Dual-functional antigen-binding molecules

Abstract

An antibody or antigen-binding fragment thereof comprising at least one immunogenic peptide inserted into the variable region of the antibody or antigen-binding fragment thereof, the insertion comprising the removal of the antibody or antigen-binding fragment sequence. A dual-function antigen-binding molecule is provided, comprising the antibody or antigen-binding fragment of the present invention and a second antibody or antigen-binding fragment thereof, and capable of binding to an antigen overexpressed on a target cell. Nucleic acid molecules encoding the dual-function antigen-binding molecule, pharmaceutical compositions comprising the dual-function antigen-binding molecule, and methods of treating cancer by administering the dual-function antigen-binding molecule are also provided. Methods of producing the antibody or antigen-binding fragment are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to Israeli Patent Application No. 287372, filed October 18, 2021, the contents of which are incorporated herein by reference in their entirety. FIELD OF THEINVENTION The present invention relates to the field of anti-cancer immunotherapy. Summary of the Invention

[0002] The present invention provides an antibody or antigen-binding fragment thereof comprising at least one immunogenic peptide inserted into the variable region of the antibody or antigen-binding fragment thereof. A dual-function antigen-binding molecule comprising the antibody or antigen-binding fragment of the present invention and a second antibody or antigen-binding fragment thereof can bind to an antigen that is overexpressed on a target cell. Nucleic acid molecules encoding the dual-function antigen-binding molecule, pharmaceutical compositions comprising the dual-function antigen-binding molecule, and methods of treating cancer by administering the dual-function antigen-binding molecule are also provided. Methods of producing the antibody or antigen-binding fragment are also provided.

[0003] In a first aspect, there is provided an antibody or antigen-binding fragment thereof comprising at least one immunogenic peptide inserted into a variable region of the antibody or antigen-binding fragment thereof, the insertion comprising a deletion of an antibody or antigen-binding fragment sequence.

[0004] In another aspect, there is provided a dual function antigen binding molecule comprising: a. a first antibody or antigen-binding fragment thereof comprising at least one immunogenic peptide inserted into a CDR of the antibody or antigen-binding fragment thereof, the insertion comprising removal of a CDR sequence; and b. A second antibody capable of binding epidermal growth factor receptor (EGFR), wherein the antibody is selected from cetuximab, panitumumab and necitumumab or an antibody comprising at least 85% sequence identity thereto.

[0005] In some embodiments, the antibody or antigen-binding fragment thereof binds to a target cell, the target cell being a cancer cell, a dendritic cell, or both. In some embodiments, the first antibody or antigen-binding fragment thereof binds to a target cell, the target cell being a cancer cell, a dendritic cell, or both.

[0006] In some embodiments, the immunogenic peptide is a cancer-specific peptide.

[0007] In some embodiments, the cancer-specific peptide is selected from the peptide sequences provided in Table 1.

[0008] In some embodiments, the immunogenic peptide is a viral peptide.

[0009] In some embodiments, the viral peptide is derived from cytomegalovirus (CMV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), adenovirus, human papillomavirus (HPV) or influenza virus (FLU).

[0010] In some embodiments, the viral peptide is selected from the peptide sequences provided in Table 2 or Table 3.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a cell-penetrating sequence that directs the antibody or antigen-binding fragment thereof to the cytoplasm of a cell that binds the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a cell-penetrating sequence that directs the first antibody or antigen-binding fragment thereof to the cytoplasm of a cell that is bound by the first antibody or antigen-binding fragment thereof.

[0012] In some embodiments, the cell penetrating sequence is an endosomal escape domain (EED).

[0013] In some embodiments, the antibody lacking the immunogenic peptide is taken up into the endosomal pathway and delivered to the cytoplasm. In some embodiments, the first antibody or antigen-binding fragment thereof is taken up into the endosomal pathway and delivered to the cytoplasm.

[0014] In some embodiments, at least one CDR or a portion thereof of the antibody or antigen-binding fragment thereof is replaced with an immunogenic peptide. In some embodiments, at least one CDR or a portion thereof of the first antibody or antigen-binding fragment thereof is replaced with an immunogenic peptide.

[0015] In some embodiments, the CDRs are inactive CDRs that contribute little or nothing to binding to the target antigen.

[0016] In some embodiments, an inactive CDR comprises no more than two amino acids that contact the target antigen.

[0017] In some embodiments, the contact comprises a distance of 5 Å or less between an amino acid of a CDR and an amino acid of the target antigen.

[0018] In some embodiments, the insertions and deletions result in no or only minimal changes in the overall conformation of the antibody or antigen-binding fragment thereof, such that the antibody or antigen-binding fragment thereof binds its target antigen with an affinity comparable to that of an antibody or antigen-binding fragment thereof lacking the immunogenic peptide. In some embodiments, the insertions and deletions result in no or only minimal changes in the overall conformation of the first antibody or antigen-binding fragment thereof, such that the first antibody or antigen-binding fragment thereof binds its target antigen with an affinity comparable to that of an antibody or antigen-binding fragment thereof lacking the immunogenic peptide.

[0019] In some embodiments, at least one inactive CDR of the antigen binding region is replaced with a cell penetrating sequence.

[0020] In some embodiments, the target cell is a dendritic cell and the dendritic cell antigen is selected from CD40, CD205, CD206, CLEC9A, CLEC12A, CD209, and CD207.

[0021] In some embodiments, the target cell is a malignant immune cell and the immune cell antigen is selected from CD20, CD19, CD21, and CD22.

[0022] In some embodiments, the target cell is a cancer cell and the cancer cell antigen is selected from HER2, EGFR, EpCAM, PSMA, BCMA, CD123, CD33, CD38, CTLA, LAG-3, ICOS, 4-1BB, and PD-L1.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof lacks a chemical linker.

[0024] In some embodiments, the antigen-binding region, the immunogenic peptide, and the cell-penetrating sequence are each separated by a linker. In some embodiments, within the first antibody or antigen-binding fragment thereof, the antigen-binding region, the immunogenic peptide, and the cell-penetrating sequence are each separated by a linker.

[0025] In some embodiments, the immunogenic peptide is recognized by CD4 T cells, CD8 T cells, or both.

[0026] In some embodiments, the antibody prior to insertion of the immunogenic peptide is selected from the following: a. The antibody TMab4 comprising the heavy chain variable region of SEQ ID NO: 1021 and the light chain variable region of SEQ ID NO: 1022; b. antibody 3E10, comprising a heavy chain variable region of SEQ ID NO: 1023 and a light chain variable region of SEQ ID NO: 1024; and c. Antibody 71F12, comprising a heavy chain variable region of SEQ ID NO:1026 and a light chain variable region of SEQ ID NO:1027.

[0027] In some embodiments, the first antibody before the immunogenic peptide is inserted is selected from the following: a. The antibody TMab4 comprising the heavy chain variable region of SEQ ID NO: 1021 and the light chain variable region of SEQ ID NO: 1022; b. antibody 3E10, comprising a heavy chain variable region of SEQ ID NO: 1023 and a light chain variable region of SEQ ID NO: 1024; and c. Antibody 71F12, comprising a heavy chain variable region of SEQ ID NO:1026 and a light chain variable region of SEQ ID NO:1027.

[0028] In some embodiments, at least one of: a. an immunogenic peptide is inserted into CDRH1, CDRH2, CDRH3 or CDRL3 of TMab4; b. the immunogenic peptide is inserted into CDRL1 or CDRL2 of 3E10; and c. An immunogenic peptide is inserted into CDRL1 of 71F12.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof is: a. a light chain variable region of SEQ ID NO: 1022 and a heavy chain variable region selected from SEQ ID NOs: 1028-1040, 1043-1045, 1047-1055, and 1058-1059; b. a heavy chain variable region of SEQ ID NO: 1021 and a light chain variable region selected from SEQ ID NOs: 1041-1042, 1046, and 1056-1057; c. a heavy chain variable region of SEQ ID NO: 1023 and a light chain variable region selected from SEQ ID NOs: 1060-1065; d. A heavy chain variable region of SEQ ID NO: 1026 and a light chain variable region of SEQ ID NO: 1066; e. a light chain variable region of SEQ ID NO: 1027 and a heavy chain variable region of SEQ ID NO: 1067; Includes at least one of the following.

[0030] In some embodiments, the first antibody is: a. a light chain variable region of SEQ ID NO: 1022 and a heavy chain variable region selected from SEQ ID NOs: 1028-1040, 1043-1045, 1047-1055, and 1058-1059; b. a heavy chain variable region of SEQ ID NO: 1021 and a light chain variable region selected from SEQ ID NOs: 1041-1042, 1046, and 1056-1057; c. a heavy chain variable region of SEQ ID NO: 1023 and a light chain variable region selected from SEQ ID NOs: 1060-1065; d. a heavy chain variable region of SEQ ID NO: 1026 and a light chain variable region of SEQ ID NO: 1066; and e. a light chain variable region of SEQ ID NO: 1027 and a heavy chain variable region of SEQ ID NO: 1067; Includes at least one of the following.

[0031] In another embodiment, a dual function antigen-binding molecule is provided comprising a first antibody or antigen-binding fragment thereof comprising an antibody or antigen-binding fragment of the present invention, and a second antibody or antigen-binding fragment thereof capable of binding an antigen that is overexpressed on a target cancer cell.

[0032] In some embodiments, the antigen overexpressed on the target cancer cell is EGFR and the second antibody is selected from cetuximab, panitumumab and necitumumab.

[0033] In some embodiments, the first antibody and the second antibody comprise at least one modification that promotes heterodimerization and inhibits homodimerization.

[0034] In some embodiments, one of the first and second antibodies comprises a heavy chain constant region comprising SEQ ID NO:1074, and the other antibody comprises a heavy chain constant region comprising SEQ ID NO:1075.

[0035] In some embodiments, the dual function antigen binding molecule comprises two heavy chains and two light chains: a. the two heavy chains are SEQ ID NOs: 1088 and 1080 and the two light chains are SEQ ID NOs: 1087 and 1079; b. the two heavy chains are SEQ ID NOs: 1088 and 1082 and the two light chains are SEQ ID NOs: 1087 and 1081; c. the two heavy chains are SEQ ID NOs: 1090 and 1080 and the two light chains are SEQ ID NOs: 1089 and 1079; d. the two heavy chains are SEQ ID NOs: 1090 and 1082 and the two light chains are SEQ ID NOs: 1089 and 1081; or e. The two heavy chains are SEQ ID NOs: 1088 and 1086, and the two light chains are SEQ ID NOs: 1087 and 1085.

[0036] In another aspect, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment of the invention or a bifunctional antigen-binding molecule of the invention and a pharma- ceutically acceptable carrier, excipient or adjuvant.

[0037] In another embodiment, a nucleic acid molecule is provided comprising at least one open reading frame, the open reading frame encoding an antibody or antigen-binding fragment thereof of the invention or a dual function antigen-binding molecule of the invention.

[0038] In another aspect, an expression vector is provided comprising at least one regulatory element operably linked to a nucleic acid molecule of the invention.

[0039] In another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the invention, thereby treating cancer in the subject.

[0040] In some embodiments, the cancer overexpresses a cancer-specific antigen.

[0041] In some embodiments, the cancer is an EGFR positive cancer.

[0042] In some embodiments, the bifunctional antigen binding molecule is a cancer vaccine and comprises an antigen binding region capable of binding a dendritic cell antigen.

[0043] In another aspect, a method of engineering an antibody or antigen-binding fragment thereof is provided, the method comprising: a. selecting an antibody or antigen-binding fragment thereof of interest; b. conducting a structural analysis of the selected antibody or antigen-binding domain bound to its target; c. determining at least one CDR of the selected antibody or antigen-binding domain that is not required for binding to the target based on structural analysis; d. replacing at least one of the determined CDRs or a portion thereof with an immunogenic peptide; thereby engineering the antibody or antigen-binding fragment thereof.

[0044] In another aspect, a method of engineering an antibody or antigen-binding fragment thereof is provided, the method comprising: a. selecting an antibody or antigen-binding fragment thereof of interest; b. obtaining a database of immunogenic peptides; c. performing pairwise alignments of peptides of the variable regions of the selected antibody of interest or antigen-binding fragment thereof with immunogenic peptides of the database; d. determining peptides and immunogenic peptides derived from the selected antibodies or antigen-binding fragments thereof having alignment scores above a predefined threshold; and e. replacing the determined peptide from the selected antibody or antigen-binding fragment thereof with the determined immunogenic peptide; thereby engineering the antibody or antigen-binding fragment thereof.

[0045] In some embodiments, the methods further comprise optimizing the substitutions to result in as little perturbation as possible in the structure of the selected antibody or antigen-binding fragment thereof of interest.

[0046] In some embodiments, the engineered antibody or antigen-binding fragment thereof is an immunogenic peptide delivery antibody.

[0047] In some embodiments, step (a) comprises selecting an antibody or antigen-binding fragment thereof that binds to the surface of a target cell.

[0048] In some embodiments, step (a) comprises selecting an antibody or antigen-binding fragment thereof that, upon binding to the surface, is internalized and delivered to the cytosol of the target cell.

[0049] In some embodiments, the method further includes confirming at least one of delivery of the immunogenic peptide to the cytosol of the target cell, delivery of the immunogenic peptide in a complex with an HLA molecule to the surface of the target cell, and specific killing of the target cell by an effector cell specific for the immunogenic peptide.

[0050] In some embodiments, the method further comprises selecting a targeting antibody that binds to a protein on the surface of a target cell, and generating a dual function antigen binding molecule by combining the engineered antibody and the targeting antibody.

[0051] In some embodiments, combining comprises engineering the heavy chain constant region of the targeting antibody and the heavy chain constant region of the engineered antibody to promote heterodimerization and suppress homodimerization.

[0052] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and that various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0053] [Figure 1] FIG. 1 is a schematic diagram showing an antibody that engages its antigen and the presence of inactive CDRs that are not involved in binding. [Diagram 2] 1 is an image of an anti-PD-L1 antibody containing an inactive CDR and a Trojan horse antibody of the invention after the inactive CDR has been replaced with an immunogenic peptide. [Diagram 3] FIG. 1 is a diagram showing a stepwise process of the dendritic cell vaccination method of the present invention. [Figure 4] FIG. 1 is a step-by-step diagram of the B cell-targeting CD4+ T cell-mediated method of the present invention. [Diagram 5] FIG. 1 is a step-by-step diagram of the CD8+ T cell-mediated cancer killing method of the present invention. [Figure 6] 1 shows an overview of an embodiment of a method for producing Trojan horse antibodies. [Figure 7A-7C]7A is a bar graph of specific killing of cancer cells contacted with (7A) a TAb containing an immunogenic peptide inserted into CDRH1 of the TMab4(P1) antibody, (7B) a TAb containing an immunogenic peptide inserted into CDRH3 of the TMab4(P1) antibody, and (7C) a TAb containing an immunogenic peptide inserted into CDRL3 or CDRH2 of the TMab4(P1) antibody, and then co-cultured with peptide-specific effector cells. [Figure 8] 1 is a bar graph of specific killing of cancer cells contacted with TAbs containing an immunogenic peptide inserted into CDRL1 of the 3E10(P2) antibody and then co-cultured with peptide-specific effector cells. [Figure 9A-9B] 9A-9B are line graphs of apoptotic cancer cells cultured with effector cells alone (cells only), (9A) parental antibody 3E10 (P2) and TAb T2_11, and (9B) parental antibody TMab4 (P1) and TAb T18. [Figure 10] Schematic diagram of bifunctional Trojan horse antibodies and corresponding controls. The bi-TAb is labeled as FTAb and the control bi-TAb is labeled as cFTAb. [Figure 11] 1 is a histogram showing the binding of bi-TAbs to EGFR on the surface of cancer cells. Therapeutic antibodies are used as positive controls and fluorescently labeled secondary antibodies are used as negative controls. The top panel shows a concentration of 0.3 nM and the bottom panel shows a concentration of 3 nM. The bi-TAbs are labeled as FTAbs. [Figure 12] 1 is a bar graph showing the percentage of cancer cells presenting HLA-peptide complexes at different time points. Each bi-TAb (white bar) is compared to its negative control (gray bar) with the same killing module but without the immunogenic peptide. The bi-TAb is labeled as FTAb and the control bi-TAb is labeled as cFTAb. [Figure 13]1 is a line graph of the percentage of cancer cells killed by effector cells at various effector to target cell ratios. Each bi-TAb (light grey line) is compared to its negative control (black line). Points with antibody only, without effector cells, are included. The bi-TAbs are labeled as FTAbs and the control bi-TAbs are labeled as cFTAbs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The present invention provides, in some embodiments, an antibody or antigen-binding fragment thereof comprising at least one immunogenic peptide inserted into the variable region of the antibody or antigen-binding fragment thereof, the insertion comprising the removal of an antibody or antigen-binding fragment sequence. A dual-function antigen-binding molecule comprising an antibody or antigen-binding fragment of the present invention and a second antibody or antigen-binding fragment thereof can bind to an antigen that is overexpressed on a target cell. Also provided are nucleic acid molecules encoding the dual-function antigen-binding molecule, pharmaceutical compositions comprising the dual-function antigen-binding molecule, and methods of treating cancer by administering the dual-function antigen-binding molecule. Also provided are methods of producing the antibody or antigen-binding fragment.

