCAM antibody that binds to an epitope different from 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 are represented as mean ± standard deviation (duplicates). Figure 1E is a diagram showing bispecific - induced ALCAM internalization when the guide - to - effector ratio exceeds a threshold. CM: cell membrane.Figure 1F shows a significant delay in the internalization of EphA2 by bispecific 3F1 / RYR when the guide-to-effector ratio is below the threshold. HEK293 cells with a low EphA2 / ALCAM ratio (less than 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-sided Student's t-test. *P < 0.05, and ***P < 0.001. Figure 1G is a diagram of the phenomenon shown in Figure 1F, where EphA2 internalization is delayed (e.g., reduced) when the ratio of EphA2 to ALCAM (E / A) is below the threshold.

[0035] [Figure 2]Figures 2A - 2F summarize the results obtained from experiments conducted to show that a bispecific antibody (3F1 / RYR) based on a guide - effector design according to some non - limiting embodiments of the present disclosure effectively removes a non - internalizing antigen (ALCAM) from the surface of pancreatic cancer cells. Figure 2A: ALCAM cell surface levels after antibody treatment. Pancreatic cancer cell lines L3.6pl (bars to the left of the X - axis), Capan - 1 (central bar), and Panc - 1 (right bar) were incubated with 3F1, 3F1 / RYR, C10 / RYR, or a mixture of 3F1 and C10 / RYR. After washing following treatment, cell surface ALCAM levels were determined using IgG labeled with Alexa® 647 that binds to an epitope on ALCAM different from 3F1. The MFI values were normalized to cells without antibody treatment. **P < 0.01, and ***P < 0.001. Duplicates. Figure 2B: Study by confocal microscopy of cell - type - selective internalization mediated by bispecific antibodies. L3.6pl (E / A ratio greater than 0.2) and Panc - 1 (E / A ratio less than 0.2) cells were incubated with 3F1, 3F1 / RYR, or C10 / RYR, and the internalizing antibody was stained with FITC - labeled anti - human IgG. Scale bar: 20 μm. Figure 2C: Co - localization of antibody and macropinosome vesicles. L3.6pl cells were incubated for 2 hours with 100 nM of 3F1, 3F1 / RYR, or C10 / RYR, and ND70 - TR (TR - Dextran, red). The antibody was 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 for 2 hours with the indicated antibody (100 nM). The internalized antibody (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: Delay of EphA2 internalization in Panc - 1 cells when targeted by bispecific antibodies. **P < 0.01, and ***P < 0.001. Duplicates.Figure 2F: Time course of the removal of EphA2 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 show that the removal of cell surface ALCAM by bispecific antibodies according to some non - limiting embodiments of the present disclosure has an anti - clonogenic effect on pancreatic tumor spheres. Figure 3A: Significant up - regulation of ALCAM in L3.6pl sphere cells compared to non - sphere tumor cells. Adherent - cultured or sphere - cultured L3.6pl cells were separated into single cells, and antigen expression was measured using 3F1 or RYR IgG followed by anti - human IgG labeled with Alexa® 647. Figure 3B: Removal of ALCAM from the surface of sphere - forming cells by 3F1 / RYR. A single - cell population of L3.6pl (200 cells / well) was incubated for 2 weeks with the indicated antibodies (100 nM) in ultra - low - attachment well plates. The cell - surface level of ALCAM after antibody treatment was determined by FACS. The MFI values were normalized to the control (without antibody treatment). **P < 0.01. Duplicate. Figure 3C: Internalization of the antibody into L3.6pl spheres. Tumor spheres incubated with the indicated antibodies were collected by centrifugation, fixed, and permeabilized for analysis by confocal microscopy. The antibody and nuclei were stained with anti - human IgG labeled with Alexa® 647 (red) and Hoechst 33342 (cyan), respectively. Scale bar: 10 μm. The intracellular antibody fluorescence intensity was quantified by Image J and shown in the right panel. ***P < 0.001. Figure 3D: Inhibition of L3.6pl tumor sphere formation by 3F1 / RYR - reduction in number. The number of tumor spheres (greater than 100 μm) was counted 14 days after antibody treatment (left), and an image of a representative well is shown (right). Error bars indicate the standard deviation of duplicates. *P < 0.05. Figure 3E: Inhibition of L3.6pl tumor sphere formation by 3F1 / RYR - reduction in 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) by site-specific conjugation in tumor cell lines having diverse EphA2 / ALCAM ratios according to some non-limiting embodiments of the present disclosure. The cytotoxicity of the ADCs or mixtures shown was studied in the L3.6pl (Figure 4A) and Capan-1 (Figure 4B) cell lines having a relatively high EphA2 / ALCAM ratio, and in the Panc-1 (Figure 4C) cell line having 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 antitumor efficacy of an exemplary bispecific 3F1 / RYR antibody-drug conjugate (ADC) 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 1×106 Capan-1 cells and randomly divided into four groups (6 mice / group) by 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 (mm3) was plotted. Figure 5B: Body weight was monitored and plotted to evaluate the toxicity of ADC treatment. No significant weight loss (e.g., greater than 15%) was seen in any of the groups studied.

[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 phages by three rounds of selection. The recombinant Fc fusion of the ALCAM-V domain was immobilized on magnetic beads and used 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 the polyclonal phages amplified from the output of each round was shown as the fold over the binding of the unselected phage library (right y-axis). Figure 6B: After three rounds of selection, FACS was performed to screen for monoclonal phages that bind to the ALCAMhigh DU145 cell line. Figure 6C: Apparent KD of 3F1 IgG for ALCAM-expressing live cells. DU145 cells were incubated overnight at 4 °C with various concentrations of 3F1 IgG and analyzed by FACS using Alexa (R) 647-conjugated anti-human IgG. The KD value was estimated by curve fitting using GraphPad Prism (GraphPad Software). Figure 6D: Study of the cellular localization of anti-ALCAM 3F1 IgG by confocal microscopy. Tumor cell lines were seeded on chamber slides and incubated with 3F1 IgG for 2 h at 37 °C. The antibody was stained with an Alexa (R) 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 microscopic images (lower panel).

[0040] [Figure 7]Figures 7A-7B illustrate the characterization of exemplary anti-ALCAM×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 (about 50 kDa) and a light chain (about 25 kDa). 3F1 / RYR or C10 / RYR bsIgG is composed of two similarly sized bands (about 50 kDa), a heavy chain and a light chain fused to an scFv. Figure 7A: FACS analysis of binding specificity. Bispecific antibodies 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 the removal of surface antigens according to some embodiments of the present disclosure. Figure 8A: Inadequate surface ALCAM removal in HEK293 cells lacking expression of the guide antigen EphA2. HEK293 cells were incubated with the indicated IgG or bsIgG at 37°C for 1, 4, and 24 hours, 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 diverse EphA2 to ALCAM ratios. Anti-ALCAM 3F1 IgG did not reduce surface EphA2 as predicted, while 3F1 / RYR and control C10 / RYR that 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: ratio of EphA2 to ALCAM. Data represent mean ± standard deviation (duplicate). *P<0.05, **P<0.01, and ***P<0.001. [Figure 8-2]Figures 8A - 8C illustrate the removal of surface antigens according to some embodiments of the present disclosure. Figure 8A: Inadequate surface ALCAM removal in HEK293 cells lacking expression of the guide antigen EphA2. HEK293 cells were incubated with the indicated IgG or bsIgG at 37°C for 1, 4, and 24 hours, 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 diverse EphA2 to ALCAM ratios. Anti - ALCAM 3F1 IgG did not reduce surface EphA2 as predicted, while 3F1 / RYR and control C10 / RYR that 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: Ratio of EphA2 to ALCAM. Data represent mean ± standard deviation (duplicate). *P < 0.05, **P < 0.01, and ***P < 0.001.

[0042] [Figure 9] Figure 9 illustrates the importance of the guide antigen in the cell - selective cytotoxicity of an exemplary antibody - drug conjugate (ADC) according to some embodiments of the present disclosure. In these experiments, various concentrations of the indicated ADC were incubated with HEK293 cells (ALCAMhighEphA2low, with 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 scFv from a yeast display mutagenesis library. Figure 10A: Apparent KD measurements of the binding affinity of four new EphA2 scFv 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 inferred by curve fitting of the normalized MFI values. Figure 10B: Apparent KD measurements of the binding affinity of four new EphA2 scFv to mouse recombinant EphA2 - Fc fusion protein. The apparent KD values were inferred by curve fitting of the normalized MFI values. [Figure 10B] Figures 10A - 10B illustrate the selection and characterization of anti - EphA2 scFv from a yeast display mutagenesis library. Figure 10A: Apparent KD measurements of the binding affinity of four new EphA2 scFv 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 inferred by curve fitting of the normalized MFI values. Figure 10B: Apparent KD measurements of the binding affinity of four new EphA2 scFv to mouse recombinant EphA2 - Fc fusion protein. The apparent KD values were inferred by curve fitting of the normalized MFI values.

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

[0045] [Figure 12] Figure 12 summarizes the results of an experiment conducted to evaluate the affinity of the IgG1 described in Figures 10A - 10B to recombinant human EphA2.

DETAILED DESCRIPTION OF THE INVENTION

[0046] DETAILED DESCRIPTION OF THE 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, e.g., for converting a non-internalizing cell surface antigen into an internalizing one and vice versa. For example, in some embodiments of the present disclosure, the conversion is achieved through a guide / effector system, where, when a set of conditions are met, the internalization properties of the guide antigen are imparted to the effector antigen. 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, where the internalization properties of the engineered antibody or a functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen. Recombinant cells, recombinant nucleic acids encoding such engineered antibodies, and pharmaceutical compositions containing the same are also provided. The present disclosure also provides methods useful for modulating intracellular trafficking in cells or in a subject, as well as methods for modulating cell-type selective signaling in a subject and / or for the treatment of health disorders and diseases, such as cancer-related diseases including solid tumors and hematological malignancies.

[0047] Considerable efforts have been made to use antibodies to transport highly toxic payloads to infected or cancerous cells, carry the drug inside the cells, and release it to act like a prodrug. 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 those that are expressed at uniformly high levels are even rarer. The compositions and methods disclosed herein address at least two unmet needs: (1) In targeted therapies where intracellular payload delivery is required, many tumor antigens are highly expressed but have insufficient internalization. By converting these into 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 into non-internalizing receptors, the signaling pathways can be continuously activated.

[0048] As described in more detail below, exemplary bispecific antibodies were 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 the bispecific antibodies are significantly affected by the relative surface expression levels (antigen density ratio) of EphA2 to ALCAM. When the ratio of EphA2 to ALCAM 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, and a small amount of the internalizing antigen EphA2 exhibits a bispecific-dependent amplification effect that induces the internalization of a large amount of the non-internalizing antigen ALCAM. When the ratio is below the threshold value, EphA2 can become non-internalizing due to the presence of excess ALCAM on the same cell surface. In some of the exemplary experiments described below, bispecific antibody-drug conjugates (ADCs) were constructed based on the above-described bispecific antibody design, and it has been found that the bispecific ADCs are more potent than monospecific ADCs with respect to 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 utilized for therapeutic targeting.

[0049] Definitions Unless otherwise defined, all technical terms, notations, and other scientific terms or terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein is not to be construed as necessarily representing a substantial difference from what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood by those of ordinary skill in the art and are widely 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 the present disclosure, the use of "or" means "and / or" unless otherwise indicated. Further, the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0051] The term "about," as used herein, has its ordinary meaning of approximately. Where the degree of approximation is not otherwise apparent from the context, "about" means within 10% of the provided value, or the value rounded to the nearest significant digit, including the provided value in all cases. Where ranges are provided, the boundary values are included.

[0052] When used in connection with cells, nucleic acids, proteins, or vectors, the terms "engineered" or "recombinant" indicate that the cell, nucleic acid, protein, or vector has been modified through human intervention, e.g., by laboratory methods or as a result thereof. 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 modifications, e.g., labeled amino acid residues. The term can include any modification to a peptide, protein, or nucleic acid sequence. Such modifications can include: any chemical modification of a peptide, protein, or nucleic acid sequence that includes one or more amino acids, deoxyribonucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more of the amino acids in a peptide or protein; and the addition, deletion, and / or substitution of one or more of the nucleic acids in a nucleic acid sequence. Thus, an engineered antibody refers to a recombinant polypeptide that includes at least an antigen-binding site derived from the variable domain of the heavy (VL) and / or light (VH) chain of the antibody and, optionally, may include all or part of the variable and / or constant domains of an antibody derived from any of the Ig classes (e.g., IgA, IgD, IgE, IgG, IgM, and IgY). The term "engineered" when used in reference to a cell is not intended to include naturally occurring cells but is intended to encompass cells that have been modified to contain or express a polypeptide or nucleic acid not present in unengineered cells.

[0053] As used herein, the term "functional fragment" refers to a molecule having a qualitative biological activity common to 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 shortened at the N-terminus and / or C-terminus, and the retention of its epitope-binding activity can be evaluated using assays known to those of skill in the art, including the exemplary assays provided herein.

[0054] As used herein, the term "operably linked" refers to a physical or functional linkage between two or more elements, such as polypeptide sequences or polynucleotide sequences, that enables them to operate in the 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 enables expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to the presence of a regulatory region and a coding sequence to be transcribed in a position such that the regulatory region is effective to regulate 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 positioned in an appropriate position relative to a sequence encoding a polypeptide or functional RNA such that the regulatory sequence directs or regulates the expression or cellular localization of an mRNA encoding a polypeptide, the polypeptide, and / or the functional RNA. Thus, a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence. Operably linked elements may be contiguous or non-contiguous. Additionally, in the context of a polypeptide, "operably linked" refers to a physical linkage (either directly or indirectly linked) between amino acid sequences (e.g., different fragments, regions, moieties, or domains) to provide the desired activity of the polypeptide. In the present disclosure, the various segments, regions, or domains of the engineered antibodies of the present disclosure can be operably linked to maintain the proper folding, processing, targeting, expression, binding, and other functional properties of the engineered antibodies in cells. Unless otherwise indicated, the various regions, domains, fragments, and moieties of the engineered antibodies of the present disclosure are operably linked to each other. The operably linked r regions, domains, fragments, and moieties of the engineered antibodies of the present disclosure may be contiguous or non-contiguous (e.g., they may be linked to each other via a linker).

[0055] The term "percent identity" refers to two or more sequences or subsequences that are the same or have the same specified percentage of 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 a comparison window or specified region) when measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI website, ncbi.nlm.nih.gov / BLAST. Such sequences are thus said to be "substantially identical." This definition also refers to, or may also apply to, the complement of a test sequence. This definition includes sequences having deletions and / or additions, as well as those having substitutions. Sequence identity typically exists over a region of at least about 20 amino acids or nucleotides, or over a region of 10 to 100 amino acids or nucleotides, or over the full 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 (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with its default parameters.

