Humanized Anti-MUC1* antibodies

Abstract

To provide antibodies that bind to MUC1* receptor effective to treat or prevent MUC1* positive cancer.SOLUTION: Provided is a human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein that binds to an extracellular domain site of an MUC1 isoform lacking the tandem repeat region or of a decomposed product.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to humanized anti-MUCl* antibodies, methods for their production, and uses. [Background technology]

[0002] The present inventors have previously discovered that a cleaved form of the MUC1 (SEQ ID NO: 1) transmembrane protein is a growth factor receptor that drives the proliferation of over 75% of all human cancers.

[0003] We call it MUCl* (pronounced mu-c-1-star), and this cleaved form of MUC1 is a potent growth factor receptor.

[0004] Cleavage and release of a large portion of the extracellular domain of MUC1 exposes the binding site for activating the ligand dimer NME1, NME6, or NME7. It is aberrantly expressed in over 75% of all cancers and is likely overexpressed in a high proportion of metastatic cancers, making it an ideal target for anticancer drugs (Fessler SP, Wotkowicz MT, Mahanta SK, and Bamdad C. (2009)). MUCl* is a determinant of trastuzumab (Herceptin) resistance in breast cancer cells. Breast Cancer Res Treat. 118(1) 113-124. Following MUC1 cleavage, most of its extracellular domain is shed from the cell surface. The remaining portion contains a truncated extracellular domain containing at least the primary growth factor receptor sequence, PSMGFR (SEQ ID NO: 2).

[0005] Antibodies are increasingly being used to treat human diseases. Antibodies generated in species other than human, such as equine antibodies, have historically been used as therapeutic tools in humans. More recently, antibodies have been engineered or selected so that they contain mostly human sequences to avoid the common rejection of xenoantibodies. The process of engineering the recognition fragment of a non-human antibody into a human antibody is commonly referred to as "humanization." The amount of non-human sequence used to replace the human antibody sequence determines whether they are called chimeric, humanized, or fully human.

[0006] Alternative technologies exist that allow for the generation of humanized or fully human antibodies. These methods involve screening libraries of human antibodies or antibody fragments and identifying those that bind to a target antigen rather than immunizing animals with the antigen. Another approach is to engineer the variable regions of antibodies into antibody-like molecules. The present invention is intended to encompass this approach, using recognition fragments of antibodies that the inventors have determined bind to the extracellular domain of MUC1*.

[0007] In addition to treating patients with antibodies, cancer immunotherapy has recently been shown to be effective in treating cancer. T cell-based cancer immunotherapy is an attractive approach to overcome cancer cell evasion from the immune system. The first immunotherapy, called CAR-T (chimeric antigen receptor T cell) therapy, relied on the expression of CARs on the surface of patient T cells for adoptive T cell therapy (Dai H, Wang Y, Lu X, Han W. (2016) Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy. J Natl Cancer Inst. 108(7): djv439). Such receptors consist of anti-cancer scFvs linked to T cell transmembrane and signaling domains. Receptor binding to cancer-associated antigens transmits a signal, resulting in T cell activation, proliferation, and targeted killing of cancer cells. In practice, patient T cells are isolated, transduced with CARs, expanded, and then infused back into the patient. When a patient's CAR T cells bind to an antigen on cancer cells, the CAR T cells expand and attack the cancer cells. The drawback of this method is that most cancer antigens are expressed in some healthy tissues, but when overexpressed in cancer tissues, there is a risk of activating the patient's immune system to destroy cells carrying the target antigen. To minimize the risk of off-tumor / on-target effects, cancer antigens should be minimally expressed in healthy tissues.

[0008] A second type of cancer immunotherapy involves BiTEs (bispecific T cell engagers). The BiTE approach was attempted to eliminate the risk of CAR T-associated off-tumor / on-target effects. Unlike CAR T, BiTEs are bispecific antibodies that may not pose any greater risks than conventional antibody-based therapies. However, unlike typical cancer treatment antibodies that bind to and block cancer antigens, BiTEs are designed to bind to antigens on tumor cells and simultaneously bind to antigens on immune cells such as T cells. In this way, BiTEs recruit T cells to tumors. BiTEs are engineered proteins that simultaneously bind to cancer-associated antigens and T cell surface proteins such as CD3 epsilon. BiTEs are antibodies created by genetically linking the scFv of an antibody that binds to a T cell antigen, such as anti-CD3 epsilon, to the scFv of a therapeutic monoclonal antibody that binds to a cancer antigen (Patrick A. Baeuerle, and CarstenReinhardt (2009) Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res. 69(12):4941-4944). Summary of the Invention

[0009] In one aspect, the invention is directed to a human or humanized anti-MUCl* antibody or antibody fragment or antibody-like protein that binds to a site on the extracellular domain of a MUCl isoform or cleavage product lacking the tandem repeat region. The human or humanized anti-MUCl* antibody or antibody fragment or antibody-like protein may specifically bind to:

[0010] (i) the PSMGFR site of MUC1;

[0011] (ii) PSMGFR peptide;

[0012] (iii) a peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620);

[0013] (iv) a peptide having the amino acid sequence SVVVQLTLAFREGTINVHDVETQFNQYKTEASRY (SEQ ID NO: 621);

[0014] (v) a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622); or

[0015] (vi) A peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623).

[0016] The human or humanized antibody may be an IgG1, IgG2, IgG3, IgG4 or IgM. The human or humanized antibody fragment or antibody-like protein may be an scFv or scFv-Fc.

[0017] Such human or humanized antibodies, antibody fragments, or antibody-like proteins may comprise heavy and light chain variable regions derived from the murine monoclonal MN-E6 antibody and have at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-E6 antibody. The heavy chain variable region may have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 13. The light chain variable region may have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 66.

[0018] A human or humanized antibody, antibody fragment or antibody-like protein according to the above may comprise complementarity determining regions (CDRs) in the heavy and light chain variable regions having at least 90%, or 95%, or 98% sequence identity at the CDR1, CDR2 or CDR3 sites, with sequences as follows:

[0019] CDR1 heavy chain SEQ ID NO: 17

[0020] CDR1 light chain SEQ ID NO: 70,

[0021] CDR2 heavy chain SEQ ID NO:21

[0022] CDR2 light chain SEQ ID NO:74,

[0023] CDR3 heavy chain SEQ ID NO:25

[0024] CDR3 light chain SEQ ID NO:78.

[0025] The human or humanized antibody, antibody fragment, or antibody-like protein described above can comprise heavy and light chain variable regions derived from the murine monoclonal MN-C2 antibody and have at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-C2 antibody. The heavy chain variable region can have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 119, and the light chain variable region can have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 169. The complementarity determining regions (CDRs) in the heavy and light chain variable regions can have at least 90%, 95%, or 98% sequence identity at the CDR1, CDR2, or CDR3 sites, with sequences as follows:

[0026] CDR1 heavy chain SEQ ID NO: 123

[0027] CDR1 light chain SEQ ID NO: 173,

[0028] CDR2 heavy chain SEQ ID NO: 127

[0029] CDR2 light chain SEQ ID NO: 177,

[0030] CDR3 heavy chain SEQ ID NO: 131

[0031] CDR3 light chain SEQ ID NO: 181.

[0032] As described above, the human or humanized antibody, antibody fragment, or antibody-like protein may comprise heavy and light chain variable regions derived from the murine monoclonal MN-C3 antibody and may have at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-C3 antibody. The heavy chain variable region may have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 414, and the light chain variable region may have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 459. The complementarity determining regions (CDRs) of the heavy and light chain variable regions may have at least 90%, 95%, or 98% sequence identity at the CDR1, CDR2, or CDR3 sites, with sequences as follows:

[0033] CDR1 heavy chain SEQ ID NO: 418,

[0034] CDR1 light chain SEQ ID NO: 463,

[0035] CDR2 heavy chain SEQ ID NO: 422,

[0036] CDR2 light chain SEQ ID NO: 467,

[0037] CDR3 heavy chain SEQ ID NO: 426,

[0038] CDR3 light chain SEQ ID NO:471.

[0039] The human or humanized antibody, antibody fragment, or antibody-like protein may comprise a heavy chain variable region and a light chain variable region derived from the murine monoclonal MN-C8 antibody and may have at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-C8 antibody. The heavy chain variable region may have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 506, and the light chain variable region may have at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 544. The complementarity determining regions (CDRs) of the heavy and light chain variable regions may have at least 90%, 95%, or 98% sequence identity to the CDR1, CDR2, or CDR3 sites, with sequences as follows:

[0040] CDR1 heavy chain SEQ ID NO: 508,

[0041] CDR1 light chain SEQ ID NO: 546,

[0042] CDR2 heavy chain SEQ ID NO: 510,

[0043] CDR2 light chain SEQ ID NO: 548,

[0044] CDR3 heavy chain SEQ ID NO: 512,

[0045] CDR3 light chain SEQ ID NO:550.

[0046] In another aspect, the invention is directed to an anti-MUCl* extracellular domain antibody composed of the sequence of humanized MN-E6, represented by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized kappa (κ) light chain or a humanized lambda (λ) light chain. The humanized IgG2 heavy chain can be SEQ ID NO: 53, the humanized IgG1 heavy chain can be SEQ ID NO: 57, the humanized kappa light chain can be SEQ ID NO: 108, and the humanized lambda light chain can be SEQ ID NO: 112, or sequences with 90%, 95%, or 98% sequence identity to each.

[0047] In another aspect, the invention is directed to an anti-MUCl* extracellular domain antibody composed of the sequence of humanized MN-C2, represented by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain, paired with a humanized kappa light chain and a humanized lambda light chain. The humanized IgG1 heavy chain MN-C2 can be SEQ ID NO: 159, and the IgG2 heavy chain can be SEQ ID NO: 164, paired with a kappa light chain (SEQ ID NO: 213) or a lambda light chain (SEQ ID NO: 219), or sequences with 90%, 95%, or 98% sequence identity to each.

[0048] In another aspect, the invention is directed to an anti-MUCl* extracellular domain antibody composed of the sequence of humanized MN-C3, represented by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized kappa light chain or a humanized lambda light chain. The humanized MN-C3 IgG1 heavy chain can be SEQ ID NO: 454, the IgG2 heavy chain can be SEQ ID NO: 456, the kappa light chain can be SEQ ID NO: 503, and the lambda light chain can be SEQ ID NO: 501, or sequences with 90%, 95%, or 98% sequence identity to each.

[0049] In another aspect, the invention is directed to an anti-MUCl* extracellular domain antibody composed of the sequence of humanized MN-C8, represented by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized kappa light chain or a humanized lambda light chain. The humanized MN-C8 IgG1 heavy chain can be SEQ ID NO: 540, the IgG2 heavy chain can be SEQ ID NO: 542, the kappa light chain can be SEQ ID NO: 582, and the lambda light chain can be SEQ ID NO: 580, or sequences with 90%, 95%, or 98% sequence identity to each.

[0050] In another aspect, the present invention is directed to a human or humanized anti-MUC1* antibody, antibody fragment, or antibody-like protein according to the above, which inhibits the binding of an NME protein to MUC1*. The NME can be NME1, NME6, NME7AB, NME7, or NME8.

[0051] In yet another aspect, the invention is directed to a single-chain variable fragment (scFv) comprising heavy and light chain variable regions connected by a linker and further comprising the CDRs of an antibody that binds to the MUCl* extracellular domain. The CDRs can be derived from the MN-E6, MN-C2, MN-C3, or MN-C8 antibody or a humanized version thereof. The scFv can have SEQ ID NOs: 233, 235, and 237 (E6); SEQ ID NOs: 239, 241, and 243 (C2); SEQ ID NOs: 245, 247, and 249 (C3); or SEQ ID NOs: 251, 253, and 255 (C8).

[0052] In yet another aspect, the present invention is directed to a chimeric antigen receptor (CAR) comprising an scFv or humanized variable region that binds to the extracellular domain of MUC1 lacking tandem repeats, a linker molecule, a transmembrane region, and a cytoplasmic region. The single chain antibody fragment may bind to:

[0053] (i) PSMGFR site of MUC1

[0054] (ii) PSMGFR peptide)

[0055] (iii) a peptide having the following amino acid sequence: SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620);

[0056] (iv) a peptide having the following amino acid sequence: SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621);

[0057] (v) a peptide having the amino acid sequence: VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622); or

[0058] (vi) A peptide having the following amino acid sequence: SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623).

[0059] In the above-described CAR, any portion of the variable region may be used as shown and described above. Alternatively, a combination thereof may be used with an extracellular domain, a transmembrane domain, and a cytoplasmic tail containing a sequence motif that signals immune system activation. The extracellular domain may be composed of a humanized single-chain antibody fragment of MN-E6 scFv, MN-C2 scFv, MN-C3 scFv, or MN-C8 scFv.

[0060] In the CARs described above, the extracellular domain comprises a humanized single-chain antibody fragment of MN-E6 scFv, designated as SEQ ID NO: 233, 235, or 237, or MN-C2 scFv (SEQ ID NO: 239, 241, or 243), MN-C3 scFv (SEQ ID NO: 245, 247, or 249), or MN-C8 scFv (SEQ ID NO: 251, 253, or 255).

[0061] In any of the CARs described above, the cytoplasmic tail can be composed of one or more of the signal sequence motifs CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7.

[0062] For any of the CARs described above, the sequence can be: CARMN-E6 CD3z (SEQ ID NO: 295); CARMN-E6 CD28 / CD3z (SEQ ID NO: 298); CARMN-E6 4-lBB / CD3z (SEQ ID NO: 301); CARMN-E6 OX40 / CD3z (SEQ ID NO: 617); CARMN-E6CD28 / 4-lBB / CD3z (SEQ ID NO: 304); CARMN-E6 CD28 / OX40 / CD3z (SEQ ID NO: 619); CAR MN-C2 CD3z (SEQ ID NO: 607); CAR MN-C2 CD28 / CD3z (SEQ ID NO: 609); CAR MN-C24-lBB / CD3z (SEQ ID NO: 611); CAR MN-C2 OX40 / CD3z (SEQ ID NO: 613); CAR MN-C2 CD28 / 4-lBB / CD3z (SEQ ID NO: 307); or CAR MN-C2 CD28 / OX40 / CD3z (SEQ ID NO: 615).

[0063] In another aspect, the CAR can have an extracellular domain unit that recognizes a peptide. The peptide can be PSMGFR (SEQ ID NO: 2). The peptide can be a peptide derived from NME7. The peptide can be:

[0064] NME7A peptide 1 (A region): MLSRKEALDFHVDHQS (SEQ ID NO: 7);

[0065] NME7A peptide 2 (A region): SGVARTDASES (SEQ ID NO: 8);

[0066] NME7B peptide 1 (B region): DAGFEISAMQMFNMDRVNVE (SEQ ID NO: 9);

[0067] NME7B peptide 2 (B region): EVYKGVVTEYHDMVTE (SEQ ID NO: 10); or

[0068] NME7B peptide 3 (B region): AIFGKTKIQNAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 11).

[0069] In another aspect, the invention is directed to a composition comprising at least two CARs with different extracellular domain units transfected into the same cell.

[0070] The at least two CARs have one CAR without a targeting recognition unit and another CAR with a targeting recognition unit. Alternatively, one of the extracellular domain recognition units can bind to the MUCl* extracellular domain. Alternatively, one of the extracellular domain recognition units can bind PD-1. Alternatively, one of the extracellular domain recognition units can be an antibody fragment and the other a peptide. Alternatively, one is an anti-MUCl* scFv selected from the group consisting of the scFv of the MN-E6 antibody, the scFv of the MN-C2 antibody, the scFv of the MN-C3 antibody, or the scFv of the MN-C8 antibody, and the other is a peptide derived from NME7 or selected from the group consisting of:

[0071] NME7A peptide 1 (A region): MLSRKEALDFHVDHQS (SEQ ID NO: 7);

[0072] NME7A peptide 2 (A region): SGVARTDASES (SEQ ID NO: 8);

[0073] NME7B peptide 1 (B region): DAGFEISAMQMFNMDRVNVE (SEQ ID NO: 9);

[0074] NME7B peptide 2 (B region): EVYKGVVTEYHDMVTE (SEQ ID NO: 10); and

[0075] NME7B peptide 3 (B region): AIFGKTKIQNAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 11).

[0076] In another aspect, the invention is directed to a cell comprising a CAR with an extracellular domain that binds to a MUCl*-transfected or transduced cell. The CAR-containing cell can be an immune system cell, preferably a T cell, a dendritic cell, or a mast cell.

[0077] In another aspect, the present invention is directed to engineered antibody-like proteins.

[0078] In another aspect, the invention is directed to a method of screening an antibody or antibody fragment library, which is human, that binds to:

[0079] (i) PSMGFR peptide;

[0080] (ii) a peptide having the following amino acid sequence: SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620);

[0081] (iii) a peptide having the following amino acid sequence: SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621);

[0082] (iv) a peptide having the following amino acid sequence: VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622);

[0083] (v) a peptide having the following amino acid sequence: SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623);

[0084] (vi) NME7 protein; or

[0085] (vii) Peptide fragments of NME7 protein.

[0086] In another aspect, the invention is directed to a method of treating a disease in a subject, wherein the subject aberrantly expresses MUC1, comprising administering to a person suffering from the disease any of the above-described antibodies. The disease can be cancer, such as breast cancer, lung cancer, colon cancer, or gastric cancer.

[0087] In another aspect, the present invention is directed to a method of treating a condition in a subject, wherein the subject aberrantly expresses MUC1, comprising administering to the person suffering from the condition an NME peptide.

[0088] In another aspect, the invention is directed to a method of growing or expanding a stem cell population comprising contacting the cells with an antibody according to any of the methods or compositions described above.

[0089] In another aspect, the present invention is directed to a method for promoting stem cell attachment to a surface, comprising coating a surface with a humanized MN-C3 or MN-C8 antibody, antibody fragment thereof, or single-chain antibody thereof, and contacting stem cells with the surface.

[0090] In another aspect, the present invention is directed to a method for in vivo or ex vivo delivery of stem cells, comprising coating a surface with a humanized MN-C3 or MN-C8 antibody, antibody fragment thereof, or single-chain antibody thereof, contacting stem cells with the surface, and transporting the stem cells to a specific location.

[0091] In another aspect, the present invention is directed to a method for isolating stem cells, comprising the steps of coating a surface with a humanized MN-C3 or MN-C8 antibody, antibody fragment, or single-chain antibody thereof, contacting the surface with a mixed population of cells, and isolating the stem cells.

[0092] In another aspect, the present invention is directed to an scFv comprising a variable region fragment derived from an antibody that binds to the extracellular domain of a MUC1 isoform or cleavage product lacking the tandem repeat region. The variable region fragment can be derived from mouse monoclonal antibody MN-E6 (SEQ ID NOs: 13 and 66), humanized MN-E6 (SEQ ID NOs: 39 and 94), or MN-E6 scFv (SEQ ID NOs: 233, 235, and 237). Alternatively, the variable region fragment can be derived from mouse monoclonal antibody MN-C2 (SEQ ID NOs: 119 and 169), humanized MN-C2 (SEQ ID NOs: 145 and 195), or MN-C2 scFv (SEQ ID NOs: 239, 241, and 243). Alternatively, the variable region fragment may be derived from mouse monoclonal antibody MN-C3 (SEQ ID NOs: 414 and 459), humanized MN-C3 (SEQ ID NOs: 440 and 487), or MN-C3 scFv (SEQ ID NOs: 245, 247, and 249). Alternatively, the variable region fragment may be derived from mouse monoclonal antibody MN-C8 (SEQ ID NOs: 505 and 544), humanized MN-C8 (SEQ ID NOs: 526 and 566), or MN-C8 scFv (SEQ ID NOs: 251, 253, and 255).

[0093] In another aspect, the present invention is directed to a method of treating a human diagnosed or suspected of also having or being at risk of developing a MUC1 or MUCl* positive cancer, comprising administering an effective amount of the scFv described above.

[0094] In another aspect, the present invention is directed to an scFv-Fc construct comprising the scFv described above. The scFv-Fc may dimerize. Alternatively, the Fc component may be mutated so that the scFv-Fc is a monomer. The mutations may include mutating or deleting the hinge region of the Fc, creating F405Q, Y407R, T366W / L368W, and T364R / L368R mutations, or combinations thereof, in the Fc, as represented by SEQ ID NOs: 281, 279, 285, and 287.

[0095] In another aspect, the invention is directed to a polypeptide comprising at least two different scFv sequences, wherein one of the scFv sequences is a sequence that binds to the extracellular domain of a MUC1 isoform or cleavage product lacking the tandem repeat region. The polypeptide is capable of binding to:

[0096] (i) the PSMGFR site of MUC1;

[0097] (ii) PSMGFR peptide;

[0098] (iii) a peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620);

[0099] (iv) a peptide having the amino acid sequence VQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621);

[0100] (v) a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622); or

[0101] (vi) A peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623).

[0102] The polypeptide may bind to a receptor on an immune cell, such as a T cell, particularly CD3 on a T cell.

[0103] In another aspect, the present invention is directed to a method for detecting the presence of cells that aberrantly express MUCl*, which cells may be cancer cells, comprising contacting the above-described scFv-Fc with a sample of cells and detecting the presence of binding of the scFv-Fc to the cells.

[0104] In another aspect, the invention is directed to a method of testing a patient's cancer for suitability for treatment with a composition comprising a portion of the variable region of MN-E6, MN-C2, MN-C3, or MN-C8, comprising the step of contacting a bodily specimen from the patient with the corresponding MN-E6 scFv-Fc, MN-C3 scFv-Fc, MN-C3 scFv-Fc, or MN-C8 scFv-Fc.

[0105] In another aspect, the present invention is directed to a method of treating a patient with a disease comprising presenting T cells from the patient with MUCl* peptides, where the T cells have developed MUCl*-specific receptors through various stages of maturation, creating adapted T cells, expanding the adapted T cells, and administering them to a donor patient diagnosed or suspected of having or being at risk of developing MUCl*-positive cancer.

[0106] These and other objects of the present invention can be more fully understood from the following detailed description of the invention, the referenced drawings disclosed therein, and the claims set forth herein.

[0107] The present invention can be more fully understood from the detailed description set forth herein below and the accompanying drawings, which are given by way of illustration only. [Brief explanation of the drawings]

[0108] [Figure 1] Figures 1A-D show cell proliferation analysis graphs of MUCl*-positive cells treated with bivalent "bv" anti-MUCl* antibodies, monovalent "mv" or Fab, NM23-H1 dimer, or NME7-AB. Bivalent anti-MUCl* antibodies stimulate cancer cell proliferation, while monovalent Fab inhibits proliferation (A, B). Classic normal curves show that ligand-induced dimerization stimulates proliferation. Dimeric NM23-H1, also known as NME1, stimulates proliferation of MUCl*-positive cancer cells, while siRNA suppressing MUC1 expression abrogates this effect (C). NME7-AB also stimulates proliferation of MUC1*-positive cells (D).

[0109] [Figure 2]Figure 2A-F shows the results of ELISA assays. The MUC1* peptide PSMGFR, PSMGFR minus 10 amino acids from the N-terminus, aka N-10, or PSMGFR minus 10 amino acids from the C-terminus, aka C-10, was immobilized on a plate and subjected to the following binding assays: NME7-AB (A), MN-C2 monoclonal antibody (B), MN-E6 monoclonal antibody (C), or dimeric NME1 (D). These assays demonstrate that NME1, NME7-AB, and monoclonal antibodies MN-C2 and MN-E6 all require the first membrane-proximal 10 amino acids of the MUC1* extracellular domain for binding. The N-terminus minus 10 amino acids from the N-terminus or PSMGFR minus 10 amino acids from the C-terminus, aka C-10, were immobilized on a plate and subjected to the following binding assays: MN-C3 (E) and MN-C8 (F).

[0110] [Figure 3] Figure 3 shows the results of a competitive ELISA assay. The PSMGFR MUC1* peptide was immobilized on a plate, and the dimeric NM23-H1 (also known as NME1) was added alone or after the addition of the MN-E6 antibody (A). A similar experiment was performed in which NM23-H7 or NME7-AB was added alone or after the addition of MN-E6 (B). The results show that MN-E6 competitively inhibits the binding of the MUC1* activating ligands NME1 and NME7. In a similar experiment (C), PSMGFR or PSMGFR minus 10 amino acids from the N-terminus (also known as N-10) was immobilized on a plate. The dimeric NM23-H1 was then added. The anti-MUC1* antibodies MN-E6, MN-C2, MN-C3, or MN-C8 were then tested for their ability to competitively block NM23-H1. The results show that all three antibodies bind to the PSMGFR peptide, but MN-E6 and MN-C2 competitively inhibit the binding of the MUCl* activating ligand.

[0111] [Figure 4]Figure 4A-F shows FACS scans demonstrating that anti-MUCl* antibodies specifically bind to MUCl*-positive cancer cells and MUCl*-transformed cells, but not to non-MUCl* or MUC1-negative cells. ZR-75-1, also known as 1500, MUCl*-positive breast cancer cells were stained with 1:2 or 1:10 dilutions of 1.5 μg / ml humanized MN-C2. After two washes, cells were stained with a secondary antibody, anti-Pent-His antibody, conjugated to Alexa488 (Qiagen) at 1:200 (A), 1:50 (B), or 1:10 dilutions to detect the 6x His tag in huMN-C2 scFv. Flow cytometry analysis revealed a concentration-dependent shift in cell subsets, indicating specific binding, that was not seen in the absence of MN-C2 scFv (A-C). In other cases, MN-E6 was used to stain MUC1-negative colon cancer cells HCT-116 transfected with empty vector, single cell clone #8 (D), HCT-116 colon cancer cells transfected with MUC1* single cell clone #10 (E), or ZR-75-1, also known as 1500, MUC1*-positive breast cancer cells. As shown by FACS, both MN-E6 and MN-C2 stained only MUC1*-positive cells, but not non-MUC1 or MUC1*-negative cells.

[0112] [Figure 5] Figure 5 shows a graph of an ELISA in which a surface was coated with either the MUCl* PSMGFR peptide or a control peptide. The humanized MN-C2 scFv was then incubated with the surface, washed, and detected by standard assays. The ELISA shows that the huMN-C2 scFv binds to the MUCl* peptide with an EC-50 of approximately 333 nM.

[0113] [Figure 6]Figures 6A-B show graphs of cancer cell growth inhibition by the MUC1* antibody variable region fragment, humanized MN-C2 scFv. hMN-C2 scFv potently inhibited the growth of ZR-75-1 (aka 1500) MUC1*-positive breast cancer cells (A) and T47D MUC1*-positive breast cancer cells (B) with the same EC-50 as in vitro ELISA.

[0114] [Figure 7] Figure 7A-B shows graphs of tumor growth in immune-tolerant mice implanted with human cancer and subsequently treated with the anti-MUCl* antibody MN-E6 or mock. Female nu / nu mice implanted with estrogen pellets for 90 days were injected with 6 million T47D human breast cancer cells formulated 50 / 50 with Matrigel. Mice bearing tumors measuring at least 150 mm3 and experiencing three consecutive increases in tumor volume were selected for treatment. Animals were injected subcutaneously twice weekly with 80 mg / kg MN-E6 Fab; an equal number of mice meeting the same selection criteria were injected with vehicle alone (A). Male NOD / SCID mice were injected with 6 million DU-145 human prostate cancer cells formulated 50 / 50 with Matrigel. Mice bearing tumors measuring at least 150 mm3 and experiencing three consecutive increases in tumor volume were selected for treatment. Animals were injected subcutaneously with 160 mg / kg MN-E6 Fab every 48 hours, and an equal number of mice meeting the same selection criteria were injected with vehicle alone (B). Tumors were measured and scored twice weekly by two investigators independently. Statistics were calculated blindly by independent statisticians, with a P value of 0.0001 for each. Anti-MUCl* Fab inhibited breast and prostate tumor growth. Treatment had no effect on weight, bone marrow cell type, or number.

[0115] [Figure 8] FIG. 8 is a graph of an ELISA analysis showing different levels of expression of humanized MN-E6 anti-MUCl* antibodies depending on whether the light chain was kappa or lambda and whether the variable portion was fused to human IgG1 or IgG2.

[0116] [Figure 9] FIG. 9 is a graph of an ELISA analysis comparing binding of the parental murine MN-E6 antibody to a humanized version of the MN-E6 antibody to a surface displaying a PSMGFR peptide derived from the MUCl* extracellular domain.

[0117] [Figure 10] FIG. 10 is a graph of an ELISA analysis showing different levels of expression of humanized MN-C2 anti-MUCl* antibodies depending on whether the light chain was kappa or lambda and whether the variable portion was fused to human IgG1 or IgG2.

[0118] [Figure 11] FIG. 11 is a graph of an ELISA analysis comparing binding of the parental murine MN-C2 antibody to a humanized version of the MN-C2 antibody to a surface displaying a PSMGFR peptide derived from the MUCl* extracellular domain.

[0119] [Figure 12] FIG. 12 is a graph of an ELISA analysis showing binding of humanized single chain (scFv) MN-C2 and MN-E6 antibodies to surfaces displaying a PSMGFR peptide derived from the MUCl* extracellular domain.

[0120] [Figure 13] Figures 13A-C show FPLC traces for the purification of MN-E6 scFv-Fc fusion protein enriched in low IgG FBS on a Protein A affinity column. A) is the flow-through trace. B) is the elution trace. C) shows the purified protein on a reducing or non-reducing gel.

[0121] [Figure 14]Figure 14A-B shows photographs of SDS-PAGE profiles of purified MN-E6 scFv-Fc fusion proteins on a non-reducing gel, in which the Fc portion fused to MN-E6 was wild-type (wt) or mutated as follows: A) F405Q, Y407R, T394D; B) T366W / L368W, T364R / L368R, T366W / L368W, or T364R / L368R. The Fc mutants F405Q, Y407R, T366W / L368W, T364R / L368R, T366W / L368W, and T364R / L368R all favored the monomer over the dimer form. The reference amino acid sequence for the indicated mutations is SEQ ID NO: 273.

[0122] [Figure 15] Figure 15A-B shows FPLC traces for the purification of MN-E6 scFv-Fc Y407Q fusion protein grown in low IgG FBS on a Protein A affinity column. A) is the flow-through trace. B) is the elution trace. The protein was further purified by size exclusion on an S200 column (C). (D) is a photograph of an SDS-PAGE gel showing which fractions had a predominance of monomer. The reference amino acid sequence for the indicated mutations is SEQ ID NO: 273.

[0123] [Figure 16] Figure 16 shows a photograph of the SDS-PAGE profile of purified MN-E6 scFv-Fc mutant fusion proteins on a non-reducing gel, in which the Fc portion fused to MN-E6 scFv was wild-type (wt) or mutated by deletion of the Fc hinge region, DHige, or carried a deletion of the Fc hinge region and an additional Y407R mutation. All Fc mutants favored the monomer over the dimeric form. The reference amino acid sequence for the indicated mutations is SEQ ID NO:273.

[0124] [Figure 17]Figures 17A-C: A and B show photographs of non-reducing SDS-PAGE profiles of large-scale expression and purification of the MN-E6 scFv-Fc hingeless mutant, demonstrating that it is monomeric. FPLC profile and purification of the MN-E6 scFv-Fc hingeless mutant is shown (C).

[0125] [Figure 18] Figures 18A-C show photographs of SDS-PAGE profiles of purified MN-C3 scFv-Fc fusion protein on a non-reducing gel (A) or a reducing gel (B). The protein was purified by size exclusion. An FPLC trace is shown (C).

[0126] [Figure 19] Figure 19A-B shows photographs of native gels of MN-C3 or MN-E6 Fabs, scFv, and scFv-Fc, where the Fc portion is wild-type or a mutant that prefers monomers or is exclusively monomeric. The native gels show that the Y407R Fc mutation (A) and the double mutant Y407R with deleted hinge (B) have the greatest preference for monomers over dimers. Note that the proteins were loaded onto the gel at concentrations much higher than typical working concentrations. The dimeric forms of the other Fc mutants may simply reflect the fact that loading concentrations were so high.

