Immunogenic peptide antibody constant region molecules

Abstract

Bispecific antibodies comprising mutations that decrease homodimerization and at least one immunogenic peptide inserted into a CHI or CL domain are provided. Proteins comprising an Fc variant of a parent Fc polypeptide wherein at least one immunogenic peptide is inserted into the parent Fc polypeptide and the insertion replaces amino acids of the parent Fc polypeptide are provided. Protein dimers and antibodies comprising the proteins of the invention are also provided as are nucleic acid molecules encoding same, pharmaceutical compositions comprising same and methods of treating cancer by administrating same. Methods of producing antibodies are also provided.

Description

IMMUNOGENIC PEPTIDE ANTIBODY CONSTANT REGION MOLECULESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 716,776, titled “IMMUNOGENIC PEPTIDE FC MOLECULES”, which was filed November 6, 2024, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (TROJ-P-003-PCT.xml; Size: 1,133,695 bytes; and Date of Creation: November 6, 2025) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of anti-cancer immunotherapy.BACKGROUND OF THE INVENTION

[0004] Delivery of immunogenic peptides into target disease cells allows for the harnessing of the immune response against those cells. International Patent Application WO2023067596 describes dual function antigen binding molecules that can bind a target protein on the surface of the target cells with one arm (targeting arm / antibody) and then penetrate into the cell cytosol with a second arm (penetrating arm / antibody). At least one immunogenic peptide is inserted into an inert CDR of one of the arms. When the molecules are brought to and digested within the cytosol, the immunogenic peptide is freed and can be brought to the cell surface by HLA proteins. In this way the target cell is made immunogenic and recognizable by the immune system. Alternative and improved dual function antigen binding molecules are needed.SUMMARY OF THE INVENTION

[0005] The present invention provides bispecific antibodies comprising mutations that decrease homodimerization and at least one immunogenic peptide inserted into a CHI or CL domain. Proteins comprising an Fc variant of a parent Fc polypeptide wherein at least one immunogenic peptide is inserted into the parent Fc polypeptide and the insertion replaces amino acids of the Fc are also provided. Protein dimers and antibodies comprising the proteins of the invention are also provided as are nucleic acid molecules encoding same, pharmaceutical compositions comprising same and methods of treating cancer by administrating same. Methods of producing antibodies are also provided.

[0006] According to a first aspect, there is provided, a bispecific antibody comprising: a. a first antibody comprising a first heavy chain and first light chain, wherein the first heavy chain comprises a CH3 domain comprising a first mutation that decreases homodimerization and wherein the first antibody comprises at least one immunogenic peptide inserted into a CHI domain, wherein the insertion replaces amino acids of the CHI domain; b. and a second antibody comprising a second heavy chain and second light chain, wherein the second heavy chain comprises a CH3 domain comprising a second mutation that decreases homodimerization and wherein the second antibody comprises at least one immunogenic peptide inserted into a CHI domain, wherein the insertion replaces amino acids of the CHI domain; wherein the first and second mutation are a pair that increases heterodimerization of the first heavy chain and the second heavy chain, and wherein the first and second antibodies are different antibodies.

[0007] According to some embodiments, the first mutation and the second mutation are selected from the mutation pairs provided in Table 5.

[0008] According to some embodiments, the first mutation is a K409R mutation and the second mutation is a F405L mutation (EU numbering).

[0009] According to some embodiments, the at least one immunogenic peptide is inserted into a CHI domain of the first heavy chain and a CHI domain of the second heavy chain.

[0010] According to some embodiments, the at least one immunogenic peptide is inserted an AB loop, a BC loop, a CD loop, a DE loop, an EF loop or an FG loop of the CHI domain.[Oi l] According to some embodiments, the at least one immunogenic peptide is inserted into the CHI AB loop which comprises amino acids 124-145 of a constant region of the heavy chain, or the at least one immunogenic peptide is inserted into the CHI CD loop which comprises amino acids 154-171 of the constant region of the heavy chain and wherein numbering is according to EU numbering.

[0012] According to another aspect, there is provided a bispecific antibody comprising: a. a first antibody comprising a first heavy chain and first light chain, wherein the first heavy chain comprises a CH3 domain comprising a first mutation that decreases homodimerization and wherein the first antibody comprises at least one immunogenic peptide inserted into a CL domain, wherein the insertion replaces amino acids of the CL domain; b. and a second antibody comprising a second heavy chain and second light chain, wherein the second heavy chain comprises a CH3 domain comprising a second mutation that decreases homodimerization and wherein the second antibody comprises at least one immunogenic peptide inserted into a CL domain, wherein the insertion replaces amino acids of the CL domain; wherein the first and second mutation are a pair that increases heterodimerization of the first heavy chain and the second heavy chain, and wherein the first and second antibodies are different antibodies.

[0013] According to some embodiments, the at least one immunogenic peptide is inserted an AB loop, a BC loop, a CD loop, a DE loop, an EF loop or an FG loop of the CL domain.

[0014] According to some embodiments, the at least one immunogenic peptide is inserted into the CL AB loop which comprises amino acids 114-133 of a constant region of the light chain and wherein numbering is according to EU numbering.

[0015] According to some embodiments, the first antibody and the second antibody are both IgGl antibodies.

[0016] According to some embodiments, the immunogenic peptide is a cancer specific peptide.

[0017] According to some embodiments, the cancer specific peptide is selected from a peptide sequence provided in Table 1.

[0018] According to some embodiments, the immunogenic peptide is a viral peptide.

[0019] According to some embodiments, the viral peptide is derived from Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Severe acute respiratory syndrome coronavirus 2 (SARS- CoV2), Adenovirus, Human papilloma virus (HPV) or Influenza virus (FLU).

[0020] According to some embodiments, the viral peptide is selected from a peptide sequence provided in Table 2 or Table 3.

[0021] According to some embodiments, the immunogenic peptide comprises or consists of NLVPMVATV (SEQ ID NO: 1).

[0022] According to some embodiments, SEQ ID NO: 1 replaces amino acids 127-135, 132- 140 or 159-167 of the heavy chain constant region, wherein the number is according to EU numbering.

[0023] According to some embodiments, SEQ ID NO: 1 replaces amino acids 124-132 of the light chain constant region, wherein the number is according to EU numbering.

[0024] According to some embodiments, the CHI domain comprises or consists of a sequence selected from SEQ ID NO: 1211-1214 or the CL domain comprises or consists of SEQ ID NO: 1170.

[0025] According to some embodiments, one of the first and the second antibodies is a cell penetrating antibody.

[0026] According to some embodiments, the cell penetrating antibody is selected from TMab4, 3E10, and 71F12.

[0027] According to some embodiments, the cell penetrating antibody is TMab4.

[0028] According to some embodiments, at least one of the first and the second antibodies further comprises at least one immunogenic peptide inserted into a CDR of the antibody, where the insertion comprises removal of CDR sequence, and wherein the insertion does not reduce binding to an antibody target.

[0029] According to some embodiments, the at least one immunogenic peptide is inserted into an inert CDR of the antibody.

[0030] According to some embodiments, the at least one immunogenic peptide is inserted into at least one of CDRH1, CDRH2, CDRH3 or CDRL3 of antibody Tmab4.

[0031] According to some embodiments, a heavy chain variable region of TMab4 comprises or consists of SEQ ID NO: 1021, 1028-1040, 1043-1045, 1047-1055, 1058 and 1082 and alight chain variable region of TMab4 comprises or consists of SEQ ID NO: 1022, 1041- 1042, 1046, and 1056-1057.

[0032] According to some embodiments, one of the first and second antibodies is an anti- epidermal growth factor receptor (EGFR) antibody selected from: cetuximab, panitumumab, necitumumab and an antibody comprising at least 85% sequence identity to cetuximab, panitumumab, or necitumumab and which binds EGFR.

[0033] According to some embodiments, one of the first and second antibodies is an CD70 antibody selected from: vorsetuzumab (also known as hlF6), cusatuzumab, and an antibody comprising at least 85% sequence identity to vorsetuzumab, or cusatuzumab and which binds CD70.

[0034] According to another aspect, there is provided a protein comprising an Fc variant of a parent Fc polypeptide, wherein the Fc variant comprises at least one immunogenic peptide inserted into the parent Fc polypeptide and wherein the insertion replaces amino acids of the parent Fc polypeptide.

[0035] According to some embodiments, the immunogenic peptide is inserted into a loop between beta sheets of the parent Fc polypeptide.

[0036] According to some embodiments, the loop is within a CH2 domain of the Fc polypeptide and is selected from the BC loop, the DE loop and the FG loop.

[0037] According to some embodiments, the CH2 BC loop comprises amino acids 263-273 of the Fc polypeptide, the CH2 DE loop comprises amino acids 292-302 of the Fc polypeptide and the FG loop comprises amino acids 323-332 of the Fc polypeptide and wherein numbering is according to the EU index.

[0038] According to some embodiments, the loop is within a CH3 domain of the Fc polypeptide and is selected from the AB loop, the BC loop, the CD loop, the DE loop and the EF loop.

[0039] According to some embodiments, the CH3 AB loop comprises amino acids 350-365 of the Fc polypeptide, the CH3 BC loop comprises amino acids 371-379 of the Fc polypeptide, the CH3 CD loop comprises amino acids 383-391 of the Fc polypeptide, the CH3 DE loop comprises amino acids 397-407 of the Fc polypeptide and the CH3 EF loop comprises amino acids 413-424 of the Fc polypeptide and wherein numbering is according to the EU index.

[0040] According to some embodiments, the Fc is a human IgGl Fc.

[0041] According to some embodiments, the immunogenic peptide is a cancer specific peptide.

[0042] According to some embodiments, the cancer specific peptide is selected from a peptide sequence provided in Table 1.

[0043] According to some embodiments, the immunogenic peptide is a viral peptide.

[0044] According to some embodiments, the viral peptide is derived from Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Severe acute respiratory syndrome coronavirus 2 (SARS- CoV2), Adenovirus, Human papilloma virus (HPV) or Influenza virus (FLU).

[0045] According to some embodiments, the viral peptide is selected from a peptide sequence provided in Table 2 or Table 3.

[0046] According to some embodiments, the immunogenic peptide comprises NLVPMVATV (SEQ ID NO: 1).

[0047] According to some embodiments, SEQ ID NO: 1 is positions 354-362 of the Fc variant.

[0048] According to some embodiments, the Fc variant comprises SEQ ID NO: 1068, 1102, 1105, 1106, 1107 or 1108.

[0049] According to some embodiments, SEQ ID NO: 1 is positions 350-358 of the Fc variant.

[0050] According to some embodiments, the Fc variant comprises SEQ ID NO: 1069.

[0051] According to some embodiments, SEQ ID NO: 1 is positions 357-365 of the Fc variant.

[0052] According to some embodiments, the Fc variant comprises SEQ ID NO: 1070.

[0053] According to some embodiments, SEQ ID NO: 1 replaces positions 384-389 in the Fc polypeptide.

[0054] According to some embodiments, the Fc variant comprises SEQ ID NO: 1071.

[0055] According to some embodiments, SEQ ID NO: 1 replaces positions 384-388 in the Fc polypeptide.

[0056] According to some embodiments, the Fc variant comprises SEQ ID NO: 1072.

[0057] According to some embodiments, SEQ ID NO: 1 replaces positions 384-387 in the Fc polypeptide.

[0058] According to some embodiments, the Fc variant comprises SEQ ID NO: 1073.

[0059] According to some embodiments, SEQ ID NO: 1 is positions 416-424 of the Fc variant.

[0060] According to some embodiments, the Fc variant comprises SEQ ID NO: 1074.

[0061] According to some embodiments, SEQ ID NO: 1 replaces positions 418-422 in the Fc polypeptide.

[0062] According to some embodiments, the Fc variant comprises SEQ ID NO: 1075, 1100, 1101 or 1102.

[0063] According to some embodiments, SEQ ID NO: 1 replaces positions 415-422 in the Fc polypeptide.

[0064] According to some embodiments, the Fc variant comprises SEQ ID NO: 1076.

[0065] According to some embodiments, SEQ ID NO: 1 is positions 414-422 of the Fc variant.

[0066] According to some embodiments, the Fc variant comprises SEQ ID NO: 1077, 1103, 1104, 1105 or 1108.

[0067] According to some embodiments, SEQ ID NO: 1 replaces positions 418-424 in the Fc polypeptide.

[0068] According to some embodiments, the Fc variant comprises SEQ ID NO: 1078.

[0069] According to some embodiments, SEQ ID NO: 1 replaces positions 418-423 in the Fc polypeptide.

[0070] According to some embodiments, the Fc variant comprises SEQ ID NO: 1079.

[0071] According to some embodiments, SEQ ID NO: 1 is positions 264-272 of the Fc variant.

[0072] According to some embodiments, the Fc variant comprises SEQ ID NO: 1080, 1100, 1103, 1106 or 1108.

[0073] According to some embodiments, SEQ ID NO: 1 is positions 265-273 of the Fc variant.

[0074] According to some embodiments, the Fc variant comprises SEQ ID NO: 1081, 1101, 1104 or 1107.

[0075] According to some embodiments, the immunogenic peptide comprises GILGFVFTL (SEQ ID NO: 3).

[0076] According to some embodiments, SEQ ID NO: 3 is positions 354-362 of the Fc variant.

[0077] According to some embodiments, the Fc variant comprises SEQ ID NO: 1123.

[0078] According to some embodiments, SEQ ID NO: 3 is positions 357-365 of the Fc variant.

[0079] According to some embodiments, the Fc variant comprises SEQ ID NO: 1124.

[0080] According to some embodiments, SEQ ID NO: 3 is positions 350-358 of the Fc variant.

[0081] According to some embodiments, the Fc variant comprises SEQ ID NO: 1125.

[0082] According to some embodiments, SEQ ID NO: 3 is positions 371-379 in the Fc variant.

[0083] According to some embodiments, the Fc variant comprises SEQ ID NO: 1126.

[0084] According to some embodiments, SEQ ID NO: 3 is positions 399-407 in the Fc variant.

[0085] According to some embodiments, the Fc variant comprises SEQ ID NO: 1127.

[0086] According to some embodiments, SEQ ID NO: 3 replaces positions 398-403 in the Fc polypeptide.

[0087] According to some embodiments, the Fc variant comprises SEQ ID NO: 1128.

[0088] According to some embodiments, SEQ ID NO: 3 replaces positions 397-402 of the Fc polypeptide.

[0089] According to some embodiments, the Fc variant comprises SEQ ID NO: 1129.

[0090] According to some embodiments, SEQ ID NO: 3 replaces positions 397-404 in the Fc polypeptide.

[0091] According to some embodiments, the Fc variant comprises SEQ ID NO: 1130.

[0092] According to some embodiments, SEQ ID NO: 3 is positions 414-422 in the Fc variant.

[0093] According to some embodiments, the Fc variant comprises SEQ ID NO: 1131.

[0094] According to some embodiments, SEQ ID NO: 3 is positions 415-423 of the Fc variant.

[0095] According to some embodiments, the Fc variant comprises SEQ ID NO: 1132.

[0096] According to some embodiments, SEQ ID NO: 3 replaces positions 418-422 in the Fc polypeptide.

[0097] According to some embodiments, the Fc variant comprises SEQ ID NO: 1133.

[0098] According to some embodiments, SEQ ID NO: 3 replaces positions 418-421 in the Fc polypeptide.

[0099] According to some embodiments, the Fc variant comprises SEQ ID NO: 1134.

[0100] According to some embodiments, SEQ ID NO: 3 replaces positions 415-421 of the Fc polypeptide.

[0101] According to some embodiments, the Fc variant comprises SEQ ID NO: 1135.

[0102] According to some embodiments, SEQ ID NO: 3 is positions 265-273 of the Fc variant.

[0103] According to some embodiments, the Fc variant comprises SEQ ID NO: 1136.

[0104] According to some embodiments, SEQ ID NO: 3 is positions 264-272 of the Fc variant.

[0105] According to some embodiments, the Fc variant comprises SEQ ID NO: 1137.

[0106] According to some embodiments, SEQ ID NO: 3 replaces positions 264-271 of the Fc polypeptide.

[0107] According to some embodiments, the Fc variant comprises SEQ ID NO: 1138.

[0108] According to some embodiments, SEQ ID NO: 3 replaces positions 263-272 of the Fc polypeptide.

[0109] According to some embodiments, the Fc variant comprises SEQ ID NO: 1139.

[0110] According to some embodiments, SEQ ID NO: 3 replaces positions 295-299 of the Fc polypeptide.

[0111] According to some embodiments, the Fc variant comprises SEQ ID NO: 1140.

[0112] According to some embodiments, SEQ ID NO: 3 replaces positions 292-298 of the Fc polypeptide.

[0113] According to some embodiments, the Fc variant comprises SEQ ID NO: 1141.

[0114] According to some embodiments, SEQ ID NO: 3 is positions 294-302 of the Fc variant.

[0115] According to some embodiments, the Fc variant comprises SEQ ID NO: 1142.

[0116] According to some embodiments, SEQ ID NO: 3 replaces positions 325-332 of the Fc polypeptide.

[0117] According to some embodiments, the Fc variant comprises SEQ ID NO: 1143.

[0118] According to some embodiments, SEQ ID NO: 3 is positions 324-332 of the Fc variant.

[0119] According to some embodiments, the Fc variant comprises SEQ ID NO: 1144.

[0120] According to some embodiments, SEQ ID NO: 3 is positions 323-331 of the Fc variant.

[0121] According to some embodiments, the Fc variant comprises SEQ ID NO: 1145.

[0122] According to some embodiments, SEQ ID NO: 3 is positions 237-245 of the Fc variant.

[0123] According to some embodiments, the Fc variant comprises SEQ ID NO: 1146.

[0124] According to some embodiments, SEQ ID NO: 3 is positions 190-198 of the Fc variant.

[0125] According to some embodiments, the Fc variant comprises SEQ ID NO: 1147.

[0126] According to some embodiments, the insertion and replacing produces no change or minimal change in the overall conformation of the Fc such that the Fc still homodimerizes and / or heterodimerizes with a second Fc.

[0127] According to some embodiments, the protein is a heavy chain of an antibody and further comprises a heavy chain variable region.

[0128] According to some embodiments, the heavy chain variable region is the heavy chain variable region selected from TMab4, 3E10, 71F12 and TMab4, 3E10, or 71F12 comprising an inert CDR replaced with an immunogenic peptide.

[0129] According to some embodiments, the heavy chain variable region comprises a sequence selected from SEQ ID NO: 1021, 1023, 1026, 1028-1040, 1043-1045, 1047-1055, 1058-1059, 1067 and 1082.

[0130] According to some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO: 1082.

[0131] According to some embodiments, the protein comprises or consists of an amino acid sequence selected from SEQ ID NO: 1083-1096 and 1109-1117.

[0132] According to some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO: 1021.

[0133] According to some embodiments, the protein comprises or consists of an amino acid sequence selected from SEQ ID NO: 1118-1122.

[0134] According to another aspect, there is provided a protein dimer comprising a first protein and a second protein dimerized together, wherein the first protein and the second protein are both a protein of the invention.

[0135] According to some embodiments, the first protein and the second protein are different proteins.

[0136] According to some embodiments, the first and second protein are the same protein.

[0137] According to another aspect, there is provided an antibody comprising two heavy chains and two light chains and wherein each heavy chain is a protein of the invention.

[0138] According to some embodiments, a heavy chain variable region comprises one of SEQ ID NO: 1021, 1028-1040, 1043-1045, 1047-1055, 1058 and 1082 and a light chain variable region comprises one of SEQ ID NO: 1022, 1041-1042, 1046, and 1056-1057.

[0139] According to some embodiments, a heavy chain variable region comprises SEQ ID NO: 1023 and a light chain variable region comprises one of SEQ ID NO: 1024-1025, and 1060-1065.

[0140] According to some embodiments, a heavy chain variable region comprises one of SEQ ID NO: 1026 and 1067 and a light chain variable region comprises one of SEQ ID NO: 1027 and 1066.

[0141] According to another aspect, there is provided a pharmaceutical composition comprising a protein of the invention, a protein dimer of the invention or an antibody of the invention and a pharmaceutically acceptable carrier excipient or adjuvant.

[0142] According to another aspect, there is provided a nucleic acid molecule comprising at least one open reading frame, wherein the open reading frame encodes a protein of the invention, a protein dimer of the invention or an antibody of the invention.

[0143] According to another aspect, there is provided an expression vector comprising at least one regulatory element operatively linked to a nucleic acid molecule of the invention.

[0144] According to 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 a subject.

[0145] According to another aspect, there is provided a method of engineering an antibody, the method comprising: a. selecting an antibody of interest; b. replacing at least one sequence in the Fc region of the selected antibody with an immunogenic peptide; thereby engineering an antibody.

[0146] According to some embodiments, the at least one sequence in the Fc region is within a loop between beta sheets of the Fc region.

[0147] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since 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

[0148] Figures 1A-1B: (1A) A table of produced CH3 or CH2 CMV peptide (NLVPMVATV-SEQ ID NO: 1) inserted antibodies. Antibodies also contained CDR peptide insertion. (IB) Bar graph of specific cancer cell killing by the CH3 or CH2 peptide inserted antibodies or their parental antibody with only CDR insertions. Wild-type TMab4 is used as a negative control. Dotted line shows the killing of the CDR only antibody TRJ134.

