Modified b cells and methods for use thereof

Abstract

B cells engineered to express B cell receptors targeting a HER2 antigen and compositions and methods for use or preparation thereof.

Description

[0001] MODIFIED B CELLS AND METHODS FOR USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 642,433, filed May 3, 2024, the entire contents of which are incorporated herein by reference.

[0004] BACKGROUND

[0005] Breast cancer (BC) is one of the most aggressive tumors and 15-20% of all BC cases harbor the overexpression or amplification of the tyrosine kinase receptor human epidermal growth factor receptor 2 (HER2) gene. Despite the fact that monoclonal antibodies (MoAbs) and antibody-drug conjugates (ADCs) directed against HER2 have greatly improved the prognosis of HER2+ BC, not all patients with limited-stage diseases are cured using these agents, and HER2+ metastatic (m)BC remains an almost incurable disease. Most therapeutic failures derive from acquired new mutations that confer resistance to tumor sub-clones that are progressively selected during the treatment. This is mainly because humoral immunity alone is not sufficient to activate strong local inflammation in the immunosuppressive tumor microenvironment (TME), and because the efficacy of the therapy depends on the direct effects of the administered agents on the target cells. Cellular adoptive immunotherapy using T cells has the potential to overcome the immunosuppressive barriers imposed by the TME to naturally occurring immune responses, thus possibly enabling better control of metastatic cells. However, previous attempts using highly activated T cells redirected towards HER2 antigen highlighted critical safety issues due to the occurrence of lethal toxicity by cytokine release syndrome (CRS).

[0006] Accordingly, there is a need for improved methods for cellular adoptive immunotherapy.

[0007] SUMMARY

[0008] As described below, the present disclosure features B cells engineered to express B cell receptors targeting a HER2 antigen and compositions and methods for use or preparation thereof.

[0009] In one aspect, the disclosure features a polynucleotide containing an insertion region to be inserted into the genome of a cell. The polynucleotide contains from 5’ to 3’: A) a left homology arm; B) an insertion region containing: a) a promoter; b) a sequence encoding a polypeptide containing: i) a heavy chain variable region of an HER2 -binding antibody (VH), a kappa light chain constant region (CLK), a peptide linker, and a light chain variable region of the HER2 -binding antibody (VL); or ii) a light chain variable region of an HER2 -binding antibody (VL), a kappa light chain constant region (CLK), a peptide linker, and a heavy chain variable region of the HER2 -binding antibody (VH); and c) a splice donor site; and C) a right homology arm. The right homology arm and the left homology arm are each capable of binding a polynucleotide strand in the genome of the cell between a region of the immunoglobulin heavy chain (IGH) encoding joining gene segments and an iEp intronic enhancer.

[0010] In another aspect, the disclosure features a vector contains the polynucleotide of any aspect of the disclosure, or embodiments thereof.

[0011] In another aspect, the disclosure features an immune cell expressing the polypeptide encoded by the polynucleotide of any aspect of the disclosure, or embodiments thereof.

[0012] In another aspect, the disclosure features a method for preparing a B cell expressing an engineered B cell receptor. The method involves I) contacting a B cell or progenitor thereof with a polynucleotide. The polynucleotide contains an insertion region to be inserted into the genome of the cell. The polynucleotide contains from 5’ to 3’ : A) a left homology arm; B) an insertion region containing: a) a promoter; b) a sequence encoding a polypeptide containing: i) a heavy chain variable region of an HER2 -binding antibody (VH), a kappa light chain constant region (CLK), a peptide linker, and a light chain variable region of the HER2 -binding antibody (VL); or ii) a light chain variable region of an HER2 -binding antibody (VL), a kappa light chain constant region (CLK), a peptide linker, and a heavy chain variable region of the HER2 -binding antibody (VH); and c) a splice donor site; and C) a right homology arm. The method further involves II) contacting the cell with a nucleic acid programmable DNA binding protein (napDNAbp) having endonuclease activity, or a polynucleotide encoding the napDNAbp, and a guide polynucleotide targeting a sequence within an immunoglobulin heavy chain (IGH) locus in the genome of the cell, thereby inserting the insertion region within the genome of the B cell at the IGH locus. The right homology arm and the left homology arm are each capable of binding a polynucleotide strand in the genome of the cell between the region of the IGH encoding joining gene segments and an iEp intronic enhancer.

[0013] In another aspect, the disclosure features a method for preparing a B cell expressing an engineered B cell receptor. The method involves I) contacting a B cell or progenitor thereof with an adeno-associated virus (AAV) vector containing a polynucleotide containing the following sequence: CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTGCGGCCGCACGCGTATGTGACGCCCGGAGACAGAAGGTCTCTGGGTGGCTGGGTTTTTGTGG GGTGAGGATGGACATTCTGCCATTGTGATTACTACTACTACTACTACATGGACGTCTGGGGCAA AGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGCCACTCTAGGGCCTTTGTTTTCTGCTAC TGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCTCGGGGCATGTTCCGAGGGGACCTGGGC

[0014] GGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTGAGGATCTGGGAGCCTCTGTGGATTTTC

[0015] CGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGTCTGATGGAGTAACTGAGCCTGGGGGCT

[0016] TGGGGAGCCACATTTGGACGAGATGCCTGAACAAACCAGGGGTCTTAGTGATGGCTGAGGAATG

[0017] TGTCTCAGGAGCGGTGTCGAATTCGTCCTGCTGGACACTCATGTAGGGTAACGAGTGGCCACCT

[0018] TTTCAGTGTTACCAGTGAGCTCTGAGTGTTCCTAATGGGACCAGGATGGGTCTAGGTGCCTGCT

[0019] CAATGTCAGAGACAGCAATGGTCCCACAAAAAACCCAGGTAATCTTTAGGCCAATAAAATGTGG

[0020] GTTCACAGTGAGGAGTGCATCCTGGGGTTGGGGTTTGTTCTGCAGCGGGAAGAGCGCTGTGCAC

[0021] AGAAAGCTTAGAAATGGGGCAAGAGATGCTTTTCCTCAGGCAGGATTTAGGGCTTGGTCTCTCA

[0022] GCATCCCACACTTGTACAGCTGATGTGGCATCTGTGTTTTCTTTCTCATCCTAGATCAGGCTTT

[0023] GAGCTGTGAAATACCCTGCCTCATGCATATGCAAATAACCTGAGGTCTTCTGAGATAAATATAG

[0024] ATATATTGGTGCCCTGAGAGCATCACATAACAACCACATTCCTCCTCTGAAGAAGCCCCTGGGA

[0025] GCACAGCTCATGCCACCATGGAAGCTCCAGCTCAGCTGCTGTTTCTGCTGCTGCTGTGGCTGCC

[0026] TGATACCACCGGCGAGATCGACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTG

[0027] GGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACGTGAACACAGCCGTGGCTTGGTATC

[0028] AGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCAGCTTTCTGTACAGCGGCGT

[0029] GCCCAGCAGATTCAGCGGCTCTAGAAGCGGCACCGACTTCACCCTGACCATAAGCAGTCTGCAG

[0030] CCCGAGGACTTCGCCACCTACTACTGTCAGCAGCACTACACCACACCTCCAACCTTTGGCCAGG

[0031] GCACCAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGA

[0032] CGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAA

[0033] GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAATAGCCAAGAGAGCGTGACCG

[0034] AGCAGGACAGCAAGGACTCTACCTACAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTA

[0035] CGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAG

[0036] AGCTTCAACCGGGGCGAGTGTTGGAGCCATCCTCAGTTCGAGAAGTGGTCACACCCACAGTTTG

[0037] AGAAATGGTCCCATCCGCAATTCGAAAAAGGCGGCAGCTCTGGCAGCGGCAGCGGATCTACTGG

[0038] AACAAGCTCTAGCGGCACAGGCACAAGCGCTGGCACAACAGGCACATCTGCCAGCACATCTGGC

[0039] TCTGGATCTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGATCAGCAGGCGGAGAAGTGCAGCTTG

[0040] TTGAATCTGGCGGTGGCCTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGG

[0041] CTTCAACATCAAGGACACCTACATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGG

[0042] GTCGCCAGAATCTACCCCACCAACGGCTACACCAGATACGCCGACTCTGTGAAGGGCAGATTCA

[0043] CCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACTCCCTGAGAGCCGAGGA

[0044] CACCGCCGTGTACTACTGTTCTAGATGGGGAGGCGACGGCTTCTACGCCATGGATGTTTGGGGA

[0045] CAGGGCACCCTGGTCACCGTGTCATCTTCAGGTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCA

[0046] GGTGTACTGGGCCAGGCAAGGGCTTTGGATCCTGTAGGACTGCAAGATCGCTGCACAGCAGCGA

[0047] ATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGCATCTTAAATTCTT TATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGACGCGTAGCCCCGGT CTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACAGTGGCTGCCGTCCTGACAGGGG CTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAAAGGATTGTTTATC TTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCTGAGAAAAATGGTT AAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTTTTAACTGTCCAAG TATTTGAAATTCTTATCATTTGATTAACACCCATTCTCGACGCTAGCGTTTCCTTCCCCTGGCT ATTCTGCTCAACCTTCCTATCAGAAAAAAAGGGGAAGCGATTCTAGGGAGCAGTCTCCATGACT GTGTGTGGAGTGTTGACAAGAGTTCGGATATTTTATTCTCTACTCAGAATCGCTGCTCCCCCTC ACTCTGTTCTGTGTTGTCATTTCCTCTTTCTTTGGTAAGCTTTTAATTTCCAGTTGCATTTTAC TAAATTAATTAAGCTGGTTATTTACTTCCCATCCTGATATCAGCTTCCCCTCCTCCTTTCCTCC CAGTCCTTCTCTCTCTCCTCTCTCTTTCTCTAATCCTTTCCTTTCCCTCAGTTCATTTCTTCTT CTTTGATCTACTTTTGTTTGTCTTTTTAAATATTGCCTTGTAACTTGCTCAGAGGACAAGGAAG ATATGTCCCTGTTTCTTCTCATAGCTCTCAAGAATAGTAGCATAATTGGCTTTTATGCCAGGGT GACAGGGGAAGAATATATTTTACATATAAATTCTGTTTGACATAGGATTCTTATAATAATTTGT CAGTAGTTTAAGGTTGCAAACCTCGAGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGG CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG. The method also involves II) contacting the cell with a Cas9 polypeptide and a guide RNA containing the sequence GUCUCAGGAGCGGUGUCUGU, thereby insertion a portion of the polynucleotide sequence of I) into the genome of the cell. The method further involves III) culturing the cell in a medium containing cluster of differentiation (CD40) ligand (CD40L).

[0048] In another aspect, the disclosure features a polynucleotide containing a homology- directed repair cassette containing the following nucleotide sequence: CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTGCGGCCGCACGCGTATGTGACGCCCGGAGACAGAAGGTCTCTGGGTGGCTGGGTTTTTGTGG GGTGAGGATGGACATTCTGCCATTGTGATTACTACTACTACTACTACATGGACGTCTGGGGCAA AGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGCCACTCTAGGGCCTTTGTTTTCTGCTAC TGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCTCGGGGCATGTTCCGAGGGGACCTGGGC GGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTGAGGATCTGGGAGCCTCTGTGGATTTTC CGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGTCTGATGGAGTAACTGAGCCTGGGGGCT TGGGGAGCCACATTTGGACGAGATGCCTGAACAAACCAGGGGTCTTAGTGATGGCTGAGGAATG TGTCTCAGGAGCGGTGTCGAATTCGTCCTGCTGGACACTCATGTAGGGTAACGAGTGGCCACCT TTTCAGTGTTACCAGTGAGCTCTGAGTGTTCCTAATGGGACCAGGATGGGTCTAGGTGCCTGCT CAATGTCAGAGACAGCAATGGTCCCACAAAAAACCCAGGTAATCTTTAGGCCAATAAAATGTGG GTTCACAGTGAGGAGTGCATCCTGGGGTTGGGGTTTGTTCTGCAGCGGGAAGAGCGCTGTGCAC

[0049] AGAAAGCTTAGAAATGGGGCAAGAGATGCTTTTCCTCAGGCAGGATTTAGGGCTTGGTCTCTCA

[0050] GCATCCCACACTTGTACAGCTGATGTGGCATCTGTGTTTTCTTTCTCATCCTAGATCAGGCTTT

[0051] GAGCTGTGAAATACCCTGCCTCATGCATATGCAAATAACCTGAGGTCTTCTGAGATAAATATAG

[0052] ATATATTGGTGCCCTGAGAGCATCACATAACAACCACATTCCTCCTCTGAAGAAGCCCCTGGGA

[0053] GCACAGCTCATGCCACCATGGAAGCTCCAGCTCAGCTGCTGTTTCTGCTGCTGCTGTGGCTGCC

[0054] TGATACCACCGGCGAGATCGACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTG

[0055] GGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACGTGAACACAGCCGTGGCTTGGTATC

[0056] AGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCAGCTTTCTGTACAGCGGCGT

[0057] GCCCAGCAGATTCAGCGGCTCTAGAAGCGGCACCGACTTCACCCTGACCATAAGCAGTCTGCAG

[0058] CCCGAGGACTTCGCCACCTACTACTGTCAGCAGCACTACACCACACCTCCAACCTTTGGCCAGG

[0059] GCACCAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGA

[0060] CGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAA

[0061] GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAATAGCCAAGAGAGCGTGACCG

[0062] AGCAGGACAGCAAGGACTCTACCTACAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTA

[0063] CGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAG

[0064] AGCTTCAACCGGGGCGAGTGTTGGAGCCATCCTCAGTTCGAGAAGTGGTCACACCCACAGTTTG

[0065] AGAAATGGTCCCATCCGCAATTCGAAAAAGGCGGCAGCTCTGGCAGCGGCAGCGGATCTACTGG

[0066] AACAAGCTCTAGCGGCACAGGCACAAGCGCTGGCACAACAGGCACATCTGCCAGCACATCTGGC

[0067] TCTGGATCTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGATCAGCAGGCGGAGAAGTGCAGCTTG

[0068] TTGAATCTGGCGGTGGCCTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGG

[0069] CTTCAACATCAAGGACACCTACATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGG

[0070] GTCGCCAGAATCTACCCCACCAACGGCTACACCAGATACGCCGACTCTGTGAAGGGCAGATTCA

[0071] CCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACTCCCTGAGAGCCGAGGA

[0072] CACCGCCGTGTACTACTGTTCTAGATGGGGAGGCGACGGCTTCTACGCCATGGATGTTTGGGGA

[0073] CAGGGCACCCTGGTCACCGTGTCATCTTCAGGTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCA

[0074] GGTGTACTGGGCCAGGCAAGGGCTTTGGATCCTGTAGGACTGCAAGATCGCTGCACAGCAGCGA

[0075] ATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGCATCTTAAATTCTT

[0076] TATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGACGCGTAGCCCCGGT

[0077] CTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACAGTGGCTGCCGTCCTGACAGGGG

[0078] CTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAAAGGATTGTTTATC

[0079] TTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCTGAGAAAAATGGTT

[0080] AAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTTTTAACTGTCCAAG

[0081] TATTTGAAATTCTTATCATTTGATTAACACCCATTCTCGACGCTAGCGTTTCCTTCCCCTGGCT

[0082] ATTCTGCTCAACCTTCCTATCAGAAAAAAAGGGGAAGCGATTCTAGGGAGCAGTCTCCATGACT GTGTGTGGAGTGTTGACAAGAGTTCGGATATTTTATTCTCTACTCAGAATCGCTGCTCCCCCTC ACTCTGTTCTGTGTTGTCATTTCCTCTTTCTTTGGTAAGCTTTTAATTTCCAGTTGCATTTTAC TAAATTAATTAAGCTGGTTATTTACTTCCCATCCTGATATCAGCTTCCCCTCCTCCTTTCCTCC CAGTCCTTCTCTCTCTCCTCTCTCTTTCTCTAATCCTTTCCTTTCCCTCAGTTCATTTCTTCTT CTTTGATCTACTTTTGTTTGTCTTTTTAAATATTGCCTTGTAACTTGCTCAGAGGACAAGGAAG ATATGTCCCTGTTTCTTCTCATAGCTCTCAAGAATAGTAGCATAATTGGCTTTTATGCCAGGGT GACAGGGGAAGAATATATTTTACATATAAATTCTGTTTGACATAGGATTCTTATAATAATTTGT CAGTAGTTTAAGGTTGCAAACCTCGAGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGG CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC

[0083] GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG.