[0055] The present invention is based on the unexpected discovery that antibodies / antigen-binding molecules can be used as a delivery system for immunogenic peptides. That is, highly immunogenic peptides can be specifically delivered to cancer cells, thereby enhancing immune surveillance against them. The antibody in this case has an antigen-binding domain against a cancer epitope that can bind a therapeutic molecule to the cancer cell. Upon endocytosis of the antibody / antigen-binding molecule, the immunogenic peptide can be delivered to the cytoplasm. This can be enhanced by the inclusion of a cell-penetrating sequence, or in particular, an endosomal escape sequence. However, endosomal escape is not essential as in some mechanisms, such as receptor-mediated transcytosis of antibodies, direct antibody delivery to the cytoplasm, etc. The therapeutic molecule is cleaved, releasing the immunogenic peptide, which can then be complexed with HLA molecules and presented on the cell surface, thus enhancing the immunogenicity of the cancer cell and enhancing immune surveillance against cancer and cancer killing.

[0056] Alternatively, the antigen-binding region can bind a dendritic cell antigen, which can deliver therapeutic molecules, including cancer cell antigens, to dendritic cells. Upon binding, the molecule is internalized, and the immunogenic cancer peptide can be cleaved from the remainder of the molecule of the invention and presented on the surface of the dendritic cell by HLA molecules. This then trains cytotoxic immune cells (T cells and NK cells) to target the immunogenic peptide and thus target the cancer.

[0057] According to a first aspect, an antigen-binding molecule is provided.

[0058] According to another aspect, a composition comprising an antigen-binding molecule of the present invention is provided.

[0059] According to another aspect, a nucleic acid molecule encoding an antigen-binding molecule of the present invention is provided.

[0060] Another aspect provides an expression vector comprising a nucleic acid molecule of the invention.

[0061] According to another aspect, a method for expressing a peptide on the surface of a target cell is provided, the method comprising contacting a target cell with an antigen-binding molecule of the present invention or a pharmaceutical composition of the present invention, thereby expressing the peptide on the surface of the target cell.

[0062] According to another aspect, a method for treating cancer in a subject is provided, the method comprising administering to the subject an antigen-binding molecule of the present invention or a pharmaceutical composition of the present invention, thereby treating cancer in a subject in need thereof.

[0063] According to another aspect, a composition of the invention is provided for use in expressing a peptide on the surface of a target cell. According to another aspect, a composition of the invention is provided for use in treating cancer. According to another aspect, a composition of the invention is provided for use in the manufacture of a medicament for the treatment of cancer.

[0064] In some embodiments, the antigen-binding molecule is a dual-function molecule. In some embodiments, the first function is to bind an antigen. In some embodiments, the second function is to enter a cell. In some embodiments, the second function is to deliver a peptide. In some embodiments, the peptide is an immunogenic peptide. In some embodiments, the second function is to deliver an immunogenic peptide into the cytoplasm of a cell.

[0065] In some embodiments, the molecule comprises an antigen binding region. In some embodiments, the region is a domain. In some embodiments, the region binds the antigen. In some embodiments, the antigen is on a target cell. In some embodiments, the antigen binding region is capable of binding to a target cell. In some embodiments, the antigen is a cancer antigen. In some embodiments, the cancer antigen is a cancer specific antigen. In some embodiments, the cancer antigen is an antigen on a cancer cell. In some embodiments, the antigen is an immune cell antigen. In some embodiments, the immune cell is selected from a dendritic cell, a B cell, a T cell, a neutrophil, a macrophage, a neutrophil, and a natural killer (NK) cell. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the antigen is a dendritic cell antigen. In some embodiments, the immune cell is a B cell. In some embodiments, the antigen is a B cell antigen. In some embodiments, the antigen is expressed on a cell. In some embodiments, the antigen is expressed on the cell surface. In some embodiments, the antigen is presented on the cell surface as an MHC molecule. In some embodiments, the MHC molecule is an MHC class I or class II molecule. In some embodiments, the MHC molecule is a protein complex of an antigen and an HLA protein. In some embodiments, the antigen is a cell surface protein. In some embodiments, the cell surface protein is a cell surface receptor. In some embodiments, the antigen binding region is capable of binding an antigen.

[0066] In some embodiments, the dendritic cell antigen is selected from CD40, CD205, CD206, CLEC9A, CLEC12A, CD209, and CD207. Markers of dendritic cells are well known in the art, and any such surface marker may be used as an antigen. In some embodiments, the dendritic cell antigen is CD40. Antigen binding domains that target dendritic cell antigens are well known in the art, and any such antigen binding domain may be used. As a non-limiting example, Fab516 specifically binds to CD40.

[0067] In some embodiments, the immune cell antigen is selected from CD20, CD19, CD21, and CD22. In some embodiments, the dendritic cell antigen is CD20. In some embodiments, the immune cell antigen is a B cell antigen. Markers for immune cells in general, and B cells in particular, are well known in the art, and any such surface marker may be used as an antigen. Antigen binding domains that target immune cell antigens are well known in the art, and any such antigen binding domain may be used. As a non-limiting example, Arzella specifically binds to CD20.

[0068] In some embodiments, the cancer cell antigen is selected from HER2, EGFR, EpCAM, PSMA, BCMA, CD123, CD33, CD38, CTLA, LAG-3, ICOS, 4-1BB and PD-L1. Cancer cell markers are well known in the art, and any such surface markers may be used as antigens. In some embodiments, the cancer cell antigen is PD-L1. Antigen binding domains that target immune cell antigens are well known in the art, and any such antigen binding domains may be used. As a non-limiting example, durvalumab specifically binds to PD-L1.

[0069] In some embodiments, the antigen binding domain is an antigen binding domain of an antibody. In some embodiments, the antigen binding domain is an antibody. In some embodiments, the antigen binding domain is an antibody or an antigen binding fragment thereof. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody is a single domain antibody. In some embodiments, the antibody is a full antibody.

[0070] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that contain at least one binding domain formed from the folding of polypeptide chains with a three-dimensional binding space with an internal surface shape and charge distribution complementary to the antigenic determinant features of the antigen. Antibodies typically have a tetrameric form, containing two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and may be antibodies that can be labeled in soluble or conjugated form, as well as fragments containing epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. Antibodies may be from any species. The term antibody also includes binding fragments, including but not limited to Fv, Fab, Fab', F(ab')2, single chain antibodies (svFC), dimeric variable regions (diabodies), and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including but not limited to an Fc region or a fragment thereof. Those skilled in the art will further appreciate that other fusion products may be produced, including but not limited to scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0071] The immunoglobulin molecule may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0072] An "antigen" is a molecule or a portion of a molecule that can induce antibody formation and be bound by an antibody. Antibody formation can occur in mice, rats, rabbits, pigs and other animals commonly used to generate antibodies, but can also occur in humans as a response to foreign antigens. An antigen can have one or more epitopes. The specific reaction referred to above is intended to indicate that an antigen will react highly selectively with its corresponding antibody and not with the numerous other antibodies that may be induced by other antigens.

[0073] The term "antigenic determinant" or "epitope" according to the present invention refers to a region of an antigen molecule that specifically reacts with a particular antibody. Epitope-derived peptide sequences can be used alone or in conjunction with a carrier moiety to immunize animals to generate additional polyclonal or monoclonal antibodies, applying methods known in the art. Immunoglobulin variable domains can also be analyzed using the IMGT information system (imgt.Cines.fr / ) (IMGT® / V-Quest) to identify variable region segments that contain CDRs. See, for example, Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0074] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence without reliance on experimental data, beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0075] The term "antibody" (also called immunoglobulin) is used in the broadest sense and specifically encompasses monoclonal antibodies and antibody fragments, so long as they exhibit the desired biological activity. In certain embodiments, the use of chimeric or humanized antibodies is also encompassed by the present invention.

[0076] The basic unit of antibody structure of natural origin is a heterotetrameric glycoprotein complex of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains, linked together by both non-covalent and disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. There are five human antibody classes (IgG, IgA, IgM, IgD, IgE), and within these classes, various subclasses are recognized based on structural differences, such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of the individual units, and differences in chain length and sequence. The class and subclass of an antibody is its isotype.

[0077] The amino-terminal regions of the heavy and light chains are more diverse in sequence than the carboxy-terminal regions and are therefore called variable domains. This part of the antibody structure confers the antigen-binding specificity of the antibody. The heavy chain variable (VH) domain and the light chain variable (VL) domain together form a single antigen-binding site, and therefore a basic immunoglobulin unit has two antigen-binding sites. Certain amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0078] The carboxy-terminal portions of the heavy and light chains form the constant domains, CH1, CH2, CH3, CL. There is much less diversity in these domains, but there are differences between animal species, and, in addition, within the same individual, there are several different antibody isotypes, each with different functions.

[0079] The term "framework region" or "FR" refers to amino acid residues in the variable domain of an antibody other than the hypervariable region amino acid residues as defined herein. The term "hypervariable region" as used herein refers to amino acid residues in the variable domain of an antibody that are involved in antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". The CDRs are primarily involved in binding to an epitope of an antigen. The extent of the FRs and CDRs has been precisely defined (see Kabat et al.). In some embodiments, the CDRs are determined using the KABAT system. In some embodiments, the CDRs are determined using Clothia. In some embodiments, the Clothia system is a modified Clothia system (Martin system).

[0080] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that lacks or does not include an immunogenic peptide. In some embodiments, the lack of or does not include an immunogenic peptide is prior to insertion of the immunogenic peptide. In some embodiments, the antibody or antigen-binding fragment thereof binds to a target cell. In some embodiments, the antibody or antigen-binding fragment thereof adheres to the surface of a target cell. In some embodiments, the antibody or antigen-binding fragment thereof is a cell-permeable antibody. In some embodiments, the antibody or antigen-binding fragment thereof is phagocytosed into the cell. In some embodiments, the antibody or antigen-binding fragment thereof is internalized into the cell. In some embodiments, into the cell is into the endosomal pathway of the cell. In some embodiments, the antibody or antigen-binding fragment thereof is incorporated into the endosomal pathway. In some embodiments, the antibody or antigen-binding fragment thereof escapes the endosomal pathway. In some embodiments, the antibody or antigen-binding fragment thereof is delivered into the cytoplasm of the cell that binds it. In some embodiments, the antibody or antigen-binding fragment thereof is a DNA-binding antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a lupus antibody.

[0081] In some embodiments, the antibody or antigen-binding fragment thereof is a Tmab4 antibody. In some embodiments, Tmab4 comprises a heavy chain variable region of SEQ ID NO: 1021 or an analog or homolog that comprises at least 85% sequence identity and is capable of binding cells and reaching the cytosol. In some embodiments, Tmab4 comprises a light chain variable region of SEQ ID NO: 1022 or an analog or homolog that comprises at least 85% sequence identity and is capable of binding cells and reaching the cytosol. In some embodiments, the antibody or antigen-binding fragment thereof is a 3E10 antibody. In some embodiments, 3E10 comprises a heavy chain variable region of SEQ ID NO: 1023 or an analog or homolog that comprises at least 85% sequence identity and is capable of binding cells and reaching the cytosol. In some embodiments, 3E10 comprises a light chain variable region of SEQ ID NO: 1024 or an analog or homolog that comprises at least 85% sequence identity and is capable of binding cells and reaching the cytosol. In some embodiments, the antibody or antigen-binding fragment thereof is a 71F12 antibody. In some embodiments, 71F12 comprises a heavy chain variable region of SEQ ID NO: 1026, or an analog or homolog that comprises at least 85% sequence identity and is capable of binding cells and accessing the cytosol. In some embodiments, 71F12 comprises a light chain variable region of SEQ ID NO: 1027, or an analog or homolog that comprises at least 85% sequence identity and is capable of binding cells and accessing the cytosol.

[0082] In some embodiments, the antigen binding molecule comprises at least one immunogenic peptide. In some embodiments, the antigen binding molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunogenic peptides. Each possibility represents a separate embodiment of the invention. In some embodiments, the immunogenic peptide is a foreign immunogenic peptide. In some embodiments, the immunogenic peptide is inserted into the antigen binding molecule. In some embodiments, the immunogenic peptide is inserted into a variable region of an antibody or antigen-binding fragment thereof. In some embodiments, the immunogenic peptide is not a naturally occurring part of the antigen binding molecule. In some embodiments, the peptide is a sequence of amino acids. In some embodiments, a sequence of immunogenic amino acids is inserted into the sequence of the antigen binding molecule. In some embodiments, the immunogenic peptide replaces an amino acid of the antigen binding molecule. In some embodiments, the insertion of the immunogenic peptide comprises the removal of an amino acid sequence. In some embodiments, the amino acid sequence is the sequence of an antibody or antigen-binding fragment thereof. In some embodiments, the immunogenic peptide is not artificially linked to the antigen binding molecule. In some embodiments, the antigen binding molecule is a recombinant molecule. In some embodiments, the recombinant molecule comprises an amino acid sequence of an immunogenic peptide. In some embodiments, the immunogenic peptide is not linked to the antigen-binding molecule by a chemical bond. In some embodiments, the chemical bond is any bond other than a peptide bond. In some embodiments, the chemical bond is any bond other than an amino acid bond. In some embodiments, the immunogenic peptide is linked to the antigen-binding molecule by a peptide bond, an amino acid bond, or both.

[0083] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, as used herein, the terms "peptide", "polypeptide" and "protein" encompass natural peptides, peptidomimetics (typically containing non-peptide bonds or other synthetic modifications) and peptide analogs peptoids and semi-peptoids or any combination thereof. In another embodiment, the described peptides, polypeptides and proteins have modifications that make them more stable in the body or more permeable into cells. In one embodiment, the terms "peptide", "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms "peptide", "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0084] As used herein, the term "recombinant protein" refers to a protein that is encoded by recombinant DNA and is therefore not of natural origin. The term "recombinant DNA" refers to a DNA molecule that has been formed by laboratory techniques of genetic recombination. Generally, this recombinant DNA is in the form of a vector, plasmid or virus that is used to express the recombinant protein in cells.

[0085] In some embodiments, the immunogenic peptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the immunogenic peptide comprises at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the immunogenic peptide is 3-20, 3-15, 3-12, 3-11, 3-10, 3-7, 5-20, 5-15, 5-12, 5-11, 5-10, 7-20, 7-15, 7-12, 7-11, or 7-10 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the immunogenic peptide is 8-11 amino acids. In some embodiments, the immunogenic peptide consists of 8 amino acids. In some embodiments, the immunogenic peptide consists of 9 amino acids. In some embodiments, the immunogenic peptide consists of 10 amino acids. In some embodiments, the immunogenic peptide consists of 11 amino acids.

[0086] As used herein, the term "immunogenic peptide" refers to an amino acid sequence that generates an immune response when exposed to the human immune system. In some embodiments, the immunogenic peptide is a non-human peptide. In some embodiments, the immunogenic peptide generates an immune response from an immune cell. In some embodiments, the immunogenic peptide is recognized in an immune response from an immune cell. In some embodiments, being recognized is being bound by it. In some embodiments, the immunogenic peptide generates an immune response from a dendritic cell. In some embodiments, the immunogenic peptide is presented on a cell surface. In some embodiments, the immunogenic peptide is presented as an MHC molecule. In some embodiments, the immunogenic peptide is presented in a complex with HLA. In some embodiments, the immunogenic peptide generates an immune response from a T cell. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is selected from a CD4 T cell and a CD8 T cell. In some embodiments, the T cell is a CD4 T cell. In some embodiments, the T cell is a CD8 T cell. In some embodiments, the immunogenic peptide comprises a CD4 epitope, a CD8 epitope, or both. In some embodiments, the immunogenic peptide generates an immune response from NK cells. In some embodiments, the immune response is an increased immune response. In some embodiments, the immunogenicity comprises increased immunogenicity. In some embodiments, increased is relative to a control peptide. In some embodiments, the control peptide is a human peptide. In some embodiments, the control peptide is a non-cancerous peptide. In some embodiments, the control peptide is a non-immunogenic peptide.