[0057] As used herein, 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 the 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 the overall treatment of a disease, reduces or avoids the symptoms or predisposition of a disease, or enhances the therapeutic efficacy 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 can also be referred to as a "therapeutically effective amount". "Reduction" of a symptom means a decrease in the severity or frequency of the symptom, or the elimination of the symptom. The exact amount of a composition containing a "therapeutically effective amount" depends on the purpose of the treatment and can be determined by one of ordinary skill 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, "subject" or "individual" includes animals, e.g., humans (e.g., human individuals) and non-human animals. In some embodiments, the "subject" or "individual" is a patient under the care of a physician. Thus, a subject can be a human patient or individual who has, is at risk of having, or is suspected of having a target disease (e.g., cancer) and / or one or more symptoms of the disease. A subject can also be an individual diagnosed at the time of diagnosis or later as being at risk of a target condition. 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 that can transfer or transport another nucleic acid molecule. The nucleic acid molecule being transferred is generally linked, e.g., inserted, into the vector nucleic acid molecule. Generally, a vector is replicable when associated with appropriate control elements. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integration vectors. An "expression vector" is a vector that contains regulatory regions by which DNA sequences and fragments can be expressed in vitro and / or in vivo. A vector can contain sequences that induce autonomous replication in a cell or sequences sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective 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 for transferring a gene into a cell.

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

[0061] All ranges disclosed herein also include any and all possible subranges and combinations of those subranges. It can be recognized that all recited ranges are sufficiently described and enabled to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. of the same range. By way of non-limiting example, each range discussed herein can be readily decomposed into lower thirds, middle thirds, and upper thirds, etc. Also, as will be understood by those skilled in the art, all terms such as "up to", "at least", "greater than", "less than", etc. include the recited numbers and refer to ranges that can later be decomposed into subranges as described above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 elements refers to a group having 1, 2, or 3 elements. Similarly, a group having 1 to 5 elements refers to a group having 1, 2, 3, 4, or 5 elements, and so on.

[0062] As will be understood, the aspects and embodiments of the present disclosure described herein include "comprising" the aspects and embodiments, "consisting" of them, and "consisting essentially of" them.

[0063] Headings, such as (a), (b), (i), etc., are presented only for ease of reading this specification and the claims. The use of headings in this specification or the claims does not require that 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 that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity may be provided separately or in any suitable sub-combination. All combinations of embodiments relevant to the present disclosure are specifically encompassed by the present disclosure and are disclosed herein such that every combination is individually and explicitly disclosed. In addition, all partial combinations of various embodiments and their elements are specifically encompassed by the present disclosure and are disclosed herein such that every such partial combination is individually and explicitly disclosed herein.

[0065] Intracellular Trafficking for Targeted Chemotherapy The high specificity of monoclonal antibodies is often utilized in the development of targeted chemotherapy. Ideally, by conjugating a potent cytotoxic agent to a cell-type selective antibody, the cytotoxic agent can be directed to target cells and preferentially accumulate in the target tissue. Antibody-drug conjugates (ADCs) are a class of targeted chemotherapy that has shown efficacy in the clinic. Intracellular trafficking 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 the cell membrane and cause a bystander effect.

[0066] Conceptually simple, the selection of targets in ADCs is hampered by the fact that so-called tumor-specific antigens are rarely discovered, and even more rarely are tumor-specific antigens found that have the properties desired for therapeutic targeting, i.e., are highly and uniformly expressed by cancer cells and efficiently internalize. 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 dual paratope design and the resulting crosslinking 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 compared to parental monospecific anti-HER2 ADCs, suggesting that important parameters regarding this design remain to be elucidated.

[0067] One important question is whether the internalization tendency of a given cell surface antigen can be affected by its neighboring surface antigen, and if so, what the parameters are that determine the conversion from non-internalizing to internalizing antigens and vice versa. Guide-effector bispecific antibody systems for achieving cell-type specific signal transduction modulation have been reported previously. The key to these designs is the guide-to-effector ratio and the threshold of guide antigen expression. Assume that internalization can be manipulated by a guide-effector based bispecific antibody approach. As described below, exemplary bispecific antibodies targeting the rapidly internalizing antigen EphA2 and the non-internalizing or slowly internalizing antigen ALCAM were developed. This bispecific antibody was found to be internalized when the ratio of EphA2 to ALCAM exceeded about 1:5. The bispecific effect, which is different from that of a simple mixture of two monoclonal antibodies, has been further shown to result in a greater number of bispecific molecules delivered to tumor cells than in the case of an antibody mixture. Thus, the guide-effector design can bring about an amplification effect where starting with a small number of internalizing seed antigens, the internalization effect spreads to more abundant non-internalizing antigens. Notably, when the ratio of EphA2 to ALCAM is below the threshold (1:5), the internalizing EphA2 can be made non-internalizing or slowly internalizing by ALCAM, and this conversion is shown to be reciprocal depending on the ratio of the guide to the effector antigen. Thus, when targeted by a bispecific antibody, the internalization of cell surface antigens can be readily manipulated by their neighboring antigens, resulting in either amplified intracellular uptake 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 signal transduction modulation to achieve modulation of cell type-selective internalization. Specifically, when the guide-to-effector ratio exceeds a threshold (e.g., 1:5 in the case of EphA2 / ALCAM), a non-internalizing antigen (ALCAM) can be made internalizing by the bispecific antibody. When the guide-to-effector ratio is below the threshold, an internalizing antigen (EphA2) can be made non-internalizing or slowly internalizing by the bispecific antibody. Thus, in the context of bispecific targeting, the internalization behavior of cell surface antigens is significantly affected by their neighboring antigens and can be readily manipulated in either direction by targeting appropriately selected guide / effector pairs based on bispecificity.

[0069] The present disclosure is relevant to therapeutic agent development. In the direction of converting non-internalizing antigens to internalizing antigens, the present disclosure has direct relevance to ADC development. ADCs are a class of anti-cancer agents that utilize the specificity of antibodies to deliver cytotoxic drugs to tumor cells. Although the concept is attractive, the clinical development of this class of anti-cancer agents has encountered various challenges. So far, only four ADCs have been approved by the FDA for clinical use. Initial problems, such as the stability of the drug and linker, have been addressed, but other problems remain. Very potent drugs, such as DNA chelating agents, are used in ADC production, but these drugs cause accumulation of toxicity and have a limited therapeutic window. Microtubule inhibitors, such as auristatin derivatives, are less potent compared to DNA chelating agents, and their toxicity, except for peripheral nerve damage, does not accumulate. Due to the low potency of auristatin and the limited amount of drug delivered to tumor cells, the therapeutic window remains narrow. Increasing the DAR can result in the delivery of more drug molecules to tumor cells in vitro, but in vivo, high-DAR ADCs are rapidly cleared from the circulation and thus have reduced efficacy and increased 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, the means 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 the tumor cells. The use of macropinocytosis antibodies in ADC construction to improve internalization has been reported previously, but the present disclosure provides an approach to increase target density through a guide-effector bispecific antibody design.

[0070] As a non-limiting example, a rapid internalization-type macropinocytosis anti-EphA2 (guide) antibody and a non / slow internalization-type anti-ALCAM (effector) antibody are used as a model system for studying the bispecific effect. When the antigen density ratio of EphA2 / ALCAM exceeds a threshold (e.g., 1:5 in the experimental system described herein), the bispecific anti-ALCAM×EphA2 antibody can induce the internalization of both EphA2 and ALCAM. In other words, the bispecific antibody can convert a non-internalizing antigen (ALCAM) into an internalizing antigen. The bispecific ADC is more potent than either of the monospecific ADCs and even a mixture of these ADCs in an in vitro cytotoxicity assay, and is consistent with an increase in the amount of ADC delivered and internalized by the bispecific antibody. Thus, there is an amplification effect inherent to the bispecific antibody rather than the monospecific antibody or a mixture thereof, where a small number of internalizing antigens (guide, EphA2) can induce the internalization of a large number of non-internalizing antigens (effector, ALCAM) when targeted by the bispecific antibody, resulting in a greater amount of ADC and drug molecules being delivered to tumor cells compared to monoclonal ADCs and mixtures thereof.

[0071] In addition to enhancing efficacy through amplified internalization, the compositions and methods disclosed herein affect the expansion of the range and types of cell surface targets of ADCs. A major challenge with current ADCs is how to deliver payloads specifically and in high amounts to target cells. In the case of monoclonal antibodies, the target antigen needs to be expressed specifically and uniformly at high levels on the tumor surface. In practice, however, it is rare to find antigens that have both absolute specificity and uniformly high levels of expression. Therefore, lineage markers expressed by the tissue from which the tumor derives are often used for tumor targeting. These lineage markers have two limitations: (1) Since they are not functionally required for tumor survival, they tend to show decreased or non-uniform expression in late-stage cancers. For example, the expression of PSMA in late-stage prostate cancer is non-uniform and downregulated in androgen signaling inhibitor-resistant small cell types. (2) They are often expressed in more than one normal tissue type. For example, mesothelin is expressed by several 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. In the monoclonal antibody approach, target selection is rather limited or suboptimal. In the context of ADCs, efforts have been made to increase the efficacy of the payload, but the therapeutic window remains narrow as described above. An alternative approach is to identify targets that amplify the difference in payload delivery between tumor cells and normal cells. The present disclosure is particularly relevant to this approach because the guide-effector bispecific antibody designs described herein enable a number of non-internalizing tumor-associated antigens to be internalized and thus contribute to increased intracellular delivery of ADCs. The amplification effect is specific to tumor cells and results from the co-expression of both the guide antigen and the effector antigen.

[0072] Guided-effector bispecific antibody designs for cell-type selective modulation of the Wnt signaling pathway have been reported previously (see, e.g., Lee NK et al., Sci Rep. 2018 Jan 15;8(1):766.), but the present disclosure extends the applicability of the bispecific approach to antigen internalization and ADCs. The essence of the guided-effector bispecific antibody system disclosed herein is that, when the ratio of guide to effector exceeds a threshold, the behavior of a given antigen (effector) can be shaped by an adjacent antigen (guide). In Wnt signaling studies, when the guide / effector ratio exceeds 5-10:1, there is a 1000-fold increase in the potency of the bispecific antibody compared to the monoclonal antibody, 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 (internalizing type) can convert a large number of effector antigens (non-internalizing type) into internalizing type antigens.

[0073] Notably, some of the experiments described below focus on the conversion of ADCs and non-internalizing antibodies to internalizing antibodies, but it has also been shown that the reverse is true, and when the ratio of internalizing antigen to non-internalizing antigen is below a threshold (e.g., 1:5 in the system described herein), the internalizing antigen EphA2 becomes a slow internalizer in the presence of the non-internalizing ALCAM. This can be useful in applications where it is desirable to leave the antigen on the cell surface to prevent degradation and extend signaling function.

[0074] There have been several recent reports on bispecific ADCs having an internalization arm that binds to either a lysosomal protein or an antigen that rapidly translocates to the lysosome. In most cases, the observations are empirical, the bispecific effect is rather moderate, and it is suggested that the important parameters affecting bispecific antibody-mediated internalization are not well described. For example, it is unclear whether a lysosomal antigen is required for this phenomenon. Also unclear is the reason why bispecific antibodies function in some cells and not in others. The present disclosure shows that an important variable in bispecific antibody design is the ratio to the guide effector antigen, and that the internalization arm requires no special properties other than internalization. The guide antigen (internalization arm) need not be a lysosomal protein to induce internalization and lysosomal trafficking. For example, in the present disclosure, macropinocytosis is utilized in the selection of a macropinocytosis antibody against the cell surface antigen EphA2 as a guide for directing the bispecific antibody to the lysosomal compartment.

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

[0076] The compositions of the present disclosure Engineered antibodies As described in more detail below, the present disclosure provides a new class of antibodies engineered to modulate the endocytic properties of cell surface molecules, e.g., to convert a non-endocytic cell surface antigen to an endocytic one and vice versa. For example, in some embodiments of the present disclosure, this conversion is achieved through a guide / effector system, where, when a set of conditions is met, the endocytic properties of the guide antigen are imparted to the effector antigen. In some embodiments of the present disclosure, the engineered antibodies disclosed herein can bind simultaneously to a cell type-selective endocytic 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 a functional fragment thereof comprising: a) a first antigen-binding portion capable of binding to a cell surface guide antigen having a first rate of cellular endocytosis; and b) a second antigen-binding portion capable of binding to a cell surface effector antigen having a second rate of cellular endocytosis, wherein the endocytic properties of the engineered antibody or its functional fragment are determined by the relative surface density ratio of the guide antigen to the effector antigen, and wherein one of the two rates of cellular endocytosis is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other rate. The term "endocytosis" refers to the transport of a moiety from the exterior to the interior of a cell. The endocytosed moiety can be located within an intracellular compartment. An antigen or antibody that is "endocytosed" or "endocytic" refers to an antigen or antibody that can be transported from the exterior to the interior of a target cell.

[0078] In the engineered antibodies disclosed herein, an antigen with a high intracellular translocation rate is defined as a rapid intracellular translocation antigen, and an antigen with a low intracellular translocation rate is defined as a slow intracellular translocation antigen. Thus, in some embodiments, the intracellular translocation rate of the rapid intracellular translocation antigen is at least 50%, at least 70%, at least 80%, or at least 90% higher than that of the slow intracellular translocation antigen. In some specific embodiments, if more than 50% of the antibodies bound to the surface are translocated intracellularly within 4 hours at 37°C, the antibody is referred to as a rapid intracellular translocation type. In some embodiments, if less than 30% of the antibodies bound to the surface are translocated intracellularly after 24 hours at 37°C, the antibody is referred to as a slow intracellular translocation type. In some embodiments, if less than 10% of the antibodies bound to the surface are translocated intracellularly after 24 hours at 37°C, the antibody is referred to as a non-intracellular translocation type.

[0079] One of ordinary skill in the art will understand that the process of intracellular trafficking generally refers to the movement of cell surface molecules across the plasma membrane from the cell surface into the interior of the cell. After internalization, the endosome can be transported to the lysosome for degradation or recycling to the cell surface. The rate of intracellular trafficking of a given cell surface molecule provides a measure of the kinetics of the movement of the molecule across the plasma membrane from the cell surface into the interior. The rates of internalization of antigens and antibodies can be experimentally monitored and / or measured by several techniques known in the art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305-17, 2008) and toxin killing assays (Pahara J. et al. Exp Cell Res., 316:2237-50, 2010 and Mazor et al., J. Immunol. Methods, 321:41-59, 2007). A number of antibody labeling techniques, dyes, and kits for antibody labeling that can be used to quantify and monitor internalization are commercially available (e.g., the pHrodo iFL antibody labeling methods, reagents, and kits sold by Thermo Fisher Scientific). For example, the intracellular trafficking kinetics of the antigens and engineered antibodies of the present disclosure can be evaluated and quantified by confocal microscopy or flow cytometry. For example, confocal laser scanning microscopy (CLSM) is widely used to verify intracellular trafficking. Another suitable technique, imaging flow cytometry (IFC) techniques that provide quantitative FACS data and images of cells, can also be used to quantify intracellular trafficking kinetics. Additional information on this can be found, for example, in Ha et al., Mol Cell Proteomics, 13(12):3320-31, 2014 and Vainshtein et al., Pharm. Res. 32:286-299, 2015.In some embodiments, the intracellular trafficking kinetics of the engineered antibodies of the present disclosure can be quantified by using the method previously described by Vainshtein et al. (Pharm Res. 2015, 32:286-299), which is incorporated herein by reference, in which an imaging technique by confocal microscopy is implemented to record the intracellular trafficking kinetics of fluorescently tagged antibodies in live cells, and a quantitative image analysis algorithm is used for the determination of the intracellular trafficking rate constant (K. int ) In some embodiments, the intracellular trafficking rate constant K int of the engineered antibodies disclosed herein is calculated from the time course of intracellular trafficking by curve fitting of 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,cyt are the initial cytoplasmic fluorescence signal and the maximum signal, respectively (see Vainshtein et al. 2015).