[0084] In another aspect, the disclosure features a pharmaceutical composition containing the B cell of any aspect of the disclosure, or embodiments thereof, or a B cell prepared according to the method of any aspect of the disclosure, or embodiments thereof, and a pharmaceutically acceptable excipient.

[0085] In another aspect, the disclosure features a cell prepared according to the method of any aspect of the disclosure, or embodiments thereof.

[0086] In another aspect, the disclosure features a method for treating a subject in need thereof having a neoplasia surface-expressing an HER2 antigen. The method involves administering to the subject a dose of B cells prepared according to the method of any aspect of the disclosure, or embodiments thereof.

[0087] In another aspect, the disclosure features a kit suitable for use in the method of any aspect of the disclosure, or embodiments thereof, where the kit contains the polynucleotide, the vector, the cell, or the pharmaceutical composition of any aspect of the disclosure, or embodiments thereof, and a container.

[0088] In any aspect of the disclosure, or embodiments thereof, the polynucleotide further contains an inverted terminal repeat at the 5’ end and an inverted terminal repeat at the 3’ end.

[0089] In any aspect of the disclosure, or embodiments thereof, the VH contains the following complementarity determining regions (CDRs): VH CDR1 : DTYIHW; VH CDR2: RIYPTNGYTRYADSVKG; and VH CDR3: WGGDGFYAMDV. In any aspect of the disclosure, or embodiments thereof, the VL contains the following CDRs: VL CDR1 : RASQDVNTA; VL CDR2: SASFLYS; and VL CDR3: QQHYTTPPT. In any aspect of the disclosure, or embodiments thereof, the VH contains the following framework (FR) regions: VH FR1 : EVQLVESGGGLVQPGGSLRLSCAASGFNIK; VH FR2: VRQAPGKGLEWVA; VH FR3: RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR; and VH FR4: WGQGTLVTVSS. In any aspect of the disclosure, or embodiments thereof, the VL contains the following FR regions: VL FR1 : DIQMTQSPSSLSASVGDRVTITC; VL FR2: VAWYQQKPGKAPKLLIY; VL FR3: GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC; and VL FR4: FGQGTKVEIK. In any aspect of the disclosure, or embodiments thereof, the VH contains an amino acid sequence with at least about 85% identity to the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS. In any aspect of the disclosure, or embodiments thereof, the VL contains an amino acid sequence with at least about 85% identity to the following sequence:

[0090] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSG SRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK. In any aspect of the disclosure, or embodiments thereof, the CLK contains an amino acid sequence having at least about 85% identity to the following sequence: RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0091] In any aspect of the disclosure, or embodiments thereof, the peptide linker is a Gly / Ser peptide linker. In any aspect of the disclosure, or embodiments thereof, the peptide linker contains an amino acid sequence having at least about 85% identity to the following sequence: GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG. In any aspect of the disclosure, or embodiments thereof, the peptide linker further contains a streptavidin tag. In any aspect of the disclosure, or embodiments thereof, the streptavidin tag is at the N-terminal end or the C-terminal end of the peptide linker. In any aspect of the disclosure, or embodiments thereof, the streptavidin tag contains a sequence having at least about 85% identity to the following sequence: WSHPQFEKWSHPQFEKWSHPQFEK.

[0092] In any aspect of the disclosure, or embodiments thereof, the splice donor site contains the following nucleotide sequence, or a fragment thereof capable of mediating splicing of RNA transcribed from the polynucleotide: GTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCAGGTGTACTGGGCCAGGCAAGGGCTTTG.

[0093] In any aspect of the disclosure, or embodiments thereof, the polynucleotide strand in the genome of the cell between the region of the IGH encoding joining gene segments and the intronic enhancer iEp contains the following nucleotide sequence, or a fragment thereof: GGCCCTCCCCGGGCTCAGTCTGAGAGGGTCCCAGGGACTTAGCGGGGTGCCAGTTCTTGCCTGG GGTCCTGGCATTGTTGTCACAATGTGACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCAGGTGAGTCCTCACCACCCCCTCTCTGAGTCCACTTAGGGAGACTCAGCTTGC CAGGGTCTCAGGGTCAGAGTCTTGGAGGCATTTTGGAGGTCAGGAAAGAAAGCTGGGGAGAGGG ACCCTTCGAATGGGAACCCAGCCTGTCCTCCCCAAGTCCGGCCACAGATGTCGGCAGCTGGGGG GCTCCTTCGGCTGGTCTGGGGTGACCTCTCTCCGCTTCACCTGGAGCATTCTCAGGGGCTGTCG TGATGATTGCGTGGTGGGACTCTGTCCCGCTCCAAGGCACCCGCTCTCTGGGACGGGTGCCCCC CGGGGTTTTTGGACTCCTGGGGGTGACTTAGCAGCCGTCTGCTTGCAGTTGGACTTCCCAGGCC GACAGTGGTCTGGCTTCTGAGGGGTCAGGCCAGAATGTGGGGTACGTGGGAGGCCAGCAGAGGG TTCCATGAGAAGGGCAGGACAGGGCCACGGACAGTCAGCTTCCATGTGACGCCCGGAGACAGAA GGTCTCTGGGTGGCTGGGTTTTTGTGGGGTGAGGATGGACATTCTGCCATTGTGATTACTACTA CTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGC CACTCTAGGGCCTTTGTTTTCTGCTACTGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCT CGGGGCATGTTCCGAGGGGACCTGGGCGGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTG AGGATCTGGGAGCCTCTGTGGATTTTCCGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGT CTGATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCACATTTGGACGAGATGCCTGAACAAACC AGGGGTCTTAGTGATGGCTGAGGAATGTGTCTCAGGAGCGGTGTCTGTAGGACTGCAAGATCGC TGCACAGCAGCGAATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGC ATCTTAAATTCTTTATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGAC GCGTAGCCCCGGTCTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACGGTGGCTGCC GTCCTGACAGGGGCTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAA AGGATTGTTTATCTTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCT GAGAAAAATGGTTAAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTT TTAACTGTCCAAGTATTTGAAATTCTTATCATTTGATTAACACCCATGAGTGATATGTGTCTGG AATTGAGGCCAAAGCAAGCTCAGCTAAGAAATACTAGCACAGTGCTGTCGGCCCCGATGCGGGA CTGCGTTTTGACCATCATAAATCAAGTTTATTTTTTTAATTAATTGAGCGAAGCTGGAAGCAGA TGATGAATTAGAGTCAAGATGGCTGCATGGGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGC AGGTCACCGCGAGAGTCTATTTTAGGAAGCAAAAAAACACAATTGGTAAATTTATCACTTCTGG TTGTGAAGAGGTGGTTTTGCCCAGGCCCAGATCTGAAAGTGCTCTACTGAGCAAAACAACACCT GGACAATTTGCGTTTCTAAAATAAGGCGAGGCTGACCGAAACTGAAAAGGCTTTTTTTAACTAT CTGAATTTCATTTCCAATCTTAGCTTATCAACTGCTAGTTTGTGCAAACAGCATATCAACTTCT AAACTGCATTCATTTTTAAAGTAAGATGTTTAAGAAATTAAACAGTCTTAGGGAGAGTTTATGA CTGTATTCAAAAAGTTTTTTAAATTAGCTTGTTATCCCTTCATGTGATAATTAATCTCAAATAC TTTTTCGATACCTCAGAGCATTATTTTCATAATGACTGTGTTCACAATCTTTTTAGGTTAACTC GTTTTC.

[0094] In any aspect of the disclosure, or embodiments thereof, the left homology arm contains the following nucleotide sequence or a fragment thereof capable of mediating insertion of the insertion region within the genome of the cell: atgtgacgcccggagacagaaggtctctgggtggctgggtttttgtggggtgaggatggacatt ctgccattgtgattactactactactactacatggacgtctggggcaaagggaccacggtcacc gtctcctcaggtaagaatggccactctagggcctttgttttctgctactgcctgtggggtttcc tgagcattgcaggttggtcctcggggcatgttccgaggggacctgggcggactggccaggaggg gatgggcactggggtgccttgaggatctgggagcctctgtggattttccgatgcctttggaaaa tgggactcaggttgggtgcgtctgatggagtaactgagcctgggggcttggggagccacatttg gacgagatgcctgaacaaaccaggggtcttagtgatggctgaggaatgtgtctcaggagcggtg tc. In any aspect of the disclosure, or embodiments thereof, the right homology arm contains the following nucleotide sequence or a fragment thereof capable of mediating insertion of the insertion region within the genome of the cell: tgtaggactgcaagatcgctgcacagcagcgaatcgtgaaatattttctttagaattatgaggt gcgctgtgtgtcaacctgcatcttaaattctttattggctggaaagagaactgtcggagtgggt gaatccagccaggagggacgcgtagccccggtcttgatgagagcagggttgggggcaggggtag cccagaaacagtggctgccgtcctgacaggggcttagggaggctccaggacctcagtgccttga agctggtttccatgagaaaaggattgtttatcttaggaggcatgcttactgttaaaagacagga tatgtttgaagtggcttctgagaaaaatggttaagaaaattatgacttaaaaatgtgagagatt ttcaagtatattaatttttttaactgtccaagtatttgaaattcttatcatttgattaacaccc at.

[0095] In any aspect of the disclosure, or embodiments thereof, the promoter is a minimal promoter. In any aspect of the disclosure, or embodiments thereof, the minimal promoter is IGHV1-69.

[0096] In any aspect of the disclosure, or embodiments thereof, the encoded polypeptide further contains an N-terminal signal peptide. In any aspect of the disclosure, or embodiments thereof, the N-terminal signal peptide contains an amino acid sequence with at least 85% identity to the following sequence, or a fragment thereof capable of mediating targeting of the encoded polypeptide to a secretory pathway and / or membrane localization: MEAPAQLLFLLLLWLPDTTGEI.

[0097] In any aspect of the disclosure, or embodiments thereof, the encoded polypeptide contains an amino acid sequence with at least about 85% identity to the following sequence: MEAPAQLLFLLLLWLPDTTGEIDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECWSHPQFEKWSHPQFEKWSHP QFEKGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGEVQLVESGGG LVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSSGESAVWG.

[0098] In any aspect of the disclosure, or embodiments thereof, the polynucleotide contains DNA. In any aspect of the disclosure, or embodiments thereof, the guide polynucleotide is a guide RNA.

[0099] In any aspect of the disclosure, or embodiments thereof, the cell is a human cell. In any aspect of the disclosure, or embodiments thereof, the cell is a B cell, or a progenitor thereof.

[0100] In any aspect of the disclosure, or embodiments thereof, the vector is a lentiviral vector or an adeno-associated virus (AAV) vector. In any aspect of the disclosure, or embodiments thereof, the AAV vector is an AAV6 vector. In any aspect of the disclosure, or embodiments thereof, the AAV vector is an AAV-DJ vector.

[0101] In any aspect of the disclosure, or embodiments thereof, the immune cell is a B cell.

[0102] In any aspect of the disclosure, or embodiments thereof, the method further involves culturing the B cell or progenitor thereof in a medium containing cluster of differentiation (CD40) ligand (CD40L). In any aspect of the disclosure, or embodiments thereof, the B cells or progenitors thereof in the medium results in a reduction or elimination of development of Bregcells relative to B cells cultured in a medium that does not contain CD40L.

[0103] In any aspect of the disclosure, or embodiments thereof, the napDNAbp is a Cas9 polypeptide or a Cas12 polypeptide.

[0104] In any aspect of the disclosure, or embodiments thereof, the gRNA contains the following nucleotide sequence at the 5’ end: GUCUCAGGAGCGGUGUCUGU.

[0105] In any aspect of the disclosure, or embodiments thereof, the polynucleotide containing the insertion region is contacted with the cell using a viral vector. In any aspect of the disclosure, or embodiments thereof, the viral vector is an adeno-associated virus (AAV) vector. In any aspect of the disclosure, or embodiments thereof, the AAV vector is an AAV6 vector or an AAV-DJ vector.

[0106] In any aspect of the disclosure, or embodiments thereof, the polynucleotide containing the insertion region further contains an inverted terminal repeat at the 5’ end and an inverted terminal repeat at the 3’ end.

[0107] In any aspect of the disclosure, or embodiments thereof, the method further involves differentiating the B cell progenitor into a B cell.

[0108] In any aspect of the disclosure, or embodiments thereof, the cell expresses a B cell receptor containing the polypeptide encoded by the insertion region and / or secretes an anti- HER2 polypeptide containing the polypeptide encoded by the insertion region. In any aspect of the disclosure, or embodiments thereof, the method involves administering to the subject only a single dose of the B cells.

[0109] In any aspect of the disclosure, or embodiments thereof, the neoplasia is a breast cancer. In any aspect of the disclosure, or embodiments thereof, the neoplasia includes a solid tumor.

[0110] In any aspect of the disclosure, or embodiments thereof, the method further involves administering to the subject one or more chemotherapeutic agents. In any aspect of the disclosure, or embodiments thereof, the subject is administered doxorubicin and / or cyclophosphamide. In any aspect of the disclosure, or embodiments thereof, the chemotherapeutic agent is administered at a low dose.

[0111] In any aspect of the disclosure, or embodiments thereof, the B cells are autologous to the subject.

[0112] Compositions and articles defined by the disclosure were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the embodiments of the disclosure will be apparent from the detailed description, and from the claims.

[0113] In any aspect of the disclosure, or embodiments thereof, the B cells remain detectable in the subject five weeks following the administering. In any aspect of the disclosure, or embodiments thereof, at least about 5% of total splenic B cells and / or at least about 2.5% of bone marrow B cells in the subject are the B cells the engineered B cell receptor following the administering. In any aspect of the disclosure, or embodiments thereof, the administering is associated with increased T cell proliferation in the solid tumor and / or in the spleen of the subject relative to a reference subject. In any aspect of the disclosure, or embodiments thereof, the administering is associated with an increase in an anti -tumor immune response in the subject relative to a reference subject.