[0087] In some embodiments, the immunogenic peptide is a cancer peptide. In some embodiments, the cancer peptide is a cancer-specific peptide. In some embodiments, the cancer peptide is an increased cancer peptide. In some embodiments, the cancer peptide is a peptide with increased surface expression in cancer cells. In some embodiments, the cancer peptide is a peptide provided in Table 1. In some embodiments, the cancer peptide is selected from the sequences provided in Table 1. In some embodiments, the cancer peptide is selected from the sequences provided in SEQ ID NOs: 702-1020. Cancer peptides are well known in the art and can be found, for example, at the cancer antigen peptide database: caped.icp.ucl.ac.be / Peptide / search. [Table 1] TIFF2024538427000003.tif242153TIFF2024538427000004.tif242153TIFF2024538427000005.tif242151TIFF20245384270 00006.tif242152TIFF2024538427000007.tif242153TIFF2024538427000008.tif242152TIFF2024538427000009.tif210159

[0088] In some embodiments, the immunogenic peptide is a non-human peptide. In some embodiments, the immunogenic peptide is a viral peptide. In some embodiments, the immunogenic peptide is a bacterial peptide. Viral peptides are well known in the art and any such peptide may be employed. Such peptides can be found, for example, in the Immune Epitope Database (IEDB) and VDJDB databases: iedb.org and vdjdb.cdr3.net. In some embodiments, the viral peptide is from a virus selected from cytomegalovirus (CMV), Epstein-Barr virus (EBV) or influenza virus (FLU). In some embodiments, the viral peptide is from a virus selected from CMV, EBV, FLU, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), adenovirus and human papillomavirus (HPV). In some embodiments, the virus is CMV. In some embodiments, the virus is EBV. In some embodiments, the virus is FLU. In some embodiments, the virus is SARS-CoV2. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is HPV. In some embodiments, the viral peptide is a peptide provided in Table 2. In some embodiments, the viral peptide is selected from the sequences provided in Table 2. In some embodiments, the viral peptide is a peptide provided in Table 3. In some embodiments, the viral peptide is selected from the sequences provided in Table 3. In some embodiments, the viral peptide is selected from SEQ ID NOs: 1-701. In some embodiments, the viral peptide is selected from SEQ ID NOs: 1-695. In some embodiments, the viral peptide is selected from SEQ ID NOs: 1-11. In some embodiments, the viral peptide is selected from SEQ ID NOs: 1-9. In some embodiments, the viral peptide is selected from SEQ ID NOs: 1-5. In some embodiments, the viral peptide is SEQ ID NO: 1. In some embodiments, the viral peptide is SEQ ID NO: 2.In some embodiments, the viral peptide is SEQ ID NO:3. In some embodiments, the viral peptide is SEQ ID NO:4. In some embodiments, the viral peptide is SEQ ID NO:5. In some embodiments, the viral peptide is SEQ ID NO:6. In some embodiments, the viral peptide is SEQ ID NO:7. In some embodiments, the viral peptide is SEQ ID NO:8. In some embodiments, the viral peptide is SEQ ID NO:9. In some embodiments, the viral peptide is SEQ ID NO:10. In some embodiments, the viral peptide is SEQ ID NO:11. In some embodiments, the viral peptide is selected from SEQ ID NOs:1, 2, 3, 4, 5, 151, 197, 471, 677, 696, 697, 698, 699, 700, and 701.

[0089] [Table 2] [Table 3] TIFF2024538427000012.tif242151TIFF2024538427000013.tif242152TIFF20245384270 00014.tif242151TIFF2024538427000015.tif242151TIFF2024538427000016.tif171159

[0090] In some embodiments, the immunogenic peptide is inserted into a complementarity determining region (CDR) of the antibody or antigen-binding fragment thereof. In some embodiments, the insertion of the immunogenic peptide comprises removal of a CDR sequence. In some embodiments, at least one CDR of the antibody or antigen-binding fragment thereof is replaced with an immunogenic peptide. In some embodiments, the CDR is the entire CDR. In some embodiments, the CDR is at least a portion of a CDR. In some embodiments, the CDR sequence comprises the sequence of at least a portion of a CDR. In some embodiments, the portion is at least 4 amino acids. In some embodiments, the portion is at least 5 amino acids. In some embodiments, the portion is at least 6 amino acids. In some embodiments, the portion is at least 7 amino acids. In some embodiments, the portion is at least 8 amino acids. In some embodiments, the portion is at least 9 amino acids. In some embodiments, the insertion of the immunogenic peptide comprises removal of a CDR. In some embodiments, the insertion of the immunogenic peptide comprises removal of at least a portion of a CDR.

[0091] In some embodiments, the replacement of a CDR or a portion of a CDR also includes replacement of at least one amino acid adjacent to the CDR. In some embodiments, the adjacent is N-terminal to the CDR. In some embodiments, the adjacent is C-terminal to the CDR. In some embodiments, the at least one amino acid is at least 1, 2, 3, 4, or 5 amino acids directly adjacent to the CDR. Each possibility represents a separate embodiment of the invention. In some embodiments, the at least one amino acid is 4-5 amino acids. In some embodiments, the at least one amino acid is 4 amino acids. In some embodiments, the at least one amino acid is 5 amino acids. In some embodiments, the flanking region is 5, 6, 7, 8, 9, or 10 amino acids or less. Each possibility represents a separate embodiment of the invention. In some embodiments, the flanking region is 5 amino acids or less. In some embodiments, the flanking region is 10 amino acids or less. In some embodiments, the flanking region comprises the stem of the CDR loop. It will be understood by those skilled in the art that in order to preserve the structure and cause as little disturbance as possible, it may be necessary to replace parts of the regions adjacent to the CDRs as well as the CDRs themselves (or parts thereof).

[0092] In some embodiments, CDRH1 of TMab4 comprises or consists of amino acids 26-33 of SEQ ID NO:1021. In some embodiments, CDRH2 of TMab4 comprises or consists of amino acids 51-58 of SEQ ID NO:1021. In some embodiments, CDRH3 of TMab4 comprises or consists of amino acids 97-109 of SEQ ID NO:1021. In some embodiments, CDRL1 of TMab4 comprises or consists of amino acids 27-38 of SEQ ID NO:1022. In some embodiments, CDRL2 of TMab4 comprises or consists of amino acids 56-58 of SEQ ID NO:1022. In some embodiments, CDRL3 of TMab4 comprises or consists of amino acids 95-103 of SEQ ID NO:1022. In some embodiments, CDRH1 of 3E10 comprises or consists of amino acids 26-33 of SEQ ID NO:1023. In some embodiments, CDRH2 of 3E10 comprises or consists of amino acids 51-58 of SEQ ID NO:1023. In some embodiments, CDRH3 of 3E10 comprises or consists of amino acids 97-105 of SEQ ID NO: 1023. In some embodiments, CDRL1 of 3E10 comprises or consists of amino acids 27-36 of SEQ ID NO: 1024. In some embodiments, CDRL2 of 3E10 comprises or consists of amino acids 54-56 of SEQ ID NO: 1024. In some embodiments, CDRL3 of 3E10 comprises or consists of amino acids 93-101 of SEQ ID NO: 1024. In some embodiments, CDRH1 of 71F12 comprises or consists of amino acids 26-33 of SEQ ID NO: 1026. In some embodiments, CDRH2 of 71F12 comprises or consists of amino acids 51-57 of SEQ ID NO: 1026. In some embodiments, CDRH3 of 71F12 comprises or consists of amino acids 96-105 of SEQ ID NO: 1026. In some embodiments, CDRL1 of 71F12 comprises, or consists of, amino acids 26 to 34 of SEQ ID NO: 1027. In some embodiments, CDRL2 of 71F12 comprises, or consists of, amino acids 52 to 54 of SEQ ID NO:1027.In some embodiments, CDRL3 of 71F12 comprises or consists of amino acids 91-100 of SEQ ID NO:1027.

[0093] In some embodiments, the antigen binding molecule comprises a cell penetrating sequence. In some embodiments, the cell penetrating sequence is a cell penetrating domain. In some embodiments, the cell penetrating sequence is a cell penetrating peptide. In some embodiments, the cell penetrating sequence directs a molecule to the interior of a cell. In some embodiments, the cell penetrating sequence delivers a molecule to the interior of a cell. In some embodiments, the cell penetrating sequence allows entry of a molecule into the interior of a cell. In some embodiments, the interior of a cell is an endosome. In some embodiments, the interior of a cell is the cytoplasm. In some embodiments, delivery to the cytoplasm includes exit from an endosome. In some embodiments, delivery to the cytoplasm includes escape from an endosome. In some embodiments, the endosome is an endosomal pathway. In some embodiments, the cell penetrating sequence is a peptide penetrating domain (PTD). In some embodiments, the cell penetrating sequence is a cell penetrating peptide (CPP). In some embodiments, the cell penetrating sequence is an endosomal escape domain (EED). Peptide sequences that allow entry into the cytoplasm, and in particular escape from the endosomal pathway after endocytosis, are well known in the art, and any such peptide sequence may be employed.

[0094] Both CDRL1 and CDRL3 of Tmab4 are known in the art to be involved in antibody penetration (see Kim et al., "Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediated endosomal escape", J Control Release, 2016, 10;235:165-175, and Choi et al., "A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells", Mabs, 2014;6(6):1402-14, which are incorporated herein by reference in their entireties). In some embodiments, the cell-penetrating sequence comprises CDRL1 of Tmab4. In some embodiments, the cell-penetrating sequence consists of CDRL1 of Tmab4. In some embodiments, the cell-penetrating sequence comprises CDRL3 of Tmab4. In some embodiments, the cell-penetrating sequence consists of CDRL3 of Tmab4. Methods for grafting cell-penetrating sequences into other antibodies (such as the method provided by Chio et al.) are known in the art.

[0095] In some embodiments, the antigen binding molecule is a bispecific antibody. In some embodiments, the antigen binding molecule is a bispecific antibody fragment of an antibody. In some embodiments, the antigen binding molecule comprises multiple antigen binding regions. In some embodiments, the antigen binding molecule comprises at least two antigen binding regions. In some embodiments, the antigen binding molecule comprises two antigen binding regions. In some embodiments, at least one antigen binding region is an antigen binding region capable of binding to a target cell. In some embodiments, at least one antigen binding region is mutated to comprise an immunogenic peptide. In some embodiments, at least one antigen binding region is mutated to comprise a cell penetrating sequence. In some embodiments, at least one antigen binding region is mutated to comprise an immunogenic peptide, a cell penetrating sequence, or both. In some embodiments, the mutation is a mutation in a complementarity determining region (CDR). In some embodiments, a CDR of the antigen binding region is mutated. In some embodiments, the mutated is a substituted. In some embodiments, the substituted is replaced by an immunogenic peptide. In some embodiments, the substituted is replaced by a cell penetrating sequence.

[0096] In some embodiments, the CDR is an inactive CDR. In some embodiments, the inactive CDR does not contribute to binding to the target antigen. In some embodiments, the binding is binding of the antigen-binding region comprising the CDR to the target antigen. In some embodiments, the inactive CDR makes little or no contribution to binding. In some embodiments, the inactive CDR comprises no more than two amino acids that contact the target antigen. In some embodiments, the inactive CDR comprises fewer than two amino acids that contact the target antigen. In some embodiments, the inactive CDR comprises 2, 1, or 0 amino acids that contact the target antigen. In some embodiments, the inactive CDR comprises 2 amino acids that contact the target antigen. In some embodiments, the inactive CDR comprises one amino acid that contacts the target antigen. In some embodiments, the inactive CDR does not comprise an amino acid that contacts the target antigen. In some embodiments, the inactive CDR does not contact the target antigen. In some embodiments, the contact comprises a distance of 3, 5, 7, 9, or 10 Å or less. Each possibility represents a separate embodiment of the invention. In some embodiments, the contact comprises a distance of 5 Å or less. In some embodiments, the distance is the distance between an amino acid of the CDR and an amino acid of the target antigen. In some embodiments, the distance is the distance that exists when the antigen-binding domain is bound to an antigen. In some embodiments, the distance is the distance during a crystallographic study of the binding.

[0097] In some embodiments, mutation or substitution of an inactive CDR does not reduce binding. In some embodiments, not reducing binding is not significantly reducing binding. In some embodiments, mutation or substitution of an inactive CDR does not abolish binding. In some embodiments, a significant reduction is a reduction in binding of more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 95, or 99%. Each possibility represents a separate embodiment of the invention. In some embodiments, a significant reduction is a reduction in binding of more than 10%. In some embodiments, a significant reduction is a reduction in binding of more than 20%. In some embodiments, mutation or substitution of an inactive CDR does not reduce binding by 100%.

[0098] In some embodiments, the mutation or substitution of the inactive CDR does not reduce cell penetration. In some embodiments, not reducing cell penetration is not significantly reducing cell penetration. In some embodiments, the mutation or substitution of the inactive CDR does not abrogate cell penetration. In some embodiments, a significant reduction is a reduction in penetration of more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 95, or 99%. Each possibility represents a separate embodiment of the invention. In some embodiments, a significant reduction is a reduction in penetration of more than 10%. In some embodiments, a significant reduction is a reduction in penetration of more than 20%. In some embodiments, the mutation or substitution of the inactive CDR does not reduce penetration by 100%. In some embodiments, the mutation or substitution of the inactive CDR does not reduce penetration to the level of a control antibody. In some embodiments, the control antibody is an antibody that does not enter cells. Antibodies that do not enter cells are well known in the art and include, for example, adalimumab and muromonab. In some embodiments, not reducing cell penetration includes retaining penetration substantially equivalent to binding of an antibody or antigen-binding fragment thereof lacking or not comprising the immunogenic peptide.

[0099] In some embodiments, the insertion of the immunogenic peptide and the removal of the CDR sequences does not result in a change in the conformation of the antibody or antigen-binding fragment thereof. In some embodiments, the insertion of the immunogenic peptide and the removal of the CDR sequences results in a minimal change in the conformation of the antibody or antigen-binding fragment thereof. In some embodiments, the conformation is the overall conformation. In some embodiments, the conformation is a 3D structure. In some embodiments, the conformation is a tertiary structure. In some embodiments, the change is a disorder. In some embodiments, the minimal change is without loss of binding. In some embodiments, the minimal change is less than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% change. Each possibility represents a separate embodiment of the invention. In some embodiments, the minimal change includes binding to the target antigen with an affinity equivalent to an antibody or antigen-binding fragment thereof lacking or not comprising the immunogenic peptide. In some embodiments, the equivalent is without a loss of affinity. In some embodiments, equivalence is no more than a 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% decrease in affinity. Each possibility represents a separate embodiment of the present invention. In some embodiments, equivalence is no more than a 10% decrease in affinity. In some embodiments, equivalence is no more than a 20% decrease in affinity.

[0100] In some embodiments, the mutated CDR is in an antigen-binding region capable of binding target cells. In some embodiments, the mutated CDR is not present in an antigen-binding region capable of binding target cells. In some embodiments, the mutated CDR is in an antigen-binding region other than the antigen-binding region capable of binding target cells. In some embodiments, an immunogenic peptide replaces a CDR in an antigen-binding region capable of binding target cells. In some embodiments, a cell-penetrating sequence replaces a CDR in an antigen-binding region other than the antigen-binding region that binds target cells. In some embodiments, the immunogenic peptide and the cell-penetrating sequence are in the same antigen-binding region. In some embodiments, the immunogenic peptide and the cell-penetrating sequence are in different antigen-binding regions.

[0101] In some embodiments, the target cell is a dendritic cell, the dendritic cell antigen is CD40, the antigen binding region capable of binding to CD40 is Fab516, and the light chain CDRL2 is replaced by the cell penetrating sequence, the immunogenic peptide, or both.

[0102] In some embodiments, the target cell is a B cell, the B cell antigen is CD20, the antigen-binding region capable of binding to CD20 is Arsela, and at least one of the heavy chain CDRH1, the light chain CDRL1, and the light chain CDRL2 is replaced by the cell-penetrating sequence, the immunogenic peptide, or both. In some embodiments, replaced is mutated to include a peptide or sequence. In some embodiments, the heavy chain CDRH1 is replaced. In some embodiments, the light chain CDRL1 is replaced. In some embodiments, the light chain CDRL2 is replaced.

[0103] In some embodiments, the target cell is a cancer, the cancer cell antigen is PD-L1, the antigen-binding region capable of binding to PD-L1 is durvalumab, and at least one of the heavy chain CDRH1, the light chain CDRL2 is replaced by the cell-penetrating sequence, the immunogenic peptide, or both. In some embodiments, replaced is mutated to include a peptide or sequence. In some embodiments, the heavy chain CDRH1 is replaced. In some embodiments, the light chain CDRL2 is replaced.