[0149] Figures 2A-2B: (2A) A table of produced CH3 and CH2 CMV peptide inserted antibodies. Antibodies also contained CDR peptide insertion. (2B) Bar graphs of specific cancer cell killing in the presence and absence of T cells when contacted the CH2 and CH3 peptide inserted antibodies and their only CH2 or only CH3 parental antibodies.

[0150] Figure 3: A table of produced antibodies when the FLU peptide (GILGFVFTL-SEQ ID NO: 3) was inserted in CH2 or CH3. No CDR insertions were present. Only CH2 inserted antibodies were successfully produced and at very low titer.

[0151] Figures 4A-4C: (4A) A table of produced CH3 or CH2 CMV peptide inserted antibodies with no CDR peptide insertion. (4B) A table of produced CHI or CL CMV peptide inserted antibodies with no CDR peptide insertion. Antibodies already contain CH3 or CH2 CMV peptide insertion (4C) Bar graphs of cancer cell killing by the CH1 / CL and CH2 / CH3 peptide inserted antibodies. Killing is compared to the parental antibodies with just CH2 / CH3 insertion. The numbers above the bars indicate the CH1 / CL modification included. Numbering is as provided in 4B.

[0152] Figures 5A-5H: (5A-5B) Table showing the production of antibodies with CH3 CMV peptide insertions and heterodimerization mutations. The F405L mutation is in (5A) the penetration arm or (5B) the targeting arm. (5C) Table showing the production of a bispecific antibody with peptide insertions in the CDRs and CH3. (5D) Table showing the production of antibodies with CH3 CMV peptide insertions and heterodimerization mutations. (5E-5F) Table showing the production of antibodies with CHI CMV peptide insertions and heterodimerization mutations. The F405L mutation is in (5E) the penetration arm or (5F) the targeting arm. (5G) Tables showing the production of bispecific antibodies with peptide insertions in the CDRs and CHI. (5H) Summary table of the 5 produced bispecific antibodies.

[0153] Figures 6A-6C: (6A) Table of the anti-CD70 bispecific antibodies tested in the cancer killing assay. Black dots represent inserted peptides. Black rectangles and notches represent heterodimerization mutations K409R and F405L, respectively. (6B-6C) Line graphs of cancer cell killing in the presence of various concentrations of Trojan anti-CD70 bispecific antibodies (6B) with and (6C) without T cells.

[0154] Figures 7A-7C: (7A) Table of the anti-EGFR bispecific antibodies tested in the cancer killing assay. Black dots represent inserted peptides. Black rectangles and notches represent heterodimerization mutations K409R and F405L, respectively. (7B-7C) Line graphs of cancer cell killing in the presence of various concentrations of Trojan anti-EGFR bispecific antibodies (7B) with and (7C) without T cells.

[0155] Figures 8A-8C: Line graphs of tumor growth over time after administration of the bispecific antibody of the invention TRJ-316. The following controls are shown (8A) PBS; (8B) anti-CD70 antibody (Vorsetuzumab, SGN-75 sequence) and (8C) TRJ-195. One-tailedtwo-way ANOVA and / or Tukey’s post-hoc test were used to determine significance. *- pval <0.05, **-pval<0.01. Data are presented as mean ± SEM.DETAILED DESCRIPTION OF THE INVENTION

[0156] The present invention, in some embodiments, bispecific antibodies comprising mutations that decrease homodimerization and at least one immunogenic peptide inserted into a CHI or CL domain. Proteins comprising an Fc variant of a parent Fc polypeptide wherein at least one immunogenic peptide is inserted into a parent Fc polypeptide and the insertion replaces amino acids of the Fc are also provided. Proteins comprising a heavy chain constant region variant of a parent heavy chain constant region polypeptide wherein at least one immunogenic peptide is inserted into a parent heavy chain constant region polypeptide and the insertion replaces amino acids of the heavy chain constant region are also provided. Protein dimers and antibodies comprising the proteins of the invention are also provided as are nucleic acid molecules encoding same, pharmaceutical compositions comprising same and methods of treating cancer by administrating same. Methods of producing antibodies are also provided.

[0157] The invention is based on the surprising finding that the constant region of antibodies can be used as a delivery system for immunogenic peptides. That is, a highly immunogenic peptide can be delivered specifically to cancer cells and thereby increase immune surveillance against them. Inclusion of the immunogenic peptide in the variable region and the constant region greatly increases the amount of peptide delivered and thus increases its effectiveness. The antibody can have an antigen binding domain to a cancer epitope which would cause the therapeutic molecule to bind the cancer cell. Upon endocytosis of the antibody the immunogenic peptide would be delivered to the cytoplasm. This can be enhanced by the inclusion of a cell penetrating sequence, or specifically an endosomal escape sequence. The therapeutic molecule would be cleaved, releasing the many copies of immunogenic peptides which would then be displayed on the cell surface in complex with an HLA molecule, thus enhancing the immunogenicity of the cancer cell and increasing immune surveillance against the cancer and cancer killing. In order to generate bispecific antibodies with a targeting arm and a penetrating arm, heterodimerization mutations must be made in the CH3 domain of the antibody. It was surprisingly found that peptide insertion in not only the CH3, but also the CH2 domains could negatively impact expression of the antibodies with heterodimerization mutations. However, insertions specifically in the CHIdomains were found to not negatively interact with heterodimerization mutations and allowed for robust protein expression with up to 5 immunogenic peptides inserted. This increase in peptide insertions directly correlated with greater cancer cell killing.

[0158] Alternatively, the antigen binding region can bind a dendritic cell antigen, which would deliver the therapeutic molecule comprising a cancer cell antigen to a dendritic cell. The molecule would be endocytosed upon binding, and the numerous copies of the immunogenic peptide will be cleaved from the rest of the molecule of the invention and displayed on the surface of the dendritic cell by HLA molecules. This will in turn train cytotoxic immune cells (T cell and NK cells) to target this immunogenic peptide and thereby the cancer.

[0159] By a first aspect, there is provided a protein comprising an Fc variant.

[0160] By another aspect, there is provided a protein comprising an antibody heavy chain or light chain constant region variant.

[0161] By another aspect, there is provided a protein dimer comprising a first protein of the invention and a second protein of the invention.

[0162] By another aspect, there is provided an antibody comprising an Fc variant of the invention.

[0163] By another aspect, there is provided an antibody comprising an antibody heavy chain or light chain constant region variant of the invention.

[0164] By another aspect, there is provided a composition comprising a protein of the invention, a protein dimer of the invention or an antibody of the invention.

[0165] By another aspect, there is provided a nucleic acid molecule encoding the protein of the invention.

[0166] By another aspect, there is provided an expression vector comprising the nucleic acid molecule of the invention.

[0167] By another aspect, there is provided a method of expressing a peptide on a surface of a target cell, the method comprising contacting the target cell with a protein of the invention, protein dimer of the invention, antibody of the invention or a pharmaceutical composition of the invention, thereby expressing a peptide on a surface of a target cell.

[0168] By another aspect, there is provided a method of treating cancer in a subject in thereof, the method comprising administering to the subject a protein of the invention,protein dimer of the invention, antibody of the invention or a pharmaceutical composition of the invention, thereby treating cancer in a subject in need thereof.

[0169] By another aspect, there is provided a protein, dimer, antibody or composition of the invention for use in expressing a peptide on a surface of a target cell.

[0170] By another aspect, there is provided a protein, dimer, antibody or composition of the invention for use in treating cancer.

[0171] By another aspect, there is provided a protein, dimer, antibody or composition of the invention for use in the production of a medicament for the treating of cancer.

[0172] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating 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 residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0173] In some embodiments, the protein is a protein dimer. In some embodiments, the protein is an antibody. As used herein, the term “antibody” refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising 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 an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab’, F(ab’)2 single stranded antibody (svFC), dimericvariable region (Diabody) and disulphide-linked variable region (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 fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions. In some embodiments, the antibody is a bispecific antibody.

[0174] The term “Fc” refers to the fragment crystallizable region or constant region of the heavy chain. The Fc contains both the CH2 and CH3 domains. It can also sometimes be considered to include the CHI constant domain or the hinge, though the Fc can be provided without the CHI domain or the hinge. imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html provides a table of the amino acid number of the CHI, hinge, CH2 and CH3 regions. For simplicity the EU numbering index is provided herein throughout, but the Kabat numbering system can also be used and the corresponding numbering is well known. In some embodiments, the CHI domain is from positions 118-215 of the heavy chain according to EU numbering. In some embodiments, the hinge is from positions 216-230 of the heavy chain according to EU numbering. In some embodiments, the CH2 domain is from positions 231-340 of the heavy chain according to EU numbering. In some embodiments, the CH3 domain is from positions 341-447 of the heavy chain according to EU numbering. In some embodiments, the Fc comprises the CH2 and CH3 domains. In some embodiments, the Fc comprises the CHI, hinge, CH2 and CH3 domains. In some embodiments, the Fc comprises positions 118-447 of the heavy chain according to EU numbering. In some embodiments, positions are amino acids. As used herein, the term Fc will be understood to refer to the heavy chain constant region and therefore include CHI. In some embodiments, the heavy chain constant region comprises positions 118-447 of the heavy chain according to EU numbering.

[0175] In some embodiments, the variant is a variant of the light chain constant region. In some embodiments, the light chain constant region comprises a CL domain. In some embodiments, the light chain constant region consists of a CL domain. In some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain. In some embodiments, the light chain constant region comprises positions 108-214 of the light chain according to EU numbering. In some embodiments, the light chain constant region comprises positions 108-216 of the light chain according to Kabatnumbering. In some embodiments, the CL domain is from positions 107-216 of the light chain according to Kabat numbering. In some embodiments, the CL domain is from positions 108-216 of the light chain according to Kabat numbering. In some embodiments, the kappa CL domain is from positions 108-216 of the light chain according to Kabat numbering. In some embodiments, the CL domain is from positions 107-216 of the light chain according to Kabat numbering. In some embodiments, the lambda CL domain is from positions 107- 216 of the light chain according to Kabat numbering.

[0176] In some embodiments, the protein comprises an Fc. In some embodiments, the Fc is a variant Fc. In some embodiments, a variant is a variant of a parent Fc polypeptide. In some embodiments, the parent Fc is a human Fc. In some embodiments, the parent Fc is an IgGl. In some embodiments, the parent Fc is a human IgGl. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. In some embodiments, the parent Fc is human IgGl, IgG2, IgG3 or IgG4. It will be understood that all IgG alleles are considered, including for example, hIgG:01 and hIgG:03. In some embodiments, the Fc is from the human IgG. In some embodiments, the Fc is from human IgG allele hIgG:01. In some embodiments, the Fc is from human IgG allele hIgG:03. hIgG:01 and hIgG:03 differ in the following mutations: L358M, K214R, and D356E according to Eu numbering. The experiments reported hereinbelow were all performed with constant regions from an hIgG:03 allele. A skilled artisan can select the allele of the IgG as needed and a skilled artisan can convert any sequences provided herein with the 03 allele sequence to one with the 01 allele sequence. Indeed, the sequences in both alleles are contemplated and are part of the invention.

[0177] In some embodiments, the variant comprises at least one peptide. In some embodiments, the peptide is an immunogenic peptide. In some embodiments, the immunogenic peptide is inserted into the Fc polypeptide. In some embodiments, the immunogenic peptide replaces sequence within the Fc polypeptide. In some embodiments, inserted is inserted in place of amino acid sequence. In some embodiments, the peptide is inserted into a loop in the Fc. In some embodiments, the immunogenic peptide is inserted into the antibody constant region polypeptide. In some embodiments, the immunogenic peptide replaces sequence within the antibody constant region polypeptide. In some embodiments, the peptide is inserted into a loop in the antibody constant region. In some embodiments, the loop is between beta-sheets. In some embodiments, sequence from a loop is replaced with the peptide.

[0178] In some embodiments, the insertion is into the CH2 domain. In some embodiments, the insertion is into a CH2 loop. In some embodiments, the insertion is into the CH2 AB loop. In some embodiments, the insertion is into the CH2 BC loop. In some embodiments, the insertion is into the CH2 CD loop. In some embodiments, the insertion is into the CH2 DE loop. In some embodiments, the insertion is into the CH2 EF loop. In some embodiments, the insertion is into the CH2 FG loop. In some embodiments, the CH2 AB loop comprises amino acids 257-271 of an antibody heavy chain by Kabat numbering or animo acids 244- 258 by EU numbering. In some embodiments, the CH2 AB loop comprises amino acids 254-274 of an antibody heavy chain by Kabat numbering or animo acids 241-261 by EU numbering. In some embodiments, the CH2 AB loop comprises amino acids 252-276 of an antibody heavy chain by Kabat numbering or animo acids 239-263 by EU numbering. In some embodiments, the CH2 BC loop comprises amino acids 264-273 of an antibody heavy chain by Eu numbering or amino acids 277-286 by Kabat numbering. In some embodiments, the CH2 BC loop comprises amino acids 265-274 of an antibody heavy chain by EU numbering or amino acids 278 to 287 by Kabat numbering. In some embodiments, the CH2 BC loop comprises amino acids 264-274 of an antibody heavy chain by EU numbering or amino acids 277 to 287 by Kabat numbering. In some embodiments, the CH2 BC loop comprises amino acids 262-276 of an antibody heavy chain by EU numbering or amino acids275 to 289 by Kabat numbering. In some embodiments, the CH2 BC loop comprises amino acids 260-278 of an antibody heavy chain by EU numbering or amino acids 273 to 291 by Kabat numbering. In some embodiments, the CH2 CD loop comprises amino acids 280-287 of an antibody heavy chain by EU numbering or amino acids 295-304 by Kabat numbering. In some embodiments, the CH2 CD loop comprises amino acids 277-290 of an antibody heavy chain by EU numbering or amino acids 292-307 by Kabat numbering. In some embodiments, the CH2 CD loop comprises amino acids 275-292 of an antibody heavy chain by EU numbering or amino acids 290-309 by Kabat numbering. In some embodiments, the CH2 DE loop comprises amino acids 295-299 of an antibody heavy chain by EU numbering or amino acids 312-318 by Kabat numbering. In some embodiments, the CH2 DE loop comprises amino acids 294-302 of an antibody heavy chain by EU numbering or amino acids 311-321 by Kabat numbering. In some embodiments, the CH2 DE loop comprises amino acids 292-302 of an antibody heavy chain by EU numbering or amino acids 309-321 by Kabat numbering. In some embodiments, the CH2 DE loop comprises amino acids 290-304 of an antibody heavy chain by EU numbering or amino acids 307-323 by Kabat numbering. In some embodiments, the CH2 EF loop comprises amino acids 307-318 of an antibody heavy chain by EU numbering or amino acids 326-337 by Kabat numbering. In someembodiments, the CH2 EF loop comprises amino acids 304-321 of an antibody heavy chain by EU numbering or amino acids 323-340 by Kabat numbering. In some embodiments, the CH2 EF loop comprises amino acids 302-323 of an antibody heavy chain by EU numbering or amino acids 321-342 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 324-332 of an antibody heavy chain by EU numbering or amino acids 343-351 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 325-332 of an antibody heavy chain by EU numbering or amino acids 344-351 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 322-332 of an antibody heavy chain by EU numbering or amino acids 341-351 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 322-334 of an antibody heavy chain by EU numbering or amino acids 341-353 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 320-336 of an antibody heavy chain by EU numbering or amino acids 339-355 by Kabat numbering. In some embodiments, numbering is according to the EU index. In some embodiments, numbering is according to the Kabat index.

[0179] In some embodiments, the insertion is into the CH3 domain. In some embodiments, the insertion is into a CH3 loop. In some embodiments, the insertion is into the CH3 AB loop. In some embodiments, the insertion is into the CH3 BC loop. In some embodiments, the insertion is into the CH3 CD loop. In some embodiments, the insertion is into the CH3 DE loop. In some embodiments, the insertion is into the CH3 EF loop. In some embodiments, the insertion is into the CH3 FG loop. In some embodiments, the CH3 AB loop comprises amino acids 354-365 of an antibody heavy chain by EU numbering or amino acids 375-388 by Kabat numbering. In some embodiments, the CH3 AB loop comprises amino acids 352- 361 of an antibody heavy chain by EU numbering or amino acids 373-384 by Kabat numbering. In some embodiments, the CH3 AB loop comprises amino acids 349-364 of an antibody heavy chain by EU numbering or amino acids 370-387 by Kabat numbering. In some embodiments, the CH3 AB loop comprises amino acids 347-366 of an antibody heavy chain by EU numbering or amino acids 368-389 by Kabat numbering. In some embodiments, the CH3 AB loop comprises amino acids 355-362 of an antibody heavy chain by EU numbering or amino acids 376-385 by Kabat numbering. In some embodiments, the CH3 AB loop comprises amino acids 353-363 of an antibody heavy chain by EU numbering or amino acids 374-386 by Kabat numbering. In some embodiments, the CH3 BC loop comprises amino acids 373-377 of an antibody heavy chain by EU numbering or amino acids 396-400 by Kabat numbering. In some embodiments, the CH3 BC loop comprises amino acids 370-380 of an antibody heavy chain by EU numbering or amino acids 393-403 byKabat numbering. In some embodiments, the CH3 BC loop comprises amino acids 368-382 of an antibody heavy chain by EU numbering or amino acids 391-405 by Kabat numbering. In some embodiments, the CH3 CD loop comprises amino acids 383-390 of an antibody heavy chain by EU numbering or amino acids 408-418 by Kabat numbering. In some embodiments, the CH3 CD loop comprises amino acids 380-393 of an antibody heavy chain by EU numbering or amino acids 405-421 by Kabat numbering. In some embodiments, the CH3 CD loop comprises amino acids 378-395 of an antibody heavy chain by EU numbering or amino acids 403-423 by Kabat numbering. In some embodiments, the CH3 DE loop comprises amino acids 399-403 of an antibody heavy chain by EU numbering or amino acids 427-434 by Kabat numbering. In some embodiments, the CH3 DE loop comprises amino acids 396-406 of an antibody heavy chain by EU numbering or amino acids 424-437 by Kabat numbering. In some embodiments, the CH3 DE loop comprises amino acids 394-408 of an antibody heavy chain by EU numbering or amino acids 422-439 by Kabat numbering. In some embodiments, the CH3 EF loop comprises amino acids 414-424 of an antibody heavy chain by EU numbering or amino acids 445-455 by Kabat numbering. In some embodiments, the CH3 EF loop comprises amino acids 414-422 of an antibody heavy chain by EU numbering or amino acids 445-453 by Kabat numbering. In some embodiments, the CH3 EF loop comprises amino acids 411-425 of an antibody heavy chain by EU numbering or amino acids 442-456 by Kabat numbering. In some embodiments, the CH3 EF loop comprises amino acids 409-427 of an antibody heavy chain by EU numbering or amino acids 440-458 by Kabat numbering. In some embodiments, the CH3 FG loop comprises amino acids 429-435 of an antibody heavy chain by EU numbering or amino acids 460-466 by Kabat numbering. In some embodiments, the CH3 FG loop comprises amino acids 426-438 of an antibody heavy chain by EU numbering or amino acids 457-469 by Kabat numbering. In some embodiments, the CH3 FG loop comprises amino acids 424-440 of an antibody heavy chain by EU numbering or amino acids 455-471 by Kabat numbering. In some embodiments, numbering is according to the EU index. In some embodiments, numbering is according to the Kabat index.

[0180] In some embodiments, the insertion is into the CHI domain. In some embodiments, the insertion is into a CHI loop. In some embodiments, the insertion is into the CHI AB loop. In some embodiments, the insertion is into the CHI BC loop. In some embodiments, the insertion is into the CHI CD loop. In some embodiments, the insertion is into the CHI DE loop. In some embodiments, the insertion is into the CHI EF loop. In some embodiments, the insertion is into the CHI FG loop. In some embodiments, the CHI AB loop comprisesamino acids 127-140 of an antibody heavy chain by EU numbering or amino acids 123-138 by Kabat numbering. In some embodiments, the CHI AB loop comprises amino acids 127- 135 of an antibody heavy chain by EU numbering or amino acids 123-133 by Kabat numbering. In some embodiments, the CHI AB loop comprises amino acids 129-139 of an antibody heavy chain by EU numbering or amino acids 125-137 by Kabat numbering. In some embodiments, the CHI AB loop comprises amino acids 126-142 of an antibody heavy chain by EU numbering or amino acids 122-140 by Kabat numbering. In some embodiments, the CHI AB loop comprises amino acids 124-144 of an antibody heavy chain by EU numbering or amino acids 120-142 by Kabat numbering. In some embodiments, the CHI BC loop comprises amino acids 148-154 of an antibody heavy chain by EU numbering or amino acids 146-152 by Kabat numbering. In some embodiments, the CHI BC loop comprises amino acids 145-157 of an antibody heavy chain by EU numbering or amino acids 143-156 by Kabat numbering. In some embodiments, the CHI BC loop comprises amino acids 143-159 of an antibody heavy chain by EU numbering or amino acids 141-162 by Kabat numbering. In some embodiments, the CHI CD loop comprises amino acids 159-167 of an antibody heavy chain by EU numbering or amino acids 162-171 by Kabat numbering. In some embodiments, the CHI CD loop comprises amino acids 159-166 of an antibody heavy chain by EU numbering or amino acids 162-169 by Kabat numbering. In some embodiments, the CHI CD loop comprises amino acids 156-169 of an antibody heavy chain by EU numbering or amino acids 154-173 by Kabat numbering. In some embodiments, the CHI CD loop comprises amino acids 154-171 of an antibody heavy chain by EU numbering or amino acids 152-175 by Kabat numbering. In some embodiments, the CHI DE loop comprises amino acids 175-179 of an antibody heavy chain by EU numbering or amino acids 179-184 by Kabat numbering. In some embodiments, the CHI DE loop comprises amino acids 172-182 of an antibody heavy chain by EU numbering or amino acids 176-187 by Kabat numbering. In some embodiments, the CHI DE loop comprises amino acids 170-184 of an antibody heavy chain by EU numbering or amino acids 174-189 by Kabat numbering. In some embodiments, the CHI EF loop comprises amino acids 189-198 of an antibody heavy chain by EU numbering or amino acids 194-206 by Kabat numbering. In some embodiments, the CHI EF loop comprises amino acids 186-201 of an antibody heavy chain by EU numbering or amino acids 191-209 by Kabat numbering. In some embodiments, the CHI EF loop comprises amino acids 184-203 of an antibody heavy chain by EU numbering or amino acids 189-211 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 205-208 of an antibody heavy chain by EU numbering or amino acids 213-216 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 202-211 ofan antibody heavy chain by EU numbering or amino acids 210-219 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 200-213 of an antibody heavy chain by EU numbering or amino acids 208-221 by Kabat numbering. In some embodiments, numbering is according to the EU index. In some embodiments, numbering is according to the Kabat index.