[0114] Definitions

[0115] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. “Administering” is referred to herein as providing one or more agents or compositions described herein to a patient or a subject. By way of example and without limitation, composition administration (e.g., injection) can be performed by intravenous (i.v.) injection, sub- cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrasternally. Alternatively, or concurrently, administration can be by the oral route.

[0116] By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof, cell, or vector. In some embodiments, the agent is a B cell expressing a B cell receptor targeting a HER2 antigen. In some cases, the agent is a polynucleotide for use as a template for insertion into the genome of a B cell. In some embodiments, the agent is a template encoding a polypeptide capable of binding a HER2 antigen. In some instances, the agent is a vector or polynucleotide of the disclosure.

[0117] “Allogeneic,” as used herein, refers to cells of the same species that differ genetically to a reference cell.

[0118] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab’, F(ab’)2, Fab, Fv, rlgG, and scFv fragments. Further non-limiting examples of antibodies include VHH domains. Unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (including, for example, Fab and F(ab’)2 fragments) that are capable of specifically binding to a target protein. As used herein, the Fab and F(ab’)2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Fragment antigen-binding (Fab) is a polypeptide containing a full light chain (variable+constant regions) and the variable region of a heavy chain linked to the full light chain (e.g., to the light chain variable domain) by a linker peptide. Antibodies (immunoglobulins) comprise two heavy chains linked together by disulfide bonds, and two light chains, with each light chain being linked to a respective heavy chain by disulfide bonds in a " Y" shaped configuration. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end. The variable domain of the light chain (VL) is aligned with the variable domain of the heavy chain (VL), and the light chain constant domain (CL) is aligned with the first constant domain of the heavy chain (CHI). The variable domains of each pair of light and heavy chains form the antigen binding site. The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa (κ) or lambda (λ)) found in all antibody classes. The terms "antibody" or "antibodies" include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic portions or fragments thereof, such as the Fab or F(ab')2 fragments, which are capable of specifically binding to a target protein. Antibodies may include chimeric antibodies; recombinant and engineered antibodies, and antigen binding fragments thereof. Exemplary functional antibody fragments comprising whole or essentially whole variable regions of both the light and heavy chains are defined as follows: (i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (ii) single-chain Fv (“scFv”), a genetically engineered single- chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker; (iii) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating an intact antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain, which consists of the variable and CHI domains thereof; (iv) Fab', a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating an intact antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are generated per antibody molecule); and (v) F(ab')2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating an intact antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds).

[0119] According to some aspects and embodiments herein, antibody fragments are understood as meaning functional parts of antibodies, such as Fc, Fab, Fab', Fv, F(ab')2, scFv. According to some aspects and embodiments herein, corresponding biologically active fragments are to be understood as meaning those parts of antibodies which are capable of binding to an antigen, such as Fab, Fab', Fv, F(ab')2, and scFv.

[0120] The term “antigen-binding fragment,” as used herein, refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to a target antigen. In an embodiment, the target antigen is an HER2 polypeptide. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, F(ab')2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and Cm domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341 :544-546, 1989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242: 423- 426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen- binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art. In some embodiments, antigen-binding fragments (e.g., .g., Fab', F(ab')2, Fab, scFab, Fv, rlgG, and scFv fragments) of an anti-HER2 antibody, which are joined by a synthetic linker, are encompassed herein.

[0121] “Autologous,” as used herein, refers to cells from the same subject.

[0122] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0123] By “alteration” is meant a change in the structure, expression levels or activity of a polynucleotide or polypeptide as detected by standard art known methods such as those described herein. The alteration can be an increase or a decrease. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels.

[0124] By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0125] By “cluster of differentiation (CD40) ligand (CD40L) polypeptide” is meant a polypeptide having at least about 85% amino acid sequence identity to Genbank Accession No. CAA48077.1, which is provided below, or a fragment thereof capable of stimulating an immune response in B cells.

[0126] >CAA48077.1 CD40 ligand [Homo sapiens]

[0127] MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFV FMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVIS EASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIAS LCLKSPGRFERILLRAANTHSSAKPCGQQS IHLGGVFELJQPGASVFVNVTDPSQVSHGTGFTSF GLLKL.

[0128] By “cluster of differentiation (CD40) ligand (CD40L) polynucleotide” is meant a nucleic acid molecule that encodes an CD40L polypeptide as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. A representative CD40L polynucleotide sequence is provided below (GenBank Accession No. X67878.1):

[0129] >X67878.1 :46-831 H. sapiens mRNA for CD40 ligand ATGATCGAAACATACAACCAAACTTCTCCCCGATCTGCGGCCACTGGACTGCCCATCAGCATGA

[0130] AAATTTTTATGTATTTACTTACTGTTTTTCTTATCACCCAGATGATTGGGTCAGCACTTTTTGC TGTGTATCTTCATAGAAGGTTGGACAAGATAGAAGATGAAAGGAATCTTCATGAAGATTTTGTA TTCATGAAAACGATACAGAGATGCAACACAGGAGAAAGATCCTTATCCTTACTGAACTGTGAGG AGATTAAAAGCCAGTTTGAAGGCTTTGTGAAGGATATAATGTTAAACAAAGAGGAGACGAAGAA AGAAAACAGCTTTGAAATGCAAAAAGGTGATCAGAATCCTCAAATTGCGGCACATGTCATAAGT GAGGCCAGCAGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAGGATACTACACCATGAGCA ACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATAT CTATGCCCAAGTCACCTTCTGTTCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGC CTCTGCCT7V\AGTCCCCCGGTAGATTCGAGAG7\ATCTTACTCAGAGCTGC7V\ATACCCACAGTT CCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGC TTCGGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTT GGCTTACTCAAACTCTGA.

[0131] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments.

[0132] As used herein, the term “complementarity determining region” (CDR) refers to a hypervariable region found both in the light chain and the heavy chain variable regions ((VL and VH domains, respectively). CDRs are noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616, 1977 and Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; by Chothia et al., (J Mol. Biol. 196:901-917, 1987), and by MacCallum et al., (J. Mol. Biol. 262:732-745, 1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term "CDR" is a CDR as defined by Kabat based on sequence comparisons. The more highly conserved portions of variable regions are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The variable regions of native heavy and light chains each comprise four framework regions (FR1, FR2, FR3, FR4) that primarily adopt a beta-sheet configuration, connected by three CDRs (CDR1, CDR2, CDR3), which form loops that connect, and in some cases form part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4. and the CDRs in each antibody chain contribute to the formation of the target binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated. In various embodiments, complementarity determining regions are identified using any of the methodologies available to one of skill in the art such as those methods described in “Antibody Structure-Function Relationships.” Therapeutic Antibody Engineering, edited by William R. Strohl and Lilia M. Strohl, Woodhead Publishing Series in Biomedicine, 2012, 37-56, 459-595, the entirety of which is incorporated herein in its entirety for all purposes, where such methods include, as non-limiting examples, those of Kabat, Chothia, Lefranc, Honegger, Martin, MacCallum, and Zhao. CDRs can be identified using sequence or structure based methods. Various software programs are available to one of skill in the art to identify CDRs for an antibody amino acid sequence. In various embodiments, a CDR as provided herein may be modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acids and / or to exclude 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids at the N-terminal and / or C-terminal end. The present disclosure contemplates that the CDRs identified for a particular antibody can vary in location or length depending upon the method by which they are determined.

[0133] The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Non- limiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained.

[0134] Amino acids generally can be grouped into classes according to the following common side-chain properties:

[0135] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0136] (3) acidic: Asp, Glu;

[0137] (4) basic: His, Lys, Arg;

[0138] (5) residues that influence chain orientation: Gly, Pro;

[0139] (6) aromatic: Trp, Tyr, Phe.

[0140] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

[0141] The term “coding sequence” or “protein coding sequence” as used interchangeably herein refers to a segment of a polynucleotide that codes for a protein. Coding sequences can also be referred to as open reading frames. The region or sequence is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon. Stop codons include the following: TAG, TAA, and TGA.

[0142] By “kappa light chain constant region (CLk; CL) polypeptide” is meant a polypeptide having at least about 85% amino acid sequence identity to the following amino acid sequence, or a functional fragment thereof: RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

[0143] TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0144] By “kappa light chain constant region (CLk; CL) polynucleotide” is meant a nucleic acid molecule that encodes an CLk polypeptide as well as the introns, exons, 3 ' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof.

[0145] By “cyclophosphamide” is meant a molecule having the structure and corresponding to CAS Number 50-18-0, and pharmaceutically acceptable salts thereof. Cyclophosphamide is an agent suitable for use as a chemotherapeutic agent and / or for suppressing the immune system. Cyclosphosphamide may be used to treat a neoplasia.

[0146] By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. In some embodiments the detectable label is a streptavidin tag (Strep-Tag) or an HA-Tag.

[0147] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasias. In some cases, the neoplasia is a breast cancer, a melanoma, or an ovarian cancer. In various instances, the neoplasia contains cells surface expressing a HER2 antigen. In some embodiments, the neoplasia is a solid tumor.

[0148] By “doxorubicin” is meant a molecule having the structure pegylated derivatives thereof, and / or pharmaceutically acceptable salts thereof. Doxorubicin may be used to treat a neoplasia. In some embodiments, doxorubicin is formulated within liposomes.

[0149] By “effective amount” is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In some embodiments, an effective amount is sufficient to slow or stop the growth of a neoplasia. In some embodiments, an effective amount is sufficient to slow or stop the spread of a neoplasia. In some embodiments, an effective amount is sufficient to induce the death of a neoplastic cell or to slow or stop that cell’s proliferation.

[0150] By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. In embodiments, portion contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. As used herein, the term “framework region” or “FR region” includes amino acid residues that are adjacent to the CDRs. FR region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single- chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others.

[0151] The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.

[0152] By “guide polynucleotide” is meant a polynucleotide or polynucleotide complex which is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl). In an embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.

[0153] By “HER2 polypeptide” is meant a polypeptide having at least about 85% amino acid sequence identity to NCBI Reference Sequence Accession No. NP_004439.2, which is provided below, or a fragment thereof capable of being bound by an anti-HER2 antibody.

[0154] >NP_004439.2 receptor tyrosine-protein kinase erbB-2 isoform a precursor [Homo sapiens] MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTY LPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTP VTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRAC HPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACL HFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQ EVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGD PASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGL GISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACH QLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTC FGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDK GCPAEQRASPLTSIISAWGILLVWLGWFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSG AMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEIL DEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKG MSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILR

[0155] RRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWM IDSECRPRFRELVSEFSRMARDPQRFWIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYL VPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDG DLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSP REGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPA FDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV.

[0156] By “HER2 polynucleotide” is meant a nucleic acid molecule that encodes an HER2 polypeptide as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. A representative HER2 polynucleotide sequence is provided below: >NM_004448.4: 176-3943 Homo sapiens erb-b2 receptor tyrosine kinase 2 (ERBB2), transcript variant 1, mRNA

[0157] ATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCCTCTTGCCCCCCGGAGC CGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAGTCCCGAGACC CACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCA CCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGT GCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACC CAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCA CCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGAT CTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGG AAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGG CCTGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTG TCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACT GACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCT GCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACAC AGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACT GCCTGTCCCTACAACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACA ACCAAGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCG AGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCAATATC CAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATG GGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGA AGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTC CAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAG GGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGTGGACTGGCCCTCAT CCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCG CACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCT GCCACCAGCTGTGCGCCCGAGGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTGCAG CCAGTTCCTTCGGGGCCAGGAGTGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAG TATGTGAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGA CCTGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCTTCTG

[0158] CGTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCA

[0159] GATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATG

[0160] ACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGTCCATCATCTCTGCGGTGGTTGG

[0161] CATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATCAAGCGACGGCAGCAGAAG

[0162] ATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTA

[0163] GCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAA

[0164] GGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAAT

[0165] GTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAA

[0166] TCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCAT

[0167] CTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCAT

[0168] GTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCA

[0169] AGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCT

[0170] GGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGAC

[0171] GAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTC

[0172] TCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGAT

[0173] GACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAG

[0174] GGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTT

[0175] GGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGC

[0176] CAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGAC

[0177] AGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCTGGTGGATGCTGAGGAGT

[0178] ATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGGCATGGTCCA

[0179] CCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACACTAGGGCTGGAGCCC

[0180] TCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCGAAGGGGCTGGCTCCGATGTATTTG

[0181] ATGGTGACCTGGGAATGGGGGCAGCCAAGGGGCTGCAAAGCCTCCCCACACATGACCCCAGCCC

[0182] TCTACAGCGGTACAGTGAGGACCCCACAGTACCCCTGCCCTCTGAGACTGATGGCTACGTTGCC

[0183] CCCCTGACCTGCAGCCCCCAGCCTGAATATGTGAACCAGCCAGATGTTCGGCCCCAGCCCCCTT

[0184] CGCCCCGAGAGGGCCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGCCCAAGAC

[0185] TCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAAC

[0186] CCCGAGTACTTGACACCCCAGGGAGGAGCTGCCCCTCAGCCCCACCCTCCTCCTGCCTTCAGCC

[0187] CAGCCTTCGACAACCTCTATTACTGGGACCAGGACCCACCAGAGCGGGGGGCTCCACCCAGCAC

[0188] CTTCAAAGGGACACCTACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTGTGA.

[0189] “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0190] By “IgHvl-69” is meant a regulatory element encoded by a polynucleotide, or a functional fragment thereof, capable of driving expression of a polynucleotide downstream of the regulatory element, where the regulatory element has at least 85% identity to the following sequence: GTCCTGCTGGACACTCATGTAGGGTAACGAGTGGCCACCTTTTCAGTGTTACCAGTGAGCTCTG AGTGTTCCTAATGGGACCAGGATGGGTCTAGGTGCCTGCTCAATGTCAGAGACAGCAATGGTCC CACAAAAAACCCAGGTAATCTTTAGGCCAATAAAATGTGGGTTCACAGTGAGGAGTGCATCCTG GGGTTGGGGTTTGTTCTGCAGCGGGAAGAGCGCTGTGCACAGAAAGCTTAGAAATGGGGCAAGA GATGCTTTTCCTCAGGCAGGATTTAGGGCTTGGTCTCTCAGCATCCCACACTTGTACAGCTGAT GTGGCATCTGTGTTTTCTTTCTCATCCTAGATCAGGCTTTGAGCTGTGAAATACCCTGCCTCAT GCATATGCAAATAACCTGAGGTCTTCTGAGATAAATATAGATATATTGGTGCCCTGAGAGCATC ACATAACAACCACATTCCTCCTCTGAAGAAGCCCCTGGGAGCACAGCTCATGCCACC.

[0191] By “IgK signal peptide” is meant a peptide having at least about 85% amino acid sequence identity to the following amino acid sequence, or a functional fragment thereof capable of mediating the targeting of a protein fused thereto to the secretory pathway and / or membrane localization of the same: MEAPAQLLFLLLLWLPDTTGEI.

[0192] By “IgK signal peptide polynucleotide” is meant a nucleic acid molecule that encodes an IgK signal peptide as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof.