[0080] Referring to the antigen-binding portion capable of binding to the cell surface guiding antigen as the "first" antigen-binding portion and the antigen-binding portion capable of binding to the cell surface effector antigen as the "second" antigen-binding portion is not intended to indicate any specific structural arrangement of the "first" and "second" antigen-binding portions within the engineered antibody. As a non-limiting example, in some embodiments of the present disclosure, the engineered antibody may include an N-terminal portion containing an antigen-binding portion capable of binding to the cell surface guiding antigen and a C-terminal portion containing an antigen-binding portion capable of binding to the cell surface effector antigen. In other embodiments, the engineered antibody may include an N-terminal portion containing an antigen-binding portion capable of binding to the cell surface effector antigen and a C-terminal portion containing an antigen-binding portion capable of binding to the cell surface guiding antigen.

[0081] As described in more detail below, the first and / or second antigen-binding portions can be multispecific, e.g., capable of binding to 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 portion can be configured to have bispecificity, i.e., to be capable of binding to two guide antigens. In some embodiments, the second antigen-binding portion can be configured to have bispecificity, i.e., to be capable of binding to two effector antigens. Additional information regarding the design of an antibody having these two functions can be found, e.g., in Schaefer G. et al., Cancer Cell. 2011 Oct 18;20(4):472-86 and Lee C V et al., MAbs. 2014;6(3):622-627.

[0082] In addition or alternatively, the engineered antibody may include more than one antigen-binding portion capable of binding to a cell surface guide antigen and / or more than one antigen-binding portion capable of binding to a cell surface effector antigen. Thus, in some embodiments, the engineered antibody can include multiple antigen-binding portions each capable of binding to a cell surface guide antigen. In some embodiments, the engineered antibody can include multiple antigen-binding portions each capable of binding to a cell surface effector antigen. In some embodiments, the engineered antibody includes multiple antigen-binding portions each capable of binding to a cell surface guide antigen and multiple antigen-binding portions each capable of binding to a cell surface effector antigen.

[0083] According to the present disclosure, by operably linking an antigen-binding portion specific for a slowly internalizing antigen to another antigen-binding portion specific for a rapidly internalizing antigen, it is possible to induce rapid internalization of such a slowly internalizing antigen. In some embodiments, by operably linking an antigen-binding portion specific for a rapidly internalizing antigen to another antigen-binding portion specific for a slowly internalizing antigen, it is possible to induce slow internalization of such a rapidly internalizing antigen.

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

[0085] In some other embodiments, the intracellular trafficking properties of the engineered antibodies disclosed herein are converted from non-intracellular trafficking types to intracellular trafficking types. In some embodiments, the intracellular trafficking properties of non-intracellular trafficking antigens (e.g., guide antigens or effector antigens) are converted from non-intracellular trafficking types to intracellular trafficking types. In some other embodiments, the intracellular trafficking properties of non-intracellular trafficking guide antigens are converted from non-intracellular trafficking types to intracellular trafficking types by using the engineered antibodies disclosed herein, including those in which an antigen-binding moiety specific for such non-intracellular trafficking guide antigens is operably linked to another antigen-binding moiety specific for an intracellular trafficking effector antigen. In some other embodiments, the intracellular trafficking properties of non-intracellular trafficking effector antigens are converted from non-intracellular trafficking types to intracellular trafficking types by using the engineered antibodies disclosed herein, including those in which an antigen-binding moiety specific for such non-intracellular trafficking effector antigens is operably linked to another antigen-binding moiety specific for an intracellular trafficking guide antigen.

[0086] In some embodiments, the guide antigen has a higher intracellular trafficking rate than the intracellular trafficking rate of the effector antigen, in which case the guide antigen is a rapid intracellular trafficking antigen and the effector antigen is a slow intracellular trafficking antigen. In some embodiments, the guide antigen has an intracellular trafficking rate that is at least about 50% higher than the intracellular trafficking rate of the effector antigen. In some embodiments, the guide antigen has an intracellular trafficking rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the intracellular trafficking rate of the effector antigen. In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a slow intracellular trafficking antigen to another antigen-binding portion specific for a rapid intracellular trafficking antigen (e.g., a guide antigen), thereby increasing the intracellular trafficking rate of the slow intracellular trafficking antigen (e.g., an effector antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% as compared to a control (e.g., a monospecific antibody comprising only the slow intracellular trafficking antigen). In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a rapid intracellular trafficking antigen to another antigen-binding portion specific for a slow intracellular trafficking antigen (e.g., an effector antigen), thereby reducing the intracellular trafficking rate of the rapid intracellular trafficking antigen (e.g., a guide antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% as compared to a control (e.g., a monospecific antibody comprising only the rapid intracellular trafficking antigen).

[0087] In some embodiments, the effector antigen has a higher intracellular translocation rate than the intracellular translocation rate of the guide antigen, in which case the effector antigen is a rapid intracellular translocation antigen and the guide antigen is a slow intracellular translocation antigen. In some embodiments, the effector antigen has an intracellular translocation rate that is at least about 50% higher than the intracellular translocation rate of the guide antigen. In some embodiments, the effector antigen has an intracellular translocation rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the intracellular translocation rate of the guide antigen. In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a slow intracellular translocation antigen to another antigen-binding portion specific for a rapid intracellular translocation antigen (e.g., an effector antigen), thereby increasing the intracellular translocation rate of the slow intracellular translocation antigen (e.g., a guide antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% as compared to a control (e.g., a monospecific antibody comprising only the slow intracellular translocation antigen). In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a rapid intracellular translocation antigen to another antigen-binding portion specific for a slow intracellular translocation antigen (e.g., a guide antigen), thereby reducing the intracellular translocation rate of the rapid intracellular translocation antigen (e.g., an effector antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% as compared to a control (e.g., a monospecific antibody comprising only the rapid intracellular translocation antigen). In some embodiments, the cell surface guide antigen is an intracellular translocation cell surface antigen. In some embodiments, the cell surface effector antigen is a non-intracellular translocation cell surface antigen.

[0088] The intracellular trafficking properties of the engineered antibodies or functional fragments thereof disclosed herein are determined by the relative surface density ratio of the guide antigen to the effector antigen. One of ordinary skill in the art can readily understand that the surface density of a given molecule, e.g., an antigen or polypeptide, refers to the number of antigens or polypeptides measured and / or inferred on a given surface area. For example, the density of an antigen present on the cell surface can be expressed as approximately 10,000 copies per cell, which means that the number of antigen molecules present on the cell surface, as measured and / or inferred, is approximately 10,000. Numerous techniques, systems, assays, and procedures for determining and / or measuring the density of molecules present on the cell surface are known in the art. Additional information regarding 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 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. The decision rule can be that any score above a given threshold indicates intracellular trafficking of the engineered antibody, while a score below the threshold indicates the absence of intracellular trafficking, e.g., non-intracellular trafficking.

[0089] In some embodiments, these scores can be compared to a threshold, and a score above the threshold indicates an increase or decrease in intracellular trafficking shown 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 derived from both intracellular trafficking and non-intracellular trafficking antigens and separating them using a linear model. The linear model can be generated by a statistical method, e.g., logistic regression or support vector machine using a linear kernel function, or the linear model can be generated by experimentation.

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

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

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

[0093] As used herein, the term "antigen-binding site" refers to a part of an antigen-binding moiety that is responsible for the specific binding between the antigen-binding moiety and an antigenic determinant. The antigen-binding site can be a single domain, such as an epitope-binding domain, or it can be a paired VH / VL domain such as found in a canonical antibody. Thus, in some embodiments, the antigen-binding site of an antibody or fragment thereof described herein is formed by the amino acid residues of the N-terminal variable regions of the heavy chain (VH) and the light chain (VL). Generally, the variable regions of VH and VL each contain three hypervariable regions, referred to as complementarity-determining regions (CDRs). The three CDRs of VH (referred to as HCDR1, HCDR2, and HCDR3) and the three CDRs of VL (referred to as LCDR1, LCDR2, and LCDR3) are arranged three-dimensionally 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). Any suitable numbering system can be used for the designated CDR regions, but in the absence of any other indication, 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] The binding of each of the first and second antigen-binding portions to its respective target can be in either a competitive or non-competitive manner with the natural ligand of the target. Thus, in some embodiments of the present disclosure, the binding of the first and / or second antigen-binding portion to its respective target can be of the ligand-blocking type. In some other embodiments, the binding of the first and / or second antigen-binding portion to its respective target does not block the 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 portion, which is linked to a second amino acid sequence encoding a second antigen-binding portion that is not naturally linked. The amino acid sequences can be present in separate proteins that come together in a fusion polypeptide, or they can be present in the same protein but placed in a new arrangement in the fusion polypeptide. The amino acid sequences encoding the first and second antigen portions can be made, for example, by chemical synthesis or by making a polynucleotide in which the peptide regions are encoded in the desired relationship and translating it.

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

[0096] In some embodiments, the first antigen-binding portion is operably linked to the second antigen-binding portion via a linker. There is no particular limitation on the linker that can be used in the engineered antibodies described herein. In some embodiments, the linker is a synthetic compound linker, such as a chemical crosslinking agent. Non-limiting examples of suitable crosslinking agents 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), dithiobis(succinimidyl tartrate) (DST), dithiobis(sulfosuccinimidyl 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 portion is operably linked to the second antigen-binding portion via a linker peptide sequence. In principle, there are no particular restrictions on the length and / or amino acid composition of the linker peptide sequence. In some embodiments, any single-stranded peptide containing from 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 the peptide linker. In some embodiments, the linker peptide sequence contains from about 5 to about 50, from about 10 to about 60, from about 20 to about 70, from about 30 to about 80, from about 40 to about 90, from about 50 to about 100, from about 60 to about 80, from about 70 to about 100, from about 30 to about 60, from about 20 to about 80, from about 30 to about 90 amino acid residues. In some embodiments, the linker peptide sequence contains from about 1 to about 10, from about 5 to about 15, from about 10 to about 20, from about 15 to about 25, from about 20 to about 40, from about 30 to about 50, from about 40 to about 60, from about 50 to about 70 amino acid residues. In some embodiments, the linker peptide sequence contains from about 40 to about 70, from about 50 to about 80, from about 60 to about 80 (about 60 to 8 about 0), from about 70 to about 90, or from about 80 to about 100 amino acid residues. In some embodiments, the linker peptide sequence contains from about 1 to about 10, from about 5 to about 15, from about 10 to about 20, from 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 achieve the desired activity of the engineered antibody in which the orientation and / or proximity of the first and second antigen-binding portions relative to each other are varied. In some embodiments, the orientation and / or proximity of the first and second antigen-binding portions 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 portions relative to each other can be optimized to create a competitive, partially competitive, or non-competitive version of the engineered antibody. In certain embodiments, the linker comprises only glycine and / or serine residues (e.g., a glycine-serine linker).

[0099] antigen In some embodiments, the engineered antibodies of the present disclosure, such as bispecific antibodies, can have binding specificities for two distinct cell surface antigens, wherein one of the two antigens has a more rapid internalization rate than the other. The bispecific antibody can comprise at least two components, namely a first component and a second component, each of which binds to its respective antigen, e.g., a first cell type-related antigen (guide antigen) and a second antigen (effector antigen) associated with the target signaling pathway, respectively. The first component can comprise a first antigen-binding portion for the first antigen, and the second component can comprise a second antigen-binding portion for the second antigen. Such bispecific antibodies enable an increase in the ability to inhibit the target signaling pathway compared to non-targeted antibodies (e.g., antibodies that do not have binding specificity for the effector antigen), and importantly, enable 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), 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), Folic Acid 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), Folic Acid-Binding Protein (Folic Acid 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-Bound (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 Signal Transducer 2 (Trop-2), and Tumor-Associated Glycoprotein 72 (TAG-72). In some embodiments, the cell surface antigens include ICAM-1, EphA2, and ALCAM.

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

[0102] In some embodiments, the guide antigen is a cancer-related antigen. Non-limiting examples of cancer-related 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, PDGFR-beta, SSEA-4, CD20, MUC1, MUC16, EGFR, ErbB2, ErbB3, ErbB4, NCAM, prostate acid phosphatase (PAP), ephrin B2, 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 antibody or functional fragment thereof disclosed herein comprises 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 related antigen (e.g., a guide antigen), the engineered antibodies of the present disclosure can be recruited to target cells associated with the guide antigen, and as a result, for example, modulate a signaling pathway in the target cells. In some embodiments, the guide antigen of the engineered antibodies of the present disclosure not only functions as a cell-type selection agent but also functions as a potency enhancer, resulting in, for example, a potent and selective inhibition of the target signaling pathway. In some embodiments, the guide antigen on the surface of the target cells has an expression threshold that (1) results in the binding affinity of the engineered antibody for the target cells and (2) enhances the occupancy of the effector antigen by the engineered antibody.

[0104] effector antigen In some embodiments, the effector antigen recognized by the engineered antibodies described herein is a molecule associated with cell activity or function, such as a molecule associated with a signaling pathway. In some embodiments, the effector antigen is expressed on the surface of the cell of interest. In some embodiments, the effector antigen recognized by the engineered antibodies described herein is a molecule associated with a signaling pathway of interest (e.g., a target signaling pathway). In some cases, the effector antigen includes 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, P-selectin. Further examples of suitable effector antigens are further described below and include EGFR, ErbB2, ErbB3, and ErbB4. In some embodiments, the engineered antibody or a functional fragment thereof disclosed herein includes 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 is above 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 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 disclosed herein. In some embodiments, the engineered antibody or functional fragment thereof described herein comprises 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 antibody or functional fragment thereof described herein comprises 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 antibody or functional fragment thereof described herein comprises an amino acid sequence having 100% sequence identity to any one of the amino acid sequences identified in Table 4. In some embodiments, the 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 substituted by different amino acid residues.

[0107] The first antigen-binding portion As outlined above, various embodiments and aspects of the present disclosure include engineered antibodies that include a first antigen-binding portion capable of binding to a cell surface guiding 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 the VH sequences 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 the VH sequences 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 by different amino acid residues. 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 by different amino acid residues. 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 substituted by different amino acid residues.

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

[0109] In some embodiments, the first antigen-binding portion comprises a variable light (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 the VL sequences 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 with 100% sequence identity to the VL sequences 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 different amino acid residues. 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 different amino acid residues. 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 different amino acid residues.

[0110] In some embodiments, the VL region of the first antigen-binding portion comprises three CDRs (e.g., LCDR1, LCDR2, and LCDR3) identified in each of the VL sequences disclosed in the Sequence Listing. In some embodiments, the LCDR1 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 107. In some embodiments, the LCDR2 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 108. In some embodiments, the LCDR3 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 109. In some embodiments, the LCDR1, LCDR2, and LCDR3 of the VL region of the first antigen-binding portion each 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 portion comprises a VL region having an amino acid sequence corresponding to any one of the VL sequences identified in Table 4, wherein 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence are substituted with different amino acid residues. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 82, wherein 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence of SEQ ID NO: 82 are substituted with different amino acid residues. In some embodiments, the first antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 97, wherein 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence of SEQ ID NO: 97 are substituted with different amino acid residues.

[0111] In some embodiments, the VL region of the first antigen-binding portion comprises 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, the LCDR1 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 107, wherein one, two, three, four, or five of the amino acid residues within SEQ ID NO: 107 are substituted with different amino acid residues. In some embodiments, the LCDR2 of the first antigen-binding portion comprises the sequence of SEQ ID NO: 108, wherein one, two, three, four, or five of the amino acid residues within 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 within SEQ ID NO: 109 are substituted with different amino acid residues.