[0181] In some embodiments, the insertion is into the light chain constant region. In some embodiments, the insertion is into the CL domain. In some embodiments, the insertion is into a CL loop. In some embodiments, the insertion is into the CL AB loop. In some embodiments, the insertion is into the CL BC loop. In some embodiments, the insertion is into the CL CD loop. In some embodiments, the insertion is into the CL DE loop. In some embodiments, the insertion is into the CL EF loop. In some embodiments, the insertion is into the CL FG loop. In some embodiments, the CL AB loop comprises amino acids 124- 132 of an antibody heavy chain by EU numbering or amino acids 124-132 by Kabat numbering. In some embodiments, the CL AB loop comprises amino acids 119-128 of an antibody heavy chain by EU numbering or amino acids 119-128 by Kabat numbering. In some embodiments, the CL AB loop comprises amino acids 116-131 of an antibody heavy chain by EU numbering or amino acids 116-131 by Kabat numbering. In some embodiments, the CL AB loop comprises amino acids 114-133 of an antibody heavy chain by EU numbering or amino acids 114-133 by Kabat numbering. In some embodiments, the CL AB loop comprises amino acids 119-133 of an antibody heavy chain by EU numbering or amino acids 119-133 by Kabat numbering. In some embodiments, the CL BC loop comprises amino acids 140-145 of an antibody heavy chain by EU numbering or amino acids 140-145 by Kabat numbering. In some embodiments, the CL BC loop comprises amino acids 137-148 of an antibody heavy chain by EU numbering or amino acids 137-148 by Kabat numbering. In some embodiments, the CL BC loop comprises amino acids 135-150 of an antibody heavy chain by EU numbering or amino acids 137-148 by Kabat numbering. In some embodiments, the CL CD loop comprises amino acids 151-158 of an antibody heavy chain by EU numbering or amino acids 151-158 by Kabat numbering. In some embodiments, the CL CD loop comprises amino acids 148-161 of an antibody heavy chain by EU numbering or amino acids 148-161 by Kabat numbering. In some embodiments, the CL CD loop comprises amino acids 146-163 of an antibody heavy chain by EU numbering or amino acids 146-163 by Kabat numbering. In some embodiments, the CL DE loop comprises amino acids 162-172 of an antibody heavy chain by EU numbering or amino acids 162-172 by Kabat numbering. In some embodiments, the CL DE loop comprises amino acids 159-175 of an antibody heavychain by EU numbering or amino acids 159-175 by Kabat numbering. In some embodiments, the CL DE loop comprises amino acids 157-177 of an antibody heavy chain by EU numbering or amino acids 157-177 by Kabat numbering. In some embodiments, the EF loop comprises amino acids 183-190 of an antibody heavy chain by EU numbering or amino acids 183-190 by Kabat numbering. In some embodiments, the EF loop comprises amino acids 180-193 of an antibody heavy chain by EU numbering or amino acids 180-193 by Kabat numbering. In some embodiments, the EF loop comprises amino acids 178-195 of an antibody heavy chain by EU numbering or amino acids 178-195 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 199-204 of an antibody heavy chain by EU numbering or amino acids 199-204 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 196-207 of an antibody heavy chain by EU numbering or amino acids 196-207 by Kabat numbering. In some embodiments, the FG loop comprises amino acids 194-209 of an antibody heavy chain by EU numbering or amino acids 194-209 by Kabat numbering. In some embodiments, numbering is according to the EU index. In some embodiments, numbering is according to the Kabat index.

[0182] In some embodiments, the antibody is a dual-function molecule. In some embodiments, the first function is binding an antigen. In some embodiments, the second function is entering a cell. In some embodiments, the second function is delivering a peptide. In some embodiments, the peptide is an immunogenic peptide. In some embodiments, the second function is delivering an immunogenic peptide into a cytoplasm of a cell.

[0183] 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, a cancer antigen is a cancer specific antigen. In some embodiments, a 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 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, an antigen is expressed on a cell. In some embodiments, an antigen is expressed on a cell surface. In some embodiments, an antigen is displayed on the cell surface as an MHC molecule. In some embodiments, an MHC molecule is an MHC class I or class II molecule. In someembodiments, an MHC molecule is a protein complex of the antigen and an HLA protein. In some embodiments, an antigen is a cell surface protein. In some embodiments, a cell surface protein is a cell surface receptor. In some embodiments, the antigen binding region is capable of binding the antigen.

[0184] In some embodiments, a 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 the 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 employed. For a non-limiting example Fab516 binds specifically to CD40.

[0185] In some embodiments, an immune cell antigen is selected from CD20, CD 19, CD21, and CD22. In some embodiments, the immune cell antigen is CD20. In some embodiments, the immune cell antigen is a B cell antigen. Markers of immune cells in general, and B cells in particular, are well known in the art and any such surface marker may be used as the antigen. Antigen binding domains that target immune cell antigens are well known in the art and any such antigen binding domain may be employed. For a non -limiting example Arzerra binds specifically to CD20

[0186] In some embodiments, a cancer cell antigen is selected from HER2, EGFR, EpCAM, PSMA, BCMA, CD123, CD33, CD38, CTLA, LAG-3, ICOS, 4-1BB and PD-L1. Markers of cancer cells are well known in the art and any such surface marker may be used as the antigen. In some embodiments, the cancer cell antigen is PD-L1. Antigen binding domains that target cancer cell antigens are well known in the art and any such antigen binding domain may be employed. For a non-limiting example Durvalumab binds specifically to PD-L1.

[0187] 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 antibody is a full antibody.

[0188] An “antigen” is a molecule or a portion of a molecule capable of eliciting antibody formation and being bound by an antibody. Antibody formation can occur in mice, rats, rabbits, pigs and other animals commonly used for generation of antibodies, but of course can also occur in humans as a response to a foreign antigen. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

[0189] The term “antigenic determinant” or “epitope” according to the invention refers to the region of an antigen molecule that specifically reacts with particular antibody. Peptide sequences derived from an epitope can be used, alone or in conjunction with a carrier moiety, applying methods known in the art, to immunize animals and to produce additional polyclonal or monoclonal antibodies. Immunoglobulin variable domains can also be analyzed using the IMGT information system (imgt. Cines.fr / ) (IMGT® / V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0190] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al, U.S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). The EU numbering system can also be used to number the variable region, though it is more commonly used when numbering the constant region of the antibody. When numbering is given herein it will be generally understood to correspond to Kabat numbering in the variable region and EU numbering in the constant region.

[0191] The term “antibody” (also referred to as an “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 a chimeric antibody or a humanized antibody is also encompassed by the invention.

[0192] The basic unit of the naturally occurring antibody structure 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 noncovalent associations and by disulfide bonds. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Five human antibody classes (IgG, IgA, IgM, IgD and IgE) exist, 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.

[0193] The amino terminal regions of the heavy and light chains are more diverse in sequence than the carboxy terminal regions, and hence are termed the variable domains. Thispart of the antibody structure confers the antigen-binding specificity of the antibody. A heavy variable (VH) domain and a light variable (VL) domain together form a single antigenbinding site, thus, the basic immunoglobulin unit has two antigen-binding sites. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0194] The carboxy terminal portion of the heavy and light chains form the constant domains i.e., CHI, CH2, CH3, CL. While there is much less diversity in these domains, there are differences from one animal species to another, and further, within the same individual there are several different isotypes of antibody, each having a different function.

[0195] The term “framework region” or “FR” refers to the amino acid residues in the variable domain of an antibody, which are other than the hypervariable region amino acid residues as herein defined. The term “hypervariable region” as used herein refers to the amino acid residues in the variable domain of an antibody, which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR”. The CDRs are primarily responsible for binding to an epitope of an antigen. The extent of FRs and CDRs has been precisely defined (see, Kabat et al.). In some embodiments, CDRs are determined using the KABAT system. In some embodiments, CDRs are determined using the Clothia system. In some embodiments, the Clothia system is the enhanced Clothia system (Martin system). In some embodiments, CDRs are determined using the EU numbering system.

[0196] In some embodiments, the parent Fc polypeptide is devoid or without the immunogenic peptide. In some embodiments, devoid of without the immunogenic peptide is before insertion of the immunogenic peptide. In some embodiments, the antibody binds to a target cell. In some embodiments, the antibody adheres to a surface of a target cell. In some embodiments, the antibody is a cell penetrating antibody. In some embodiments, the antibody is phagocytosed into the cell. In some embodiments, the antibody is endocytosed into the cell. In some embodiments, into the cell is into the endosomal pathway of the cell. In some embodiments, the antibody is brought into the endosomal pathway. In some embodiments, the antibody escapes from the endosomal pathway. In some embodiments, the antibody is delivered into the cytoplasm of a cell that binds it. In some embodiments, the antibody is a DNA binding antibody. In some embodiments, the antibody is a lupus antibody.

[0197] In some embodiments, the antibody is the Tmab4 antibody. In some embodiments, the Tmab4 comprises a heavy chain variable region of SEQ ID NO: 1021 or an analog or homolog comprising at least 85% sequence identity and being capable of binding cells and reaching the cytosol. In some embodiments, the Tmab4 comprises a light chain variable region of SEQ ID NO: 1022 or an analog or homolog comprising at least 85% sequence identity and being capable of binding cells and reaching the cytosol. In some embodiments, the antibody is the 3E10 antibody. In some embodiments, the 3E10 comprises a heavy chain variable region of SEQ ID NO: 1023 or an analog or homolog comprising at least 85% sequence identity and being capable of binding cells and reaching the cytosol. In some embodiments, the 3E10 comprises a light chain variable region of SEQ ID NO: 1024 or an analog or homolog comprising at least 85% sequence identity and being capable of binding cells and reaching the cytosol. In some embodiments, the antibody is the 71F12 antibody. In some embodiments, the 71F12 comprises a heavy chain variable region of SEQ ID NO: 1026 or an analog or homolog comprising at least 85% sequence identity and being capable of binding cells and reaching the cytosol. In some embodiments, the 71F12 comprises a light chain variable region of SEQ ID NO: 1027 or an analog or homolog comprising at least 85% sequence identity and being capable of binding cells and reaching the cytosol.

[0198] In some embodiments, the variant Fc 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 an exogenous immunogenic peptide. In some embodiments, the immunogenic peptide is inserted into the variant Fc. In some embodiments, the immunogenic peptide is inserted into a constant region of the antibody. In some embodiments, the immunogenic peptide is not a natural part of the Fc. In some embodiments, the peptide is a sequence of amino acids. In some embodiments, the sequence of immunogenic amino acids is inserted into the sequence of the Fc polypeptide. In some embodiments, the immunogenic peptide replaces amino acids of the Fc polypeptide. In some embodiments, insertion of the immunogenic peptide comprises removal of amino acid sequence. In some embodiments, the amino acid sequence is sequence of the Fc polypeptide. In some embodiments, the immunogenic peptide is not artificially linked to Fc polypeptide. In some embodiments, the variant Fc is a recombinant molecule. In some embodiments, the recombinant molecule comprises an amino acid sequence of the immunogenic peptide. In some embodiments, the immunogenic peptide is not linked by a chemical linkage to the Fc polypeptide. In some embodiments, the immunogenic peptide isnot linked by a chemical linkage to the variant Fc. In some embodiments, a chemical linkage is any linkage other than a peptide linkage. In some embodiments, a chemical linkage is any linkage other than an amino acid linkage. In some embodiments, the immunogenic peptide is linked to the Fc by a peptide bond, an amino acid linkage or both.

[0199] As used herein, the term “recombinant protein” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring. The term “recombinant DNA” refers to DNA molecules formed by laboratory methods of genetic recombination. Generally, this recombinant DNA is in the form of a vector, plasmid or virus used to express the recombinant protein in a cell.

[0200] 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 between 3 and 20, 3 and 15, 3 and 12, 3 and 11, 3 and 10, 3 and 7, 5 and 20, 5 and 15, 5 and 12, 5 and 11, 5 and10, 7 and 20, 7 and 15, 7 and 12, 7 and 11, or 7 and 10 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the immunogenic peptide is between 8 and 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.

[0201] As used herein, the term “immunogenic peptide” refers to an amino acids sequence that produces an immune response when exposed to the human immune system. In some embodiments, an immunogenic peptide is a non-human peptide. In some embodiments, the immunogenic peptide produces an immune response from an immune cell. In some embodiments, the immunogenic peptide is recognized an immune response from an immune cell. In some embodiments, recognized is bound by. In some embodiments, an immunogenic peptide produces an immune response from a dendritic cell. In some embodiments, an immunogenic peptide is displayed on the cell surface. In some embodiments, an immunogenic peptide is displayed as an MHC molecule. In some embodiments, an immunogenic peptide is displayed in complex with HLA. In some embodiments, the immunogenic peptide produces 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, a T cell is selected from a CD4 T cell and a CD8 T cell. In someembodiments, 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 produces an immune response from an NK cell. In some embodiments, an immune response is an elevated immune response. In some embodiments, immunogenic comprises increase immunogenicity. In some embodiments, increased is as compared to a control peptide. In some embodiments, the control peptide is a human peptide. In some embodiments, a control peptide is a non- cancerous peptide. In some embodiments, a control peptide is a non-immunogenic peptide.

[0202] 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 a cancer elevated peptide. In some embodiments, the cancer peptide is a peptide with increased surface expression in cancer cells. In some embodiments, a cancer peptide is a peptide provided in Table 1. In some embodiments, a cancer peptide is selected from a sequence provided in Table 1. In some embodiments, a cancer peptide is selected from the sequences provided in SEQ ID NO: 702-1020. Cancer peptides are well known in the art and can be found, for example at the Cancer Antigenic Peptide Database: caped.icp.ucl.ac.be / Peptide / search.

[0203] Table 1 : Cancer peptidesembodiments, 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 found, for example, at the Immune Epitope Database (IEDB) and VDJDB database: iedb.org and vdjdb.cdr3.net. In some embodiments, the viral peptide is derived from a virus selected from Cytomegalovirus (CMV), Epstein-Barr virus (EB V) or Influenza virus (FLU). In some embodiments, the viral peptide is derived from a virus selected from CMV, EBV, FLU, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), Adenovirus and Human Papilloma virus (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 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 a sequence 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 a sequence provided in Table 3. In some embodiments, the viral peptide is selected from SEQ ID NO: 1-701. In some embodiments, the viral peptide is selected fromSEQ ID NO: 1-695. In some embodiments, the viral peptide is selected from SEQ ID NO: 1-11. In some embodiments, the viral peptide is selected from SEQ ID NO: 1-9. In some embodiments, the viral peptide is selected from SEQ ID NO: 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 NO: 1, 2, 3, 4, 5, 151, 197, 471, 677, 696, 697, 698, 699, 700, and 701.

[0205] Table 2: Viral peptides0206] Table 3: Full list of viral peptides

[0207] In some embodiments, the immunogenic peptide is inserted into the Fc, such that there is Fc sequence both N-terminal and C-terminal to the peptide. In some embodiments, the immunogenic peptide is inserted into a loop of the Fc. In some embodiments, the immunogenic peptide is inserted into an antibody heavy chain constant region, such that there is constant region sequence both N-terminal and C-terminal to the peptide. In some embodiments, the immunogenic peptide is inserted into a loop of the antibody heavy chain constant region. In some embodiments, the immunogenic peptide is inserted into an antibody light chain constant region, such that there is constant region sequence both N-terminal and C-terminal to the peptide. In some embodiments, the immunogenic peptide is inserted into a loop of the antibody light chain constant region. In some embodiments, insertion of the immunogenic peptide comprises removal of loop sequence. In some embodiments, at least one loop is replaced with the immunogenic peptide. In some embodiments, the loop is the whole loop. In some embodiments, the loop is at least a portion of the loop. In some embodiments, loop sequence comprises sequence of at least a portion of the loop. In some embodiments, a portion is at least 4 amino acids. In some embodiments, a portion is at least 5 amino acids. In some embodiments, a portion is at least 6 amino acids. In some embodiments, a portion is at least 7 amino acids. In some embodiments, a portion is at least 8 amino acids. In some embodiments, a portion is at least 9 amino acids. In some embodiments, insertion of the immunogenic peptide comprises removal of the loop. In someembodiments, insertion of the immunogenic peptide comprises removal of at least a portion of the loop.

[0208] In some embodiments, replacement of the loop or a portion of the loop also comprises replacement of at least one amino acid flanking the loop. In some embodiments, flanking is N-terminal to the loop. In some embodiments, flanking is C-terminal to the loop. In some embodiments, at least one amino acid is at least the 1, 2, 3, 4 or 5 amino acids directly flanking the loop. Each possibility represents a separate embodiment of the invention. In some embodiments, at least one amino acid is 4-5 amino acids. In some embodiments, at least one amino acid is 4 amino acids. In some embodiments, at least one amino acid is 5 amino acids. In some embodiments, the flanking region is not more than 5, 6, 7, 8, 9 or 10 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the flanking region is not more than 5 amino acids. In some embodiments, the flanking region is not more than 10 amino acids. It will be understood by a skilled artisan that in order to preserve antibody structure and to produce as little perturbation as possible it may be necessary to also replace a portion of the loop flanking regions along with the loop itself (or a portion thereof).

[0209] In some embodiments, the peptide is inserted into the CHI domain. In some embodiments, the peptide replaces a loop in CHI. In some embodiments, the peptide replaces part of a loop in CHI. In some embodiments, the loop is the CHI EF loop. In some embodiments, the CHI EF loop comprises or consists of amino acids 190-198. In some embodiments, the numbering is according to the EU index numbering. In some embodiments, the peptide replaces positions 190-198 of the parent Fc polypeptide. In some embodiments, the peptide is positions 190-198 of the Fc variant.

[0210] In some embodiments, the peptide is inserted into the CH2 domain. In some embodiments, the peptide replaces a loop in CH2. In some embodiments, the peptide replaces part of a loop in CH2. In some embodiments, the loop is the CH2 AB loop. In some embodiments, the CH2 AB loop comprises or consists of amino acids 246-258. In some embodiments, the loop is the CH2 BC loop. In some embodiments, the CH2 BC loop comprises or consists of amino acids 264-273. In some embodiments, the loop is the CH2 CD loop. In some embodiments, the CH2 CD loop comprises or consists of amino acids 279- 288. In some embodiments, the loop is the CH2 DE loop. In some embodiments, the CH2 DE loop comprises or consists of amino acids 292-299. In some embodiments, the loop is the CH2 EF loop. In some embodiments, the CH2 EF loop comprises or consists of amino acids 308-318. In some embodiments, the loop is the CH2 FG loop. In some embodiments,the CH2 FG loop comprises or consists of amino acids 324-331. In some embodiments, the numbering is according to the EU index numbering.

[0211] In some embodiments, the peptide is inserted into the CH3 domain. In some embodiments, the peptide replaces a loop in CH3. In some embodiments, the peptide replaces part of a loop in CH3. In some embodiments, the loop is the CH3 AB loop. In some embodiments, the CH3 AB loop comprises or consists of amino acids 352-363. In some embodiments, the loop is the CH3 BC loop. In some embodiments, the CH3 BC loop comprises or consists of amino acids 373-378. In some embodiments, the loop is the CH3 CD loop. In some embodiments, the CH3 CD loop comprises or consists of amino acids 382- 391. In some embodiments, the loop is the CH3 DE loop. In some embodiments, the CH3 DE loop comprises or consists of amino acids 398-403. In some embodiments, the loop is the CH3 EF loop. In some embodiments, the CH3 EF loop comprises or consists of amino acids 413-424. In some embodiments, the loop is the CH3 FG loop. In some embodiments, the CH3 FG loop comprises or consists of amino acids 429-436. In some embodiments, the numbering is according to the EU index numbering.