[0193] By “immunoglobulin heavy chain (IGH) locus polynucleotide” is meant a region, or a fragment thereof, of a genome containing a polynucleotide sequence encoding heavy chains of antibodies. In various embodiments, the IGH locus is a region on human choromosome 14 that contains a gene for the heavy chains of human antibodies. The IGH locus includes polynucleotide sequences encoding V (variable), D (diversity), J (joining), and C (constant) portions of an heavy chain antibody polypeptide. In various embodiments, an IGH locus has at least 85% sequence identity to NCBI Reference Sequence Accession No. NC_000014.9:cl06879844-105586437. A representative IGH polynucleotide sequence is provided below, where the sequence corresponding to a left homology arm of SEQ ID NO: 1 is shown in bold, the sequence corresponding to the right homology arm of SEQ ID NO: 1 is shown in bold, italic text, and the sequence corresponding to the site targeted by the IgH296 sgRNA of the disclosure is underlined: GGCCCTCCCCGGGCTCAGTCTGAGAGGGTCCCAGGGACTTAGCGGGGTGCCAGTTCTTGCCTGG

[0194] GGTCCTGGCATTGTTGTCACAATGTGACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTC

[0195] ACCGTCTCCTCAGGTGAGTCCTCACCACCCCCTCTCTGAGTCCACTTAGGGAGACTCAGCTTGC

[0196] CAGGGTCTCAGGGTCAGAGTCTTGGAGGCATTTTGGAGGTCAGGAAAGAAAGCTGGGGAGAGGG

[0197] ACCCTTCGAATGGGAACCCAGCCTGTCCTCCCCAAGTCCGGCCACAGATGTCGGCAGCTGGGGG

[0198] GCTCCTTCGGCTGGTCTGGGGTGACCTCTCTCCGCTTCACCTGGAGCATTCTCAGGGGCTGTCG

[0199] TGATGATTGCGTGGTGGGACTCTGTCCCGCTCCAAGGCACCCGCTCTCTGGGACGGGTGCCCCC

[0200] CGGGGTTTTTGGACTCCTGGGGGTGACTTAGCAGCCGTCTGCTTGCAGTTGGACTTCCCAGGCC

[0201] GACAGTGGTCTGGCTTCTGAGGGGTCAGGCCAGAATGTGGGGTACGTGGGAGGCCAGCAGAGGG

[0202] TTCCATGAGAAGGGCAGGACAGGGCCACGGACAGTCAGCTTCCATGTGACGCCCGGAGACAGAA

[0203] GGTCTCTGGGTGGCTGGGTTTTTGTGGGGTGAGGATGGACATTCTGCCATTGTGATTACTACTA

[0204] CTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGC

[0205] CACTCTAGGGCCTTTGTTTTCTGCTACTGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCT

[0206] CGGGGCATGTTCCGAGGGGACCTGGGCGGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTG

[0207] AGGATCTGGGAGCCTCTGTGGATTTTCCGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGT

[0208] CTGATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCACATTTGGACGAGATGCCTGAACAAACC

[0209] AGGGGTCTTAGTGATGGCTGAGGAATGTGTCTCAGGAGCGGTGTCTGTAGGACTGCAAGATCGC

[0210] TGCACAGCAGCGAATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGC

[0211] ATCTTAAATTCTTTATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGAC

[0212] GCGTAGCCCCGGTCTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACGGTGGCTGCC

[0213] GTCCTGACAGGGGCTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAA

[0214] AGGATTGTTTATCTTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCT

[0215] GAGAAAAATGGTTAAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTT

[0216] TTAACTGTCCAAGTATTTGAAATTCTTATCATTTGATTAACACCCATGAGTGATATGTGTCTGG

[0217] AATTGAGGCCAAAGCAAGCTCAGCTAAGAAATACTAGCACAGTGCTGTCGGCCCCGATGCGGGA

[0218] CTGCGTTTTGACCATCATAAATCAAGTTTATTTTTTTAATTAATTGAGCGAAGCTGGAAGCAGA

[0219] TGATGAATTAGAGTCAAGATGGCTGCATGGGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGC

[0220] AGGTCACCGCGAGAGTCTATTTTAGGAAGCAAAAAAACACAATTGGTAAATTTATCACTTCTGG

[0221] TTGTGAAGAGGTGGTTTTGCCCAGGCCCAGATCTGAAAGTGCTCTACTGAGCAAAACAACACCT

[0222] GGACAATTTGCGTTTCTAAAATAAGGCGAGGCTGACCGAAACTGAAAAGGCTTTTTTTAACTAT

[0223] CTGAATTTCATTTCCAATCTTAGCTTATCAACTGCTAGTTTGTGCAAACAGCATATCAACTTCT

[0224] AAACTGCATTCATTTTTAAAGTAAGATGTTTAAGAAATTAAACAGTCTTAGGGAGAGTTTATGA

[0225] CTGTATTCAAAAAGTTTTTTAAATTAGCTTGTTATCCCTTCATGTGATAATTAATCTCAAATAC

[0226] TTTTTCGATACCTCAGAGCATTATTTTCATAATGACTGTGTTCACAATCTTTTTAGGTTAACTC

[0227] GTTTTC (SEQ ID NO: 2). In various embodiments, the methods of the disclosure involve inserting a polynucleotide sequence within the region of a genome corresponding to SEQ ID NO: 2. In some embodiments, the methods of the disclosure involve inserting a polynucleotide sequence within the region of a genome corresponding to the region of SEQ ID NO: 2 shown in bold and italics.

[0228] By “increase” is meant to alter positively relative to a reference. An increase may be by 1%, 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more, or by 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more.

[0229] By “inverted terminal repeats (ITRs)” is meant the polynucleotide sequences at the 5’ and 3’ ends of a polynucleotide to be packaged within an adeno-associated virus (AAV) capsid and that facilitate packaging of the polynucleotide within the AAV capsid. In various embodiments, a 5’ ITR comprises a sequence with at least about 85% sequence identity to the following nucleotide sequence, or a fragment thereof capable of mediating packaging of a polynucleotide to which said 5’ ITR is fused at the 5’ end thereof within an AAV capsid: CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTC

[0230] GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT.

[0231] In various embodiments, a 3’ ITR comprises a sequence with at least about 85% sequence identity to the following nucleotide sequence, or a fragment thereof capable of mediating packaging of a polynucleotide to which said 3’ ITR is fused at the 3’ end thereof within an AAV capsid: AGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAG GTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGC AGG.

[0232] The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from an original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0233] By “isolated polynucleotide” is meant a nucleic acid that is free of the genes which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0234] By an “isolated polypeptide” is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, and or at least 99%, by weight, a polypeptide of the disclosure. An isolated polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0235] The term “linker,” as used herein, refers to a molecule that links two moieties. In one embodiment, the term “linker” refers to a covalent linker (e.g., covalent bond) or a non-covalent linker. In some embodiments, a linker is a peptide. In some embodiments, a linker contains an amino acid sequence with at least 85% identity to the following sequence, or a fragment thereof GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG.

[0236] By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a developmental state, condition, disease, or disorder.

[0237] The term “nucleic acid programmable DNA binding protein” or “napDNAbp” may be used interchangeably with “polynucleotide programmable nucleotide binding domain” to refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain. Non-limiting examples of nucleic acid programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9), Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, and Casl2j / CasΦ (Casl2j / Casphi). Non-limiting examples of Cas enzymes include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csnl or Csxl2), CaslO, CaslOd, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Casl2j / Cas, Cpfl, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1:325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / science.aav7271, the entire contents of each are hereby incorporated by reference.

[0238] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0239] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0240] By “polynucleotide” or “nucleic acid molecule” is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof. This term includes oligomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced stability in the presence of nucleases. By “polypeptide” or “amino acid sequence” is meant any chain of amino acids, regardless of length or post-translational modification. In various embodiments, the post- translational modification is glycosylation or phosphorylation. In various embodiments, conservative amino acid substitutions may be made to a polypeptide to provide functionally equivalent variants, or homologs of the polypeptide. In some aspects, the disclosure embraces sequence alterations that result in conservative amino acid substitutions. In some embodiments, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the conservative amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In various embodiments, conservative amino acid substitutions can be made to the amino acid sequence of the proteins and polypeptides disclosed herein.

[0241] By “reduce” is meant to alter negatively relative to a reference. A reduction may be by 1%, 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more, or by 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more.

[0242] By “reference” is meant a standard or control condition. In various embodiments, a reference is a wild-type B cell, or a B cell not altered to express a B cell receptor of the disclosure. In some cases, a reference is a healthy subject, or a subject not administered a treatment according to the methods of the disclosure. In some cases, a reference is a subject having a neoplasia that does not surface-express a HER2 antigen. A reference may be a subject prior to being administered a treatment according to the methods of the disclosure (e.g., being administered a B cell engineered to express a B cell receptor capable of binding a HER2 antigen and / or that has been engineered to secrete an anti-HER2 antibody).

[0243] A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 nucleotides, or any integer thereabout or therebetween.

[0244] By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the disclosure, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the disclosure.

[0245] By “splice donor site” is meant a site in an RNA molecule capable of mediating RNA splicing, or a DNA sequence encoding said site. In some embodiments, a splice donor site contains the following nucleotide sequence, a fragment thereof suitable for use as a splice donor site, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide alterations and suitable for use as a splice donor site: GTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCAGGTGTACTGGGCCAGGCAAGGGCTTTG.

[0246] Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0247] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, about less than about 500 mM NaCl and 50 mM trisodium citrate, or about less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, of at least about 37° C, or of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0248] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, or of at least about 68° C. In an embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0249] By “StrepTag polypeptide” is meant a polypeptide having at least about 85% amino acid sequence identity to the following amino acid sequence, or a functional fragment thereof capable of being bound by an antibody: WSHPQFEKWSHPQFEKWSHPQFEK.

[0250] By “StrepTag polynucleotide” is meant a nucleic acid molecule that encodes an StrepTag polypeptide as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In embodiments, such a sequence is at least 60%, at least 80% or 85%, or at least about 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. In various embodiments, a polypeptide or polynucleotide suitable for use in compositions or methods of the disclosure comprises an amino acid or polynucleotide sequence having about or at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to a sequence provided herein.

[0251] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence.

[0252] By “subject” is meant an animal. The animal can be a mammal. The mammal can be a human or non-human mammal, such as a bovine, equine, canine, ovine, rodent, or feline.

[0253] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0254] By “Trastuzumab polypeptide” is meant an antibody or antigen binding fragment thereof having at least about 85% amino acid sequence identity to an antibody sequence of Trastuzumab, wherein the antibody or antigen binding fragment thereof binds to a HER2 antigen. In embodiments, the Trastuzumab polypeptide comprises VH and / or VL CDRs 1-3 of Trastuzumab, wherein the VH and / or VL CDRs, and antigen binding fragments thereof, bind to an HER2 antigen. In embodiments, the antibody or antigen binding fragment thereof has at least 90%, 93%, 95%, 98%, 99% or 100% amino acid sequence identity to an antibody sequence of Trastuzumab. Exemplary heavy chain and light chain sequences for Trastuzumab are provided below, where the variable regions are in plain text and complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR2, are underlined:

[0255] Trastuzumab variable heavy chain (HC):

[0256] E VQLVE S GGGLVQ PGGS LRL S CAAS GEN I KDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS Trastuzumab variable light chain (LC):

[0257] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSG

[0258] SRSGTDFTLTI SSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK.

[0259] The three CDRs of the Trastuzumab VH region are as follows:

[0260] VH CDR1 : DTYIHW;

[0261] VH CDR2: RIYPTNGYTRYADSVKG; and

[0262] VH CDR3: WGGDGFYAMDV.

[0263] The three CDRs of the Trastuzumab antibody VL region are as follows:

[0264] VL CDR1 : RASQDVNTA;

[0265] VL CDR2: SAS FLYS; and

[0266] VL CDR3: QQHYTTPPT.

[0267] The four framework (FR) regions, i.e., FR1, FR2, FR3, and FR4, of the Trastuzumab antibody are located on either side of each of the CDRs in VH and VL region sequences shown supra. In particular, the four FRs of the Trastuzumab antibody VH region are as follows:

[0268] VH FR1 : EVQLVESGGGLVQPGGSLRLSCAASGFNIK;

[0269] VH FR2: VRQAPGKGLEWVA;

[0270] VH FR3: RFTI SADTSKNTAYLQMNSLRAEDTAVYYCSR; and

[0271] VH FR4: WGQGTLVTVSS.

[0272] The four FRs of the Trastuzumab antibody VL region are as follows:

[0273] VL FR1 : DIQMTQSPSSLSASVGDRVTITC;

[0274] VL FR2: VAWYQQKPGKAPKLLIY;

[0275] VL FR3: GVPSRFSGSRSGTDFTLTI SSLQPEDFATYYC; and

[0276] VL FR4: FGQGTKVEIK.

[0277] By “Trastuzumab polynucleotide” is meant a nucleic acid molecule encoding at least a fragment of a Trastuzumab antibody. In an embodiment, the Trastuzumab polynucleotide is DNA, RNA, or a hybrid thereof. In an embodiment, the Trastuzumab polynucleotide contains a sequence having at least 85% identity to the following sequence: GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCA

[0278] CCTGTAGAGCCAGCCAGGACGTGAACACAGCCGTGGCTTGGTATCAGCAGAAGCCTGGCAAGGC CCCTAAGCTGCTGATCTACAGCGCCAGCTTTCTGGAGAGCGGCGTGCCCAGCAGATTCAGCGGC TCTGGAAGCGGCACCGACTTCACCCTGACCATAAGCAGTCTGCAGCCCGAGGACTTCGCCACCT ACTACTGTCAGCAGCACTACACCACACCTCCAACCTTTGGCCAGGGCACCAAGGTGGAAATCAA GCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGC ACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAGG TGGACAACGCCCTGCAGAGCGGCAATAGCCAAGAGAGCGTGACCGAGCAGGACAGCAAGGACTC TACCTACAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTAC GCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGT

[0279] GTTGGAGCCATCCTCAGTTCGAGAAGTGGTCACACCCACAGTTTGAGAAATGGTCCCATCCGCA ATTCGAAAAAGGCGGCAGCTCTGGCAGCGGCAGCGGATCTACTGGAACAAGCTCTAGCGGCACA GGCACAAGCGCTGGCACAACAGGCACATCTGCCAGCACATCTGGCTCTGGATCTGGCGGAGGCG GAGGTTCTGGTGGCGGAGGATCAGCAGGCGGAGAAGTGCAGCTTGTTGAATCTGGCGGTGGCCT GGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCAACATCAAGGACACC TACATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGAATCTACCCCA CCAACGGCTACACCAGATACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCGCCGACACCAG CAAGAACACCGCCTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGT

[0280] TCT.

[0281] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0282] By “upstream” and “downstream” it is intended to define relevant positions at least two regions or sequences in a nucleic acid molecule orientated in a 5 '-to-3 ' direction. For example, a first sequence is upstream of a second sequence in a DNA molecule where the first sequence is positioned 5' to the second sequence. Accordingly, the second sequence is downstream of the first sequence.