[0112] The second antigen-binding portion As outlined above, various embodiments and aspects of the present disclosure include engineered antibodies that include 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 the VH sequences 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 the VH sequences 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 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence are substituted with different amino acid residues. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence corresponding to SEQ ID NO: 73, wherein 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence of SEQ ID NO: 73 are substituted with different amino acid residues. In some embodiments, the second antigen-binding portion comprises a VH region having an amino acid sequence corresponding to SEQ ID NO: 75, wherein 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence of SEQ ID NO: 75 are substituted with different amino acid residues.

[0113] In some embodiments, the VH region of the second antigen-binding portion comprises three CDRs (e.g., HCDR1, HCDR2, and HCDR3) identified in each of the VH sequences disclosed in the Sequence Listing. In some embodiments, the HCDR1 of the second antigen-binding portion comprises the sequence of SEQ ID NO: 98. In some embodiments, the HCDR2 of the second antigen-binding portion comprises the sequence of SEQ ID NO: 99. In some embodiments, the HCDR3 of the second antigen-binding portion comprises the sequence of SEQ ID NO: 106 or the sequence of SEQ ID NO: 100. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the VH region of the first antigen-binding portion each 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 portion 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, the HCDR1 of the second antigen-binding portion comprises the sequence of SEQ ID NO: 98, wherein one, two, three, four, or five of the amino acid residues within SEQ ID NO: 98 are substituted with different amino acid residues. In some embodiments, the HCDR2 of the second antigen-binding portion comprises the sequence of SEQ ID NO: 99, wherein one, two, three, four, or five of the amino acid residues within SEQ ID NO: 99 are substituted with different amino acid residues. In some embodiments, the HCDR3 of the second antigen-binding portion comprises the sequence of SEQ ID NO: 100, wherein one, two, three, four, or five of the amino acid residues within SEQ ID NO: 100 are substituted with 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 the VL sequences 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 the VL sequences 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 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence are substituted with different amino acid residues. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 74, wherein 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence of SEQ ID NO: 74 are substituted with different amino acid residues. In some embodiments, the second antigen-binding portion comprises a VL region having an amino acid sequence corresponding to SEQ ID NO: 76, wherein 1, 2, 3, 4, or 5 of the amino acid residues within the amino acid sequence of SEQ ID NO: 76 are substituted with different amino acid residues.

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

[0116] Target portion In some embodiments of the present disclosure, the antibody or a functional fragment thereof is conjugated or covalently bound to at least one moiety of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a moiety that improves pharmacokinetics. 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 antibacterial agent, an antimicrobial agent, an antibiotic, an anti-infectious disease agent, and an antiviral agent. In some embodiments, the at least one MOI is selected from the group consisting of a cytotoxic anti-cancer agent, a DNA chelating agent, 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 anti-cancer agent is selected from the group consisting of auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, anthracycline, calicheamicin, camptothecin, irinotecan, SN-38, combretastatin, duocarmycin, enediyne, epothilone, ethyleneimine, mitomycin, pyrrolobenzodiazepine (PBD), and calicheamycin.

[0118] In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the constant region of an engineered antibody or a 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 an antibody or a functional fragment thereof. In some embodiments, at least one moiety of interest (MOI) is conjugated or covalently attached to the CH1 region of an antibody or a 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 an antibody or a functional fragment thereof. In principle, there is no particular limitation on 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 number of MOIs per antibody (i.e., average drug-to-antibody ratio, DAR) in the range of 1 to 20. In some embodiments, the engineered antibodies of the present disclosure have an average number of MOIs per antibody in the range of 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] One of ordinary skill in the art will understand that a reverse-translated gene can be constructed using the complete amino acid sequence of the engineered antibodies disclosed herein. For example, a DNA oligomer containing the nucleotide sequence encoding a given antibody can be synthesized. For example, a plurality of small oligonucleotides encoding portions of the desired antibody can be synthesized and then ligated together. The individual oligonucleotides typically include 5' or 3' overhangs for complementary assembly.

[0120] In addition to generating engineered antibodies engineered through the expression of nucleic acid molecules altered by recombinant molecular biology techniques, the engineered antibodies or functional fragments thereof according to the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those skilled 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 the expression of the engineered antibody or functional fragment thereof in the desired transformed host. Appropriate 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, in order to obtain high expression levels 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 the engineered antibodies or functional fragments thereof of the present disclosure can be assayed by any suitable method known in the art. An antibody or polypeptide that "binds preferentially" or "binds specifically" (used interchangeably herein) to a target antigen or target epitope is a term well understood in the art, and methods for determining such specific or preferential binding are also known in the art. An antibody or polypeptide exhibits "specific binding" or "preferential binding" when it reacts or associates with a particular antigen or epitope more frequently, more rapidly, for a longer period of time, and / or with a higher affinity than with alternative antigens or epitopes. An antibody or polypeptide "binds specifically" or "binds preferentially" to a target when it binds with a higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other substances. Also, an antibody or polypeptide "binds specifically" or "binds preferentially" to a target when it binds with a higher affinity, avidity, more readily, and / or for a longer period of time to the target in a sample than it binds to other substances present in the sample. For example, an antibody or polypeptide that binds specifically or preferentially to an EphA2 epitope is an antibody or polypeptide that binds to this epitope with a higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other EphA2 epitopes or non-EphA2 epitopes. By reading this definition, it is also understood that, for example, an antibody or polypeptide (or portion or epitope) that binds specifically or preferentially to a first target may or may not bind if it also binds specifically or preferentially to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but may include) exclusive binding.

[0123] Using various assay formats, antibodies or polypeptides that specifically bind to a molecule of interest can be selected. For example, among the numerous assays that can be used to identify antibodies that specifically react with an antigen or its ligand-binding portion and specifically bind to a homologous ligand or binding partner, particularly 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. Generally, a specific or selective reaction is at least 2-fold, more typically greater than 10-fold, even more typically greater than 50-fold, more typically greater than 100-fold, still more typically greater than 500-fold, even more typically greater than 1000-fold, and even more typically greater than 10,000-fold the background signal or noise. Also, in some embodiments, an antibody is said to "specifically bind" to an antigen if the 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 the non-covalent interaction between two molecules, e.g., between an antibody or a portion thereof and an antigen. The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity). The binding affinity between two molecules can be quantified by determination of the dissociation constant (K D ). Next, K D can be determined, for example, using a surface plasmon resonance (SPR) method (Biacore) by measuring the kinetics of complex formation and dissociation. The rate constants corresponding to the binding and dissociation of a monovalent complex are the association rate constant k a (or k on ) and the dissociation rate constant kd (or k off ) is called. 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 target antigens 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 evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), for example, using the 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 that include 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 non-conventional nucleotides or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence" when used interchangeably herein refer to the sequence of a polynucleotide molecule. The nomenclature of nucleotide bases described in 37 CFR §1.822 is used herein.

[0127] The nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, which generally include nucleic acid molecules of 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., nucleic acid molecules of 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] As used herein, the term "recombinant" nucleic acid molecule refers to a nucleic acid molecule that has been altered through human intervention. By way of non-limiting example, cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been produced by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been incorporated into a vector, such as a cloning or expression vector. By way of non-limiting example, recombinant nucleic acid molecules include those that have been: 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques or recombination of nucleic acid molecules; 2) joined to nucleotide sequences that are not joined in nature; 3) engineered using molecular cloning techniques such that one or more nucleotides are absent as compared to a naturally occurring nucleic acid molecule sequence; and / or 4) engineered using molecular cloning techniques such that they have one or more sequence changes or rearrangements as compared to a naturally occurring nucleic acid sequence.

[0129] In some embodiments disclosed herein, the nucleic acid molecules of the present disclosure comprise 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 the engineered antibodies disclosed herein. In some embodiments, the nucleic acid molecules of the present disclosure comprise 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, the nucleic acid molecules of the present disclosure comprise 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, the nucleic acid molecules of the present disclosure comprise 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 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 engineered antibodies disclosed herein. As used herein, the term "expression cassette" refers to a construct of genetic material that includes a coding sequence, as well as regulatory information sufficient to direct proper transcription and / or translation of the coding sequence in recipient cells, 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. Thus, the term "expression cassette" can be used interchangeably with the term "expression construct." As used herein, the term "construct" refers to any recombinant nucleic acid molecule, such as 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, that is derived from any source, is capable of genomic integration or autonomous replication, and has 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 that include one or more of the nucleic acid molecules encoding any of the engineered antibodies disclosed herein. The nucleic acid molecules described above can be included, for example, in a vector that can induce its expression in a cell into which the vector has been transformed / transduced. Vectors suitable for use in eukaryotic and prokaryotic cells are known in the art, commercially available, or can be readily prepared by those of skill in the art. Additional vectors can also be found, for example, in Ausubel, F. M., 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 will function equally well to express the DNA sequences described herein. Nor will all hosts function equally well in the same expression system. However, one of ordinary skill in the art can make a selection among these vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host must be considered because the vector must be replicated 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 the DNA encoding the engineered antibodies of the present disclosure to be amplified in copy number units. Such amplifiable vectors are known in the art.

[0133] Thus, in some embodiments, the engineered antibodies described herein can be expressed from a vector, such as an expression vector. The vector can be 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 being replicated with the host genome (e.g., a non-episomal mammalian vector). An expression vector can induce the expression of a coding sequence to which it is operably linked. In general, expression vectors useful in recombinant DNA techniques are often in the form of a plasmid (vector). 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 can be selected based on the host cell 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 by 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 sequences encoding the engineered antibodies of the present disclosure can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to the average level of a given cell host, calculated with reference to known genes expressed in the host cell. Methods for codon optimization are known in the art. The codon usage frequency within the coding sequences of the engineered antibodies disclosed herein can be optimized to enhance expression in the 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 particular host cell.

[0136] Vectors suitable for use include T7-based vectors for use in bacteria, the pMSXND expression vector for use in mammalian cells, and baculovirus-derived vectors for use in insect cells. In some embodiments, the nucleic acid insert encoding the engineered antibody in such vectors can 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 in particular, its compatibility with the actual DNA sequence encoding the polypeptide of interest with respect to potential secondary structure. The host should be selected considering their compatibility with the selected vector, the toxicity of the product encoded by the DNA sequences of the present disclosure, their secretion characteristics, their ability to correctly fold the polypeptide, their fermentation or culture requirements, and the ease of purification of the product encoded by the DNA sequence.

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

[0139] The selection of the expression control sequence and the expression vector depends, in some embodiments, on the selection of the host. A wide range of expression host / vector combinations can be utilized. Non-limiting examples of expression vectors useful for eukaryotic hosts include, for example, vectors having 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, such as plasmids derived from E. coli, including col El, pCRI, pER32z, pMB9, and their derivatives, broad-host-range plasmids, such as RP4, phage DNA, such as phage lambda, such as NM989, and many derivatives of other DNA phages, such as 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 pVL 941 and pFastBac™ 1.

[0140] In addition to the sequences that promote transcription of the inserted nucleic acid molecule, the vector may contain an origin of replication and other genes encoding selectable markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to the cells in which it is expressed, thereby enabling phenotypic selection of transfected cells. One of ordinary skill 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, and adeno-associated viral vectors, lentiviral vectors, herpesviruses, simian virus 40 (SV40), and bovine papillomavirus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). In some embodiments, the vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0142] Recombinant prokaryotic or eukaryotic cells that contain an engineered antibody or a functional fragment thereof disclosed herein and / or a nucleic acid molecule encoding any one of the engineered antibodies or functional fragments thereof disclosed herein and express the same are also a feature of the present disclosure. In some embodiments, the recombinant cells of the present disclosure are transfected cells, e.g., cells 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. The progeny of such cells are also considered to be within the scope of the present disclosure. Cell cultures containing at least one recombinant cell disclosed herein are also within the scope of the present disclosure. The terms "cell", "cell culture", "cell line", "recombinant cell", "recipient cell", and "host cell" as used herein include primary subject cells and any progeny thereof, regardless of the number of transfers. Not all progeny are identical to the parental cells (due to intentional or unintentional mutations or differences in the environment), however, such altered progeny are included in these terms as long as the progeny retain the same functionality as the original transformed cells.

[0143] The exact composition of the expression system is not critical. For example, the engineered antibodies disclosed herein can be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell (e.g., Sf21 cells), or a mammalian cell (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 only important that the components are compatible with each other. One of ordinary skill in the art can make such a determination. Further, if guidance is needed in selecting an expression system, one of ordinary skill in the art may refer to Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

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

[0145] In some embodiments, the resulting engineered antibody is glycosylated or not glycosylated depending on the host organism used to produce the engineered antibody. When bacteria are selected as the host, the resulting engineered antibody is not glycosylated. On the other hand, eukaryotic cells glycosylate the engineered antibody, although perhaps not in the same manner as native polypeptides are glycosylated. The recombinant antibodies produced by the transformed host can be purified according to any suitable method known in the art. The produced recombinant antibodies can be isolated from inclusion bodies produced in bacteria, e.g., E. coli, or from the conditioned media 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] In addition to, or alternatively, another exemplary method of constructing a DNA sequence encoding an engineered antibody of the present disclosure is by chemical synthesis. This includes the direct synthesis of peptides by chemical means of the amino acid sequences encoding engineered antibodies that exhibit the described characteristics. This method can incorporate both natural and non-natural amino acids at positions that affect the binding affinity of the engineered antibody to its target protein. Alternatively, the gene encoding the desired engineered antibody can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are generally designed by selecting codons preferred by the host cell in which the engineered antibody of the present disclosure is to be produced, based on the amino acid sequence of the desired engineered antibody. 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, TTC or TTT, Tyr (Y) is encoded by TAC or TAT, His (H) is encoded by CAC or CAT. Trp (W) is encoded by a single codon, TGG. Thus, one of ordinary skill in the art will understand that for a given DNA sequence encoding a particular engineered antibody, there are a number of DNA degenerate sequences encoding that engineered antibody. For example, in addition to the DNA sequences of the engineered antibodies provided herein, it is understood that there are a number of degenerate DNA sequences encoding the engineered antibodies disclosed herein. These degenerate DNA sequences are considered to be within the scope of the present disclosure. Thus, in the context of the present disclosure, "its degenerate variant" means all DNA sequences that encode a particular engineered antibody and thereby enable its expression.

[0147] The DNA sequence encoding the engineered antibody of the subject matter may include a DNA sequence encoding a signal sequence, regardless of whether it is prepared by site-directed mutagenesis, chemical synthesis, or other methods. Such a signal sequence, if present, needs to be recognized by the cells selected for the expression of the engineered antibody. It can be of prokaryotic origin, eukaryotic origin, or a combination of the two. Generally, whether to include a signal sequence depends on whether it is desired to secrete the engineered antibody disclosed herein from the recombinant cells in which it is made. If the selected cells are of prokaryotic origin, the DNA sequence generally does not encode a signal sequence. If the selected cells are of eukaryotic origin, a signal sequence is generally included.

[0148] The provided nucleic acid molecules may contain naturally occurring sequences or may be different from naturally occurring sequences but, due to the degeneracy of the genetic code, may contain sequences encoding the same polypeptide, e.g., an antibody. These nucleic acid molecules can consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modified forms of nucleotides within these types of nucleic acids. Additionally, the nucleic acid molecules can be double-stranded or single-stranded (e.g., either the sense strand or the antisense strand).