[0212] In some embodiments, the antigen binding molecule comprises a cell penetration sequence. In some embodiments, the cell penetration sequence is a cell penetration domain. In some embodiments, the cell penetration sequence is a cell penetration peptide. In some embodiments, the cell penetration sequence targets the molecule to the inside of the cell. In some embodiments, the cell penetration sequence delivers the molecule to the inside of the cell. In some embodiments, the cell penetration sequence enables entrance of the molecule to the inside of the cell. In some embodiments, the inside of the cell is an endosome. In some embodiments, the inside of the cell is the cytoplasm. In some embodiments, delivery to the cytoplasm comprises exit from an endosome. In some embodiments, delivery to the cytoplasm comprises escape from an endosome. In some embodiments, an endosome is the endosomal pathway. In some embodiments, the cell penetration sequence is a peptide transduction domain (PTD). In some embodiments, the cell penetration sequence is a cell penetrating peptide (CPP). In some embodiments, the cell penetration sequence is an endosomal escape domain (EED). Peptide sequences that allow entrance 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.

[0213] In some embodiments, SEQ ID NO: 1 is the immunogenic peptide. In some embodiments, the Fc variant comprises SEQ ID NO: 1. In some embodiments, the antibody heavy chain constant region variant comprises SEQ ID NO: 1. In some embodiments, theantibody light chain constant region comprises SEQ ID NO: 1. In some embodiments, the peptide replaces positions 354-362 of the parent Fc polypeptide. In some embodiments, the peptide is positions 354-362 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1068. In some embodiments, the Fc variant consists of SEQ ID NO:1068. In some embodiments, the Fc variant comprises SEQ ID NO: 1102. In some embodiments, the Fc variant consists of SEQ ID NO: 1102. In some embodiments, the Fc variant comprises SEQ ID NO: 1105. In some embodiments, the Fc variant consists of SEQ ID NO: 1105. In some embodiments, the Fc variant comprises SEQ ID NO: 1106. In some embodiments, the Fc variant consists of SEQ ID NO: 1106. In some embodiments, the Fc variant comprises SEQ ID NO: 1107. In some embodiments, the Fc variant consists of SEQ ID NO: 1107. In some embodiments, the Fc variant comprises SEQ ID NO: 1108. In some embodiments, the Fc variant consists of SEQ ID NO: 1108. In some embodiments, the peptide replaces positions 350-358 of the parent Fc polypeptide. In some embodiments, the peptide is positions 350-358 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1069. In some embodiments, the Fc variant consists of SEQ ID NO:1069. In some embodiments, the peptide replaces positions 357-365 of the parent Fc polypeptide. In some embodiments, the peptide is positions 357-365 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1070. In some embodiments, the Fc variant consists of SEQ ID NO: 1070. In some embodiments, the peptide replaces positions 384-389 of the parent Fc polypeptide. In some embodiments, the peptide is positions 384-389 of the Fc variant. In the case where the peptide is larger than the region it replaces it may be said that the peptide is still that smaller region as the positioning of the other amino acids after it and their number will not change. This is similar to how a longer or shorter CDR will not change the framework numbering of the antibody. Thus, though SEQ ID NO: 1 consists of 9 amino acids it can still be said to be positions 384-389 of the Fc, because the 9 amino acids have replaced the 6 amino acids of the parent Fc polypeptide. In some embodiments, the Fc variant comprises SEQ ID NO: 1071. In some embodiments, the Fc variant consists of SEQ ID NO: 1071. In some embodiments, the peptide replaces positions 384-388 of the parent Fc polypeptide. In some embodiments, the peptide is positions 384-388 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1072. In some embodiments, the Fc variant consists of SEQ ID NO: 1072. In some embodiments, the peptide replaces positions 384-387 of the parent Fc polypeptide. In some embodiments, the peptide is positions 384-387 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1073. In some embodiments, the Fc variant consists of SEQ ID NO: 1073. In some embodiments, the peptide replaces positions 416-424 of theparent Fc polypeptide. In some embodiments, the peptide is positions 416-424 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1074. In some embodiments, the Fc variant consists of SEQ ID NO: 1074. In some embodiments, the peptide replaces positions 418-422 of the parent Fc polypeptide. In some embodiments, the peptide is positions 418-422 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1075. In some embodiments, the Fc variant consists of SEQ ID NO:1075. In some embodiments, the Fc variant comprises SEQ ID NO: 1100. In some embodiments, the Fc variant consists of SEQ ID NO: 1100. In some embodiments, the Fc variant comprises SEQ ID NO: 1101. In some embodiments, the Fc variant consists of SEQ ID NO: 1101. In some embodiments, the Fc variant comprises SEQ ID NO: 1102. In some embodiments, the Fc variant consists of SEQ ID NO: 1102. In some embodiments, the peptide replaces positions 415-422 of the parent Fc polypeptide. In some embodiments, the peptide is positions 415-422 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1076. In some embodiments, the Fc variant consists of SEQ ID NO:1076. In some embodiments, the peptide replaces positions 414-422 of the parent Fc polypeptide. In some embodiments, the peptide is positions 414-422 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1077. In some embodiments, the Fc variant consists of SEQ ID NO: 1077. In some embodiments, the Fc variant comprises SEQ ID NO: 1103. In some embodiments, the Fc variant consists of SEQ ID NO: 1103. In some embodiments, the Fc variant comprises SEQ ID NO: 1104. In some embodiments, the Fc variant consists of SEQ ID NO: 1104. In some embodiments, the Fc variant comprises SEQ ID NO: 1105. In some embodiments, the Fc variant consists of SEQ ID NO: 1105. In some embodiments, the Fc variant comprises SEQ ID NO: 1108. In some embodiments, the Fc variant consists of SEQ ID NO: 1108. In some embodiments, the peptide replaces positions 418-424 of the parent Fc polypeptide. In some embodiments, the peptide is positions 418-424 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1078. In some embodiments, the Fc variant consists of SEQ ID NO: 1078. In some embodiments, the peptide replaces positions 418-423 of the parent Fc polypeptide. In some embodiments, the peptide is positions 418-423 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1079. In some embodiments, the Fc variant consists of SEQ ID NO: 1079. In some embodiments, the peptide replaces positions 264-272 of the parent Fc polypeptide. In some embodiments, the peptide is positions 264-272 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1080. In some embodiments, the Fc variant consists of SEQ ID NO: 1080. In some embodiments, the Fc variant comprises SEQ ID NO: 1100. In some embodiments, the Fc variant consists of SEQID NO: 1100. In some embodiments, the Fc variant comprises SEQ ID NO: 1103. In some embodiments, the Fc variant consists of SEQ ID NO: 1103. In some embodiments, the Fc variant comprises SEQ ID NO: 1106. In some embodiments, the Fc variant consists of SEQ ID NO: 1106. In some embodiments, the Fc variant comprises SEQ ID NO: 1108. In some embodiments, the Fc variant consists of SEQ ID NO: 1108. In some embodiments, the peptide replaces positions 265-273 of the parent Fc polypeptide. In some embodiments, the peptide is positions 265-273 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1081. In some embodiments, the Fc variant consists of SEQ ID NO: 1081. In some embodiments, the Fc variant comprises SEQ ID NO: 1101. In some embodiments, the Fc variant consists of SEQ ID NO: 1101. In some embodiments, the Fc variant comprises SEQ ID NO: 1104. In some embodiments, the Fc variant consists of SEQ ID NO: 1104. In some embodiments, the Fc variant comprises SEQ ID NO: 1107. In some embodiments, the Fc variant consists of SEQ ID NO: 1107.

[0214] In some embodiments, SEQ ID NO: 1 replaces positions 375-385, by Kabat numbering (354-362 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 375-385, by Kabat numbering (354-362 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1149 (the CH3 domain of TRJ175). In some embodiments, SEQ ID NO: 1 replaces positions 371-381, by Kabat numbering (350-358 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 371-381, by Kabat numbering (350-358 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1150 (the CH3 domain of TRJ176). In some embodiments, SEQ ID NO: 1 replaces positions 378-388, by Kabat numbering (357-365 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 378-388, by Kabat numbering (357-365by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1151 (the CH3 domain of TRJ177). In some embodiments, SEQ ID NO: 1 replaces positions 410-417, by Kabat numbering (384-389 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 410-417, by Kabat numbering (384-389 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1152 (the CH3 domain of TRJ178). In some embodiments, SEQ ID NO: 1 replaces positions 410-416, by Kabat numbering (384-388 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1is positions 410-416, by Kabat numbering (384-388 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1153 (the CH3 domain of TRJ179). In some embodiments, SEQ ID NO: 1 replaces positions 410-415, by Kabat numbering (384-387 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 410-415, by Kabat numbering (384-387 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1154 (the CH3 domain of TRJ180). In some embodiments, SEQ ID NO: 1 replaces positions 449-453, by Kabat numbering (418-422 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 449-453, by Kabat numbering (418-422 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1156 (the CH3 domain of TRJ182). SEQ ID NO: 1155 corresponds to the CH3 domain of TRJ181. In some embodiments, SEQ ID NO: 1 replaces positions 446-453, by Kabat numbering (415-422 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 446-453, by Kabat numbering (415-422 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1157 (the CH3 domain of TRJ183). In some embodiments, SEQ ID NO: 1 replaces positions 445-453, by Kabat numbering (414-422 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 445-453, by Kabat numbering (414-422 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1158 (the CH3 domain of TRJ184). In some embodiments, SEQ ID NO: 1 replaces positions 449-455, by Kabat numbering (418-424 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 449-455, by Kabat numbering (418-424 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1159 (the CH3 domain of TRJ185). In some embodiments, SEQ ID NO: 1 replaces positions 449-454, by Kabat numbering (418-423 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 449-454, by Kabat numbering (418-423 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH3 domain comprising or consisting of SEQ ID NO: 1160 (the CH3 domain of TRJ186).

[0215] It will be understood that SEQ ID NO: 1149-1160 can further comprise other mutations in the CH3 domain. In some embodiments, any one of SEQ ID NO: 1149-1160further comprises the F405L mutation according to EU numbering. In some embodiments, any one of SEQ ID NO: 1149-1160 further comprises a leucine at position 405 according to EU numbering. In some embodiments, F405 is F65 in the CH3. In some embodiments, F405 is F65 in any one of SEQ ID NO: 1149-1160. In some embodiments, any one of SEQ ID NO: 1149-1160 further comprises the K409R mutation according to EU numbering. In some embodiments, any one of SEQ ID NO: 1149-1160 further comprises an arginine at position 409 according to EU numbering. In some embodiments, K409 is K69 in the CH3. In some embodiments, K409 is K69 in any one of SEQ ID NO: 1149-1160. In some embodiments, the CH3 domain comprises an aspartic acid or a glutamic acid at position 356 according to EU numbering. In some embodiments, the CH3 domain comprises a glutamic acid at position 356 according to EU numbering. In some embodiments, any one of SEQ ID NO: 1149-1160 further comprises a glutamic acid at position 356 according to EU numbering. In some embodiments, any one of SEQ ID NO: 1149-1160 further comprises the D356E mutation according to EU numbering. In some embodiments, D356 is D16 in the CH3. In some embodiments, D356 is D16 in any one of SEQ ID NO: 1149-1160. In some embodiments, the CH3 domain comprises a leucine or a methionine at position 358 according to EU numbering. In some embodiments, the CH3 domain comprises a methionine at position 358 according to EU numbering. In some embodiments, any one of SEQ ID NO: 1149-1160 further comprises a methionine at position 358 according to EU numbering. In some embodiments, any one of SEQ ID NO: 1149-1160 further comprises the L358M mutation according to EU numbering. In some embodiments, L358 is LI 8 in the CH3. In some embodiments, L358 is L18 in any one of SEQ ID NO: 1149-1160.

[0216] In some embodiments, the CH3 domain is selected from SEQ ID NO: 1149-1160 and a sequence with at least 85, 90, 92, 95, 97 or 99% sequence identity thereto which comprises the at least one immunogenic peptide. Each possibility represents a separate embodiment of the invention. In some embodiments, the CH3 domain is a sequence with at least 85% sequence identity to any one of SEQ ID NO: 1149-1160. In some embodiments, the CH3 domain is a sequence with at least 95% sequence identity to any one of SEQ ID NO: 1149- 1160. In some embodiments, the CH3 domain is a sequence with at least 99% sequence identity to any one of SEQ ID NO: 1149-1160.

[0217] In some embodiments, SEQ ID NO: 1 replaces positions 277-285, by Kabat numbering (264-272 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 277-285, by Kabat numbering (264-272 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH2 domaincomprising or consisting of SEQ ID NO: 1161 (the CH2 domain of TRJ187). In some embodiments, SEQ ID NO: 1 replaces positions 278-286, by Kabat numbering (265-273 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 278-286, by Kabat numbering (265-273 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH2 domain comprising or consisting of SEQ ID NO: 1162 (the CH2 domain of TRJ188). In some embodiments, the CH2 domain is selected from SEQ ID NO: 1161-1162 and a sequence with at least 85, 90, 92, 95, 97 or 99% sequence identity thereto which comprises the at least one immunogenic peptide. Each possibility represents a separate embodiment of the invention. In some embodiments, the CH2 domain is a sequence with at least 85% sequence identity to any one of SEQ ID NO: 1161-1162. In some embodiments, the CH2 domain is a sequence with at least 95% sequence identity to any one of SEQ ID NO: 1161-1162. In some embodiments, the CH2 domain is a sequence with at least 99% sequence identity to any one of SEQ ID NO: 1161-1162.

[0218] In some embodiments, the Fc variant comprises a CH3 selected from SEQ ID NO: 1149-1160 and a CH2 selected from SEQ ID NO: 1161-1162. In some embodiments, the heavy chain constant region variant comprises a CH3 selected from SEQ ID NO: 1149-1160 and a CH2 selected from SEQ ID NO: 1161-1162. In some embodiments, the Fc variant comprises a CH3 of SEQ ID NO: 1156 (TRI 182) and a CH2 of SEQ ID NO: 1161 (TRJ187). In some embodiments, the heavy chain constant region comprises a CH3 of SEQ ID NO: 1156 (TRJ182) and a CH2 of SEQ ID NO: 1161 (TRJ187). In some embodiments, the Fc variant comprises a CH3 of SEQ ID NO: 1158 (TRJ184) and a CH2 of SEQ ID NO: 1161 (TRJ187). In some embodiments, the heavy chain constant region comprises a CH3 of SEQ ID NO: 1158 (TRJ184) and a CH2 of SEQ ID NO: 1161 (TRJ187). In some embodiments, the Fc variant comprises a CH3 of SEQ ID NO: 1149 (TRJ175) and a CH2 of SEQ ID NO: 1161 (TRJ187). In some embodiments, the heavy chain constant region comprises a CH3 of SEQ ID NO: 1149 (TRJ175) and a CH2 of SEQ ID NO: 1161 (TRJ187).

[0219] In some embodiments, SEQ ID NO: 3 is the immunogenic peptide. In some embodiments, the Fc variant comprises SEQ ID NO: 3. In some embodiments, the peptide replaces positions 354-362 of the parent Fc polypeptide. In some embodiments, the peptide is positions 354-362 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1123. In some embodiments, the peptide replaces positions 357-365 of the parent Fc polypeptide. In some embodiments, the peptide is positions 357-365 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1124. In some embodiments, the peptide replaces positions 350-358 of the parent Fc polypeptide. In some embodiments, thepeptide is positions 350-358 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1125. In some embodiments, the peptide replaces positions 371-379 of the parent Fc polypeptide. In some embodiments, the peptide is positions 371-379 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1126. In some embodiments, the peptide replaces positions 399-407 of the parent Fc polypeptide. In some embodiments, the peptide is positions 399-407 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1127. In some embodiments, the peptide replaces positions 398-403 of the parent Fc polypeptide. In some embodiments, the peptide is positions 398-403 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1128. In some embodiments, the peptide replaces positions 397-402 of the parent Fc polypeptide. In some embodiments, the peptide is positions 397-402 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1129. In some embodiments, the peptide replaces positions 397-404 of the parent Fc polypeptide. In some embodiments, the peptide is positions 397-404 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1130. In some embodiments, the peptide replaces positions 414-422 of the parent Fc polypeptide. In some embodiments, the peptide is positions 414-422 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1131. In some embodiments, the peptide replaces positions 415-423 of the parent Fc polypeptide. In some embodiments, the peptide is positions 415-423 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1132. In some embodiments, the peptide replaces positions 418-422 of the parent Fc polypeptide. In some embodiments, the peptide is positions 418-422 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1133. In some embodiments, the peptide replaces positions 418-421 of the parent Fc polypeptide. In some embodiments, the peptide is positions 418-421 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1134. In some embodiments, the peptide replaces positions 415-421 of the parent Fc polypeptide. In some embodiments, the peptide is positions 415-421 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1135. In some embodiments, the peptide replaces positions 265-273 of the parent Fc polypeptide. In some embodiments, the peptide is positions 265-273 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1136. In some embodiments, the peptide replaces positions 264-272 of the parent Fc polypeptide. In some embodiments, the peptide is positions 264-272 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1137. In some embodiments, the peptide replaces positions 264-271 of the parent Fc polypeptide. In some embodiments, the peptide is positions 264-271 of the Fc variant. In some embodiments, the Fc variant comprises SEQID NO: 1138. In some embodiments, the peptide replaces positions 263-272 of the parent Fc polypeptide. In some embodiments, the peptide is positions 263-272 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1139. In some embodiments, the peptide replaces positions 295-299 of the parent Fc polypeptide. In some embodiments, the peptide is positions 295-299 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1140. In some embodiments, the peptide replaces positions 292-298 of the parent Fc polypeptide. In some embodiments, the peptide is positions 292-298 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1141. In some embodiments, the peptide replaces positions 294-302 of the parent Fc polypeptide. In some embodiments, the peptide is positions 294-302 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1142. In some embodiments, the peptide replaces positions 325-332 of the parent Fc polypeptide. In some embodiments, the peptide is positions 325-332 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1143. In some embodiments, the peptide replaces positions 324-332 of the parent Fc polypeptide. In some embodiments, the peptide is positions 324-332 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1144. In some embodiments, the peptide replaces positions 323-331 of the parent Fc polypeptide. In some embodiments, the peptide is positions 323-331 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1145. In some embodiments, the peptide replaces positions 237-245 of the parent Fc polypeptide. In some embodiments, the peptide is positions 237-245 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1146. In some embodiments, the peptide replaces positions 190-198 of the parent Fc polypeptide. In some embodiments, the peptide is positions 190-198 of the Fc variant. In some embodiments, the Fc variant comprises SEQ ID NO: 1147.

[0220] In some embodiments, SEQ ID NO: 3 replaces positions 278-286, by Kabat numbering (265-273 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 3 is positions 278-286, by Kabat numbering (265-273 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH2 domain comprising or consisting of SEQ ID NO: 1163 (the CH2 domain of TRJ224). In some embodiments, SEQ ID NO: 3 replaces positions 311-321, by Kabat numbering (294-302 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 3 is positions 311-321, by Kabat numbering (294-302 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH2 domain comprising or consisting of SEQ ID NO: 1164 (the CH2 domain of TRJ232). In some embodiments, SEQ ID NO: 3 replacespositions 344-351, by Kabat numbering (325-332 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 3 is positions 344-351, by Kabat numbering (325-332 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH2 domain comprising or consisting of SEQ ID NO: 1165 (the CH2 domain of TRJ233). In some embodiments, SEQ ID NO: 3 replaces positions 343-351, by Kabat numbering (324-332 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 3 is positions 343-351, by Kabat numbering (324-332 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CH2 domain comprising or consisting of SEQ ID NO: 1166 (the CH2 domain of TRJ234).

[0221] It will be understood that SEQ ID NO: 1161-1166 can further comprise other mutations in the CH2 domain. In some embodiments, a mutation of the CH2 is a mutation that reduces antibody cytotoxicity. In some embodiments, antibody cytotoxicity is antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). In some embodiments, the mutation is an Fc silencing mutation. Mutations that reduce cytotoxicity are known in the art and any may be included in the variant heavy chain constant region (i.e., in the CH2 domain). Examples of such mutations include the LALA mutations (L234A and L235A, by EU numbering), the LALAPG mutations (L234A, L235A and P329G, by EU numbering) and the FEA mutations (L234F, L235E and D265A by EU numbering). In some embodiments, any one of SEQ ID NO: 1161-1166 further comprises the FEA mutations according to EU numbering. In some embodiments, any one of SEQ ID NO: 1161-1166 further comprises a phenylalanine at position 324, a glutamic acid at position 325 and an alanine at position 265, according to EU numbering. In some embodiments, L324 is L4 in the CH2. In some embodiments, L324 is L4 in any one of SEQ ID NO: 1161-1166. In some embodiments, L325 is L5 in the CH2. In some embodiments, L325 is L5 in any one of SEQ ID NO: 1161-1162. In some embodiments, P329 is P9 in the CH2. In some embodiments, P329 is P9 in any one of SEQ ID NO: 1161-1166. In some embodiments, the CH2 domain is selected from SEQ ID NO: 1161-1166 and a sequence with at least 85, 90, 92, 95, 97 or 99% sequence identity thereto which comprises the at least one immunogenic peptide. Each possibility represents a separate embodiment of the invention. In some embodiments, the CH2 domain is a sequence with at least 85% sequence identity to any one of SEQ ID NO: 1161-1166. In some embodiments, the CH2 domain is a sequence with at least 95% sequence identity to any one of SEQ ID NO: 1161-1166. In some embodiments, the CH2 domain is a sequence with at least 99% sequence identity to any one of SEQ ID NO: 1161-1166.