[0127] [Figure 20] Figure 20 shows graphs of ELISAs in which surfaces were immobilized with PSMGFR peptide, PSMGFR minus 10 amino acids from the N-terminus, or PSMGFR minus 10 amino acids from the C-terminus. hu MN-E6 scFv-Fc binds to the PSMGFR peptide and the PSMGFR N-10 peptide, but not to the PSMGFR C-10 peptide. The parent MN-E6 antibody and humanized MN-E6 require the C-terminal 10 amino acids of PSMGFR for binding.

[0128] [Figure 21]Figure 21A-B shows ELISA graphs of several anti-MUCl* scFv-Fc fusion proteins in which the Fc region has been either removed or mutated. hu MN-E6 scFv-Fc-wt, hu MN-E6 scFv-Fc-Y407R, hu MN-E6 scFv-Fc-hingeless, and hu MN-E6 scFv-Fc Y407R-hingeless are shown. All mutants bind to the PSMGFR peptide of the MUCl* extracellular domain (A). ELISA graphs of several anti-MUCl* scFv-Fc fusion proteins in which the Fc region is wild-type or mutated are shown. hu MN-E6 scFv-Fc-wt, hu MN-E6 scFv-Fc-hingeless, and hu MN-C3 scFv-Fc are shown (B). All bind to the PSMGFR peptide of the MUCl* extracellular domain.

[0129] [Figure 22] Figures 22A-C show graphs of an ELISA in which the assay plate surface was immobilized with the PSMGFR peptide, PSMGFR minus 10 amino acids from the N-terminus, or C-terminus minus 10 amino acids. MN-C3 antibody variants were then analyzed for binding to various MUCl* peptides: A) purified mouse monoclonal MN-C3 antibody; B) impure humanized MN-C3 antibody; and C) humanized MN-C3 scFv-Fc. The ELISA shows binding to the PSMGFR peptide as well as to certain deletion peptides.

[0130] [Figure 23] FIG. 23 shows a graph of an ELISA analysis quantifying binding of humanized MN-E6 scFv-Fc delta hinge, akAD hinge or hingeless, and humanized MN-E6 scFv to the MUCl* peptide PSMGFR.

[0131] [Figure 24]Figure 24 shows photographs of immunofluorescence experiments in which humanized MN-C2 scFv or MN-E6 scFv specifically bind to MUCl*-positive breast cancer cells in the same concentration-dependent manner. A-G: hu MN-C2 scFv binding to T47D breast cancer cells at the indicated concentrations. H-N: fluorescently labeled scFv and DAPI. O-U: hu MN-E6 scFv binding to T47D breast cancer cells at the indicated concentrations. V-B': fluorescently labeled scFv and DAPI. C' is a secondary antibody control.

[0132] [Figure 25] Figure 25A-L shows photographs of 1500 MUCl*-positive breast cancer cells cultured in normal medium or in the presence of humanized MN-E6 scFv. A-D are bright-field images obtained at 4X magnification. E-H are calcein fluorescence images obtained at 4X magnification. I-L are calcein fluorescence images obtained at 10X magnification. A, E, and I show control cells cultured in normal RPMI medium. B, F, and J show control cells cultured in normal RPMI medium plus a volume of PBS equal to the amount of MN-E6 scFv in PBS added to experimental wells. C, G, and K show cells cultured in normal RPMI medium plus 500 μg / mL MN-E6 scFv. D, H, and L show cells cultured in normal RPMI medium plus 5 μg / mL MN-E6 scFv. The photographs show the killing and / or growth inhibition of MUCl* positive cells by MN-E6 scFv at 500 μg / mL and at 5 μg / mL with greater efficacy. Images were obtained 96 hours post addition of the test molecules.

[0133] [Figure 26]Figure 26, A-L, shows photographs of 1500 MUCl*-positive breast cancer cells cultured in normal medium or in the presence of humanized MN-E6 scFv-Fc D-hinge, either hingeless or delta-hinge mutant. A-F are bright-field images obtained at 20X magnification. G-L are calcein fluorescence images obtained at 4X magnification. A and G show control cells cultured in normal RPMI medium. B and H show cells cultured in normal RPMI medium plus 100 μg / mL hMN-E6 scFv-Fc D-hinge. C and I show cells cultured in normal RPMI medium plus 50 μg / mL hMN-E6 scFv-Fc D-hinge. D and J show cells cultured in normal RPMI medium plus 5 μg / mL hMN-E6 scFv-Fc D-hinge. E and K show cells cultured in normal RPMI medium supplemented with 0.5 μg / mL hMN-E6 scFv-Fc D-hinge. F and L show cells cultured in normal RPMI medium supplemented with 500 μg / mL MN-E6 Fab. Photographs show the killing and / or growth inhibition of MUCl*-positive cells by hMN-E6 scFv-Fc D-hinge at 100 μg / mL, 50 μg / mL, and 5 μg / mL, respectively. Compared to control cells, cancer cells grown with MN-E6 Fab or an effective dose of hMN-E6 scFv-Fc D-hinge exhibit cell rounding, a morphological change that occurs before cell death. Images were obtained 96 hours after addition of the test molecules.

[0134] [Figure 27] Figure 27 shows a graph of the image analysis of the fluorescence images of Figures 25 and 26. Image J was used to calculate the number of cells remaining after 96 hours of treatment with humanized MN-E6 scFv or MN-E6 scFv-Fc delta-hinge, akA D-hinge. The analysis software uses pixel fluorescence intensity and pixel counts to calculate the amount of cells in each photograph. Analysis was performed on the entire image, a 512x512 pixel, 8-bit image. For comparison, the inhibition of murine monoclonal MN-E6 Fab was also analyzed.

[0135] [Figure 28] Figures 28A-C show a schematic of the CAR sequence components.

[0136] [Figure 29] Figure 29 is a graph of an experiment measuring IL-2 cytokine secretion by urkat T cells transduced with a panel of CARs including MN-E6-CD8-3z, MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, MN-E6-CD4-CD28-3z, and MN-E6-CD4-CD28-41BB-3z when the CAR T cells were exposed to K562-wt cells or K562 cells transfected with MUCl*.

[0137] [Figure 30] Figure 30 is a graph of an experiment measuring IL-2 cytokine secretion by Jurkat T cells transduced with a panel of CARs including MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, MN-E6-CD4-CD28-3z, and MN-E6-CD4-41BB-3z when the CAR T cells were exposed to K562-wt cells or K562 cells transfected with MUCl*.

[0138] [Figure 31] Figure 31 is a graph of an experiment measuring IL-2 cytokine secretion by primary human T cells, isolated from human blood, transduced with a panel of CARs including MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, and MN-E6-CD4-41BB-3z when the CAR T cells were exposed to K562-wt cells or K562 cells transfected with MUCl*.

[0139] [Figure 32]Figure 32 is a graph of an experiment measuring interferon gamma (IFNg) secretion by primary human T cells, isolated from human blood, transduced with a panel of CARs including MN-E6-CD8-CD28-3z and MN-E6-CD4-41BB-3z when the CAR T cells were exposed to K562-wt cells or K562 cells transfected with MUCl*.

[0140] [Figure 33] Figure 33 is a graph of an experiment measuring interferon gamma (IFNg) secretion by primary human T cells, isolated from human blood, transduced with a panel of CARs including MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, and MN-E6-CD8-CD28-41BB-3z when the CAR T cells were exposed to K562-wt cells, K562 cells transfected with MUCl*, or MUCl*-positive cancers of the prostate, breast, or pancreas.

[0141] [Figure 34] Figure 34 is a graph of an experiment measuring target cell killing in primary human T cells, isolated from human blood, transduced with a panel of CARs including MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, and MN-E6-CD4-41BB-3z when the CAR T cells were exposed to K562-wt cells or K562 cells transfected with MUCl*. The T cell to target cell ratio was 1:1, and the cells were co-cultured for 24 hours.

[0142] [Figure 35]Figure 35A-B are FACS graphs measuring the time course of target cell survival from day 1 to day 3. Primary human T cells isolated from blood samples were transduced with a panel of CARs, including humanized MN-E6-CD8-3z, MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. CAR T cells were then exposed to K562-wt cells, which naturally express low levels of MUCl*, or K562 cells transfected with MUCl* high. The ratio of MUCl* targeting CAR T cells to target cells was 1:1, 10:1, or 20:1. Viable cells were detected and measured on day 1 (A) or day 3 (B).

[0143] [Figure 36] Figure 36 is a graph of FACS measurements of target cell survival on day 3 of a coculture experiment. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-3z, MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. The CAR T cells were then exposed to MUCl*-positive T47D breast cancer cells or MUCl*-positive 1500 aka ZR-75-1 breast cancer cells. The ratio of MUCl*-targeting CAR T cells to target cells was 1:1 or 10:1. As can be seen from the graph, T cells transduced with MUCl*-targeting CARs have a much greater killing effect on MUCl* cancer cells than untransduced control T cells. Furthermore, the killing effect is further increased when the T cell:target cell ratio is increased.

[0144] [Figure 37]Figure 37 is a graph of FACS measurements of target cell survival on day 1 of the coculture experiment. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-41BB-3z, MN-E6-CD4-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. The CAR T cells were then exposed to the following MUCl*-positive cancer cells: T47D breast cancer; capan2 pancreatic cancer; or DU-145 prostate cancer. The ratio of MUCl*-targeting CAR T cells to target cells was 5:1. As can be seen from the graph, T cells transduced with MUCl*-targeting CARs have a much greater killing effect on MUCl* cancer cells than untransduced control T cells. Note that measurements were performed after 24 hours at a 5:1 (T cell to target cell) ratio. Furthermore, it is noted that a MUCl* targeting CAR with a CD4 extracellular domain-transmembrane-cytoplasmic tail, fully equivalent to the CD8 construct, also works.

[0145] [Figure 38]Figure 38 is a graph of FACS measurements of target cell survival on day 3 of a coculture experiment. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-41BB-3z, MN-E6-CD4-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. CAR T cells were then exposed to the following MUCl*-positive cancer cells: MUCl*-transfected K562 leukemia cells; T47D breast cancer; 1500 aka ZR-75-1 breast cancer cells; or CAPAN-2 pancreatic cancer cells. In addition to untransduced T cell controls, analysis was also performed with PC3 MUCl*-negative prostate cancer cells. The ratio of MUCl*-targeting CAR T cells to target cells was 1:1. As can be seen from the graph, T cells transduced with MUCl* targeting CARs have a much greater killing effect on MUCl* cancer cells than untransduced control T cells. Furthermore, the killing is specific to MUCl* positive cells. Note that MUCl* targeting CARs with CD4 extracellular domain-transmembrane-cytoplasmic tails work equally well as CD8 constructs.

[0146] [Figure 39] Figure 39 is a graph of FACS measurements of CAR T cell proliferation over 24 hours in co-culture with target cells at a 5:1 ratio of CAR T cells to target cells. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-41BB-3z, MN-E6-CD4-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. CAR T cells were co-cultured with MUC1*-positive T47D breast cancer cells, MUC1*-positive Capan pancreatic cancer cells, and MUC1-negative HCT-116 colon cancer cells and HEK-293 human embryonic kidney cells. As can be seen from the graph, the CAR T population is expanded in the presence of MUC1*-positive cells.

[0147] [Figure 40]Figure 40 shows a photograph of a Western blot of MUCl* targeting CARs. 1 to 9 are as follows: 1. E6scFv-Fc-8-41BB-CD3z (human Fc as hinge region with CD8 TM); 2: E6scFv-FcH-8-41BB-CD3z (human Fc as hinge region with CD8 TM, hingeless); 3: E6scFv-Fc-4-41BB-CD3z (human Fc as hinge region with CD4 TM); 4: E6scFv-FcH-4-41BB-CD3z (human Fc as hingeless hinge region with CD4 TM); 5: E6scFv-IgD-8-41BB-CD3z (hinge region from human IgD with CD8 TM); 6: E6scFv-IgD-4-41BB-CD3z (CD4 (hinge region from human IgD with TM) 7: E6scFv-X4-8-41BB-CD3z (long flexible linker as hinge region with CD8 TM) 8: E6scFv-X4-4-41BB-CD3z (long flexible linker as hinge region with CD4 TM) 9: E6scFv-8-4-41BB-CD3z (hinge region from CD8 and CD4 with CD4 TM).

[0148] [Figure 41] Figure 41 shows a graph of a FACS scan of T47D breast cancer cells co-cultured with human T cells transduced with MN-E6scFv-Fc-8-41BB-CD3z, MN-E6scFv-FcH-8-41BB-CD3z, MN-E6scFv-Fc-4-41BB-CD3z, MN-E6scFv-IgD-8-41BB-CD3z, MN-E6scFv-X4-8-41BB-CD3z, and MN-E6scFv-X4-4-41BB-CD3z. T cells and cancer cells were co-cultured at a 1:1 ratio for 48 hours. T cell counts were normalized to the average of all untransduced T cells, and target cells were normalized to each specific cell type when co-cultured with untransduced T cells. The graph shows that when CAR T cells are co-cultured with MUCl*-positive cancer cells, the T cell population expands and the targeted cancer cell population decreases.

[0149] [Figure 42] Figure 42 shows graphs of FACS scans of T47D breast cancer cells, Capan-2 pancreatic cancer cells, K562-MUC1* transfected cells, and K562-wt cells co-cultured with human T cells transduced with MN-E6scFv-Fc-8-41BB-CD3z, MN-E6scFv-FcH-8-41BB-CD3z, MN-E6scFv-Fc-4-41BB-CD3z, MN-E6scFv-IgD-8-41BB-CD3z, MN-E6scFv-X4-8-41BB-CD3z, and MN-E6scFv-X4-4-41BB-CD3z. T cells and cancer cells were co-cultured at a 1:1 ratio for 48 hours. T cell counts were normalized to the average of all untransduced T cells. Target cell counts were also normalized to individual specific cell types when co-cultured with untransduced T cells. T cell counts were normalized to the average of all untransduced T cells, and target cells were normalized to each specific cell type when co-cultured with untransduced T cells. The graph shows that when CAR T cells are co-cultured with MUCl*-positive cancer cells, the T cell population expands and the targeted cancer cell population decreases.

[0150] [Figure 43] Figure 43 A and B are photographs of breast cancer tissue arrays. A) Stained with VU4H5, which recognizes MUC1-FL (full-length); B) Stained with MN-C2, a mouse monoclonal antibody that recognizes cancerous MUC1*. By automated staining (Clarient Diagnostics), tissue staining was scored using the Allred scoring method, which combines intensity and distribution scores. C, D, E, and F are color-coded graphs showing the scores calculated for MUC1 full-length staining in each patient's tissue. G, H, I, and J are color-coded graphs showing the scores calculated for MUC1* staining in each patient's tissue.

[0151] [Figure 44]Figure 44 A and B are photographs of breast cancer tissue arrays. A) Stained with VU4H5, which recognizes MUC1-FL (full-length); B) Stained with MN-C2, a mouse monoclonal antibody that recognizes cancerous MUC1*. By automated staining (Clarient Diagnostics), tissue staining was scored using the Allred scoring method, which combines intensity and distribution scores. C, D, E, and F are color-coded graphs showing the scores calculated for MUC1 full-length staining in each patient's tissue. G, H, I, and J are color-coded graphs showing the scores calculated for MUC1* staining in each patient's tissue.

[0152] [Figure 45] Figure 45 shows photographs of breast cancer tissue and normal breast tissue stained with 2.5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody followed by a secondary streptavidin HRP antibody. A) Normal breast tissue. B) Breast cancer tissue from the patient as indicated in the figure. E) H) Photographs of corresponding serial sections stained with the secondary antibody alone.

[0153] [Figure 46] Photographs of breast cancer tissue and normal breast tissue stained with 2.5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody followed by secondary streptavidin HRP antibody are shown. A) Normal breast tissue. BC) Breast cancer tissue from the patient as indicated in the figure. DF) Photographs of corresponding serial sections stained with secondary antibody alone.

[0154] [Figure 47] Figure 47 shows photographs of breast cancer tissues stained with 10 μg / mL of MN-E6 anti-MUCl* antibody and then with a rabbit anti-mouse secondary HRP antibody. A-D are breast cancer tissues from patient #300. E-H are breast cancer tissues from metastatic cancer patient #291.

[0155] [Figure 48]Figure 48 shows photographs of lung cancer tissue and normal lung stained with 2.5 μg / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody followed by secondary streptavidin HRP antibody. A) Normal lung tissue. B and C) Lung cancer tissue from the patient indicated in the figure. D-F) Photographs of corresponding serial sections stained with secondary antibody alone.

[0156] [Figure 49] Figure 49 shows photographs of lung cancer tissue and normal lung stained with 2.5 μg / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody followed by secondary streptavidin HRP antibody. A) Normal lung tissue. B and C) Lung cancer tissue from the patient indicated in the figure. D-F) Photographs of corresponding serial sections stained with secondary antibody alone.

[0157] [Figure 50] Figure 50 shows photographs of lung cancer tissue and normal lung stained with 25 μg / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody followed by secondary streptavidin HRP antibody. A) Normal lung tissue. B and C) Lung cancer tissue from the patient indicated in the figure. D-F) Photographs of corresponding serial sections stained with secondary antibody alone.

[0158] [Figure 51] Figure 51 shows photographs of lung cancer tissue and normal lung stained with 25 μg / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody followed by secondary streptavidin HRP antibody. A) Normal lung tissue. B and C) Lung cancer tissue from the patient indicated in the figure. D-F) Photographs of corresponding serial sections stained with secondary antibody alone.

[0159] [Figure 52]Figure 52 shows photographs of small intestinal cancer tissue and normal small intestine stained with 5 μg / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody and then stained with a secondary streptavidin HRP antibody. A) Normal small intestinal tissue. B) Small intestinal cancer tissue from the patient indicated in the figure. C and D are photographs of corresponding serial sections stained with the secondary antibody alone.

[0160] [Figure 53] Figure 53 shows photographs of small intestinal cancer tissue and normal small intestine stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody and then stained with a secondary goat anti-human HRP antibody. A to D are normal small intestinal tissues. E to H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0161] [Figure 54] Figure 54 shows photographs of small intestinal cancer tissue and normal small intestine stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody and then stained with a secondary goat anti-human HRP antibody. A-D are small intestinal cancer tissues from the patients indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0162] [Figure 55] Figure 55 shows photographs of small intestine cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are small intestine cancer tissues from the patients indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0163] [Figure 56] Figure 56 shows photographs of normal colon tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by secondary goat anti-human HRP antibody. A-D are normal colon tissues. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0164] [Figure 57] Figure 57 shows photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are colon cancer tissues from patients with metastatic cancer as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0165] [Figure 58] Figure 58 shows photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are colon cancer tissues from patients with grade 2 cancer as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0166] [Figure 59] Figure 59 shows photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by secondary goat anti-human HRP antibody. A-D are colon cancer tissues from patients with metastatic cancer as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0167] [Figure 60] Figure 60 shows photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0168] [Figure 61]Figure 61 shows photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0169] [Figure 62] Figure 62 shows photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. DETAILED DESCRIPTION OF THE INVENTION

[0170] In the present invention, "a" and "an" are used to refer to both singular and plural objects.

[0171] As used herein, "h" or "hu" preceding an antibody construct is an abbreviation for humanized.

[0172] As used herein, the term "antibody-like" refers to an engineered molecule that contains antibody portions but is not a naturally occurring antibody. Examples include, but are not limited to, CAR (chimeric antigen receptor) T cell technology and Ylanthia™ technology. CAR technology uses antibody epitopes fused to portions of T cells, thereby directing the body's immune system to attack specific target proteins or cells. Ylanthia™ technology consists of an antibody-like library, a collection of synthetic human Fabs, which are screened for binding to peptide epitopes from target proteins. Selected Fab portions are engineered into a scaffold or framework so that they resemble antibodies.

[0173] As used herein, the antibodies MN-C2, MN-E6, MN-C3, and MN-C8 are also referred to as C2, E6, C3, and C8, respectively.

[0174] As used herein, "PSMGFR" is an abbreviation for the primary sequence of the MUC1 growth factor receptor identified by SEQ ID NO:2, and thus is not to be confused with the six amino acid sequence. A "PSMGFR peptide" or "PSMGFR portion" refers to a peptide or portion that includes the primary sequence of the MUC1 growth factor receptor (SEQ ID NO:2).

[0175] As used herein, the "MUCl*" extracellular domain is defined primarily by the PSMGFR sequence (GTINVHD VETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2)). The exact site of MUC1 cleavage depends on the enzyme that excises it, and the exact sequence of the MUCl* extracellular domain can vary at the N-terminus because the cleavage enzyme varies depending on the cell type, tissue type, or time in the evolution of the cell.

[0176] Other truncated amino acid sequences may include: SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621).

[0177] As used herein, the term "PSMGFR" is an acronym for the primary sequence of the MUC1 growth factor receptor, set forth as GTINVHDVETQFNQYKTE AASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2). In this regard, the "N-number," as in "N-10 PSMGFR," "N-15 PSMGFR," or "N-20 PSMGFR," refers to the number of amino acid residues deleted at the N-terminus of PSMGFR. Similarly, the "C-number," as in "C-10 PSMGFR," "C-15 PSMGFR," or "C-20 PSMGFR," refers to the number of amino acid residues deleted at the C-terminus of PSMGFR.

[0178] As used herein, the MUCl* extracellular domain refers to the extracellular portion of the MUC1 protein that lacks the tandem repeat region. In most cases, MUCl* is a cleavage product, in which the MUCl* portion consists of a short extracellular domain that lacks the tandem repeats, transmembrane region, and cytoplasmic tail. The exact location of the cleavage site in MUC1 is unknown, as it is likely that it can be cleaved by more than one enzyme. The MUCl* extracellular domain contains most of the PSMGFR sequence, but may also contain an additional 10-20 N-terminal amino acids.

[0179] As used herein, "sequence identity" means homology of a particular polypeptide or nucleic acid sequence to a reference sequence of nucleic acids or amino acids, such that the function of the homologous peptide is the same as the reference peptide or nucleic acid. Such homology is so close to the reference peptide that the two sequences are 90%, 95%, or 98% identical and may still possess the same function in binding or other biological activity.

[0180] MUC1* antibodies (anti-PSMGFR) for the treatment or prevention of cancer

[0181] The present inventors have discovered that a cleaved form of the MUC1 (SEQ ID NO: 1) transmembrane protein is a growth factor receptor that drives the growth of more than 75% of all human cancers. The cleaved form of MUC1, which the inventors call MUCl* (MUC1-star), is a potent growth factor receptor. Enzymatic cleavage releases a large portion of the MUC1 extracellular domain. This remnant, including the truncated extracellular domain, transmembrane, and cytoplasmic tail, is called MUCl*. Cleavage and release of the large portion of the MUC1 extracellular domain exposes binding sites for activating the ligand dimers NME1, NME6, NME8, NME7-AB, or NME7. Cell proliferation assays indicate that it is the ligand-induced dimerization of the MUCl* extracellular domain that promotes proliferation (Figure 1A-D). MUC1*-positive cells were treated with bivalent "bv" anti-MUC1* antibodies, monovalent "mv" or Fab, NM23-H1 dimer, or NME7-AB. Bivalent anti-MUC1* antibodies stimulate cancer cell proliferation, while monovalent Fab inhibits proliferation. Classic normal curves show that ligand-induced dimerization stimulates proliferation. Dimeric NM23-H1, also known as NME1, stimulates proliferation of MUC1*-positive cancer cells, whereas siRNA suppressing MUC1 expression abrogates the effect (C). NME7-AB also stimulates proliferation of MUC1*-positive cells (D).

[0182] MUC1* is aberrantly expressed in over 75% of all cancers and is likely overexpressed in an even higher proportion of metastatic cancers, making it an ideal target for anticancer drugs (Fessler SP, Wotkowicz MT, Mahanta SK and Bamdad C. (2009)). MUC1* is a determinant of trastuzumab (Herceptin) resistance in breast cancer cells (Breast Cancer Res Treat. 118(1): 113-124). After MUC1 cleavage, most of its extracellular domain is released from the cell surface. The remaining portion contains a truncated extracellular domain that contains at least the primary growth factor receptor sequence, PSMGFR (SEQ ID NO: 2). Antibodies that competitively inhibit the binding of activating ligands, particularly NME proteins including NME1, NME6, NME8, and NME7, that bind to the PSMGFR sequence are ideal therapeutic agents and may be used as unique antibodies, antibody fragments or variable region fragments thereof incorporated into bispecific antibodies, or chimeric antigen receptors, also known as CARs, to treat or prevent MUC1- or MUC1*-positive cancers. Therapeutic anti-MUCl* antibodies can be monoclonal, polyclonal, antibody mimics, engineered antibody-like molecules, whole antibodies, or antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, scFv, and scFv-Fc. Human or humanized antibodies are preferred for use in cancer treatment or prevention. In these antibody-like molecules, mutations can be introduced to prevent or minimize dimer formation. Because MUC1* function is activated by ligand-induced dimerization, monovalent or bispecific anti-MUCl* antibodies are preferred. Representative binding assays show that NME1 and NME7 bind to the PSMGFR peptide portion of MUCl* (Fig. 2A–D). Furthermore, they demonstrate that these activated growth factors bind to the membrane-proximal portion of MUCl*, since they do not bind to the PSMGFR peptide if the 10 C-terminal amino acids are deleted. Similarly, the anti-MUCl* antibodies MN-C2 and MN-E6 bind to the PSMGFR peptide only when the 10 C-terminal amino acids are present (Fig. 2B, C).The antibodies, MN-C3 and MN-C8, bind to a distinct epitope from MN-C2 and MN-E6, and they are independent of the presence of the 10 C-terminal amino acids of the PSMGFR peptide (Figure 2E, F). As unique antibodies or incorporated into chimeric antigen receptors expressed by immune cells, also known as bispecific antibodies, BiTEs, or CARs, the antibodies, MN-C2, MN-E6, MN-C3, or MN-C8, or fragments derived from them, are all potential cancer therapeutics.

[0183] Therapeutic anti-MUCl* antibodies to be used as stand-alone antibody therapeutics or for incorporation into BiTEs or CARs can be selected based on criteria of specificity. Parent antibodies can be generated using typical methods for generating monoclonal antibodies in animals. Alternatively, they can be selected by screening antibody and antibody fragment libraries for the ability to bind to the MUCl* peptide, which can be the PSMGFR peptide (SEQ ID NO: 2), SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621).

[0184] The resulting antibodies or antibody fragments thus generated or selected can then be further selected by additional screening. For example, antibodies or antibody fragments may be more suitable based on their ability to bind to MUCl*-positive cancer cells or tissues but not to MUC1-negative cancer cells or tissues. Furthermore, anti-MUCl* antibodies or antibody fragments can be screened as anti-cancer therapeutics if they bind to stem or progenitor cells. Anti-MUCl* antibodies or antibody fragments may be more suitable if they have the ability to competitively inhibit the binding of activating ligands to MUCl*. 3A-C show that MN-E6 and MN-C2 competitively inhibit the binding of activating ligands NME1 and NME7 to MUCl*. The selection process for anti-MUCl* antibodies to be used in the treatment of patients diagnosed with MUCl-positive cancer and those at risk for or suspected of having MUCl-positive cancer involves one or more of the following steps to select antibodies or antibody fragments: 1) binding to the PSMGFR peptide; 2) binding to the N-10 PSMGFR peptide; 3) binding to cancer cells; 4) not binding to stem or progenitor cells; and 5) competitively inhibiting the binding of dimeric NME1 or NME7-AB to the PSMGFR peptide. For example, Figures 3A–C show that monoclonal antibodies MN-E6 and MN-C2 fulfill all five criteria, while monoclonal antibodies MN-C3 and MN-C8 do not competitively inhibit the binding of activating ligands NME1 and NME7 (Figure 3C). However, antibodies or antibody fragments derived from MN-C3 and MN-C8 are equally potent anticancer agents when integrated into BiTEs or CARs, because in these approaches, immune cell killing outweighs the ability to inhibit activating ligand binding. Furthermore, toxic agents conjugated to MN-E6, MN-C2, MN-C3, or MN-C8 are potent cancer therapeutics. It is important to note that the MUCl* growth factor receptor is activated by ligand-induced dimerization of its extracellular domain. Therefore, an ideal therapeutic antibody should not dimerize the MUCl* extracellular domain. Rather, suitable antibodies in this regard include monovalent antibodies such as those generated in llamas and camels, Fabs, scFvs, single domain antibodies (sdAbs), and scFv-Fc, so long as the Fc portion is constructed so that it does not homodimerize.

[0185] FACS scans show that the anti-MUCl* antibodies MN-C2, MN-E6, MN-C3, and MN-C8 bind to MUCl*-positive cancer cells and MUCl*-transfected cells but not to non-MUCl* or MUCl*-negative cells. In one example, humanized MN-C2 scFv was shown to bind to ZR-75-1, also known as 1500, MUCl*-positive breast cancer cells (Figure 4A-C). MN-E6 was shown to bind to MUCl*-negative HCT-116 colon cancer cells only when they were transfected with MUCl*. MN-E6 also binds to MUCl*-positive cancer cells, such as ZR-75-1, also known as 1500, MUCl*-positive breast cancer cells (Figure 4D-F). Binding assays such as ELISA and immunofluorescence all demonstrate that MN-C2 and MN-E6 bind to PSMGFR peptides and live MUCl-positive cancer cells. Humanized anti-MUCl* antibodies were also selected based on their ability to bind to the PSMGFR peptide or MUC1-positive cancer cells. Figure 5 shows that humanized MN-C2 scFv binds with high affinity to the MUC1* peptide PSMGFR with an EC-50 of approximately 333 nM. Like the Fab, humanized MN-C2 scFv potently inhibits the proliferation of MUC1*-positive cancer cells, as shown in an example in Figure 6A and B.

[0186] The Fabs of MN-E6 and MN-C2, or comparable single-chain variable regions derived from them, potently inhibit the growth of MUCl*-positive cancers in vivo and in vitro. In some instances, the Fabs of anti-MUCl* antibodies inhibited the growth of human MUCl*-positive cancers in vivo. In one case, immune-compromised mice were implanted with human breast cancer and treated with Fab MN-E6 after implantation. Figure 7A shows that FabMN-E6 potently inhibited the growth of MUCl*-positive breast cancer. Female Nu / Nu mice, injected with estrogen pellets for 90 days, were implanted with 6 million T47D human breast cancer cells formulated 50 / 50 in Matrigel. Tumor-bearing mice with three consecutive increases in tumor volume of at least 150 mm3 were selected for treatment. Animals were injected subcutaneously twice weekly with 80 mg / kg MN-E6 Fab; an equal number of mice meeting the same selection criteria were injected with vehicle alone (A). In another study, MN-E6 was shown to halt prostate cancer growth. Figure 7B shows that MN-E6Fab potently inhibited the growth of MUCl*-positive prostate cancer. Male NOD / SCID mice were injected with 6 million DU-145 human prostate cancer cells formulated 50 / 50 in Matrigel. Tumor-bearing mice with three consecutive increases in tumor volume of at least 150 mm3 were selected for treatment. Animals were injected subcutaneously with 160 mg / kg MN-E6Fab every 48 hours, and an equal number of mice meeting the same selection criteria were injected with vehicle alone (B). Tumors were measured and scored twice weekly by two investigators independently. Statistics were calculated blindly by independent statisticians, each giving a P value of 0.0001. Anti-MUCl* Fab inhibited breast and prostate cancer growth. Treatment had no effect on weight, bone marrow cell type, or number. FabMN-E6 effectively inhibited tumor growth, while control tumors continued to grow until sacrifice. No side effects of treatment were observed or detected.