[0283] As used herein, the term “vector” refers to a means of introducing a nucleic acid into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, lipid nanoparticles, and episomes. “Expression vectors” are nucleic acid sequences comprising the nucleotide sequence to be expressed in the recipient cell. Expression vectors contain a polynucleotide sequence as well as additional nucleic acid sequences to promote and / or facilitate the expression of the introduced sequence, such as start, stop, enhancer, promoter, and secretion sequences, into the genome of a mammalian cell. Examples of vectors include nucleic acid vectors, e.g., DNA vectors, such as plasmids, RNA vectors, viruses or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 11026; incorporated herein by reference. Certain vectors contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors of some aspects and embodiments herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0284] As used herein, the term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (VH) followed by a number of constant domains. Each light chain of a native antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus.

[0285] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0286] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art. In some cases, a range of normal tolerance in the art is within 1 or 2 standard deviations of the mean. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0287] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0288] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0289] BRIEF DESCRIPTION OF THE DRAWINGS

[0290] FIGs. 1A to II provide a schematic diagram, plots, stacked bar graphs, flow cytometry scatter plots, and a histogram relating to a murine B-cell culture protocol and phenotype analysis. FIG. 1A provides a schematic diagram showing a culture and editing protocol for primary murine B-cells. FIG. IB provides a plot showing a growth curve of B-cells cultured in IL4-M and CpG-M. FIG. 1C provides representative flow cytometry scatter plots for IL4-M and CpG- M cultured B-cells. FIG. ID provides stacked bar graphs showing the phenotype of murine B- cells in IL4-M (left) and CpG-M (right) medium. FIG. IE provides a plot showing frequency of Germinal Centers (GC) B-cells CD95+ GL7+ in IL-4 cultured B-cells. FIG. IF provides stacked bar graphs showing surface immunoglobulin expression in IL4-M and CpG-M cultured B-cells over time. FIG. 1G provides a plot showing frequency of Plasma cells (PC) CD 138+ TACH within the CD 19+ cells in IL4-M and CpG-M cultured B-cells. FIG. 1H provides plots showing activation markers mean fluorescent intensity (MFI) over time in IL4-M and CpG-M cultured B- cells. FIG. II provides a stacked bar graph and a histogram plot showing percent (%) of proliferating CD4+ T-cells co-cultured with B-cells or BM-DC. Different generations of proliferating cells are indicated.

[0291] FIGs. 2A to 21 provide a schematic diagram, contour plots, bar graphs, dot plots, scatter plots, and plots relating to optimization of the murine B-cell editing procedure. FIG. 2A provides a schematic diagram showing a homology-directed repair (HDR) cassette and murine immunoglobulin heavy chain (IGH) locus structure. FIG. 2B provides representative flow cytometry contour plots for Unedited (left) and Edited (Right) murine B-cells. FIG. 2B also provides bar graphs showing the frequency of Edited B-cells (Tag+) determined by flow cytometry 2 days after editing for samples edited after different times of pre-stimulation. FIG. 2D provides a bar graph showing frequency of edited alleles analyzed by droplet digital PCR (ddPCR) on B-cells edited after different times of pre-stimulation. FIG. 2D provides a bar graph showing a comparison between AAV6 and AAVD-J editing efficiency, where editing efficiency was measured as frequency of edited B-cells (Tag+) by flow cytometry (left) or ddPCR (right). FIG. 2E provides a dot plot showing frequency of Edited B-cells(Tag+) determined by flow cytometry, where the cells were analyzed 4 days after editing. FIG. 2E also provides representative flow cytometry plots for Unedited (left) and Edited (right) murine B-cells. FIG. 2F provides a bar graph showing cell growth of edited or unedited B-cells expressed as fold increase compared to the previous time point. FIG. 2G provides a bar graph showing the levels of anti-HER2 IgM and IgG measured in culture supernatant by ELISA. FIG. 2H provides a plot showing frequency of Germinal Centers (GC) B-cells CD95+ GL7+ in Edited or unedited B- cells. Fig. 21 provides plots showing levels of activation markers measured as mean fluorescent intensity (MFI) over time in Edited or Unedited B-cells. In FIG. 2F, each pair of bars represents from left-to-right “Untreated” and “Edited B Cells”. In FIG. 2G, each pair of bars represents from left-to-right, “Unedited B cells” and “Edited B cells.”

[0292] FIGs. 3A to 3C provide a schematic diagram, a plot, and images relating to a spontaneous HER2+ Breast Cancer Model. FIG. 3A provides a schematic diagram showing a protocol for doxycycline-diet tumor induction in an MMTV-rtTA / tetO-HER2 mouse model (HER2 mice). FIG. 3B provides a plot showing tumor volume over time in individual HER2 mice over time. FIG. 3C provides images showing representative pictures of tumors isolated from 3 individual HER2 mice. In FIGs. 2F and 2G, each pair of bars from left-to-right represents “Untreated” and “Edited B cells,” respectively.

[0293] FIGs. 4A to 41 provide plots and a schematic diagram relating to chemo regimen selection using an EO771 murine Breast Cancer Model. FIG. 4A provides schematic diagrams and a plot showing tumor volume over time in mice bearing EO771-WT or EO771 HER2-GFP, which contained a construct described in the upper schematic diagram provided above the plot, or EO771-HER2-OVA-Luc, which contained a construct described in the lower schematic diagram provided above the plot. FIG. 4B provides a plot of tumor volume over time in EO771- HER2-GFP bearing mice treated with Edited or Unedited B-cells at day 14. FIG. 4C provides a plot of tumor volume in EO771 HER2- OVA-Luc tumor-bearing mice receiving weekly injections of Doxo 5mg / kg intravenously (iv) compared to untreated Tumor Only mice. FIG. 4D provides a plot of tumor volume in EO771 HER2-0VA-Luc tumor-bearing mice receiving weekly injection of CPA 116mg / kg by intraperitoneal injection (ip) compared to untreated Tumor Only mice. FIG. 4E provides a plot of tumor volume in EO771 HER2-0VA-Luc tumor- bearing mice receiving a combination of Doxo + / - CPA compared to untreated Tumor Only mice. FIG. 4F provides a plot of tumor volume in EO771 HER2-GFP bearing mice receiving a combination of Doxo + / - CPA or PD1 ip compared to untreated Tumor Only mice. FIG. 4G provides a schematic diagram showing an experimental scheme involving a low-intensity chemo regimen and B-cells injection in EO771 tumor-bearing mice. FIG. 4H provides a plot showing results from hematocytometer analysis of EO771 HER2-0VA-Luc tumor-bearing mice to determine total White Blood Cells (WBCs) count over time. FIG. 41 provides a set of plots showing results from hematocytometer analysis of EO771 HER2-0VA-Luc tumor-bearing mice to determine frequency and absolute count of lymphocytes (LY), Neutrophils (NE), and Monocytes (MO).

[0294] FIGs. 5A to 5F provide flow cytometry plots, plots, images, bar graphs, and dot plots demonstrating that edited B cells were effective in reducing tumor (TM) burden in an EO771- GFP breast cancer model. FIG. 5A provides representative flow cytometry plots and bar graphs showing the frequency of donor B cells (CD45.1) versus host B cells (CD45.2) within (w / in) the total CD 19+ cells (top and bottom right panels) or the GC+ B-cells (middle and bottom left panels) in the tumor-draining lymph nodes (TLN) of untreated mice (TM only), mice treated with chemotherapy (TM+chemo), mice receiving B cells (TM+B), and mice receiving B cells in combination with chemotherapy (TM+B+chemo). FIG. 5B provides a plot showing quantification of the bioluminescent signals (Total Flux in photons per second (p / s)) in different treatment groups measured over time. As shown in the right panel of FIG. 5B, signal was measured on a region of interest (ROI) encompassing the entire mouse on the side view using bioluminescence imaging (BLI) scans of mice. FIG. 5C provides a plot showing tumor volume in EO771 HER2-GFP tumor-bearing mice over time. Chemo was administered at day 12 and a single B-cell injection was administered at day 14. FIG. 5D provides a bar graph showing frequency of germinal centers (GC+) (GL7+ CD95+) B-cells in the spleen of EO771 HER2-GFP tumor-bearing mice receiving different treatments. FIG. 5E provides plots of tumor volume (left) and tumor weight at sacrifice (right) in EO771 HER2-GFP tumor-bearing mice over time. Chemo was administered at day 12 and two injections of B-cell injection were performed at day 14 and day 21. FIG. 5F provides representative FACS plot showing hHER2 and GFP expression in cultured tumor cells harvested from treated mice at sacrifice (right), compared to their bulk population at the time of injection (left). In the lower panels of FIG. 5A, the bars in the bar graphs represent, from left-to-right, “Tumor Only,” “Edited B cells only,” “Edited B cells + Chemo,” and “Chemo only.”

[0295] FIGs. 6A to 6G provide plots, fluorescence activated cell sorting (FACS) plots, and bar graphs relating to the effect of B cells on EO771-OVA tumor cells in mice. FIG. 6A provides a plot of tumor volume in EO771 HER2-0va-Luc tumor-bearing mice over time. Chemo was administered at day 12 and two injections of B-cell injection were performed at day 14 and day 21. FIG. 6B provides a bar graph showing the frequency of hHER2 expression analyzed by FACS in cultured tumor cells harvested from treated mice at sacrifice. FIG. 6B also provides representative FACS plots showing hHER2 and GFP expression in cultured tumor cells harvested from treated mice at sacrifice (Post-Tx), compared to their bulk population at the time of injection (Pre-Tx). FIG. 6C provides a plot showing a correlation between the tumor volume and the frequency of hHER2+ cells analyzed by flow cytometry. FIG. 6D provides a bar graph showing the frequency of T cells (CD3+), B-cells (CD19+), Monocytes (CD1 lb+ Ly6G-) and Neutrophils (CD1 lb+ Ly6G+) within the host CD45 2+ cells in the EO771 HER2-0VA-Luc tumors at sacrifice. FIG. 6E provides bar graphs showing the frequency of GC+ (GL7+ CD95+) (left) and the CD80 median fluorescence intensity (MFI) (right) within the host CD45-2+ B-cells in the EO771 HER2-0VA-Luc tumors at sacrifice. FIG. 6F provides a bar graph showing frequency of GC+ (GL7+ CD95+) within the host CD45-2+ B-cells in the TDLN of EO771 HER2-0VA-Luc tumor-bearing mice. FIG. 6G provides a bar graph showing MFI for activation markers on host CD45- 2+ B-cells in the TDLN of EO771 HER2-0VA-Luc tumor-bearing mice. In FIGs. 6D and 6G, each set of four bars correspond, from left-to-right, to “TM only,” “Chemo only,” “B cell Edited + Chemo,” and “B cell Unedited + Chemo.” In the leftmost plot of FIG. 6E, the bars correspond, from left-top-right, to “TM only,” “Chemo only,” and “B cell Edited + Chemo,” and in the rightmost plot of FIG. 6E, the bars correspond from left-to-right, to “TM only,” “Chemo only,” “B cell Untreated + Chemo,” and “B cell Edited + Chemo.” In FIG. 6F, the bars correspond, from left-to-right, to “TM only,” “Chemo only,” “B cell Untreated + Chemo,” and “B cell Edited + Chemo.”

[0296] FIGs. 7A to 7E provide plots, a schematic diagram, stacked bar graphs, and a bar graph relating to the effect of B cells on EO771-OVA tumor cells in mice. FIG. 7A provides plots of tumor volume in mice transplanted with EO771 HER2-0va-Luc single clones (C3, C18, C8) or an EO771 HER2- Ova-Luc Bulk population over time. FIG. 7B provides a schematic diagram showing an experimental procedure. FIG. 7C provides a stacked bar graph showing the percent (%) of proliferating CTV+ OT-I donor CD8+ T-cells isolated form TDLN at sacrifice and analyzed by Flow cytometry. Different generations (DO, DI, D2, D3, etc.) of proliferating cells are indicated. FIG. 7D provides a stacked bar graph showing frequency of Naive (CD44- CD62L+), Central Memory (CM; CD44hlghCD62L+) and Effector Memory (EF; CD44hlghCD62Llow) OT-I donor CD8+ T-cells in TDLN at sacrifice, as determined using Flow cytometry. FIG. 7E provides a bar graph showing the frequency of Activated CD69+ OT-I donor CD8+ T-cells in TDLN at sacrifice, as determined using Flow cytometry.

[0297] FIGs. 8A to 8D provide a schematic diagram, plots, a bar graph, and flow cytometry plots relating to efficacy of B cells in a cold tumor (TM) or the B 16 melanoma model. FIG. 8A provides a schematic diagram showing the experimental procedure. B16 cells were injected subcutaneously (SQ) 4 days before an intravenous (IV) B-cell injection. Tumor volume in mice transplanted with B 16 HER2+GFP+ was compared to B16 WT over time. FIG. 8B provides a plot of tumor volume in mice transplanted with B 16 HER2+GFP+ and treated with Edited or Unedited B-cells IV at day 4. FIG. 8C provides a bar graph showing absolute number of host CD45 2 per grams of tumor at sacrifice. The infiltration was evaluated by flow cytometry. FIG. 8D provides representative fluorescence activated cell sorting (FACS) plots showing hHER2 and GFP expression in cultured tumor cells harvested from treated mice at sacrifice (Post-Tx), compared to their bulk population at the time of injection (Pre-Tx).

[0298] FIGs. 9A to 9K provide a schematic diagram, plots, bar graphs, stacked bar graphs, and FACS plots showing that B cells were effective in the B16-0VA melanoma model independently of their specificity. FIG. 9A provides a schematic diagram showing an experimental procedure. B16 cells were injected subcutaneously (SQ) 4 days before the B-cell intravenous (IV) injection. Tumor volume in mice transplanted with B 16 HER2+0VA+Luc+ was compared to B16 wild-type (WT) over time. FIG. 9A also provides a representative plot of data gathered using the experiment and a schematic diagram showing an expression construct contained in the B 16 HER2+0VA+Luc+ cells. FIG. 9B provides a plot of tumor volume over time in mice transplanted with B16 HER2+0va+Luc+ and treated with Edited or Unedited B- cells IV at day 4. FIG. 9C provides a bar graph showing absolute number of host CD45 2 per grams of tumor at sacrifice. The infiltration was evaluated by flow cytometry. FIG. 9D provides a bar graph showing frequency of GC (CD95+GL7+) within the host B- cells analyzed by flow cytometry in the SP at sacrifice. FIG. 9E provides a bar graph showing frequency of GC (CD95+GL7+) within the host B-cells analyzed by flow cytometry in the tumor draining lymph nodes (TDLN) at sacrifice. FIG. 9F provides a plot showing frequency of CD 19+ host B-cells analyzed by flow cytometry in the TDLN at sacrifice. FIG. 9G provides a bar graph showing frequency of CD 19+ host B-cells analyzed by flow cytometry in the tumor at sacrifice. FIG. 9H provides stacked bar graphs showing frequency of host Naive (CD44-CD62L+), Central Memory (CM; CD44highCD62L+) and Effector Memory (EF; CD44highCD62Llow) CD4+ (right) and CD8+ (left) T-cells in SP at sacrifice, as determined using flow cytometry. FIG. 91 provides stacked bar graphs showing frequency of host Naive (CD44-CD62L+), Central Memory (CM; CD44highCD62L+) and Effector Memory (EF; CD44highCD62Llow) CD4+ (right) and CD8+ (left) T-cells in TDLN at sacrifice, as determined using flow cytometry. FIG. 9J provides stacked bar graphs showing frequency of host Naive (CD44-CD62L+), Central Memory (CM; CD44highCD62L+) and Effector Memory (EF; CD44highCD62Llow) CD4+ (right) and CD8+ (left) T-cells in tumors at sacrifice, as determined using Flow cytometry. FIG. 9K provides representative FACS plots showing hHER2 and GFP expression in cultured tumor cells harvested from treated mice at sacrifice (Post-Tx), compared to their bulk population at the time of injection (Pre-Tx). FIGs. 9C to 9J show 1 out of 3 representative experiments. The data plotted in FIG. 9F correspond, from left-to-right, to “TM Only,” “B cell Untreated,” and “B cell Edited.” In each of FIGs. 9H to 9J, the stacked bars correspond, from bottom-to-top, to the cells listed in the legend, from left-to-right, respectively.