[0222] In some embodiments, SEQ ID NO: 1 replaces positions 123-133, by Kabat numbering (127-135 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 123-133, by Kabat numbering (127-135 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CHI domain comprising or consisting of SEQ ID NO: 1167 (the CHI domain of TRJ256 / TRJ257 / TRJ242). In some embodiments, SEQ ID NO: 1 replaces positions 128- 138, by Kabat numbering (132-140 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 128-138, by Kabat numbering (132-140 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CHI domain comprising or consisting of SEQ ID NO: 1168 (the CHI domain of TRJ259 / TRJ260 / TRJ243). In some embodiments, SEQ ID NO: 1 replaces positions 162- 171, by Kabat numbering (159-167 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 162-171, by Kabat numbering (159-167 by EU) of the heavy chain variant. In some embodiments, the variant comprises a CHI domain comprising or consisting of SEQ ID NO: 1169 (the CHI domain of TRJ265 / TRJ266 / TRJ246). In some embodiments, the variant comprises a CHI selected from SEQ ID NO: 1167-1169. It will be understood that SEQ ID NO: 1167-1169 can further comprise other mutations in the CHI domain. In some embodiments, the CHI domain comprises lysine or an arginine at position 214 according to EU numbering. In some embodiments, the CHI domain comprises an arginine at position 356 according to EU numbering. In some embodiments, the CHI domain comprises a lysine at position 356 according to EU numbering. In some embodiments, any one of SEQ ID NO: 1167-1169 further comprises an arginine at position 214 according to EU numbering. In some embodiments, any one of SEQ ID NO: 1167-1169 further comprises the K214R mutation according to EU numbering. In some embodiments, any one of SEQ ID NO: 1167-1169 further comprises an arginine at position 214 according to EU numbering. In some embodiments, K214 is K97 in the CHI. In some embodiments, K214 is K97 in any one of SEQ ID NO: 1167-1169. In some embodiments, the CMV peptide inserted CHI domains are selected from SEQ ID NO: 1209-1211. In some embodiments, the CMV peptide inserted CHI domains are selected from SEQ ID NO: 1212-1214 wherein the X at position 97 is R or K. In some embodiments, the CMV peptide inserted CHI domains are selected from SEQ ID NO: 1167-1169 and 1209-1211.

[0223] In some embodiments, the CHI domain is selected from SEQ ID NO: 1167-1169 and a sequence with at least 85, 90, 92, 95, 97 or 99% sequence identity thereto whichcomprises the at least one immunogenic peptide. Each possibility represents a separate embodiment of the invention. In some embodiments, the CHI domain is a sequence with at least 85% sequence identity to any one of SEQ ID NO: 1167-1169. In some embodiments, the CHI domain is a sequence with at least 95% sequence identity to any one of SEQ ID NO: 1167-1169. In some embodiments, the CHI domain is a sequence with at least 99% sequence identity to any one of SEQ ID NO: 1167-1169.

[0224] In some embodiments, SEQ ID NO: 1 replaces positions 124-132, by Kabat numbering (124-132 by EU) of the light chain constant region polypeptide. In some embodiments, SEQ ID NO: 1 is positions 124-132, by Kabat numbering (124-132 by EU) of the light chain variant. In some embodiments, the variant comprises a CL domain comprising or consisting of SEQ ID NO: 1170 (the CL domain of TRJ258 / TRJ259 / TRJ252). In some embodiments, the light chain is the kappa light chain. In some embodiments, the light chain is the lambda light chain. In some embodiments, the CL domain is selected from SEQ ID NO: 1170 and a sequence with at least 85, 90, 92, 95, 97 or 99% sequence identity thereto which comprises the at least one immunogenic peptide. Each possibility represents a separate embodiment of the invention. In some embodiments, the CL domain is a sequence with at least 85% sequence identity to any one of SEQ ID NO: 1170. In some embodiments, the CL domain is a sequence with at least 95% sequence identity to any one of SEQ ID NO: 1170. In some embodiments, the CL domain is a sequence with at least 99% sequence identity to any one of SEQ ID NO: 1170.

[0225] In some embodiments, YLQPRTFLL (SEQ ID NO: 373) is the immunogenic peptide. In some embodiments, the Fc variant comprises SEQ ID NO: 373. In some embodiments, the peptide replaces positions 123-133 of the parent Fc polypeptide, by Kabat numbering (127-135 by EU). In some embodiments, the peptide is positions 127-135 of the Fc variant by EU numbering. In some embodiments, SEQ ID NO: 373 replaces positions 123-133, by Kabat numbering (127-135 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 3 is positions 123-133, by Kabat numbering (127-135 by EU) of the heavy chain variant. In some embodiments, the variant comprises a SEQ ID NO: 373 inserted into the CHI domain.

[0226] In some embodiments, LLMWEAVTV (SEQ ID NO: 310) is the immunogenic peptide. In some embodiments, the Fc variant comprises SEQ ID NO: 310. In some embodiments, the peptide replaces positions 445-453 of the parent Fc polypeptide, by Kabat numbering (414-422 by EU). In some embodiments, the peptide is positions 414-422 of the Fc variant by EU numbering. In some embodiments, SEQ ID NO: 310 replaces positions445-453, by Kabat numbering (414-422 by EU) of the heavy chain constant region polypeptide. In some embodiments, SEQ ID NO: 310 is positions 445-453, by Kabat numbering (414-422 by EU) of the heavy chain variant. In some embodiments, the variant comprises a SEQ ID NO: 310 inserted into the CH3 domain.

[0227] In some embodiments, the antibody is a bi-specific antibody. In some embodiments, the protein comprises a plurality of antigen binding regions. In some embodiments, the protein comprises at least two antigen binding regions. In some embodiments, the antibody comprises two antigen binding regions. In some embodiments, at least one of the antigen binding regions is the antigen binding region that is capable of binding to a target cell. In some embodiments, at least one of the antigen binding regions is mutated to comprise the cell penetration sequence. In some embodiments, the Fc is mutated to comprises the cell penetration sequence. In some embodiments, at least one of the antigen binding regions is mutated to comprises the immunogenic peptide, the cell penetration sequence or both. In some embodiments, the mutation is mutation of a complementarity determining region (CDR). In some embodiments, a CDR of an antigen binding region is mutated. In some embodiments, mutated is replaced. In some embodiments, replaced is replaced with the immunogenic peptide. In some embodiments, replaced is replaced with the cell penetration sequence.

[0228] In some embodiments, mutation or replacement in the Fc does not diminish binding. In some embodiments, mutation or replacement in the constant region does not diminish binding. In some embodiments, not diminishing binding is not significantly diminishing binding. In some embodiments, mutation or replacement in the Fc does not abrogate binding, n some embodiments, mutation or replacement in the constant region does not abrogate binding. In some embodiments, a significant diminishment 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 diminishment is a reduction in binding of more than 10%. In some embodiments, a significant diminishment is a reduction in binding of more than 20%. In some embodiments, mutation or replacement of an inert CDR does not reduce binding by 100%.

[0229] In some embodiments, mutation or replacement in the Fc does not diminish cell penetrance. In some embodiments, mutation or replacement in the constant region does not diminish cell penetrance. In some embodiments, not diminishing cell penetrance is not significantly diminishing cell penetrance. In some embodiments, mutation or replacement of an inert CDR does not abrogate cell penetrance. In some embodiments, a significantdiminishment is a reduction in penetrance 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 diminishment is a reduction in penetrance of more than 10%. In some embodiments, a significant diminishment is a reduction in penetrance of more than 20%. In some embodiments, mutation or replacement in the Fc does not reduce penetrance by 100%. In some embodiments, mutation or replacement of an inert CDR does not reduce penetrance to the level of a control antibody. In some embodiments, a control antibody is an antibody that does not enter a cell. Antibodies that do not enter cells are well known in the art, and include for example adalimumab and muromonab. In some embodiments, not diminishing cell penetrance comprises retaining penetrance that is substantially equal to the binding of the antibody devoid of or without the immunogenic peptide.

[0230] In some embodiments, insertion of the immunogenic peptide and removal of loop sequence produces no change in the conformation of the antibody. In some embodiments, insertion of the immunogenic peptide and removal of loop sequence produces minimal change in the conformation of the antibody. In some embodiments, conformation is overall conformation. In some embodiments, conformation is 3D structure. In some embodiments, conformation is tertiary structure. In some embodiments, change is perturbation. In some embodiments, minimal change is without a loss of a bond. In some embodiments, minimal change is a change of less than 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%. Each possibility represents a separate embodiment of the invention. In some embodiments, minimal change comprises binding of a target antigen at an equivalent affinity to the antibody devoid or without the immunogenic peptide. In some embodiments, equivalent is with no reduction in affinity. In some embodiments, equivalent is with a reduction in affinity of not more than 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%. Each possibility represents a separate embodiment of the invention. In some embodiments, equivalent is with a reduction in affinity of not more than 10%. In some embodiments, equivalent is with a reduction in affinity of not more than 20%

[0231] In some embodiments, the mutated CDR is in the antigen binding region capable of binding the target cell. In some embodiments, the mutated CDR is not in the antigen binding region capable of binding the target cell. In some embodiments, the mutated CDR is in an antigen binding region other than the antigen binding region capable of binding the target cell. In some embodiments, the immunogenic peptide replaces a CDR in the antigen binding region capable of binding the target cell. In some embodiments, the cell penetrating sequencereplaces a CDR in an antigen binding region that is not the antigen binding region that binds the target cell. In some embodiments, the immunogenic peptide and the cell penetration sequence are in the same antigen binding region. In some embodiments, the immunogenic peptide and the cell penetration sequence are in different antigen binding regions.

[0232] 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 light chain CDR L2 is replaced with the cell penetrating sequence, the immunogenic peptide or both.

[0233] 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 Arzerra and at least one of heavy chain CDR Hl, light chain CDR LI and light chain CDR L2 is replaced with the cell penetrating sequence, the immunogenic peptide or both. In some embodiments, replaced is mutated to include the peptide or sequence. In some embodiments, heavy chain CDR Hl is replaced. In some embodiments, the light chain CDR LI is replaced. In some embodiments, the light chain CDR L2 is replaced.

[0234] In some embodiments, the target cell is a cancer cell, the cancer cell antigen is PD- Ll, the antigen binding region capable of binding to PD-L1 is Durvalumab and at least one of heavy chain CDR Hl, and light chain CDR L2 is replaced with the cell penetrating sequence, the immunogenic peptide or both. In some embodiments, replaced is mutated to include the peptide or sequence. In some embodiments, heavy chain CDR Hl is replaced. In some embodiments, the light chain CDR L2 is replaced.

[0235] 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 someembodiments, 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 comprises replacing amino acids 25-33 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 26-33 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 26-32 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 27-33 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 28-33 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 22-30 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 22- 29 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 26-31 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 23-32 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 23-31 of the heavy chain. In some embodiments, into CDRH1 comprises replacing amino acids 28-35 of the heavy chain. In some embodiments, into CDRH3 comprises replacing amino acids 100-108 of the heavy chain. In some embodiments, into CDRH3 comprises replacing amino acids 99-106 of the heavy chain. In some embodiments, into CDRH3 comprises replacing amino acids 99-105 of the heavy chain. In some embodiments, into CDRH3 comprises replacing amino acids 100-106 of the heavy chain. In some embodiments, into CDRH3 comprises replacing amino acids 100-105 of the heavy chain. In some embodiments, into CDRH3 comprises replacing amino acids 100-104 of the heavy chain. In some embodiments, into CDRH3 comprises replacing amino acids 99-107 of the heavy chain. In some embodiments, into CDRH2 comprises replacing amino acids 52-59 of the heavy chain. In some embodiments, into CDRH2 comprises replacing amino acids 52-60 of the heavy chain. In some embodiments, into CDRL3 comprises replacingamino acids 97-103 of the light chain. In some embodiments, into CDRL3 comprises replacing amino acids 98-103 of the light chain. In some embodiments, into CDRL3 comprises replacing amino acids 96-104 of the light chain. In some embodiments, into CDRL3 comprises replacing amino acids 98-104 of the light chain. In some embodiments, the CHI domains of the invention are used to replace the CHI domain of Tmab4. In some embodiments, the immunogenic peptide is inserted into a CHI domain of Tmab4.

[0236] In some embodiments, the antibody is 3E10 and the immunogenic peptide is inserted into any 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 of3E10. In some embodiments, SEQ ID NO: 9 is inserted into CDRL2 of3E10. In some embodiments, into CDRL1 comprises replacing amino acids 27-35 of the light chain. In some embodiments, into CDRL1 comprises replacing amino acids 28-36 of the light chain. In some embodiments, into CDRL2 comprises replacing amino acids 50-58 of the light chain. In some embodiments, the CHI domains of the invention are used to replace the CHI domain of 3E10. In some embodiments, the immunogenic peptide is inserted into a CHI domain of 3E10.

[0237] 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 replacing amino acids 28-36 of the light chain. In some embodiments, into CDRL1 comprises replacing amino acids 26-34 of the light chain. In some embodiments, into CDRL2 comprises replacing amino acids 50-58 of the light chain. In some embodiments, the CHI domains of theinvention are used to replace the CHI domain of 71F12. In some embodiments, the immunogenic peptide is inserted into a CHI domain of 71F12.

[0238] In some embodiments, the antibody is a commercially available antibody. In some embodiments, the antibody penetrates into a bound cell at a level comparable to any one of TMab4, 3E10 and 71F12. In some embodiments, the antibody penetrates into a bound cell at a level comparable to TMab4. In some embodiments, the antibody penetrates into a bound cell at a level comparable to 3E10. In some embodiments, the antibody penetrates into a bound cell at a level comparable to 71F12. In some embodiments, comparable is with a penetrance 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, comparable is with a penetrance that is at least 80% of the original antibody. In some embodiments, comparable is with a penetrance that is at least 90% of the original antibody.

[0239] 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 penetration sequence are part of the same amino acid chain. In some embodiments, the immunogenic peptide and the cell penetration sequence are part of the same amino acid chain. In some embodiments, the antigen binding molecule of the invention is a single fusion protein. In some embodiments, the antigen binding molecule of the invention is a single amino acid chain.

[0240] In some embodiments, the protein is a heavy chain of an antibody. In some embodiments, the protein is an antibody heavy chain. In some embodiments, the protein comprises a heavy chain variable region. In some embodiments, the protein comprises an antigen binding region. In some embodiments, the antigen binding region comprises the heavy chain variable region. In some embodiments, the heavy chain variable region is the heavy chain variable region of TMab4. In some embodiments, the heavy chain variable region is the heavy chain variable region of TMab4 comprising an immunogenic peptide. In some embodiments, the heavy chain variable region is the heavy chain variable region of TMab4 comprising an immunogenic peptide inserted or replacing an inert CDR. In some embodiments, the heavy chain variable region is the heavy chain variable region of 3E10. In some embodiments, the heavy chain variable region is the heavy chain variable region of 3E10 comprising an immunogenic peptide. In some embodiments, the heavy chain variable region is the heavy chain variable region of 3E10 comprising an immunogenic peptide inserted or replacing an inert CDR. In some embodiments, the heavy chain variable regionis the heavy chain variable region of 71F12. In some embodiments, the heavy chain variable region is the heavy chain variable region of 71F12 comprising an immunogenic peptide. In some embodiments, the heavy chain variable region is the heavy chain variable region of 71F12 comprising an immunogenic peptide inserted or replacing an inert CDR.

[0241] In some embodiments, the heavy chain variable region is selected from heavy chain variable regions of T1-T19, T1 30-T1 35, T1 39-T1 45 and T1 47. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising a sequence selected from SEQ ID NO: 1028-1040, 1043-1045, 1047-1055, and 1058-1059 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, an analog or homolog comprises the immunogenic peptide. In some embodiments, an analog or homolog is capable of binding cells. In some embodiments, an analog or homolog is delivered to the cytosol upon cell binding. In some embodiments, reaching the cytosol comprises displaying the immunogenic peptide on the surface of the bound cell. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1028 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1029 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1030 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1031 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1032 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1033 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1034 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1035 or analogs orhomologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1036 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1037 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1038 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1039 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1040 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1043 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1044 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1045 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1047 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1048 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1049 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1050 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1051 or analogs or homologs comprising at least85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1052 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1053 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1054 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1055 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1058 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 1022 and a heavy chain variable region comprising SEQ ID NO: 1059 or analogs or homologs comprising at least 85% sequence identity.

[0242] In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising a sequence selected from SEQ ID NO: 1041-1042, 1046, and 1056-1057 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1041 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1042 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1046 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1056 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1021 and a light chain variable region comprising SEQ ID NO: 1057 or analogs or homologs comprising at least 85% sequence identity.

[0243] In some embodiments, the heavy chain variable region is selected from heavy chain variable regions of T2_6, T2 11-T2 13, T2 20 and T2 23. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising a sequence selected from SEQ ID NO: 1060-1065 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1060 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1061 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1062 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1063 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1064 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1023 and a light chain variable region comprising SEQ ID NO: 1065 or analogs or homologs comprising at least 85% sequence identity.

[0244] In some embodiments, the heavy chain variable region is selected from heavy chain variable regions of T4_l and T4_3. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1026 and a light chain variable region comprising SEQ ID NO: 1066 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 1027 and a light chain variable region comprising SEQ ID NO: 1067 or analogs or homologs comprising at least 85% sequence identity.

[0245] 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. It will be understood by a skilled artisan that while certain regions in the antibodies are required for function, such as the antigen binding CDRs, the immunogenic peptide and the cell penetrating moiety, other regions may bear alterations without altering function. Other inert CDRs are such a region as are many inter-CDR sequences as well as sequences within theconstant region of an antibody. Analogs and homologs that retain function but contain alterations with these other regions are also encompassed within the invention.

[0246] In some embodiments, the heavy chain variable region comprises a sequence selected from SEQ ID NO: 1021, 1023, 1026, 1028-1040, 1043-1045, 1047-1055, 1058-1059, 1067 and 1082. In some embodiments, the heavy chain variable region comprises a sequence selected from SEQ ID NO: 1021, 1023, 1026, 1028-1040, 1043-1045, 1047-1055, 1058- 1059, 1067 and 1082 and a sequence with at least 85% identity thereto. In some embodiments, the heavy chain variable region comprises SEQ ID NO: 1082. In some embodiments, the heavy chain variable region consists of SEQ ID NO: 1082. In some embodiments, the protein comprises a sequence selected from SEQ ID NO: 1083-1096. In some embodiments, the protein consists of a sequence selected from SEQ ID NO: 1083- 1096. In some embodiments, the heavy chain comprises a sequence selected from SEQ ID NO: 1083-1096. In some embodiments, the heavy chain consists of a sequence selected from SEQ ID NO: 1083-1096. In some embodiments, the protein comprises a sequence selected from SEQ ID NO: 1109-1117. In some embodiments, the protein consists of a sequence selected from SEQ ID NO: 1109-1117. In some embodiments, the heavy chain comprises a sequence selected from SEQ ID NO: 1109-1117. In some embodiments, the heavy chain consists of a sequence selected from SEQ ID NO: 1109-1117. In some embodiments, the protein comprises a sequence selected from SEQ ID NO: 1118-1122. In some embodiments, the protein consists of a sequence selected from SEQ ID NO: 1118-1122. In some embodiments, the heavy chain comprises a sequence selected from SEQ ID NO: 1118-1122. In some embodiments, the heavy chain consists of a sequence selected from SEQ ID NO: 1118-1122. In some embodiments, the protein comprises a sequence selected from SEQ ID NO: 1109-1122. In some embodiments, the protein consists of a sequence selected from SEQ ID NO: 1109-1122. In some embodiments, the heavy chain comprises a sequence selected from SEQ ID NO: 1109-1122. In some embodiments, the heavy chain consists of a sequence selected from SEQ ID NO: 1109-1122. In some embodiments, the protein comprises a sequence selected from SEQ ID NO: 1083-1096 and 1109-1122. In some embodiments, the protein consists of a sequence selected from SEQ ID NO: 1083-1096 and 1109-1122. In some embodiments, the heavy chain comprises a sequence selected from SEQ ID NO: 1083- 1096 and 1109-1122. In some embodiments, the heavy chain consists of a sequence selected from SEQ ID NO: 1083-1096 and 1109-1122.

[0247] In some embodiments, the protein is a protein dimer. In some embodiments, the dimer comprises a first protein and a second protein. In some embodiments, the proteins areproteins of the invention. In some embodiments, the first protein and second protein are dimerized together. In some embodiments, the first protein and second protein are dimerized to each other. In some embodiments, the first protein and the second protein are the same protein. In some embodiments, the first protein and the second protein are different proteins. Mutations that can be inserted to produce heterodimerization are well known in the art and examples are provided hereinbelow in Table 5. Any mutation can be made so long as it is not within an immunogenic peptide inserted into the variant Fc.