[0104] In some embodiments, the antibody is TMab4 and the immunogenic peptide is inserted into any one of CDRH1, CDRH2, CDRH3 and CDRL3. In some embodiments, the antibody is TMab4 and the immunogenic peptide is inserted into CDRH1. In some embodiments, the antibody is TMab4 and the immunogenic peptide is inserted into CDRH2. In some embodiments, the antibody is TMab4 and the immunogenic peptide is inserted into CDRH3. In some embodiments, the antibody is TMab4 and the immunogenic peptide is inserted into CDRL3. In some embodiments, SEQ ID NO:1 is inserted into CDRH1 of TMab4. In some embodiments, SEQ ID NO:2 is inserted into CDRH1 of TMab4. In some embodiments, SEQ ID NO:3 is inserted into CDRH1 of TMab4. In some embodiments, SEQ ID NO:4 is inserted into CDRH1 of TMab4. In some embodiments, SEQ ID NO:5 is inserted into CDRH1 of TMab4. In some embodiments, SEQ ID NO:1 is inserted into CDRH2 of TMab4. In some embodiments, SEQ ID NO:2 is inserted into CDRH2 of TMab4. In some embodiments, SEQ ID NO:3 is inserted into CDRH2 of TMab4. In some embodiments, SEQ ID NO:4 is inserted into CDRH2 of TMab4. In some embodiments, SEQ ID NO:5 is inserted into CDRH2 of TMab4. In some embodiments, SEQ ID NO:1 is inserted into CDRH3 of TMab4. In some embodiments, SEQ ID NO:2 is inserted into CDRH3 of TMab4. In some embodiments, SEQ ID NO:3 is inserted into CDRH3 of TMab4. In some embodiments, SEQ ID NO:4 is inserted into CDRH3 of TMab4. In some embodiments, SEQ ID NO:5 is inserted into CDRH3 of TMab4. In some embodiments, SEQ ID NO:1 is inserted into CDRL3 of TMab4. In some embodiments, SEQ ID NO:2 is inserted into CDRL3 of TMab4. In some embodiments, SEQ ID NO:3 is inserted into CDRL3 of TMab4.In some embodiments, SEQ ID NO:4 is inserted into CDRL3 of TMab4. In some embodiments, SEQ ID NO:5 is inserted into CDRL3 of TMab4. In some embodiments, SEQ ID NO:6 is inserted into CDRH1 of TMab4. In some embodiments, SEQ ID NO:7 is inserted into CDRH23 of TMab4. In some embodiments, SEQ ID NO:8 is inserted in place of amino acids 14-22 of the light chain of TMab4. In some embodiments, into CDRH1, a substitution of amino acids 25-33 of the heavy chain is included. In some embodiments, into CDRH1, a substitution of amino acids 26-33 of the heavy chain is included. In some embodiments, into CDRH1, a substitution of amino acids 26-32 of the heavy chain is included. In some embodiments, into CDRH1, a substitution of amino acids 27-33 of the heavy chain is included. In some embodiments, into CDRH1, a substitution of amino acids 28-33 of the heavy chain is included. In some embodiments, into CDRH1, a substitution of amino acids 22-30 of the heavy chain is included. In some embodiments, into CDRH1, substitutions are made of amino acids 22-29 of the heavy chain. In some embodiments, into CDRH1, substitutions are made of amino acids 26-31 of the heavy chain. In some embodiments, into CDRH1, substitutions are made of amino acids 23-32 of the heavy chain. In some embodiments, into CDRH1, substitutions are made of amino acids 23-31 of the heavy chain. In some embodiments, into CDRH1, substitutions are made of amino acids 28-35 of the heavy chain. In some embodiments, into CDRH3, substitutions are made of amino acids 100-108 of the heavy chain. In some embodiments, into CDRH3, substitutions are made of amino acids 99-106 of the heavy chain. In some embodiments, into CDRH3, substitutions are made of amino acids 99-105 of the heavy chain. In some embodiments, into CDRH3, substitutions are made of amino acids 100-106 of the heavy chain. In some embodiments, into CDRH3, a substitution is made of amino acids 100-105 of the heavy chain. In some embodiments, into CDRH3, a substitution is made of amino acids 100-104 of the heavy chain.In some embodiments, into CDRH3, it comprises substituting amino acids 99-107 of the heavy chain. In some embodiments, into CDRH2, it comprises substituting amino acids 52-59 of the heavy chain. In some embodiments, into CDRH2, it comprises substituting amino acids 52-60 of the heavy chain. In some embodiments, into CDRL3, it comprises substituting amino acids 97-103 of the light chain. In some embodiments, into CDRL3, it comprises substituting amino acids 98-103 of the light chain. In some embodiments, into CDRL3, it comprises substituting amino acids 96-104 of the light chain. In some embodiments, into CDRL3, it comprises substituting amino acids 98-104 of the light chain.

[0105] In some embodiments, the antibody is 3E10 and the immunogenic peptide is inserted into either one of CDRL1 and CDRL2. In some embodiments, the antibody is 3E10 and the immunogenic peptide is inserted into CDRL1. In some embodiments, the antibody is 3E10 and the immunogenic peptide is inserted into CDRL2. In some embodiments, SEQ ID NO:1 is inserted into CDRL1 of 3E10. In some embodiments, SEQ ID NO:2 is inserted into CDRL1 of 3E10. In some embodiments, SEQ ID NO:3 is inserted into CDRL1 of 3E10. In some embodiments, SEQ ID NO:4 is inserted into CDRL1 of 3E10. In some embodiments, SEQ ID NO:5 is inserted into CDRL1 of 3E10. In some embodiments, SEQ ID NO:1 is inserted into CDRL2 of 3E10. In some embodiments, SEQ ID NO:2 is inserted into CDRL2 of 3E10. In some embodiments, SEQ ID NO:3 is inserted into CDRL2 of 3E10. In some embodiments, SEQ ID NO:4 is inserted into CDRL2 of 3E10. In some embodiments, SEQ ID NO:5 is inserted into CDRL2 of 3E10. In some embodiments, SEQ ID NO:6 is inserted into CDRL1 of 3E10. In some embodiments, SEQ ID NO:9 is inserted into CDRL2 of 3E10. In some embodiments, into CDRL1 comprises substituting amino acids 27-35 of the light chain. In some embodiments, into CDRL1 comprises substituting amino acids 28-36 of the light chain. In some embodiments, into CDRL2 comprises substituting amino acids 50-58 of the light chain.

[0106] In some embodiments, the antibody is 71F12 and the immunogenic peptide is inserted into CDRL1. In some embodiments, SEQ ID NO:1 is inserted into CDRL1 of 71F12. In some embodiments, SEQ ID NO:2 is inserted into CDRL1 of 71F12. In some embodiments, SEQ ID NO:3 is inserted into CDRL1 of 71F12. In some embodiments, SEQ ID NO:4 is inserted into CDRL1 of 71F12. In some embodiments, SEQ ID NO:5 is inserted into CDRL1 of 71F12. In some embodiments, into CDRL1 comprises substituting amino acids 28-36 of the light chain. In some embodiments, into CDRL1 comprises substituting amino acids 26-34 of the light chain. In some embodiments, into CDRL2 comprises substituting amino acids 50-58 of the light chain.

[0107] In some embodiments, the antibody is a commercially available antibody. In some embodiments, the antibody enters into cells bound at a level equivalent to any one of TMab4, 3E10, and 71F12. In some embodiments, the antibody enters into cells bound at a level equivalent to TMab4. In some embodiments, the antibody enters into cells bound at a level equivalent to 3E10. In some embodiments, the antibody enters into cells bound at a level equivalent to 71F12. In some embodiments, equivalent involves penetration that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% of the original antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, equivalent involves penetration that is at least 80% of the original antibody. In some embodiments, equivalent involves penetration that is at least 90% of the original antibody.

[0108] In some embodiments, the antigen-binding region and the immunogenic peptide are part of the same amino acid chain. In some embodiments, the antigen-binding region and the cell-penetrating sequence are part of the same amino acid chain. In some embodiments, the immunogenic peptide and the cell-penetrating sequence are part of the same amino acid chain. In some embodiments, the antigen-binding molecule of the present invention is a single fusion protein. In some embodiments, the antigen-binding molecule of the present invention is a single amino acid chain.

[0109] In some embodiments, the antigen binding region and the immunogenic peptide are separated by a linker. In some embodiments, the antigen binding region and the cell penetrating sequence are separated by a linker. In some embodiments, the immunogenic and cell penetrating sequences are separated by a linker. In some embodiments, the linker is not a chemical linker. In some embodiments, the linker is not an artificial linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker comprises or consists of at least 1, 2, 3, 4, or 5 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises or consists of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Each possibility represents a separate embodiment of the invention.

[0110] In some embodiments, the antibody is selected from antibodies T1-T19, T1_30-T1_35, T1_39-T1_45 and T1_47. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 1028-1040, 1043-1045, 1047-1055, and 1058-1059 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the analog or homolog comprises an immunogenic peptide. In some embodiments, the analog or homolog is capable of binding cells. In some embodiments, the analog or homolog is delivered to the cytosol upon cell binding. In some embodiments, delivery to the cytosol comprises presentation of the immunogenic peptide on the surface of the bound cell. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1028 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1029 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1030 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1031 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1032 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1033, or an analog or homolog comprising at least 85% sequence identity.In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1034 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1035 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1036 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1037 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1038 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1039, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1040, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1043, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1044, or an analog or homolog comprising at least 85% sequence identity.In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1045 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1047 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1048 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1049 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1050 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1051, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1052, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1053, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1054, or an analog or homolog comprising at least 85% sequence identity.In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1055, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1058, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1059, or an analog or homolog comprising at least 85% sequence identity.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising a sequence selected from SEQ ID NOs: 1041-1042, 1046, and 1056-1057, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1041, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1042, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1046, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1056, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1057, or an analog or homolog comprising at least 85% sequence identity.

[0112] In some embodiments, the antibody is selected from antibodies T2_6, T2_11-T2_13, T2_20 and T2_23. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising a sequence selected from SEQ ID NOs: 1060-1065, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1060, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1061, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1062, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1063, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1064, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1065, or an analog or homolog comprising at least 85% sequence identity.

[0113] In some embodiments, the antibody is selected from antibodies T4_1 and T4_3. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1026 and a light chain variable region comprising SEQ ID NO: 1066, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 1027 and a light chain variable region comprising SEQ ID NO: 1067, or an analog or homolog comprising at least 85% sequence identity.

[0114] In some embodiments, at least 85% identity is at least 90, 92, 95, 97, 99, or 100% identity. Each possibility represents a separate embodiment of the invention. Those skilled in the art will understand that certain regions in an antibody are required for function, such as antigen-binding CDRs, immunogenic peptides, and cell-penetrating moieties, while other regions can tolerate changes without altering function. Other inactive CDRs are such regions, such as many inter-CDR sequences, as well as sequences within the constant region of an antibody. Analogs and homologs that retain function but contain changes in these other regions are also encompassed by the invention.

[0115] In some embodiments, the antigen-binding region is a single chain antibody. In some embodiments, the antigen-binding molecule of the present invention comprises a first single chain antibody and a second single chain antibody. In some embodiments, the first single chain antibody can bind an antigen on a target cell. In some embodiments, the second single chain antibody is not functional. In some embodiments, the second single chain antibody comprises a cell-penetrating sequence. In some embodiments, the first single chain antibody comprises an immunogenic peptide. In some embodiments, the second single chain antibody comprises an immunogenic peptide. In some embodiments, the first single chain antibody comprises an inactive CDR substituted with an immunogenic peptide. In some embodiments, the first single chain antibody comprises an inactive CDR substituted with a cell-penetrating sequence. In some embodiments, the first single chain antibody and the second single chain antibody are separated by a linker. In some embodiments, the linker is a peptide linker or an amino acid linker. In some embodiments, the peptide linker is an amino acid linker. In some embodiments, the peptide linker is a peptide bond.

[0116] In some embodiments, the antigen-binding molecule is an antibody. In some embodiments, the antigen-binding molecule comprises a first heavy chain and a first light chain. In some embodiments, the first antigen-binding region comprises a first heavy chain and a first light chain. In some embodiments, the first heavy chain and the first light chain can bind an antigen on a target cell. In some embodiments, the CDRs of the first heavy chain are inactive. In some embodiments, the CDRs of the first light chain are inactive. In some embodiments, the inactive CDRs of the first heavy chain are replaced with an immunogenic peptide. In some embodiments, the inactive CDRs of the first light chain are replaced with an immunogenic peptide. In some embodiments, the inactive CDRs of the first heavy chain are replaced with a cell-penetrating sequence. In some embodiments, the inactive CDRs of the first light chain are replaced with a cell-penetrating sequence.

[0117] In some embodiments, the antigen binding molecule comprises a second heavy chain. In some embodiments, the antigen binding molecule comprises a second light chain. In some embodiments, the antigen binding molecule comprises a second heavy chain and a second light chain. In some embodiments, the second antigen binding region comprises a second heavy chain. In some embodiments, the second antigen binding region comprises a second light chain. In some embodiments, the second antigen binding region comprises a second heavy chain and a second light chain. In some embodiments, the CDRs of the second heavy chain are inert. In some embodiments, the CDRs of the second light chain are inert. In some embodiments, the second heavy chain comprises an immunogenic peptide. In some embodiments, the second light chain comprises an immunogenic peptide. In some embodiments, the second heavy chain comprises a cell penetrating sequence. In some embodiments, the second light chain comprises a cell penetrating sequence. In some embodiments, the CDRs of the second light chain are substituted. In some embodiments, the CDRs of the second heavy chain are substituted. In some embodiments, the inactive CDRs of the second heavy chain are replaced with an immunogenic peptide. In some embodiments, the inactive CDRs of the second light chain are replaced with an immunogenic peptide. In some embodiments, the inactive CDRs of the second heavy chain are replaced with a cell-penetrating sequence. In some embodiments, the inactive CDRs of the second light chain are replaced with a cell-penetrating sequence.

[0118] In some embodiments, the antigen-binding molecule is a bifunctional antigen-binding molecule. In some embodiments, the composition comprises a bifunctional antigen-binding molecule. In some embodiments, the bifunctional antigen-binding molecule comprises an antibody or antigen-binding fragment of the invention. In some embodiments, the bifunctional antigen-binding molecule is a bispecific antibody. In some embodiments, the bifunctional antigen-binding molecule comprises a first antibody or antigen-binding molecule and a second antibody or antigen-binding molecule. In some embodiments, the first antibody comprises a first heavy chain and a first light chain, and the second antibody comprises a second heavy chain and a second light chain. In some embodiments, the heavy and light chains are hybridized between the CH1 domain of the heavy chain and the CL domain of the light chain. In some embodiments, hybridized is linked. In some embodiments, hybridized comprises a disulfide bond. In some embodiments, the second antibody or antigen-binding fragment thereof can bind an antigen that is overexpressed on a target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the antigen is a cancer antigen.

[0119] Examples of cancer antigens include, but are not limited to, epidermal growth factor (EGFR), receptor tyrosine protein kinase erbB2 (HER2), nectin cell adhesion molecule 4 (nectin-4), tumor-associated calcium signaling molecule 2 (TROP-2 / TACSTD2), tissue factor (TF / F3), B cell maturation antigen (BCMA / TNFRSF17), programmed cell death ligand 1 (PDL-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), epithelial cell adhesion molecule (EpCAM), tumor necrosis factor receptor superfamily member 8 (CD30 / TNFRSF8), B lymphocyte antigen CD19 (CD19), cluster of differentiation-22 (CD22), These include siglec-3 (CD33), cluster of differentiation 38 (CD38), cluster of differentiation 79 (CD79), lymphocyte activation gene 3 (LAG-3), CC motif chemokine receptor 4 (CCR4), vascular endothelial growth factor 2 (VEGFR2 / KDR), folate receptor 1 (FOLR1), Campus-1 antigen (CD52), platelet-derived growth factor receptor A (PDGFRα), disialoganglioside GD2, monosialodihexosylganglioside (GM3), insulin-like growth factor 1 (IGF-1) receptor (IGF1R), SLAM family member 7 (SLAMF7), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Antibodies that bind to these cancer antigens are well known in the art, and any such antibody may be used as a targeting module of the present invention. In some embodiments, the cancer antigen targeted by the targeting molecule is selected from HER2, EGFR, EpCAM, BCMA, CD33, CD38, CTLA, LAG-3, and PD-L1. Examples of antibodies that can be used for the targeting moiety are provided in Table 4. [Table 4] TIFF2024538427000018.tif97159

[0120] In some embodiments, the antigen is epidermal growth factor receptor (EGFR). In some embodiments, the second antibody is selected from cetuximab, panitumumab, and necitumumab. In some embodiments, the second antibody is cetuximab. In some embodiments, the second antibody is panitumumab. In some embodiments, the second antibody is necitumumab. In some embodiments, the cetuximab comprises a heavy chain comprising SEQ ID NO: 1069 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the cetuximab comprises a light chain comprising SEQ ID NO: 1068 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the panitumumab comprises a heavy chain comprising SEQ ID NO: 1071 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the panitumumab comprises a light chain comprising SEQ ID NO: 1070 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, necitumumab comprises a heavy chain comprising SEQ ID NO: 1073 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, necitumumab comprises a light chain comprising SEQ ID NO: 1072 or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the analog or homolog retains target binding function. In some embodiments, the analog or homolog retains the CDRs of the antibody. In some embodiments, the CDRs are all CDRs that are not inactive.

[0121] In some embodiments, at least one inactive CDR of the second antibody is replaced with an immunogenic peptide. In some embodiments, the second antibody is a targeting antibody. In some embodiments, the CDRH1 of cetuximab is replaced. In some embodiments, the CDRL1 of cetuximab is replaced. In some embodiments, the CDRL2 of cetuximab is replaced. In some embodiments, the CDRL1 of panitumumab is replaced. In some embodiments, the CDRL2 of panitumumab is replaced. In some embodiments, the CDRL1 of necitumumab is replaced. In some embodiments, the CDRL2 of necitumumab is replaced. In some embodiments, the CDR is replaced with a peptide selected from those provided in Tables 1-3. In some embodiments, the CDR is replaced with a peptide selected from those provided in Table 1. In some embodiments, the CDR is replaced with a peptide selected from those provided in Table 2. In some embodiments, the CDR is replaced with a peptide selected from those provided in Table 3. In some embodiments, the CDRs are replaced with a peptide selected from SEQ ID NOs: 1-11. In some embodiments, the CDRs are replaced with a peptide selected from SEQ ID NOs: 1-5. In some embodiments, the CDRs are replaced with SEQ ID NO: 1. In some embodiments, the second antibody comprises an immunogenic peptide in place of the amino acid sequence from the inactive CDR. In some embodiments, the inactive CDRs are provided in Table 14.

[0122] In some embodiments, the first antibody comprises at least one modification that promotes heterodimerization. In some embodiments, the first antibody comprises at least one modification that inhibits homodimerization. In some embodiments, the second antibody comprises at least one modification that promotes heterodimerization. In some embodiments, the second antibody comprises at least one modification that inhibits homodimerization. In some embodiments, the modification is a knobs-in-holes modification. In some embodiments, the modification is a mutation. In some embodiments, the modification is in the constant region. In some embodiments, the constant region is an Fc region.