[0149] The nucleic acid molecules are not limited to sequences encoding polypeptides (e.g., antibodies) and may also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence of the engineered antibody). Those skilled in the art of molecular biology are proficient in routine techniques for isolating nucleic acid molecules. They can be generated, 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 their native state. Accordingly, the present disclosure encompasses recombinant molecules, e.g., nucleic acid sequences (e.g., sequences encoding engineered antibodies disclosed herein) that are incorporated into a vector (e.g., a plasmid or viral vector) or the genome of a heterologous cell (or the genome of a homologous cell at a location different from its native 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 that include a pharmaceutical composition. Such compositions generally include 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., anti-cancer agents) can also be incorporated into the compositions.

[0152] Suitable pharmaceutical compositions for use in injection include sterile aqueous solutions (in the case of water-soluble substances) or dispersion solutions, and sterile powders (sterile powder) for the immediate preparation of sterile injection solutions or dispersion solutions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. The composition needs to be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersion solutions, by maintaining the required particle size, and by the use of surfactants such as sodium dodecyl sulfate. Protection from the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, one or more isotonic agents such as saccharides, polyhydric alcohols such as mannitol, sorbitol, and sodium chloride are included in the composition. Sustained absorption of the injection composition can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0153] The sterile injectable solutions can be prepared by incorporating the active compound in the required amounts into a suitable solvent, with one or a combination of the above-mentioned ingredients, and, if necessary, subsequently filtering and sterilizing. Generally, the dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the other necessary ingredients other than those mentioned above. In the case of sterile powders and powders for the preparation of sterile injectable solutions, an exemplary method of preparation is to obtain a powder, which is the active ingredient with any additional desired ingredients added thereto, from their previously sterile-filtered solutions by means of vacuum drying and lyophilization.

[0154] Oral compositions, when used, generally contain an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound (for example, the engineered antibodies and / or nucleic acid molecules of the present disclosure) can be incorporated with excipients and used in the form of tablets, troches, or capsules, such as 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 similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel (trademark), or corn starch; lubricants, such as magnesium stearate or Sterotes (trademark); glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavor.

[0155] In the case of administration by inhalation, the engineered antibodies of the subject disclosure are delivered in the form of an aerosol spray from a suitable propellant, such as a pressurized container or dispenser containing a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0156] Systemic administration of the engineered antibodies of the subject disclosure can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, suitable penetration enhancers are used in the formulation to the barrier to be permeated. Such penetration enhancers are generally known in the art and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated in ointments, plasters, gels, or creams, as generally known in the art.

[0157] In some embodiments, the engineered antibodies of the subject disclosure can also be prepared in the form of suppositories (using, for example, 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 subject 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. (Nature Biotechnol. 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 present disclosure are prepared using carriers that protect the engineered antibodies from rapid in vitro clearance, such as controlled release formulations that include implants and microencapsulation delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells by monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those of skill 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 may be further modified to extend their half-lives in vivo and / or ex 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"), to 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 a stable protein such as albumin. For example, human albumin is known as one of the most effective proteins for enhancing the stability of polypeptides fused thereto, and numerous such fusion proteins have been reported.

[0161] In some embodiments, the engineered antibodies of the present disclosure are chemically modified using one or more polyethylene glycol moieties, e.g., are PEGylated or modified similarly, e.g., PASylated. In some embodiments, the PEG or PAS molecule is conjugated to one or more amino acid side chains of the interferon. In some embodiments, the PEGylated or PASylated antibody contains a PEG or PAS moiety on only one amino acid. In other embodiments, the PEGylated or PASylated antibody contains a PEG or PAS moiety on two or more amino acids, e.g., is 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 5000 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 directly linked to PEG or PAS (e.g., without using a linker) through an amino group, sulfhydryl group, hydroxyl group, or carboxyl group.

[0162] In some embodiments, the pharmaceutical composition of the present disclosure comprises 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 is attached. PEG or PEG derivatives of a certain size range, 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 various chemical reactions. In some embodiments, the PEGylation reagent is selected from methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butyrate (mPEG-SBA), mPEG-succinimidyl succinate (mPEG-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG-succinimidyl glutarate (mPEG-SG), mPEG-N-hydroxyl-succinimide (mPEG-NHS), mPEG-tresylate, and mPEG-aldehyde. In some embodiments, the PEGylation reagent is methoxypolyethylene glycol-succinimidyl propionate, for example, the PEGylation reagent is methoxypolyethylene glycol-succinimidyl propionate 5000 with an average molecular weight of 5,000 Daltons.

[0163] The method of the present disclosure Methods for modulating intracellular trafficking and for modulating cell type-selective signaling 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 the same are used to modulate the intracellular trafficking of cell surface molecules. The term "modulate" refers to decreasing, reducing, inhibiting, increasing, inducing, activating, or otherwise affecting the intracellular trafficking of cell surface molecules.

[0164] In one aspect, some embodiments of the present disclosure are methods for modulating intracellular trafficking, the method comprising administering to a cell one or more of the following: (a) an engineered antibody or a 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 are methods for modulating intracellular trafficking, the method comprising administering an engineered antibody or a functional fragment thereof comprising: (a) a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first intracellular trafficking rate, and (b) a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second intracellular trafficking rate, wherein the intracellular trafficking characteristics of the engineered antibody or the functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen, and one of the two intracellular trafficking 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 will be discussed in more detail below, some embodiments of the present disclosure relate to methods for treating a healthy condition or a disease (e.g., cancer) in a subject using an engineered antibody or a conjugate thereof disclosed herein.

[0167] According to the methods disclosed herein, it is possible to induce rapid internalization of such slowly internalizing antigens 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 such rapidly internalizing antigens 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 characteristics of the engineered antibody are converted from an internalizing type to a non-internalizing type. In some embodiments, the internalization characteristics of the guide antigen and / or effector antigen are converted from an internalizing type to a non-internalizing type. In some embodiments, the internalization characteristics of an internalizing antigen (e.g., a guide antigen or an effector antigen) are converted from an internalizing type to a non-internalizing type by using an engineered antibody disclosed herein, which 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 of the disclosed methods, the internalization characteristics of an internalizing guide antigen are converted from an internalizing type to a non-internalizing type by using an engineered antibody disclosed herein, which 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 characteristics of an internalizing effector antigen are converted from an internalizing type to a non-internalizing type by using an engineered antibody disclosed herein, which comprises an antigen-binding portion specific for the internalizing effector antigen operably linked to another antigen-binding portion specific for a non-internalizing guide antigen. In some embodiments of the disclosed methods, the internalization characteristics of the engineered antibody are converted from a non-internalizing type to an internalizing type. In some embodiments, the internalization characteristics of a non-internalizing antigen (e.g., a guide antigen or an effector antigen) are converted from a non-internalizing type to an internalizing type.In some other embodiments, the intracellular trafficking properties of a non-intracellular trafficking guide antigen are converted from non-intracellular trafficking to intracellular trafficking by using an engineered antibody disclosed herein, including those in which an antigen-binding portion specific for such a non-intracellular trafficking guide antigen is operably linked to another antigen-binding portion specific for an intracellular trafficking effector antigen. In some other embodiments, the intracellular trafficking properties of a non-intracellular trafficking effector antigen are converted from non-intracellular trafficking to intracellular trafficking by using an engineered antibody disclosed herein, including those in which an antigen-binding portion specific for such a non-intracellular trafficking effector antigen is operably linked to another antigen-binding portion specific for an intracellular trafficking guide antigen.

[0168] In some embodiments, the guide antigen has an intracellular translocation rate that is higher than the intracellular translocation rate of the effector antigen, in which case the guide antigen is a rapid intracellular translocation antigen and the effector antigen is a slow intracellular translocation antigen. In some embodiments, the guide antigen has an intracellular translocation rate that is at least about 50% higher than the intracellular translocation rate of the effector antigen. In some embodiments, the guide antigen has an intracellular translocation rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the intracellular translocation rate of the effector antigen. In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a slow intracellular translocation antigen to another antigen-binding portion specific for a rapid intracellular translocation antigen (e.g., a guide antigen), thereby increasing the intracellular translocation rate of the slow intracellular translocation antigen (e.g., an effector antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% as compared to a control (e.g., a monospecific antibody containing only the slow intracellular translocation antigen). In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a rapid intracellular translocation antigen to another antigen-binding portion specific for a slow intracellular translocation antigen (e.g., an effector antigen), thereby reducing the intracellular translocation rate of the rapid intracellular translocation antigen (e.g., a guide antigen) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% as compared to a control (e.g., a monospecific antibody containing only the rapid intracellular translocation antigen).

[0169] In some embodiments, the effector antigen has a higher intracellular translocation rate than the intracellular translocation rate of the guide antigen, in which case the effector antigen is a rapid intracellular translocation antigen and the guide antigen is a slow intracellular translocation antigen. In some embodiments, the effector antigen has an intracellular translocation rate that is at least about 50% higher than the intracellular translocation rate of the guide antigen. In some embodiments, the effector antigen has an intracellular translocation rate that is at least about 50%, 60%, 70%, 80%, or 90% higher than the intracellular translocation rate of the guide antigen. In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a slow intracellular translocation antigen to another antigen-binding portion specific for a rapid intracellular translocation antigen (e.g., an effector antigen), thereby increasing the intracellular translocation rate of the slow intracellular translocation 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 the slow intracellular translocation antigen). In some embodiments, the engineered antibody of the present disclosure operably links an antigen-binding portion specific for a rapid intracellular translocation antigen to another antigen-binding portion specific for a slow intracellular translocation antigen (e.g., a guide antigen), thereby reducing the intracellular translocation rate of the rapid intracellular translocation 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 rapid intracellular translocation antigen).

[0170] 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. In some embodiments, the relative surface density ratio of the guide antigen to the 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 the guide antigen to the effector antigen is above about 1:1, above about 1:2, above about 1:3, above about 1:4, above about 1:5, above about 1:10, above about 1:20, or above about 1:30. In some particular embodiments, the relative surface density ratio of the guide antigen to the effector antigen is above about 1:5. In some embodiments, the relative surface density ratio of the guide antigen to the 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 the guide antigen to the effector antigen is below about 1:1, below about 1:2, below about 1:3, below about 1:4, below about 1:5, below about 1:10, below about 1:20, or below about 1:30. In some particular embodiments, the relative surface density ratio of the guide antigen to the effector antigen is below about 1:5.

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

[0172] In some embodiments, the method of the present disclosure further comprises modulating the cell surface density of the guide antigen and / or the cell surface density of the effector antigen. One of ordinary skill in the art can readily understand that it is possible to modulate the guide-to-effector ratio by using techniques known in the art for modulating the expression and / or function of a target gene or target protein. Non-limiting examples of such techniques include gene silencing, small interfering RNA, partial gene knockout, small molecules or proteins / peptides having signal transduction functions that alter cell metabolism, proliferation, migration, death, aging, differentiation, and immune regulation.

[0173] In yet another aspect, some embodiments of the present disclosure are methods for modulating cell-type selective signaling in a subject, the method comprising administering to the cell: (a) a first antigen-binding portion 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 intracellular trafficking rate, and (b) a second antigen-binding portion capable of binding to a cell surface effector antigen having a second intracellular trafficking rate, wherein the intracellular trafficking characteristics 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 intracellular trafficking rates is at least 50%, at least 70%, at least 80%, or at least 90% higher than the other. In some embodiments, the engineered antibodies described herein modulate a signaling pathway, which can be an upregulation or downregulation of such a signaling pathway. In some embodiments, the engineered antibodies of the present disclosure can function as agonists and upregulate (enhance, stimulate, promote, activate, or increase) the signaling pathway of interest, i.e., the target pathway. In some embodiments, the engineered antibodies described herein increase the activity of the 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, upregulation of the target signaling pathway includes activating or initiating a pathway that was stopped or substantially inactive. In another example, the engineered antibodies described herein can function as antagonists and downregulate (suppress, inhibit, reduce, decrease, or attenuate) the target pathway. In some embodiments, the engineered antibodies disclosed herein decrease the activity of the 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, downregulation of a target signaling pathway includes stopping or substantially blocking a pathway that was operative or substantially active.

[0174] Method of treatment As described above, the experimental results presented herein demonstrate that the guide-effector bispecific antibody designs disclosed herein can be used for the development of 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, is significantly affected by its neighboring antigen(s), and can be readily manipulated in either direction through targeting based on the bispecificity of an appropriately selected guide / effector pair, a phenomenon that can be exploited for therapeutic targeting.

[0175] Some embodiments of the disclosure relate to methods for treating a healthy condition or a 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, conjugate thereof, or pharmaceutical composition comprising the same, alone (e.g., as a monotherapy) or in combination with one or more additional agents, such as a pharmaceutically acceptable excipient (e.g., as a combination therapy). In certain aspects, the engineered antibody or pharmaceutical composition administered to the subject specifically targets cells, where the signaling pathway is modulated as a result of the treatment.

[0176] In one aspect, some embodiments of the present disclosure are methods for treating a health condition or a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered antibody or a functional fragment thereof comprising (a) a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first rate of intracellular trafficking, and (b) a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second rate of intracellular trafficking, wherein the intracellular trafficking characteristics of the engineered antibody or the functional fragment thereof are determined by the relative surface density ratio of the guide antigen to the effector antigen, and wherein one of the two rates of intracellular trafficking 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 are methods for killing cancer cells, the method comprising administering to the cells an engineered antibody or a functional fragment thereof disclosed herein. In some embodiments, the engineered antibody or the functional fragment thereof comprises (a) a first antigen-binding moiety capable of binding to a cell surface guide antigen having a first rate of intracellular trafficking, and (b) a second antigen-binding moiety capable of binding to a cell surface effector antigen having a second rate of intracellular trafficking.

[0178] In yet another aspect, some embodiments of the present disclosure are methods for killing tumor cells, the method comprising administering to the tumor cells an engineered antibody or a functional fragment thereof disclosed herein. In some embodiments of the disclosed methods, the engineered antibody or the functional fragment thereof comprises a first antigen-binding moiety capable of binding to Ephrin receptor A2 (EphA2) expressed on the surface of the tumor cells, and a second antigen-binding moiety 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 exceeds a threshold of about 1:5.

[0179] As used herein, the terms "administer" and "administering" 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 combinations thereof. The term includes, but is not limited to, administration by a healthcare provider and self-administration.

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

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

[0182] In some embodiments, the health 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 containing the same are administered to an individual (e.g., a human patient) to, for example, reduce the viability and / or invasiveness of cancerous cells, e.g., to reduce tumor size or metastasis, to reduce tumor burden, and / or to improve the clinical prognosis of the patient. In certain aspects, the antibody composition is used to disrupt the cell cycle of cancer cells and thereby promote the cells to enter apoptosis, for example, by inducing the cancerous cells to enter the pre-G0 phase of the cell cycle. 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 situations of treating or preventing a wide range of cancers. In one aspect, cancer refers to a general term encompassing primary cancer and metastatic cancer. In some embodiments, primary cancer may mean a group of tumor cells that have acquired at least one characteristic property of cancer cells, but have not invaded adjacent tissues and are located together in a tumor at the site of the primary origin. In some other embodiments, metastatic cancer may mean a group of tumor cells that originate from the cells of a primary cancer, invade the tissues surrounding the primary cancer, spread throughout the body, attach to a new location far away, and grow into 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, 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 containing the same are used in anti-cancer therapy, where the cancerous cells exhibit cell-specific markers that can function as the guiding antigens of the bispecific antibodies of the present disclosure on the cell surface accessible from the outside of the cell. Cancers particularly suitable for treatment using the bispecific antibodies of the present disclosure include those targeted by the antibodies through binding to the guiding antigens. In some embodiments, the presence or expression level of such guiding antigens in normal human tissues or cells is transient and may be present at low abundance compared to cancer cells that overexpress the guiding antigens. The guiding antigens may be present predominantly in abnormal cells, such as cancer cells. Since high levels of expression of the guiding antigens may be present predominantly in cancer cells, treatment with the bispecific antibodies of the present disclosure or compositions containing such antibodies can be used to treat cancer cells with high specificity or selectivity and minimize non-specific cytotoxicity to non-cancerous or healthy cells.