[0248] In some embodiments, a first constant region of a heavy chain of the bispecific antibody comprises a F405L mutation and a second constant region of a heavy chain of the bispecific antibody comprises a K409R mutation. In some embodiments, the first constant region of a heavy chain comprises a CH3 domain comprising SEQ ID NO: 1171 and the second constant region of a heavy chain comprises a CH3 domain comprising SEQ ID NO: 1173. In some embodiments, the first constant region of a heavy chain comprises or consists of SEQ ID NO: 1172 and the second constant region of a heavy chain comprises or consists of SEQ ID NO: 1174 (constant region / Fc of TRJ278). In some embodiments, the first constant region of a heavy chain comprises a CHI domain selected from SEQ ID NO: 1167- 1169. In some embodiments, the second constant region of a heavy chain comprises a CHI domain selected from SEQ ID NO: 1167-1169. In some embodiments, the CHI of the first and second constant regions of a heavy chain are the same CHI domain. In some embodiments, the first constant region of a heavy chain comprises or consists of SEQ ID NO: 1175 and the second constant region of a heavy chain comprises or consists of SEQ ID NO: 1176 (constant region / Fc of TRJ315). In some embodiments, the first constant region of a heavy chain comprises or consists of SEQ ID NO: 1177 and the second constant region of a heavy chain comprises or consists of SEQ ID NO: 1178 (constant region / Fc of TRJ316). In some embodiments, the first constant region of a heavy chain comprises or consists of SEQ ID NO: 1179 and the second constant region of a heavy chain comprises or consists of SEQ ID NO: 1180 (constant region / Fc of TRJ317). In some embodiments, the first constant region of a heavy chain comprises or consists of SEQ ID NO: 1181 and the second constant region of a heavy chain comprises or consists of SEQ ID NO: 1182 (constant region / Fc of TRJ318). In some embodiments, any or all of SEQ ID NO: 1172-1182 further comprise the FEA mutations. In some embodiments, the CH3 domain is selected from SEQ ID NO: 1172- 1182 and a sequence with at least 85, 90, 92, 95, 97 or 99% sequence identity thereto which comprises the at least one immunogenic peptide. Each possibility represents a separate embodiment of the invention. In some embodiments, the CH3 domain is a sequence with atleast 85% sequence identity to any one of SEQ ID NO: 1172-1182. In some embodiments, the CH3 domain is a sequence with at least 95% sequence identity to any one of SEQ ID NO: 1172-1182. In some embodiments, the CH3 domain is a sequence with at least 99% sequence identity to any one of SEQ ID NO: 1172-1182.

[0249] In some embodiments, the protein is an antibody. In some embodiments, the antigen biding 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 are capable of binding the antigen on a target cell. In some embodiments, a CDR of the first heavy chain is inert. In some embodiments, a CDR of the first light chain is inert. In some embodiments, an inert CDR of the first heavy chain is replaced with the immunogenic peptide. In some embodiments, an inert CDR of the first light chain is replaced with the immunogenic peptide. In some embodiments, an inert CDR of the first heavy chain is replaced with the cell penetration sequence. In some embodiments, an inert CDR of the first light chain is replaced with the cell penetration sequence.

[0250] In some embodiments, the antigen biding molecule comprises a second heavy chain. In some embodiments, the antigen biding molecule comprises a second light chain. In some embodiments, the antigen biding 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, a CDR of the second heavy chain is inert. In some embodiments, a CDR of the second light chain is inert. In some embodiments, the second heavy chain comprises the immunogenic peptide. In some embodiments, the second light chain comprises the immunogenic peptide. In some embodiments, the second heavy chain comprises the cell penetrating sequence. In some embodiments, the second light chain comprises the cell penetrating sequence. In some embodiments, a CDR of the second light chain is replaced. In some embodiments, a CDR of the second heavy chain is replaced. In some embodiments, an inert CDR of the second heavy chain is replaced with the immunogenic peptide. In some embodiments, an inert CDR of the second light chain is replaced with the immunogenic peptide. In some embodiments, an inert CDR of the second heavy chain is replaced with the cell penetration sequence. In some embodiments, an inert CDR of the second light chain is replaced with the cell penetration sequence.

[0251] In some embodiments, the protein is a dual-function protein. In some embodiments, the composition comprises a dual-function protein. In some embodiments, the dual-function protein comprises an antibody or antigen binding fragment of the invention. In some embodiments, the dual-function protein is a bi-specific antibody. In some embodiments, the dual function protein comprises a first antibody or protein and a second antibody or protein. In some embodiments, the first antibody is a first heavy chain and a first light chain and the second antibody and a second heavy chain and a second light chain. In some embodiments, the heavy chain and light chain are hybridized between the CHI domain of the heavy chain and the CL domain of the light chain. In some embodiments, hybridized is bonded. In some embodiments, hybridized comprises disulfide bonds. In some embodiments, the second antibody is capable of binding an antigen overexpressed on a target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the antigen is a cancer antigen.

[0252] 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 signal transducer 2 (TROP-2 / TACSTD2), Tissue Factor (TF / F3), B-cell maturation antigen (BCMA / TNFRSF17), Programmed death-ligand 1 (PDL-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), Epithelial cell adhesion molecule (EpCAM), TNF receptor superfamily member 8 (CD30 / TNFRSF8), B- lymphocyte antigen CD19 (CD19), cluster of differentiation-22 (CD22), Siglec-3 (CD33), cluster of differentiation 38 (CD38), Cluster of differentiation 79 (CD79), Lymphocyteactivation gene 3 (LAG-3), C-C Motif Chemokine Receptor 4 (CCR4), vascular endothelial growth factor receptor 2 (VEGFR2 / KDR), Folate receptor 1 (FOLR1), CAMPATH-1 antigen (CD52), platelet-derived growth factor receptor A (PDGFRa), 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 can be used as the targeting module of the 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.

[0253] Table 4 targeting moiety antibodiesAdebrelimab PD-L10254] 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.

[0255] In some embodiments, a CH2 domain comprises the amino acid sequence SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 1097) or analogs or homologs 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 the IgGl CH2 domain. In some embodiments, at least 85% is at least 95%.

[0256] In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1098) or analogs or homologs comprising at least 85% sequence identity. In some embodiments, a CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1099) or analogs or homologs 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 the IgGl CH3 domain. In some embodiments, SEQ ID NO: 1099 is the IgGl CH3 domain. In some embodiments, the SEQ ID NO: 1098 sequence is the sequence found predominantly is humans of European and American descent. In some embodiments, SEQ ID NO: 1099 is the sequence found predominantly in humans of Asian descent. In some embodiments, at least 85% is at least 95%.

[0257] In some embodiments, a 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, a 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 mutations. In some embodiments, the first CH2 domain and first CH3 domains 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 mutations inhibit homodimerization of the first polypeptide chain. In some embodiments, the first mutation inhibits homodimerization of the first polypeptide chain. In some embodiments, the mutations inhibit homodimerization of the second polypeptide chain. In some embodiments, the second mutation inhibits homodimerization of the second polypeptide chain. In some, embodiments, the mutations permit heterodimerization. In some embodiments, the mutations permit heterodimerization of the first and second chains. In some embodiments, permitting is promoting. In some embodiments, permitting is enhancing.

[0258] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutations or alterations may be used for constructing the polypeptides of the invention. In some embodiments, a region from an IgG is replaced with a region from an IgA. In some embodiments, a region from a TCRa is inserted into the first CH3 domain and a region from TCRb is inserted in to the second CH3 domain. In some embodiments, the mutation is insertion of a region from a TCR. In some embodiments, the TCR is selected from TCRa and TCRb. In some embodiments, the mutation is insertion of a region from a different Ig. Examples of these mutations can be found in Table 5. In some embodiments, the mutation is selected from a mutation in Table 5. In some embodiments, the first mutation is selected from a group of mutation provided in a row and the second column of Table 5 and the second mutation is the group of mutations provided in that same row of Table 5 in the third column. The mutations in Table 5 are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in 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 IgG2, and 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 F405 A 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 someembodiments, 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 IgG2, and 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 analogs or homologs 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 analogs or homologs 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 analogs or homologs 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 retains the mutations that promote heterodimerization and inhibit homodimerization.

[0259] Table 5: Mutations for enhancing heterodimerization and inhibiting homodimerization of CH3 domains (Eu numbering).

[0260] In some embodiments, the antibody comprises two heavy chains and two light chains. In some embodiments, the two heavy chains are selected from SEQ ID NO: 1021, 1028-1040, 1043-1045, 1047-1055, 1058 and 1082 and the two light chains are selected from SEQ ID NO: 1022, 1041-1042, 1046, and 1056-1057 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the two heavy chains comprise SEQ ID NO: 1023 and the two light chains are selected from SEQ ID NO: 1024-1025, and 1060- 1065 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the two heavy chains are selected from SEQ ID NO: 1026 and 1067 and the two light chains are selected from SEQ ID NO: 1027 and 1066 or analogs or homologs comprising at least 85% sequence identity. In some embodiments, the heavy and light chains are the recited sequences. In some embodiments, the heavy and light chains consists of the recited seqeunces. It will be understood that analogs or homologs will target to the target protein by the targeting module, bind the cell and be delivered to the cytosol by the killing module and they will contain the immunogenic peptide which is displayed on the cell surface in complex with HLA.

[0261] In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1183 (penetrating arm), a first light chain comprising or consisting of SEQ ID NO: 1184 (penetrating arm), a second heavy chain comprising or consisting of SEQ ID NO: 1185 (targeting arm) and a second light chaincomprising or consisting of SEQ ID NO: 1186 (targeting arm) (TRJ315). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1187 (penetrating arm), a first light chain comprising or consisting of SEQ ID NO: 1188 (penetrating arm), a second heavy chain comprising or consisting of SEQ ID NO: 1189 (targeting arm) and a second light chain comprising or consisting of SEQ ID NO: 1190 (targeting arm) (TRJ316). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1191 (targeting arm), a first light chain comprising or consisting of SEQ ID NO: 1192 (targeting arm), a second heavy chain comprising or consisting of SEQ ID NO: 1193 (penetrating arm) and a second light chain comprising or consisting of SEQ ID NO: 1194 (penetrating arm) (TRJ317). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1195 (targeting arm), a first light chain comprising or consisting of SEQ ID NO: 1196 (targeting arm), a second heavy chain comprising or consisting of SEQ ID NO:1197 (penetrating arm) and a second light chain comprising or consisting of SEQ ID NO:1198 (penetrating arm) (TRJ318). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1199 (targeting arm), a first light chain comprising or consisting of SEQ ID NO: 1200 (targeting arm), a second heavy chain comprising or consisting of SEQ ID NO: 1201 (penetrating arm) and a second light chain comprising or consisting of SEQ ID NO: 1202 (penetrating arm) (TRJ278).

[0262] In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1183 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto, a first light chain comprising or consisting of SEQ ID NO: 1184 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto, a second heavy chain comprising or consisting of SEQ ID NO: 1185 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto and a second light chain comprising or consisting of SEQ ID NO: 1186 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto (TRJ315). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1187 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto, a first light chain comprising or consisting of SEQ ID NO: 1188 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto, a second heavy chain comprising or consisting of SEQ ID NO: 1189 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto and a second light chain comprising or consisting of SEQ ID NO: 1190 (targeting arm) or a homolog thereofcomprising at least 85% sequence identity thereto (TRJ316). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1191 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto, a first light chain comprising or consisting of SEQ ID NO: 1192 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto, a second heavy chain comprising or consisting of SEQ ID NO: 1193 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto and a second light chain comprising or consisting of SEQ ID NO: 1194 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto (TRJ317). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1195 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto, a first light chain comprising or consisting of SEQ ID NO: 1196 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto, a second heavy chain comprising or consisting of SEQ ID NO: 1197 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto and a second light chain comprising or consisting of SEQ ID NO: 1198 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto (TRJ318). In some embodiments, the bispecific antibody comprises a first heavy chain comprising or consisting of SEQ ID NO: 1199 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto, a first light chain comprising or consisting of SEQ ID NO: 1200 (targeting arm) or a homolog thereof comprising at least 85% sequence identity thereto, a second heavy chain comprising or consisting of SEQ ID NO: 1201 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto and a second light chain comprising or consisting of SEQ ID NO: 1202 (penetrating arm) or a homolog thereof comprising at least 85% sequence identity thereto (TRJ278). In some embodiments, at least 85% is at least 95%. It will be understood that a sequence with less than 100% identity will still retain the immunogenic peptide.

[0263] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically 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.

[0264] As used herein, the term “carrier,” “excipient,” or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically 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 carboxymethyl cellulose, ethyl cellulose 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, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, 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. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically 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 components, as well as effective formulations and administration procedures, are well known in the art and are 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 by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0265] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0266] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as 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 can include parenteral, subcutaneous, oral, intramuscular, intratumoral or intraperitoneal.

[0267] In some embodiments, the nucleic acid molecule comprises an open reading frame. In some embodiments, the open reading frame encodes the antigen binding molecule of the invention. In some embodiments, the nucleic acid molecule comprises a plurality of open reading frames which collectively encode the antigen binding molecule of the invention.

[0268] In some embodiments, the vector is an expression vector. In some embodiments, the vector comprises at least one regulatory element operatively linked to a nucleic acid molecule of the invention. In some embodiments, the vector comprises at least one regulatory element operatively linked to an open reading frame encoding the antigen binding molecule of the invention. In some embodiments, the vector comprises a plurality ofregulatory elements each operatively linked to an open reading frame which collectively encode the antigen binding molecule of the invention. In some embodiments, a composition comprises a plurality of vectors each comprising at least one regulatory element operatively linked to an open reading frame wherein the plurality of open reading frames collectively encodes the antigen binding molecule of the invention.

[0269] The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or translation of RNA into a precursor or mature protein (polypeptide).

[0270] Expressing of a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector.

[0271] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.

[0272] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.

[0273] 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 the regulatory element or elements in a manner that allows for 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).

[0274] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and / or the like.

[0275] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0276] In some embodiments, nucleic acid sequences are 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 snRNA and microRNA.

[0277] 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, pNMTl, 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 their derivatives.

[0278] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0279] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0280] Various methods can be used to introduce the expression vector of the present 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 at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0281] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0282] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0283] In some embodiments, the method is a method of treating cancer. In some embodiments, the antigen binding molecule of the invention is for use in treating cancer. In some embodiments, the dual function antigen binding molecule of the invention is for use in treating cancer. In some embodiments, the composition of the invention is for use in treating cancer. In some embodiments, the cancer is a cancer that expresses the cancer specificantigen. In some embodiments, the cancer is a cancer that overexpresses the cancer specific antigen. In some embodiments, overexpresses is expresses at a level higher than in non- cancerous cells. In some embodiments, the non-cancerous cells are of the same cell type or tissue as the cancerous cells. In some embodiments, the cancer is a cancer that expresses the immunogenic cancer peptide. In some embodiments, the cancer is a hematopoietic cancer. In some embodiments, the cancer comprises a malignant immune cell. In some embodiments, the immune cell is a B cell. 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 an EGFR overexpressing cancer.

[0284] As used herein "cancer" refers to diseases associated with cell proliferation. Nonlimiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma and germ cells tumors. In one embodiment, carcinoma refers to tumors derived from epithelial cells including but not limited to breast cancer, prostate cancer, lung cancer, pancreas cancer, and colon cancer. In one embodiment, sarcoma refers of tumors derived from mesenchymal cells including but not limited to sarcoma botryoides, chondrosarcoma, ewings sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcomas. In one embodiment, lymphoma refers to tumors derived from hematopoietic cells that leave the bone marrow and tend to mature in the lymph nodes including but not limited to hodgkin lymphoma, non-hodgkin lymphoma, multiple myeloma and immunoproliferative diseases. In one embodiment, leukemia refers to tumors derived from hematopoietic cells that leave the bone marrow and tend to mature in the blood including but not limited to acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous 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 tumors refers to tumors derived from germ cells including but not limited to germinomatous or seminomatous germ cell tumors (GGCT, SGCT) and nongerminomatous or nonseminomatous germ cell tumors (NGGCT, NSGCT). In one embodiment, germinomatous or seminomatous tumors include but not limited to germinoma, dysgerminoma and seminoma. In one embodiment,nongerminomatous or nonseminomatous tumors refers to pure and mixed germ cells tumors including but not limited to embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, tearoom, polyembryoma, gonadoblastoma and teratocarcinoma.

[0285] In some embodiments, the protein of the invention is a cancer vaccine. In some embodiments, cancer vaccine is an protein comprising an antibody capable of binding a dendritic cell antigen. It will be understood by a skilled artisan that upon entrance into a dendritic cell, the immunogenic cancer peptide will be cleaved from the rest of the molecule of the invention and displayed on the surface of the dendritic cell by HLA molecules. This will in turn train cytotoxic immune cells (T cell and NK cells) to target this immunogenic peptide and thereby the cancer.

[0286] In some embodiments, treating further comprises administering effector cells specific to the immunogenic peptide to the subject. In some embodiments, an effector cell is an immune cell. In some embodiments, an effector cell is a cytotoxic cell. In some embodiments, an effector cell is a lymphocyte. In some embodiments, an effector cell is a CD8 T cell. In some embodiments, an 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 presenting the peptide in complex with an HLA.

[0287] In some embodiments, the treating further comprises providing 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 suffers from cancer. In some embodiments, the subject is suitable to be treated by a method 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 by the pathogen from which the immunogenic peptide originates. In some embodiments, a pathogen is a virus. In some embodiments, the subject is capable of mounting an immune response against 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.

[0288] In some embodiments, the method is a method of producing surface display of the peptide in a target cell. In some embodiments, the surface display is display of HLAcomplexed with the peptide. In some embodiments, expressing is surface display. In some embodiments, expressing is expressing in an HLA complex.

[0289] By another aspect, there is provided a method of engineering an antibody, the method comprising: a. selecting an antibody of interest; and b. replacing at least one sequence of the Fc region with a peptide; thereby engineering an antibody.

[0290] In some embodiments, the at least one sequence is a loop of the Fc. In some embodiments, the engineered antibody is an antibody of the invention. In some embodiments, the engineered antibody is a protein of the invention. In some embodiments, the engineered antibody is a dual-function antigen binding molecule of the invention. In some embodiments, the engineered antibody is an immunogenic peptide delivery antibody. In some embodiments, the engineered antibody is for use in a method of the invention. In some embodiments, the antibody before engineering is a cell penetrating antibody. In some embodiments, the selecting is selecting an antibody that penetrates into cells to which it binds. In some embodiments, the antibody before engineering is a commercially available antibody.

[0291] In some embodiments, the antibody before engineering 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 that binds to a surface of a target cell. In some embodiments, binding the surface is binding a surface antigen. In some embodiments, antibody before engineering is a DNA binding antibody. In some embodiments, antibody before engineering is endocytosed into the endocytic pathway of a cell. In some embodiments, antibody before engineering is transported into the cytoplasm of a cell to which it binds. In some embodiments, antibody before engineering is capable of endosomal escape. In some embodiments, antibody before engineering is delivered to the cytosol of a cell to which it binds. In some embodiments, the method further comprises inserting into the antibody a cell penetrating moiety. In some embodiments, the cell penetrating moiety is inserted before the immunogenic peptide. In some embodiments, the cell penetrating moiety is inserted after the immunogenic peptide. In some embodiments, the cell penetrating moiety is inserted concomitantly to the insertion of the immunogenic peptide. In some embodiments, step (a) comprises selecting an antibody that upon binding to a surface of a target cell is internalized and delivered to a cytosol of the target cell.

[0292] In some embodiments, the antibody before engineering penetrates / intemalizes into a bound cell at a level comparable to any one of TMab4, 3E10 and 71F12. In some embodiments, the antibody before engineering penetrates / intemalizes into a bound cell at a level comparable to TMab4. In some embodiments, the antibody before engineering penetrates / intemalizes into a bound cell at a level comparable to 3E10. In some embodiments, the antibody before engineering penetrates / intemalizes into a bound cell at a level comparable to 71F12. In some embodiments, comparable is with a penetrance 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, comparable is with a penetrance that is at least 80% of the original antibody. In some embodiments, comparable is with a penetrance that is at least 90% of the original antibody.

[0293] 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 peptide comprises between 8 and 11 amino acids. In some embodiments, the peptide is an immunogenic peptide provided hereinabove. In some embodiments, peptide is from the variable region of the antibody. In some embodiments, peptide is from a CDR of the antibody. Methods of determining the position of CDRs are well known in the art and the Clothia and Kabat systems may be used. Given the sequence of a variable domain of an antibody a skilled artisan will be readily able to identify the CDRs.