[0187] Like Fab, recombinant MN-E6 was constructed as a monomer. In this case, MN-E6 was humanized. There are many methods for antibody humanization known to those skilled in the art. In addition to humanization, human antibody libraries can be screened to identify other fully human antibodies that bind to PSMGFR. A single-chain humanized MN-E6 variable region, called an scFv, was genetically engineered and attached to the Fc portion of the antibody (SEQ ID NOs: 256 and 257). The Fc portion confers certain benefits to antibody fragments for therapeutic use. The Fc portion of an antibody recruits complement and, in a general sense, recruits other aspects of the immune system, thus amplifying the anti-tumor response beyond simply inhibiting the target. The addition of an Fc portion further increases the half-life of antibody fragments (Czajkowsky DM, Hu J, Shao Z and Pleass RJ. (2012) Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 4(10): 1015-1028). However, the Fc portion of antibodies homodimerizes, which is suboptimal in the case of therapeutic anti-MUCl* antibodies, since ligand-induced dimerization of the MUCl* receptor stimulates proliferation. As can be seen in Figure 13B, humanized MN-E6scFv-Fc is a dimer, in part due to disulfide bonds. Therefore, mutations in the Fc region that prevent dimerization are suitable for anti-MUCl* anti-cancer therapeutics. Deletions of the hinge region (hingeless is also called delta hinge or Dhinge in some figures and examples, SEQ ID NOs: 288 and 289) and other mutations in the Fc region were made that result in dimerization-resistant Fc mutants. To prepare monomeric scFv-Fc fusion proteins, the following mutations were made in the CH3 region: Y407R (SEQ ID NOs: 278 and 279), F405Q (SEQ ID NOs: 280 and 281), T394D (SEQ ID NOs: 282 and 283), T366W / L368W (SEQ ID NOs: 284 and 285), T364R / L368R (SE ID NOs: 286 and 287). Figure 14 shows a photograph of the SDS-PAGE profile of purified MN-E6 scFv-Fc fusion protein in a non-reducing gel, in which the Fc portion fused to MN-E6 was wild-type (wt) or mutated as follows: A) F405Q, Y407R, T394D; B) T366W / L368W, T364R / L368R, T366W / L368W, or T364R / L368R. The Fc mutants F405Q, Y407R, T366W / L368W, T364R / L368R, T366W / L368W, and T364R / L368R all favored the monomer over the dimeric form. Figure 15 shows FPLC traces of the purification of MN-E6 scFv-Fc Y407Q fusion protein grown in low-IgG FBS on a Protein A affinity column. A) is the flow-through trace. B) is the elution trace. The protein was further purified by size exclusion on an S200 column (C). (D) is a photograph of an SDS-PAGE gel showing which fractions had a predominance of monomer. Figure 16 shows a photograph of the SDS-PAGE profile of purified MN-E6 scFv-Fc mutant fusion proteins on a non-reducing gel, in which the Fc portion fused to MN-E6 scFv was wild-type (wt) or mutated by deletion of the Fc hinge region ("DHinge") or deletion of the Fc hinge region and carrying the Y407R mutation. All Fc mutants favored the monomer over the dimeric form. The reference construct amino acid sequence for the indicated mutations is SEQ ID NO: 273. Other suitable sequences are SEQ ID NOs: 289 and 279. Figures 17A-C. A and B show photographs of non-reducing SDS-PAGE profiles of large-scale expression and purification of the MN-E6 scFv-Fc hingeless mutant, demonstrating that it is a monomer. FPLC profile and purification of the MN-E6 scFv-Fc hingeless mutant is shown in (C). Figures 18A-C show photographs of SDS-PAGE profiles of purified MN-C3 scFv-Fc fusion protein on a non-reducing gel (A) or a reducing gel (B). The protein was purified by size exclusion. The FPLC trace is shown in (C). Figure 19A-B shows photographs of native gels of MN-C3 or MN-E6 Fab, scFv, and scFv-Fc, where the Fc region is wild-type or a mutant that prefers or is exclusively monomeric. The native gel also favors monomeric versus dimeric forms, with the Y407R Fc mutation (A) and the double mutant Y407R and deleted hinge (B) being the best. Note that the proteins were loaded onto the gel at concentrations much higher than typical working concentrations. The dimeric forms of the other Fc mutants may simply reflect the fact that loading concentrations were so high.

[0188] Some mutations or deletions are so effective that they resist dimer formation, even when loaded onto a gel at high concentrations (Figure 14A, B). The Y407R mutation results in an almost pure population of dimeric scFv-Fc (Figure 10). Similarly, deletion of the hinge region of the Fc results in a fusion protein that is monomeric rather than dimeric. Combinations of mutations can result in more effective resistance to dimer formation (Figures 16 and 17). These and other mutations and their combinations were introduced into CH2-CH3 (SEQ ID NOS: 274 and 275) and CH3 (SEQ ID NOS: 276 and 277) fusion proteins such as scFvs, or into hingeless Fc fusion proteins such as scFvs, and were shown to eliminate or minimize dimerization.

[0189] Like the parental murine monoclonal antibody, human or humanized antibodies, as well as single-chain constructs scFv, scFv-Fc fusions, or scFv-Fc mutants, specifically bind the synthetic MUC1* peptide (Figures 20-22). Figure E23 shows a graph of an ELISA analysis quantifying binding of humanized MN-E6 scFv-Fc delta-hinge, akA Dhinge, or hingeless, humanized MN-E6 scFv to the MUC1* peptide PSMGFR.

[0190] The human or humanized anti-MUCl* antibody fragments described herein specifically bind to MUC1- and MUCl*-positive cancer cells. Figure 24 shows photographs of immunofluorescence experiments in which humanized MN-C2 scFv or MN-E6 scFv specifically bind to MUCl*-positive breast cancer cells in the same concentration-dependent manner. AG: hu MN-C2 scFv binding to T47D breast cancer cells at the indicated concentrations. HN: fluorescently labeled scFv and DAPI. OU: hu MN-E6 scFv binding to T47D breast cancer cells at the indicated concentrations. V-B': fluorescently labeled scFv and DAPI. C': secondary antibody control.

[0191] In addition to binding to MUCl*-positive cancer cells, the anti-MUCl* antibody variable region fragments, scFv', scFv-Fc', and scFv-Fc mutants inhibited the proliferation of MUCl*-positive cancer cells. Figures 25A-L show photographs of MUCl*-positive breast cancer cells cultured in normal medium or in the presence of humanized MN-E6 scFv. The photographs demonstrate killing and / or growth inhibition of MUCl*-positive cells by MN-E6 scFv, with greater effect at 5 μg / mL and 500 μg / mL. Figures 26A-L show photographs of MUCl*-positive breast cancer cells cultured in the presence of normal medium or humanized MN-E6 scFv Dhinge, either hingeless or delta-hinge mutant. The photographs demonstrate killing and / or growth inhibition of MUCl*-positive cells by MN-E6 scFv Dhinge at 5 μg / mL and with greater effect at 50 μg / mL and with even greater effect at 100 μg / mL. Figure 27 shows a graph of the image analysis of the fluorescence images of Figures 25 and 26. Image J was used to quantify the number of cells remaining after 96 hours of treatment with humanized MN-E6 scFv or MN-E6 scFv-Fc delta hinge, akA Dhinge. The analysis software uses pixel counting and pixel fluorescence intensity to quantify the number of cells in each photograph. Analysis was performed on the entire image, 512x512 pixels, 8-bit. For comparison, the inhibition of the murine monoclonal MN-E6Fab was also analyzed.

[0192] These data demonstrate that human or humanized MN-E6 antibody or antibody fragment, Fab, MN-E6 scFv, or hu MN-E6 scFv-Fcmut are effective anticancer agents that can be administered to individuals diagnosed with, suspected of having, or at risk for developing MUCl- or MUCl*-positive cancer.

[0193] In each of these examples, the dimer-resistant Fc fused to the antibody fragment or scFv is the hu MN-E7 scFv. However, any of these Fc site mutations, or combinations thereof, that eliminate or minimize dimerization can be fused to variable fragments or single-chain constructs of the MN-E6, MN-C2, MN-C3, MN-C8, or other antibodies that have been identified as selectively binding to MUCl* as they are present in cancer cells or tissues. Furthermore, the Fabs of these antibodies can be used as anti-cancer therapeutics. In one aspect of the invention, a person diagnosed with, suspected of having, or at risk of developing MUCl or MUCl* positive cancer can be treated with an effective amount of human or humanized MN-E6 scFv, MN-C2 scFv, MN-C3 scFv, or MN-C8 scFv. In another aspect of the invention, a person diagnosed with, suspected of having, or at risk of developing MUCl or MUCl* positive cancer can be treated with an effective amount of human or humanized MN-E6SCFV-FCY407R and MN-C2 scFv-Fcy407R and MN-C3 scFv-Fcy407R. In another aspect of the invention, a person diagnosed with MUCl or MUCl* positive cancer, suspected of having MUCl or MUCl* positive cancer, or at risk of developing MUCl or MUCl* positive cancer can be treated with an effective amount of human or humanized MN-E6 scFv-Fc mutantDhinge, MN-C2 scFv-Fc mutantDhinge, MN-C3 scFv-Fc mutantDhinge, or MN-C8 scFv-Fc mutantDhinge. In yet another aspect of the invention, a person diagnosed with, suspected of having, or at risk of developing MUCl or MUCl* positive cancer can be treated with an effective amount of human or humanized MN-E6 scFv-Fc mutanty407R-Dhinge, MN-C2 scFv-Fc mutantY407R-Dhinge, MN-C3 scFv-Fc mutantY407R-Dhinge, or MN-C8 scFv-Fc mutantY407R-Dhinge. In one aspect of the invention, a method of treating a person diagnosed with, suspected of having, or at risk for developing a MUCl or MUCl* positive cancer, in which the patient is administered an effective amount of monomeric MN-E6 scFv, MN-C2 scFv, MN-C3 scFv, MN-C8 scFv, or MN-E6 scFv-Fc, MN-C2 scFv-Fc, MN-C3 scFv-Fc, MN-C8 scFv-Fc, in which the Fc portion of the antibody-like protein is displaced so that it is resistant to dimer formation.

[0194] Humanization

[0195] Humanized antibodies or antibody fragments, or fully humanized antibodies that bind to the MUCl* extracellular domain, are suitable for therapeutic use. The techniques described herein for humanizing antibodies are only a few of the various methods known to those of skill in the art. It is not intended that the present invention be limited by the technique used to humanize an antibody.

[0196] Humanization is the process of replacing non-human regions of a therapeutic antibody, usually a murine monoclonal antibody, with human ones without altering its binding specificity and affinity. The primary goal of humanization is to reduce the immunogenicity of therapeutic monoclonal antibodies when administered to humans. Three distinct types of humanization are possible. First, chimeric antibodies are created by replacing the non-human constant regions of an antibody with human constant regions. Such antibodies contain mouse Fab regions and approximately 80–90% human sequence. Second, humanized antibodies are created by grafting mouse CDR regions (responsible for binding specificity) onto the variable regions of a humanized antibody in exchange for human CDRs (CDR grafting). Such antibodies contain approximately 90–95% human sequence. Third, and finally, fully human antibodies (100% human sequence) can be prepared by phage display, in which a library of humanized antibodies is screened to select antigen-specific human antibodies, or by immunizing transgenic mice expressing humanized antibodies.

[0197] A typical technique for humanizing antibodies is roughly as follows: A monoclonal antibody is generated in a host animal, typically a mouse. The monoclonal antibody is then screened for affinity and specificity for binding to a target. Once a monoclonal antibody with the desired effect and desired properties is identified, its sequence is characterized. The sequence of the antibody generated in the animal is then aligned with the sequences of many humanized antibodies to find the humanized antibody with the most homologous sequence to the animal antibody. Biochemical techniques are used to join the humanized antibody sequence and the animal antibody sequence together. Typically, non-human CDRs are grafted onto the humanized antibody with the most homology to the non-human antibody. This process can generate many candidate humanized antibodies needed to identify which antibody or antibodies have the desired affinity and specificity.

[0198] Once a human or humanized antibody is generated, it can be further modified for use as an antibody-like substance, such as a Fab fragment, a whole antibody, or a single-chain molecule containing the variable region, such as scFv or scFv-Fc. In some cases, it is preferable to have the Fc portion of the antibody or antibody-like molecule mutated so that it does not dimerize.

[0199] In addition to introducing human sequences into antibodies generated in non-human species, fully human antibodies can be obtained by screening humanized antibody libraries with peptide fragments of the antigen. Fully human antibodies that function like MN-E6 or MN-C2 can be generated by screening human antibody libraries with peptides having the sequence of the PSMGFR N-10 peptide. Fully human antibodies that function like MN-C3 or MN-C8 can be generated by screening human antibody libraries with peptides having the sequence of the PSMGFR C-10 peptide.

[0200] Humanized anti-MUCl* antibodies have been generated based on the sequences of murine monoclonal antibodies MN-E6, MN-C2, MN-C3, and MN-C8. In one aspect of the invention, patients diagnosed with MUCl*-positive cancer are treated with an effective amount of humanized MN-E6, MN-C2, MN-C3, or MN-C8. In a preferred embodiment, patients diagnosed with MUCl*-positive cancer are treated with an effective amount of humanized MN-E6, MN-C2. In another aspect of the invention, patients diagnosed with MUCl*-positive cancer are treated with an effective amount of humanized monovalent MN-E6, MN-C2, MN-C3, or MN-C8, where monovalent refers to the corresponding Fab fragment, corresponding scFv, or corresponding scFv-Fc fusion. In a preferred embodiment, patients diagnosed with MUCl*-positive cancer are treated with an effective amount of humanized scFv or monovalent humanized scFv-Fc of MN-E6 or MN-C2. Because the MUCl* growth factor receptor is activated by ligand-induced dimerization of its extracellular domain and because the Fc portion of antibodies homodimerizes, constructs containing Fc portions preferably use variant Fc regions that prevent or minimize dimerization.

[0201] Antibodies that bind to the PSMGFR (SEQ ID NO: 2) peptide of the extracellular domain of the MUCl* receptor are potential cancer therapeutics that are effective in treating or preventing MUCl*-positive cancers. They have been shown to inhibit the binding of activating ligand dimers NME1 (SEQ ID NOs: 3 and 4) and NME7 (SEQ ID NOs: 5 and 6) to the MUCl* extracellular domain. Anti-MUCl* antibodies that specifically bind to the PSMGFR sequence inhibit the proliferation of MUCl*-positive cancer cells if they block ligand-induced receptor dimerization. Fab fragments of anti-MUCl* antibodies have been demonstrated to block tumor growth in animals. Therefore, antibodies or antibody fragments that bind to the MUCl* extracellular domain are useful for treating cancers in which the cancerous tissue expresses MUCl*.

[0202] Antibodies that bind to the PSMGFR site of MUC1* or to synthetic PSMGFR peptides are preferred. The present inventors have identified several monoclonal antibodies that bind to the MUC1* extracellular domain. This group includes murine monoclonal antibodies MN-E6, MN-C2, MN-C3, and MN-C8, whose variable regions have been sequenced and are given in SEQ ID NOS: 12-13 and 65-66 for MN-E6, 118-119 and 168-169 for MN-C2, 413-414 and 458-459 for MN-C3, and 505-506 and 543-554 for MN-C8. The CDRs of these antibodies are the most important portions of the murine antibodies to be retained when constructing antibody recognition units and ligating them into humanized antibodies. The CDR sequences of each mouse monoclonal are as follows, with the light chain following the heavy chain sequence: MN-E6 CDR1 (SEQ ID NOs: 16-17 and 69-70), CDR2 (SEQ ID NOs: 20-21 and 73-74), CDR3 (SEQ ID NOs: 24-25 and 77-78); MN-C2 CDR1 (SEQ ID NOs: 122-123 and 172-173), CDR2 (SEQ ID NOs: 126-127 and 176-177), CDR3 (SEQ ID NOs: 130-131 and 180-181); MN-C3 CDR1 (SEQ ID NOs: 417-418 and 462-463), CDR2 (SEQ ID NOs: 421-422 and 466-467), CDR3 (SEQ ID NOs: 425-426 and 470-471); MN-C8 CDR1 (SEQ ID NOs: 507-508 and 545-546), CDR2 (SEQ ID NOs: 509-510 and 547-548), CDR3 (SEQ ID NOs: 511-512 and 549-550). In some cases, portions of the framework regions that, through modeling, are believed to be important for the three-dimensional structure of the CDRs are also introduced from the murine sequence.

[0203] Monoclonal antibodies MN-C6 and MN-C2 have greater affinity for MUC1*, which is expressed on stem cells. Monoclonal antibodies MN-C3 and MN-C8 have greater similarity to MUC1* as it is expressed on stem cells. By sequence alignment, the following human antibodies were selected to be sufficiently homologous to the murine antibodies so that replacement of the murine CDRs would result in antibodies that retained the ability to recognize the target. The murine MN-E6 heavy chain variable region is homologous to the human IGHV3-21*03 heavy chain variable region (SEQ ID NOs: 26-27), and the light chain variable region is homologous to the human IGKV3-11*02 light chain variable region (SEQ ID NOs: 79-80). The murine MN-C2 heavy chain variable region is homologous to the human IGHV3-21*04 heavy chain variable region (SEQ ID NOs: 132-133), and the light chain variable region is homologous to the human IGKV7-3*01 light chain variable region (SEQ ID NOs: 182-183). The murine MN-C3 heavy chain variable region is homologous to the human IGHV1-18*04 heavy chain variable region (SEQ ID NO: 427-428), and the light chain variable region is homologous to the human IGKV2-29*03 light chain variable region (SEQ ID NO: 472-473). The murine MN-C8 heavy chain variable region is homologous to the human IGHV3-21*04 heavy chain variable region (SEQ ID NO: 513-514), and the light chain variable region is homologous to the human Z00023 light chain variable region (SEQ ID NO: 551-552).

[0204] All four antibodies were humanized, and the process involved several humanization formats for each antibody: CDRs derived from the variable regions of murine antibodies were biochemically grafted to homologous human antibody variable region sequences. The humanized variable regions of MN-E6 (SEQ ID NOS: 38-39 and 93-94), MN-C2 (SEQ ID NOS: 144-145 and 194-195), MN-C3 (SEQ ID NOS: 439-440 and 486-487), and MN-C8 (SEQ ID NOS: 525-526 and 543-544) were generated by grafting murine CDRs onto the variable regions of homologous humanized antibodies. The humanized heavy chain variable constructs were then fused to either the human IgG1 heavy chain constant region (SEQ ID NOS: 58-59) or the human IgG2 heavy chain constant region (SEQ ID NOS: 54-55). These were then paired with a humanized light chain variable construct fused to the human κ chain (SEQ ID NOS: 109-110) or the human λ chain (SEQ ID NOS: 113-114) constant region. Other IgG isotypes can be used as constant regions, including IgG3 or IgG4.

[0205] Examples of humanized MN-E6 variable regions into IgG2 heavy chains (SEQ ID NOs: 52-53) and into IgG1 heavy chains (SEQ ID NOs: 56-57), humanized MN-C2 variable regions into IgG1 heavy chains (SEQ ID NOs: 158-159) or into IgG2 heavy chains (SEQ ID NOs: 163-164) paired with either lambda light chains (SEQ ID NOs: 111-112 and 216-219) or kappa chains (SEQ ID NOs: 107-108 and 210-213), and humanized MN-C3 (SEQ ID NOs: 455-456, 453-454 and 500-501, 502-503) and MN-C8 (SEQ ID NOs: 541-542, 539-540 and 579-580, 581-582) antibodies were generated. Because each isotype has its own characteristic activity, the choice of which IgG constant region to graft to the humanized variable region depends on the desired effect. The isotype of the human constant region is selected based on considerations such as whether antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) is desired, as well as the reliance on yield of antibody produced in a cell-based protein expression system. In a preferred embodiment, a humanized anti-MUCl* antibody or antibody fragment is administered to an individual diagnosed with or at risk of developing a MUCl-positive cancer.

[0206] One way to test and select humanized anti-MUCl* antibodies that will be useful in treating individuals with or at risk of developing cancer is to test them for their ability to inhibit the binding of activating ligands to the MUCl* extracellular domain. Dimeric NME1 can bind to and dimerize the MUCl* extracellular domain, thereby stimulating cancer cell growth. Therefore, antibodies and antibody fragments that compete with NME1 for binding to the MUCl* extracellular domain are anti-cancer agents. NME7 is another activating ligand for MUCl*. In some cases, it is desirable to identify antibodies that inhibit the binding of NME7 or NME7 deletion or degradation products to the MUCl* extracellular domain. Antibodies and antibody fragments that compete with NME7 and NME7 variants for binding to the MUCl* extracellular domain are useful as cancer therapeutics. These antibodies include, but are not limited to, MN-E6, MN-C2, MN-C3, and MN-C8, as well as single-chain versions, such as scFv, of these antibodies and their humanized versions. Other NME proteins also bind to MUCl* or MUC1, including NME6 and NME8. Antibodies that compete with these proteins for binding to MUCl* may also be useful as therapeutic agents. In a preferred embodiment, a humanized anti-MUCl* antibody or antibody fragment is administered to an individual diagnosed with or at risk for developing a MUCl-positive cancer. In a more preferred embodiment, single-chain antibody fragments or monomeric scFv-Fc fusions derived from the humanized sequences of MN-E6 and MN-C2 are administered to an individual diagnosed with or at risk for developing a MUCl-positive cancer.

[0207] Single-chain variable fragments, scFvs, or other forms resulting in monovalent antibodies or antibody-like proteins are also useful. In some cases, it is preferable to prevent dimerization of the MUCl* extracellular domain. Single-chain variable fragments, Fabs, and other monovalent antibody-like proteins have been shown to be effective in binding to the extracellular domain of MUCl* and blocking MUCl* dimerization. These single-chain variable fragments, Fabs, and other monovalent antibody-like molecules effectively block cancer growth in vitro and in animals xenografted with human MUCl-positive cancer cells. Therefore, humanized single-chain variable fragments or monovalent anti-MUCl* antibodies or antibody-like molecules are highly useful as cancer therapeutics. Such humanized single-chain antibodies, Fabs, or other monovalent antibody-like molecules that bind to the MUCl* extracellular domain or to PSMGFR peptides are therefore useful as anti-cancer therapeutics. Anti-MUCl* single-chain variable fragments are generated by grafting non-human CDRs of antibodies that bind to the MUCl* extracellular domain or PSMGFR peptide to the framework of a homologous human antibody variable region. The resulting humanized heavy and light chain variable regions are then connected to each other by a suitable linker, which is flexible and long enough to allow heavy chain binding to light chain, allowing heavy chain binding to light chain, but preventing binding of the heavy chain of one molecule to the light chain of another. For example, a linker of about 10-15 residues. Preferably, the linker contains [(Glycine)4(Serine)1]3 (SEQ ID NO: 401-402), but is not limited to this sequence, as other sequences are possible.

[0208] In one aspect, the humanized variable regions of MN-E6 (SEQ ID NOS: 38-39 and 93-94), MN-C2 (SEQ ID NOS: 144-145 and 194-195), MN-C3 (SEQ ID NOS: 439-440 and 486-487), and MN-C8 (SEQ ID NOS: 525-526 and 565-566) were biochemically linked into a construct connecting the heavy and light chains via a linker. Examples of humanized single-chain anti-MUCl* antibodies have been generated that contain humanized sequences from the variable regions of MN-E6, MN-C2, MN-C3, and MN-C8. Several humanized MN-E6 single-chain proteins have been generated (SEQ ID NOS: 232-237). Several humanized MN-C2 single-chain proteins have been generated (SEQ ID NOS: 238-243). Several humanized MN-C3 single-chain proteins have been generated (SEQ ID NOS: 244-249). Several humanized MN-C8 single-chain proteins have been produced (SEQ ID NOS: 250-255). Within preferred embodiments, humanized anti-MUCl* antibody fragments, scFv antibody fragments MN-E6 scFv, MN-C2 scFv, MN-C3 scFv, or MN-C8 scFv, containing variable construct fragments, are administered to individuals diagnosed with or at risk for developing MUCl-positive cancer. In even more preferred embodiments, single-chain antibody fragments, such as variable fragments derived from the humanized sequences of MN-E6 and MN-C2, are administered to individuals diagnosed with or at risk for developing MUCl-positive cancer.

[0209] In another aspect, the humanized variable regions of MN-E6 (SEQ ID NOS: 38-39 and 93-94), MN-C2 (SEQ ID NOS: 144-145 and 194-195), MN-C3 (SEQ ID NOS: 439-440 and 486-487), and MN-C8 (SEQ ID NOS: 525-526 and 565-566) were biochemically linked to single-chain variable fragments, scFvs, which further comprise the Fc portion of an antibody. Exemplary humanized single-chain variable fragments of MN-E6, MN-C2, MN-C3, and MN-C8 fused to the Fc portion of an antibody were generated (SEQ ID NOS: 256-257, 260-261, 264-265, and 268-269). The inclusion of an Fc region serves several purposes. It increases the molecular weight of the antibody fragment, slowing its decay and increasing its half-life. The Fc region also recruits immune system complement to tumor sites. Furthermore, the addition of an antibody Fc region makes scFvs useful diagnostic tools, as a secondary antibody can detect and label the Fc region. However, the Fc region homodimerizes. Thus, scFv-Fc is bivalent and dimerizes and activates the MUCl* growth factor receptor. To obtain the benefits of having an Fc attached to an anti-MUCl* scFv without the drawbacks of causing MUCl* dimerization, the Fc portion can be mutated to minimize or eliminate Fc homodimerization. To prepare monomeric scFv-Fc fusion proteins, the following mutations were made in the CH3 region: Y407R (SEQ ID NOs: 278 and 279), F405Q (SEQ ID NOs: 280 and 281), T394D (SEQ ID NOs: 282 and 283), T366W / L368W (SEQ ID NOs: 284 and 285), and T364R / L368R (SEQ ID NOs: 286 and 285). Any combination of these mutations could be tested and introduced into Fc (SEQ ID NOs: 272-273), CH2-CH3 (SEQ ID NOs: 274-275) or CH3 (SEQ ID NOs: 276-277) fusion proteins, or in hingeless Fc fusion proteins (SEQ ID NOs: 288-289).

[0210] One aspect of the invention relates to a method of treating a person diagnosed with or at risk of developing a MUC1-positive cancer, in which an effective amount of monomeric MN-E6 scFv, MN-C2 scFv, MN-C3 scFv, MN-C8 scFv, or MN-E6 scFv-Fc and MN-C2 scFv-Fc and MN-C3 scFv-Fc and MN-C8 scFv-Fc is administered to the patient, wherein the antibody variable fragment portion is human or humanized, and wherein the Fc portion of the antibody-like protein has been altered so that it is resistant to dimer formation.

[0211] CAR T and cancer immunotherapy technology

[0212] In another aspect of the invention, some or all of the anti-MUCl* antibody fragment single chain portions are biochemically fused onto immune system molecules using several different chimeric antigen receptor, 'CAR', strategies. The idea is to fuse the recognition portion of an antibody, typically a single-chain variable fragment, to an immune system molecule with a cytoplasmic tail and transmembrane domain that can transmit a signal that activates the immune system. The recognition unit can be an antibody fragment, a single-chain variable fragment, an scFv, or a peptide. In one aspect, the recognition portion of the extracellular domain of the CAR comprises sequences from the humanized variable regions of MN-E6 (SEQ ID NOs: 38-39 and 93-94), MN-C2 (SEQ ID NOs: 144-145 and 194-195), MN-C3 (SEQ ID NOs: 439-440 and 486-487), and MN-C8 (SEQ ID NOs: 525-526 and 565-566). In another aspect, it comprises sequences from single-chain variable fragments. Examples of single-chain constructs are provided. Several humanized MN-E6 single-chain proteins, scFvs, have been generated (SEQ ID NOs: 232-237). Several humanized MN-C2 single-chain proteins, scFvs, have been generated (SEQ ID NOs: 238-243). Several humanized MN-C3 single-chain proteins, scFvs, have been generated (SEQ ID NOs: 244-249). Several humanized MN-C8 single chain proteins, scFv, were generated (SEQ ID NOs: 250-255). The transmembrane domain of the CAR can be derived from CD8, CD4, antibody domains, or other transmembrane domains, including the transmembrane domain of the proximal cytoplasmic costimulatory domain. The cytoplasmic tail of the CAR contains one or more motifs that signal immune system activation. This group of cytoplasmic signaling motifs, sometimes referred to as costimulatory cytoplasmic domains, includes, but is not limited to, CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and Fc receptor gamma domains. A minimal CAR can have a CD3-zeta or Fc receptor gamma domain, with one or two of these domains in tandem in the cytoplasmic tail. In one aspect, the cytoplasmic tail includes CD3-ZETA, CD28, 4-1BB, and / or OX40. Several examples of humanized MN-E6 CARs have been generated: CAR MN-E6 CD3z (SEQ ID NOs: 294-295); CAR MN-E6 CD28 / CD3z (SEQ ID NOs: 297-298); CAR MN-E6 4-1BB / CD3z (SEQ ID NOs: 300-301); CAR MN-E6 OX40 / CD3z (SEQ ID NOs: 616-617); CAR MN-E6 CD28 / OX40 / CD3z (SEQ ID NOs: 618-619); CAR MN-E6 CD28 / 4-1BB / CD3z (SEQ ID NOs: 303-304). Several examples of humanized MN-C2 CARs have been generated: CAR MN-C2 CD3z (SEQ ID NOs: 606-607); CAR MN-C2 CD28 / CD3z (SEQ ID NOs: 608-609); CAR MN-C2 4-1BB / CD3z (SEQ ID NOs: 610-611); CAR MN-C2 OX40 / CD3z (SEQ ID NOs: 612-613); CAR MN-C2 CD28 / 4-1BB / CD3z (SEQ ID NOs: 306-307); CAR MN-C2 CD28 / OX40 / CD3z (SEQ ID NOs: 614-615). A humanized MN-C3 CAR has been generated: CAR MN-C3 4-1BB / CD3z (SEQ ID NOs: 600-601).

[0213] Several examples of humanized MN-E6 CARs with different hinge regions (SEQ ID NOs: 345-360) were generated: CAR MN-E6-Fc / 8 / 41BB / CD3z (SEQ ID NOs: 310-311); CAR MN-E6 FcH / 8 / 41BB / CD3z (SEQ ID NOs: 315-316); CAR MN-E6 Fc / 4 / 41BB / CD3z (SEQ ID NOs: 318-319); CAR MN-E6 FcH / 4 / 41BB / CD3z (SEQ ID NOs: 321-322); CAR MN-E6 IgD / 8 / 41BB / CD3z (SEQ ID NOs: 323-324); CAR MN-E6 IgD / 4 / 41BB / CD3z (SEQ ID NOs: 327-328); CAR MN-E6 X4 / 8 / 41BB / CD3z (SEQ ID NOs: CAR MN-E6 X4 / 4 / 4 1BB / CD3z (SEQ ID NO: 333-334); CAR MN-E6 8+4 / 4 / 4 1BB / CD3z (SEQ ID NO: 336-337). In addition, several humanized MN-C3 single-chain variable fragments and humanized MN-C8 single-chain variable fragments were also generated.

[0214] The MUCl*-targeting CAR extracellular domain recognition unit can comprise the variable region of a humanized MN-E6, MN-C2, MN-C3, or MN-C8 or other antibody that binds to the PSMGFR portion of the MUCl* or PSMGFR peptide. In one aspect, the CAR extracellular domain recognition unit consists essentially of a humanized MN-E6, MN-C2, MN-C3, or MN-C8 single-chain variable fragment scFv. The CAR transmembrane domain can be derived from CD8 (SEQ ID NO: 363-364) or can be the transmembrane domain of CD3-ZETA, CD28, 41bb, OX40, or other transmembrane domains (SEQ ID NO: 361-372). The cytoplasmic domain of the CAR bearing an antibody fragment targeting the MUCl* extracellular domain can comprise one or two selected from the group consisting of immune system costimulatory cytoplasmic domains. The group of immune system costimulatory regions includes, but is not limited to, CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and Fc receptor gamma region (SEQ ID NOs: 373-382). Alternatively, the recognition unit portion of the CAR can comprise a peptide that binds to a target. NME7 binds to and activates MUCl*. In one aspect of the present invention, the recognition unit of the CAR is a peptide derived from NME7 (SEQ ID NO: 5-6) or a peptide derived from NME7, including, but not limited to, NME7 peptide A1 (SEQ ID NO: 7), NME7 peptide A2 (SEQ ID NO: 8), NME7 peptide Bl (SEQ ID NO: 9), NME7 peptide B2 (SEQ ID NO: 10) and NME7 peptide B3 (SEQ ID NO: 11).