[0299] FIGs. 10A to 10H provide a schematic diagram, plots, and bar graphs showing that edited B cells were effective in the B16-0VA melanoma model when HER2 expression was maintained. FIG. 10A provides a schematic diagram showing the experimental procedure used to obtain and culture B 16 HER2-0VA-Luc single clones. FIG. 10B provides a plot of tumor volume over time in mice transplanted with B16 HER2-0va-Luc single clones (C8, CD14, C17) or a B 16 HER2-0va-Luc Bulk population. FIG. IOC provides a bar graph comparing the expression of HER2 in the TM in single clone tumors and in mice transplanted with bulk unsorted tumor cells (i.e., not single clones) at time of sacrifice of the mice. FIG. 10D provides a plot of tumor volume in B16 HER2-0VA-Luc Clone8 tumor-bearing mice treated with Edited or Unedited B-cells injected IV at day 4. FIG. 10E provides a bar graph showing frequency of hHER2 expression, as determined by FACS, in cultured tumor cells harvested from treated mice at sacrifice. FIG. 10F provides a plot showing a correlation between the tumor volume and the frequency of HER2 expression in cultured tumor cells according to the treatment received. FIG. 10G provides a bar graph showing absolute number of host CD45 2 per grams of tumor at sacrifice. The infiltration was evaluated by flow cytometry. FIG. 10H provides a plot of tumor volume in B16 HER2-0VA-Luc Clone8 tumor-bearing mice treated with Edited or Unedited B- cells injected IV at dayO.

[0300] FIGs. 11A to 11D provide plots and a schematic diagram relating to a strategy involving the use of murinized HER2 to avoid immune selection of hHER2+ cells. FIGs. 11A to 11C provide plots showing results from an ELISA assay on murine plasma to evaluate anti-HER2 IgM (left) and IgG (right). The amount of Antibody is expressed as optical density (OD) at 405 nm. The assay was run on mice bearing the following tumors: EO771 HER2-GFP (FIG. 11A), EO771 HER2 OVA-Luc (FIG. 11B) and B 16 HER2-OVA-Luc (FIG. 11C). FIG. 11D provides a schematic diagram showing a murinized HER2 construct (top panels). The expression of the construct was monitored by using Trastuzumab antibody (binding HER2 domain 4) and a different Anti-HER2 Ab binding HER2 Domain 1. The bottom panels of FIG. 11D provide representative FACS plots of an A20 cell line expressing the different murinized HER2 proteins shown above each respective FACS plot, where the cells measured in the leftmost plot did not express any murinized construct. FIGs. 12A to 12C provide images and stacked bar graphs showing that EO771 and B16 can be a suitable model to evaluate the formation of lung metastasis. FIG. 12A provides representative images of fixed (4% v / w PF A) lungs at 1, 2 or 3 weeks post-injection. FIGs. 12B and 12C provide stacked bar graphs showing frequency of B cells (“B”; CD19+, B220+), classical monocytes (“classical”; Ly6Chlghclassical inflammatory), non-classical monocytes (“non classical”; Ly6Clownon-classical anti-inflammatory), granulocytes (“granule”; Ly6G+), CD4+ and / or CD8+ T-cells, and double negative T cells (“DN”; CD4- and CD8-) in lungs at sacrifice and analyzed by Flow cytometry. The stacked bars in FIGs. 12B and 12C correspond, from bottom-to-top, to the cells listed in the legend from top-to-bottom, respectively.

[0301] FIGs. 13A to 13N provide schematic diagrams, plots, stacked bar graphs, flow cytometry plots, and bar graphs relating to a human B-cell culture protocol and a B cell phenotype analysis. FIG. 13A provides a schematic diagram showing the culture and editing protocol for primary human B-cells. FIG. 13B provides a plot showing a growth curve of primary human B-cells in CD40L-based culture. FIG. 13C provides a plot showing expression of activation markers over time in culture in primary human B cells, as determined using FACS. FIG. 13D provides stacked bar graphs showing (left panel) frequencies of Transitional (CD27- CD38+), Naive (CD27- CD38-), Memory (CD27+ CD38-), Plasmablasts (PB; CD27+CD38highCD 138-), Plasmacells (PC; CD27+CD38hlghCD138+) in cultured primary B-cells (Left panel), as determined using FACS and (right panel) expression of surface IgM and IgG in cultured primary B-cells, as determined by FACS. FIG. 13E provides a schematic diagram showing a homology-directed repair (HDR) cassette and human immunoglobulin heavy chain (IGH) locus structure. FIG. 13F provides a plot of the frequency IgH296 mediated NHEJ-alleles prepared using AAVS1 gRNA or IgH296 gRNA, as measured by the Interference of CRISPR Edits (ICE) bioinformatics tool in Synthego in K562 cells (left panel) and frequency of IgH296 gRNA mediated NHEJ-alleles measured by ICE in K562, JY, and JAKO1 cell lines (right panel). FIG. 13G provides a plot showing frequency of NHEJ-alleles in primary human B-cells prepared using AAVS1 gRNA or IgH296 gRNA, as measured by ICE. FIG. 13H provides a plot showing a growth curve of Edited and Unedited cells following the editing procedure. FIG. 131 provides representative flow cytometry plots for Unedited (left) and Edited (Right) human B-cells (Anti-Tag+ / HER2+) showing frequency of Edited B-cells(Tag+) determined by flow cytometry and droplet-based digital PCR (ddPCR) (% of edited alleles) 2 days after editing. FIG. 13J provides a stacked bar graph showing the frequency of Naive, Memory, PB and PC within the CD 19+ Edited and Unedited B-cells, as determined 2 days after editing using FACS. FIG. 13K provides stacked bar graphs showing frequency of Naive, Memory, PB and PC (left panel) and expression of surface IgM and IgG (right panel), within the CD19+Tag+HER2+ or CD19+Tag-HER2- Edited B-cells. FIG. 13L provides stacked bar graphs showing Frequency of Naive, Memory, PB, and PC within the CD 19+ B-cells cultured in different CD40L-based media over time. FIG. 13M provides a bar graph showing frequency of Edited cells (Tag+ HER2+) within the CD19+ B- cells cultured in different CD40L-based B-cell growth medium, as determined using FACS 2 days after editing. FIG. 13N provides a stacked bar graph showing frequency of Edited alleles within the CD 19+ B-cells cultured in different CD40L-based B-cell growth medium, as determined using ddPCR 2 days after editing. Each bar of the stacked bar graph of FIG. 13N corresponds to, in order from left-to-right, “Non-edited,” “HDR,” and “NHEJ.” In FIG. 13E, the term “iEu” represents the intronic enhancer iEp, the term “CSR” indicates the site of class switch recombination, and the term “SA” indicates a splice acceptor site. The bars of FIG. 13N correspond to the same experimental conditions listed for the corresponding bars listed in FIG. 13M

[0302] FIGs. 14A to 14C provide histograms, plots, and bar graphs demonstrating that edited B cells were functional. FIG. 14A provides a histogram and a plot presenting representative flow cytometry data showing phosphorylated extracellular signal-regulated kinase (pERK) expression in Edited (right curve in histogram of the left panel) or Unedited (left curve in histogram of the left panel) cells. The right panel of FIG. 14A provides a plot showing phosphorylated extracellular signal-regulated kinase (pERK) median fluorescent intensity (MFI) ratio between Edited (Tag+) and Unedited (Tag-) cells. FIG. 14B provides results from an ELISA assay on culture supernatant of Edited (right bar of each pair of bars) or Unedited (left bar of each pair of bars) primary human B-cells 2 and 10 days after editing. Anti-HER2 IgM (left panels) is expressed raw as OD per 100k cells, while Anti-HER2 IgG (right panels) is expressed as ng / ml per 100k cells after interpolation of the OD measurement with a standard curve. FIG. 14C provides a plot of frequencies of live (7-AADnegDAPIneg) target cells (MDA-MB-453 corresponding to the first four bars from the left and SK-OV3 corresponding to the four bars closest to the right) when co- cultured with different B-cell conditions. Each set of four bars in FIG. 14C correspond to, from left-to-right, “Edited B cells,” “Electroporated only B cells,” “Unedited B cells,” and “No B cells.”

[0303] FIGs. 15A to 15K provide images, plots, a schematic diagram, a bar graph, and stacked bar graphs demonstrating that edited B-cells could engraft in a xenotransplantation NOD scid gamma mouse (NSG™) breast cancer model. FIG. 15A provides images of bioluminescence imaging (BLI) scans of representative mice - side view. FIG. 15B provides a plot showing quantification of the bioluminescent signal at the tumor site in different treatment groups over time expressed as Total Flux in photons per second (p / s). Signal was measured on a region of interest (ROI) encompassing the tumor side on the side view. FIG. 15C provides a schematic diagram showing the experimental procedure in NSG™ mice. FIG. 15D provides a stacked bar graph showing frequency of homology-directed repair (HDR) and non-homologous end joining (NHEJ) edited alleles measured using ddPCR or the ICE bioinformatic tool, respectively. FIG. 15E provides a bar graph showing editing efficiency in the total CD 19+ cells and Naive and Memory subsets in Edited Primary human B-cells 2 days after editing, as determined using FACS. FIGs. 15F and 15G provide stacked bar graphs showing composition of syngeneic peripheral blood mononuclear cells (PBMC). FIG. 15H provides a plot showing human cells expansion across the experimental groups in the peripheral blood. FIG. 151 provides stacked bar graphs showing the composition of human cells in the peripheral blood 3 and 4 weeks after B- cell injection. FIG. 15 J provides plots showing a time course of secreted HER2-specific IgM (left) and IgG (right) antibody levels, as determined by using ELISA to evaluate NSG™ plasma samples. FIG. 15K provides a plot showing a correlation between the IgM titer in the plasma and the human engraftment in the spleen of the NSG™ mice.

[0304] FIGs. 16A to 16K provide plots, bar graphs, and stacked bar graphs demonstrating that edited B-cells could significantly reduce the tumor (TM) burden in a xenotransplantation NOD scid gamma mouse (NSG™) breast cancer model. FIG. 16A provides a plot showing tumor burden over time in MDA-MB-453 tumor-bearing mice, as determined using caliper measurements. B cells were injected IV at day 14. FIG. 16B provides a bar graph showing tumor weight at sacrifice. FIG. 16C provides a stacked bar graph showing frequency of mice with a detectable human infiltration (>3%). FIG. 16D provides a stacked bar graph showing cellular composition of the human CD45 infiltrate in the tumors at the day of sacrifice. FIG. 16E provides a plot showing human engraftment (%hCD45+ cells) in the spleen of mice at sacrifice. FIG. 16F provides a stacked bar graph showing cellular composition of the human CD45 infiltrate in the spleen at the day of sacrifice. FIG. 16G provides a plot showing human engraftment (%hCD45+ cells) in the bone marrow of mice at sacrifice. FIG. 16H provides a stacked bar graph showing cellular composition of the human CD45 infiltrate in the bone marrow at the day of sacrifice. FIG. 161 provides a plot showing human T cells growth in culture. FIG. 16J provides a stacked bar graph showing composition of human T cells after 12 days of in vitro stimulation. FIG. 16K provides a bar graph showing enzyme-linked immunosorbent spot (ELISPOT) data measuring specific activation of human splenic T cells against HER2+ target cells. The graph shows IFN-γ+ area per lx105cells. FIGs. 17A to 17G provide schematic diagrams, plots, and flow cytometry plots demonstrating that trastuzumab alone did not reduce the tumor (TM) burden in a xenotransplantation NOD scid gamma mouse (NSG™) breast cancer model. FIGs. 17A and D provide schematic diagrams showing protocols followed for Trastuzumab in vivo administration. FIGs. 17B and 17E provide plots showing tumor burden over time in MDA-MB- 453 tumor- bearing treated and control mice, as measured using calipers. FIG. 17C provides a plot showing a time course of secreted HER2-specific IgG antibody levels, as determined by using ELISA to evaluate NSG™ plasma samples. FIG. 17F provides a schematic diagram showing the structure of a homology-directed repair (HDR) cassette encoding membrane-bound anti-HER2 IgM and also showing the human immunoglobulin heavy chain (IGH) locus structure. FIG. 17G provides representative flow cytometry plots measuring the classic HER2-Fab Edited B-cells (left) and the membrane-bound HER2 BCR (right).

[0305] FIGs. 18A and 18B provide a plot and a bar graph assessing the impact of the editing procedure of the disclosure on the anti-TM efficacy of the BCR Edited B cells. FIG. 18A provides a plot of tumor burden over time in MDA-MB-453 tumor-bearing mice receiving B cell receptor (BCR) Edited B-cells, AAVS1 Edited B-cells, or unedited B-cells at day 14. FIG. 18B provides a bar graph showing tumor weight at sacrifice.

[0306] FIGs. 19A and 19B provide a plot and a bar graph demonstrating that chemotherapy conditioning may be a suitable add-on to the B-cell effect in the xenotransplantation NOD scid gamma mouse (NSG™) breast cancer model. FIG. 19A provides a plot of tumor burden over time in MDA-MB-453 tumor-bearing mice treated with different doses of Doxo IV at day 12 in parallel to untreated control mice. FIG. 19B provides a bar graph showing tumor weight at sacrifice.

[0307] FIGs. 20A to 20H provide schematic diagrams, images, and plots demonstrating that SK-0V3 may be a suitable model to evaluate the formation of lung metastasis in the NOD scid gamma mouse (NSG™) model. FIGs. 20A and 20E provide schematic diagrams showing the metastatic model of SK-OV3. FIGs. 20B and 20F provide images of bioluminescent imaging (BLI) scans of representative mice - ventral view. FIGs. 20C and 20G provide plots of tumor burden measured by BLI scans and expressed as photons per second, per square centimeter, per steradian (ph / s / cm2 / sr). FIGs. 20D and 20H provide plots showing expansion of human cells across the experimental groups in the peripheral blood.