[0294] In some embodiments, the at least one loop is removed. In some embodiments, a portion of the at least one loop is removed. In some embodiments, the removed amino acids are replaced with the immunogenic peptide. In some embodiments, the replacing is optimized. In some embodiments, the method further comprises optimizing the replacement. In some embodiments, optimizing is structural optimization. In some embodiments, optimizing comprises performing an optimization algorithm. In some embodiments, optimizing comprises producing as little perturbation in the structure of the selected antibody as possible. In some embodiments, optimizing comprises in silico analysis or the insertion of the immunogenic peptide into the at least one loop with removal of all possible portions of the loop and selected the insertion and removal of a portion that produces the least perturbation. In some embodiments, optimization comprise insertion of at least one filler amino Acid, some embodiments, optimization comprise producing at least onecompensatory mutation outside the determined loop. In some embodiments, the optimization algorithm is a minimal perturbation replacement (MBR) algorithm. In some embodiments, optimization comprises optimization of loop flanking sequence. In some embodiments, optimization of flanking sequence comprises antibody stem preservation. In some embodiments, the structure of the sheets flanking the loop are maintained. In some embodiments, the stems are the ends of the beta sheets flanking the loop. In some embodiments, the end comprises the 1, 2, 3, 4, or 5 amino acids directly adjacent to the loop. Each possibility represents a separate embodiment of the invention.

[0295] In some embodiments, the method further comprises confirming binding of the engineered antibody to the same target that was bound by the selected antibody. In some embodiments, the method further comprises measuring binding of the engineered antibody to the same target that was bound by the selected antibody. In some embodiments, the method further comprises determining binding of the engineered antibody to the same target that was bound by the selected antibody. In some embodiments, binding is equivalent to the binding of the selected antibody. In some embodiments, the measuring further comprises determining that the binding is not significantly reduced as compared to the binding of the selected antibody. In some embodiments, significantly is statistically significantly. In some embodiments, significantly reduced comprises a greater than 10% reduction. In some embodiments, significantly reduced comprises a greater than 20% reduction. In some embodiments, significantly reduced comprises a greater than 50% reduction. In some embodiments, significantly reduced is abolished.

[0296] In some embodiments, the method further comprises measuring levels of 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.

[0297] In some embodiments, the method further comprises confirming delivery of the peptide to a cytosol of the target cell. In some embodiments, the method further comprises measuring levels of 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.

[0298] In some embodiments, the method further comprises confirming delivery of the peptide to the surface of the target cell. In some embodiments, delivery is surface display of the peptide. In some embodiments, delivery is delivery of the peptide in complex with anHLA molecule to the surface of the target cell. In some embodiments, the method further comprises measuring levels of peptide on the surface of the target cell. In some embodiments, levels is levels of the peptide in 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.

[0299] In some embodiments, the method further comprises confirming killing of the target cell by effector cells. In some embodiments, killing is specific killing. In some embodiments, effector cells are specific to the peptide. In some embodiments, effector cells are immune cells. In some embodiments, the method further comprises measuring killing of the target cell by effector cells. In some embodiments, the method further comprises determining killing of the target cell by effector cells. In some embodiments, the method is an in vitro method. In some embodiments, the confirming, measuring and determining is performed in vitro. In some embodiments, in vitro is ex vivo. Examples of methods of performing the confirming, measuring and determining are provided hereinbelow, but any assay known in the art for such measuring / confirming / determining may be used.

[0300] Databases of peptides and specifically immunogenic peptides are known in the art and any such database may be used. In some embodiments, the peptide is a cancer peptide. In some embodiments, database comprises or consists of Table 3. In some embodiments, the alignment is a pairwise alignment. In some embodiments, the alignment is alignment of one peptide from the selected antibody and one peptide of the database to produce an alignment pair. In some embodiments, an alignment pair with an alignment score above a predetermined threshold is used for replacement in step (e)

[0301] In some embodiments, the method further comprises inserting the engineered antibody into a dual-function antigen binding molecule. In some embodiments, the dualfunction 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 a target cell. In some embodiments, on a target cell is on the surface of a 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 a protein only expressed on cancer cells and not counterpart healthy cell. In some embodiments, the protein is a protein overexpressed on cancer cells as compared to counterpart healthy cells. In some embodiments, counterpart cell are cells of the same tissueor cell type as the cancer cells. In some embodiments, counterpart cells are control cells. In some embodiments, the selected targeting antibody is combined with the engineered antibody. In some embodiments, the combining produces the dual-function antigen binding molecule. In some embodiments, a dual-function antigen binding molecule is a bi-specific antibody.

[0302] 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, a 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 / or 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 regions of the engineered antibody and / or the targeting antibody. In some embodiments, the engineering is producing a set of mutations provided in Table 5.

[0303] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0304] It is 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, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0305] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" wouldinclude but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or claims, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0306] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0307] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily 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 delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0308] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0309] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols inMolecular Biology" Volumes I-III Ausubel, R. M., 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 (1998); methodologies as set forth in U.S. Pat. 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, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., 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 by reference. Other general references are provided throughout this document.Materials and Methods

[0310] Anti-viral specific T cell expansion (for example CMV specific T cells): CMV specific T cells are expended from PBMCs of healthy HLA-A2.02.01 positive, CMV positive donors. PBMCs are expanded in Grex6M open system plates for 12 days in the presence of CMV peptide loaded monocytes and cytokines (IL2, IL7, IL15). Cells are boosted with CMV peptide loaded on fresh PBMCs on day 0 and 7. Cytokines and media are added freshly every 3 days.

[0311] T cell mediated killing of cancer cells: Target cancer cells are labeled with CFSE to allow detection by flow cytometry. Labeled cells are co-cultured with viral specific effector T cells in the presence of Trojan antibodies being tested, or control agents. Viral Pepl is used as a positive control indicated. Cells are co-cultured in effector to target cell ratios of 8: 1 for 48-96 hours (as indicated per experiment). Following co-culture, cells are stained with viability dye (zombie) and analyzed by flow cytometry.

[0312] Mice in-vivo study: Immunodeficient NSG mice (NOD.Cg- PrkdcscidIL2rgtm IWjl / / SzJ), 8 weeks old females, were obtained from Jackson Laboratory. All mice were inoculated with A498, a kidney carcinoma human cell line (ATCC). Prior to injection l*10A6 cells were mixed 1 : 1 with Matrigel. When tumor volume reached approximately 85-90mm3, mice were randomized to treatment groups (n=6) and intraperitoneally (IP) injected with Antibodies (6mg / kg) plus IL15 / IL15Ra (15 pg) or PBSplus IL15 / IL15Ra (15pg). The next day all mice were intravenously (IV) injected with 10*10A6 viral specific effector T cells derived from human PBMCs (detailed in expansion method). The study included 3 rounds of treatment every 7 days. During the entire study mice were weighed and tumor volume was evaluated every 2-3days. Tumor growth curves were analyzed by one-tailed two-way ANOVA for time-point comparisons (*p < 0.05, **p < 0.01), and overall group differences were assessed by Tukey’s post-hoc test. Data presented as mean ± SEM.Example 1: Trojan antibody design

[0313] It is known that immunogenic peptides that are presented by cancer cells can lead to immune cell activation and cancer cell killing. Lymphocytes within the subject recognize the immunogenic peptide and the subject’s immune system is brought to bear against the cancer cell. This mechanism can be harnessed by actively delivering the immunogenic peptide into cancer cells, who intern display the immunogenic peptide on their cell surface. Two classes of immunogenic peptides can be employed: 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 are mutated or otherwise altered to provide cancer specificity so that the immune system is not activated against healthy cells.

[0314] It has previously been hypothesized that the immunogenic peptide can be delivered via an antibody. 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 made use of a full-sized antibody conjugated to a peptide via a disulfide bond. However, due to the conjugation with a linker the peptide is exposed to proteinases which risks degradation and / or deconjugation. This in turn leads to poor delivery to the target cells, reduced potency and high off target effects (side effects). Gaston et al., 2019, “Intracellular delivery of therapeutic antibodies into specific cells using antibody - peptide fusions”, Sci. Rep., Dec 10;9(l): 18688 employed a fusion protein construct in which the peptide was integrated into an end of the amino acid chain of the heavy or light chain or was placed next to the hinge domain within the chain. This method led to decreased production yield, increased aggregation and reduced in vitro stability.

[0315] To alleviate the problems inherent to these methods, rather than insert the immunogenic peptide at the end of the chain or between structures International Patent Application WO2023067596 disclosed inserting the immunogenic peptide into an inert CDR of the antibody variable region. Three antibodies were selected that were known tointernalize upon binding a cell: TMab4, 3E10 and 71F12. These antibodies were engineered to replace inert CDRs with immunogenic peptides.

[0316] The first antibody selected for insertion of an immunogenic peptide was a DNA hydrolyzing antibody 3D8, also known as TMab4. The mouse 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, the contents of which are hereby incorporated by reference in their entirety, and its humanized form was provided in International Patent Application WO2019 / 244086, the contents of which is hereby incorporated by reference in its entirety. TMab4 was shown to bind to and penetrate into cancer cells and reach the cytoplasm via endosomal escape, thus it was selected for conversion into a Trojan antibody. It was found that CDRs 1, 2 and 3 of the heavy chain (CDRH1, CDRH2, CDRH3) and CDR 3 of the light chain (CDRL3) were inert CDRs. TMab4 heavy chain and light chain variable region variants with immunogenic peptide insertions are summarized in Table 6,

[0317] Table 6: TMab4 variants comprising viral peptide insertions.0318] 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, hereby incorporated by reference in its entirety) was examined for inert CDRs and it was determined that CDRL1 and CDRL2 both did notengage the antigen during binding. 3E10 light chain variable region variants with immunogenic peptide insertions are summarized in Table 7.

[0319] Table 7: 3E10 variants comprising viral peptide insertions.0320] A third 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, hereby incorporated by reference in its entirety), was analyzed for inert CDRs and it was determined that CDRL1 was inert. A 71F12 light chain variable region variant and a heavy chain variant with an insertion between CDRs with immunogenic peptide insertions are summarized in Table 8.

[0321] Table 8: 71F12 variants comprising viral peptide insertions.

[0322] A new TMab4 variant heavy chain was designed in which the immunogenic peptide NLVPMVATV (SEQ ID NO: 1) was inserted into both the CDRH1 and CDRH2, thus increasing the amount of immunogenic peptide delivered to the cell. The resulting heavy chain variable region produced was SEQ ID NO: 1082. This new heavy chain variable region was still able to bind cells as part of an antibody and to facilitate delivery into the cell. However, it was decided that it would be advantageous to deliver even more copies of the immunogenic peptide to the cell and further insertions into the variable region were determined to produce a decrease in binding. Thus, the constant regions of the antibody were examined for locations in which the viral peptide could be inserted.Example 2: Constant region insertion design

[0323] The constant region of the heavy chain is made up of the CHI, CH2 and CH3 domains that partake in heavy chain dimerization and are recognized by Fc receptors (FCR) and by compliment (Clq). The constant region of the light chain contains only the CL1 domain. Each of the CH2 and CH3 domains comprise 7 beta-sheets that are connected byloops of various lengths and compositions. The beta-sheets are identified from the N- terminus to the C-terminus as sheets A to G. The loops are thus referred to by the sheets that they connect. Thus, loop AB connects sheet A to sheet B, for example. The CHI and CL domains similarly have 6 loops (AB, BC, CD, DE, EF and FG). Loops in all domains were examined for being capable of accommodating mutations / insertions and still fold and function properly. In particular, regions that impact FcRn binding or TRIM binding were avoided so as not to impair antibody half-life or trafficking after internalization, respectively, whereas mutations that might affect complement or effector function (FcRgamma binding) were not avoided. Additionally, regions which play a role in homodimerization / heterodimerization were avoided, although most of these are found in the beta-sheets themselves.

[0324] Within the CH2 domain loop boundaries were determined based on crystal structure, and the following loops were identified as potential peptide insertion sites: loop BC (264- 273), loop DE (292-299) and loop FG (324-331). Within the CH3 domain the following loops were identified as potential peptide insertion sites: loop AB (354-365), loop CD (383- 391) and loop EF (413-424). All positions are given according to the EU index numbering system of antibody positions. Constructs were generated with insertion of SEQ ID NO: 1 into a variety of locations in these loops. The constructs produced are summarized in Table 9.

[0325] Table 9: Produced constructs with SEQ ID NO: 1

[0326] The various constructs were all successfully synthesized, indicating that none of the mutations abolished protein production. Yields were high for most of the constructs with high purity as well. However, TRJ181 showed a greatly decreased purity and TRJ183 produced a much lower yield (Fig. 1A). The two mutations in the CH2 domain were also found to negatively impact FcRgamma and Clq binding, however, this is a potential benefit rather than a drawback.

[0327] The anticancer efficacy of the new antibodies was tested in vitro. Cancer cells were cultured with viral peptide specific effector T cells and cell killing was measured after 48- 96 hours later (Fig. IB). The unmodified TMab4 antibody did not significantly increase killing while TRJ134, with peptides inserted in two CDRs, produced almost 60% cell killing. All of the antibodies with single constant region peptide insertions increased cell killing beyond that of TRJ134 (Fig. IB, below dotted line). TRJ175, TRJ182, TRJ184, and TRJ187 were selected for further study.

[0328] Several combinations of peptide insertions were investigated. These combinations were TRJ182+TRJ187; TRJ182+TRJ175; TRJ184+TRJ187; TRJ184+TRJ175;TRJ187+TRJ175; and TRJ184+TRJ175+TRJ187. The synthesized molecules are summarized in Table 10. Surprisingly, all three molecules with a double insertion in the CH3 domain produced very low yields, so much so that purity could not be assessed (Fig. 2A). Molecules with a combination of a CH2 insertion and a CH3 insertion were successfully produced and with acceptable purity. These molecules were tested for their ability to induce cancer cell killing and compared to the parental single insertions that had been combined. All three molecules were superior to the single insertions, and TRJ203 in particular was found highly effective (Fig. 2B).

[0329] Table 10: Multi-peptide Fc variant constructs

[0330] Additional constructs were generated with insertion of a different viral peptide. SEQ ID NO: 3 (GILGFVFTL) a highly immunogenic peptide from flu virus was inserted into avariety of locations in the selected loops and in a location in a beta-sheet. The constructs produced are summarized in Table 11.

[0331] Table 11 : Produced constructs with SEQ ID NO: 3

[0332] Surprisingly, most of the insertions impaired protein production, with only some of the insertions into the CH2 domain successfully producing protein (Fig. 3). The protein that was produced showed acceptable purity. Cancer killing is tested as above and the produced antibodies increase specific cancer cell killing.

[0333] The constant regions of TRJ175, TRJ184 and TRJ187 were transferred to a WT TMab4 without any peptides inserted into the CDRs. These molecules (TRJ273, TRJ274 and TRJ275) were produced and had acceptable purity (Fig. 4A). Since combination of mutations in CH2 and CH3 had been successful, combination of the CH2 / CH3 mutations with mutations in CHI and CL were tested. Most of the combinations were successfully produced and with very high purity, however, insertions into the BC loop of CHI and the EF loop of CL produced either very poor yields or unacceptably low purity (Fig. 4B). Thecancer killing ability of the produced molecules was tested. It was found that the double insertions produced greater killing as compared to the CH3 insertions alone (Fig. 4C). The combination of insertions in CH1 / CL with CH2 / CH3 and with insertions already in the CDRs allows for the insertion of 6 immunogenic peptides into a single antibody (insertions are doubled as every antibody has two heavy chains and two light chains).Example 3: Generation of full Trojan antibodies with constant region insertions

[0334] In order to produce full Trojan antibodies, bispecific antibodies must be generated using one arm as the penetrating arm (TMab4) and one art as the targeting arm (anti-CD70, anti-EGFR, etc.). The production of such antibodies is accomplished using complementary mutations in the Fc regions of each antibody which increase heterodimerization while decreasing homodimerization. Examples of such mutations are provided in Table 5. The F405L / K409R mutations, referred to in Table 5 as the DouBody (L-R) strategy, was employed.

[0335] Two antibodies, the anti-EGFR antibody panitumumab (PaniVl) and the anti-CD70 antibody vorsetuzumab (SGN-75 sequence), were tested as the targeting arm. Immunogenic peptides were not inserted into the CDRs of vorsetuzumab but panitumumab has a viral peptide inserted into an inert CDR (heavy chain variable region is SEQ ID NO: 1203 and light chain variable region is SEQ ID NO: 1204). The TMab4 penetrating arm included two CDR insertions. The CH3 insertions of TRJ175 and TRJ176 (peptide inserted into the AB loop of CH3) were tested in both CH3 of both the targeting heavy chain and the penetration heavy chain. The F405L mutation inserted into the TRJ175 or TRJ176 heavy chains of TMab4, surprisingly produced almost no protein (titers of less than 0.1 mg / ml) (Fig. 5A). The K409R mutation in the targeting arm heavy chain did allow for protein production. When the K409R mutation was inserted into the penetrating arm protein was also produced, but again the F405L was poorly compatible with the TRJ175 and TRJ176 heavy chains (Fig. 5B) Insertion into the TRJ176 heavy chain with the panitumumab variable region (with a peptide insertion) produced essentially no protein (less than 0.1 mg / ml) while insertion into TRJ175 produced a small amount of protein though certainly below what would be considered optimal (Fig. 5B). Combining the one targeting arm produced with its penetrating arm counterpart (both with the TRJ175 heavy chain constant region) resulted in a bispecific anti-EGFR antibody: TRJ278. This antibody had high purity, though very little of it could be produced (Fig. 5C).

[0336] It was hypothesized that though insertion to the CH3 was not directly contacting the heterodimerization mutations, the proximity of the peptide insertion to the CH3 heterodimerization mutations, specifically F405L, led to the poor yields and overall production problems. In an effort to remedy this issue, the heterodimerization mutations were made in TMab4s with CH2 peptide insertions. Quite surprisingly this was not found to remedy the problem. CH2 peptide insertions still led to very low protein production and very poor purity when combined with the F405L mutation, though they were compatible with the K409R mutation (Fig. 5D). A bispecific antibody was not successfully produced.

[0337] Finally, CHI peptide insertions were combined with the heterodimerization mutations. This was done in molecules with CDR peptide insertions. CHI insertions are far from the heterodimerization mutations in CH3 but are very close to the variable region insertions and the compatibility of CDR and CHI peptide insertion had yet to be confirmed (previous CHI mutations had been made in wild-type TMab4). Quite surprisingly, all of the tested CHI mutations produced very robust amounts of protein with very high purity (Fig. 5E). This was observed both with insertions into the AB loop (mutation 242) and into the CD loop (mutation 246) of CHI. Antibodies could also be robustly produced when the F405L mutation was in the targeting arm (Fig. 5F). TMab4 with two heavy chain CDR insertions (TRJ109 / PDB2-T18+T12) or with one heavy chain CDR insertion (T7) and one light chain CDR insertion (T16), whether with the F405L or K409K mutation could all be produced. CL insertions are also able to combine with the heterodimerization mutations, as they are on a separate chain.

[0338] Four different bispecific antibodies were able to be produced from the CHI peptide inserted antibodies (TRI 315-318) (Fig. 5G). Two anti-CD70 antibodies (TRJ315-316) and two anti-EGFR antibodies (TRJ317-318) were produced at high titer and with the highest possible purity. Thus, a total of 5 bispecific antibodies were produced (Fig. 5H). It is notable that the yields from the 4 antibodies with CHI insertions as opposed to the one with a CH3 insertion were two orders of magnitude higher. This indicates that while CH3 or possibly CH2 insertions can be compatible with heterodimerization mutations, it’s a greatly superior strategy to couple CH1 / CL insertion with the mutations. CH1 / CL insertions are compatible with CDR insertions, even insertions into CDRH3 and they can be easily produced in conjunction with the heterodimerization mutations needed to make a bispecific antibody.

[0339] The cell killing assay was performed as before but with Trojan bispecific antibodies with varying numbers of peptide insertions. The tested anti-CD70 molecules are summarized in Figure 6A. TRJ196 was a control as it had no peptides inserted. It produced a smallamount of killing as compared to the untreated control (Fig. 6B) or the no T cell control (Fig. 6C). This killing is likely due to the antibody binding the surface and inducing antibody-dependent cellular cytotoxicity (ADCC). TRJ193, which contains two total peptides, both inserted into heavy chain CDRs, greatly improved the killing, but produced less than 80% killing even at the highest concentration. The addition of two more peptides by including them in each CHI domain (molecules TRJ315 and 316) significantly increased killing even over TRJ193 (Fig. 6B). Even at a concentration of 0.5 uM killing reached 80% and at the highest concentration the killing surpassed even this threshold. Thus, the four peptide bispecific antibodies are significantly superior to the two peptide bispecific antibodies, and their production was enabled by the inclusion of CHI domains containing peptide.