[0215] Some strategies for generating CARs involve a portion of the molecule that dimerizes with itself. In some cases, target dimerization is undesirable. Therefore, CARs can be constructed so that they heterodimerize. In one case, the recognition unit of a first CAR binds to a first target, while the recognition unit of a second CAR binds to a second target. Both recognition units can be antibody fragments, both can be peptides, or one can be an antibody fragment and the other a peptide. The primary target of the CAR can be the MUCl* extracellular domain. The recognition unit of the CAR can be composed of an antibody fragment that binds to the MUCl* extracellular domain or to a PSMGFR peptide. Alternatively, the recognition unit of the CAR can be composed of a peptide that binds to the MUCl* extracellular domain, and such peptides include peptides derived from NME proteins such as NME1 or NME7, particularly the NME7-derived peptides listed as SEQ ID NOs:7-11. The secondary target of the heterodimeric CAR can be a peptide or antibody fragment that binds to NME7. Alternatively, the secondary target of the heterodimeric CAR can be a peptide or antibody fragment that binds to PD1 or other targets on MUC1*-presenting cells. The secondary target can be a peptide or antibody fragment that binds to NME1. Because it is desirable to prevent CAR-induced MUC1 dimerization, heterodimeric CARs can be constructed such that only the extracellular domain of one molecule has an extracellular recognition unit that binds to the target (SEQ ID NOs: 584-587). The other molecule can have a deleted extracellular domain lacking the target recognition unit or antibody fragment (SEQ ID NOs: 588-599). The described CARs can be transfected or transduced into cells of the immune system. In a preferred embodiment, a MUC1*-targeting CAR can be transfected or transduced into T cells. In one aspect, the T cells are CD3+ / CD28+ T cells. In another, they are dendritic cells. In another, they are B cells. In another, they are mast cells. The recipient cells are obtained from the patient or a donor. If from a donor, they can be engineered to remove molecules that cause rejection. Cells transfected or transduced with the CAR of the invention are expanded ex vivo or in vivo and then administered to a patient. The route of administration is selected from the group including, but not limited to, bone marrow transplant, intravenous injection, in situ infusion, or transplantation. In a preferred embodiment, a MUC1*-targeting CAR is administered to a person diagnosed with or at risk for developing a MUC1-positive cancer.

[0216] There are many possible anti-MUCl* CAR constructs that can be transduced into T cells or other immune cells for the treatment or prevention of MUCl*-positive cancers. CARs are composed of multiple modules, the identities of some of the modules being relatively unimportant while the identities of other modules are critical.

[0217] Our experiments have demonstrated that the antibody recognition fragment at the outermost part of the CAR is crucial because it targets CAR-carrying immune cells to tumor sites. Furthermore, intracellular signaling motifs are crucial, but can also be exchanged. Figure 28 shows a model of the CAR and various sequence components that can be included in a CAR. See Figure 28.

[0218] Rl is: nothing; or

[0219] a fragment of or a ligand of a cancer-associated antigen; or

[0220] a fragment of or a ligand of MUC1 or MUC1*; or

[0221] or an antibody or antibody fragment, wherein the antibody or antibody fragment binds to MUC1 or MUC1*; or an antibody or antibody fragment, wherein the antibody or antibody fragment binds to PSMGFR*, wherein the antibody is human or humanized; or an antibody or antibody fragment of MN-E6, MN-C2, MN-C3, or MN-C8, or humanized MN-E6, MN-C2, MN-C3, or MN-C8; or an antibody, scFv, single chain variable fragment that binds to cleaved MUC1 or MUC1*; or an scFv of MN-E6, MN-C2, MN-C3, or MN-C8, which may be humanized; or a peptide that binds to MUC1* or a PSMGFR peptide; or an antibody fragment, scFv, or peptide that binds the PSMGFR portion of MUC1*; or an antibody fragment, scFv, or peptide that binds the PSMGFR portion of MUC1*; or an antibody fragment, scFv, or peptide that binds to MN-E6 (SEQ ID NOs: 38-39 and 93-94), MN-C2 (SEQ ID NOs: 144-145 and 194-195), MN-C3 (SEQ ID NOs: The sequences comprise the humanized variable regions of MN-C8 (SEQ ID NOs: 439-440 and 486-487) and MN-C8 (SEQ ID NOs: 525-526 and 565-566). In one aspect, R1 is an scFv that binds the PSMGFR portion of MUCl* composed of sequences from humanized MN-E6 scFv (SEQ ID NOs: 232-237), humanized MN-C2 scFv (SEQ ID NOs: 238-243), humanized MN-C3 scFv (SEQ ID NOs: 244-249), or humanized MN-C8 scFv (SEQ ID NOs: 250-255). In another aspect, R1 is an scFv that binds the PSMGFR portion of MUCl* composed of sequences from humanized MN-E6 scFv (SEQ ID NOs: 232-237) or humanized MN-C2 scFv (SEQ ID NOs: 238-243). In one example, R1 is an scFv that binds the PSMGFR portion of MUC1* composed of sequences from humanized MN-E6 scFv (SEQ ID NOs: 232-237).

[0222] R2 is a polypeptide flexible linker that connects the recognition moiety to the transmembrane region of the CAR. In one aspect, R2 can be a polypeptide linker of different lengths, from 5 to 250 amino acids. In another aspect, R2 is a polypeptide linker of human origin. In one aspect, R2 can be made from or be a mutation of the Fc region of a human immunoglobulin (IgG, IgA, IgE, IgM, or IgD). In another aspect, R2 can be a hinge region or a mutation of the hinge region of a human immunoglobulin (IgG, IgA, IgE, IgM, or IgD). In one aspect, R2 can be a hinge region or a mutation of the hinge region of a T cell receptor (CD8a, CD28, or CD4). In one example, R2 is the hinge region of CD8a, the hinge region of human IgD, or the Fc region of human IgG1.

[0223] R3 is a transmembrane region. In one aspect, R3 can be a transmembrane region or a mutation in the transmembrane region of any transmembrane human protein. In another aspect, R3 can be a transmembrane region or a mutation in the transmembrane region from a human cell receptor. In one aspect, R3 can be a transmembrane region or a mutation in the transmembrane region of a T cell receptor (CD8a, CD4, CD28, CD3z, OX40, or 41-BB). In another aspect, R3 is a transmembrane region from the first cytoplasmic costimulatory region of the CAR. In one aspect, R3 can be a mutation in the transmembrane region of a T cell receptor extended by 1, 2, 3, 4, or 5 amino acids in the transmembrane region or the cytoplasmic region associated with the transmembrane region. In another aspect, R3 can be a mutation in the transmembrane region of a T cell receptor extended by 1, 2, 3, 4, or 5 amino acids in the transmembrane region or the cytoplasmic region associated with the transmembrane region where cysteines for disulfide bond formation occur. In one example, R3 is the transmembrane region of CD8a or CD4.

[0224] R4 is a signaling region from the T cell receptor. In one aspect, R4 can be the cytoplasmic signaling region of CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and the Fc receptor gamma region. In one example, R4 is the cytoplasmic region of CD3-zeta. Several examples of humanized CARs with a single signaling region (CAR I) have been regenerated: CAR MN-E6 CD3z (SEQ ID NO: 294-295); CAR MN-C2 CD3z (SEQ ID NO: 606-607).

[0225] R5 is a costimulatory region from the T cell receptor. In one aspect, R5 can be the cytoplasmic signaling region of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and the Fc receptor gamma region. R5 is distinct from R4 and R6. In one example, R5 is the cytoplasmic region of CD28, 4-1BB, or OX40. Several examples of humanized CARs with two signaling regions (CAR II) were regenerated: CAR MN-E6 CD28 / CD3z (SEQ ID NOs: 297-298); CAR MN-E6 4-1BB / CD3z (SEQ ID NOs: 300-301); CAR MN-E6 OX40 / CD3z (SEQ ID NOs: 616-617); CAR MN-C2 CD28 / CD3z (SEQ ID NOs: 608-609); CAR MN-C2 4-1BB / CD3z (SEQ ID NOs: 610-611); CAR MN-C2 OX40 / CD3z (SEQ ID NOs: 612-613); CAR MN-C3 4-1BB / CD3z (SEQ ID NOs: 600-601); CAR MN-E6-Fc / 8 / 41BB / CD3z (SEQ ID NOs: 310-311); CAR MN-E6 FcH / 8 / 41BB / CD3z (SEQ ID NO: 315-316); CAR MN-E6 Fc / 4 / 41BB / CD3z (SEQ ID NO: 318-319); CAR MN-E6 FcH / 4 / 41BB / CD3z (SEQ ID NO: 321-322); CAR MN-E6 IgD / 8 / 41BB / CD3z (SEQ ID NO: 323-324); CAR MN-E6 IgD / 4 / 41BB / CD3z (SEQ ID NO: 327-328); CAR MN-E6 X4 / 8 / 41BB / CD3z (SEQ ID NO: 330-331); CAR MN-E6 X4 / 4 / 41BB / CD3z (SEQ ID NO: 333-334); CAR MN-E6 8+4 / 4 / 4 IBB / CD3z (SEQ ID NO: 336-337).

[0226] R6 is a costimulatory domain from the T cell receptor. In one aspect, R6 can be the cytoplasmic signaling domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and the Fc receptor gamma domain. R6 is distinct from R4 and R5. In one example, R5 is the cytoplasmic domain of CD28. Several examples of humanized CARs with two signaling regions (CAR III) were regenerated: CAR MN-E6 CD28 / OX40 / CD3z (SEQ ID NOs: 618-619); CAR MN-E6 CD28 / 4-1BB / CD3z (SEQ ID NOs: 303-304); CAR MN-C2 CD28 / 4-1BB / CD3z (SEQ ID NOs: 306-307); CAR MN-C2 CD28 / OX40 / CD3z (SEQ ID NOs: 614-615).

[0227] The present inventors and others are cited [Pule MA, Straathof KC, Dotti G, Heslop HE, Rooney CM and Brenner MK. (2005)): Chimeric T cell antigen receptors increase cytokine release and support clonal expansion of primary human T cells. Mol Ther. 12(5):933-941; Hombach AA, Heiders J, Foppe M, Chmielewski M and Abken H. (2012)1; OX40 costimulation with a chimeric antigen receptor suppresses IL-2-induced IL-10 secretion and CD28 expression by redirected CD4(+) T cells. Oncoimmunology.1(4):458-466; Kowolik CM, Topp MS, Gonzalez S, Pfeiffer T, Olivares S, GonzalezN, Smith DD, Forman SJ, Jensen MC and Cooper LJ. (2006): CD28 costimulation provided by a CD19-specific chimeric antigen receptor enhances the in vivo persistence and antitumor efficacy of adaptively transferred T cells. Cancer Res. 66(22): 10995-11004; Loskog A, Giandomenico V, Rossig C, Pule M, Dotti Gand Brenner MK. (2006): Addition of the CD28 signaling domain to a chimeric T cell receptor enhances chimeric T cell resistance to T regulatory cells. Leukemia.20(10): 1819- 1828; Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK,Teachey D, Samanta M, Lakhal M, Gloss B, Danet-Desnoyers G, Campan A-D, Riley JL, Grupp SA and June CH. (2009): CD 137; a chimeric receptor containing the signaling domain mediator enhances T cell survival and increases anti-leukemic efficacy in vivo.Mol Ther.17(8): 1453-1464; Song DG, Ye Q, Carpenito C, Poussin M, Wang LP, Ji C, Figini M, June CH, Coukos G, Powell DJ Jr. (2011): During in vivo persistence, tumor localization and antitumor activity of CAR-engineered T cells are enhanced by costimulatory signaling through CD137 (4-1BB). Cancer Res. 71(13):4617-4627; Intracellular signaling modules such as CD3-zeta (SEQ ID NO: 373-376), CD28 (SEQ ID NO: 377-378), and 41BB (SEQ ID NO: 379-380), alone or together, stimulate immune cell expansion, cytokine secretion, and immune cell-mediated killing of target tumor cells. Of lesser importance is the identity of the short extracellular piece representing the antibody fragment, the transmembrane domain, and the short cytoplasmic tail preceding the intracellular signaling motif.

[0228] The identity of the recognizing antibody fragment that targets the CAR to the tumor is critical. For the treatment of MUCl-positive or MUCl*-positive cancers, the antibody recognizing fragment must bind to the extracellular domain of the portion of MUCl remaining after cleavage and shedding of most of the extracellular domain, including the tandem repeat region. In one aspect of the invention, the remaining portion comprises the PSMGFR sequence. In another aspect of the invention, the portion of MUCl remaining after cleavage and shedding comprises the PSMGFR sequence plus more than 9 amino acids extended at the N-terminus. In another aspect of the invention, the portion of MUCl remaining after cleavage and shedding comprises the PSMGFR sequence plus more than 21 amino acids extended at the N-terminus. In one aspect, the antibody recognizing fragment binds to the PSMGFR peptide. In another aspect of the invention, the antibody recognizing fragment binds to a peptide comprising the sequence SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621). As a demonstration, single-chain antibody fragments containing the variable regions of monoclonal anti-MUCl* antibodies, designated MN-E6 or MN-C2, were engineered into a panel of CARs. The MUCl*-targeted CARs were then transduced into immune cells, either separately or in combination. When challenged with surfaces displaying MUCl* peptides, MUCl*-transfected antigen-presenting cells, or MUCl*-positive cancer cells, the MUCl*-targeted CAR-transduced immune cells elicited an immune response, including cytokine release, target cell killing, and immune cell expansion. In one case, human Jurkat cells were transduced with the MUCl*-targeted CAR and, upon exposure to surfaces displaying PSMGFR peptides, with MUCl* or MUCl*-transfected K562 antigen-presenting cells, and the Jurkat cells secreted IL-2. In another case, purified human T cells were transduced with a MUCl*-targeted CAR and with MUCl* or MUCl*-transfected K562 antigen-presenting cells by exposure to surface-presenting PSMGFR peptide; the T cells secreted IL-2, interferon gamma, and killed the targeted antigen-presenting cells and cancer cells while the T cells expanded. As demonstrated, CARs comprising antibody fragments, where the antibody fragments can bind to a PSMGFR peptide, a transmembrane domain, and a cytoplasmic tail bearing a costimulatory domain, elicit an immune system anti-tumor cell response when transduced into immune cells, including T cells. Therefore, other antibodies, antibody fragments, or antibody mimetics capable of binding to a PSMGFR peptide can similarly perform and be used to treat or prevent cancer. Those skilled in the art will recognize that there are many techniques available for transfecting or transducing cells with CARs, and the present invention is not limited by the method used to express a MUCl*-targeted CAR in immune cells. For example, retroviruses, adenoviruses, lentiviruses, etc. can be used. Similarly, the identity of the molecules making up portions of the CAR, such as the non-targeting portion, extracellular domain, transmembrane domain, and membrane-proximal portion of the cytoplasmic domain, is not essential to the function of the MUCl*-targeted CAR. For example, the extracellular domain, transmembrane domain, and membrane-proximal portion of the cytoplasmic domain can be composed of portions of CD8, CD4, CD28, or generic antibody domains such as Fc, CH2CH3, or CH3. Additionally, the non-targeting portion of the CAR can be a composite of one or more portions of these molecules or other family members.

[0229] One aspect of the invention is a method of treating a patient diagnosed or suspected of having or at risk of developing a MUCl-positive or MUCl*-positive cancer, in which the patient is administered an effective amount of immune cells transduced with a MUCl*-targeting CAR. In another aspect of the invention, the immune cells are T cells isolated from the patient, which are transduced with a CAR whose targeting head binds to MUCl*, and after expansion of the transduced T cells, the CAR T cells are administered to the patient in an effective amount. In yet another aspect of the invention, the immune cells are T cells isolated from the patient, which the targeting head of the CAR comprises a huMN-E6, huMN-C2, huMN-C3, or huMN-C8 portion, and after optional expansion of the transduced T cells, the CAR T cells are administered to the patient in an effective amount.

[0230] CAR CHARACTERISTICS BUILT AND TESTED

[0231] A number of MUCl*-targeting CARs were generated, in which the targeting antibody fragment at the distal end of the CAR was MN-E6, MN-C2, MN-C3, or MN-C8. The DNA of each CAR was sequenced to confirm accurate cloning. Each construct was then mixed into an expression plasmid and transfected into cells. Successful insertion of the construct was then confirmed by Western blot. Surface expression was confirmed by FACS. The MUCl*-targeting CARs were then virally transduced into immune cells. In one aspect, they were transduced into Jurkat cells. In another aspect, they were transduced into primary human T cells purified from blood. A series of functional analyses were performed to confirm that the CARs were functional. The functional analyses showed that both Jurkat cells and primary T cells transduced with MUCl*-targeting CARs secreted the cytokine IL-2 when challenged with MUCl*-expressing cells. Figure 29 is a graph experimentally measuring IL-2 cytokine secretion by Jurkat cells transduced with a panel of CARs, including MN-E6 CD8 / CD3z, MN-E6 CD8 / CD28 / CD3z, MN-E6 CD8 / 41BB / CD3z, MN-E6 CD4 / CD28 / CD3z, and MN-E6 CD4 / CD28 / 41BB / CD3z. CAR Jurkat cells secreted IL-2 only when exposed to K562 cells or K562-wt cells transfected with MUCl*. It should be noted that the parental K562-wt cells express very low levels of MUCl*. Another group of CARs transfected into Jurkat cells was similarly tested for cytokine secretion. Figure 30 shows IL-2 secretion by Jurkat T cells transduced with MN-E6 CD8 / CD28 / CD3z, MN-E6 CD8 / 41BB / CD3z, MN-E6 CD4 / CD28 / CD3z, or MN-E6 CD4 / 41BB / CD3z when the CAR T cells were exposed to K562 cells or K562-wt cells transfected with MUCl*. Similarly, Figure 31 shows IL-2 cytokine secretion by primary human T cells transduced with MN-E6 CD8 / CD28 / CD3z, MN-E6 CD8 / 41BB / CD3z, or MN-E6 CD4 / 41BB / CD3z. Cytokine secretion occurred only when MUCl*-targeted CAR T cells were exposed to K562 cells or K562-wt cells transfected with MUCl*. Another cytokine secreted by activated T cells when they see target cells is interferon gamma (IFNg). Figure 32 shows that primary human T cells transduced with a panel of CARs including MN-E6 CD8 / CD28 / CD3z and MN-E6 CD4 / 41BB / CD3z secreted interferon-γ when the CAR T cells were exposed to MUCl*-transfected K562 cells or K562-wt cells. Interferon-γ is secreted when primary human T cells transduced with a panel of CARs including MN-E6 CD8 / CD28 / CD3z, MN-E6 CD8 / 41BB / CD3z, and MN-E6 CD8 / CD28 / 41BB / CD3z, and MUCl*-targeted CAR T cells are exposed to MUCl*-positive cancer cells from prostate (DU145), breast (1500), or pancreatic (Capan) cancer, or to MUCl*-transfected K562 or K562-wt cells (Figure 33).

[0232] Another measure of CAR T cell function is whether they cause target cell killing. T cells transfected with various CARs containing antibody fragments that bind to the PSMGFR sequence of MUCl* killed MUCl*-expressing cells in coculture assays. In one assay, target MUCl*-expressing cells were cultured with calcein. When they were mixed with CAR T cells containing antibody fragments such as MN-E6, MN-C2, MN-C3, or MN-C8, the CAR T cells killed the MUCl*-presenting cells, which lysed the target cells and released calcein into the supernatant. Figure 34 is a graph of experimentally measured target cell death when primary human T cells isolated from blood samples were transduced with a panel of CARs including MN-E6 CD8 / 41BB / CD3z and MN-E6 CD4 / 41BB / CD3z, and the CAR T cells were exposed to MUCl*-transfected K562 cells or K562-wt cells. The T cell to target cell ratio was 1:1, and the cells were co-cultured for 24 hours. Figure 35A-B are FACS graphs measuring the time course of target cell survival from day 1 to day 3. Primary human T cells isolated from blood samples were transduced with a panel of CARs, including humanized MN-E6-CD8-3z, MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. CAR T cells were then exposed to K562-wt cells, which naturally express low levels of MUCl*, or to K562 cells transfected with high MUCl*. The ratio of MUCl*-targeted CAR T cells to target cells was 1:1, 10:1, or 20:1. Viable cells were detected and measured on day 1 (A) or day 3 (B).

[0233] Figure 36 is a graph of FACS measurements of target cell survival on day 3 of a coculture experiment. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-3z, MN-E6-CD8-CD28-3z, MN-E6-CD8-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. The CAR T cells were then exposed to MUCl*-positive T47D breast cancer cells or MUCl*-positive 1500 aka ZR-75-1 breast cancer cells. The ratio of MUCl*-targeted CAR T cells to target cells was 1:1 or 10:1. As can be seen from the graph, T cells transduced with MUCl*-targeted CARs were much more effective at killing MUCl* cancer cells than untransduced control T cells. Furthermore, the killing effect was much greater as the ratio of T cells to target cells increased. Figure 37 is a graph of FACS measurements of target cell survival on day 1 of the coculture experiment. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-41BB-3z, MN-E6-CD4-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. The CAR T cells were then exposed to the following MUCl*-positive cancer cells: T47D breast cancer; capan2 pancreatic cancer; or DU-145 prostate cancer. The ratio of MUCl*-targeted CAR T cells to target cells was 5:1. As can be seen from the graph, T cells transduced with MUCl*-targeted CARs have a much greater killing effect on MUCl* cancer cells than untransduced control T cells. Note that measurements were taken after 24 hours only at a T cell:target cell ratio of 5:1. Furthermore, it should be noted that, like the CD8 construct, a MUCl*-targeted CAR with a CD4 extracellular domain transmembrane cytoplasmic tail works equally well. Figure 38 is a graph of FACS measurements of target cell survival on day 3 of a coculture experiment. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-41BB-3z, MN-E6-CD4-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. The CAR T cells were then exposed to the following MUCl*-positive cancer cells: MUCl*-transfected K562 leukemia cells; T47D breast cancer; 1500aka ZR-75-1 breast cancer cells; or CAPAN-2 pancreatic cancer cells. In addition to untransduced T cell controls, analysis was performed with PC3 MUCl*-negative prostate cancer cells. The ratio of MUCl*-targeted CAR T cells to target cells was 1:1. As can be seen from the graph, T cells transduced with MUCl*-targeted CARs have a much greater killing effect on MUCl* cancer cells than untransduced control T cells. Furthermore, the killing effect is specific to MUCl*-positive cells. Furthermore, it should be noted that, like the CD8 construct, MUCl*-targeted CARs with CD4 extracellular domain transmembrane cytoplasmic tails work equally well. Figure 39 is a graph of FACS measurements of CAR T cell expansion over 24 hours in co-culture with target cells at a 5:1 CAR T cell to target cell ratio. Primary human T cells were transduced with a panel of CARs, including humanized MN-E6-CD8-41BB-3z, MN-E6-CD4-41BB-3z, and MN-E6-CD8-CD28-41BB-3z. CAR T cells were co-cultured with MUCl*-positive T47D breast cancer cells, MUCl*-positive Capan pancreatic cancer cells, and MUCl-negative HCT-116 colon cancer cells and HEK-293 human embryonic kidney cells. As can be seen from the graph, the CAR T population is expanded in the presence of MUCl*-positive cells. Figure 40 shows a photograph of a Western blot of MUCl*-targeting CAR. Numbers 1 to 9 are as follows: 1: MN-E6scFv-Fc-8-41BB-CD3z (human Fc as hinge with CD8 TM); 2: MN-E6scFv-FcH-8-41BB-CD3z (human Fc hingeless as hinge with CD8 TM); 3: MN-E6scFv-Fc-4-41BB-CD3z (human Fc as hinge with CD4 TM); 4: MN-E6scFv-FcH-4-41BB-CD3z (human Fc hingeless hinge with CD4 TM); 5: MN-E6scFv-IgD-8-41BB-CD3z (hinge from human IgD with CD8 TM); 6: MN-E6scFv-IgD-4-41BB-CD3z (hinge region from human IgD with CD4 TM); 7: MN-E6scFv-X4-8-41BB-CD3z (long flexible linker as hinge region with CD8 TM); 8: MN-E6scFv-X4-4-41BB-CD3z (long flexible linker as hinge region with CD4 TM); 9: MN-E6scFv-8-4-41BB-CD3z (hinge region from CD8 and CD4 with CD4 TM).

[0234] Figure 41 shows a graph of FACS scans of T47D breast cancer cells co-cultured with human T cells transduced with MN-E6scFv-Fc-8-41BB-CD3z, MN-E6scFv-FcH-8-41BB-CD3z (hingeless), MN-E6scFv-Fc-4-41BB-CD3z, MN-E6scFv-IgD-8-41BB-CD3z, MN-E6scFv-X4-8-41BB-CD3z, and MN-E6scFv-X4-4-41BB-CD3z. T cells and cancer cells were co-cultured at a 1:1 ratio for 48 hours. T cell counts were normalized to the average of all untransduced T cells, and target cells were normalized to each specific cell type when co-cultured with untransduced T cells. The graph shows that when CAR T cells are co-cultured with MUCl*-positive cancer cells, the T cell population expands and the targeted cancer cell population decreases.

[0235] Figure 42 shows graphs of FACS scans of T47D breast cancer cells, Capan-2 pancreatic cancer cells, K562-MUC1* transfected cells, and K562-wt cells cocultured with human T cells transduced with MN-E6scFv-Fc-8-41BB-CD3z, MN-E6scFv-FcH-8-41BB-CD3z, MN-E6scFv-Fc-4-41BB-CD3z, MN-E6scFv-IgD-8-41BB-CD3z, MN-E6scFv-X4-8-41BB-CD3z, and MN-E6scFv-X4-4-41BB-CD3z. T cells and cancer cells were cocultured at a 1:1 ratio for 48 hours. T cell counts were normalized to the average of all untransduced T cells, and target cells were normalized to each specific cell type when co-cultured with untransduced T cells. The graph shows that when CAR T cells are co-cultured with MUCl*-positive cancer cells, the T cell population expands and the target cancer cell population decreases.

[0236] As these experiments demonstrate, the key part of a CAR is the antibody fragment that targets immune cells to tumor cells. As we show in the next section, MN-E6 and MN-C2 are specific for the form of MUCl* expressed on tumor cells. The next most important part of a CAR is the cytoplasmic tail, which carries immune system costimulatory domains. The identity of these domains modulates the magnitude of the immune response but in no way affects specificity. As shown, the identity of the CAR's transmembrane domain is not critical. As long as the transmembrane domain has some flexibility and is long enough to allow the antibody fragment to reach its cognate receptor on tumor cells, this appears to be sufficient. This is demonstrated in Figures 40-42. CARs with a variety of different extracellular, transmembrane, and short cytoplasmic tails, including the MN-E6-targeting antibody fragment and the intracellular costimulatory domains 4-1BB and CD3-zeta, all worked, specifically killing target cells while stimulating the expansion of host T cells. These CARs with variable intermediate regions are: 1: MN-E6scFv-Fc-8-41BB-CD3z (human Fc as hinge region with CD8 TM); 2: MN-E6scFv-FcH-8-41BB-CD3z (human Fc as hinge region with CD8 TM hingeless); MN-E6scFv-Fc-4-41BB-CD3z (human Fc as hinge region with CD4 TM); 4: MN-E6scFv-FcH-4-41BB-CD3z (human Fc as hinge region with CD4 TM hingeless hinge region); 5: MN-E6scFv-IgD-8-41BB-CD3z (hinge region from human IgD with CD8 TM); 6: MN-E6scFv-IgD-4-41BB-CD3z (hinge region from human IgD with CD4 TM); 7: MN-E6scFv-X4-8-41BB-CD3z (long flexible linker as hinge region with CD8 TM); 8: MN-E6scFv-X4-4-41BB-CD3z (long flexible linker as hinge region with CD4 TM); 9: MN-E6scFv-8-4-41BB-CD3z (hinge region from CD8 and CD4 with CD4 TM).

[0237] One aspect of the invention is a method of treating a patient diagnosed with, or suspected of having, a MUCl-positive or MUCl*-positive cancer, or at risk of developing a MUCl-positive or MUCl*-positive cancer, wherein the patient is administered an effective amount of immune cells transduced with a MUCl*-targeting CAR, wherein the CAR is selected from the group consisting of: MN-E6-CD8-3z; MN-E6-CD4-3z;MN-E6-CD8-CD28-3z;MN-E6-CD4-CD28-3z;MN-E6-CD8-41BB-3z;MN-E6-CD4-41BB-3z;MN-E6-CD8-CD28-41 BB-3z;MN-E6-CD4-CD28-41BB-3z;MN-E6scFv-Fc-8-41BB-CD3z;MN-E6scFv-FcH-8-41BB-CD3z;MN-E6scFv-Fc-4-41BB-CD 3z; MN-E6scFv-FcH-4-41BB-CD3z; MN-E6scFv-IgD-8-41BB-CD3z; MN-E6scFv-IgD-4-41BB-CD3z; MN-E6scFv-X4-8-41BB-CD3z; MN-E6scFv-X4-4-41BB-CD3z; MN-E6scFv-8-4-41BB-CD3z, or any of the above CARs in which MN-E6 is replaced with MN-C2, MN-C3, or MN-C8. Another aspect of the invention is a method of treating a patient diagnosed with, suspected of having, or at risk of developing cancer, wherein the patient is administered an effective amount of immune cells transduced with one of the above-described CARs, wherein the NM-E6 has an antibody variable domain fragment replaced with a peptide that is specific for a cancer antigen. In any of the above methods, the immune cells can be T cells and can be further isolated from the patient being treated.

[0238] Specificity of anti-MUCl*-targeting antibodies

[0239] The most accurate way to demonstrate antibody specificity is to test the antibody on normal human tissue specimens compared with cancer tissue specimens. MN-C2 and MN-E6 were shown to specifically bind to MUCl or MUCl*-positive cancer cells. Several breast cancer arrays were analyzed using several anti-MUCl or MUCl* antibodies. Essentially, a study of serial sections of breast cancer tissue specimens from over 1,200 different breast cancer patients showed that very little full-length MUCl remains in breast cancer tissue. The majority of expressed MUCl is MUCl*, which is stained by MN-C2. The analysis was performed using Clarient diagnostics, and tissue staining was scored using the Alfred method. For example, Figure 43 shows serial sections of a breast cancer tissue array stained with VU4H5, a commercially available anti-MUCl antibody that binds to tandem repeats, or MN-C2, which binds MUCl*. Figures 43 and 44 are photographs of breast cancer tissue arrays stained with either VU4H5, which recognizes MUC1-FL (full-length), or MN-C2, which recognizes cancer MUCl*. Tissue staining was scored using the Alfred scoring method, which combines intensity and distribution scores. Below the tissue array photograph is a color-coded graph displaying the results. As can be seen, the VU4H5-stained array was very light, and many tissues showed no staining at all, despite published reports that MUCl is aberrantly expressed in over 96% of all breast cancers as evidenced by nucleic acid-based diagnostics. In contrast, the MN-C2-stained array was very dark (red or white in the graphs, compared to yellow). Furthermore, many tissues showed no staining with anti-full-length MUCl and very dark staining with MN-C2 (see green boxes in the graph). Similarly, we stained normal and cancerous breast tissues with humanized MN-E6 scFv-Fc. The antibody fragment was biotinylated so that it could be visualized with a second streptavidin-based antibody. As can be seen in Figure 45, hMN-E6 scFv-Fc does not stain normal breast tissue but does stain cancerous breast tissue. Furthermore, the intensity and homogeneity of staining increases with tumor grade and / or patient metastatic grade (Figures 45 and 46). Similarly, hMN-E6 scFv-Fc did not stain normal lung tissue but did stain lung cancer tissue (Figures 47-51), and the staining intensity and distribution increased as tumor grade or metastatic grade increased. Figure 52 shows photographs of normal and cancerous small intestinal tissues stained with 5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody and then stained with a secondary streptavidin HRP antibody. A) Normal small intestinal tissue. B) Small intestinal cancer from the patient indicated in the figure. C and D are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 53 shows photographs of normal small intestinal tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody and then stained with a secondary goat anti-human HRP antibody. A-D are normal small intestinal tissues. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 54 shows photographs of cancerous small intestine tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are cancerous small intestine tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 55 shows photographs of cancerous small intestine tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody; A-D are cancerous small intestine tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 56 shows photographs of normal colon tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are normal colons. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 57 shows photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are colon cancer tissues from patients with metastases as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 58 shows photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are colon cancer tissues from grade 2 patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 59 shows photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are colon cancer tissues from metastatic patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 60 shows photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 61 shows photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 62 shows photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUCl* antibody followed by a secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated in the figure. E-H are photographs of corresponding serial sections stained with the secondary antibody alone.