[0184] In some embodiments, the mode of treatment is to modulate the signaling pathway using the engineered antibodies of the present disclosure. Dysregulation of signaling pathways is often associated with the development and / or progression of a disease or condition in that modulation of such signaling pathways can result in effective treatment of the disease or condition. In some examples, a disease or condition may be associated with dysregulation of one or more signaling pathways, and such dysregulation can be alleviated or attenuated by modulation of another signaling pathway. In such situations, upregulation or downregulation of the signaling pathway using the engineered antibodies of the present disclosure that can antagonize or reduce the activity of the dysregulated signaling pathway can provide an effective treatment means.

[0185] In some embodiments, the cells to be treated using the engineered antibodies of the present disclosure or compositions containing such antibodies are not limited to cancer cells and include all cells in which modulation of intracellular trafficking and signaling may be desired. Such cells include, but are 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 techniques in cell cultures or experimental animals, for example, to determine the LD50 (the dose at which 50% of the population dies) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage ratio between the toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio of LD50 / ED50. For example, compounds exhibiting a high therapeutic index are generally preferred. Compounds with toxic side effects can be used, but care must be taken to design a delivery system that targets such compounds to the site of the affected tissue in order to minimize the potential for damage to non-infected cells and thereby reduce side effects.

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

[0188] Individual doses generally do not fall below the amount required to produce a measurable effect in an individual and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion ("ADME") of the antibody and thus can be determined based on the tendency of the composition within the individual. This includes consideration of the route of administration as well as the dose, which can be adjusted, for example, for parenteral (applied by a route other than through the gastrointestinal tract for systemic or local action) application. For example, administration of the engineered antibody or composition thereof is generally by injection and often by intravenous, intramuscular, intratumoral, or combinations thereof.

[0189] The engineered antibody or composition thereof can be administered by infusion or local injection, for example, at a rate of about 10 mg / hour to about 200 mg / hour, about 50 mg / hour to about 400 mg / hour, including, for example, 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, about 225 mg / hour to about 275 mg / hour. By way of exemplary infusion rates, for example, about 1 mg / m 2 / day to about 9 mg / m2 / day, about 2 mg / m 2 / day to about 8 mg / m 2 / day, about 3 mg / m 2 / day to about 7 mg / m 2 / day, about 4 mg / m 2 / day to about 6 mg / m 2 / day, about 4.5 mg / m 2 / day to about 5.5 mg / m 2 / day, including about 0.5 mg / m 2 / day to about 10 mg / m 2 / day, the desired therapeutic dose can be achieved. Administration (e.g., by infusion) can be repeated over a desired period, for example, over a period of about 1 day to about 5 days, or for several days, for example, once every about 5 days, about 1 month, about 2 months. It may be administered before, at the time of, or after other therapeutic interventions, such as surgical intervention to remove cancerous cells. The engineered antibody or composition thereof can also be administered as part of a combination therapy, in which case 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 disclosed herein, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions containing the same can be formulated to be compatible with their intended route of administration. For example, the engineered antibodies, conjugates thereof, and functional fragments thereof disclosed herein, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions containing the same may be provided orally or by inhalation, but they are likely to be administered via a parenteral route. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. The solutions or suspensions used for parenteral application may contain the following components: a sterile diluent, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate, and agents for adjusting isotonicity, such as sodium chloride or dextrose. The pH can be adjusted using an acid or a base, such as monosodium and / or disodium phosphate, hydrochloric acid, or sodium hydroxide (for example, to a pH of about 7.2 to 7.8, for example, 7.5). Parenteral preparations can be enclosed in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0191] When practicing the methods, the route of administration (the route by which the engineered antibodies, conjugates thereof, and functional fragments thereof, nucleic acids encoding such engineered antibodies, and / or pharmaceutical compositions containing them are administered to an individual or subject) can vary. The engineered antibody or composition thereof can be administered systemically (e.g., parenterally, e.g., by the intravenous route) or locally (e.g., to the local tumor site, e.g., by intratumoral administration (e.g., to solid tumors, to the relevant lymph nodes in the case of lymphoma or leukemia), by administration to the blood vessels supplying the 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, intraparenchymal, intraventricular, or intrathecal administration. In some cases, the engineered antibodies are administered to the subject as an injection. In other cases, the engineered antibodies are administered to the subject as an infusion.

[0193] Formulations suitable for parenteral administration may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, aqueous and non-aqueous isotonic sterile injection solutions, and aqueous and non-aqueous sterile suspensions that may contain suspending, solubilizing, thickening, stabilizing, and preserving agents. The formulations 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 that requires only the addition of a sterile liquid excipient for injection, for example water, immediately prior to use. Immediate injection solutions and suspensions can be prepared from sterile powders, powders, granules, and tablets of the aforementioned types.

[0194] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined quantity of a compound of the present disclosure calculated to provide the desired effect, together with a pharmaceutically acceptable diluent, carrier, or vehicle. The specification of the novel unit dosage form depends on the specific compound used, the effect to be achieved, the pharmacokinetics associated with each compound in the 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, and written instructions for making and using them. For example, in some embodiments, provided herein are systems and / or kits 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 prefilled syringes) and / or catheters (including prefilled syringes) that are used to administer any one of the provided engineered antibodies, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, the kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the components of the other kits for a desired purpose, e.g., to modulate intracellular trafficking, to modulate cell type-selective signaling in a subject, or to effect treatment of a disease in a subject in need thereof.

[0196] Any of the systems and kits described above may further include one or more additional reagents, where such additional reagents may be selected from dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control antibodies, positive control antibodies, reagents for in vitro production of engineered antibodies.

[0197] In some embodiments, the system or kit may further include instructions for practicing the method using the components of the kit. 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 an accompanying document, or may be present, for example, on a label of the kit or a container of the kit or its components (i.e., associated with the packaging or sub-packaging). The instructions may be present as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not present in the kit, but means may be provided for obtaining the instructions from a remote source (e.g., via the Internet). An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. Similar to 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 hereby incorporated by reference herein 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 of the references cited in this specification constitutes prior art. The discussion of references is provided to describe what the authors claim, and the inventors reserve the right to dispute the accuracy and appropriateness of the cited documents. Several information sources, including scientific journal articles, patent documents, and textbooks, are referenced herein, but it is clearly understood that these references do not establish the recognition that any of these documents form part of the widely general knowledge in the relevant technical field.

[0200] The discussion of the general methods provided herein is intended to be for illustrative purposes only. Other alternative methods and alternatives will be apparent to those skilled in the art from the discussion of the present disclosure and are within the spirit and scope of the present application.

Examples

[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 present disclosure or the scope of the claims in any way.

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

[0203] A tetravalent bispecific IgG-scFv (bsIgG) was constructed, which consisted 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 and here, high-content analysis was used to identify macropinocytosis-type antibodies. For the control, a non-binding C10 IgG was used to construct a control C10 / RYR bsIgG (which binds to EphA2 only). SDS-PAGE analysis showed the electrophoretic patterns of the predicted monoclonal and bispecific antibodies (Figure 7A). The binding specificity of the bsIgG was then studied using a HEK293 cell line expressing ALCAM and an engineered HEK293 cell line (HEK293-EphA2#2) that stably expressed high levels of EphA2. As shown in Figure 7B, the anti-ALCAM 3F1 IgG bound to both HEK293 and HEK293-EphA2#2 cells as predicted. The 3F1 / RYR bsIgG bound to HEK293-EphA2#2 (ALCAM high EphA2 low ) at a higher level compared to HEK293 (ALCAM high EphA2 high) was conjugated. The control C10 / RYR bsIgG that binds to EphA2 showed specific binding only to HEK293-EphA2#2 cells and no binding 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, which was internalized by HEK293-EphA2#2 but not by the HEK293 cell line. The control C10 / RYR bsIgG was internalized by HEK293-EphA2#2 but not by HEK293. The results indicate that in the guide-effector bispecific antibody design described herein, the internalization arm (EphA2, guide) can confer internalization characteristics to the non-internalization arm (ALCAM, effector) and the bispecific antibody as a whole.

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

[0205] (Example 3) Internalization and non-internalization are interconvertible properties in bispecific antibody design Non-internalizing antigen (ALCAM) has been shown to be induced to internalize by an anti-EphA2 / ALCAM bispecific antibody when the ratio of EphA2 to ALCAM exceeds a threshold. In this experiment, it is investigated whether rapidly internalizing EphA2 can be made slow or non-internalizing by the presence of ALCAM at a specific ratio of EphA2 to ALCAM. Using the HEK293 cell line (ALCAM high EphA2 low ) as a model, it was found that when the ratio of EphA2 to ALCAM is below 0.2, EphA2 internalization is significantly delayed and targeting with 3F1 / RYR bsIgG results in a high proportion of surface-bound EphA2, but not with the control C10 / RYR (Figure 1F), suggesting that internalization and non-internalization are interconvertible properties and that the relative abundance of internalizing versus non-internalizing antigens significantly affects the turnover of cell surface antigens when targeted by bispecific antibodies (summarized in Figure 1G).

Table 1

[0206] (Example 4) Expansion beyond model cell lines: Regulation of internalization kinetics in tumor cells by guide / effector-based bispecific antibodies This example shows the kinetics of bispecific antibody-induced surface antigens in a panel of pancreatic cancer cell lines with diverse 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-internalizing ALCAM is converted to an internalizing antigen by bispecific antibodies at EphA2 to ALCAM ratios above the threshold (above 0.2), and (2) how rapidly internalizing EphA2 becomes a slowly internalizing form by bispecific antibodies at EphA2 to ALCAM ratios below the threshold (below 0.2). The internalization kinetics of EphA2 and ALCAM were studied by measuring the surface antigen levels by FACS after antibody treatment. For ALCAM, bispecific 3F1 / RYR was effective in removing approximately 60% of cell surface ALCAM in both L3.6pl and Capan-1 cells with guide-to-effector ratios above 0.2, but not in Panc-1 cells with a ratio of 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 the removal of ALCAM is a bispecificity-dependent phenomenon that cannot be achieved by an oligoclonal antibody mixture. The above-described bispecificity 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 (about 0.31), anti-ALCAM 3F1 was most frequently detected on the cell surface, while bispecific 3F1 / RYR was detected mainly in the cytoplasm, with some cell membrane staining. The control C10 / RYR that binds to EphA2 was detected mainly in the cytoplasm, consistent with its ability to induce rapid EphA2 internalization. In contrast, for the Panc-1 cell line with an EphA2 / ALCAM ratio less than 0.2 (about 0.08), bispecific 3F1 / RYR was detected mainly on the cell surface (Figure 2B), again suggesting that the internalization of the bispecific antibody is dependent on the EphA2 / ALCAM ratio. These data confirm that in the guide-effector bispecific antibody design described herein, the ability of a non-internalizing bispecific antibody to convert to an internalizing effector antigen is dependent on the guide-to-effector ratio.

Table 2

[0208] To evaluate the internalization pathway and transport to lysosomes, L3.6pl cells were used for studies by confocal microscopy. As shown in Figure 2C, the bispecific 3F1 / RYR co-localizes with 70 kDa neutral dextran (ND70), a macropinocytosis marker, suggesting that the mode of internalization is macropinocytosis. After internalization, 3F1 / RYR and the control C10 / RYR co-localize with lysosome-associated membrane protein 1 (LAMP1), a lysosome marker (Figure 2D), suggesting that the bispecific antibody guided by the anti-EphA2 antibody is transported to lysosomes.

[0209] To investigate the opposite directionality of the interconversion between internalizing and non-internalizing, i.e., the conversion from rapidly internalizing antibodies to slowly or non-internalizing antibodies, the surface removal of EphA2 by bispecific antibodies in the presence of the adjacent non-internalizing antigen ALCAM was studied. As shown in Figure 2E, in Panc-1 cells where the EphA2 to ALCAM ratio is below 0.2 (about 0.08), EphA2 mainly remains on the cell surface when targeted by the bispecific 3F1 / RYR. The control C10 / RYR removes EphA2 from the cell surface. A mixture of C10 / RYR and 3F1 does not delay EphA2 internalization, suggesting that this phenomenon is dependent on the bispecific antibody. Time-course internalization studies showed that 3F1 / RYR significantly delayed the EphA2 internalization kinetics, while the control C10 / RYR induced rapid EphA2 internalization (Figure 2F). These studies show that EphA2 internalization can be significantly delayed by bispecific antibodies when ALCAM is present on the same cell in an amount above the EphA2 / ALCAM ratio threshold.

[0210] (Example 5) The bispecific 3F1 / RYR inhibits pancreatic tumor spheroid formation The effect of anti-EphA2-guided bsIgG(3F1 / RYR) on the survival and expansion of pancreatic tumor spheres was investigated to evaluate the functional consequences of surface antigen removal. Previous studies have shown that cancer cells overexpressing ALCAM actively form tumor spheres, suggesting that ALCAM plays a role in tumor clonogenicity. In this example, therefore, experiments were conducted to investigate whether ALCAM removal by tetravalent ALCAM×EphA2 bsIgG could inhibit pancreatic tumor sphere formation. First, antigen expression was evaluated in L3.6pl tumor spheres, and ALCAM was found to be significantly upregulated on the surface of tumor cells forming the spheres (Figure 3A). There was no difference in the EphA2 surface levels of L3.6pl cells between monolayer and sphere states (Figure 3A). After incubating L3.6pl tumor spheres with the antibody for 2 weeks, bispecific 3F1 / RYR reduced the ALCAM surface density by 70%, while the control C10 / RYR that binds only to anti-ALCAM mAb 3F1 or EphA2 had no effect on the ALCAM surface level (Figure 3B). Next, studies by confocal microscopy were performed to confirm antibody internalization. As shown in Figure 3C, 3F1 / RYR was effectively internalized in L3.6pl sphere-forming cells. In contrast, the monoclonal anti-ALCAM antibody 3F1 showed mainly surface staining (Figure 3C). The control bispecific C10 / RYR was internalized, consistent with its single specific binding to EphA2 (Figure 3C). Notably, based on the fluorescence signal intensity per cell, a greater amount of 3F1 / RYR was taken up by tumor sphere-forming cells compared to the control bispecific C10 / RYR (Figure 3C, right panel), suggesting an amplification effect inherent to bispecific antibodies. Regarding the functional effect on tumor clonogenic activity, the number (Figure 3D) and size (Figure 3E) of L3.6pl spheres were significantly decreased by treatment with 3F1 / RYR, but not with 3F1 or C10 / RYR, consistent with previous studies on the role of ALCAM in tumor sphere formation and growth.Therefore, a bispecific antibody having one arm that binds to the internal migrating antigen EphA2 can effectively remove the non-internal migrating antigen ALCAM from the surface of tumor cells and can result in inhibition of pancreatic tumor sphere growth.