[0340] The tested anti-EGFR molecules are summarized in Figure 7A. TRJ153 was a control as it had no peptides inserted. It produced a small amount of killing as compared to the untreated control (Fig. 7B) or the no T cell control (Fig. 7C). As before this killing is likely due to the antibody binding the surface and inducing ADCC. TRJ132, which contains three total peptides, one inserted into a heavy chain CDR of TMab4 and one inserted into a light chain CDR of both antibodies, greatly improved the killing, and produced about 80% killing even at the highest concentration. The addition of two more peptides by including them in each CHI domain (molecules TRJ278 and 318) significantly increased killing even over TRJ132 (Fig. 7B). Even at a concentration of 0.5 uM killing reached about 90% and at the highest concentration the killing increased even more. Thus, the five peptide bispecific antibodies are significantly superior to the three peptide bispecific antibodies, and their production was enabled by the inclusion of CHI domains containing peptide.Example 4: In vivo testing of full Trojan antibodies with constant region insertions

[0341] NSG mice with T cells enriched for CMV specificity (see Materials and Methods) were inoculated with A498 carcinoma cells (CD70 positive) and tumors were allowed to form (85-90 mmA3). Mice were then administered by intraperitoneal injection either TRJ- 316 or a control. The initial controls contained either PBS (PBS) or contained the anti-CD70 antibody that is the targeting arm of TRJ-316 (vorsetuzumab). TRJ-316 significantly reduced tumor size as compared to both the PBS (Fig. 8A) and as compared to the anti-CD70 (Fig.8B)

[0342] Next, the four viral peptide bispecific antibody TRJ316 was compared to a bispecific antibody with only 2 viral peptides: TRJ195. TRJ195 is based off the PDB15 Tmab4penetration arm, with SEQ ID NO: 1 inserted in an inert CDR of the heavy chain and the light chain (heavy chain variable region is SEQ ID NO: 1205 and light chain variable region is SEQ ID NO: 1206, full heavy chain is SEQ ID NO: 1207 and full light chain is SEQ ID NO: 1208). Both were tested in the same mouse model and TRJ-316 was found to be significantly superior to TRJ-195 (Fig. 8C), demonstrating the surprising usefulness of immunogenic peptide insertion into the CHI domain. The superiority of the other bispecific antibodies of the invention comprising constant region peptide insertions is also demonstrated in a mouse model.

[0343] In order to make the full Trojan antibodies more suitable for regulatory approval, the FEA silencing mutations were generated in TRJ301 and TRJ306 (TRJ316 when together). The three mutations are located in the CH2 domain (L234F, L235E and DI 65 A). Mutations in the CH2 domain were found to also be compatible with the CHI peptide insertions and both polypeptides could be robustly produced (see Table 12). Thus, insertion into the CHI domain is compatible with both CH2 and CH3 mutations.

[0344] Table 12: Production of FEA silenced full Trojan Antibodies

[0345] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A bispecific antibody comprising: a. a first antibody comprising a first heavy chain and first light chain, wherein said first heavy chain comprises a CH3 domain comprising a first mutation that decreases homodimerization and wherein said first antibody comprises at least one immunogenic peptide inserted into a CHI domain, wherein said insertion replaces amino acids of said CHI domain; b. and a second antibody comprising a second heavy chain and second light chain, wherein said second heavy chain comprises a CH3 domain comprising a second mutation that decreases homodimerization and wherein said second antibody comprises at least one immunogenic peptide inserted into a CHI domain, wherein said insertion replaces amino acids of said CHI domain; wherein said first and second mutation are a pair that increases heterodimerization of said first heavy chain and said second heavy chain, and wherein said first and second antibodies are different antibodies.

2. The bispecific antibody of claim 1, wherein said first mutation and said second mutation are selected from the mutation pairs provided in Table 5.

3. The bispecific antibody of claim 1 or 2, wherein said first mutation is a K409R mutation and said second mutation is a F405L mutation (EU numbering).

4. The bispecific antibody of any one of claims 1 to 3, wherein said at least one immunogenic peptide is inserted into a CHI domain of said first heavy chain and a CHI domain of said second heavy chain.

5. The bispecific antibody of any one of claims 1 to 4, wherein said at least one immunogenic peptide is inserted an AB loop, a BC loop, a CD loop, a DE loop, an EF loop or an FG loop of said CHI domain.

6. The bispecific antibody of claim 5, wherein said at least one immunogenic peptide is inserted into said CHI AB loop which comprises amino acids 124-145 of a constant region of said heavy chain, or said at least one immunogenic peptide is inserted into said CHI CD loop which comprises amino acids 154-171 of said constant region of said heavy chain and wherein numbering is according to EU numbering.

7. A bispecific antibody comprising: a. a first antibody comprising a first heavy chain and first light chain, wherein said first heavy chain comprises a CH3 domain comprising a first mutation thatdecreases homodimerization and wherein said first antibody comprises at least one immunogenic peptide inserted into a CL domain, wherein said insertion replaces amino acids of said CL domain; b. and a second antibody comprising a second heavy chain and second light chain, wherein said second heavy chain comprises a CH3 domain comprising a second mutation that decreases homodimerization and wherein said second antibody comprises at least one immunogenic peptide inserted into a CL domain, wherein said insertion replaces amino acids of said CL domain; wherein said first and second mutation are a pair that increases heterodimerization of said first heavy chain and said second heavy chain, and wherein said first and second antibodies are different antibodies.

8. The bispecific antibody of claim 7, wherein said at least one immunogenic peptide is inserted an AB loop, a BC loop, a CD loop, a DE loop, an EF loop or an FG loop of said CL domain.

9. The bispecific antibody of claim 8, wherein said at least one immunogenic peptide is inserted into said CL AB loop which comprises amino acids 114-133 of a constant region of said light chain and wherein numbering is according to EU numbering.

10. The bispecific antibody of any one of claims 1 to 9, wherein said first antibody and said second antibody are both IgGl antibodies.

11. The bispecific antibody of any one of claims 1 to 10, wherein said immunogenic peptide is a cancer specific peptide.

12. The bispecific antibody of claim 11, wherein said cancer specific peptide is selected from a peptide sequence provided in Table 1.

13. The bispecific antibody of claims 1 to 12, wherein said immunogenic peptide is a viral peptide.

14. The bispecific antibody of claim 13, wherein said viral peptide is derived from Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), Adenovirus, Human papilloma virus (HPV) or Influenza virus (FLU).

15. The bispecific antibody of claim 14, wherein said viral peptide is selected from a peptide sequence provided in Table 2 or Table 3.

16. The bispecific antibody of any one of claims 1 to 15, wherein said immunogenic peptide comprises or consists of NLVPMVATV (SEQ ID NO: 1).

17. The bispecific antibody of claim 16, wherein SEQ ID NO: 1 replaces amino acids 127- 135, 132-140 or 159-167 of said heavy chain constant region, wherein said number is according to EU numbering.

18. The bispecific antibody of claim 16 or 17, wherein said CHI domain comprises or consists of a sequence selected from SEQ ID NO: 1211-1214 or said CL domain comprises or consists of SEQ ID NO: 1170.

19. The bispecific antibody of any one of claims 1 to 18, wherein one of said first and said second antibodies is a cell penetrating antibody.

20. The bispecific antibody of claim 19, wherein said cell penetrating antibody is selected from TMab4, 3E10, and 71F12.

21. The bispecific antibody of claim 20, wherein said cell penetrating antibody is TMab4.

22. The bispecific antibody of any one of claims 1 to 21, wherein at least one of said first and said second antibodies further comprises at least one immunogenic peptide inserted into a CDR of said antibody, where said insertion comprises removal of CDR sequence, and wherein said insertion does not reduce binding to an antibody target.

23. The bispecific antibody of claim 22, wherein said at least one immunogenic peptide is inserted into an inert CDR of said antibody.

24. The bispecific antibody of claim 23, wherein said at least one immunogenic peptide is inserted into at least one of CDRH1, CDRH2, CDRH3 or CDRL3 of antibody Tmab4.

25. The bispecific antibody of claim 21 or 24, wherein a heavy chain variable region of TMab4 comprises or consists of SEQ ID NO: 1021, 1028-1040, 1043-1045, 1047- 1055, 1058 and 1082 and a light chain variable region of TMab4 comprises or consists of SEQ ID NO: 1022, 1041-1042, 1046, and 1056-1057.

26. The bispecific antibody of any one of claims 19 to 25, wherein one of said first and second antibodies is an anti-epidermal growth factor receptor (EGFR) antibody selected from: cetuximab, panitumumab, necitumumab and an antibody comprising at least 85% sequence identity to cetuximab, panitumumab, or necitumumab and which binds EGFR.

27. The bispecific antibody of any one of claims 19 to 25, wherein one of said first and second antibodies is an CD70 antibody selected from: vorsetuzumab (also known as hlF6), cusatuzumab, and an antibody comprising at least 85% sequence identity to vorsetuzumab, or cusatuzumab and which binds CD70.

28. A protein comprising an Fc variant of a parent Fc polypeptide, wherein said Fc variant comprises at least one immunogenic peptide inserted into said parent Fc polypeptide and wherein said insertion replaces amino acids of said parent Fc polypeptide.

29. The protein of claim 28, wherein said immunogenic peptide is inserted into a loop between beta sheets of said parent Fc polypeptide.

30. The protein of claim 29, wherein said loop is within a CH2 domain of said Fc polypeptide and is selected from the BC loop, the DE loop and the FG loop.

31. The protein of claim 30, wherein said CH2 BC loop comprises amino acids 263-273 of said Fc polypeptide, said CH2 DE loop comprises amino acids 292-302 of said Fc polypeptide and said FG loop comprises amino acids 323-332 of said Fc polypeptide and wherein numbering is according to the EU index.

32. The protein of claim 29, wherein said loop is within a CH3 domain of said Fc polypeptide and is selected from the AB loop, the BC loop, the CD loop, the DE loop and the EF loop.

33. The protein of claim 32, wherein said CH3 AB loop comprises amino acids 350-365 of said Fc polypeptide, said CH3 BC loop comprises amino acids 371-379 of said Fc polypeptide, said CH3 CD loop comprises amino acids 383-391 of said Fc polypeptide, said CH3 DE loop comprises amino acids 397-407 of said Fc polypeptide and said CH3 EF loop comprises amino acids 413-424 of said Fc polypeptide and wherein numbering is according to the EU index.

34. The protein of any one of claims 28 to 33, wherein said Fc is a human IgGl Fc.

35. The protein of any one of claims 28 to 34, wherein said immunogenic peptide is a cancer specific peptide.

36. The protein of claim 35, wherein said cancer specific peptide is selected from a peptide sequence provided in Table 1.

37. The protein of claims 28 to 34, wherein said immunogenic peptide is a viral peptide.

38. The protein of claim 37, wherein said viral peptide is derived from Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), Adenovirus, Human papilloma virus (HPV) or Influenza virus (FLU).

39. The protein of claim 38, wherein said viral peptide is selected from a peptide sequence provided in Table 2 or Table 3.

40. The protein of any one of claims 37 to 39, wherein said immunogenic peptide comprises NLVPMVATV (SEQ ID NO: 1).

41. The protein of claim 40, wherein SEQ ID NO: 1 is positions 354-362 of said Fc variant.

42. The protein of claim 41, wherein said Fc variant comprises SEQ ID NO: 1068, 1102, 1105, 1106, 1107 or 1108.

43. The protein of claim 40, wherein SEQ ID NO: 1 is positions 350-358 of said Fc variant.

44. The protein of claim 43, wherein said Fc variant comprises SEQ ID NO: 1069.

45. The protein of claim 40, wherein SEQ ID NO: 1 is positions 357-365 of said Fc variant.

46. The protein of claim 45, wherein said Fc variant comprises SEQ ID NO: 1070.

47. The protein of claim 40, wherein SEQ ID NO: 1 replaces positions 384-389 in said Fc polypeptide.

48. The protein of claim 47, wherein said Fc variant comprises SEQ ID NO: 1071.

49. The protein of claim 40, wherein SEQ ID NO: 1 replaces positions 384-388 in said Fc polypeptide.

50. The protein of claim 49, wherein said Fc variant comprises SEQ ID NO: 1072.

51. The protein of claim 40, wherein SEQ ID NO: 1 replaces positions 384-387 in said Fc polypeptide.

52. The protein of claim 51, wherein said Fc variant comprises SEQ ID NO: 1073.

53. The protein of claim 40, wherein SEQ ID NO: 1 is positions 416-424 of said Fc variant.

54. The protein of claim 53, wherein said Fc variant comprises SEQ ID NO: 1074.

55. The protein of claim 40, wherein SEQ ID NO: 1 replaces positions 418-422 in said Fc polypeptide.

56. The protein of claim 55, wherein said Fc variant comprises SEQ ID NO: 1075, 1100, 1101 or 1102.

57. The protein of claim 40, wherein SEQ ID NO: 1 replaces positions 415-422 in said Fc polypeptide.

58. The protein of claim 57, wherein said Fc variant comprises SEQ ID NO: 1076.

59. The protein of claim 40, wherein SEQ ID NO: 1 is positions 414-422 of said Fc variant.

60. The protein of claim 59, wherein said Fc variant comprises SEQ ID NO: 1077, 1103, 1104, 1105 or 1108.

61. The protein of claim 40, wherein SEQ ID NO: 1 replaces positions 418-424 in said Fc polypeptide.

62. The protein of claim 61, wherein said Fc variant comprises SEQ ID NO: 1078.

63. The protein of claim 40, wherein SEQ ID NO: 1 replaces positions 418-423 in said Fc polypeptide.

64. The protein of claim 53, wherein said Fc variant comprises SEQ ID NO: 1079.

65. The protein of claim 40, wherein SEQ ID NO: 1 is positions 264-272 of said Fc variant.

66. The protein of claim 65, wherein said Fc variant comprises SEQ ID NO: 1080, 1100, 1103, 1106 or 1108.

67. The protein of claim 40, wherein SEQ ID NO: 1 is positions 265-273 of said Fc variant.

68. The protein of claim 67, wherein said Fc variant comprises SEQ ID NO: 1081, 1101, 1104 or 1107.

69. The protein of any one of claims 37 to 39, wherein said immunogenic peptide comprises GILGFVFTL (SEQ ID NO: 3).

70. The protein of claim 69, wherein SEQ ID NO: 3 is positions 354-362 of said Fc variant.

71. The protein of claim 70, wherein said Fc variant comprises SEQ ID NO: 1123.

72. The protein of claim 69, wherein SEQ ID NO: 3 is positions 357-365 of said Fc variant.

73. The protein of claim 72, wherein said Fc variant comprises SEQ ID NO: 1124.

74. The protein of claim 69, wherein SEQ ID NO: 3 is positions 350-358 of said Fc variant.

75. The protein of claim 74, wherein said Fc variant comprises SEQ ID NO: 1125.

76. The protein of claim 69, wherein SEQ ID NO: 3 is positions 371-379 in said Fc variant.

77. The protein of claim 76, wherein said Fc variant comprises SEQ ID NO: 1126.

78. The protein of claim 70, wherein SEQ ID NO: 3 is positions 399-407 in said Fc variant.

79. The protein of claim 78, wherein said Fc variant comprises SEQ ID NO: 1127.

80. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 398-403 in said Fc polypeptide.

81. The protein of claim 80, wherein said Fc variant comprises SEQ ID NO: 1128.

82. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 397-402 of said Fc polypeptide.

83. The protein of claim 82, wherein said Fc variant comprises SEQ ID NO: 1129.

84. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 397-404 in said Fc polypeptide.

85. The protein of claim 84, wherein said Fc variant comprises SEQ ID NO: 1130.

86. The protein of claim 69, wherein SEQ ID NO: 3 is positions 414-422 in said Fc variant.

87. The protein of claim 86, wherein said Fc variant comprises SEQ ID NO: 1131.

88. The protein of claim 68, wherein SEQ ID NO: 3 is positions 415-423 of said Fc variant.

89. The protein of claim 88, wherein said Fc variant comprises SEQ ID NO: 1132.

90. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 418-422 in said Fc polypeptide.

91. The protein of claim 90, wherein said Fc variant comprises SEQ ID NO: 1133.

92. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 418-421 in said Fc polypeptide.

93. The protein of claim 92, wherein said Fc variant comprises SEQ ID NO: 1134.

94. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 415-421 of said Fc polypeptide.

95. The protein of claim 94, wherein said Fc variant comprises SEQ ID NO: 1135.

96. The protein of claim 69, wherein SEQ ID NO: 3 is positions 265-273 of said Fc variant.

97. The protein of claim 96, wherein said Fc variant comprises SEQ ID NO: 1136.

98. The protein of claim 69, wherein SEQ ID NO: 3 is positions 264-272 of said Fc variant.

99. The protein of claim 98, wherein said Fc variant comprises SEQ ID NO: 1137.

100. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 264-271 of said Fc polypeptide.

101. The protein of claim 100, wherein said Fc variant comprises SEQ ID NO: 1138.

102. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 263-272 of said Fc polypeptide.

103. The protein of claim 102, wherein said Fc variant comprises SEQ ID NO: 1139.

104. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 295-299 of said Fc polypeptide.

105. The protein of claim 104, wherein said Fc variant comprises SEQ ID NO: 1140.

106. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 292-298 of said Fc polypeptide.

107. The protein of claim 106, wherein said Fc variant comprises SEQ ID NO: 1141.

108. The protein of claim 69, wherein SEQ ID NO: 3 is positions 294-302 of said Fc variant.

109. The protein of claim 108, wherein said Fc variant comprises SEQ ID NO: 1142.

110. The protein of claim 69, wherein SEQ ID NO: 3 replaces positions 325-332 of said Fc polypeptide.

111. The protein of claim 110, wherein said Fc variant comprises SEQ ID NO: 1143.

112. The protein of claim 68, wherein SEQ ID NO: 3 is positions 324-332 of said Fc variant.

113. The protein of claim 111, wherein said Fc variant comprises SEQ ID NO: 1144.

114. The protein of claim 69, wherein SEQ ID NO: 3 is positions 323-331 of said Fc variant.

115. The protein of claim 114, wherein said Fc variant comprises SEQ ID NO: 1145.

116. The protein of claim 69, wherein SEQ ID NO: 3 is positions 237-245 of said Fc variant.

117. The protein of claim 116, wherein said Fc variant comprises SEQ ID NO: 1146.

118. The protein of claim 69, wherein SEQ ID NO: 3 is positions 190-198 of said Fc variant.

119. The protein of claim 118, wherein said Fc variant comprises SEQ ID NO: 1147.

120. The protein of any one of claims 28 to 119, wherein said insertion and replacing produces no change or minimal change in the overall conformation of said Fc such that said Fc still homodimerizes and / or heterodimerizes with a second Fc.

121. The protein of any one of claims 28 to 120, wherein said protein is a heavy chain of an antibody and further comprises a heavy chain variable region.

122. The protein of claim 121, wherein said heavy chain variable region is the heavy chain variable region selected from TMabO4TMab4, 3E10, 71F12 and TmabO4TMab4, 3E10, or 71F12 comprising an inert CDR replaced with an immunogenic peptide.

123. The protein of claim 122, wherein said heavy chain variable region comprises a sequence selected from SEQ ID NO: 1021, 1023, 1026, 1028-1040, 1043-1045, 1047-1055, 1058-1059, 1067 and 1082.

124. The protein of claim 123, wherein said heavy chain variable region comprises or consists of SEQ ID NO: 1082.

125. The protein of claim 124, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 1083-1096 and 1109-1117.

126. The protein of claim 123, wherein said heavy chain variable region comprises or consists of SEQ ID NO: 1021.

127. The protein of claim 126, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 1118-1122.

128. A protein dimer comprising a first protein and a second protein dimerized together, wherein said first protein and said second protein are both a protein of any one of claims 28 to 127.

129. The protein dimer of claim 128, wherein said first protein and said second protein are different proteins.

130. The protein dimer of claim 128, wherein said first and second protein are the same protein.

131. An antibody comprising two heavy chains and two light chains and wherein each heavy chain is a protein of any one of claims 123 to 127.

132. The antibody of claim 131, wherein a heavy chain variable region comprises one of SEQ ID NO: 1021, 1028-1040, 1043-1045, 1047-1055, 1058 and 1082 and a light chain variable region comprises one of SEQ ID NO: 1022, 1041-1042, 1046, and 1056-1057.

133. The antibody of claim 131, wherein a heavy chain variable region comprises SEQ ID NO: 1023 and a light chain variable region comprises one of SEQ ID NO: 1024-1025, and 1060-1065.

134. The antibody of claim 131, wherein a heavy chain variable region comprises one of SEQ ID NO: 1026 and 1067 and a light chain variable region comprises one of SEQ ID NO: 1027 and 1066.

135. A pharmaceutical composition comprising a bispecific antibody of any one of claims 1 to 27, a protein of any one of claims 28 to 127, a protein dimer of any one of claims 128 to 130 or an antibody of any one of claims 131 to 134 and a pharmaceutically acceptable carrier excipient or adjuvant.

136. A nucleic acid molecule comprising at least one open reading frame, wherein said open reading frame encodes a bispecific antibody of any one of claims 1 to 27, a protein of any one of claims 28 to 127, a protein dimer of any one of claims 128 to 130 or an antibody of any one of claims 131 to 134.

137. An expression vector comprising at least one regulatory element operatively linked to a nucleic acid molecule of claim 136.

138. A method of treating cancer in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition of claim 135, thereby treating cancer in a subj ect.

139. A method of engineering a bispecific antibody, the method comprising: a. selecting a bispecific antibody comprising heavy chains comprising CH3 domains comprising mutations that decrease homodimerization and increase heterodimerization; and b. replacing at least one sequence in a CHI or CL domain of said selected bispecific antibody with an immunogenic peptide; thereby engineering a bispecific antibody.

140. A method of engineering an antibody, the method comprising: a. selecting an antibody of interest; b. replacing at least one sequence in the Fc region of said selected antibody with an immunogenic peptide; thereby engineering an antibody.

141. The method of claim 140, wherein said at least one sequence in the Fc region is within a loop between beta sheets of said Fc region.

142. The method of any one of claims 139 to 141, further comprising confirming said bispecific antibody retains binding to its target after said replacing.

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