[0240] One aspect of the invention is a method of treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, in which a specimen is obtained from the patient's cancer and tested for reactivity with antibodies that bind to PSMGFR SEQ ID NO:2, SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620), or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621). The patient is then treated with an scFv, scFv-Fc, or CAR T comprising antibody variable construct fragments from antibodies that reacted with the cancer specimen. Another aspect of the invention is a method of treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, in which specimens are obtained from the patient's cancer and tested for reactivity with MN-E6-scFv, MN-C2-scFv, MN-C3-scFv, or MN-C8-scFv; the patient is then treated with an scFv, scFv-Fc-mut, or CAR T containing the portion of the antibody that reacted with the cancer specimen.

[0241] BiTES

[0242] Divalent (divalent or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by creating a single peptide chain with two VH and two VL domains, resulting in tandem scFvs. Another possibility is to generate scFvs with a linker peptide (approximately five amino acids) that is too short to allow the two variable regions to fold together, forcing the scFvs to dimerize. This type of scFv is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than comparable scFvs, meaning they have much higher affinity for their targets. Therefore, diabody drugs can be administered at much lower doses than other therapeutic antibodies and are capable of highly specific targeting of cancers in vivo. Even shorter linkers (one or two amino acids) allow the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. They exhibit even higher affinity for their targets than diabodies.

[0243] All of these formats can be composed of variable fragments with specificity for two different antigens, in which case they are a type of bispecific antibody. The earliest developed are bispecific tandem di-scFvs, known as bispecific T cell engagers (BiTE antibody constructs). BiTEs are fusion proteins consisting of two scFvs of different antibodies on a single peptide chain of approximately 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule, such as aberrantly expressing MUC1*.

[0244] Another aspect of the invention is a method of treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, in which an effective amount of a BiTE is administered, wherein one antibody variable fragment of the BiTE binds to a T cell surface antigen and another antibody variable fragment of the BiTE binds to PSMGFR SEQ ID NO:2, SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620), or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621). In one case, the antibody variable fragment of the BiTE that binds MUC1* comprises a portion of huMN-E6, huMN-C2, huMN-C3, or huMN-C8.

[0245] In another aspect of the invention, MUC1* peptides containing all or part of PSMGFR (SEQ ID NO: 2), SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620), or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621) are used in an adoptive T cell approach. In this case, a patient's T cells are exposed to the MUC1* peptide, and through various rounds of maturation, the T cells develop MUC1*-specific receptors. The adapted T cells are then expanded and administered to patients diagnosed with, suspected of having, or at risk of developing MUC1*-positive cancer.

[0246] Other MUC1 cleavage sites

[0247] However, MUC1 is cleaved into the growth factor receptor form, MUCl*, in some normal cells, in addition to cancer cells. For example, MUC1 is cleaved into MUCl* in healthy stem and progenitor cells. A large percentage of bone marrow cells are MUCl* positive. Portions of the intestine are MUCl* positive.

[0248] The present inventors have discovered that MUC1 can be cleaved at different positions relatively close to each other, but the position of cleavage alters the fold of the remainder of the extracellular domain. As a result, monoclonal antibodies can be identified that bind to MUC1* cleaved at one position but not to MUC1* cleaved at another position. This discovery is disclosed in WO 2014 / 028668, filed August 14, 2013, the contents of which are incorporated herein by reference in their entirety. The present inventors have identified a set of anti-MUCl* monoclonal antibodies that bind to MUC1* as it appears on cancer cells but not to MUC1* as it appears on stem and progenitor cells. Conversely, the present inventors have identified another set of monoclonal antibodies that bind to stem and progenitor cells but not to cancer cells. One method used to identify stem-specific antibodies is as follows: Supernatants from monoclonal hybridomas were adsorbed separately onto two multiwell plates. Stem cells, which are nonadherent cells, were placed on one plate, and adherent cancer cells were placed on the same plate. After incubation, the plates were rinsed and inverted. If the nonadherent stem cells attached to the plate, the monoclonal antibody in that particular well would recognize the stem cells and not the cancer cells. Antibodies that did not capture stem cells or that captured cancer cells were identified as cancer-specific stem cells. FACS analysis confirmed this method of investigation. Antibodies MN-E6 and MN-C2 are examples of cancer-specific antibodies. Antibodies MN-C3 and MN-C8 are examples of stem-specific antibodies. Although both sets of antibodies can bind to a peptide containing the PSMGFR sequence, FACS analysis shows that the anti-MUCl* polyclonal antibody and MN-C3 bind to MUCl*-positive bone marrow cells, but not MN-E6. The MUCl* polyclonal antibody was generated by immunizing rabbits with the PSMGFR peptide. Similarly, MN-C3 binds to intestinal crypt stem cells, but not MN-E6. Conversely, the MN-E6 antibody binds to cancer tissue, whereas the stem-specific MN-C3 does not. Competitive ELISA experiments show that the C-terminal 10 amino acids of the PSMGFR peptide are required for binding to MN-E6 and MN-C2, but not to MN-C3 and MN-C8. Therefore, another method for identifying cancer-specific antibodies is to immunize with a peptide containing the sequence of the PSMGFR peptide minus the 10 N-terminal amino acids and use that peptide to screen for cancer-specific antibodies or antibody fragments. An antibody that binds to a peptide having a sequence of the PSMGFR peptide minus the N-terminal 10 amino acids, but does not bind to a peptide having a sequence of the PSMGFR peptide minus the C-terminal 10 amino acids, is a cancer-specific antibody that can be used to treat or prevent cancer.

[0249] The extracellular domain of MUC1 is also cleaved in stem cells and some progenitor cells, where activation of cleaved MUC1 by the ligand NME1, either in a dimeric form or NME7, promotes proliferation and pluripotency and inhibits differentiation. The transmembrane portion of MUC1 that remains after cleavage is called MUCl*, and its extracellular domain consists essentially of the primary sequence of the MUC1 growth factor receptor (PSMGFR) sequence. However, the exact site of cleavage may vary depending on which cell type, tissue type, or individual expresses or overexpresses the cleavage enzyme. In addition to the cleavage site previously identified by the present inventors, which discards the transmembrane portion of MUCl* containing most or all of PSMGFR SEQ ID NO:2, other cleavage sites result in an extended MUCl* composed of most or all of SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:620); or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:621). The site of MUC1 cleavage affects how the remaining extracellular domain folds. The present inventors have identified a monoclonal antibody that binds to cleaved MUCl* in cancer cells but not to cleaved MUCl* as present in healthy stem and progenitor cells.

[0250] An anti-MUCl* antibody or antibody-like molecule can be very effective if it competitively inhibits the binding of NMEl, NME6, NME8, NME7, or NME7-AB to MUC1*, for example, if the antibody cannot bind to the PSMGFR peptide, or if the 10 C-terminal amino acids are deleted. Antibodies that bind to the PSMGFR sequence, particularly antibodies or antibody-like molecules bearing a payload, need not competitively inhibit the binding of the MUCl* ligand to be effective as anti-cancer agents. For example, antibodies or antibody-like molecules that are conjugated to a toxin can be effective in killing target cancer cells without necessarily inhibiting the binding of the activating ligand. For example, antibody-like molecules or antibodies, such as CARs or BiTEs, that recruit the immune system to a patient's tumor can be effective as anti-cancer agents, provided that the antibody fragment targets a portion of MUCl* such that the antibody fragment binding does not competitively inhibit the binding of NMEl, NME6, NME8, NME7-AB, or NME7. In a preferred embodiment, the antibody fragment incorporated into the CAR, adaptive T cell receptor, or BiTE competitively inhibits binding of NME1, NME6, NME8, NME7-AB, or NME7 to MUCl*.

[0251] Antibodies capable of binding to the extracellular domain of the remaining transmembrane segment block the interaction between the MUCl* extracellular domain and an activating ligand and, in this manner, can be used as therapeutic agents, for example, to treat cancer. Furthermore, anti-MUCl* antibodies are useful for the proliferation, transport, identification, or isolation of stem cells both in vitro and in vivo.

[0252] A general strategy for using antibodies, antibody fragments, and CARs targeting the extracellular domain of MUC1*

[0253] The monoclonal antibodies MN-C3 and MN-C8 have greater binding affinity for stem cells than for cancer cells. Humanized antibodies and antibody fragments containing sequences derived from the variable regions of humanized MN-C3 and MN-C8 can be used as adhesive surface coatings for human stem cells.

[0254] Alternatively, humanized antibodies and antibody fragments containing sequences derived from the variable regions of MN-C3 and MN-C8 can be used to transport stem cells to specific locations for in situ human therapy. In one case, a substrate coated with humanized MN-C3- or MN-C8-derived antibodies or antibody fragments is loaded with stem cells and then administered to a patient. In another case, a substrate coated with humanized MN-C3- or MN-C8-derived antibodies or antibody fragments is administered to a patient to recruit the patient's own stem cells to a specific area for therapy. Human therapies in which antibodies that bind to human stem cells are useful include spinal cord repair. Substrates coated with humanized MN-C3- or MN-C8-derived antibodies or antibody fragments are used to identify or isolate human antibodies. Humanized MN-C3- or MN-C8-derived antibodies can also be used to stimulate stem cell proliferation.