[0123] In some embodiments, the Fc region comprises an Ig CH2 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH2 domain. In some embodiments, the Fc region comprises an Ig CH3 domain. In some embodiments, the Fc region comprises an Ig heavy chain CH3 domain. In some embodiments, the Fc region comprises, or consists of, both an Ig CH2 and an Ig CH3 domain. In some embodiments, the Fc region comprises, or consists of, both an Ig heavy chain CH2 and an Ig heavy chain CH3 domain. In some embodiments, the first chain comprises a first portion of an Fc region and the second chain comprises a second portion of an Fc region. In some embodiments, the first portion comprises a CH2 domain, a CH3 domain, or both. In some embodiments, the second portion comprises a CH2 domain, a CH3 domain, or both. In some embodiments, the boundary between the first portion of the Fc region and the second portion of the Fc region generates a functional Fc region. In some embodiments, the boundary comprises a contact. In some embodiments, the boundary comprises an adjacent positioning. In some embodiments, the interface comprises the formation of a protein complex of the invention. In some embodiments, the interface comprises dimerization of the first and second dimerization domains. In some embodiments, the CH2 domain is an Ig CH2 domain. In some embodiments, the CH2 domain is a heavy chain CH2 domain. In some embodiments, the CH3 domain is an Ig CH3 domain. In some embodiments, the CH3 domain is a heavy chain CH3 domain.

[0124] In some embodiments, the CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 1097) or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the CH2 domain consists of SEQ ID NO: 1097. In some embodiments, SEQ ID NO: 1097 is an IgG1 CH2 domain.

[0125] In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1098) or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1099) or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the CH3 domain consists of SEQ ID NO: 1098. In some embodiments, the CH3 domain consists of SEQ ID NO: 1099. In some embodiments, SEQ ID NO: 1098 is an IgG1 CH3 domain. In some embodiments, SEQ ID NO: 1099 is an IgG1 CH3 domain. In some embodiments, SEQ ID NO: 1098 is a sequence found primarily in humans of European and American descent. In some embodiments, SEQ ID NO: 1099 is a sequence found primarily in humans of Asian descent.

[0126] In some embodiments, the CH3 domain comprises a mutation. In some embodiments, the first CH3 domain comprises a first mutation. In some embodiments, the second CH3 domain comprises a second mutation. In some embodiments, the CH2 domain comprises a mutation. In some embodiments, the first CH2 domain comprises a first mutation. In some embodiments, the second CH2 domain comprises a second mutation. In some embodiments, the CH2 and CH3 domains both comprise a mutation. In some embodiments, the first CH2 domain and the first CH3 domain each comprise a first mutation. In some embodiments, the second CH2 domain and the second CH3 domain each comprise a second mutation. In some embodiments, the mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the first mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the second mutation inhibits homodimerization of the second polypeptide chain. In some embodiments, the mutation allows for heterodimerization. In some embodiments, the mutation allows for heterodimerization of the first and second chains. In some embodiments, allowing is promoting. In some embodiments, allowing is enhancing.

[0127] Mutations that promote heavy chain heterodimerization and / or suppress homodimerization are well known in the art. Any such mutations or changes may be used to construct the polypeptides of the invention. In some embodiments, a region from IgG is replaced with a region from IgA. In some embodiments, a region from TCRa is inserted into the first CH3 domain and a region from TCRb is inserted into the second CH3 domain. In some embodiments, the mutation is an insertion of a region from a TCR. In some embodiments, the TCR is selected from TCRa and TCRb. In some embodiments, the mutation is an insertion of a region from a different Ig. Examples of these mutations are found in Table 5. In some embodiments, the mutation is selected from the mutations in Table 5. In some embodiments, the first mutation is selected from the group of mutations provided in the row and second column of Table 5, and the second mutation is the group of mutations provided in the same row in the third column of Table 5. The mutations in Table 5 are provided with Kabat numbering for IgG1 unless otherwise stated; corresponding mutations can be made in other IGs, particularly other IgGs. In some embodiments, the first mutation is T366Y and the second mutation is Y407T. In some embodiments, the first mutation is S354C and T366W and the second mutation is Y349C, T366S, L368A, and Y407V. In some embodiments, the first mutation is S364H and F405A and the second mutation is Y349T and T392F. In some embodiments, the first mutation is T350V, L351Y, F405A, and Y407V and the second mutation is T350V, T366L, K392L, and T394W. In some embodiments, the first mutation is K392D and K409D, and the second mutation is E356K and D399K. In some embodiments, the first mutation is D221E, P228E, and L368E, and the second mutation is D221R, P228R, and K409R. In some embodiments, the first mutation is K360E and K409W, and the second mutation is Q347R, D399V, and F405T.In some embodiments, the first mutation is K360E, K409W, and Y349C, and the second mutation is Q347R, D399V, F405T, and S354C. In some embodiments, the first mutation is F405L, and the second mutation is K409R. In some embodiments, the first mutation is K360D, D399M, and Y407A, and the second mutation is E345R, Q347R, T366V, and K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, and K409V, and the second mutation is E356G, E357D, S364Q, and Y407A. In some embodiments, the first mutation is T366K and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, and Y349D and R355D. In some embodiments, the first mutation is T366K and C351K and the second mutation is selected from C351D, Y349E, Y349D, L368E, L368D, Y349E and R355E, Y349E and R355D, Y349D and R355E, and Y349D and R355D. In some embodiments, the first mutation is L351D and L368E, and the second mutation is L351K and T366K. In some embodiments, the first mutation is L368D and K370S, and the second mutation is E357Q and S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A, and Y407V. In some embodiments, the Ig is an IgG2, the first mutation is C223E, P228E, and L368E, and the second mutation is C223R, E225R, P228R, and K409R. In some embodiments, the first mutation is S354C or T366W, and the second mutation is Y349C, T366S, L368A, or Y407V. In some embodiments, the first mutation is S364H or F405A and the second mutation is Y349T or T392F.In some embodiments, the first mutation is T350V, L351Y, F405A, or Y407V, and the second mutation is T350V, T366L, K392L, or T394W. In some embodiments, the first mutation is K392D or K409D, and the second mutation is E356K or D399K. In some embodiments, the first mutation is D221E, P228E, or L368E, and the second mutation is D221R, P228R, or K409R. In some embodiments, the first mutation is K360E or K409W, and the second mutation is Q347R, D399V, or F405T. In some embodiments, the first mutation is K360E, K409W, or Y349C, and the second mutation is Q347R, D399V, F405T, or S354C. In some embodiments, the first mutation is K360D, D399M, or Y407A, and the second mutation is E345R, Q347R, T366V, or K409V. In some embodiments, the first mutation is Y349S, K370Y, T366M, or K409V, and the second mutation is E356G, E357D, S364Q, or Y407A. In some embodiments, the first mutation is L351D or L368E, and the second mutation is L351K or T366K. In some embodiments, the first mutation is L368D or K370S, and the second mutation is E357Q or S364K. In some embodiments, the first mutation is T366W, and the second mutation is T366S, L368A, or Y407V. In some embodiments, the Ig is an IgG2, the first mutation is C223E, P228E, or L368E, and the second mutation is C223R, E225R, P228R, or K409R. In some embodiments, the first heavy chain constant region comprises or consists of SEQ ID NO: 1074, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the second heavy chain constant region comprises or consists of SEQ ID NO: 1074.In some embodiments, the first heavy chain constant region comprises or consists of SEQ ID NO: 1075, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the second heavy chain constant region comprises or consists of SEQ ID NO: 1075. It will be understood that SEQ ID NO: 1074 and SEQ ID NO: 1075 heterodimerize with each other but inhibit homodimerization. In some embodiments, the CL domain comprises or consists of SEQ ID NO: 1076, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the light chain constant region comprises or consists of SEQ ID NO: 1076. In some embodiments, the analog or homolog carries a mutation that promotes heterodimerization and inhibits homodimerization. [Table 5]

[0128] In some embodiments, the dual-function antigen-binding molecule comprises two heavy chains and two light chains. In some embodiments, the two heavy chains are SEQ ID NOs: 1088 and 1080, and the two light chains are SEQ ID NOs: 1087 and 1079, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the two heavy chains are SEQ ID NOs: 1088 and 1082, and the two light chains are SEQ ID NOs: 1087 and 1081, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the two heavy chains are SEQ ID NOs: 1090 and 1082, and the two light chains are SEQ ID NOs: 1089 and 1079, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the two heavy chains are SEQ ID NOs: 1090 and 1082, and the two light chains are SEQ ID NOs: 1089 and 1081, or an analog or homolog comprising at least 85% sequence identity. In some embodiments, the two heavy chains are SEQ ID NOs: 1088 and 1086 and the two light chains are SEQ ID NOs: 1087 and 1085, or an analog or homolog comprising at least 85% sequence identity. It will be understood that the analog or homolog comprises an immunogenic peptide that is targeted to a target protein by the targeting module, binds to the cell, is delivered to the cytosol by the killing module, and is presented on the cell surface in a complex with HLA.

[0129] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharma- ceutically acceptable carrier, excipient, or adjuvant. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for administration to a tumor. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the subject is a human.

[0130] As used herein, the term "carrier", "excipient" or "auxiliary agent" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium such as physiological saline. Some examples of substances which may function as pharma- ceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; glycols such as glycerin, sorbitol, mannitol and polyethylene glycol polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffers, as well as other non-toxic, compatible substances used in pharmaceutical preparations. Some non-limiting examples of materials that can function as carriers herein include sugar, starch, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, alginic acid, polyols, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers and other non-toxic pharmacologic compatible materials used in other pharmaceutical preparations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants and preservatives may also be present. Any non-toxic, inert and effective carrier may be used to formulate the compositions contemplated herein.In this regard, suitable pharma- ceutically acceptable carriers, excipients and diluents are well known to those skilled in the art and are described, for example, in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management. The contents of all of these are incorporated herein by reference in their entirety. Examples of pharma-ceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety.The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, sustained release particles, and other vehicles that extend the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, and lamellar layers. Liposomes for use with the peptides described herein are formed from standard vehicle-forming lipids, which usually include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipid is usually determined by considerations such as liposome size and stability in blood. Various methods are available for the preparation of liposomes, for example as reviewed by Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York. See also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0131] The carriers may in total constitute from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0132] As used herein, the terms "administer", "administration" and the like refer to any method of delivering a composition containing an active agent to a subject in a sound medical practice to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, intratumoral or intraperitoneal.

[0133] In some embodiments, the nucleic acid molecule comprises an open reading frame. In some embodiments, the open reading frame encodes an antigen-binding molecule of the invention. In some embodiments, the nucleic acid molecule comprises multiple open reading frames that collectively encode an antigen-binding molecule of the invention.

[0134] In some embodiments, the vector is an expression vector. In some embodiments, the vector comprises at least one regulatory element operably linked to the nucleic acid molecule of the present invention. In some embodiments, the vector comprises at least one regulatory element operably linked to an open reading frame encoding the nucleic acid molecule of the present invention. In some embodiments, the vector comprises multiple regulatory elements each operably linked to an open reading frame that collectively encodes the antigen binding molecule of the present invention. In some embodiments, the composition comprises multiple vectors each comprising at least one regulatory element operably linked to an open reading frame, and the multiple open reading frames collectively encode the nucleic acid molecule of the present invention.

[0135] The term "expression" as used herein refers to the biosynthesis of a gene product and includes transcription and / or translation of said gene product. Thus, expression of a nucleic acid molecule can refer to the transcription of a nucleic acid fragment (e.g., transcription to produce an mRNA or other functional RNA) and / or the translation of an RNA into a precursor or mature protein (polypeptide).

[0136] The expression of a gene in a cell is well known to those skilled in the art. It can be carried out by gene transfer, viral infection, or direct modification of the genome of a cell, among other methods. In some embodiments, the gene is in an expression vector, such as a plasmid or a viral vector.

[0137] A vector nucleic acid sequence usually contains at least an origin of replication for propagation in a cell, and optionally additional elements, such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), selectable markers (e.g., antibiotic resistance), polyadenine sequences, etc.

[0138] The vector can be a DNA plasmid that is delivered by non-viral or viral methods.The viral vector can be a retroviral vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector or a pox virus vector.The promoter can be active in mammalian cells.The promoter can be a viral promoter.

[0139] In some embodiments, the gene is operably linked to a promoter. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to regulatory elements that allow expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0140] In some embodiments, vectors are introduced into cells by standard methods such as electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with viral vectors, high velocity ballistic penetration by small particles with nucleic acid within the matrix or on the surface of small beads or particles (Klein et al., Nature 327.70-73 (1987)).

[0141] As used herein, the term "promoter" refers to a group of transcriptional control modules clustered around the initiation site for RNA polymerase, i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, that contain one or more recognition sites for transcriptional activator or repressor proteins.

[0142] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0143] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (which are available from Invitrogen), pCI (which is available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (which are available from Strategene), pTRES (which is available from Clontech) and derivatives thereof.

[0144] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses, such as retroviruses, are used in the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0145] In some embodiments, recombinant viral vectors that offer advantages such as horizontal infection and target specificity are used for in vivo expression. In one embodiment, horizontal infection is inherent in, for example, the life cycle of retroviruses, a process by which a single infected cell produces many progeny virions that detach from the mother and infect neighboring cells. In one embodiment, the result is that a large area quickly becomes infected, most of which were not initially infected by the original viral particle. In one embodiment, a viral vector that cannot be horizontally transmitted is generated. In one embodiment, this feature can be useful when the desired goal is to introduce a specific gene into only a limited number of target cells.

[0146] A variety of methods can be used to introduce the expression vectors of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4(6):504-512, 1986] and include, for example, stable or transient gene transfer with recombinant viral vectors, lipofection, electroporation and infection. See also US Pat. Nos. 5,464,764 and 5,487,992 regarding positive-negative selection methods.

[0147] In one embodiment, a plant expression vector is used. In one embodiment, the expression of the polypeptide coding sequence is driven by several promoters. In some embodiments, viral promoters are used, such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter for TMV [Takamatsu et al., EMBO J.6:307-311 (1987)]. In another embodiment, a plant promoter is used, such as the small subunit of RUBISCO [Coruzzi et al., EMBO J.3:1671-1680 (1984) and Brogli et al., Science 224:838-843 (1984)] or a heat shock promoter, such as soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol.6:559-565 (1986)]. In one embodiment, the construct is introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation, and other techniques known to those skilled in the art.See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)].Other expression systems, such as insect and mammalian host cell systems, known in the art, may also be used according to the present invention.

[0148] It will be understood that, besides containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the present invention may also contain sequences that have been engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0149] In some embodiments, the method is a method of treating cancer. In some embodiments, the antigen binding molecule of the present invention is for use in treating cancer. In some embodiments, the bifunctional antigen binding molecule of the present invention is for use in treating cancer. In some embodiments, the composition of the present invention is for use in treating cancer. In some embodiments, the cancer is a cancer that expresses a cancer specific antigen. In some embodiments, the cancer is a cancer that overexpresses a cancer specific antigen. In some embodiments, the overexpression is at a higher level than in a non-cancer cell. In some embodiments, the non-cancer cell is the same cell type or tissue as the cancer cell. In some embodiments, the cancer is a cancer that expresses an immunogenic cancer peptide. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer comprises malignant immune cells. In some embodiments, the immune cells are B cells. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a PD-L1 positive cancer. In some embodiments, the cancer is an EGFR positive cancer. In some embodiments, the cancer is a cancer that overexpresses EGFR.

[0150] As used herein, "cancer" refers to a disease associated with cell proliferation. Non-limiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma, and germ cell tumors. In one embodiment, carcinoma refers to tumors derived from epithelial cells, including, but not limited to, breast cancer, prostate cancer, lung cancer, pancreatic cancer, and colon cancer. In one embodiment, sarcoma refers to tumors derived from mesenchymal cells, including, but not limited to, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, and soft tissue sarcoma. In one embodiment, lymphoma refers to tumors derived from hematopoietic cells that tend to leave the bone marrow and mature in lymph nodes, including, but not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, and immunoproliferative disorders. In one embodiment, leukemia refers to tumors derived from hematopoietic cells that tend to leave the bone marrow and mature in the blood, including but not limited to acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, and adult T-cell leukemia. In one embodiment, blastoma refers to tumors derived from immature precursor cells or embryonic tissue, including but not limited to hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, and glioblastoma multiforme. In one embodiment, germ cell tumor refers to tumors derived from germ cells, including but not limited to germinoma or seminoma germ cell tumors (GGCT, SGCT) and non-germinoma or non-seminomatous germ cell tumors (NGGCT, NSGCT). In one embodiment, germinoma or seminoma tumors include, but are not limited to, germinoma, dysgerminoma, and seminoma. In one embodiment, non-germinoma or non-seminomatous tumors refer to pure germ cell and mixed germ cell tumors, including, but are not limited to, embryonal carcinoma, yolk sac tumor, choriocarcinoma, tearoom, polygerminoma, gonadoblastoma, and teratocarcinoma.