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

Table 3

[0212] (Example 7) In Vivo Antitumor Efficacy of ALCAM×EphA2 Bispecific ADC In this example, experiments conducted to study the in vivo efficacy of the bispecific 3F1 / RYR ADC, together with a control ADC, in pancreatic cancer xenografts are summarized. Capan-1 cells were implanted subcutaneously in NSG mice. When the tumors reached an average volume of 110 mm3, 3F1 / RYR, 3F1, or C10 / RYR ADC was injected at 3 mg / kg every 4 days for a total of 4 times. Tumor status was monitored by caliper measurement. Apparent 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. For any of the ADCs studied, there was no significant change in body weight during the study (Figure 5B). These data indicate that in the guide-effector bispecific antibody design described herein, the rapid internalization-type anti-guide (EphA2) scFv can induce the internalization of the effector antigen (ALCAM), which is non-internalization-type without it, resulting in more uptake of the bispecific ADC into tumor cells compared to the single-specific ADC, and thus showing enhanced antitumor efficacy in vivo.

[0213] (Example 8) Cell Lines and Plasmids The human embryonic kidney (HEK) cell lines HEK293 and HEK293A, the prostate cancer cell lines DU145 and PC3, and the 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 was maintained in the laboratory. The cells were maintained at 37°C and 5% CO2 in DMEM or RPMI1640 supplemented with 10% FBS (Fisher Scientific) and 100 μg / ml penicillin / streptomycin (Axenia BioLogix). The full-length human EphA2 cDNA cloned into pCMV-Entry (Origene) or pLV202 (Origene) was used for transient or stable EphA2 expression, respectively.

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

[0215] (Example 10) Generation of anti-EphA2 scFv antibody This example describes experiments conducted to identify a new version of the EphA2-binding scFv antibody 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 named HCA-F1 and the germline version of RYR was named RYRgerm. To identify a new version of the EphA2-binding scFv antibody with improved binding affinity for 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 for EphA2 were identified and named RYRgerm_102019_14, RYRgerm_102919_15, RYRgerm_102919_22, and RYRgerm_102919_33, respectively. The VH and VL regions, as well as the amino acid sequences of the CDRs, of these newly identified EphA2 scFvs are presented in Tables 4-5 and the Sequence Listing. In these experiments, recombinant EphA2 proteins from both human and mouse were used in the selection to maintain cross-species binding. The apparent affinity for binding to both human and mouse EphA2 was measured by flow cytometry. As shown in Figure 10A, the new version of the EphA2-binding scFv antibody identified in the yeast display mutagenesis library showed enhanced binding affinity for human EphA2 compared to the original EphA2-binding scFv RYR, and the increase in binding affinity was from about 8-fold to about 70-fold. The apparent K of human EphA2 binding affinity DThe values were 354.9 nM for RYRgerm (original EphA2 scFv), 21.27 nM for RYRgerm_102019_14, 5.58 nM for RYRgerm_102919_15, 28.13 nM for RYRgerm_102919_22, and 42.49 nM for RYRgerm_102919_33. Similarly, as shown in Figure 10B, the new versions of the EphA2-binding scFv antibodies identified in the yeast display mutagenesis library showed improved binding affinity for mouse recombinant EphA2-Fc compared to the original EphA2-binding scFv RYR, and the increase in binding affinity was about 40-fold to about 80-fold. The apparent K of the binding affinity of the mouse recombinant EphA2-Fc fusion 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 precede the evaluation of scFv binding activity in yeast cells, additional recombinant human IgG1 was designed and constructed using the original EphA2 scFv (RYR) and the newly improved RYRgerm_102919_15, and their binding affinities for 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 the recombinant IgG1 between the original RYR and the newly improved RYR-binding scFv RYRgerm_102919_15 described in Figures 10A - 10B. In these experiments, the apparent binding affinity in DU145 cells comparing RYR IgG1 and RYRgerm_102919_15 IgG1 was evaluated. In these experiments, DU145 cells were incubated with RYR or RYRgerm_102919_15 at a concentration range of 40 pM to 125 nM for 1 hour at 25°C, washed, and binding was detected using anti-human Alexa Fluor 647. The MFI values were curve-fitted to determine the apparent K DValues were generated. The K values of RYR IgG1 and RYRgerm_102919_15 were 23.7 nM and 0.23 nM, respectively, indicating an approximately 100-fold increase in binding affinity. D The values are 23.7 nM and 0.23 nM, respectively, indicating an approximately 100-fold increase in binding affinity.

[0217] In addition, additional experiments were conducted to evaluate the binding affinity of the new EphA2 scFv RYRgerm_102919_15 to recombinant human EphA2 (see, for example, Figure 12). In these experiments, unlabeled Biolayer interferometry (BLI) analysis was performed using a Probe Life Gator instrument. An anti-human Fc probe was loaded with RYR or RYRgerm_102919_15 IgG1, and 100 nM of recombinant human EphA2 (R&D System) was used for the binding assay at 25°C. The apparent affinity K of RYR IgG1 D was approximately 28 nM (K off / K on = 1.45E-02 / 5.18E+05), while the apparent affinity K of the improved RYRgerm_102919_15 IgG1 D was 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) Production of recombinant antibodies The VH and VL antibody genes were amplified by PCR from the candidate scFv phagemids and subcloned into the Abvec Ig-γ and -λ expression vectors, respectively. To produce the bispecific IgG-scFv, anti-ALCAM 3F1 or the non-binding control C10 was utilized as the IgG backbone, and the internal translocation scFv was introduced into the C-terminus of the λ light chain constant region (CL) by fusion with the (Gly4Ser)3 linker. HEK293A cells were transfected for 24 h with the antibody expression plasmids mixed with polyethyleneimine (Sigma Aldrich) in Opti-MEM (Life Technologies). The transfection medium was replaced with Freestyle™ 293 (Gibco), and the cells were cultured for an additional up to 8 days. The secreted antibodies were purified from the culture supernatant with protein A agarose (Thermo Scientific) and analyzed by SDS-PAGE gradient gels (4–20%).

[0219] (Example 12) Generation of a stable HEK293-EphA2 cell line 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 goat anti-human IgG labeled with Alexa Fluor® 647 (Jackson ImmunoResearch). The stable clones were further screened by FACS to obtain those expressing various levels of EphA2.

[0220] (Example 13) Measurement of cell surface antigen copy number The copy number of cell surface antigen (or antigen density) was measured as described above. 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 antibody conjugated with Alexa Fluor® 647 using a monoclonal antibody labeling kit (Molecular Probes) to detect EphA2 or ALCAM respectively, and analyzed by BD Accuri C6 (BD Biosciences). The 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, the E / A (EphA2 / ALCAM) ratio was calculated by dividing the copy number of EphA2 by the copy number of ALCAM.

[0221] (Example 14) Apparent K D Determination of Dissociated cells (about 2×10 5 cells) were incubated with various concentrations of human IgG at 4°C for 16 hours. After washing three times with ice-cold PBS, the IgG bound to the cells was detected with goat anti-human IgG labeled with Alexa Fluor® 647 (Jackson ImmunoResearch) and analyzed by FACS. The apparent K D value was calculated by the curve fitting method 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 (at approximately 80% confluence) for 24 hours, and the remaining EphA2 or ALCAM on the cell surface was determined using L1A1 anti-EphA2 human IgG or L50 anti-ALCAM mouse IgG (Fisher Scientific) labeled with Alexa Fluor® 647, respectively. The cell surface copy number was calculated using the method described above and normalized to the control group without antibody treatment.

[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 evaluate the pathway of internalization (macropinocytosis), cells were co-incubated with 70 kDa neutral dextran conjugated with TexasRed (ND70-TR, Life Technologies), a marker of macropinocytosis. Cells after incubation were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS / 1% FBS / 0.2% Triton-X100. Antibodies associated with the cells were stained with goat anti-human IgG labeled with Alexa Fluor® 488 or 647 (Jackson ImmunoResearch) for 1 hour at room temperature. Lysosomes were detected with rabbit anti-lysosomal associated membrane protein 1 (LAMP1) antibody (Cell Signaling) and subsequently incubated with goat anti-rabbit IgG labeled with Alexa Fluor® 647 (Jackson ImmunoResearch). For analysis of the localization of antibodies in tumor spheres, the spheres were recovered by centrifugation at 500×g for 5 minutes, washed, fixed, permeabilized, and immunolabeled using the antibodies described above. The spheres were fixed on 8-well chamber slides for microscopic analysis using CyGEL™ (Abcam). For imaging, cells or spheres were counterstained with Hoechst 33342 (Thermo Scientific) and imaged with a FluoView® FV10i laser confocal microscope (Olympus) equipped with a 60× phase contrast water immersion objective lens from Olympus.

[0224] (Example 17) Formation of tumor spheres In an ultra-low attachment 24-well plate (Corning), tumor spheres 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) at 37 °C / 5% CO2. For the sphere growth assay, the first generation of spheres was 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 an ultra-low attachment 24-well plate (Corning) at 37 °C / 5% CO2 for 24 hours and treated with the indicated antibodies for 2 weeks. The cells were given 100 μl of SFM every 3-4 days. Each well was scanned for cross-sections using a BIOREVO digital microscope (BZ-9000, Keyence), and these were integrated 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 Cysteine residues were introduced at position 116 (T116C) of the heavy chain of IgG or bsIgG, and site-specific ADCs with modifications were generated as described above. Briefly, the antibody in PBS was reduced by incubating with a 10-fold molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Thermo Scientific) at 37 °C for 2 hours, purified by a Zeba spin desalting column (Thermo Scientific), and buffer-exchanged into PBS / 5 mM EDTA. To re-oxidize the inter-chain 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-exchanging into 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 at 25 °C for 1 hour. The final conjugation product was purified by passing it through a Zeba™ spin desalting column (Fisher Scientific) twice 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 Cells were seeded overnight at 2×10 3 cells / well in a 96-well cell culture plate 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 study All animal studies were approved by the UCSF Animal Care and Use Committee (AN092211) and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. NOD / SCID / IL-2Rγ - / - (NSG) female mice were engrafted with 1 × 10 6 individual Capan-1 cells and randomly divided into four groups on day 5 (n = 6 mice per group). Mice were intravenously treated with vehicle PBS or a single-specificity or dual-specificity ADC by a total of four injections at 3 mg / kg every 4 days. Tumor size was measured with calipers and tumor volume was calculated using the formula V = (width 2 × length) / 2. Body weight was monitored throughout the study.

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 5

[0228] Although specific alternative methods of the present disclosure are disclosed, various modifications and combinations are possible and are intended to be within the true spirit and scope of the appended patent claims. Accordingly, there is no intention to limit the exact summaries and disclosures presented herein. References 1. de Goeij BE, Lambert JM. New developments for antibody-drug conjugate-based therapeutic approaches. Curr Opin Immunol 2016;40:14-23 doi 10.1016 / j.coi.2016.02.008. 2. Polakis P. Antibody Drug Conjugates for Cancer Therapy. Pharmacol Rev 2016;68(1):3-19 doi 10.1124 / pr.114.009373. 3. Gebleux R, Stringhini M, Casanova R, Soltermann A, Neri D. Non-internalizing antibody-drug conjugates display potent anti-cancer activity upon proteolytic release of monomethyl auristatin E in the subendothelial extracellular matrix. Int J Cancer 2017;140(7):1670-9 doi 10.1002 / ijc.30569. 4. Li JY, Perry SR, Muniz-Medina V, Wang X, Wetzel LK, Rebelatto MC, et al. A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy. Cancer Cell 2016;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 Payload Delivery by a Bispecific Antibody-Drug Conjugate Targeting HER2 and CD63. Mol Cancer 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. Bispecific Antibodies and Antibody-Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs. Mol Cancer Ther 2017;16(4):681-93 doi 10.1158 / 1535-7163.MCT-16-0658. 7. DeVay RM, Delaria K, Zhu G, Holz C, Foletti D, Sutton J, et al. Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates. Bioconjug Chem 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-type specific potent Wnt signaling blockade by bispecific antibody. Sci Rep 2018;8(1):766 doi 10.1038 / s41598-017-17539-z. 9. Bruns CJ, Harbison MT, Kuniyasu H, Eue I, Fidler IJ. In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using 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 both adenocarcinoma and neuroendocrine prostate cancer. JCI Insight 2018;3(17):pii:121497 doi 10.1172 / jci.insight.121497. 11. Lee NK, Bidlingmaier S, Su Y, Liu B. Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes. Methods Mol Biol 2018;1701:61-82 doi 10.1007 / 978-1-4939-7447-4_4. 12. Rogosch T, Kerzel S, Hoi KH, Zhang Z, Maier RF, Ippolito GC, et al. Immunoglobulin analysis tool: a novel tool for the analysis of human and mouse heavy and light chain transcripts. Front Immunol 2012;3:176 doi 10.3389 / fimmu.2012.00176. 13. Smith K, Garman L, Wrammert J, Zheng NY, Capra JD, Ahmed R, et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 2009;4(3):372-84 doi 10.1038 / nprot.2009.3. 14. Sherbenou DW, Aftab BT, Su Y, Behrens CR, Wiita A, Logan AC, et al. Antibody-drug conjugate targeting CD46 eliminates multiple myeloma cells. J Clin Invest 2016;126(12):4640-53 doi 10.1172 / JCI85856. 15. Orcutt KD, Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL, Frangioni JV, et al. A modular IgG-scFv bispecific antibody topology. Protein Eng Des Sel 2010;23(4):221-8 doi 10.1093 / protein / gzp077. 16. Ha KD, Bidlingmaier SM, Zhang Y, Su Y, Liu B. High-content analysis of antibody phage-display library selection outputs identifies tumor selective macropinocytosis-dependent rapidly internalizing antibodies. Mol Cell Proteomics 2014;13(12):3320-31 doi 10.1074 / mcp.M114.039768. 17. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic 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 of CD166 as a surface marker for enriching prostate stem / progenitor and cancer initiating cells. PLoS One 2012;7(8):e42564 doi 10.1371 / journal.pone.0042564. 19. Yan M, Yang X, Wang L, Clark D, Zuo H, Ye D, et al. Plasma membrane proteomics of tumorspheres identify CD166 as a novel marker for cancer stem-like cells in head and neck squamous cell carcinoma. Mol Cell Proteomics 2013;12(11):3271-84 doi 10.1074 / mcp.M112.025460. 20. Moon BS, Jeong WJ, Park J, Kim TI, Min do S, Choi KY. Role of oncogenic K-Ras in cancer stem cell activation by aberrant Wnt / beta-catenin signaling. J Natl Cancer Inst 2014;106(2):djt373 doi 10.1093 / jnci / djt373. 21. Behrens CR, Liu B. Methods for site-specific drug conjugation to antibodies. MAbs 2014;6(1):46-53 doi 10.4161 / mabs.26632. 22. Fu Y, Ho M. DNA damaging agent-based antibody-drug conjugates for cancer therapy. 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 drug conjugates for cancer therapy. MAbs 2014;6(1):34-45 doi 10.4161 / mabs.27022. 24. Ha KD, Bidlingmaier SM, Su Y, Lee NK, Liu B. Identification of Novel Macropinocytosing Human Antibodies by Phage Display and High-Content Analysis. Methods Enzymol 2017;585:91-110 doi 10.1016 / bs.mie.2016.10.004. 25. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci U S A 1996;93(1):136-40. 26. Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res 1997;3(1):81-5. 27. Kinoshita Y, Kuratsukuri K, Landas S, Imaida K, Rovito PM, Jr., Wang CY, et al. Expression of prostate-specific membrane antigen in normal and malignant human tissues. World J Surg 2006;30(4):628-36 doi 10.1007 / s00268-005-0544-5. 28. Wang K, Wei G, Liu D. CD19: a biomarker for B cell development, lymphoma diagnosis and therapy. Exp Hematol Oncol 2012;1(1):36 doi 10.1186 / 2162-3619-1-36. 29. Vainshtein I, Roskos LK, Cheng J, Sleeman MA, Wang B, Liang M. Quantitative measurement of the target-mediated internalization kinetics of biopharmaceuticals. Pharm Res. 2015 Jan;32(1):286-99. 30. Li N, Hill KS, Elferink LA. Analysis of receptor tyrosine kinase internalization using flow cytometry. Methods Mol Biol. 2008;457:305-17. 31. Pahara J, Shi H, Chen X, Wang Z. Dimerization drives PDGF receptor endocytosis through a C-terminal hydrophobic motif shared by EGF receptor. Exp Cell Res. 2010;316:2237-50. 32. Mazor Y, Barnea I, Keydar I, Benhar I. Antibody internalization studied using a novel IgG binding toxin fusion. J Immunol Methods. 2007;321:41-59. 33. Schaefer G et al. A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies. Cancer Cell. 2011 Oct 18;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 humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions. MAbs. 2014;6(3):622-627. doi:10.4161 / mabs.28483)