[0308] FIGs. 21A to 21G provide a schematic diagram, plots, and flow cytometry plots relating to the transplantation of B cell receptor (BCR) Edited CD34+ cells in NOD scid gamma mouse (NSG™) mice. FIG. 21A provides a schematic diagram showing a protocol for CD34+ pre- engraftment in the MDa-MB-453 tumor model. FIG. 21B provides a plot showing the frequency of human CD45+ cells in the peripheral blood of transplanted mice over time. FIGs. 21C and 21D provide plots showing the composition of the human CD45+ cells in the peripheral blood: BCR Edited CD34+ transplanted mice on the left and AAVS1 Edited CD34 transplanted mice on the right. FIG. 21E provides a plot showing persistence of edited cells in vivo measured in the peripheral blood of transplanted mice as percent (%) of AAVS1 or BCR+ within the CD 19+ cells. FIG. 21F provides a plot showing tumor volume over time, as measured using calipers. FIG. 21G provides flow cytometry plots showing edited B-cells (Tag+) at sacrifice in the bone marrow (BM) and spleen (SP) of mice transplanted with BCR Edited CD34+ cells.

[0309] FIGs. 22A and 22B provide a schematic diagram and flow cytometry plots presenting a representative B-cells culture protocol FIG. 22A provides a schematic diagram showing the B- cells culture protocol. FIG. 22B provides flow cytometry plots showing B cell phenotype in the B cells growth according to the protocol.

[0310] FIG. 23 provides a schematic diagram showing insertion of a region of an HDR cassette of the disclosure into a target site, where the target site is the endogenous immunoglobulin heavy chain (IGH) locus in a genome.

[0311] FIGs. 24A to 24E provide plots showing that the anti-tumor effect of engineered B cells was mediated by antigen-specific T cell activation. FIG. 24A provides a pair of plots showing the lack of a correlation between plasma titers of anti-HER2 IgG (left panels) or IgM (right panels) antibodies and tumor weight at endpoint (40-50 days post tumor transplant) in mice transplanted with edited B cells and peripheral blood mononuclear cells (PBMCs). FIGs. 24B to 24D each provide pairs of plots showing a correlation between tumor weight at endpoint in mice transplanted with edited B-cells+PBMCs and either i) the percentage of human CD45+ within total live cells measured by fluorescence activated cell sorting (FACS) in the spleen (left panel of FIG. 24B) or in the tumor (right panel of FIG. 24B); ii) the percentage of human CD3+ cells within total live cells measured by FACS in the spleen (left panel of FIG. 24C) or in the tumor (right panel of FIG. 24C); iii) the percentage of human CD8+ cells within total hCD45 measured by FACS in the tumor (left panel of FIG. 24D) or in the spleen (right panel of FIG. 24D). FIG. 24E provides a pair or plots showing a correlation between the tumor weight at the endpoint in mice transplanted with unedited B-cells+PBMCs and the percentage of human CD45+ within total live cells measured by FACS in the tumor (left panel) or in the spleen (right panel).

[0312] FIGs. 25A to 250 provide bar graphs, stacked bar graphs, schematics, plots, and flow cytometry contour plots showing that CD4 T cells were central orchestrators of B and CD8 T cell responses in T cell-dependent tumor control. FIG. 25A provides a schematic representation of the experimental treatment scheme of NSG™ mice injected in the mammary fat pad (MFP) with MDA-MB-453 HER2+ tumors and treated by intravenous (IV) transplant of edited or unedited B cells + / - syngeneic CD4 and / or CD8 collected from the same healthy donor. FIG. 25B provides a plot showing the percentage of human hematopoietic cells (hCD45+) in mice treated with edited B cells and CD8 T cells at the time of sacrifice in the tumor (TM), bone marrow (BM), spleen (SP) and Blood (BL, measured 4 weeks after injection). FIG. 25C provides a bar graph showing tumor weight at endpoint in MDA-MB-453 tumor-bearing mice, treated with either unedited or B cell receptor (BCR) edited B cells + Syngeneic PBMCs, and BCR Edited B cells plus syngeneic CD8 T cells only. FIG. 25D provides percentage of human hematopoietic cells (hCD45+) in mice treated with unedited or B cell receptor (BCR) Edited B cells and CD4 T cells at the time of sacrifice in the TM, BM, SP and BL (BL was measured 4 weeks after injection). Percentage of hCD45 reported previously in mice treated with unedited or BCR edited B cells plus syngeneic PBMCs is reported for comparison. FIG. 25E provides a bar graph showing tumor weight at endpoint in MDA-MB-453 tumor-bearing mice treated with either unedited or BCR edited B cells + syngeneic PBMCs (left two bars) or CD4 T cells only (middle two bars) or a combination of CD4 and CD8 T cells (right two bars). FIG. 25F provides a plot showing percentage of human hematopoietic cells (hCD45+) in mice treated with unedited or BCR edited B cells and CD4 T+CD8 T cells (ratio 2:1) at the time of sacrifice in the TM, BM, SP and BL (BL measured 4 weeks after injection). Percentage of hCD45 reported previously in mice treated with unedited or BCR Edited B cells plus syngeneic PBMCs is reported for comparison. FIG. 25G provides a flow cytometry contour plot showing Edited human B cells (Tag +) in the spleen of treated mice at the time of sacrifice. FIG. 25H provides bar graph showing frequency of edited Tag+ human B cells within the total CD 19+ B cells population analyzed at endpoint by flow cytometry in the spleen and in the bone marrow of mice treated with BCR edited B cells plus CD4 only or CD4+CD8 T cells (2: 1 ratio). FIGs. 251 and 25J provide plots showing plasma level of anti-human HER2 IgM (FIG. 251) or IgG (FIG. 25J) assessed by ELISA on NSG™ mice plasma. Anti-HER2 IgM levels are expressed as blank-normalized optical density (OD); anti-HER2 IgG levels are expressed as ng / ml of plasma after interpolation with a Trastuzumab standard curve. FIGs. 25K and 25L provide plots showing a correlation between plasma titers of anti-HER2 IgM (FIG. 25K) or IgG (FIG. 25L) antibodies and tumor weight at endpoint in mice transplanted with Edited B cells+PBMCs or different T cell fractions. FIG. 25M provides a stacked bar graph showing composition of human engraftment in the TM, SP and BM at endpoint in mice transplanted with unedited or BCR edited B cells plus CD4+CD8 T cells (2: 1 ratio). FIG. 25N provides a stacked bar graph showing percentage of mice with an IFNγ+ enzyme-linked immunospot (ELISPOT) signal. Human T cells, isolated from the spleen of transplanted mice were re-challenged in vitro with HER2 MDA-MB-453 and IFNγ production was assessed via ELISPOT assay. FIG. 250 provides a bar graph showing interferon gamma (IFNγ) ELISPOT signal measured as IFNy+ signal area (mm2) per 100,000 plated T-cells isolated from the spleen of transplanted mice. In FIGs. 25K and 25L, “Edited B cells + PBMCs” data points are represented by light grey circles, “Edited B cells + CD4 ” data points are represented by dark grey circles, and “Edited B cells + CD4+CD8+” data points are represented by empty circles.

[0313] DETAILED DESCRIPTION

[0314] The disclosure features B cells engineered to express B cell receptors targeting a HER2 antigen and compositions and methods for use or preparation thereof.

[0315] The present disclosure is based, at least in part, upon the discovery that engineered B cells expressing B cell receptors targeting a HER2 antigen can be used to treat HER2+ breast cancer. To engineer the B cells, a CRISPR / Cas9 gene editing protocol was developed to insert the trastuzumab antigen-binding fragment (Fab) into the endogenous B cell receptor (BCR) locus of primary B cells, thus redirecting their specificity towards the HER2 tumor-associated antigen (TAA). This strategy combines the efficacy of anti-HER2 monoclonal antibodies (MoAbs) with the power of cellular therapy, thus enabling local delivery and on demand production of antitumor immunity. Not intending to be bound by theory, since B cell activation and proliferation rates after antigen encountering are slower compared to T cells, a B cell approach may avoid the risk of systemic toxicity by cytokine release syndrome (CRS). Moreover, tumor- specific B cells may cross-activate T cell effector / memory responses by their antigen presenting cell (APC) function and by inducing local inflammation, thus broadening the antigenic specificity of the therapy and possibly overcoming tumor resistance to alternative anti-HER2 therapies. The ease of collection of B cells, their capacity to expand with already-established clinical-grade procedures, and their resistance to oncogenic transformation after genetic manipulation, due to their terminally differentiated nature, make B cells suitable for a straightforward human testing. The B cell editing strategy of the present disclosure will open new avenues for improved cellular immunotherapy of BC and several other malignant diseases.

[0316] The methods of the present disclosure utilize the potential of B-cell editing to harness both humoral and cellular immunity against an HER2 Tumor-Associated Antigen (TAA). B cells can be conveniently collected from peripheral blood, survive in ex vivo culture, home to tumors and tumor draining lymph nodes (TDLNs) upon transplantation, and respond to antigen stimulation, making them an ideal immunotherapy target. While CAR cell therapies have made substantial progress, current strategies primarily focus on T cells, which have shown suboptimal efficacy in solid tumors, with limited attention to NK or γδ T cells. A recent study introduced anti-HER2 CAR macrophages, demonstrating the induction of a pro-inflammatory TME and enhanced anti-tumor T cell activity. However, generating sufficient CAR macrophages remains challenging. The present disclosure seeks to expand the scope of immunotherapies by introducing a potentially more effective tool to the breast cancer (BC) treatment arsenal. The Examples of the present disclosure shed new light on the role of antigen-specific B cell responses in cancer and reveal that active B cell immunity can significantly impact tumor progression.

[0317] B Cells and the B Cell Receptor

[0318] B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or inserted into the plasma membrane where they serve as a part of B-cell receptors. When a naive or memory B cell is activated by an antigen, it proliferates and differentiates into an antibody-secreting effector cell, known as a plasmablast or plasma cell. In addition, B cells present antigens (they are also classified as professional antigen-presenting cells, APCs) and secrete cytokines. In mammals, including marsupials, B cells mature in the bone marrow. In birds, B cells mature in the bursa of Fabricius, a lymphoid organ where they were first discovered by Chang and Glick, which is why the B stands for bursa and not bone marrow, as commonly believed.

[0319] B cells, unlike the other two classes of lymphocytes, T cells and natural killer cells, express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a foreign antigen (e.g., HER2), against which it will initiate an antibody response. B cell receptors are extremely specific, with all BCRs on a B cell recognizing the same epitope.

[0320] A B-cell receptor (BCR) is a transmembrane protein expressed on the surface of a B cell that recognizes and binds to a specific foreign antigen (e.g., HER2). A BCR is contains a membrane-bound immunoglobulin molecule (e.g., an anti-HER2 immunoglobulin molecule) and a signal transduction moiety. The BCR stimulates the activation of the B cell by intracellular signaling, and initiates an antibody response against the antigen. A B cell is activated by its first encounter with an antigen (e.g., HER2) that binds to its receptor, resulting in cell proliferation and differentiation to generate a population of antibody-secreting plasma B cells and memory B cells. The antibody-secreting plasma B cells secrete antibodies containing the antigen-binding portion of the BCR and capable of binding the antigen. The B cell receptor (BCR) has two crucial functions upon interaction with the antigen. One function is signal transduction, involving changes in receptor oligomerization, and the second function is to mediate internalization for subsequent processing of the antigen and presentation of peptides to helper T cells.

[0321] Tumor-infiltrating B cells and plasma cells (collectively referred to as tumor-infiltrating B lymphocytes (TIBs)) can produce antibodies directed against tumor antigens in the tumor- draining lymph nodes (TDLNs) facilitating antibody-dependent cellular toxicity (ADCC) and antibody-depdendent cellular phagocytosis (ADCP). TIBs can kill cancer cells via Granzyme B, FasL, TRAIL (TNF superfamily). TIBs form Tertiary Lymphoid structures (TLS), lymph-node- like structures that: A) arise de novo in the stroma of hot tumors in response to antigen (Ag) and inflammatory stimuli; B) promote Ag-driven clonal expansion, class switching, and affinity maturation; and B) are considered prognostic value to immuno-therapy responses. TIBs can produce immunostimulatory cytokines. TIBs are more efficient than dendritic cells and Macrophages in presenting low-abundance Antigens. TIBs are the only professional APCs capable of clonal expansion. The epitope spreading mechanism could mitigate the challenge of tumor heterogeneity and lead to overcoming tumor monoresistance. TIBs presence has been associated with favorable prognosis in many solid tumors. Anti-CD20 antibody-mediated B-cell depletion has been shown to decrease T-cell infiltrates and anti-tumor immunity. TIBs may overcome the Ab difficulty of infiltrating the tumor (TM) matrix barrier. TIBs may trigger both humoral and cellular-mediated anti-TM responses.

[0322] Accordingly, the present disclosure features B cells B cells modified to target the tumor- associated antigen (TAA) HER2 through engineering of the B cell receptor.

[0323] Anti-HER2 B Cells

[0324] High levels of anti-HER2 autoreactive Abs are associated with a reduced risk of ductal carcinoma in situ or invasive breast carcinoma, and some breast cancer (BC) patients that spontaneously produce anti-HER2 autoantibodies have shown improved survival. This suggests that the generation of HER2 autoreactive B cells can confer a protective effect on the recurrence of BC. Tumor-infiltrating B cells (TIBs) can be observed in various solid tumors, including breast cancer (BC), and constitute the third most abundant tumor- infiltrating immune cells. Heavy B cell infiltration in medullary BC is associated with a more favorable prognosis, implying a positive role in the elimination of tumor cells. In addition to their role in the production of antibodies, naturally occurring cancer-specific B cells may directly participate in tumor suppression by i) direct killing of tumor cells via granzyme B, FasL, and TRAIL expression and ii) by triggering robust T cell immunity, through activation of inflammatory responses and through their professional antigen-presenting cell (APC) function. Indeed, TIBs often form immune compartments called tertiary lymphoid structures (TLS), which are sites for the differentiation of effector and memory T and B cells. TLS presence in human tumors is associated with better outcomes in both HER2+ and triple-negative BC patients, as well as in other types of tumors. Moreover, the TLS presence is more accurate than currently used T cell signatures as predictive biomarkers for immunotherapy response with checkpoint inhibitors. However, despite their role in TLS, TIBs could also exert pro-tumoral functions by acquiring a B regulatory cell (Breg) phenotype in the TME. In both human and mouse studies, Bregs display their characteristic immunosuppressive effect by secreting cytokines such as IL-10 and TGF-β and / or upregulating immune regulatory ligands such as PD-L1 and CTLA-4. Accordingly, to overcome this issue, the present disclosure provides compositions and methods exploiting in vitro activated B cells as new cellular immunotherapy agents. Once activated in vitro by a CD40L-based stimulation, B cells i) do not induce tolerance by themselves, ii) become insensitive to tumor- derived immunosuppressive mechanisms, and iii) are well tolerated upon infusion. Interesting preclinical studies using a 4T1 mBC model have shown that adoptive transplant of B cells, collected from the tumor draining lymph nodes (TDLN) of BC bearing mice and activated in vitro with CD40L stimulation, are able to i) produce tumor-specific Abs, ii) infiltrate the orthotopically implanted primary tumor and the secondary metastatic lung tissues, iii) trigger the establishment of tumor specific T cell immunity and iv) mediate reduction of lung metastases. However, tumor-specific B cells within TDLN are rare and their selection and expansion from the extensive polyclonal repertoire of patient B cells would require highly complex, time- consuming and low yielding procedures.