[0151] In some embodiments, the antigen-binding molecule of the present invention is a cancer vaccine. In some embodiments, the cancer vaccine is an antigen-binding molecule that comprises an antigen-binding region that can bind dendritic cell antigens. Those skilled in the art will understand that upon entry into dendritic cells, immunogenic cancer peptides are cleaved from the remainder of the molecule of the present invention and presented on the surface of dendritic cells by HLA molecules. This then trains cytotoxic immune cells (T cells and NK cells) to target this immunogenic peptide and thus target cancer.

[0152] In some embodiments, treating further comprises administering to the subject an effector cell specific for the immunogenic peptide. In some embodiments, the effector cell is an immune cell. In some embodiments, the effector cell is a cytotoxic cell. In some embodiments, the effector cell is a lymphocyte. In some embodiments, the effector cell is a CD8 T cell. In some embodiments, the effector cell is a natural killer (NK) cell. In some embodiments, the effector cell has been exposed to the peptide. In some embodiments, the effector cell has been exposed to an antigen presenting cell that presents the peptide in complex with HLA.

[0153] In some embodiments, treating comprises administering a vaccine comprising the immunogenic peptide. In some embodiments, the subject has previously received a vaccine comprising the immunogenic peptide. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is afflicted with cancer. In some embodiments, the subject is suitable for treatment by the methods of the invention. In some embodiments, the subject has previously been exposed to the immunogenic peptide. In some embodiments, the subject has previously been infected with a pathogen from which the immunogenic peptide is derived. In some embodiments, the pathogen is a virus. In some embodiments, the subject is capable of mounting an immune response to the peptide. In some embodiments, the subject comprises T cells comprising a TCR that recognizes the peptide or a portion thereof. In some embodiments, the subject comprises memory B cells comprising a BCR that recognizes the peptide or a portion thereof.

[0154] In some embodiments, the method is a method of generating surface presentation of a peptide in a target cell. In some embodiments, the surface presentation is presentation of HLA complexed with the peptide. In some embodiments, the expression is surface presentation. In some embodiments, the expression is expression in an HLA complex.

[0155] In another aspect, a method of engineering an antibody or antigen-binding fragment thereof is provided, the method comprising: a. selecting an antibody or antigen-binding fragment thereof of interest; b. determining that at least one CDR of the selected antibody or antigen-binding domain thereof is not required for binding to the target; c. replacing at least one of the determined CDRs or a portion thereof with a peptide; thereby engineering the antibody or antigen-binding fragment thereof.

[0156] In another aspect, a method of engineering an antibody or antigen-binding fragment thereof is provided, the method comprising: a. selecting an antibody or antigen-binding fragment thereof of interest; b. receiving a database of peptides; c. performing an alignment of peptides of the variable regions of the selected antibody or antigen-binding fragment thereof with peptides of a database; d. determining peptides from the selected antibody or antigen-binding fragment thereof and peptides from the database that have an alignment score above a predefined threshold; and e. replacing the peptide determined from the selected antibody or antigen-binding fragment thereof with a peptide determined from a database; thereby engineering the antibody or antigen-binding fragment thereof.

[0157] In some embodiments, the engineered antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment of the invention. In some embodiments, the engineered antibody or antigen-binding fragment thereof is an antigen-binding molecule of the invention. In some embodiments, the engineered antibody or antigen-binding fragment thereof is a dual-function antigen-binding molecule of the invention. In some embodiments, the engineered antibody or antigen-binding fragment thereof is an immunogenic peptide delivery antibody. In some embodiments, the engineered antibody or antigen-binding fragment thereof is for use in a method of the invention. In some embodiments, the antibody or antigen-binding fragment thereof prior to engineering is a cell-permeable antibody. In some embodiments, the selecting is to select an antibody that enters into the cell to which it binds. In some embodiments, the antibody or antigen-binding fragment thereof prior to engineering is a commercially available antibody.

[0158] In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof binds to the surface of a target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, step (a) comprises selecting an antibody or antigen-binding fragment thereof that binds to the surface of a target cell. In some embodiments, binding to the surface comprises binding to a surface antigen. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof is a DNA-binding antibody. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof is internalized into the endocytic pathway of a cell. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof is transported into the cytoplasm of a cell to which it binds. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof is capable of endosomal escape. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof is delivered to the cytosol of a cell to which it binds. In some embodiments, the method further comprises inserting a cell-permeable moiety into the antibody or antigen-binding fragment thereof. In some embodiments, the cell-permeable moiety is inserted before the immunogenic peptide. In some embodiments, the cell-permeable moiety is inserted after the immunogenic peptide. In some embodiments, the cell-permeable moiety is inserted simultaneously with the insertion of the immunogenic peptide. In some embodiments, step (a) comprises selecting an antibody or antigen-binding fragment thereof that, upon binding to the surface of a target cell, is internalized and delivered to the cytosol of the target cell.

[0159] In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof enters / internalizes into cells that are bound at a level equivalent to any one of TMab4, 3E10, and 71F12. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof enters / internalizes into cells that are bound at a level equivalent to TMab4. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof enters / internalizes into cells that are bound at a level equivalent to 3E10. In some embodiments, the pre-engineered antibody or antigen-binding fragment thereof enters / internalizes into cells that are bound at a level equivalent to 71F12. In some embodiments, equivalent involves penetration that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% of the original antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, equivalent involves penetration that is at least 80% of the original antibody. In some embodiments, equivalent involves penetration that is at least 90% of the original antibody.

[0160] In some embodiments, the determining includes at least one inactive CDR. In some embodiments, the determining includes determining at least one inactive CDR that is not involved in trafficking into the cell. In some embodiments, the determining includes determining at least one CDR that is not involved in endosomal escape. In some embodiments, the determining is based on a structural analysis of the antibody or antigen-binding fragment thereof bound to its target. In some embodiments, the structural analysis is an in silico analysis. In some embodiments, the structural analysis is a crystallographic analysis. In some embodiments, the method further includes undergoing a structural analysis. In some embodiments, the method further includes performing a structural analysis. In some embodiments, the analysis provides a distance of each amino acid of the antibody or antigen-binding fragment thereof to the amino acid of the target. In some embodiments, the analysis provides a distance of each amino acid of the CDR of the antibody or antigen-binding fragment thereof to the amino acid of the target. In some embodiments, the determining is determining a distance of each amino acid of the CDR to the target. In some embodiments, a CDR with two or fewer amino acids in contact with the antigen is determined to not be required for binding. In some embodiments, the determining is determining a distance of each amino acid of the CDR to the target. In some embodiments, CDRs that have one or less amino acids that contact the antigen are determined not to be required for binding. In some embodiments, CDRs that are not required for binding are inactive CDRs. In some embodiments, CDRs that are not required for binding are CDRs in which mutations have been shown not to affect antibody function. In some embodiments, the function is binding.

[0161] In some embodiments, the peptide is an immunogenic peptide. In some embodiments, the peptide comprises at least 5, 6, 7, 8, 9, 10, or 11 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the peptide comprises at least 7 amino acids. In some embodiments, the peptide comprises at least 8 amino acids. In some embodiments, the immunogenic peptide comprises 8-11 amino acids. In some embodiments, the peptide is an immunogenic peptide provided herein. In some embodiments, the peptide is derived from a variable region of an antibody or antigen-binding fragment thereof. In some embodiments, the peptide is derived from a CDR of an antibody or antigen-binding fragment thereof. Methods for determining the location of CDRs are well known in the art and may use the Clothia and Kabat system. Given the sequence of the variable domain of an antibody, one skilled in the art would be able to readily identify the CDRs.

[0162] In some embodiments, at least one CDR is removed. In some embodiments, a portion of at least one CDR is removed. In some embodiments, the removed amino acid is replaced with an immunogenic peptide. In some embodiments, the replacing is optimized. In some embodiments, the method further comprises optimizing the replacement. In some embodiments, the optimizing is a structural optimization. In some embodiments, the optimizing comprises performing an optimization algorithm. In some embodiments, the optimizing comprises generating as little disturbance as possible in the structure of the selected antibody or antigen-binding fragment thereof. In some embodiments, the optimizing comprises in silico analysis or insertion of the immunogenic peptide into at least one determined CDR with the removal of all possible portions of the CDR and the selected insertion, and removal of the portion that generates the least disturbance. In some embodiments, the optimization comprises the insertion of at least one filler amino acid. In some embodiments, the optimization comprises generating at least one compensatory mutation outside the determined CDR. In some embodiments, the optimization algorithm is a minimal perturbation replacement (MBR) algorithm. In some embodiments, the optimization comprises optimization of the CDR flanking sequences. In some embodiments, optimization of the flanking sequences includes preservation of the antibody stem. In some embodiments, the structure of the stem adjacent to the CDR is maintained. In some embodiments, the stem is the end of the beta sheet adjacent to the CDR. In some embodiments, the end includes at least 1, 2, 3, 4, or 5 amino acids directly adjacent to the CDR. Each possibility represents a separate embodiment of the invention.

[0163] In some embodiments, the method further comprises confirming binding of the engineered antibody or antigen-binding fragment thereof to the same target bound by the selected antibody or antigen-binding fragment thereof. In some embodiments, the method further comprises measuring binding of the engineered antibody or antigen-binding fragment thereof to the same target bound by the selected antibody or antigen-binding fragment thereof. In some embodiments, the method further comprises determining binding of the engineered antibody or antigen-binding fragment thereof to the same target bound by the selected antibody or antigen-binding fragment thereof. In some embodiments, the binding is equivalent to binding of the selected antibody or antigen-binding fragment thereof. In some embodiments, measuring further comprises determining that binding is not significantly reduced compared to the selected antibody or antigen-binding fragment thereof. In some embodiments, significantly is statistically significant. In some embodiments, greatly reduced comprises a reduction of more than 10%. In some embodiments, significantly reduced comprises a reduction of more than 20%. In some embodiments, significantly reduced comprises a reduction of more than 50%. In some embodiments, significantly reduced is abolished.

[0164] In some embodiments, the method further comprises measuring the level of the peptide in the cytosol of the target cell. In some embodiments, the method further comprises determining delivery of the peptide to the cytosol. In some embodiments, the method further comprises measuring delivery of the peptide to the cytosol.

[0165] In some embodiments, the method further comprises confirming delivery of the peptide to the cytosol of the target cell. In some embodiments, the method further comprises measuring the level of the peptide in the cytosol of the target cell. In some embodiments, the method further comprises determining delivery of the peptide to the cytosol. In some embodiments, the method further comprises measuring delivery of the peptide to the cytosol.

[0166] In some embodiments, the method further comprises confirming delivery of the peptide to the surface of the target cell. In some embodiments, the delivery is surface presentation of the peptide. In some embodiments, the delivery is delivery of the peptide in a complex with an HLA molecule to the surface of the target cell. In some embodiments, the method further comprises measuring the level of the peptide on the surface of the target cell. In some embodiments, the level is the level of the peptide in a complex with HLA. In some embodiments, the method further comprises determining delivery of the peptide to the surface of the target cell. In some embodiments, the method further comprises measuring delivery of the peptide to the surface of the target cell.

[0167] In some embodiments, the method further comprises confirming killing of the target cell by the effector cell. In some embodiments, the killing is specific killing. In some embodiments, the effector cell is specific for the peptide. In some embodiments, the effector cell is an immune cell. In some embodiments, the method further comprises measuring killing of the target cell by the effector cell. In some embodiments, the method further comprises determining killing of the target cell by the effector cell. In some embodiments, the method is an in vitro method. In some embodiments, the confirming, measuring and determining are performed in vitro. In some embodiments, the in vitro is ex vivo. Examples of methods for performing the confirming, measuring and determining are provided herein, although any assay known in the art for such measuring / confirming / determining may be used.

[0168] Databases of peptides, and in particular immunogenic peptides, are well known in the art, and any such database may be employed. In some embodiments, the immunogenic peptide is a cancer peptide. In some embodiments, the database comprises or consists of Table 3. In some embodiments, the alignment is a pairwise alignment. In some embodiments, the alignment is an alignment of one peptide from the selected antibody or its antigen-binding fragment with one peptide from the database to generate an alignment pair. In some embodiments, the alignment pair with an alignment score above a predefined threshold is used for replacement in step (e).

[0169] In some embodiments, the method further comprises inserting the engineered antibody into the bifunctional antigen binding molecule. In some embodiments, the bifunctional antigen binding molecule is a molecule of the invention. In some embodiments, the method further comprises selecting a targeting antibody that binds to a protein on the target cell. In some embodiments, the bifunctional antigen binding molecule is on the surface of the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the protein is a receptor. In some embodiments, the protein is a cancer antigen. In some embodiments, the protein is expressed only on the cancer cell and not on a corresponding healthy cell. In some embodiments, the protein is a protein that is overexpressed on the cancer cell compared to a corresponding healthy cell. In some embodiments, the corresponding cell is a cell of the same tissue or cell type as the cancer cell. In some embodiments, the corresponding cell is a control cell. In some embodiments, the selected targeting antibody is combined with the engineered antibody. In some embodiments, the combining generates a bifunctional antigen binding molecule. In some embodiments, the bifunctional antigen binding molecule is a bispecific antibody.

[0170] In some embodiments, the engineered antibody comprises one heavy chain and one light chain. In some embodiments, the targeting antibody or antigen-binding fragment comprises one heavy chain and one light chain. In some embodiments, the engineered antibody is a single chain antibody. In some embodiments, the targeting antibody is a single chain antibody. In some embodiments, the single chain antibody comprises a heavy chain variable domain and a light chain variable domain linked in a single polypeptide by an amino acid linker. In some embodiments, the heavy chain constant region of the targeting antibody is engineered to promote heterodimerization and inhibit homodimerization, in some embodiments, the heavy chain constant region of the engineered antibody is engineered to promote heterodimerization and / or inhibit homodimerization, in some embodiments, engineered is modified. In some embodiments, modified is mutated. In some embodiments, the method comprises engineering the constant region of the engineered antibody and / or the targeting antibody. In some embodiments, engineering is generating a set of mutations provided in Table 5. In some embodiments, the heavy chain constant region used comprises or consists of SEQ ID NOs: 1074 and 1075 or analogs or homologs containing 85% sequence identity. In some embodiments, the analogs or homologs carry a knob-in-hole mutation.

[0171] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0172] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a polynucleotide" includes a plurality of such polynucleotides, a reference to "the polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those of skill in the art, and so forth. It should be further noted that the claims may be written to exclude any optional element. Thus, this statement is intended to serve as a prerequisite for using exclusive language, such as "sole," "only," and the like, in connection with the recitation of claim elements or the use of "negative" limitations.

[0173] In instances where a convention similar to "at least one of A, B, and C, etc." is used, such a structure is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but be limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It would further be understood by one of ordinary skill in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B."

[0174] It is understood that certain features of the invention 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 invention that are described for brevity as a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the invention are specifically embraced by the invention and disclosed herein as if all combinations were individually and explicitly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and disclosed herein as if all such subcombinations were individually and explicitly disclosed herein.

[0175] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0176] Various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below are found experimentally supported in the following examples.

[0177] Working Example In general, the nomenclature used herein and the laboratory techniques used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained in detail in the literature, see, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methodologies described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated herein by reference. Other general references are set forth throughout the specification.

[0178] material and method Cell penetration assay: The penetration ability of all Abs is screened by intracellular antibody detection followed by flow cytometry analysis. Intracellular antibodies are detected using fluorescently labeled anti-Fc-FITC. Briefly, breast cancer cells are seeded in 24-well plates and incubated with 0.5-1 μM of control antibody or Trojan Ab for 6 h. Cells are then washed with PBS and low pH buffer (glycine, pH=2.5) to remove the antibody bound to the cell surface. Cells are then fixed, permeabilized, and intracellularly fluorescently labeled with anti-Fc-FITC antibody (0.1% saponin, 1% BSA in PBS, 2 h at room temperature). Labeled cells are analyzed by flow cytometry for FITC, and unlabeled cells are used as a negative control. To determine the level of proteasomal degradation of intracellular Trojan Abs, the proteasome inhibitor MG132 is added to the cells prior to the addition of Trojan Abs.

[0179] Luciferase killing assay: Target cancer cells (T) expressing luciferase are seeded in 96-well plates and treated with Trojan Abs at various concentrations (0.5-4 μM). After 3-24 hours, antigen-specific effector cells (E), specific for the relevant peptide in the Trojan Ab, are added at various E:T ratios. Cells are then co-cultured for 18-24 hours, followed by addition of luciferase substrate (Bio-Glo; Promega). Luminescence intensity (lum) is recorded to quantify the number of viable cells. Killing is assessed by calculating % Kill = 1-lum (treated target + effector) / Lum (untreated control), which can also be calculated against other controls.

[0180] Image-based killing assay (IncuCyte Imaging System): Target cells (T) are first labeled with a cytosolic red dye (IncuCyte® Cytolight Rapid Red) to allow tracking of target cell proliferation with the IncuCyte imager. The labeled target cells are then seeded in 96-well plates and treated with Trojan Abs at various concentrations (0.5-4 μM). After 3-24 hours, antigen-specific effector cells (E), specific for the relevant peptide within the Trojan Ab, are added at various E:T ratios. The plates are then placed in an IncuCyte (37°C; 5% CO2) and imaged at 2-hour intervals for 48 hours. Alternatively, caspase 3 / 7 green dye is added to the medium to allow for apoptosis assessment of target cells. Data is analyzed by the IncuCyte software analysis tool, which compares two main parameters: proliferation: red dye confluency normalized to time 0h; and apoptosis: (red + green area) / red area normalized to time 0h.