Claims

1. An engineered antibody or a functional fragment thereof, comprising a first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell, wherein the first antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 81, 83, 85, 87, 89 or 96, and the VH region of the first antigen-binding portion (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 and comprises HCDR1, HCDR2 and HCDR3 consisting thereof, wherein the first antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 82, 84, 86, 88, 90 or 97, and the VL region of the first antigen-binding portion comprises LCDR1, LCDR2 and LCDR3 consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, respectively, a first antigen-binding portion; a second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cell, wherein the second antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 73 or SEQ ID NO: 75, and the VH region of the second antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 consisting of SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, respectively, wherein the second antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 74 or SEQ ID NO: 76, and the VL region of the second antigen-binding portion comprises LCDR1, LCDR2 and LCDR3 consisting of SEQ ID NO: 101, SEQ ID NO: 102 and SEQ ID NO: 103, respectively, a second antigen-binding portion and comprising, wherein the engineered antibody or a functional fragment thereof is mobilized to target cells when binding to the EphA2, An engineered antibody or a functional fragment thereof, wherein the intracellular trafficking property of the engineered antibody or the functional fragment thereof is determined by the relative surface density ratio of the EphA2 to the ALCAM.

2. The engineered antibody or a functional fragment thereof according to claim 1, wherein the relative surface density ratio of the first antigen to the second antigen exceeds a threshold value.

3. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

30.

4. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

20.

5. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

10.

6. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

5.

7. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

4.

8. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

3.

9. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

2.

10. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 2, wherein the threshold value is 1:

1.

11. The engineered antibody or a functional fragment thereof according to any one of claims 1 to 10, 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.

12. An engineered antibody or a functional fragment thereof according to any one of claims 1 to 11, which is conjugated or covalently bound to at least one moiety of interest (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a moiety that improves pharmacokinetics. **Claim 13** The engineered antibody or a functional fragment thereof according to claim 12, 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 antibacterial agent, an antimicrobial agent, an antibiotic, an anti-infectious disease agent, and an antiviral agent. **Claim 14** The engineered antibody or a functional fragment thereof according to claim 13, wherein the at least one MOI is selected from the group consisting of a cytotoxic anti-cancer agent, a DNA chelating agent, 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. **Claim 15** The engineered antibody or a functional fragment thereof according to claim 14, wherein the cytotoxic anti-cancer agent is selected from the group consisting of auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, anthracycline, calicheamicin, camptothecin, irinotecan, SN-38, combretastatin, duocarmycin, enediyne, epothilone, ethyleneimine, mitomycin, pyrrolobenzodiazepine (PBD), and calicheamicin. **Claim 16** The engineered antibody or a functional fragment thereof according to any one of claims 12 to 15, wherein the at least one moiety of interest (MOI) is conjugated or covalently bound to the constant region of the engineered antibody or a functional fragment thereof. **Claim 17** The manipulated antibody or functional fragment thereof according to claim 16, wherein the at least one moiety of interest (MOI) is conjugated to or covalently bound to the heavy chain constant (CH1) region of the manipulated antibody or functional fragment thereof. **Claim 18** The manipulated antibody or functional fragment thereof according to claim 16, wherein the at least one moiety of interest (MOI) is conjugated to or covalently bound to the light chain constant (CL) region of the manipulated antibody or functional fragment thereof. **Claim 19** The manipulated antibody or functional fragment thereof according to any one of claims 12 to 18, wherein the average number of MOIs per antibody (average DAR) ranges from 1 to 20. **Claim 20** The manipulated antibody or functional fragment thereof according to claim 19, wherein the average DAR is 0.9 to 5, 5 to 10, or 10 to 22. **Claim 21** The manipulated antibody or functional fragment thereof according to claim 19, wherein the average DAR is 2 to 6. **Claim 22** The manipulated antibody or functional fragment thereof according to claim 19, wherein the average DAR is DAR 10 to 15. **Claim 23** The manipulated antibody or functional fragment thereof according to claim 19, wherein the average DAR is DAR 15 to 20. **Claim 24** A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the manipulated antibody or functional fragment thereof according to any one of claims 1 to 23. **Claim 25** The recombinant nucleic acid molecule according to claim 24, which is operably linked to a heterologous nucleic acid sequence. **Claim 26** The recombinant nucleic acid molecule according to any one of claims 24 to 25, further defined as an expression cassette or vector. **Claim 27** The manipulated antibody or functional fragment thereof according to any one of claims 1 to 23, and / or A nucleic acid molecule according to any one of claims 24 to 26 A recombinant cell comprising the same.

28. The recombinant cell according to claim 27, which is a prokaryotic cell or a eukaryotic cell.

29. A cell culture comprising at least one recombinant cell according to any one of claims 27 to 28 and a culture medium.

30. An engineered antibody or a functional fragment thereof according to any one of claims 1 to 23, A nucleic acid molecule according to any one of claims 24 to 26, and A recombinant cell according to any one of claims 27 to 28 one or more of the above, and a pharmaceutically acceptable carrier A pharmaceutical composition comprising the same.

31. A composition for modulating intracellular trafficking, comprising an engineered antibody or a functional fragment thereof according to any one of claims 1 to 23, a nucleic acid molecule according to any one of claims 24 to 26, and the pharmaceutical composition according to claim 30 one or more of the above, characterized in that the composition is administered to a cell.

32. A composition for modulating intracellular trafficking, comprising a first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell, wherein the first antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 81, 83, 85, 87, 89 or 96, and the VH region of the first antigen-binding portion is (a) SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, respectively; or (b) consisting of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 110 comprising HCDR1, HCDR2, and HCDR3 consisting thereof, wherein the first antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 82, 84, 86, 88, 90, or 97, and the VL region of the first antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 consisting of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively, a first antigen-binding portion; a second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cell, wherein the second antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 73 or SEQ ID NO: 75, and the VH region of the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 consisting of SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively, wherein the second antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 74 or SEQ ID NO: 76, and the VL region of the second antigen-binding portion comprises LCDR1, LCDR2, and LCDR3 consisting of SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, respectively, a second antigen-binding portion comprising an engineered antibody or a functional fragment thereof, wherein the engineered antibody or a functional fragment thereof is mobilized to target cells when binding to the EphA2, wherein the internalization property of the engineered antibody or a functional fragment thereof is determined by the relative surface density ratio of the EphA2 to the ALCAM, and the composition is administered to cells. A composition characterized by that.

33. A composition for modulating cell type-selective signal transduction in a subject, a first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell, The first antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 81, 83, 85, 87, 89 or 96, and the VH region of the first antigen-binding portion is (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 comprising HCDR1, HCDR2 and HCDR3 consisting of, the first antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 82, 84, 86, 88, 90 or 97, and the VL region of the first antigen-binding portion comprises LCDR1, LCDR2 and LCDR3 consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, respectively, a first antigen-binding portion, a second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cell, the second antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 73 or SEQ ID NO: 75, and the VH region of the second antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 consisting of SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, respectively, the second antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 74 or SEQ ID NO: 76, and the VL region of the second antigen-binding portion comprises LCDR1, LCDR2 and LCDR3 consisting of SEQ ID NO: 101, SEQ ID NO: 102 and SEQ ID NO: 103, respectively, a second antigen-binding portion comprising an engineered antibody or a functional fragment thereof, the engineered antibody or a functional fragment thereof is mobilized to target cells when binding to the EphA2, a composition, wherein the internalization property of the engineered antibody or a functional fragment thereof is determined by the relative surface density ratio of the EphA2 to the ALCAM.

34. A composition for use in treating a health condition or disease in a subject in need thereof, a first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of a cell, wherein the first antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 81, 83, 85, 87, 89 or 96, and the VH region of the first antigen-binding portion (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 comprising HCDR1, HCDR2 and HCDR3 consisting of, wherein the first antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 82, 84, 86, 88, 90 or 97, and the VL region of the first antigen-binding portion comprises LCDR1, LCDR2 and LCDR3 consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, respectively, a first antigen-binding portion, a second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cell, wherein the second antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 73 or SEQ ID NO: 75, and the VH region of the second antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 consisting of SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, respectively, wherein the second antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 74 or SEQ ID NO: 76, and the VL region of the second antigen-binding portion comprises LCDR1, LCDR2 and LCDR3 consisting of SEQ ID NO: 101, SEQ ID NO: 102 and SEQ ID NO: 103, respectively, a second antigen-binding portion comprising an engineered antibody or a functional fragment thereof, wherein the engineered antibody or a functional fragment thereof is mobilized to target cells when binding to the EphA2, A composition wherein the intracellular translocation property of the manipulated antibody or its functional fragment is determined by the relative surface density ratio of the EphA2 to the ALCAM.

35. The composition according to claim 34, wherein the health state or disease is cancer.

36. A composition for killing cancer cells, a first antigen-binding portion capable of binding to ephrin receptor A2 (EphA2) expressed on the surface of cells, wherein the first antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 81, 83, 85, 87, 89 or 96, and the VH region of the first antigen-binding portion is (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 comprising HCDR1, HCDR2 and HCDR3 consisting of, wherein the first antigen-binding portion comprises a VL region having at least 90% sequence identity to SEQ ID NO: 82, 84, 86, 88, 90 or 97, and the VL region of the first antigen-binding portion comprises LCDR1, LCDR2 and LCDR3 consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 respectively, a first antigen-binding portion; a second antigen-binding portion capable of binding to activated leukocyte cell adhesion molecule (ALCAM) expressed on the surface of the same cells, wherein the second antigen-binding portion comprises a VH region having at least 90% sequence identity to SEQ ID NO: 73 or SEQ ID NO: 75, and the VH region of the second antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 consisting of SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100 respectively, The second antigen-binding portion includes a VL region having at least 90% sequence identity to SEQ ID NO: 74 or SEQ ID NO: 76, and the VL region of the second antigen-binding portion includes LCDR1, LCDR2, and LCDR3 consisting of SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, respectively, and a second antigen-binding portion and includes an engineered antibody or a functional fragment thereof, wherein the engineered antibody or the functional fragment thereof is mobilized to target cells when binding to EphA2, A composition, characterized in that the composition is administered to the cells.

37. The composition according to any one of claims 35 to 36, 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.

38. The composition according to any one of claims 31 to 37, wherein the relative surface density ratio of the first antigen to the second antigen exceeds a threshold value.

39. The composition according to any one of claims 31 to 38, wherein the threshold value is 1:

30.

40. The engineered antibody or a functional fragment thereof according to any one of claims 31 to 38, wherein the threshold value is 1:

20.

41. The engineered antibody or a functional fragment thereof according to any one of claims 31 to 38, wherein the threshold value is 1:

10.

42. The engineered antibody or a functional fragment thereof according to any one of claims 31 to 38, wherein the threshold value is 1:

5.

43. The engineered antibody or a functional fragment thereof according to any one of claims 31 to 38, wherein the threshold value is 1:

4. **Claim 44**: The engineered antibody or a functional fragment thereof according to any one of claims 31 to 38, wherein the threshold value is 1:

3. **Claim 45**: The engineered antibody or a functional fragment thereof according to any one of claims 31 to 38, wherein the threshold value is 1:

2. **Claim 46**: The engineered antibody or a functional fragment thereof according to any one of claims 31 to 38, wherein the threshold value is 1:

1. **Claim 47** The composition according to any one of claims 31 to 46, wherein the cell surface density of the first antigen and / or the cell surface density of the second antigen is modulated. **Claim 48** The composition according to any one of claims 31 to 47, wherein the intracellular trafficking property of the engineered antibody or a functional fragment thereof is converted from a non-intracellular trafficking type to an intracellular trafficking type. **Claim 49** The composition according to any one of claims 31 to 47, wherein the intracellular trafficking property of the engineered antibody or a functional fragment thereof is converted from an intracellular trafficking type to a non-intracellular trafficking type. **Claim 50** The composition according to any one of claims 31 to 49, 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. **Claim 51** The composition according to any one of claims 31 to 50, wherein the expression of the first antigen and / or the second antigen is cell-type selective. **Claim 52** The composition according to any one of claims 31 to 51, wherein the antibody or a functional fragment thereof is conjugated or covalently bound to at least one purposeful moiety (MOI) selected from the group consisting of a therapeutic moiety, a diagnostic agent, and a moiety that improves pharmacokinetics.

53. The composition according to claim 52, 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 antibacterial agent, an antimicrobial agent, an antibiotic, an anti-infectious disease agent, and an antiviral agent.

54. The composition according to claim 53, wherein the at least one MOI is selected from the group consisting of a cytotoxic anti-cancer agent, a DNA chelating agent, 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.

55. The composition according to claim 54, wherein the cytotoxic anti-cancer agent is selected from the group consisting of auristatin, dolastatin, tubulysin, maytansinoid, taxane, vinca alkaloid, amatoxin, anthracycline, calicheamicin, camptothecin, irinotecan, SN-38, combretastatin, duocarmycin, enediyne, epothilone, ethyleneimine, mitomycin, pyrrolobenzodiazepine (PBD), and calicheamycin.

56. The composition according to any one of claims 52 to 55, wherein the at least one moiety of interest (MOI) is conjugated or covalently bound to the constant region of the engineered antibody or a functional fragment thereof.

57. The composition according to claim 56, wherein the at least one moiety of interest (MOI) is conjugated or covalently bound to the CH1 region of the engineered antibody or a functional fragment thereof.

58. The composition according to claim 56, wherein the at least one moiety of interest (MOI) is conjugated or covalently bound to the CL region of the engineered antibody or a functional fragment thereof.

59. The composition according to any one of claims 31 to 58, wherein the average number of moles of the payload per antibody (DAR) ranges from 1 to 20.

60. The composition according to claim 59, wherein the average DAR is 0.9 - 5, 5 - 10, or 10 - 22.

61. The engineered antibody or a functional fragment thereof according to claim 59, wherein the average DAR is 2 - 6.

62. The engineered antibody or a functional fragment thereof according to claim 59, wherein the average DAR is 10 - 15.

63. The engineered antibody or a functional fragment thereof according to claim 59, wherein the average DAR is 15 - 20.

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