[0255] Free text in sequence listing

[0256] For the use of nucleotide symbols other than a, g, c, and t, they follow the conventions set out in WIPO Standard ST.25, Appendix 2, Table 1, where k represents t or g; n represents a, c, t, or g; m represents a or c; r represents a or g; s represents c or g; w represents a or t; and y represents c or t. MUC1 Receptor(Mucin 1 precursor, GenbankAccession nuMber: P15941) PSMGFR GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2) Human NME1(DNA) atggccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaaggattccgccttgttggtctg aaattcatgcaagcttccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgcactcagggccggtagttgccatgg tctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactccaagcctgggaccatccgtggagacttctgcatacaagttggcaggaa cattatacatggcagtgattctgtggagagtgcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatctatgaatga (Sequence number: 3) (amino acid) MANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEKEIGLWFHPEELVDYTSCAQNWIYE- (SEQ ID NO: 4) Human NME7(DNA) (amino acid) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPI IAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN- (SEQ ID NO: 6) NME7 peptide NME7A peptide 1 (A domain) MLSRKEALDFHVDHQS (SEQ ID NO: 7) NME7A peptide 2 (A domain) SGVARTDASES (SEQ ID NO: 8) NME7B peptide 1 (B domain) DAGFEISAMQMFNMDRVNVE (SEQ ID NO: 9) NME7B peptide 2 (B domain) EVYKGVVTEYHDMVTE (SEQ ID NO: 10) NME7B peptide 3 (B domain) AIFGKTKIQNAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 11) Mouse E6 heavy chain, variable region sequence: (DNA) gaggtgaaggtggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgtagtctctggattcactttcagtagatatggcatgtcttgggttcgccagactccaggcaagaggctggagtgggtcgcaaccattagtggtggcggtacttacatctactatccagac agtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatcactgtacaagggataactacggtaggaactacgactacggtatggactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO: 12) (amino acid) EVKVVESGGDLVKPGGSLKLSCWSGFTFSRYGMSWVRQTPGKRLEWVATISGGGTYIYYPDSVKGRFTISRD NAKNTLYLQMSSLKSEDTAMYHCTRDNYGRNYDYGMDYWGQGTSVTVSS (SEQ ID NO: 13) Mouse E6 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtgaaggtggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgtagtctct(SEQ ID NO: 14) (amino acid) EVKVVESGGDLVKPGGSLKLSCWSGFTFS (SEQ ID NO: 15) Mouse E6 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) ggattcactttcagtagatatggcatgtct (SEQ ID NO: 16) (amino acid) RYGMS (SEQ ID NO: 17) Mouse E6 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggttcgccagactccaggcaagaggctggagtgggtcgca (SEQ ID NO: 18) (amino acid) WVRQTPGKRLEWVA (SEQ ID NO: 19) Mouse E6 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) accattagtggtggcggtacttacatctactatccagacagtgtgaagggg (SEQ ID NO: 20) (amino acid) TISGGGTYIYYPDSVKG (SEQ ID NO: 21) Mouse E6 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatcactgtacaagg (SEQ ID NO: 22) (amino acid) RFTISRDNAKNTLYLQMSSLKSEDTAMYHCTR (SEQ ID NO: 23) Mouse E6 heavy chain variable complementarity determining region 3 (CDR3) sequence: (DNA) gataactacggtaggaactacgactacggtatggactac (SEQ ID NO: 24) (amino acid) DNYGRNYDYGMDY (SEQ ID NO: 25) IGHV3-21*03 heavy chain variable region sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatcc attagtagtagtagttacatatactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaga(SEQ ID NO: 26) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 27) IGHV3-21*01 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagt (SEQ ID NO: 28) (Amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 29) IGHV3-21*01 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agctatagcatgaac (SEQ ID NO: 30) (amino acid) SYSMN (SEQ ID NO: 31) IGHV3-21*01 heavy chain variable framework region 2 (FWR2) sequence: (DNA)tgggtccgccaggctccagggaaggggctggagtgggtctca (SEQ ID NO: 32) (Amino acid) WVRQAPGKGLEWVS (SEQ ID NO: 33) IGHV3-21*01 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) tccattagtagtagtagtagttacatatactacgcagactcagtgaagggc (SEQ ID NO: 34) (amino acid)SISSSSSSYIYYADSVKG (SEQ ID NO: 35) IGHV3-21*01 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaga (SEQ ID NO: 36) (amino acid) RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 37) Humanized E6 heavy chain variable region sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaaccattagtggcggaggcacctacatatactaccca gactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagc (Sequence number: 38) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRD NAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSS (SEQ ID NO: 39) Humanized E6 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagt (SEQ ID NO: 40) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 41) Humanized E6 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) aggtatggcatgagc (SEQ ID NO: 42) (amino acid) RYGMS (SEQ ID NO: 43) Humanized E6 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggtccgccaggctccagggaagaggctggagtgggtctca (SEQ ID NO: 44) (amino acid) WVRQAPGKRLEWVS (SEQ ID NO: 45) Humanized E6 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) accattagtggcggaggcacctacatatactacccagactcagtgaagggc (SEQ ID NO: 46) (amino acid )TISGGGTYIYYPDSVKG (SEQ ID NO: 47) Humanized E6 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaacgccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccaga (SEQ ID NO: 48) (amino acid) RFTISRDNAKNTLYLQMNSLRAEDTAVYYCTR (SEQ ID NO: 49) Humanized E6 heavy chain variable complementarity determining region 3 (CDR3) sequence: (DNA) gataactatggccgcaactatgattatggcatggattat (SEQ ID NO: 50) (amino acid) DNYGRNYDYGMDY (SEQ ID NO: 51) Humanized E6 IgG2 heavy chain synthesized by Genescript: (DNA) gaattctaagcttgggccaccatggaactggggctccgctgggttttccttgttgctattttagaaggtgtccagtgtgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaaccattagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcgcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgac(SEQ ID NO: 52) (amino acid) EF*AWATMELGLRWVFLVAILEGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* *V*TLX(SEQ ID NO: 53) Human IgG2 heavy chain constant region sequence: (DNA) gcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcagg actctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttctcttt ccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagecacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtca gcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccccgggaggagatgaccaagaaccaggtcagcc tgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatatag (sequence number: 54) (amino acid) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55) Humanized E6 IgG1 heavy chain sequence: (DNA) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNPLYLQMNSLRAEDTAVYYCPRDNYGRNYDYGMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* * (SEQ ID NO: 57) Human IgG1 heavy chain constant region sequence: (DNA) gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgacagtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataa(SEQ ID NO: 58) (amino acid) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* * (SEQ ID NO: 59) Human IgGl heavy chain constant region gBLOCK#l sequence: (DNA) atggcatggattattggggccagggcaccctggtgaccgtgagcagcgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgacagtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaag (SEQ ID NO: 60) Human IgG1 heavy chain constant region gBLOCK#2 sequence: (DNA) tacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtct ccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaa aggcttctatcccagcgacatcgccgtggagtggggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcagg tggcagcaggggaacgtcttcteatgetccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataagtttaaacccgctgatcagcctcgactgtgccttctagttg (Sequence number: 61) E6 heavy chain variable region overlap sequence: (DNA) atggcatggattattggggccagggcaccct (SEQ ID NO: 62) IgGl heavy chain constant region overlap region sequence: (DNA) tacgtggacggcgtggaggtgcataatgccaag (SEQ ID NO: 63) pCDNA3.1 V5 and pSECTag2: (DNA) ccgctgatcagcctcgactgtgccttctagttg (SEQ ID NO: 64) Mouse E6 light chain variable region sequence: (DNA) caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggaggaggtcaccctaacctgcagtgccacctcaagtgtaagttacatacactggttccagcagaggccaggcacttctcccaaactctggatttatagcacatccaacctg gcttctggagtccctgttcgcttcagtggcagtggatatgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgccagcaaaggagtagttccccattcacgttcggctcggggacaaagttggaaataaaa (Sequence number: 65) (amino acid) QIVLTQSPAIMSASPGEEVTLTCSATSSVSYIHWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIK (SEQ ID NO: 66) Mouse E6 light chain variable framework region 1 (FWR1) sequence: (DNA) caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggaggaggtcaccctaacctgc (SEQ ID NO: 67) (amino acid) QIVLTQSPAIMSASPGEEVTLTC (SEQ ID NO: 68) Mouse E6 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) AGTGCCACCTCAAGTGTAAGTTACATACAC (SEQ ID NO: 69) (amino acid) SATSSVSYIH (SEQ ID NO: 70) Mouse E6 light chain variable framework region 2 (FWR2) sequence: (DNA) tggttccagcagaggccaggcacttctcccaaactctggatttat (SEQ ID NO: 71) (amino acid) WFQQRPGTSPKLWIY (SEQ ID NO: 72) Mouse E6 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) agcacatccaacctggcttct (SEQ ID NO: 73) (amino acid) STSNLAS (SEQ ID NO: 74) Mouse E6 light chain variable framework region 3 (FWR3) sequence: (DNA) ggagtccctgttcgcttcagtggcagtggatatgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgc (SEQ ID NO: 75) (amino acid) GVPVRFSGSGYGTSYSLTISRMEAEDAATYYC (SEQ ID NO: 76) Mouse E6 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) cagcaaaggagtagttccccattcacg (SEQ ID NO: 77) (amino acid) QQRSSSPFT (SEQ ID NO: 78) IGKV3-11*02 light chain variable region sequence: (DNA) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagectggtaccaacagaaacctggccaggctcccaggctccteateta tgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggagagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctcc (SEQ ID NO: 79) (amino acid) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGRDFTLTISSLEPEDFAVYYCQQRSNWPP (SEQ ID NO: 80) IGKV3-11*02 light chain variable framework region 1 (FWR1) sequence: (DNA) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgc (SEQ ID NO: 81) (amino acid) EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 82) IGKV3-11*02 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agggccagtcagagtgttagcagctacttagcc (SEQ ID NO: 83) (amino acid) RASQSVSSYLA (SEQ ID NO: 84) IGKV3-11*02 light chain variable framework region 2 (FWR2) sequence: (DNA) tggtaccaacagaaacctggccaggctcccaggctcctcatctat (SEQ ID NO: 85) (amino acid) WYQQKPGQAPRLLIY (SEQ ID NO: 86) IGKV3-11*02 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA)gatgcatccaacagggccact (SEQ ID NO: 87) (amino acid) DASNRAT (SEQ ID NO: 88) IGKV3-11*02 light chain variable framework region 3 (FWR3) sequence: (DNA) ggcatcccagccaggttcagtggcagtgggtctggggagagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgt (SEQ ID NO: 89) (amino acid) GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 90) IGKV3-11*02 light chain variable complementarity-determining region 3 (CDR3) sequence (DNA) cagcagcgtagcaactggcctcc (SEQ ID NO: 91) (amino acid) QQRSNWPP (SEQ ID NO: 92) Humanized E6 light chain variable region sequence: (DNA) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacctccaacctg gccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagttattactgtcagcagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaa (Sequence number: 93) (amino acid) EIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO: 94) Humanized E6 light chain variable framework region 1 (FWR1) sequence: (DNA) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgc (SEQ ID NO: 95) (amino acid) EIVLTQSPATLSLSPGERATLTC (SEQ ID NO: 96) Humanized E6 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agcgccaccagcagtgttagctacatccac (SEQ ID NO: 97) (amino acid) SATSSVSYIH (SEQ ID NO: 98) Humanized E6 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tggtaccaacagaggcctggccagagccccaggctcctcatctat (SEQ ID NO: 99) (amino acid) WYQQRPGQSPRLLIY (SEQ ID NO: 100) Humanized E6 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) agcacctccaacctggccagc (SEQ ID NO: 101) (amino acid) STSNLAS (SEQ ID NO: 102) Humanized E6 light chain variable framework region 3 (FWR3) sequence: (DNA) ggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgt (SEQ ID NO: 103) (amino acid) GIPARFSGSGSGSDYTLTISSLEPEDFAVYYC (SEQ ID NO: 104) Humanized E6 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) cagcagcgtagcagctcccctttcacc (SEQ ID NO: 105) (amino acid) QQRSSSPFT (SEQ ID NO: 106) Humanized E6 kappa light chain, synthesized by Genescript: (DNA) gaattctaagcttgggccaccatggaagccccagcgcagcttctcttctcctgctactctggctcccagataccactggagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactggta ccaacagaggcctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttatactgtcagcagcgtagcagctcccctttcacct ttggcagcggcaccaaagtggaaattaaaggacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacaggacaggacacctacagcctcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggagtgttagtaagtttaaactctaga (sequence number: 107) (amino acid) EF*AWATMEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSS PFTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* *V*TLX (array number: 108) Human kappa light chain constant region sequence: (DNA) aaggacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccagga gagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (Sequence number: 109) (amino acid) RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 110) Humanized E6 λ light chain sequence: (DNA) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatagtaa (SEQ ID NO: 111) (Amino acid) EIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS * * (SEQ ID NO: 112) Humanized lambda light chain constant region sequence: (DNA) ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggag ccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagc ctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatagtaa (SEQ ID NO: 113) (amino acid) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS* * (SEQ ID NO: 114) Human lambda light chain constant region gBLOCK#3 sequence: (DNA) (SEQ ID NO: 115) E6 light chain variable region overlap sequence: (DNA) agcgccaccagcagtgttagctacatccact (SEQ ID NO: 116) pCDNA3.1 V5 and pSECTag2 overlapping sequences: (DNA) ccgctgatcagcctcgactgtgccttctagttg (SEQ ID NO: 117) Mouse C2 heavy chain variable region sequence: (DNA) gaggtccagctggaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtggctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattagtagtggtggtacttatatctactatccagacagtgtgaaggg gcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagacttgggggggataattactacgaatacttcgatgtctggggcgcagggaccacggtcaccgtctcctccgccaaaacgacacccccatctgtctat(SEQ ID (array number: 118) (amino acid) EVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVATISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLGGDNYYEYFDVWGAGTTVTVSSAKTTPPSVY (SEQ ID NO: 119) Mouse C2 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtccagctggaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagt (SEQ ID NO: 120) (amino acid) EVQLEESGGGLVKPGGSLKLSCAASGFTFS (SEQ ID NO: 121) Mouse C2 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) ggctatgccatgtct (SEQ ID NO: 122) (Amino acid) GYAMS (SEQ ID NO: 123) Mouse C2 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggttcgccagactccggagaagaggctggagtgggtcgca (SEQ ID NO: 124) (amino acid) WVRQTPEKRLEWVA (SEQ ID NO: 125) Mouse C2 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) accattagtagtggtggtacttatatctactatccagacagtgtgaagggg (SEQ ID NO: 126) (amino acid) TISSGGTYIYYPDSVKG (SEQ ID NO: 127) Mouse C2 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaaga (SEQ ID NO: 128) (amino acid) RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR (SEQ ID NO: 129) Mouse C2 heavy chain variable complementarity determining region 3 (CDR3) sequence: (DNA) cttgggggggataattactacgaatacttcgatgtc (SEQ ID NO: 130) (amino acid) LGGDNYYEYFDV (SEQ ID NO: 131) IGHV3-21*04 heavy chain variable region sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatcc attagtagtagtagttacatatactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaga(SEQ ID NO: 132) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 133) IGHV3-21*04 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagt (SEQ ID NO: 134) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 135) IGHV3-21*04 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agctatagcatgaac (SEQ ID NO: 136) (amino acid) SYSMN (SEQ ID NO: 137) IGHV3-21*04 heavy chain variable framework region 2 (FWR2) sequence: (DNA) gggtccgccaggctccagggaaggggctggagtgggtctca (SEQ ID NO: 138) (amino acid) WVRQAPGKGLEWVS (SEQ ID NO: 139) IGHV3-21*04 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA)tccattagtagtagtagtagttacatatactacgcagactcagtgaagggc (SEQ ID NO: 140) (amino acid) SISSSSSYIYYADSVKG (SEQ ID NO: 141) IGHV3-21*04 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaga (SEQ ID NO: 142) (amino acid) RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 143) Humanized C2 heavy chain variable region sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatactacc ccgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctcc (Sequence number: 144) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSS (SEQ ID NO: 145) Humanized C2 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagt (SEQ ID NO: 146) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 147) Humanized C2 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) ggctatgccatgagc (SEQ ID NO: 148) (amino acid) GYAMS (SEQ ID NO: 149) Humanized C2 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggtccgccaggctccagggaaggggctggagtgggtctcaa (SEQ ID NO: 150) (amino acid) WVRQAPGKGLEWVS (SEQ ID NO: 151) Humanized C2 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) accattagtagtggcggaacctacatatactaccccgactcagtgaagggc (SEQ ID NO: 152) (amino acid) TISSGGTYIYYPDSVKG (SEQ ID NO: 153) Humanized C2 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaga (SEQ ID NO: 154) (amino acid) RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 155) Humanized C2 heavy chain variable complementarity determining region 3 (CDR3) sequence: (DNA) cttgggggggataattactacgaatacttcgatgtc (SEQ ID NO: 156) (amino acid) LGGDNYYEYFDV (SEQ ID NO: 157) Humanized C2 IgG1 heavy chain sequence (DNA) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWG KGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* * (SEQ ID NO: 159) Humanized C2 gBLOCK#4 sequence: (DNA) actcactataggagacccaagctggctagttaagcttgggccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgaggtgcagctggtggagtctgggggaggcctggtcaagcctggg gggtccctgagactctcctgtgcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatactacccccgactcagtgaagggcc gattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcac cgtctcctccgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccacc (Sequence number: 160) pCDNA3.1 V5 overlap sequence: (DNA) actcactatagggagacccaagctggctagtt (SEQ ID NO: 161) Human IgG1 constant region overlap sequence: (DNA) gacggtgtcgtggaactcaggcgccctgaccagc (SEQ ID NO: 162) Humanized C2 IgG2 heavy chain sequence (DNA) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* * (SEQ ID NO: 164) Humanized C2 gBLOCK#5 sequence: (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggccgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtggc tatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatactacccgactcagtgaagggccgattcaccatctccagagacaacgccaagaa ctcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctccgcct ccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgacca (Sequence number: 165) pSEC Tag2 overlap sequence: (DNA) tgctctgggttccaggttccactggtgacgc (SEQ ID NO: 166) Human IgG2 constant region overlap sequence: (DNA) gacggtgtcgtggaactcaggcgctctgacca (SEQ ID NO: 167) Mouse C2 light chain variable region sequence: (DNA) gacattgtgatcacacagtctacagcttccttaggtgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctatatagttatatgcactggtaccaacagagaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatct ggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgttcacgttcggaggggggaccaagctggagataaaacgggctgatgctgcaccaactgtatcc (Sequence number: 168) (amino acid )DIVITQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGGGTKLEIKRADAAPTVS (SEQ ID NO: 169) Mouse C2 light chain variable framework region 1 (FWR1) sequence: (DNA) gacattgtgatcacacagtctacagcttccttaggtgtatctctggggcagagggccaccatctcatgc (SEQ ID NO: 170) (amino acid) DIVITQSTASLGVSLGQRATISC (SEQ ID NO: 171) Mouse C2 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agggccagcaaaagtgtcagtacatctggctatagttatatgcac (SEQ ID NO: 172) (amino acid) RASKSVSTSGYSYMH (SEQ ID NO: 173) Mouse C2 light chain variable framework region 2 (FWR2) sequence: (DNA) tggtaccaacagagaccaggacagccacccaaactcctcatctat (SEQ ID NO: 174) (amino acid) WYQQRPGQPPKLLIY (SEQ ID NO: 175) Mouse C2 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) cttgcatccaacctagaatc (SEQ ID NO: 176) (amino acid) LASNLES (SEQ ID NO: 177) Mouse C2 light chain variable framework region 3 (FWR3) sequence: (DNA) tggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgt (SEQ ID NO: 178) (amino acid) GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 179) Mouse C2 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) cagcacagtagggagcttccgttcacg (SEQ ID NO: 180) (amino acid) QHSRELPFT (SEQ ID NO: 181) IGKV7-3*01 light chain variable region sequence: (DNA) gacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtgagagtgtcagtttcttgggaataaacttaattcactggtatcagcagaaaccaggacaacctcctaaactcctga tttaccaagcatccaataaagacactggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtctgcagagtaagaattttcctcccaca (SEQ ID NO: 182) (amino acid) DIVLTQSPASLAVSPGQRATITCRASESVSFLGILIHWYQQKPGQPPKLLIYQASNKDTGVPARFSGSGSGTDFTLTINPVEANDTANYYCLQSKNFPPT (SEQ ID NO: 183) IGKV7-3*01 light chain variable framework region 1 (FWR1) sequence: (DNA) gacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgc (SEQ ID NO: 184) (amino acid) DIVLTQSPASLAVSPGQRATITC (SEQ ID NO: 185) IGKV7-3*01 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agagccagtgagagtgtcagtttcttgggaataaacttaattcac (SEQ ID NO: 186) (amino acid) RASESVSFLGINLIH (SEQ ID NO: 187) IGKV7-3*01 light chain variable framework region 2 (FWR2) sequence: (DNA) tggtatcagcagaaaccaggacaacctcctaaactcctgatttac (SEQ ID NO: 188) (amino acid) WYQQKPGQPPKLLIY (SEQ ID NO: 189) IGKV7-3*01 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) caagcatccaataaagacact (SEQ ID NO: 190) (amino acid) QASNKDT (SEQ ID NO: 191) IGKV7-3*01 light chain variable framework region 3 (FWR3) sequence: (DNA) ggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgt (SEQ ID NO: 192) (amino acid) GVPARFSGSGSGTDFTLTINPVEANDTANYYC (SEQ ID NO: 193) Humanized C2 light chain variable region sequence: (DNA) gacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatc tggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaact (SEQ ID NO: 194) (amino acid) DIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO: 195) Humanized C2 light chain variable framework region 1 (FWR1) sequence: (DNA) gacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgc (SEQ ID NO: 196) (amino acid) DIVLTQSPASLAVSPGQRATITC (SEQ ID NO: 197) Humanized C2 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agagccagtaagagtgtcagtaccagcggatactcctacatgcac (SEQ ID NO: 198) (amino acid) RASKSVSTSGYSYMH (SEQ ID NO: 199) Humanized C2 heavy / light variable framework region 2 (FWR2) sequence: (DNA) tggtatcagcagaaaccaggacaacctcctaaactcctgatttac (SEQ ID NO: 200) (amino acid) WYQQKPGQPPKLLIY (SEQ ID NO: 201) Humanized C2 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) ctggcatccaatctggagagc (SEQ ID NO: 202) (amino acid) LASNLES (SEQ ID NO: 203) Humanized C2 light chain variable framework region 3 (FWR3) sequence: (DNA) ggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgt (SEQ ID NO: 204) (amino acid) GVPARFSGSGSGTDFTLTINPVEANDTANYYC (SEQ ID NO: 205) Humanized C2 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) cagcacagtagggagctgcctttcaca (SEQ ID NO: 206) (amino acid) QHSRELPFT (SEQ ID NO: 207) Humanized C2 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) ctgcagagtaagaattttcctcccaca (SEQ ID NO: 208) (amino acid) LQSKNFPPT (SEQ ID NO: 209) Humanized C2 gBLOCK#6 sequence (kappa light chain in pCDNA3.1 V5): (DNA) actcactatagggagacccaagctggctagttaagcttgggccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagetcgcccgtcacaaagagcttcaacaggggagagtgttagtaagtttaaacccgctgatcaccctcgactgtgccttctagttg(SEQ ID NO: 210) pCDNA3.1 V5 5'-Repeat Sequence: (DNA)actcactatagggagacccaagctggctagtt(SEQ ID NO: 211) pCDNA3.1 V5 3'-Repeat Sequence: (DNA) ccgctgatcagcctcgactgtgccttctagttg (SEQ ID NO: 212) Humanized C2 gBLOCK#7 sequence (κ light chain in pSEC Tag2): (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggccgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagetcgcccgtcacaaagagcttcaacaggggagagtgttagtaagtttaaacccgctgatcaccetcgactgtgccttctagttg (SEQ ID NO: 213) pSEC Tag2 5' repeat sequence: (DNA) tgctctgggttccaggttccactggtgacgc (SEQ ID NO: 214) pSEC Tag2 3' overlap sequence: (DNA) ccgctgatcagcctcgactgtgccttctagttg (SEQ ID NO: 215) Humanized C2 gBLOCK#8 sequence (lambda light chain in pCDNA3.1 V5) (DNA) actcactatagggagacccaagctggctagttaagcttgggccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatagtaagtttaaacccgctgatcaccctcgactgtgccttctagttg (SEQ ID NO: 216) pCDNA3.1 V5 5'repeat sequence: (DNA) actcactatagggagacccaagctggctagtt (SEQ ID NO: 217) pCDNA3.1 V5 3’ Repeat Sequence: (DNA) ccgctgatcagcctcgactgtgccttctagttg (SEQ ID NO: 218) Humanized C2 gBLOCK#9 Sequence (λ light chain in pSEC Tag2): (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggccgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatagtaagtttaaacccgctgatcagcctcgactgtgccttctagttg (SEQ ID NO: 219) pSEC Tag2 5' Repeat Sequence: (DNA) tgctctgggttccaggttccactggtgacgc (SEQ ID NO: 220) pSEC Tag2 3' overlap sequence: (DNA) ccgctgatcagcctcgactgtgccttctagttg (SEQ ID NO: 221) Murine Ig kappa chain leader sequence: (DNA) atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac (SEQ ID NO: 222) (amino acid) METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 223) Interleukin 2 (IL-2) leader sequence (DNA) atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagt (SEQ ID NO: 224) (amino acid) MYRMQLLSCIALSLALVTNS (SEQ ID NO: 225) CD33 leader sequence (DNA) atgcctcttctgcttctgcttcctctgctttgggctggagctcttgct (SEQ ID NO: 226) (amino acid) MPLLLLLPLLWAGALA (SEQ ID NO: 227) IGHV3-21*03 leader sequence (DNA) atggaactggggctccgctgggttttccttgttgctattttagaaggtgtccagtgt (SEQ ID NO: 228) (amino acid) MELGLRWVFLVAILEGVQC (SEQ ID NO: 229) IGHV3-11*02 leader sequence (DNA) atggaagccccagcgcagcttctcttcctcctgctactctggctcccagataccactgga (SEQ ID: 230) (Amino acid) MEAPAQLLFLLLLWLPDTTG (SEQ ID: 231) Humanized E6 single-stranded GS3 (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaaccattagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcggcggtggcggatccggcggtggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaa (SEQ ID: 232) (Amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO: 233) Humanized E6 single-chain IgGlnoC (DNA) (SEQ ID NO: 234) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRD NAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSDKTHTKPPKPAPELLGGPGTGEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO: 235) Humanized E6 single chain X4 (linker is IgG1 and IgG2 modified hinge region) (DNA) (SEQ ID NO: 236) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRD NAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSDKTHTKPPKPAPELLGGPGTGTGGPTIKPPKPPKPAPNLLGGPEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO: 237) Humanized C2 single chain GS3 (DNA) (SEQ ID NO: 238) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO: 239) Humanized C2 single-chain IgG (cysteine-free) (DNA) (SEQ ID NO: 240) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSDKTHTKPPKPAPELLGGPGTGDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO: 241) Humanized C2 single chain X4 (linker is IgG1 and IgG2 modified hinge region) (DNA) (SEQ ID NO: 242) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSDKTHTKPPKPAPELLGGPGTGTGGPTIKPPKPPKPAPNLLGGPDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO: 243) Humanized C3 single chain GS3 (DNA) (SEQ ID NO: 244) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRT (SEQ ID NO: 245) Humanized C3 single chain IgGl (no cysteine) (DNA) (SEQ ID NO: 246) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQGTTLTVSSDKTHTKPPKPAPELLGGPGTGDIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRT (SEQ ID NO: 247) Humanized C3 single chain X4 (linker is IgG1 and IgG2 modified hinge region) (DNA) (SEQ ID NO: 248) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQGTTLTVSSDKTHTKPPKPAPELLGGPGTGTGGPTIKPPKPPKPAPNLLGGPDIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRT (SEQ ID NO: 249) Humanized C8 single-chain GS3 (linker is [Gly4Ser1]3) (DNA) (SEQ ID NO: 250) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGKGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHIRELTRSEFGGGTKVEIKRT (SEQ ID NO: 251) Humanized C8 single chain IgGl (no cysteine) (DNA) (SEQ ID NO: 252) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGKGTTVTVSSDKTHTKPPKPAPELLGGPGTGDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHIRELTRSEFGGGTKVEIKRT (SEQ ID NO: 253) Humanized C8 single chain X4 (linker is IgG1 and IgG2 modified hinge region) (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagactgggcggcgacaattactatgagtattggggcaaagggaccacggtcaccgtctcctccgataaaacccatactaaaccgccaaaaccggcgccggaactgctgggtggtcctggtaccggtactggtggtccgactattaaacctccgaaacctccgaaacctgctccgaacctgctgggtggtccggacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcagggccagcaagagtgttagcaccagcggctacagctacatgcactggtaccagcagaaaccaggacagcctcctaagctgctcatttacctggtgtctaacctggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacacattcgggaactgaccaggagtgaattcggcggagggaccaaggtggagatcaaacgaact (SEQ ID NO: 254) (Amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGKGTTVTVSSDKTHTKPPKPAPELLGGPGTGTGGPTIKPPKPPKPAPNLLGGPDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHIRELTRSEFGGGTKVEIKRT (SEQ ID NO: 255) pSECTag2 E6 scFV-FC (DNA) (amino acid) METDTLLLWVLLLWVPGSTGDAAQPAEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTI SGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLT CSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGGSGTKVEIKEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** (SEQ ID NO: 257) E6 scFC-FC 1 gBLOCk sequence: (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggccgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaaccattagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcggcggtggcggatccggcggtggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccaccctgtctttgtc (SEQ ID NO: 258) E6 scFC-FC 2 gBLOCk sequence: (DNA) aattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagc(SEQ ID NO: 259) pSECTag2 C2 scFV-FC (DNA) (amino acid) METDTLLLWVLLLWVPGSTGDAAQPAEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGG DNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYC QHSRELPFTFGGGTKVEIKRTEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* *(SEQ ID NO: 261) C2 scFV-FC 1 gBLOCk sequence: (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggccgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctccggcggtggcggatccggcggtggcggatccggcggtggcggatccgacattgtgctgacccagtctccagcctccttggc(SEQ ID NO: 262) C2 scFV-FC 2 gBLOCk sequence: (DNA) cattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagc(SEQ ID NO: 263) pSECTag2 C3 scFV-FC (DNA) (Amino acid) METDTLLLWVLLLWVPGSTGDAAQPAQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRTEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* *(SEQ ID NO: 265) C3 GS scFV FC1 gBLOCk sequence: (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggcccaggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtgatcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggcagagtcaecatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgactactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagcggcggtggcggatccggcggtggcggatccggcggtggcggatccgatattgtgatgacccagactccactctctctgt(SEQ ID NO: 266) C3 scFV FC2 gBLOCk sequence: (DNA) tattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtctagtcagaccattgtccatagtaatggaaacacctatttggagtggtacctgcagaagccaggccagtctccacagct cctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgettccaaggtagccacgtgcctttc accessory tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggt cagc (SEQ ID NO: 267) pSECTag2 C8 scFV-FC (DNA) (amino acid) METDTLLLWVLLLWVPGSTGDAAQPAEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLG GDNYYEYWGKGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ HIRELTRSEFGGGTKVEIKRTEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* * (SEQ ID NO: 269) C8 scFV FC 1 gBLOCk sequence: (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggccgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatactaccctgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagactgggcggcgataactattatgaatattggggcaaagggaccacggtcaccgtctcctccggcggtggcggatccggcggtggcggatccggcggtggcggatccgacatcgtgatgacccagtctccagactccctgg (SEQ ID NO: 270) C8 scFV FC2 gBLOCk sequence: (DNA)catcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcagggccagcaagagtgttagcaccagcggctacagctacatgcactggtaccagcagaaaccaggacagcctcctaagctgctcatttacctggtgtctaacctggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacacattcgggaactgaccaggagtgaattcggcggagggaccaaggtggagatcaaacgaactgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagc(SEQ ID NO: 271) Human IgGl Fc sequence: (DNA) (SEQ ID NO: 272) (amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK* *(request number: 273) Human IgGl CH2-CH3 region sequence: (DNA) ccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataa (SEQ ID NO: 274) (Amino acid) PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK**(SEQ ID NO: 275) Human IgGl CH3 region sequence: (DNA) gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctc ccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataa (Sequence number: 276) (amino acid) GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* *(SEQ ID NO: 277) Human IgGl Fc Y407R sequence: (DNA) (SEQ ID NO: 278) (amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLRSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK* *(request number: 279) Human IgGl Fc F405Q sequence: (DNA) gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttccagctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataa (SEQ ID NO: 280) (Amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* *(SEQ ID NO: 281) Human IgGl Fc T394D sequence: (DNA) (SEQ ID NO: 282) (amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTDPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK* 283) Human IgGl Fc T366W / L368W sequence: (DNA) gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgtggtgctgggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataa (SEQ ID NO: 284) (Amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCWVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* *(SEQ ID NO: 285) Human IgGl Fc T364R / L368R sequence: (DNA) (SEQ ID NO: 286) (amino acid) EPKSCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVRLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK* 287) Human IgGl Fc hinge-less sequence: (DNA) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataa (SEQ ID NO: 288) (Amino acid) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* * (SEQ ID NO: 289) Human IgG1 G237A FC sequence: (DNA) (SEQ ID NO: 290) (amino acid) EPKSCDKTTHTCPPCPAPELLGAPSVFLFPKPKDTLMISRTPEVTVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS(request number: 291) Human IgGl L234A / L235A FC sequence: (DNA) gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaagccgccgggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcaga agagcctctccctgtctccgggtaaa (SEQ ID NO: 292) (Amino acid) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS(SEQ ID NO: 293) CAR-T E6 CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAED TAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSG SGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* *** (SEQ ID NO: 295) CAR-T E6 CD3z gBLOCK sequence: (DNA) tggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgetcctcctctccctggtgattaccctgtactgccgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataagtttaaacccgctgatcagcctcgactgtgc (SEQ ID NO: 296) CAR-T E6 CD28 / CD3z sequence: [[ID=*5*]](DNA) It should be noted that there seems to be an incorrect "tgetc" in the original sequence in the part "tggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgetcctcctctccctggtgattaccctgtactgccgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataagtttaaacccgctgatcagcctcgactgtgc". It might be a typo. (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSDYTLTISSLEPEDFAVY YCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIYWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 298) CAR-T E6 CD28 / CD3z g BLOCK sequence: (DNA) tggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcagaagcaagcggtctcggctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgctccgccacgcgacttcgctgcctaccggtcccgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataagtttaaacccgctgatcagcctcgactgtgc (SEQ ID NO: 299) CAR-T E6 4-1BB / CD3z sequence: (DNA) (Amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* *(SEQ ID NO: 301) CAR-T E6 4-1BB / CD3z gBLOCK sequence: (DNA) tggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataagtttaaacccgctgatcagcctcgactgtgc (SEQ ID NO: 302) CAR-T E6 CD28 / 4-1BB / CD3z sequence: (DNA) (amino acid) * * (SEQ ID NO: 304) CAR-T E6 CD28 / 4-lBB / CD3z gBLOCK sequence: CAR-T C2 CD28 / 4-lBB / CD3z sequence: (DNA) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGGSGG GGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRTTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR** (SEQ ID NO: 307) CAR-T C2-1 gBLOCK sequence: (DNA) atagggagacccaagctggctagttaagcttggtaccgagggccaccatggccttgccagtgacggccctgctgctgccattggctcttctgttgcacgctgccaggcctgaagtgcagctcgtagagagtggcgggggactggtgaagcccggtggaagcctcagactcagttgcgccgcctcaggtttcactttttcaggttacgccatgtcctgggtaagacaggcaccggggaaaggactcgagtgggtgtctactatcagctcaggaggcacttatatatattatcctgactctgtaaaaggccgatttacgatttctcgcgacaatgcaaagaactccctctacctccaaatgaacagtcttagggcagaagacactgctgtatactattgtgcacgcctcggcggcgacaactactacgagtactttgacgtgtgggggaaagggactaccgtgacagtttcaagcggaggaggtggctcaggtggaggcgggtcaggggggggaggaagtgatattgtgctcacacaatccccagcctccctggc (SEQ ID NO: 308) 9CAR-T C2-2 gBLOCK sequence: (DNA) aagtgatattgtgctcacacaatccccagcctccctggctgtgtctcccggccaacgcgctacaattacatgtcgggcctccaaaagcgtgagcaccagcggctacagctacatgcactggtatcaacagaaaccaggacaaccccccaaactgttgatttatctcgcttcaaacttggagtccggcgtgcctgcgcgcttttcagggagtgggagcggcacagattttacgctgactatcaaccccgtagaagcaaacgatacagcgaattattattgtcaacattcccgggaactcccctttacgttcggcgggggcacaaaggtcgaaattaagagaaccacgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgt ggagtgc(SEQ ID NO: 309) CAR, E6 Fc / 8 / 4-lBB / CD3z sequence: (DNA) (amino acid) ** (SEQ ID NO: 311) E6 CAR pCDH gBLOCK sequence: (DNA) acgctgttttgacctccatagaagattctagagctagctgtagagcttggtaccgagggccaccatggccctgcccgtgaccgctttgctgctccccctggcgctgctgctgcacgccgccaggccagaggtccagctggttgagagtggcggtgggctggttaagcctggcggctccctgcggctgagetgcgccgcgagtggatttactttcagccgatatgggatgagttgggtgcggcaagctcccgggaagaggctggaatgggtctcaacaatctccggggggggcacttacatctattaccccgactcagtcaaggggagatttaccatttcacgagacaacgctaagaataccctgtatttgcagatgaattctctgagagcagaggacacagctgtttactattgtacccgcgacaactatggcaggaactacgactacggtatggactattggggacaagggacattggttacagtgagcagtggcggcgggggcagcggaggaggaggcagcggtggcggaggcagcgagatagtgctcacgcagtcacccgcgactctcagtctctcacctggggaacgagctaccctgacgtgctctgctacctcctcagtgtcatatattcactggtatcagcaacggcccgggcagtcccctagattgctcatttatagtacctctaatctggcctcaggtatccctgc (SEQ ID NO: 312) E6 CAR Fc pCDH gBlock Sequence:<00X1917>(DNA) It should be noted that in the translation of sequence-related content, it is best to double-check with relevant biological knowledge and professional requirements to ensure the accuracy and compliance of the translation. Also, for the tag, it seems there might be a typo in the original "00X1917", and it is translated as is according to the rules. If it is an incorrect tag in the original, it may need to be corrected in the source material for a more accurate translation.agtacctctaatctggcctcaggtatccctgcacgattttctggatctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg(SEQ ID NO: 313) E6 CAR 8BB3 pCDH gBlock Sequence: (DNA) agaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataagtttaaacccgctgatcaggcggccgcgaaggatctgcgatcgctccggtgcccgtcag(SEQ ID NO: 314) CAR E6 FcH / 8 / 4-lBB / CD3z sequence: (DNA) (amino acid) * * (SEQ ID NO: 316) E6 CAR FcH pCDH gBLOCK sequence: (DNA) agtacctctaatctggcctcaggtatccctgcacgattttctggatctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg(SEQ ID NO: 317) CAR E6 Fc / 4 / 4-lBB / CD3z sequence: (DNA) (amino acid) ** (SEQ ID NO: 319) E6 CAR 44BB3 pCDH gBLOCK sequence: (DNA) agaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaatggccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataagtttaaacccgctgatcaggcggccgcgaaggatctgcgatcgctccggtgcccgtcag (SEQ ID NO: 320) CAR E6 FcH / 4 / 4-lBB / CD3z sequence: (DNA) (amino acid) * * (SEQ ID NO: 322) CAR E6 IgD / 8 / 4-lBB / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRN YDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSS SPFTFGSGTKVEIKESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 324) E6 CAR IgD8 pcDH gBLOCK sequence: (DNA) agtacctctaatctggcctcaggtatccctgcacgattttctggatctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaagagtctccaaaggcacaggcctcctcagtgcccactgcacaaccccaagcagagggcagcctcgccaaggcaaccacagccccagccaccacccgtaacacaggaagaggcggcgaagagaagaaaaaggagaaggagaaagaggaacaagaagagagagagacaaagacaccaatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatga ggccag (SEQ ID NO: 325) E6 CAR BB 3 pCDH gBlock Sequence: (DNA) acatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataagtttaaacccgctgatcaggcggccgcgaaggatctgcgatcgctccggtgcccgtcag(SEQ ID NO: 326) CAR E6 IgD / 4 / 4-lBB / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGR NYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRS SSPFTFGSGTKVEIKESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 328) E6 CAR IgD4 pcDH gBLOCK sequence: (DNA) agtacctctaatctggcctcaggtatccctgcacgattttctggatctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaagagtctccaaaggcacaggcctcctcagtgcccactgcacaaccccaagcagagggcagcctcgccaaggcaaccacagccccagccaccacccgtaacacaggaagaggcggcgaagagaagaaaaaggagaaggagaaagaggaacaagaagagagagagacaaagacaccaatggccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccag(SEQ ID NO: 329) CAR E6 X4 / 8 / 4-lBB / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDN YGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVY YCQQRSSSPFTFGSGTKVEIKDKTHTKPPKPAPELLGGPGTGTGGPTIKPPKPPKPAPNLLGGPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 331) E6 CAR X48 pCDH gBLOCK sequence: (DNA) agtacctctaatctggcctcaggtatccctgcacgattttctggatctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaagacaagacgcacaccaagccacctaaaccagctccagaactgctcggaggtcctggcaccggaaccggaggacctaccatcaaaccacctaagc cacctaagcctgctcctaacctgctcggaggacctatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccag (SEQ ID NO: 332) CAR E6 X4 / 4 / 4-lBB / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDN YGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAV YYCQQRSSSPFTFGSGTKVEIKDKTHTKPPKPAPELLGGPGTGTGGPTIKPPKPPKPAPNLLGGPMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 334) E6 CAR X44 pCDH gBLOCK sequence: (DNA) agtacctctaatctggcctcaggtatccctgcacgattttctggatctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaagacaagacgcacaccaagccacctaaaccagctccagaactgctcggaggtcctggcaccggaaccggaggacctaccatcaaaccacctaagccacctaagcctgctcctaacctgctcggaggacctatggccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccag (SEQ ID NO: 335) CAR E6 8+4 / 4 / 4-lBB / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDN YGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVY YCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 337) E6 CAR CD844 pCDH gBLOCK sequence: (DNA) agtacctctaatctggcctcaggtatccctgcacgattttctggatctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaaacgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatggccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccag (SEQ ID NO: 338) Humanized C2 scFV sequence in CAR: (DNA) (SEQ ID NO: 339) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO: 340) Humanized E6 scFv sequence in CAR: (DNA) (SEQ ID NO: 341) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO: 342) CD8 leader sequence: (DNA) atggccctgcccgtgaccgctttgctgctccccctggcgctgctgctgcacgccgccaggcca (SEQ ID NO: 343) (amino acid) MALPVTALLLPLALLLHAARP (SEQ ID NO: 344) CD8 hinge region sequence: (DNA) acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgat (SEQ ID NO: 345) (amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 346) CD4 hinge region sequence: (DNA) tcgggacaggtcctgctggaatccaacatcaaggttctgcccacatggtccacccggtgcagcca (SEQ ID NO: 347) (Amino acid) SGQVLLESNIKVLPTWSTPVQP (SEQ ID NO: 348) CD28 hinge region sequence: (DNA) aaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 349) (amino acid) KHLCPSPLFPGPSKP (SEQ ID NO: 350) CD8+CD4 hinge region sequence: (DNA) acgacaaccccggccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctg tgcacacaagaggactggatttcgcctgtgattcgggacaggtcctgctggaatccaacatcaaggttctgcccacatggtccacccggtgcagcca(SEQ ID NO: 351) (amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSGQVLLESNIKVLPTWSTPVQP (SEQ ID NO: 352) CD8+CD28 hinge region sequence: (DNA) acgacaaccccggccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgataaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 353) (amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDKHLCPSPLFPGPSKP (SEQ ID NO: 354) CD28+CD4 hinge region sequence: (DNA) aaacacctttgtccaagtcccctatttcccggaccttctaagccctcgggacaggtcctgctggaatccaacatcaaggttctgcccacatggtccacccggtgcagcca (SEQ ID NO: 355) (amino acid) KHLCPSPLFPGPSKPSGQVLLESNIKVLPTWSTPVQP (SEQ ID NO: 356) Human IgD hinge region sequence: (DNA) gagtctccaaaggcacaggcctcctcagtgcccactgcacaaccccaagcagagggcagcctcgccaaggcaaccacagccccagccaccaccgtaacacaggaagaggcggcgaagagaagaaaaaggagaaggagaaagaggaacaagaagagagagagacaaagacacca (SEQ ID NO: 357) (amino acid) ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTP (SEQ ID NO: 358) X4 linker (IgG1 and IgG2 modified hinge region) sequence: (DNA) gacaagacgcacaccaagccacctaaaccagctccagaactgctcggaggtcctggcaccggaaccggaggacctaccatcaaaccacctaagccacctaagcctgctcctaacctgctcggaggacct (SEQ ID NO: 359) (amino acid) DKTHTKPPKPAPELLGGPGTGTGGPTIKPPKPPKPAPNLLGGP (SEQ ID NO: 360) CD3 ZETA transmembrane domain sequence: (DNA) ctctgctacctgctggatggaatcctcttcatctatggtgtcattctcactgccttgttcctg (SEQ ID NO: 361) (amino acid) LCYLLDGILFIYGVILTALFL (SEQ ID NO: 362) CD8 transmembrane domain sequence: (DNA) atctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgc (SEQ ID NO: 363) (amino acid) IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 364) CD4 transmembrane domain sequence: (DNA) atggccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttc (SEQ ID NO: 365) (amino acid) MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 366) CD28 transmembrane domain sequence: (DNA) ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg (SEQ ID NO: 367) (amino acid) FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 368) 4-1BB transmembrane domain sequence: (DNA) atcatctccttctttcttgcgctgacgtcgactgcgttgctcttcctgctgttcttcctcacgctccgtttctctgttgtt (Sequence number: 369) (amino acid) IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 370) OX40 transmembrane domain sequence: (DNA) gttgccgccatcctgggcctgggcctggtgctggggctgctgggccccctggccatcctgctggccctgtacctgctc (SEQ ID NO: 371) (Amino acid) VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO: 372) CD3 ZETA domain sequence: (DNA) cgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccgaggga ctgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacgg (SEQ ID NO: 373) (amino acid) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 374) CD3 ZETA domain variant sequences: (DNA) agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggc ctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO: 375) (amino acid) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 376) CD28 domain sequence: (DNA) agaagcaagcggtctcggctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgctccgccacgcgacttcgctgcctaccggtcc (SEQ ID NO: 377) (amino acid) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 378) 4-1BB region sequence: (DNA) aaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactg (SEQ ID NO: 379) (amino acid) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 380) 0x40 Area Array: (DNA)c ggagggaccagaggctgccccccgatgcccacaagccccctgggggaggcagtttccggaccccccatccaagaggagcaggccgacgcccactccaccctggccaagatc (SEQ ID NO: 381) (amino acid) RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 382) Humanized anti-CD3 scFV clone 12F6 (VH-VL) sequence: (DNA) (SEQ ID NO: 383) (amino acid) QVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSGYTKYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFCARWQDYDVYFDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTMTCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITR (SEQ ID NO: 384) Humanized anti-CD3 scFV clone 12F6 (VL-VH) sequence: (DNA) (SEQ ID NO: 385) (amino acid) DIQMTQSPSSLSASVGDRVTMTCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITRGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSGYTKYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFCARWQDYDVYFDYWGQGTPVTVSS (SEQ ID NO: 386) Humanized anti-CD3 scFV clone OKT3 (VH-VL) sequence: (DNA) caggtgcagctggtgcagagcggaggcggagtggtgcagcctggaagaagcctgcgcctgagctgcaaagcgagcggctatacctttacccgctataccatgcattgggtgcgccaggcgccgggcaaaggcctggaatggattggctatattaacccgagccgcggctataccaactataaccagaaagtgaaagatcgctttaccattagcaccgataaaagcaaaagcaccgcgtttctgcagatggatagcctgcgcccggaagataccgcggtgtattattgcgcgcgctattatgatgatcattattgcctggattattggggccagggcaccaccctgaccgtgagcagcggcggtggcggatccggcggtggcggatccggcggtggcggatccgatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcagcgcgagcagcagcgtgagctatatgaactggtatcagcagaccccgggcaaagcgccgaaacg ctggatttatgataccagcaaactggcgagcggcgtgccgagccgctttagcggcagcggcagcggcaccgattatacctttaccattagcagcctgcagccggaagatattgcgacctattattgccagcagtggagcagcaacccgtttacctttggccagggcaccaaactgcagattacccgc(SEQ ID NO: 387) (Amino acid) QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 388) Humanized anti-CD3 scFV clone OKT3 (VH-VL) sequence: (DNA) (SEQ ID NO: 389) (amino acid) DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 390) Humanized E6 scFV (VH-VL) sequence: (DNA) (SEQ ID NO: 391) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO: 392) Humanized E6 scFV (VL-VH) sequence: (DNA) (SEQ ID NO: 393) (amino acid) EIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSS (SEQ ID NO: 394) Humanized C2 scFV (VH-VL) sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctgggggtccctgagactctcctgtgcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatatactacccg actcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctccgggcgg tggcggatccgggggtggcggatccggggggtggcggatccgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccacatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattatactgtcagcacagtagggagctgcctttcacattcggcggagggaccaaggtggagat caaacgaact(sequence number: 395) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRT(SEQ ID NO: 396)<00M2175><00M2176>Humanized E6 scFV (VL-VH) sequence:<00M2177>(DNA)<00M2178> Please note that the tags , , , are not standard English alphanumeric characters and are likely specific to a particular system or encoding. I've kept them as close as possible while following your rules. If there's a specific way these should be translated or handled in the context of your work, you may need to adjust accordingly. Also, the "配列番号: 396" is translated as "SEQ ID NO: 396" which is a common way to represent sequence identifiers in English in the context of biological sequences.(SEQ ID NO: 397) (amino acid) DIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRTGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSS (SEQ ID NO: 398) G4S1 linker sequence: (DNA) ggcggtggcggatcc (SEQ ID NO: 399) (amino acid) GGGGS (SEQ ID NO: 400) [G4S1] x3 linker sequence: (DNA) ggcggtggcggatccggcggtggcggatccggcggtggcggatccggcggtggcggatcc (SEQ ID NO: 401) (amino acid) GGGGSGGGGSGGGGS (SEQ ID NO: 402) 8 aa GS linker sequence: (DNA) ggcggttccggcggtggatccgga (SEQ ID NO: 403) (amino acid) GGSGGGSG (SEQ ID NO: 404) 12 aa GS linker sequence: (DNA) ggcggttccggcggtggatccggcggtggcggatccgga (SEQ ID NO: 405) (amino acid) GGSGGGSGGGSG (SEQ ID NO: 406) 13 aa GS linker sequence: (DNA) ggcggtggatccggcggtggcggatccggcggtggatcc (SEQ ID NO: 407) (amino acid) GGGSGGGGSGGGS (SEQ ID NO: 408) 22 aa GS linker sequence: (DNA) ggcggtggaagcggcggtggcggatccggcagcggcggaagcggcggtggcggatccggcggtgga (SEQ ID NO: 409) (amino acid) GGGSGGGGSGSGGSGGGGSGGG (SEQ ID NO: 410) 24 aa GS linker sequence: (DNA) ggcggttccggcggtggatccggcggtggcggatccggaggcggttccggcggtggatccggcggtggcggatccgga (SEQ ID NO: 411) (amino acid) GGSGGGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 412) Mouse C3 heavy chain variable region sequence: (DNA) caggtccagctgcagcagtctgggcctgagctggtgaggcctggggtctcagtgaagatttcctgcaagggttccggctacagattcactgattatgctatgaactgggtgaagcagagtcatgcaaagagtctagagtggattggagttattagtactttctctggtaatacaaacttca accagaagtttaagggcaaggccacaatgactgtagacaaatcctccagcacagcctatatggaacttgccagattgacatctgaggattctgccatgtattactgtgcaagatcggattactacggcccatactttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 413) (amino acid) QVQLQQSGPELVRPGVSVKISCKGSGYRFTDYAMNWVKQSHAKSLEWIGVISTFSGNTNFNQKFKGKATMTVDKSSSTAYMELARLTSEDSAMYYCARSDYYGPYFDYWGQGTTLTVSS (SEQ ID NO: 414) Mouse C3 heavy chain variable framework region 1 (FWR1) sequence: (DNA) caggtccagctgcagcagtctgggcctgagctggtgaggcctggggtctcagtgaagatttcctgcaagggttccggctacagattcact (SEQ ID NO: 415) (amino acid) QVQLQQSGPELVRPGVSVKISCKGSGYRFT (SEQ ID NO: 416) Mouse C3 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) gattatgctatgaac (SEQ ID NO: 417) (amino acid) DYAMN (SEQ ID NO: 418) Mouse C3 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggtgaagcagagtcatgcaaagagtctagagtggattgga (SEQ ID NO: 419) (amino acid) WVKQSHAKSLEWIG (SEQ ID NO: 420) Mouse C3 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) gttattagtactttctctggtaatacaaacttcaaccagaagtttaagggc (SEQ ID NO: 421) (amino acid) VISTFSGNTNFNQKFKG (SEQ ID NO: 422) Mouse C3 heavy chain variable framework region 3 (FWR3) sequence: (DNA) aaggccacaatgactgtagacaaatcctccagcacagcctatatggaacttgccagattgacatctgaggattctgccatgtattactgtgcaaga (SEQ ID NO: 423) (amino acid) KATMTVDKSSSTAYMELARLTSEDSAMYYCAR (SEQ ID NO: 424) Mouse C3 heavy chain variable complementarity determining region 3 (CDR3) sequence: (DNA) tcggattactacggcccatactttgactac (SEQ ID NO: 425) (amino acid) SDYYGPYFDY (SEQ ID NO: 426) IGHV1-18*04 heavy chain variable region sequence: (DNA) caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccagctacggtatcagctgggtgcgacaggcccctggacaagggct tgagtggatgggatggatcagcgcttacaatggtaacacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacg aacacggccgtgtattactgtgcgagaga (SEQ ID NO: 427) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR (SEQ ID NO: 428) IGHV1-18*04 heavy chain variable framework region 1 (FWR1) sequence: (DNA) caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttacc (SEQ ID NO: 429) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 430) IGHV1-18*04 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agctacggtatcagc (SEQ ID NO: 431) (amino acid) SYGIS (SEQ ID NO: 432) IGHV1-18*04 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggtgcgacaggcccctggacaagggcttgagtggatggga (SEQ ID NO: 433) (amino acid) WVRQAPGQGLEWMG (SEQ ID NO: 434) IGHV1-18*04 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) tggatcagcgcttacaatggtaacacaaactatgcacagaagctccagggc (SEQ ID NO: 435) (amino acid) WISAYNGNTNYAQKLQG (SEQ ID NO: 436) IGHV1-18*04 heavy chain variable framework region 3 (FWR3) sequence: (DNA) agagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgaga (SEQ ID NO: 437) (amino acid) RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR (SEQ ID NO: 438) Humanized C3 heavy chain variable region sequence: (DNA) caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtgatcagcaccttcagcggtaacacaaacttca accagaagttcaagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacggccgtgtattactgtgcgagaagcgactactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagc (SEQ ID NO: 439) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQGTTLTVSS (SEQ ID NO: 440) Humanized C3 heavy chain variable framework region 1 (FWR1) sequence: (DNA) caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttacc (SEQ ID NO: 441) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 442) Humanized C3 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) gactacgccatgaac (SEQ ID NO: 443) (amino acid) DYAMN (SEQ ID NO: 444) Humanized C3 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggtgcgacaggcccctggacaagggcttgagtggatggga (SEQ ID NO: 445) (amino acid) WVRQAPGQGLEWMG (SEQ ID NO: 446) Humanized C3 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) gtgatcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggc (SEQ ID NO: 447) (amino acid) VISTFSGNTNFNQKFKG (SEQ ID NO: 448) Humanized C3 heavy chain variable framework region 3 (FWR3) sequence: (DNA) agagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgaga (SEQ ID NO: 449) (amino acid) RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR (SEQ ID NO: 450) Humanized C3 heavy chain variable complementarity determining region 3 (CDR3) sequence: (DNA) agcgactactacggcccatacttcgactac (SEQ ID NO: 451) (amino acid) SDYYGPYFDY (SEQ ID NO: 452) Humanized C3 IgG1 heavy chain sequence (DNA) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQG TTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* *(SEQ ID NO: 454) Humanized C3 IgG2 heavy chain sequence (DNA) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQ GTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** (SEQ ID NO: 456) Humanized C3 heavy chain IgGl gBLOCK sequence: (DNA) tgetctgggttccaggttccactggtgacgcggcccagccggcccaggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtgatcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgactactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagcgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagc(SEQ ID NO: 457) Mouse C3 light chain variable region sequence: (DNA) gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagaccattgtacatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcaacagagtggaggctgaggatctgggagtttattactgctttcaaggttcacatgttccattcacgttcggctcggggacaaagttggaaataaaa(SEQ ID NO: 458) (Amino acid) DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 459) Mouse C3 light chain variable framework region 1 (FWR1) sequence: (DNA) gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgc (Sequence number: 460) (amino acid) DVLMTQTPLSLPVSLGDQASISC (SEQ ID NO: 461) Mouse C3 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agatctagtcagaccattgtacatagtaatggaaacacctatttagaa (SEQ ID NO: 462) (amino acid) RSSQTIVHSNGNTYLE (SEQ ID NO: 463) Mouse C3 light chain variable framework region 2 (FWR2) sequence: (DNA) tggtacctgcagaaaccaggccagtctccaaagctcctgatctac (SEQ ID NO: 464) (amino acid) WYLQKPGQSPKLLIY (SEQ ID NO: 465) Mouse C3 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) aaagtttccaaccgattttct (SEQ ID NO: 466) (amino acid) KVSNRFS (SEQ ID NO: 467) Mouse C3 light chain variable framework region 3 (FWR3) sequence: (DNA) ggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcaacagagtggaggctgaggatctgggagtttattactgc (SEQ ID NO: 468) (amino acid) GVPDRFSGSGSGTDFTLKINRVEAEDLGVYYC (SEQ ID NO: 469) Mouse C3 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) tttcaaggttcacatgttccattcacg (SEQ ID NO: 470) (amino acid) FQGSHVPFT (SEQ ID NO: 471) IGKV2-29*03 light chain variable region sequence: (DNA) gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaagtctagtcagagcctcctgcatagtgatggaaagacctatttgtattggtacctgcagaagccaggccagtctccacagctc ctgatctatgaagtttccagccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgcatgcaaggtatacaccttcct (Sequence number: 472) (amino acid) DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQSPQLLIYEVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGIHLP (SEQ ID NO: 473) IGKV2-29*03 light chain variable framework region 1 (FWR1) sequence: (DNA) gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgc (SEQ ID NO: 474) (amino acid) DIVMTQTPLSLSVTPGQPASISC (SEQ ID NO: 475) IGKV2-29*03 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) aagtctagtcagagcctcctgcatagtgatggaaagacctatttgtat (SEQ ID NO: 476) (amino acid) KSSQSLLHSDGKTYLY (SEQ ID NO: 477) IGKV2-29*03 light chain variable framework region 2 (FWR2) sequence: (DNA) tggtacctgcagaagccaggccagtctccacagctcctgatctat (SEQ ID NO: 478) (amino acid) WYLQKPGQSPQLLIY (SEQ ID NO: 479) IGKV2-29*03 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) gaagtttccagccggttc (SEQ ID NO: 480) (amino acid) EVSSRFS (SEQ ID NO: 481) IGKV2-29*03 light chain variable framework region 3 (FWR3) sequence: (DNA) ggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgc (SEQ ID NO: 482) (amino acid) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 483) IGKV2-29*03 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) atgcaaggtatacaccttcct (SEQ ID NO: 484) (amino acid) MQGIHLP (SEQ ID NO: 485) Humanized C3 light chain variable region sequence: (DNA) gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtctagtcagaccattgtccatagtaatggaaacacctatttggagtggtacctgcagaagcc aggccagtctccacagctcctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttgggg tttattactgcttccaaggtagccacgtgcctttcaccttcggcggagggaccaaggtggagatcaaacgaact (SEQ ID NO: 486) (amino acid) DIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRT (SEQ ID NO: 487) Humanized C3 light chain variable framework region 1 (FWR1) sequence: (DNA) gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgc (SEQ ID NO: 488) (amino acid) DIVMTQTPLSLSVTPGQPASISC (SEQ ID NO: 489) Humanized C3 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) ggtctagtcagaccattgtccatagtaatggaaacacctatttggag (SEQ ID NO: 490) (amino acid) RSSQTIVHSNGNTYLE (SEQ ID NO: 491) Humanized C3 light chain variable framework region 2 (FWR2) sequence: (DNA) tggtacctgcagaagccaggccagtctccacagctcctgatctat (SEQ ID NO: 492) (amino acid) WYLQKPGQSPQLLIY (SEQ ID NO: 493) Humanized C3 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) aaggtttccaaccggttctct (SEQ ID NO: 494) (amino acid) KVSNRFS (SEQ ID NO: 495) Humanized C3 light chain variable framework region 3 (FWR3) sequence: (DNA) ggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgc (SEQ ID NO: 496) (amino acid) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 497) Humanized C3 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) ttccaaggtagccacgtgcctttcacc (SEQ ID NO: 498) (amino acid) FQGSHVPFT (SEQ ID NO: 499) Humanized C3λ light chain sequence (DNA) (SEQ ID NO: 500) (amino acid) DIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRTGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS** (SEQ ID NO: 501) Humanized C3κ light chain (DNA) gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtctagtcagaccattgtccatagtaatggaaacacctatttggagtggtacctgcagaagccaggccagtctccacagctcctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgcttccaaggtagccacgtgcctttcaccttcggcggagggaccaaggtggagatcaaacgaactacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaaca caaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagtaa (SEQ ID NO: 502) (Amino acid) DIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRTTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC** (SEQ ID NO: 503) Humanized C3 kappa light gBLOCK sequence: (DNA) agctggctaggtaagcttggtaccgagctcggatccacgccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtctagtcagaccattgtccatagtaatggaaacacctatttggagtggtacctgcagaagccaggccagtctccacagctcctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgettccaaggtagccacgtgcctttcaccttcggcggagggaccaaggtggagatcaaacgaactacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagtaagtttaaacccgctgatcagcctcgactgtgccttctagttgc(SEQ ID NO: 504) Mouse C8 heavy chain variable region sequence: (DNA) gaagtgatggtcgtggaaagcggcggtggtctggtaaagccggggggatcccttaagctttcttgcgccgcatccgggttcacgttctccggctatgccatgtcctgggtccgacagactcccgaaaagcgctt ggaatgggtggccactatctcctccggggggacgtacatctactacccgacagtgtgaaaggaagatttacaatatctcgcgacaacgcaaaaaataccttgtatcttcaaatgagctccctgcggtcagagg acactgccatgtactattgcgcccgcctgggcggcgacaattactatgagtat (SEQ ID NO: 505) (amino acid) EVMVVESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVATISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLGGDNYYEY (SEQ ID NO: 506) Mouse C8 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) ggctatgccatgtcc (SEQ ID NO: 507) (amino acid) GYAMS (SEQ ID NO: 508) Mouse C8 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) actatctcctccggggggacgtacatctactaccccgacagtgtgaaagga (SEQ ID NO: 509) (amino acid) TISSGGTYIYYPDSVKG (SEQ ID NO: 510) Mouse C8 heavy chain variable complementarity-determining region 3 (CD3: (DNA)c tgggcggcgacaattactatgagtat (SEQ ID NO: 511) (amino acid) LGGDNYYEY (SEQ ID NO: 512) IGHV3-21*04 heavy chain variable region sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatc cattagtagtagtagtagttacatatactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcga(SEQ ID NO: 513) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 514) IGHV3-21*04 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagt (SEQ ID NO: 515) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 516) IGHV3-21*04 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agctatagcatgaac (SEQ ID NO: 517) (amino acid) SYSMN (SEQ ID NO: 518) IGHV3-21*04 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggtccgccaggctccagggaaggggctggagtgggtc (SEQ ID NO: 519) (amino acid) WVRQAPGKGLEWV (SEQ ID NO: 520) IGHV3-21*04 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) tcatccattagtagtagtagtagttacatatactacgcagactcagtgaagggc (SEQ ID NO: 521) (amino acid) SSISSSSSYIYYADSVKG (SEQ ID NO: 522) IGHV3-21*04 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcga (SEQ ID NO: 523) (amino acid) RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 524) Humanized C8 heavy chain variable region sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattagtagtggcggaacctacatatactac cctgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagactgggcggcgataactattatgaatattggggcaaagggaccacggtcaccgtctcctcc(SEQ ID NO: 525) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGKGTTVTVSS (SEQ ID NO: 526) Humanized C8 heavy chain variable framework region 1 (FWR1) sequence: (DNA) gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagt (SEQ ID NO: 527) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 528) Humanized C8 heavy chain variable complementarity determining region 1 (CDR1) sequence: (DNA) ggctatgccatgagc (SEQ ID NO: 529) (amino acid) GYAMS (SEQ ID NO: 530) Humanized C8 heavy chain variable framework region 2 (FWR2) sequence: (DNA) tgggtccgccaggctccagggaaggggctggagtgggtctca (SEQ ID NO: 531) (amino acid) WVRQAPGKGLEWVS (SEQ ID NO: 532) Humanized C8 heavy chain variable complementarity determining region 2 (CDR2) sequence: (DNA) accattagtagtggcggaacctacatatactaccctgactcagtgaagggc (SEQ ID NO: 533) (amino acid) TISSGGTYIYYPDSVKG (SEQ ID NO: 534) Humanized C8 heavy chain variable framework region 3 (FWR3) sequence: (DNA) cgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaga (SEQ ID NO: 535) (amino acid) RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 536) Humanized C8 heavy chain variable complementarity determining region 3 (CDR3) sequence: (DNA) ctgggcggcgataactattatgaatat (SEQ ID NO: 537) (amino acid) LGGDNYYEY (SEQ ID NO: 538) Humanized C8 IgG1 heavy chain sequence (DNA) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGKGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** (SEQ ID NO: 540) Humanized C8 IgG2 heavy chain sequence (DNA) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGK GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** (SEQ ID NO: 542) Mouse C8 light chain variable region sequence (DNA) gacatcgtcattacgcagacccctgccagtcttgccgtttctctgggccagaggggccactatcagttacagggcgagtaagtctgtgagtaccagcggctatagttacatgcattggaaccagcagaaaccgggacagccaccacgcctgcttatt tatctggtgtctaatcttgagtccggggtgcccgccaggttcagcggcagcggctctgggaccgacttcacactcaacattcatccagtggaagaagaggacgctgctacatactactgtcaacacattcgggaactgaccaggagtgaa (SEQ ID NO: 543) (amino acid) DIVITQTPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSE (SEQ ID NO: 544) Mouse C8 light chain variable complementarity-determining region 1 (CD1: (DNA) agggcgagtaagtctgtgagtaccagcggctatagttacatgcat (SEQ ID NO: 545) (amino acid) RASKSVSTSGYSYMH (SEQ ID NO: 546) Mouse C8 light chain variable complementarity-determining region 2 (CD2: (DNA) ctggtgtctaatcttgagtcc (SEQ ID NO: 547) (amino acid) LVSNLES (SEQ ID NO: 548) Mouse C8 light chain variable complementarity-determining region 3 (CD3): (DNA) caacacattcgggaactgaccaggagtgaa (SEQ ID NO: 549) (amino acid) QHIRELTRSE (SEQ ID NO: 550) NCBI germline z00023 light chain variable region sequence: (DNA) gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagaggggccaccatcaactgcaagtccagccagagtgttttacagctccaacaataagaactacttagcttggtaccagcagaaaccaggacagcctcctaagc tgctcatttactgggcatctacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaatattatagtactcct (SEQ ID NO: 551) (amino acid) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQYYSTP (SEQ ID NO: 552) NCBI germline z00023 light chain variable framework region 1 (FWR1) sequence: (DNA) gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgc (SEQ ID NO: 553) (amino acid) DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 554) NCBI germline z00023 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) aagtccagccagagtgttttatacagctccaacaataagaactacttagct (SEQ ID NO: 555) (amino acid) KSSQSVLYSSNNKNYLA (SEQ ID NO: 556) NCBI germline z00023 light chain variable framework region 2 (FWR2) sequence: (DNA) tggtaccagcagaaaccaggacagcctcctaagctgctcatttac (SEQ ID NO: 557) (Amino acid) WYQQKPGQPPKLLIY (SEQ ID NO: 558) NCBI germline z00023 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) tgggcatctacccgggaatcc (SEQ ID NO: 559) (amino acid) WASTRES (SEQ ID NO: 560) NCBI germline z00023 light chain variable framework region 3 (FWR3) sequence: (DNA) ggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgt (SEQ ID NO: 561) (amino acid) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 562) NCBI germline z00023 light chain variable complementarity determining region 3 (CD3) sequence: (DNA) cagcaatattatagtactcct (SEQ ID NO: 563) (amino acid) QQYYSTP (SEQ ID NO: 564) Humanized C8 light chain variable region sequence (DNA) gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcagggccagcaagagtgttagcaccagcggctacagctacatgcactggtaccagcagaaaccaggacagcctcctaagctgctcatttacctggtgtctaacctg gaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacacattcgggaactgaccaggagtgaattcggcggagggaccaaggtggagatcaaacgaact (SEQ ID NO: 565) (amino acid) DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQHIRELTRSEFGGGTKVEIKRT (SEQ ID NO: 566) Humanized C8 light chain variable framework region 1 (FWR1) sequence: (DNA) gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgc (SEQ ID NO: 567) (amino acid) DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 568) Humanized C8 light chain variable complementarity determining region 1 (CDR1) sequence: (DNA) agggccagcaagagtgttagcaccagcggctacagctacatg (SEQ ID NO: 569) (amino acid) RASKSVSTSGYSYM (SEQ ID NO: 570) Humanized C8 light chain variable framework region 2 (FWR2) sequence: (DNA) cactggtaccagcagaaaccaggacagcctcctaagctgctcatttac (SEQ ID NO: 571) (amino acid) HWYQQKPGQPPKLLIY (SEQ ID NO: 572) Humanized C8 light chain variable complementarity determining region 2 (CDR2) sequence: (DNA) ctggtgtctaacctggaatcc (SEQ ID NO: 573) (amino acid) LVSNLES (SEQ ID NO: 574) Humanized C8 light chain variable framework region 3 (FWR3) sequence: (DNA) ggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgt (SEQ ID NO: 575) (amino acid) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 576) Humanized C8 light chain variable complementarity determining region 3 (CDR3) sequence: (DNA) caacacattcgggaactgaccaggagtgaa (SEQ ID NO: 577) (amino acid) QHIRELTRSE (SEQ ID NO: 578) Humanized C8 λ light chain sequence (DNA) gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcagggccagcaagagtgttagcaccagcggctacagctacatgcactggtaccagcagaaaccaggacagcctcctaagctgctcatttacctggtgtctaacctggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacacattcgggaactgaccaggagtgaattcggcggagggaccaaggtggagatcaaacgaactggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaecacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatagtaa(SEQ ID NO: 579) (Amino acid) DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHIRELTRSEFGGGTKVEIKRTGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS**(SEQ ID NO: 580) Humanized C8κ light chain sequence (DNA) gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcagggccagcaagagtgttagcaccagcggctacagctacatgcactggtaccagcagaaaccaggacagcctcctaagctgctcatttacctggtgtctaacctggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacacattcgggaactgaccaggagtgaattcggcggagggaccaaggtggagatcaaacgaactacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagtaa(SEQ ID NO: 581) (Amino acid) DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHIRELTRSEFGGGTKVEIKRTTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC** (SEQ ID NO: 582) Humanized C8κ light chain gBLOCk sequence: (DNA) agctggctaggtaagcttggtaccgagctcggatccacgccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcagggccagcaagagtgttagcaccagcggctacagctacatgcactggtaccagcagaaaccaggacagcctcctaagctgctcatttacctggtgtctaacctggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacacattcgggaactgaccaggagtgaattcggcggagggaccaaggtggagatcaaacgaactacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagtaagtttaaacccgctgatcagcctcgactgtgccttctagttgc(SEQ ID NO: 583) CAR-T E6 CD8 sequence: (DNA) (SEQ ID NO: 584) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERA TLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC* *(SEQ ID NO: 585) CAR-T C2 CD8 sequence: (DNA) gaagtgcagctcgtagagagtggcggggactggtgaagcccggtggaagcctcagactcagttgcgccgcctcaggtttcactttttcaggttacgccatgtcctgggtaagacaggcaccgggaaaggactcgagtgggtgtctactatcagctcaggaggcacttatatattatcctgactctgtaaaaggccgatttacgatttctcgcgacaatgcaaagaactccctctacctccaaatgaacagtcttagggcaag acactgctgtatactattgtgcacgctcggcggcgacaactactacgagtactttgacgtgtgggggaaagggactaccgtgacagtttcaagcggaggggtggctcaggtggaggcgggtcagggggggaggagagtgatattgtgctcacacaatccccagcctccctggctgtgtctcccggccaacgcgctacaattacatgtcgggcctccaaaagcgtgagcaccagcctacagctacatgcactggtatcaacagaa accaggacaaccccccaaactgttgatttatctcgcttcaaacttggagtccggcgtgcctgcgcgctttcagggagtgggagcggcacagattttacgctgactatcaacccccgtagaagcaaacgatacagcgaattattgtcaacattcccgggaactcccctttacgttcggcggggcacaaaggtcgaaattaagaaccacgacaaccccggccccagaccaacgccacccaccatcgccaaccctgtctctgagaccagaagcctgtagcctgccgccggggggggagctgtgcacacaagaggactggatttcgcctgtgatatctacattgggccccgctcgcaggcacatgtggagtgctctctccctggtgattaccctgtactgctgataa(sequence number: 586) (amino acid) EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC* *(SEQ ID NO: 587) CD8 / 4-1BB sequence (DNA) acgacaaccccggccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggag tgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactgtgataa (Sequence number: 588) (amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL* *(SEQ ID NO: 589) CD8 / CD28 sequence (DNA) acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcagaagcaagcggtctcggctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgctccgccacgcgacttcgctgcctaccggtcctgataa (SEQ ID NO: 590) (Amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS * * (SEQ ID NO: 591) CD8 / CD3ζ sequence: (DNA) acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgccgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataa(SEQ ID NO: 592) (Amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 593) CD8 / CD28 / CD3z sequence: (DNA) acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcagaagcaagcggtctcggctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgctccgccacgcgacttcgctgcctaccggtcccgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgat aa(SEQ ID NO: 594) (Amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 595) CD8 / 4-lBB / CD3z sequence: (DNA) acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataa(SEQ ID NO: 596) (Amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 597) CD8 / CD28 / 4-1BB / CD3ζ array: (DNA) acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcagaagcaagcggtctcggctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgctccgccacgcgacttcgctgcctaccggtccaaaaggggccgcaaaaaactcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataa (SEQ ID NO: 598) (Amino acid) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 599) CAR-T C3 4-lBB / CD3z sequence: (DNA) (amino acid) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNTNFNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGQGTTLTV SSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP FTFGGGTKVEIKRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR** (SEQ ID NO: 601) C3 CAR gBLOCK 1 Layout: (DNA) atccacgctgttttgacctccatagaagattctagagctagctgtagagcttggtaccgagggccaccatggccctgcccgtgaccgctttgctgctccccctggcgctgctgctgcacgccgccaggccacaggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtgatcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgactactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagcggcggtggcggatccggcggtggcggatccggcggtggcggatccgat attgtgatgacccagactccactctctctgt(SEQ ID NO: 602) C3 CAR gBlock 2 Sequence: (DNA) tattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtctagtcagaccattgtccatagtaatggaaacacctatttggagtggtacctgcagaagccaggccagtctccacagctcctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgettccaaggtagccacgtgcctttcaccttcggcggagggaccaaggtggagatcaaacgaactacgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtg (SEQ ID NO: 603) E6 scFV gBLOCK 1 sequence: (DNA) tgctctgggttccaggttccactggtgacgcggcccagccggccgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaaccattagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcggcggtggcggatccggcggtggcggatccggcggtggcggatcc(SEQ ID NO: 604) E6 scFV gBLOCK 2 Sequence: (DNA) ggcggtggcggatccggcggtggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaaaccggtcatcatcaecatcaccactgataagtttaaacccgctgatcagcctcgactgtgccttctagt(SEQ ID NO: 605) CAR-T C2 CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGS GSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 607) CAR-T C2 CD28 / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTTVVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGTDFTLTINPVEANDTAN YYCQHSRELPFTFGGGTKVEIKRTTTTPAPPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 609) CAR-T C2 4-lBB / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGD NYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDTANY YCQHSRELPFTFGGGTKVEIKRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 611) CAR-T C2 OX40 / CD3z sequence: (DNA) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGG DNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEANDT ANYYCQHSRELPFTFGGGTKVEIKRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 613) CAR-T C2 CD28 / OX40 / CD3z sequence: (DNA) (amino acid) * * (SEQ ID NO: 615) CAR-T E6 0X40 / CD3z sequence: (DNA) atggccctgcccgtgaccgctttgctgctccccctggcgctgctgctgcacgccgccaggccagaggtccagctggttgagagtggcggtgggctggttaagcctggcggctccctgcggctgagctgcgccgcgagtggatttactttcagccgatatgggatgagttgggtgcggcaagctcccgggaagaggctggaatgggtctcaacaatctccggggggggcacttacatctattaccccgactcagtcaaggggagatttaccatttcacgagacaacgctaagaataccctgtatttgcagatgaattctctgagagcagaggacacagctgtttactattgtacccgcgacaactatggcaggaactacgactacggtatggactattggggacaagggacattggttacagtgagcagtggcggcgggggcagcggaggaggaggcagcggtggggggggcagcgagatagtgctcacgcagtcacccgcgactctcagtctctcacctggggaacgagctaccct(SEQ ID NO: 616) (amino acid) MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSDYTLTISSLEPEDFA VYYCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIYWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* * (SEQ ID NO: 617) CAR-T E6 CD28 / OX40 / CD3z sequence: (DNA) (amino acid) ** (SEQ ID NO: 619) MUC1 deletion cytoplasmic sequence (amino acid) SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620) MUC1 deletion cytoplasmic sequence (amino acid) SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621) MUC1 deletion cytoplasmic sequence (amino acid) VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622) MUC1 deletion cytoplasmic sequence (amino acid) SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623) Primer attctaagcttgggccaccatggaactg (SEQ ID NO: 624) tctagagtttaaacttactatttacccggagacagggagag (SEQ ID NO: 625) agtatggcccagccggccgaggtgcagctggtggagtctgg (SEQ ID NO: 626) tagaaggcacagtcgaggctgatcag (SEQ ID NO: 627) attctaagcttgggccaccatggaagc (SEQ ID NO: 628) tctagagtttaaacttactaacactctcccctgttgaagc (SEQ ID NO: 629) agtatggcccagccggccgaaattgtgttgacacagtctccag (SEQ ID NO: 630) tagaaggcacagtcgaggctgatcag (SEQ ID NO: 631) actgtcatatggaggtgcagctggtggagtctg (SEQ ID NO: 632) actgtctcgagtttaatttccactttggtgccgctgc (SEQ ID NO: 633) actgtcatatggaggtgcagctggtggagtctg (SEQ ID NO: 634) actgtaccggttttaatttccactttggtgccgctgc (SEQ ID NO: 635) cttcttcctcaggagcaagctcaccgtgg (SEQ ID NO: 636) gagccgtcggagtccagc (SEQ ID NO: 637) gcacctgaactcctgggg (SEQ ID NO: 638) tttaatttccactttggtgccg (SEQ ID NO: 639) cgcggctagcttaagcttggtaccgagggcca (SEQ ID NO: 640) cgcggcggccgcctgatcagcgggtttaaacttatc (SEQ ID NO: 641)