[0181] EGFR Binding Assay: A flow cytometry binding assay was established to evaluate the ability of full Trojan Horse antibodies (FTAb) to bind EGFR on the cancer cell surface. Pre-seeded cells were trypsinized, washed, and then incubated for 1 h on ice with 0.3 or 3 nM of FTAb or an EGFR bivalent therapeutic antibody serving as a positive control. Cells are then washed three times with 2% FBS in PBS buffer and labeled with a fluorescent secondary antibody (anti-human Fc-FITC) for 30 min on ice. Cells are then washed three times with the same buffer and analyzed by flow cytometry. FTAb binding is proportional to FITC fluorescence levels.

[0182] Presentation of HLA-peptide complexes on cancer cell surface: To quantify the level of presentation of HLA-peptide complexes on cancer cells upon incubation with FTAb, a TCR-like antibody that specifically binds HLA-peptide complexes was used. Briefly, cells are seeded in 24-well plates and incubated with 1 μM FTAb or cFTAb (control FTAb) for different durations (4 h, 8 h, and 10 h) at 37° C. and 5% CO2. The cells are then incubated with C1-17 antibody conjugated to fluorescein (FITC) (see Lee et al. “Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for a Cytomegalovirus pp65-Derived Peptide Presented by HLA-A*02:01” Int. J. Mol. Sci. 2021, 22, 2349, which is incorporated herein by reference in its entirety) on ice for 30 minutes and analyzed by flow cytometry.

[0183] Example 1: Trojan horse antibody design It is known that immunogenic peptides presented by cancer cells can lead to immune cell activation and cancer cell killing. Lymphocytes in a subject recognize the immunogenic peptides, and the subject's immune system is directed against the cancer cells. This mechanism can be exploited by actively delivering immunogenic peptides into cancer cells, which in turn present the immunogenic peptides on their cell surface. Two classes of immunogenic peptides can be used: 1) non-self peptides / peptides not found in humans, such as viral or bacterial peptides, and 2) modified self peptides, which are human peptides that have been mutated or modified to confer cancer specificity so that the immune system is not activated against healthy cells.

[0184] It has been previously hypothesized that immunogenic peptides can be delivered via antibodies. Sefrin et al. utilized full-sized antibodies conjugated to peptides via disulfide bonds (Sefrin et al., 2019 “Sensitization of tumors for attack by virus-specific CD8+ T-cells through antibody-mediated delivery of immunogenic T cell epitopes”, Front. Immunol., Aug 21; 10: 1962). However, this construct has very low stability and can undergo high rates of deconjugation in serum. This in turn can lead to poor delivery to target cells, reduced efficacy and high off-target effects. Gaston et al. employed fusion protein constructs in which peptides were incorporated at the end of the amino acid chain of the heavy or light chain, or placed adjacent to the hinge domain within the chain (Gaston et al., 2019, “Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions”, Sci. Rep., Dec 10; 9(1): 18688). This method resulted in lower production yields, increased aggregation, and reduced in vitro stability.

[0185] To alleviate the problems inherent in these methods, immunogenic peptides are inserted into antibody variable regions, rather than inserting them at the ends of the chains or between structures. In particular, immunogenic peptides are used to replace all or part of the "inactive", non-essential CDRs in the heavy or light chains of an antibody (Figure 1). In silico modeling is used to identify the inactive CDR loops in a known therapeutic antibody. The inactive CDRs are then replaced by the immunogenic peptide (Figure 2). In this conformation, the engineered antibody maintains high stability and antigen binding affinity. If the immunogenic peptide is shorter than the CDR, filler amino acids can be added so as not to alter the antibody conformation. Similarly, if the CDR is longer than the immunogenic peptide, only a portion of the CDR is removed. The adjacent regions of the inactive CDR can also be replaced, but these regions are often important to the overall antibody conformation, so their overall structure must be preserved. These engineered antibodies are called Trojan Horse antibodies.

[0186] Example 2: Use of Trojan Horse Antibodies One application of the Trojan horse antibody of the present invention is dendritic cell vaccination. Antibodies against dendritic cell surface markers (e.g., CD40) are used for engineering. In this case, anti-human CD40 antibody Fab516 is employed, and modified self-antigens expressed by target cancer are inserted into the inactive CDR. After antibody binding to CD40, the antibody is digested, and short peptides containing immunogenic peptides are presented on the cell surface by MHCI molecules. CD8+ T cells are activated by these dendritic cells and become tumor-specific, resulting in enhanced tumor cell killing (Figure 3).

[0187] The Trojan Horse antibodies of the present invention are also used to treat B-cell malignancies such as leukemia. In this case, an antibody against a B-cell surface marker (e.g., CD20) is used. For example, the Arzera antibody has an inactive CDR replaced with a viral epitope (e.g., a CD4 viral epitope / epitope recognized by CD4 T cells). After endocytosis, the antibody is processed and presented on the surface of the B cell by MHCII molecules. Cytotoxic T cells recognize the viral epitope and kill the malignant cell (Figure 4).

[0188] The Trojan horse antibodies of the present invention are also used to treat solid tumors or general cancers. Antibodies that bind to cancer-specific epitopes such as HER2, EGFR, EpCAM and PSMA are used, and in particular bispecific antibodies are used. In the case of bispecific molecules, only one antigen-binding domain is required to deliver the drug to the cancer cells; the other can be replaced with one or more peptides recognized by CD8+ T cells and / or cell-penetrating moieties. It will of course be understood that cell-penetrating moieties can also be inserted into non-bispecific antibodies if two inactive CDRs are present (one for the penetrating moiety and one for the immunogenic peptide). Alternatively, antibodies known to internalize can be used, as long as the insertion of the immunogenic peptide does not prevent internalization. After binding to the target protein, the antibody is digested and released into the cytosol (endosomal escape). From there, it is presented on the surface of the cancer cells via MHCII molecules (Figure 5).

[0189] Example 3: Trojan Horse Antibody Pathway with Inactive CDR Identification (Figure 6, Right Pathway) Trojan horse antibodies are designed by first selecting an antibody of interest. A structural analysis of the binding of the antibody to its target is performed and inactive CDRs are identified. Inactive CDRs are CDRs that do not directly contact the protein target (more than 5 Å away from the protein target). A suitable immunogenic peptide that can replace the CDR is identified and inserted into the engineered antibody. The immunogenic peptide may be preselected or the immunogenic peptide may be selected based on its similarity to the CDR to be removed. Computer modeling is used to select a suitable peptide and confirm the correct antibody conformation. This computer modeling is called minimal perturbation substitution (MBR), which can be applied to optimize the substitution position within the CDR. For example, if the CDR is longer than the immunogenic peptide, filler amino acids may need to be added for minimal perturbation of antigen binding. Similarly, if the CDR is shorter than the immunogenic peptide, amino acids from the inter-CDR region may also be removed. Adjacent amino acids are also often involved in binding and antibody conformation, and antibody conformation and structural similarity / minimal perturbation in the adjacent regions are also desirable. MBR optimizes the positioning of immunogenic peptides.

[0190] When an antibody is selected without obviously inactive CDRs, the MBR is applied to rank possible substitution positions within the antibody, particularly within the CDRs. If a position is found that does not disrupt binding (or disrupts it less than a given threshold), the immunogenic peptide is inserted at this position.

[0191] MBR analysis considers the canonical structure of an antibody and selects the positions / substitution positions that most closely maintain this structure. Antibody stem conservation is highly conserved. Stem refers to the beta sheet portion directly adjacent to the CDR loop. This region is also called the CDR flanking region. These 1-5 amino acids are important for the loop position and tend to be highly rigid, while the loop itself, especially the inactive CDR loop, is less regular. Stem conservation is therefore important during MBR analysis. MBR also considers the position frequency matrix of other known antibodies. The computer model has hundreds of known antibodies imputed and considers the frequency of each amino acid at each position. The peptide replacement that most closely matches the position frequency of the known antibodies is preferred. Finally, MBR outputs the optimal position within the antibody for peptide replacement, or if there are two or more acceptable positions, it outputs a hierarchical position.

[0192] Example 4: Trojan Horse Antibody Pathway by Sequence Similarity (Figure 6, Left Pathway) Trojan horse antibodies can alternatively be generated based on sequence similarity. Target antibodies are selected as in the list of immunogenic peptides (see, for example, Tables 1-3). Pairwise sequence alignments are performed between various viral peptides and antibodies. Overlapping peptides in the antibody of 8-11 amino acids are compared with the immunogenic peptides and alignment scores are given for each pair (one peptide from the antibody and one immunogenic peptide). Scores were calculated based on blosum62 and a penalty of -3 per gap opening and per gap extension. A threshold was set for acceptable alignment (e.g., >25 was used in the examples below). Peptides in the antibody to be replaced with the immunogenic peptides that fit the alignment score threshold are selected and MBR is applied to optimize the replacement positions.

[0193] Example 5: Functional validation of Trojan horse antibodies (inactive CDRs) Substitution based on sequence similarity is not limited to CDR, but can be carried out anywhere in antibody.Because of the similarity in sequence, minimal disturbance is expected.Nevertheless, this method and the inactive CDR substitution method still require confirmation that the antibody maintains its functionality.In particular, it needs to be determined that the antibody still binds to its target and still internalizes into cells.If binding / internalization is lost, the new antibody is useless.

[0194] The first antibody selected for insertion of immunogenic peptides was the DNA hydrolysis antibody 3D8, also known as TMab4. The murine antibody 3D8 was first disclosed in Kim et al., “Heavy and Light Chain Variable Single Domains of an Anti-DNA Binding Antibody Hydrolyze Both Double- and Single-stranded DNAs without Sequence Specificity”, J Biol Chem., 2006, Jun 2; 281(22): 15287-9, and its humanized version was provided in International Publication No. WO2019 / 244086. TMab4 was shown to bind to and enter cancer cells, reaching the cytoplasm via endosomal escape, and therefore it was selected for conversion into a Trojan horse antibody. CDR1, 2, 3 (CDRH1, CDRH2, CDRH3) of the heavy chain and CDR3 (CDRL3) of the light chain were found to be inactive CDRs.

[0195] First, the ability of TMab4 to replace CDRH1 was tested. Two peptides from CMV, two from EBV, and one from influenza were inserted at various positions in CDRH1 (see Table 6). Superior penetration over the negative control (adalimumab) was maintained for all inserted peptides regardless of their position in CDRH1. However, some insertions showed superior penetration while others had reduced penetration compared to the parent TMab4. Some constructs contained substitutions of amino acids adjacent to the CDRs. This was done because these additional substitutions were predicted to reduce perturbation of the overall antibody conformation. Specifically, in construct T1, an "S" was also substituted directly N-terminal to the CDR. In constructs T_32, T_33, and T_34, a "CAAS" was also substituted directly N-terminal to the CDR. In constructs T_42, T_43, an "AAS" was also substituted directly N-terminal to the CDR. One construct, T1_34, contained a compensatory mutation in the heavy chain (T30S) that was predicted to reduce antibody promiscuity, thus the entire alteration of the inactive CDR did not abolish penetration as predicted, but did have an effect on the overall ability to enter cells. [Table 6]

[0196] Some of the generated antibodies were tested for their ability to kill target cells in the presence of effector cells specific for the immunogenic peptide present in the Trojan horse antibody. As can be seen in Figure 7A, all antibodies were still able to enter the cells, but their ability to induce specific killing was highly variable. Of the five insertions of the NLVPMVATV (SEQ ID NO: 1) peptide, two did not result in specific killing, while the other three did. For the peptide CLGGLLTMV (SEQ ID NO: 2), the two insertions that produced good penetration also produced good killing.

[0197] Next, insertion of SEQ ID NO:1 and SEQ ID NO:3 into CDRH3 was tested (Table 7). SEQ ID NO:3 shows some structural similarity to the CDR it replaces, in particular the "G" near the beginning of the CDR is maintained, so it was tested together with SEQ ID NO:1. CDRH3 was previously reported to be completely dispensable for antibody binding and cell penetration (see Lee et al., "Functional Consequences of Complementarity-determining Region Deactivation in a Multifunctional Anti-nucleic Acid Antibody", J Biol Chem. 2013 Dec 13;288(50):35877-85, which is incorporated herein by reference in its entirety). However, insertion of SEQ ID NO:1 significantly impaired penetration, making it only slightly better than the negative control antibody. In contrast, insertion of SEQ ID NO:3 always resulted in penetration as good, if not better, than the parent antibody at the two insertion positions. All this suggests that the selection of an immunogenic peptide that matches the insertion site is crucial. [Table 7]

[0198] Interestingly, when specific killing was tested using a Trojan Horse antibody containing SEQ ID NO:1 inserted in CDRH3, unexpected results were observed. The two Tabs with the lowest measured penetration showed specific killing (Figure 7B). The low detection within cells could have been due to rapid antibody processing and presentation on the cell surface leading to effective killing.

[0199] Insertions of SEQ ID NO:1 and SEQ ID NO:5 into CDRL3 and SEQ ID NO:1 into CDRH2 were also tested (Table 8). SEQ ID NO:5 has very high sequence alignment with CDRL3 and was therefore selected for testing. Some of the constructs contained substitutions of amino acids adjacent to the CDRs. This was done because these additional substitutions were predicted to reduce perturbation of the overall antibody conformation. Specifically, in constructs T1_44 and T1_45, an "F" was also substituted directly C-terminal to the CDR. In construct T18, a "Y" was also substituted directly C-terminal to the CDR. All tested insertions resulted in good penetration, essentially equivalent to the parent antibody. This result is crucial since CDRL3 has been reported to be important for cell penetration, and this function was not perturbed by the replacement of this CDR with the immunogenic peptide tested. [Table 8]

[0200] Specific killing was tested for Trojan Horse antibodies containing SEQ ID NO:1, where all antibodies were found to induce killing (FIG. 7C). This indicates that this insertion results in high measured penetration and high levels of specific killing. Trojan Horse antibodies containing combinations of inactive CDR substitutions are also generated. Since four CDRs are inactive, combinations of two, three and four immunogenic peptides are generated. The inserted peptides can be repeats of the same peptide or different peptides.

[0201] A second DNA-binding antibody capable of penetrating into cells was also tested. Antibody 3E10 (see Weisbart et al., "DNA-dependent targeting of cell nuclei by a lupus autoantibody", Sci Rep. 2015 Jul 9;5:12022, which is incorporated herein by reference in its entirety) was probed for inactive CDRs and it was determined that both CDRL1 and CDRL2 were not involved in antigen binding. SEQ ID NO:1 was inserted into either CDRL1 or CDRL2, and SEQ ID NO:3 was also inserted into CDRL1 (Table 9). One of the constructs contained substitutions adjacent to amino acids adjacent to the CDRs. This was done because additional substitutions were predicted to reduce perturbation of the overall antibody conformation. Specifically, construct T2_13 was similarly substituted with "LLIK" directly N-terminal to the CDR and "YL" directly C-terminal to the CDR. All SEQ ID NO:1 insertions resulted in penetration above the negative control, but again the levels of penetration varied. [Table 9]

[0202] Killing by peptide-specific effector cells was similarly tested on cancer cells treated with a Trojan Horse antibody derived from 3E10 and containing SEQ ID NO:1. Both tested TAbs caused strong specific killing (FIG. 8). These results demonstrate the generality of the Trojan Horse system. Immunogenic peptides can be inserted into a variety of antibodies that can penetrate into cells and cause peptide-specific killing.

[0203] All TAbs generated are incubated with cancer cells and permeation is measured. Permeation is observed for all peptides determined to maintain antibody conformation upon substitution of inactive CDRs. All TAbs are tested for their ability to induce specific cancer cell killing in the presence of peptide-specific effector cells. Those skilled in the art will understand that the specific type of cancer tested is not important, since all cancer cells can carry HLA complexes to the cell surface and thereby present the immunogenic peptide of the TAb. The TAb can produce enhanced specific cell killing beyond that of the parent antibody without the immunogenic peptide. Trojan horse antibodies containing combinations of inactive CDR substitutions are also generated. Since two CDRs are inactive, combinations of two immunogenic peptides are generated. The peptides inserted can be repeats of the same peptide or different peptides.