[0257] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing detailed description, examples, and drawings. All such modifications are intended to be within the scope of the appended claims. The following examples are offered by way of illustration of the invention, not by way of limitation. [Example]

[0258] Example 1 - ELISA competition with NME1 and NME7

[0259] The PSMGFR peptide was covalently linked to BSA using the Imject Maleimide-activated BSA kit (Thermo Fisher). PSMGFR peptide-conjugated BSA was diluted to 7.5 μg / mL in 0.1 M carbonate / bicarbonate buffer, pH 9.6, and 50 μL was added to each well of a 96-well plate. After overnight incubation at 4°C, the plate was washed twice with PBS-T. A 3% BSA solution was then added to block any remaining binding sites in the wells. After 1 h at room temperature (RT), the plate was washed twice with PBS-T, and NME1 or NME7 diluted in PBS-T + 1% BSA was added to a saturating concentration. After 1 h at room temperature, the plate was washed 3x with PBS-T, and anti-MUCl* antibody (or antibody fragment) diluted in PBS-T + 1% BSA was added (5x molar excess over NME1 / NME7). After 1 h at room temperature, the plates were washed 3x with PBS-T, and goat anti-HisTag-HRP diluted in PBS-T + 1% BSA was added at a dilution of 1 / 10,000. After 1 h at room temperature, the plates were washed 3x with PBS-T, and the remaining NME1 or NME7 bound to the PSMGFR peptide was measured at 415 nm using ABTS solution (Thermo Fisher).

[0260] Example 2 – Humanization of anti-MUCl* extracellular domain monoclonal antibody

[0261] The present inventors have generated humanized antibodies that bind to the extracellular domain of MUC1* through a process called complementarity-determining region (CDR) grafting. First, a homology search was performed to independently align the heavy and light chain variable region nucleotide sequences of mouse monoclonal anti-MUC1* antibodies (E6 HC SEQ ID NOs: 12-13; LC SEQ ID NOs: 65-66 and MN-C2 HC SEQ ID NOs: 118-119; LC SEQ ID NOs: 168-169) against a repertoire of humanized antibody sequences (IMGT, the international ImMunoGeneTics information system). The sequence with the highest homology was selected. IGHV3-21*01 is a human IgG heavy chain variable region sequence with 82.9% (DNA) and 74.5% (amino acid) identity to the mouse MN-E6 heavy chain variable region. IGKV3-11*02 is a human IgG light chain variable region sequence with 68.8% (DNA) and 61.1% (amino acid) identity to the mouse MN-E6 light chain variable region. IGHV3-21*04 is a human IgG heavy chain variable region sequence with 85% (DNA) and 81.6% (amino acid) identity to the mouse MN-C2 heavy chain variable region. IGKV7-3*01 is a human IgG light chain variable region sequence with 76.9% (DNA) and 71.3% (amino acid) identity to the mouse MN-C2 light chain variable region. Next, a model of the mouse scFv was generated to select and maintain mouse residues important for CDR and framework stability. Finally, CDRs from the human germline were replaced with the corresponding mouse CDRs.

[0262] Humanized MN-E6 IgG2 heavy chain cloning

[0263] The IGHV3-21*03 leader sequence, humanized MN-E6 heavy chain variable region, and Kozak consensus sequence followed by the constant region of human IgG2 were synthesized at the inventor's request by GenScript, NJ (SEQ ID NOs: 52-53). The cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5'-ATTCTAAGCTTGGGCCACCATGGAACTG-3' (SEQ ID NO: 624) and 5'-TCTAGAGTTTAAACTTACTATTTACCCGGAGACAGGGAGAG-3' (SEQ ID NO: 625). After digestion with Hindlll and Pmel restriction enzymes (New England Biolabs), the purified fragment was digested with the same restriction enzymes and cloned into the pCDNA 3.1V5 vector (Life Technologies).

[0264] Humanized MN-E6 heavy chain cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5'-AGTATGGCCCAGCCGGCCGAGGTGCAGCTGGTGGAGTCTGG-3' (SEQ ID NO: 626) and 5'-TAGAAGGC ACAGTCGAGGCTGATC AG-3' (SEQ ID NO: 627). After digestion with Sfil and Pmel restriction enzymes (New England Biolabs), the purified fragment was digested with the same restriction enzymes and cloned into the pSECTag2 vector (Life Technologies).

[0265] Humanized MN-E6 κ light chain cloning

[0266] The IGHV3-11*02 leader sequence, humanized MN-E6 light chain variable region, and human kappa light chain constant region followed by a Kozak consensus sequence were synthesized by GenScript, NJ at our request (SEQ ID NOs: 107-108). The cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5'-ATTCTAAGCTTGGGCC ACC ATGGAAGC-3' (SEQ ID NO: 628) and 5'-TCTAGAGTTTAAACTTACTAACACTCTCCCCTGTTGAAGC-3' (SEQ ID NO: 629). After digestion with Hindlll and Pmel restriction enzymes (New England Biolabs), the purified fragment was cloned into pCDNA 3.1V5 vector (Life Technologies) digested with the same restriction enzymes.

[0267] Humanized MN-E6 light chain cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5'-AGTATGGCCCAGCCGGCCGAAATTGTGTTGACACAGTCTCCAG-3' (SEQ ID NO: 630) and 5'-TAGAAGGCACAGTCGAGGCTGATCAG-3' (SEQ ID NO: 631). After digestion with Sfil and Pmel restriction enzymes (New England Biolabs), the purified fragment was cloned into the pSECTag2 vector (Life Technologies) digested with the same restriction enzymes.

[0268] Humanized MN-E6 IgG1 heavy chain cloning

[0269] To remove the IgG2 heavy chain constant region, the humanized MN-E6 IgG2 constructs (pCDNA 3.1 V5 and pSECTag2) were digested with BstEII and Pmel (New England Biolabs). The vector carrying the humanized MN-E6 heavy chain variable region was purified. The human IgG1 heavy chain constant region was synthesized at the inventor's request by IDT, IA (SEQ ID NOs: 60-61). Both gBLOCKS and the purified vector carrying the humanized MN-E6 variable region were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0270] Humanized MN-E6 λ light chain cloning

[0271] The humanized MN-E6 kappa light chain constructs (pCDNA 3.1 V5 vector and pSECTag2 vector) were digested with Kpnl and Pmel (New England Biolabs) to remove the kappa light chain constant region. The vector carrying the humanized MN-E6 light chain variable region was purified. The human lambda light chain constant region was synthesized by IDT, IA at the inventor's request (SEQ ID NO: 115). Both the gBLOCK and purified vector carrying the humanized MN-E6 light chain variable region were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0272] Humanized MN-C2 IgG1 and IgG2 heavy chain cloning

[0273] Humanized MN-E6 IgG1 and IgG2 heavy chains in pSECTag2 were digested with Sfil and Agel to remove the MN-E6 variable region. Humanized MN-E6 IgG1 and IgG2 heavy chains in pCDNA 3.1 V5 were digested with Hindlll and Agel to remove the MN-E6 variable region, and vectors carrying human IgG1 or IgG2 constant regions were purified. Humanized MN-C2 heavy chains were synthesized at the inventor's request by IDT, IA (SEQ ID NOs: 160 and 165). The sequence cloned into pCDNA 3.1 V5 contains the 5' murine Ig κ chain leader sequence (SEQ ID NO: 160). Both gBLOCK and the purified vectors carrying the human IgG1 or IgG2 constant regions were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0274] Humanized MN-C2 κ / λ light chain cloning

[0275] Two humanized MN-C2 variable regions linked to kappa light chain constant regions and two humanized MN-C2 variable regions linked to lambda light chain constant regions were synthesized by IDT (IA) at the inventor's request (SEQ ID NOS: 210 and 213, and SEQ ID NOS: 216 and 219, respectively). pCDNA 3.1 V5 was digested with Hindlll and Pmel restriction enzymes (New England Biolabs), and pSEC Tag2 was digested with Sfil and Pmel restriction enzymes (New England Biolabs). Both plasmids were then purified. SEQ ID NOS: 210 and 216 were ligated into the digested pCDNA 3.1 V5, and SEQ ID NOS: 213 and 219 were ligated into the digested pSEC Tag2 using the Gibson Assembly Cloning Kit (New England Biolabs).

[0276] Humanized C3 IgG1 heavy chain cloning

[0277] The humanized E6 IgG1 construct (pSECTag2) was digested with Sfil and Agel (New England Biolabs) to remove the E6 heavy chain variable region. The vector was purified without the humanized E6 heavy chain variable region. The humanized C3 heavy chain variable region was synthesized at the inventor's request by IDT, IA (SEQ ID NO: 457). The gBLOCK and purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0278] Humanized C3κ light chain cloning

[0279] pEF V5-His was digested with BamHI and Pmel (New England Biolabs) and purified. Humanized C3 κ light chain (SEQ ID NO: 504) was synthesized by IDT (IA) at the request of the inventors. Both gBLOCK and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0280] Humanized C8κ light chain cloning

[0281] pEF V5-His was digested with BamHI and Pmel (New England Biolabs) and purified. Humanized C8 kappa light chain (SEQ ID NO: 583) was synthesized by IDT, IA at the inventor's request. Both gBLOCK and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0282] Example 3 – Cloning of humanized scFV of anti-MUCl* extracellular domain antibody

[0283] Humanized E6 scFV cloning:

[0284] pSEC Tag2 was digested with Sfil and Pmel (New England Biolabs) and purified. Humanized E6 scFV gBLOCKS (SEQ ID NO: 604-605) was synthesized by IDT, IA at the request of the inventors. Both gBLOCKS and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0285] Humanized E6 scFV cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5-ACTGTCATATGGAGGTGCAGCTGGTGGAGTCTG-3' (SEQ ID NO: 632) and 5'-ACTGTCTCGAGTTTAATTTCCACTTTGGTGCCGCTGC-3' (SEQ ID NO: 633). After digestion with Ndel and Xhol restriction enzymes (New England Biolabs), the purified fragment was cloned into pET21b vector (Novagen) digested with the same restriction enzymes. The humanized E6 scFv cDNA was cloned 5' of a histidine tag for protein purification.

[0286] Humanized E6 scFV cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5-ACTGTCATATGGAGGTGCAGCTGGTGGAGTCTG-3' (SEQ ID NO: 634) and 5'-ACTGTACCGGTTTTAATTTCCACTTTGGTGCCGCTGC-3' (SEQ ID NO: 635). After digestion with Ndel and Agel restriction enzymes (New England Biolabs), the purified fragment was cloned into a modified pET21b vector (Novagen) digested with the same restriction enzymes. The vector was modified to include the StrepTag2 sequence followed by two stop codons 5′ of a histidine tag. A humanized E6 scFv cDNA was cloned 5′ of StrepTag2 for protein purification.

[0287] Humanized E6, C2, C3 and C8 scFV-Fc cloning

[0288] To remove portions of the IgG1 heavy chain constant region and the E6 heavy chain variable region, the humanized E6 IgG1 construct (pSECTag2) was digested with Sfil and SacII (New England Biolabs). The vector without the humanized E6 heavy chain variable region was purified. Humanized E6, C2, C3, and C8 scFV gBLOCKS were synthesized by IDT, IA at the inventor's request (SEQ ID NOs: 258-259, 262-263, 266-267, and 270-271). The E6, C2, C3, and C8 gBLOCKS and purified vector were ligated using a Gibson Assembly Cloning Kit (New England Biolabs) to assemble the corresponding scFV in frame with the human IgG1 Fc region.

[0289] Humanized E6 scFV-Fc Y407R cloning

[0290] Tyrosine 407 of the humanized E6 scFV-Fc was changed to arginine (Y407R) by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (NEB) with the following primers: 5'-CTTCTTCCTCAGGAGCAAGCTCACCGTGG-3' (SEQ ID NO: 636) and 5'-GAGCCGTCGGAGTCC AGC-3' (SEQ ID NO: 637).

[0291] Humanized E6 scFV-Fc hingeless cloning

[0292] The hinge region of the humanized E6 scFv-Fc was removed by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (NEB) with the following primers: 5'-GCACCTGAACTCCTGGGG-3' (SEQ ID NO: 638) and 5'-TTTAATTTCCACTTTGGTGCCG3' (SEQ ID NO: 639).

[0293] Example 4 – Cloning of anti-MUCl* extracellular domain antibody CAR-T

[0294] CAR E6 CD28 / 4 1BB / CD3z cloning:

[0295] pCDNA 3.1 V5 was digested with Kpnl and Pmel (New England Biolabs) and purified. The complete CAR-T E6 (CD8 / CD28 / 4-lBB / CD3z) gBLOCK was synthesized at the inventor's request by IDT, IA (SEQ ID NO: 305). Both the gBLOCK and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0296] CAR E6 CD3z cloning:

[0297] pCDNA 3.1 V5 CAR-T E6CD8 / CD28 / 4-lBB / CD3z was digested with EcoRV and Pmel (New England Biolabs) to remove the cytoplasmic domain. The vector without the cytoplasmic domain was purified. CAR-T E6 CD8 / CD3z gBLOCK was synthesized by IDT, IA at the inventor's request (SEQ ID NO: 296). Both the gBLOCK and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0298] CAR E6 CD28 / CD3z cloning:

[0299] pCDNA 3.1 V5 CAR-T E6CD8 / CD28 / 4-lBB / CD3z was digested with EcoRV and Pmel (New England Biolabs) to remove the cytoplasmic region. The vector without the cytoplasmic region was purified. CAR-T E6 CD8 / CD28 / CD3z gBLOCK was synthesized by IDT, IA at the inventor's request (SEQ ID NO: 299). Both the gBLOCK and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0300] CAR E6 4-1BB / CD3z cloning:

[0301] pCDNA 3.1 V5 CAR-T E6CD8 / CD28 / 4-lBB / CD3z was digested with EcoRV and Pmel (New England Biolabs) to remove the cytoplasmic region. The vector without the cytoplasmic region was purified. CAR-T E6 CD8 / 4-lBB / CD3z gBLOCK was synthesized by IDT, IA at the inventor's request (SEQ ID NO: 302). Both the gBLOCK and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0302] CAR C2 CD28 / 4 1BB / CD3z cloning:

[0303] pCDNA 3.1 V5 CAR-T E6CD8 / CD28 / 4-lBB / CD3z was digested with Kpnl and EcoRV (New England Biolabs) to remove the E6 scFV. The vector without the E6 scFV was purified. CAR-T C2 gBLOCKs were synthesized by IDT, IA at the inventor's request (SEQ ID NO: 308-309). Both gBLOCKs and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0304] CAR subcloning into lentiviral vectors:

[0305] The entire pcDNA 3.1 V5 CAR cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5-CGCGGCTAGCTTAAGCTTGGTACCGAGGGCCA-3' (SEQ ID NO: 640) and 5'-CGCGGCGGCCGCCTGATCAGCGGGTTTAAACTTATC-3' (SEQ ID NO: 641). After digestion with Nhel and Notl restriction enzymes (New England Biolabs), the purified fragment was cloned into lentiviral vectors (pCDH-EFl-MCS-IRESGFP and pCDH-CMV-MCS-EFl-copGFP+puro, SBI) digested with the same restriction enzymes.

[0306] CAR-E6-Fc / 8 / 41BB / CD3z cloning:

[0307] pCDH-CMV-MCS-EF1-copGFP+puro (SBI) was digested with Nhel and Notl (New England Biolabs), and the vector was purified. gBLOCKs were synthesized at the request of the inventors by IDT, IA (SEQ ID NOs: 312, 313, and 314). gBLOCKs and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0308] CAR-E6-FcH / 8 / 41BB / CD3z cloning:

[0309] pCDH-CMV-MCS-EF1-copGFP+puro (SBI) was digested with Nhel and Notl (New England Biolabs), and the vector was purified. gBLOCKs were synthesized at the request of the inventors by IDT, IA (SEQ ID NOs: 312, 317, and 314). gBLOCKs and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0310] CAR-E6-Fc-4-41BB-CD3z Cloning:

[0311] pCDH-CMV-MCS-EF1-copGFP+puro (SBI) was digested with Nhel and Notl (New England Biolabs), and the vector was purified. gBLOCKs were synthesized at the request of the inventors by IDT, IA (SEQ ID NOs: 312, 313, and 320). gBLOCKs and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0312] CAR-E6 FcH / 4 / 41BB / CD3z cloning:

[0313] pCDH-CMV-MCS-EF1-copGFP+puro (SBI) was digested with Nhel and Notl (New England Biolabs), and the vector was purified. gBLOCKs were synthesized at the request of the inventors by IDT, IA (SEQ ID NOs: 312, 317, and 320). gBLOCKs and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0314] CAR-E6 IgD / 8 / 41BB / CD3z cloning:

[0315] pCDH-CMV-MCS-EF1-copGFP+puro (SBI) was digested with Nhel and Notl (New England Biolabs), and the vector was purified. gBLOCKs (SEQ ID NOs: 312, 325, and 326) were synthesized by IDT, IA at the inventor's request. gBLOCKs and the purified vector were ligated using the Gibson Assembly Cloning Kit (New England Biolabs).

[0316] CAR...

Claims

1. A human or humanized anti-MUCl* antibody, antibody fragment, or antibody-like protein that binds to a site in the extracellular domain of a MUCl isoform or degradation product lacking the tandem repeat region.

2. 2. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 1, which specifically binds to: (i) the PSMGFR site of MUC1; (ii) a PSMGFR peptide; (iii) a peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); (iv) a peptide having the amino acid sequence SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621); (v) a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622), or (vi) A peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623).

3. 2. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 1, wherein the human or humanized antibody is IgG1, IgG2, IgG3, IgG4 or IgM.

4. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 1, wherein the human or humanized antibody fragment or antibody-like protein is an scFv or scFv-Fc.

5. The human or humanized antibody, antibody fragment, or antibody-like protein of claim 1, which comprises a heavy chain variable region and a light chain variable region derived from the murine monoclonal MN-E6 antibody and has at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-E6 antibody.

6. 6. A human or humanized antibody or antibody fragment or antibody-like protein according to claim 5, wherein the heavy chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 13, and the light chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO:

66.

7. 6. The human or humanized antibody or antibody fragment or antibody-like protein of claim 5, comprising complementarity determining regions (CDRs) in the heavy and light chain variable regions having at least 90%, 95%, or 98% sequence identity to the CDR1, CDR2, or CDR3 regions having the following sequences: CDR1 heavy chain SEQ ID NO: 17, CDR1 light chain SEQ ID NO: 70, CDR2 heavy chain SEQ ID NO: 21, CDR2 light chain SEQ ID NO: 74, CDR3 heavy chain SEQ ID NO: 25, CDR3 light chain SEQ ID NO:

78.

8. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 5, wherein the human or humanized antibody is IgG1, IgG2, IgG3, IgG4 or IgM.

9. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 5, wherein the human or humanized antibody fragment or antibody-like protein is an scFv or scFv-Fc.

10. The human or humanized antibody, antibody fragment, or antibody-like protein of claim 1, which comprises a heavy chain variable region and a light chain variable region derived from the murine monoclonal MN-C2 antibody and has at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-C2 antibody.

11. 11. The human or humanized antibody or antibody fragment or antibody-like protein of claim 10, wherein the heavy chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 119, and the light chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO:

169.

12. 11. The antibody of claim 10, comprising complementarity determining regions (CDRs) in the heavy and light chain variable regions that have at least 90%, 95%, or 98% sequence identity to the CDR1, CDR2, or CDR3 regions having the following sequences: CDR1 heavy chain SEQ ID NO: 123, CDR1 light chain SEQ ID NO: 173, CDR2 heavy chain SEQ ID NO: 127, CDR2 light chain SEQ ID NO: 177, CDR3 heavy chain SEQ ID NO: 131, CDR3 light chain SEQ ID NO:

181.

13. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 10, wherein the human or humanized antibody is IgG1, IgG2, IgG3, IgG4 or IgM.

14. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 10, wherein the human or humanized antibody fragment or antibody-like protein is an scFv or scFv-Fc.

15. The human or humanized antibody, antibody fragment, or antibody-like protein of claim 1, which comprises a heavy chain variable region and a light chain variable region derived from the murine monoclonal MN-C3 antibody and has at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-C3 antibody.

16. 16. The human or humanized antibody or antibody fragment or antibody-like protein of claim 15, wherein the heavy chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 414, and the light chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO:

459.

17. 16. The antibody of claim 15, comprising complementarity determining regions (CDRs) in the heavy and light chain variable regions having at least 90%, 95%, or 98% sequence identity to the CDR1, CDR2, or CDR3 regions with the following sequences: CDR1 heavy chain SEQ ID NO: 418, CDR1 light chain SEQ ID NO: 463, CDR2 heavy chain SEQ ID NO: 422, CDR2 light chain SEQ ID NO: 467, CDR3 heavy chain SEQ ID NO: 426, CDR3 light chain SEQ ID NO:

471.

18. 16. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 15, wherein the human or humanized antibody is IgG1, IgG2, IgG3, IgG4 or IgM.

19. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 15, wherein the human or humanized antibody fragment or antibody-like protein is an scFv or scFv-Fc.

20. The human or humanized antibody, antibody fragment, or antibody-like protein of claim 1, which comprises a heavy chain variable region and a light chain variable region derived from a murine monoclonal MN-C8 antibody and has at least 80%, 90%, 95%, or 98% sequence identity to the murine monoclonal MN-C8 antibody.

21. 21. The human or humanized antibody or antibody fragment or antibody-like protein of claim 20, wherein the heavy chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO: 506, and the light chain variable region has at least 90%, 95%, or 98% sequence identity to SEQ ID NO:

544.

22. 21. The antibody of claim 20, comprising complementarity determining regions (CDRs) in the heavy and light chain variable regions that have at least 90%, 95%, or 98% sequence identity to the CDR1, CDR2, or CDR3 regions with the following sequences: CDR1 heavy chain SEQ ID NO: 508, CDR1 light chain SEQ ID NO: 546, CDR2 heavy chain SEQ ID NO: 510, CDR2 light chain SEQ ID NO: 548, CDR3 heavy chain SEQ ID NO: 512, CDR3 light chain SEQ ID NO:

550.

23. 21. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 20, wherein the human or humanized antibody is IgG1, IgG2, IgG3, IgG4 or IgM.

24. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 20, wherein the human or humanized antibody fragment or antibody-like protein is an scFv or scFv-Fc.

25. An anti-MUC1* extracellular domain antibody comprising the sequence of humanized MN-E6 displayed by a humanized IgG2 or IgG1 heavy chain paired with a humanized κ or λ light chain.

26. 26. The antibody of claim 25, wherein the humanized IgG2 heavy chain is SEQ ID NO: 53, the humanized IgG1 heavy chain is SEQ ID NO: 57, the humanized κ light chain is SEQ ID NO: 108, and the humanized λ light chain is SEQ ID NO: 112, or sequences with 90%, 95%, or 98% sequence identity thereto.

27. An anti-MUC1* extracellular domain antibody comprising the sequence of humanized MN-C2 expressed by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized κ or a humanized λ light chain.

28. 28. The antibody of claim 27, comprising a humanized IgG1 heavy chain MN-C2 (SEQ ID NO: 159) or IgG2 heavy chain (SEQ ID NO: 164) paired with a κ light chain (SEQ ID NO: 213) or a λ light chain (SEQ ID NO: 219), or a sequence with 90%, 95% or 98% sequence identity thereto.

29. An anti-MUC1* extracellular domain antibody comprising the sequence of humanized MN-C3 expressed by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized κ light chain or a humanized λ light chain.

30. 30. The antibody of claim 29, wherein the humanized MN-C3 IgG1 heavy chain is SEQ ID NO: 454, the IgG2 heavy chain is SEQ ID NO: 456, the κ light chain is SEQ ID NO: 503, and the λ light chain is SEQ ID NO: 501, or sequences with 90%, 95%, or 98% sequence identity thereto.

31. An anti-MUC1* extracellular domain antibody comprising the sequence of humanized MN-C8 expressed by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized κ light chain or a humanized λ light chain.

32. 32. The antibody of claim 31, wherein the humanized MN-C8 IgG1 heavy chain is SEQ ID NO: 540, the IgG2 heavy chain is SEQ ID NO: 542, the κ light chain is SEQ ID NO: 582, and the λ light chain is SEQ ID NO: 580, or sequences with 90%, 95%, or 98% sequence identity thereto.

33. 2. The human or humanized anti-MUC1* antibody, antibody fragment, or antibody-like protein of claim 1, which inhibits the binding of an NME protein to MUC1*.

34. 34. The human or humanized anti-MUC1* antibody, antibody fragment, or antibody-like protein of claim 33, wherein the NME is NME7 or NME1.

35. 34. The human or humanized anti-MUC1* antibody, antibody fragment, or antibody-like protein of claim 33, wherein the NME is NME6 or NME8.

36. 34. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 33, wherein the human or humanized antibody is IgG1, IgG2, IgG3, IgG4 or IgM.

37. The human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein of claim 33, wherein the human or humanized antibody fragment or antibody-like protein is an scFv or scFv-Fc.

38. A single chain variable fragment (scFv) comprising heavy and light chain variable regions joined via a linker, further comprising the CDRs of an antibody that binds to the MUCl* extracellular domain.

39. The scFv of claim 38, wherein the CDRs of the anti-MUC1* antibody are derived from the MN-E6, MN-C2, MN-C3, or MN-C8 antibody.

40. The scFv of claim 39, wherein the CDRs of the anti-MUC1* antibody are derived from a humanized MN-E6, MN-C2, MN-C3, or MN-C8 antibody.

41. 39. The scFv of claim 38 selected from the group of SEQ ID NOs: 233, 235 and 237 (E6).

42. 39. The scFv of claim 38, selected from the group of SEQ ID NOs: 239, 241 and 243 (C2).

43. 39. The scFv of claim 38 selected from the group of SEQ ID NOs: 245, 247 and 249 (C3).

44. 39. The scFv of claim 38 selected from the group of SEQ ID NOs: 251, 253 and 255 (C8).

45. A chimeric antigen receptor (CAR) comprising an scFv or humanized variable region that binds to the extracellular domain of MUC1 lacking tandem repeats, a linker molecule, a transmembrane region, and a cytoplasmic region.

46. 46. ​​The CAR of claim 45, wherein the single chain antibody fragment binds to: (i) the PSMGFR site of MUC1; (ii) a PSMGFR peptide; (iii) a peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); (iv) a peptide having the amino acid sequence of SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621); (v) a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622), or (vi) A peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623).

47. The CAR according to claim 46, comprising any part of a variable region according to any one of claims 25 to 32, or a combination thereof in an extracellular domain, a transmembrane domain, and a cytoplasmic tail comprising a sequence motif that signals immune system activation.

48. The CAR of claim 45, wherein the extracellular domain comprises a humanized single-chain antibody fragment of MN-E6 scFv, MN-C2 scFv, MN-C3 scFv, or MN-C8 scFv.

49. 4. The antibody of claim 4, comprising a humanized single-chain antibody fragment of MN-E6 scFv (SEQ ID NO: 233, 235, or 237), MN-C2 scFv (SEQ ID NO: 239, 241, or 243), MN-C3 scFv (SEQ ID NO: 245, 247, or 249), or MN-C8 scFv (SEQ ID NO: 251, 253, or 254).

8. A CAR according to claim 8.

50. The CAR of claim 45, wherein the cytoplasmic tail comprises one or more of the signal sequence motifs CD3-ZETA, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7.

51. The sequences are CARMN-E6 CD3z (SEQ ID NO: 295), CARMN-E6 CD28 / CD3z (SEQ ID NO: 298), CARMN-E6 4-lBB / CD3z (SEQ ID NO: 301), CARMN-E6 OX40 / CD3z (SEQ ID NO: 617), CARMN-E6 CD28 / 4-lBB / CD3z (SEQ ID NO: 304), CARMN-E6 CD28 / OX40 / CD3z (SEQ ID NO: 619), CAR MN-C2 CD3z (SEQ ID NO: 607), CAR MN-C2 CD28 / CD3z (SEQ ID NO: 609), CAR MN-C2 4-lBB / CD3z (SEQ ID NO: 611), CAR MN-C2 The CAR of claim 45, which is OX40 / CD3z (SEQ ID NO: 613), CAR MN-C2 CD28 / 4-lBB / CD3z (SEQ ID NO: 307), or CAR MN-C2 CD28 / OX40 / CD3z (SEQ ID NO: 615).

52. A cell containing a CAR with an extracellular domain that binds to MUC1* transformed or transduced cells.

53. The cell of claim 52, wherein the cell comprising the CAR is an immune system cell.

54. The cell of claim 53, wherein the immune system cell is a T cell.

55. The cell of claim 53, wherein the immune system cell is a dendritic cell.

56. 54. The cell of claim 53, wherein the immune system cell is a mast cell.

57. A CAR molecule in which the extracellular domain unit recognizes a certain peptide.

58. The CAR molecule of claim 57, wherein the peptide is PSMGFR (SEQ ID NO: 2).

59. The CAR molecule of claim 57, wherein the peptide is a peptide derived from NME7.

60. 60. The CAR molecule of claim 59, wherein the peptide is selected from the following: NME7A peptide 1 (A region): MLSRKEALDFHVDHQS (SEQ ID NO: 7), NME7A peptide 2 (A region): SGVARTDASES (SEQ ID NO: 8), NME7B peptide 1 (B region): DAGFEISAMQMFNMDRVNVE (SEQ ID NO: 9), NME7B peptide 2 (B region): EVYKGVVTEYHDMVTE (SEQ ID NO: 10), or NME7B peptide 3 (B region): AIFGKTKIQNAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 11).

61. A composition comprising at least two CARs having different extracellular domain units transfected into the same cell.

62. A composition comprising at least two CARs with different extracellular domain units transfected into the same cell, wherein one CAR does not have a target recognition unit and another CAR has a target recognition unit.

63. 62. A composition comprising at least two CARs according to claim 61, wherein one of the extracellular domain recognition units binds to the MUC1* extracellular domain.

64. The composition comprising at least two CARs according to claim 61, wherein one of the extracellular domain recognition units binds to PD-1.

65. 62. The composition comprising at least two CARs of claim 61 , wherein one of the extracellular domain recognition units is an antibody fragment and the other is a peptide.

66. The composition comprising at least two CARs according to claim 61, wherein one is an anti-MUC1* scFv selected from the group consisting of scFv of MN-E6 antibody, scFv of MN-C2 antibody, scFv of MN-C3 antibody, or scFv of MN-C8 antibody, and the other is derived from NME7 or a peptide selected from the group below: NME7A peptide 1 (A region): MLSRKEALDFHVDHQS (SEQ ID NO: 7), NME7A peptide 2 (A region): SGVARTDASES (SEQ ID NO: 8), NME7B peptide 1 (B region): DAGFEISAMQMFNMDRVNVE (SEQ ID NO: 9), NME7B peptide 2 (B region): EVYKGVVTEYHDMVTE (SEQ ID NO: 10), and NME7B peptide 3 (B region): AIFGKTKIQNAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 11).

67. The antibody of claim 1 , wherein the antibody is an engineered antibody-like protein.

68. 1. A method for screening a library of antibodies or antibody fragments that are human, comprising: , which binds to (i) a PSMGFR peptide; (ii) a peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); (iii) a peptide having the amino acid sequence of SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621); (iv) a peptide having the amino acid sequence VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622); (v) a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623); (vi) NME7 protein, or (vii) Peptide fragments of the NME7 protein.

69. 69. A method of treating a disease in a subject, the method comprising administering to a human suffering from said disease an antibody according to any one of claims 1 to 68, wherein said subject abnormally expresses MUC1.

70. 70. The method of claim 69, wherein the disease is cancer.

71. A method of treating a disease in a subject comprising administering an NME peptide to a human suffering from said disease, wherein said subject aberrantly expresses MUC1.

72. 69. A method of growing or expanding a stem cell population, comprising contacting the cells with an antibody according to any one of claims 1 to 68.

73. A method for promoting stem cell attachment to a surface, comprising coating a surface with a humanized MN-C3 or MN-C8 antibody, antibody fragment thereof, or single-chain antibody thereof, and contacting the surface with stem cells.

74. A method for transporting stem cells in vitro or in vivo, comprising the steps of coating a surface with a humanized MN-C3 or MN-C8 antibody, its antibody fragment, or its single-chain antibody, contacting stem cells with the surface, and transporting the stem cells to a specific site.

75. A method for isolating stem cells, comprising the steps of coating a surface with a humanized MN-C3 or MN-C8 antibody, antibody fragment thereof, or single-chain antibody thereof, contacting a mixed population of cells with the surface, and isolating the stem cells.

76. An scFv comprising a variable region fragment derived from an antibody that binds to the extracellular domain of a MUC1 isoform or cleavage product lacking the tandem repeat region.

77. The scFv of claim 76, wherein the variable region fragment is derived from mouse monoclonal antibody MN-E6 (SEQ ID NOs: 13 and 66), humanized MN-E6 (SEQ ID NOs: 39 and 94), or MN-E6 scFv (SEQ ID NOs: 233, 235, and 237).

78. The scFv of claim 76, wherein the variable region fragment is derived from mouse monoclonal antibody MN-C2 (SEQ ID NOs: 119 and 169), humanized MN-C2 (SEQ ID NOs: 145 and 195), or MN-C2 scFv (SEQ ID NOs: 239, 241, and 243).

79. The scFv of claim 76, wherein the variable region fragment is derived from mouse monoclonal antibody MN-C3 (SEQ ID NOs: 414 and 459), humanized MN-C3 (SEQ ID NOs: 440 and 487), or MN-C3 scFv (SEQ ID NOs: 245, 247, and 249).

80. The scFv of claim 76, wherein the variable region fragment is derived from mouse monoclonal antibody MN-C8 (SEQ ID NOs: 505 and 544), humanized MN-C8 (SEQ ID NOs: 526 and 566), or MN-C8 scFv (SEQ ID NOs: 251, 253, 255).

81. A method of treating a human diagnosed or suspected of also having or at risk of developing a MUC1 or MUCl* positive cancer, comprising administering to said human an effective amount of an scFv of any one of claims 76 to 80.

82. An scFv-Fc construct comprising an scFv according to any one of claims 76 to 80.

83. The scFv-Fc construct of claim 82, which is dimerized.

84. 83. The scFv-Fc construct of claim 82, wherein the Fc component is mutated so that the scFv-Fc is monomeric.

85. The scFv-Fc construct of claim 84, wherein the mutations include creating F405Q, Y407R, T366W / L368W and T364R / L368R mutations or combinations thereof in the Fc represented by SEQ ID NOs: 281, 279, 285 and 287, or mutating or deleting the hinge region of the Fc.

86. A polypeptide comprising at least two different scFv sequences, wherein one of the scFv sequences is a sequence that binds to the extracellular domain of a MUC1 isoform or degradation product lacking the tandem repeat region.

87. 87. The polypeptide of claim 86, wherein the polypeptide binds to: (i) the PSMGFR site of MUC1; (ii) a PSMGFR peptide; (iii) a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); (iv) a peptide having the amino acid sequence VQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621); (v) a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 622), or (vi) A peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 623).

88. 87. The polypeptide of claim 86, wherein the polypeptide binds to a receptor on an immune cell.

89. 89. The polypeptide of claim 88, wherein the polypeptide binds to a receptor on a T cell.

90. 90. The polypeptide of claim 89, wherein the polypeptide binds to CD3 on a T cell.

91. 83. A method for detecting the presence of cells that aberrantly express MUC1*, comprising contacting a sample of cells with the scFv-Fc of claim 82 and detecting the presence of binding of the scFv-Fc to the cells.

92. 92. The method of claim 91, wherein the cell is a cancer cell.

93. A method of testing a subject cancer for suitability for treatment with a composition comprising a portion of the variable region of MN-E6, MN-C2, MN-C3, or MN-C8, comprising contacting a portion of a body sample from the patient with the corresponding MN-E6 scFv-Fc, MN-C3 scFv-Fc, MN-C3 scFv-Fc, or MN-C8 scFv-Fc.

94. A method of treating a subject with a disease comprising exposing T cells from the subject to MUC1* (wherein T cells at various stages of maturation develop MUC1*-specific receptors), generating and expanding adaptive T cells, and administering the adaptive T cells to a donor patient diagnosed or suspected of also having or being at risk of developing MUC1*-positive cancer.

95. A method of treating a patient diagnosed or suspected of also having or at risk of developing MUC1-positive or MUC1*-positive cancer, comprising administering an effective amount of immune cells transduced with a MUC1*-targeting CAR.

96. 96. The method of claim 95, wherein the immune cells are T cells isolated from a patient, which are transduced with CARs in which the targeting head of the CAR binds to MUC1*, and after expansion of the transduced T cells, the CAR T cells are administered to the patient in an effective amount.

97. 96. The method of claim 95, wherein the immune cells are T cells isolated from a patient, which are transduced with CARs, wherein the targeting head of the CAR comprises a portion of huMN-E6, huMN-C2, huMN-C3, or huMN-C8, and after optional expansion of the transduced T cells, the CAR T cells are administered to the patient in an effective amount.

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