[0204] Example 6: Functional validation of Trojan horse antibodies (sequence similarity) Substitutions based on sequence similarity were also performed on the TMab4 antibody. Screening of immunogenic peptides (see Table 3) found that only 0.0057% of peptides tested exceeded the alignment score threshold (>25) for possible substitutions. The three with the highest similarity to the TMab4 antibody were selected for substitution. SEQ ID NO:6 from EBV corresponded to amino acids 28-35 in the heavy chain of the antibody. It contains two amino acids ("MH") adjacent to CDRH1 at its C-terminus. A compensatory mutation (Y27D) in the heavy chain was also made to reduce antibody promiscuity. SEQ ID NO:7 from SARS-CoV2 corresponds to amino acids 52-60 in the heavy chain of the antibody. It contains two amino acids ("YY") adjacent to CDRH2 at its C-terminus. SEQ ID NO:8 from EBV corresponds to amino acids 14-22 in the first framework region of the light chain of the antibody. These three TAbs were generated and penetration tested as previously described (Table 10). All three showed penetration beyond that of the negative control, but SEQ ID NO:7 and SEQ ID NO:8 produced penetration equivalent to or better than the parent antibody, while SEQ ID NO:6 produced poorer penetration. [Table 10]

[0205] Sequence similarity methods were also used to modify the 3E10 antibody. Screening of immunogenic peptides (see Table 3) found that only 0.0093% of peptides tested exceeded the alignment score threshold (>25) for possible substitutions. SEQ ID NO:6 from EBV corresponds to amino acids 28-36 within CDRL1 of the antibody. SEQ ID NO:9 from SARS-CoV2 corresponds to amino acids 50-58 within the heavy chain of the antibody. It contains four amino acids ("LLIK") adjacent to CDRL2 at its N-terminus and two amino acids ("YL") adjacent to CDRL2 at its C-terminus. Both TAbs were generated and were found to have penetration into cells that was comparable to the parent antibody (Table 11). [Table 11]

[0206] Finally, a third antibody, a DNA-binding antibody, 71F12 (see Sakakibara et al., “Clonal evolution and antigen recognition of anti-nuclear antibodies in acute systemic lupus erythematosus”, Sci Rep. 2017;7:16428, which is incorporated herein by reference in its entirety), was analyzed for inactive CDRs and CDRL1 was determined to be inactive. Substitutions were made based on sequence similarity, with SEQ ID NO:10 from adenovirus corresponding to amino acids 26-34 within CDRL1 of the antibody (termed T4_1). SEQ ID NO:11 from HPV corresponding to amino acids 17-24 in the first framework region of the heavy chain of the antibody (termed T4_3). Both TAbs were generated and were found to have penetration into cells that was at least equivalent to the parental antibodies.

[0207] All generated TAbs were also tested for specific cell killing as described above. TAbs that successfully entered cells were able to induce cell killing superior to the killing-free parent antibody.

[0208] Example 7: Further confirmation of killing by Trojan horse antibodies Killing by the Trojan Horse antibodies was confirmed by a second method. Cancer cells were tracked and apoptosis quantified in the presence of caspase 3 / 7 dye using an IncuCyte imaging system (see Materials and Methods). TAbs derived from TMab4 and 3E10 antibodies (T18 and T2_11) were selected for further investigation as both showed good killing. As can be seen in Figures 9A-9B, both TAbs caused specific cell killing well beyond that caused by effector cells alone or by effector cells co-cultured with parental antibody-treated cells. This indicates that the immunogenic peptide is not only imported into the target cells but is also processed and presented on the cell surface, which allows specific killing by effector cells.

[0209] Other TAbs are also tested using the IncuCyte imaging assay. Specific killing beyond that induced by effector cells alone and / or the parental antibody is observed with the TAbs of the invention.

[0210] The sequences of the various TAbs produced, and the parent antibodies, are summarized in Table 12. It will be understood that if the heavy chain contains an immunogenic peptide, the parent light chain is used to produce the TAb, and if the light chain contains an immunogenic peptide, the parent heavy chain is used to produce the TAb. In particular, the 3E10 antibody has two variants (called P2 and P3) that contain different light chains. Either can be used to generate TAbs. [Table 12]

[0211] Various known therapeutic antibodies were investigated for inactive CDRs. A crystal structure analysis of each CDR in contact with its antigen was performed, and the number of amino acids in contact with the antigen was counted. A distance of less than 5 Å between the amino acids of the CDR and the antigen was considered as a contact. Three or more amino acids in contact with the antigen indicate a CDR involved in binding. Fewer than two indicate a definite inactive CDR. Exactly two indicate a CDR that is unlikely or poorly involved in binding. For evaluation, all such CDRs are considered inactive. Numerous other antibodies were found to contain inactive CDRs that can be replaced with immunogenic peptides such as those provided in Tables 1-3. MBRs are used to optimize positioning. Alternatively, TAbs are designed by identifying peptides from Table 3 that have sequence homology to known antibodies, especially those with inactive CDRs. Peptides with high homology are swapped into the antibody to generate TAbs. All generated Tabs are tested for cell penetration and maintain permeability. The TAbs are also tested for specific cell killing as described above to observe the level of killing that exceeds that obtained with the parent antibody.

[0212] Example 8: Trojan horse antibodies combined with targeting antibodies (bispecific Trojan horse antibodies, bi-TAbs). The aforementioned TAbs may be considered as killing modules, as they have been shown to enter target cells and induce specific killing by effector cells primed against the immunogenic peptide. However, to increase the specificity of these molecules, target them to cancer cells, and reduce off-target effects, a targeting module was added. Three EGFR antibodies were selected to be used as targeting modules: cetuximab, panitumumab, and necitumumab. All three are known to bind specifically to EGFR and target cancers that overexpress EGFR. Antibodies that target other cancer-specific / overexpressed molecules are also possible. Since this is just a targeting module and is different from the killing module, any known cancer targeting antibody can be used. EGFR was used simply as a proof of concept.

[0213] T18 and T2_11 TAbs were used for the generation of bi-TAbs as proof of principle. Control bifunctional antibodies were also generated using TMab4 or 3E10 parent antibodies. Bispecific molecules were generated using the known knob-in-hole approach to limit homodimerization of the heavy chains. Unique modifications were made in the constant regions of the heavy chains of both the killing and targeting modules (see Table 13; see the method provided in Shatz et al., "Knob-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity", 2013, mAbs 5:6, 872-881, which is incorporated herein by reference in its entirety). This promotes heterodimerization between different heavy chains and prevents homodimerization. The generated bi-TAbs were found to have a purity of at least 90% of the desired bifunctional molecules. A schematic of the final bi-TAbs and their controls is shown in FIG. 10 and the full sequences are provided in Table 13. [Table 13] TIFF2024538427000028.tif16159

[0214] The bi-TAbs were tested for their ability to bind to surface EGFR on breast cancer cells expressing high levels of EGFR. It was predicted that the additional killing module should not affect surface binding of the targeting module, which was indeed observed (Figure 11). The bi-TAbs successfully bound to surface EGFR at either 0.3 nM or 3 nM concentration, at levels comparable to the control original anti-EGFR antibody. Thus, the bi-TAbs can effectively target cancer cells based on the selected targeting module.

[0215] Next, the ability of bi-TAbs to deliver immunogenic peptides onto the surface of cancer cells in complex with HLA molecules was tested. HLA peptide presentation was measured as previously described herein (Materials and Methods). A time course experiment was performed to monitor surface presentation over time. All five bi-TAbs successfully delivered peptides to the cell surface by 10 hours of incubation (Figure 12). All bi-TAbs outperformed their control counterparts.

[0216] Finally, cancer cell killing was investigated at various effector cell concentrations. bi-TAbs 3-5, each with a different targeting module, were tested at effector:target cell (E:T) concentrations of 1:1, 3:1, and 6:1. At a ratio of 6:1, all three antibodies induced specific killing beyond that observed with the control antibody (Figure 13). bi-TAbs 4 and 5 (one containing TAb-derived P1 and one containing TAb-derived P2) also showed increased killing at ratios of 3:1 and even 1:1. Cultures without effector cells were used as controls. These results indicate a high level of efficacy of bi-TAbs, since they combine cancer cell targeting with a high level of specific killing.

[0217] The three anti-EGFR antibodies used contain inactive CDRs themselves. A summary of these inactive CDRs is provided in Table 14. CDRH1, CDRL1 and CDRL2 of cetuximab and CDRL1 and CDRL2 of panitumumab and CDRL1 and CDRL2 of necitumumab were all found to be inactive. These CDRs are also replaced with immunogenic peptides. The targeting of the replaced antibodies is confirmed, and the ability to bind to EGFR and target EGFR overexpressing cancer cells is maintained. The combination of the modified targeting module with the killing module increases the number of immunogenic peptides delivered. The peptides used in the killing module and the peptides used in the targeting module can be the same or different. [Table 14]

[0218] Bi-TAbs are generated using other killing modules and various targeting modules. The bi-TAbs are evaluated as described above for cancer cell targeting, HLA peptide surface presentation and specific killing. The bi-Tabs outperform control bifunctional molecules in being able to target cancer, induce peptide presentation, and ultimately enhance killing of target cells by effector cells specific for the immunogenic peptide.

[0219] In vivo validation is also performed. The engineered bi-TAbs are injected into immune-competent mice expressing tumors that can be targeted by the targeting module. Control bifunctional molecules that lack the immunogenic peptide and / or bind to non-cancer-related targets are also administered. Animal survival over time is monitored as well as tumor size. Trojan horse antibodies are found to statistically significantly shrink tumors and / or extend survival time, indicating that they activate the immune system against cancer. Mice can be vaccinated with the immunogenic peptide beforehand.

[0220] While the present invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. a. a first antibody or antigen-binding fragment thereof comprising at least one immunogenic peptide inserted into a CDR of the antibody or antigen-binding fragment thereof, wherein the insertion comprises removal of a CDR sequence, and optionally the immunogenic peptide is recognized by CD4 T cells, CD8 T cells, or both; and b. a second antibody capable of binding epidermal growth factor receptor (EGFR), wherein the second antibody is selected from cetuximab, panitumumab, and necitumumab, or an antibody comprising at least 85% sequence identity thereto; A dual-function antigen-binding molecule comprising: Claim 2: a. the first antibody or antigen-binding fragment thereof and the second antibody comprise at least one modification that promotes heterodimerization and inhibits homodimerization; b. the first and second antibodies comprise a heavy chain constant region comprising SEQ ID NO: 1074, and the other antibody comprises a heavy chain constant region comprising SEQ ID NO: 1075; c. the first antibody or antigen-binding fragment thereof binds to a target cell, and the target cell is a cancer cell, a dendritic cell, or both; d. The immunogenic peptide is a cancer-specific peptide. e. The cancer-specific peptide is a cancer-specific peptide selected from the peptide sequences provided in Table 1. f. The immunogenic peptide is a viral peptide. g. The immunogenic peptide is a viral peptide derived from cytomegalovirus (CMV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), adenovirus, human papillomavirus (HPV), or influenza virus (FLU); h. the immunogenic peptide is a viral peptide selected from the peptide sequences provided in Table 2 or Table 3; and i. the first antibody or antigen-binding fragment thereof binds to a target cell; the target cell is a dendritic cell, and the dendritic cell antigen is selected from CD40, CD205, CD206, CLEC9A, CLEC12A, CD209, and CD207; the target cell is a malignant immune cell and the immune cell antigen is selected from CD20, CD19, CD21, and CD22; or the target cells are cancer cells and the cancer cell antigens are selected from HER2, EGFR, EpCAM, PSMA, BCMA, CD123, CD33, CD38, CTLA, LAG-3, ICOS, 4-1BB, and PD-L1; The dual-function antigen-binding molecule of claim 1, wherein the dual-function antigen-binding molecule is at least one.

3. 3. The dual-function antigen-binding molecule of claim 1, further comprising a cell-penetrating sequence that directs the first antibody or its antigen-binding fragment to the cytoplasm of a cell bound by the first antibody or its antigen-binding fragment, and optionally the cell-penetrating sequence is an endosomal escape domain (EED), such that the first antibody or its antigen-binding fragment is taken up into the endosomal pathway and delivered to the cytoplasm or both.

4. The dual-function antigen-binding molecule of claim 1 or 2, wherein the CDR is an inactive CDR that contributes little or nothing to binding to the target antigen.

5. The dual-function antigen-binding molecule of claim 1, wherein at least one inactive CDR in the antigen-binding region is replaced with a cell-penetrating sequence.

6. The dual-function antigen-binding molecule of claim 4, wherein the inactive CDR comprises no more than two amino acids that contact the target antigen, and the contact comprises a distance of no more than 5 Å between an amino acid of the CDR and an amino acid of the target antigen.

7. The dual function antigen-binding molecule of claim 1 or 2, wherein the insertions and deletions result in no or minimal change in the overall conformation of the first antibody or antigen-binding fragment thereof such that the first antibody or antigen-binding fragment thereof binds its target antigen with an affinity equivalent to that of the first antibody or antigen-binding fragment lacking the immunogenic peptide.

8. Before the immunogenic peptide is inserted, the first antibody a. the antibody TMab4 comprising the heavy chain variable region of SEQ ID NO: 1021 and the light chain variable region of SEQ ID NO: 1022; b. antibody 3E10 comprising the heavy chain variable region of SEQ ID NO: 1023 and the light chain variable region of SEQ ID NO: 1024; and c. Antibody 71F12 comprising the heavy chain variable region of SEQ ID NO: 1026 and the light chain variable region of SEQ ID NO: 1027 is selected from the immunogenic peptide is inserted into CDRH1, CDRH2, CDRH3 or CDRL3 of the TMab4, into CDRL1 or CDRL2 of the 3E10, or into CDRL1 of the 71F12; The dual-function antigen-binding molecule of claim 1 or 2.

9. The first antibody is a. a light chain variable region of SEQ ID NO: 1022 and a heavy chain variable region selected from SEQ ID NOs: 1028-1040, 1043-1045, 1047-1055, and 1058-1059; b. a heavy chain variable region of SEQ ID NO: 1021 and a light chain variable region selected from SEQ ID NOs: 1041-1042, 1046, and 1056-1057; c. a heavy chain variable region of SEQ ID NO: 1023 and a light chain variable region selected from SEQ ID NOs: 1060-1065; d. a heavy chain variable region of SEQ ID NO: 1026 and a light chain variable region of SEQ ID NO: 1066; and e. a light chain variable region of SEQ ID NO: 1027 and a heavy chain variable region of SEQ ID NO: 1067; and The bifunctional antigen-binding molecule comprises two heavy chains and two light chains, f. the two heavy chains are SEQ ID NOs: 1088 and 1080 and the two light chains are SEQ ID NOs: 1087 and 1079; g. the two heavy chains are SEQ ID NOs: 1088 and 1082 and the two light chains are SEQ ID NOs: 1087 and 1081; h. the two heavy chains are SEQ ID NOs: 1090 and 1080 and the two light chains are SEQ ID NOs: 1089 and 1079; i. the two heavy chains are SEQ ID NOs: 1090 and 1082 and the two light chains are SEQ ID NOs: 1089 and 1081; or j. The two heavy chains are SEQ ID NOs: 1088 and 1086 and the two light chains are SEQ ID NOs: 1087 and 1085; or both; The dual-function antigen-binding molecule of claim 8.

10. An antibody or antigen-binding fragment thereof comprising at least one immunogenic peptide inserted into a variable region of said antibody or antigen-binding fragment, said insertion comprising a deletion of an antibody or antigen-binding fragment sequence.

11. A pharmaceutical composition comprising the dual-function antigen-binding molecule of claim 2 or the antibody or antigen-binding fragment thereof of claim 10 and a pharmaceutically acceptable carrier, excipient or adjuvant.

12. The pharmaceutical composition of claim 11 for use in treating cancer in a subject in need thereof, optionally wherein the cancer is an EGFR-positive cancer.

13. 11. A nucleic acid molecule comprising at least one open reading frame encoding the dual-function antigen-binding molecule of claim 1 or 2 or the antibody or antigen-binding fragment thereof of claim 10, optionally wherein the nucleic acid molecule is an expression vector and comprises at least one regulatory element operably linked to the nucleic acid molecule.

14. 1. A method of engineering an antibody or antigen-binding fragment thereof, comprising: I. a. selecting an antibody or antigen-binding fragment thereof of interest; b. receiving a structural analysis of the selected antibody or antigen-binding domain bound to its target; c. determining at least one CDR of the selected antibody or antigen-binding domain that is not required for binding to the target based on the structural analysis; d. replacing said determined at least one CDR or portion thereof with an immunogenic peptide; or II. a. selecting an antibody or antigen-binding fragment thereof of interest; b. receiving a database of immunogenic peptides; c. performing pairwise alignments of peptides from the variable regions of the selected antibody or antigen-binding fragment thereof of interest with immunogenic peptides from the database; d. Determining peptides and immunogenic peptides derived from the selected antibodies or antigen-binding fragments thereof that have alignment scores above a predetermined threshold; and e. replacing the determined peptide from the selected antibody or antigen-binding fragment thereof with the determined immunogenic peptide; Including, This method allows the antibody or antigen-binding fragment thereof to be engineered.

15. The method further comprising optimizing the substitution to cause as little perturbation as possible in the structure of the selected antibody or antigen-binding fragment thereof of interest. b. The engineered antibody or antigen-binding fragment thereof is an immunogenic peptide delivery antibody; c. step (a) comprises selecting an antibody or antigen-binding fragment thereof that binds to the surface of a target cell; d. step (a) comprises selecting an antibody or antigen-binding fragment thereof that binds to the surface of a target cell and, upon binding to the surface, is internalized and delivered to the cytosol of said target cell; e. the method further comprises confirming at least one of: delivery of the immunogenic peptide to the cytosol of the target cell, delivery of the immunogenic peptide in a complex with an HLA molecule to the surface of the target cell, and specific killing of the target cell by an effector cell specific for the immunogenic peptide; f. The method further comprises selecting a targeting antibody that binds to a protein on the surface of a target cell, and combining the engineered antibody with the targeting antibody to generate a bifunctional antigen-binding molecule, optionally comprising engineering the heavy chain constant region of the targeting antibody and the heavy chain constant region of the engineered antibody to promote heterodimerization and suppress homodimerization; The method of claim 14, wherein there is at least one.

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