[0325] Accordingly, the present disclosure features the use of genetic engineering technologies to redirect the specificity of B cell receptor (BCR) towards tumor-associated antigens (TAAs) to enable the development a combined humoral and cellular adoptive immunotherapy for HER2+ BC. The embodiments of the present disclosure pioneer the use of B cells in reducing tumor burden in vivo, offering a promising avenue for solid tumor immunotherapy.

[0326] Preparation Modified B Cells Targeting HER2

[0327] Therapeutic gene editing is a major focus of biomedical research, embracing the interface between basic and clinical science. The methods of the disclosure involve engineering a B cell to express a B cell receptor capable of binding a HER2 antigen. In various embodiments, the methods of the disclosure involve isolating B cells, or progenitors thereof, from a subject, engineering the B cells or B cell progenitors according to the methods provided herein, and subsequently administering the B cells or B cell progenitors to the subject. In some cases, the B cell progenitors are differentiated into mature B cells prior to administration to the subject. In some cases the B cell progenitors are primary B cells (e.g., CD43- resting B cells). The B cells may be isolated from the peripheral blood mononuclear cells (PBMCs) of a subject.

[0328] In various embodiments, the methods of the disclosure involve insertion at the immunoglobulin heavy chain (IGH) locus in a genome a polynucleotide encoding a polypeptide capable of binding an HER2 antigen (see, e.g., FIG. 13E). Insertion of the polynucleotide involves introducing into a B cell to be edited a homology-directed repair (HDR) cassette containing the polynucleotide to be inserted into the genomic locus. Non-limiting examples of such cassettes include polynucleotides having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more sequence identity to SEQ ID NO: 1 or derivatives or fragments thereof suitable for use in the methods of the disclosure. In some cases, SEQ ID NO: 1 is modified to remove the Murine HBB intronic sequence. In some instances, SEQ ID NO: 1 is modified to remove the 5’ UTR and / or 3’ UTR sequences or replace the 5’ UTR and / or 3’ UTR with alternative polynucleotide sequences. The HDR cassette may be codon optimized.

[0329] In some instances, the HDR cassette encodes, in order from 5’ to 3’ end, a heavy-chain variable region of an anti-HER2 antibody, a peptide linker optionally containing a streptavidin tag, and a light-chain variable region of the anti-HER2 antibody. In some instances, the HDR cassette encodes, in order from 5’ to 3’ end, a light-chain variable region of an anti-HER2 antibody, a peptide linker optionally containing a streptavidin tag, and a heavy-chain variable region of the anti-HER2 antibody. The streptavidin tag may be a 3x streptavidin tag (i.e., three tandem repeats of a streptavidin tag). In some embodiments, the streptavidin tag is used for detection and / or sorting of B cells expressing engineered B cell receptors of the disclosure. In various embodiments, the peptide linker is about or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length. In various embodiments, the peptide linker is no more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length. The linker may be a Gly-Ser linker optionally containing between 1 and 5 instances of a Strep- Tag sequence motif. In embodiments, the linker prevents coupling of the light-chain variable region and / or heavy-chain variable region with an endogenous L chain.

[0330] In some cases, the HDR cassette further contains a minimal promoter (e.g., an IGHV1-69 minimal promoter) upstream of the heavy-chain and light-chain variable regions of the anti- HER2 antibody. It may be advantageous to select the promoter such that it becomes active only when the enhancer located before each V-fragment is placed in close proximity upon variable- diversity -joining (V(D)J) recombination. The minimal promoter mediates expression of the polypeptide encoded by the HDR cassette.

[0331] In various embodiments, the HDR cassette contains regions homologous to a target site for insertion in a genome, where the HDR cassette contains two said regions, and where the regions flank the region of the HDR to be inserted within the genome (e.g., the region encoding the promoter and polypeptide). In various embodiments, the homology arms flank a site cut by a nuclease. FIG. 23 provides a schematic diagram showing insertion of a region of an HDR cassette of the disclosure into a target site. In some embodiments, a homologous region upstream of the region of the HDR cassette encoding the promoter and polypeptide comprises the following nucleotide sequence, a fragment thereof suitable for use in HDR, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide alterations and suitable for use in HDR: atgtgacgcccggagacagaaggtctctgggtggctgggtttttgtggggtgaggatggacatt ctgccattgtgattactactactactactacatggacgtctggggcaaagggaccacggtcacc gtctcctcaggtaagaatggccactctagggcctttgttttctgctactgcctgtggggtttcc tgagcattgcaggttggtcctcggggcatgttccgaggggacctgggcggactggccaggaggg gatgggcactggggtgccttgaggatctgggagcctctgtggattttccgatgcctttggaaaa tgggactcaggttgggtgcgtctgatggagtaactgagcctgggggcttggggagccacatttg gacgagatgcctgaacaaaccaggggtcttagtgatggctgaggaatgtgtctcaggagcggtg tc. In some embodiments, a homologous region downstream of the region of the HDR cassette encoding the promoter and polypeptide comprises the following nucleotide sequence, a fragment thereof suitable for use in HDR, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide alterations and suitable for use in HDR: tgtaggactgcaagatcgctgcacagcagcgaatcgtgaaatattttctttagaattatgaggt gcgctgtgtgtcaacctgcatcttaaattctttattggctggaaagagaactgtcggagtgggt gaatccagccaggagggacgcgtagccccggtcttgatgagagcagggttgggggcaggggtag cccagaaacagtggctgccgtcctgacaggggcttagggaggctccaggacctcagtgccttga agctggtttccatgagaaaaggattgtttatcttaggaggcatgcttactgttaaaagacagga tatgtttgaagtggcttctgagaaaaatggttaagaaaattatgacttaaaaatgtgagagatt ttcaagtatattaatttttttaactgtccaagtatttgaaattcttatcatttgattaacaccc at. In various embodiments, the methods of the disclosure involve insertion of the contiguous region of the HDR cassette encoding the promoter and the splice donor site into a region of the immunoglobulin heavy chain (IGH) locus between the region of the IGH encoding joining gene segments (“J”) and the intronic enhancer iEp (“iEu”).

[0332] In some embodiments, the HDR cassette further encodes a signal peptide (e.g., an Igk signal peptide or other signal peptide suitable to garget the encoded protein to the secretory pathway and membrane localization) fused to the N-terminus of the polypeptide encoded by the cassette (e.g., at the N-terminus of the light-chain or heavy-chain variable region of the HER2 antibody).

[0333] In some cases, the HDR cassette contains a splice donor site (SD) downstream of the portion of the cassette encoding the heavy-chain and light-chain variable regions of the anti- HER2 antibody. The splice donor site may be used for splicing in an endogenous IGH locus. In various embodiments, the splice donor site is downstream of the region of the HDR cassette encoding the promoter and polypeptide and upstream of the downstream homologous region described above, and contains the following nucleotide sequence, a fragment thereof suitable for use as a splice donor site, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide alterations and suitable for use as a splice donor site: GTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCAGGTGTACTGGGCCAGGCAAGGGCTTTG.

[0334] In some embodiments, the HDR cassette is flanked by inverted terminal repeats (ITRs) suitable for mediating packaging of the HDR cassette within an AAV capsid. The HDR cassette may be introduced to a cell using an AAV vector, such as an AAV6 vector or an AAV-DJ vector. In some cases, the C-terminus of the variable light chain of the anti-HER2 antibody is fused to a constant region of kappa light chain.

[0335] In various embodiments, following homology-directed recombination at a target site of a genome with the HDR cassette, the encoded recombinant antibody will 1) be expressed only if the Fab (Fragment antigen-binding, which is a polypeptide containing a full light chain (variable+constant regions) and the variable region of a heavy chain linked by a linker peptide) is spliced into the endogenous IGH, 2) be expressed in both transmembrane and secreted forms, 3) undergo class-switching, and / or 4) will undergo somatic hypermutation.

[0336] It may be advantageous to culture the edited B cells in a CD40L-based medium to stimulate the cells and generate anti-tumoral specific cells. In some embodiments, CD40L- stimulated cells A) do not acquire a B-reg phenotype or show reduced tendency to form the B- reg phenotype relative to cells cultured in the absence of CD40L stimulation, B) become insensitive toward tumor microenvironment (TME) immunosuppression relative to cells cultured in the absence of CD40L stimulation, and / or C) are well tolerated upon being transplanted into a subject relative to cells cultured in the absence of CD40L stimulation. A representative protocol for preparation of B cells according to the methods of the disclosure is provided in FIGs. 22A and 22B.

[0337] The development of novel “gene editing” tools provide the ability to manipulate the DNA sequence of a cell (e.g., to knock out a target gene) at a specific chromosomal locus, without introducing deleterious mutations at other sites of the genome. This technology effectively enables the researcher to manipulate the genome of a subject’s cells.

[0338] In some embodiments, a heterologous polynucleotide may be inserted into the genome of a cell using a transposable element such as a transposon, as described, for example, in Tipanee, et al. Human Gene Therapy, Nov. 2017, 1087-1104, DOI: 10.1089 / hum.2017.128. Transposable elements are divided into two categories: retrotransposons and DNA transposons. Transposable elements can alter the genome of the host cells through insertions, duplications, deletions, and translocations. Retrotransposons are described as mobile elements that employ an RNA intermediate that is first reverse transcribed into a complementary single-stranded (c) DNA strand by a reverse transcriptase encoded by the retrotransposon. Subsequently, the single- stranded DNA is converted into a double-stranded DNA that then integrates into the host genome. This so-called “replicative mechanism” yields several new copies of retrotransposons expanding throughout the target genome over evolutionary time. Retrotransposons are categorized into many subtypes according to the DNA sequences of the long terminal repeats and its open reading frames. Retrotransposons were employed to enable transgene integration into the target cell DNA, in some cases relying on adenoviral delivery. Alternatively, DNA transposons translocate via a “non-replicative mechanism,” whereby two Terminal Inverted Repeats (TIRs) are recognized and cleaved by a transposase enzyme, releasing the cognate DNA transposons with free DNA ends. The excised DNA transposons then integrate into a new genomic region where target sites are recognized and cut by the same transposase. This cut-and- paste mechanism usually duplicates DNA target sites upon insertion, leaving target site duplications (TSDs). Non-limiting examples of transposons include the Sleeping Beauty (SB) transposon, the piggyBac (PB) transposon, and Tol2 transposable elements.

[0339] In one embodiment, gene editing involves targeting an endonuclease (an enzyme that causes DNA breaks internally within a DNA molecule) to a specific site of the genome and thereby triggering formation of a chromosomal double strand break (DSB) at the chosen site. If, concomitant with the introduction of the chromosome breaks, a donor DNA molecule (e.g., an HDR cassette) may be introduced (for example, by plasmid or oligonucleotide introduction, such as through the use of a vector, such as an AAV vector), interactions between the broken chromosome and the introduced DNA can occur, especially if the two sequences share homology. In this instance, a process termed “gene targeting” can occur, in which the DNA ends of the chromosome invade homologous sequences of the donor DNA by homologous recombination (HR). By using the HDR cassette as a template for HR, a seamless repair of the chromosomal DSB can be accomplished.

[0340] Current genome editing tools use the induction of double strand breaks (DSBs) to enhance gene manipulation of cells. Such methods include zinc finger nucleases (ZFNs; described for example in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, and U.S. Pat. Publ. Nos. 20030232410 and US2009020314, which are incorporated herein by reference), Transcription Activator-Like Effector Nucleases (TALENs; described for example in U.S. Patent Nos. 8,440,431, 8,440,432, 8,450,471, 8,586,363, and 8,697,853, and U.S. Pat. Publ. Nos. 20110145940, 20120178131, 20120178169, 20120214228, 20130122581, 20140335592, and 20140335618, which are incorporated herein by reference), and the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system (described for example in U.S. Patent Nos. 8,697,359, 8,771,945, 8,795,965, 8,871,445, 8,889,356, 8,906,616, 8,932,814, 8,945,839, 8,993,233, and 8,999,641, and U.S. Pat. Publ. Nos. 20140170753, 20140227787, 20140179006, 20140189896, 20140273231, 20140242664, 20140273232, 20150184139, 20150203872, 20150031134, 20150079681, 20150232882, and 20150247150, which are incorporated herein by reference). In some embodiments a CRISPR / Casl2 system can be used for gene editing. In some embodiments, the Casl2 polypeptide is Casl2b. In some embodiments any Cas polypeptide can be used for gene editing (e.g., CasX). In various embodiments, the Cas polypeptide is selected so that a nucleotide encoding the Cas polypeptide can fit within an adeno- associated virus (AAV) capsid. ZFNs and TALENs entail use of modular sequence-specific DNA binding proteins to generate specificity for ~18 bp sequences in the genome. CRISPR / Cas9, TALENs, and ZFNs have all been used in clinical trials (see, e.g., Li., H, etal., “Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects”, Signal Transduct Target Ther.. 5: 1 (2020), DOI: 10.1038 / s41392-019-0089-y).

[0341] RNA-guided nucleases-mediated genome editing, based on Type 2 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR Associated) systems, offers a valuable approach to alter the genome. In brief, Cas9, a nuclease guided by single-guide RNA (sgRNA), binds to a targeted genomic locus next to the protospacer adjacent motif (PAM) and generates a double-strand break (DSB). The DSB is then repaired either by non-homologous end joining (NHEJ), which leads to insertion / deletion (indel) mutations, or by homology-directed repair (HDR), which requires an exogenous template and can generate a precise modification at a target locus (Mali et al., Science. 2013 Feb 15;339(6121):823-6). Genetic manipulation using engineered nucleases has been demonstrated in tissue culture cells and rodent models of diseases.

[0342] CRISPR has been used in a wide range of organisms including baker’s yeast (5. cerevisiae), zebra fish, nematodes (C. elegans), plants, mice, and several other organisms. Additionally, CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes. Libraries of tens of thousands of guide RNAs are now available.

[0343] Since 2012, the CRISPR / Cas system has been used for gene editing (silencing, enhancing or changing specific genes) that even works in eukaryotes like mice and primates. By inserting a plasmid containing Cas genes and specifically designed CRISPRs, an organism's genome can be cut at any desired location.

[0344] CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote's genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.

[0345] CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Casl appears to be ubiquitous among different CRISPR / Cas systems. Particular combinations of Cas genes and repeat structures have been used to define 8 CRISPR subtypes (E. coli, Y. pest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR / Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

[0346] Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (about 30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype E. coif) proteins (called CasA-E in E. coll) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coll requires Cascade and Cas3, but not Casl and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes. See also U.S. Patent Publication 2014 / 0068797, which is incorporated by reference in its entirety.

[0347] Nucleic Acid Programmable DNA Binding Proteins (napDNAbps)

[0348] Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. linek et al. (2012) combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. It has been proposed that such synthetic guide RNAs might be able to be used for gene editing ( inek et al., Science. 2012 Aug 17;337(6096):816-21). Cas9 is one of many representative nucleic acid programmable DNA binding proteins suitable for use in the methods of the disclosure, such as Casl2.

[0349] Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR / Cas- mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR / Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. Coinjection of Cas9 mRNA and sgRNAs into the germline (zygotes) generated mice with mutations. Delivery of Cas9 DNA sequences also is contemplated.

[0350] Cas9 variants have been developed or discovered that can fit into an adeno-associated virus (AAV) capsid with sgRNA. Non-limiting examples of such variants (e.g., Cas9 orthologs) suitable for use in embodiments of the disclosure of the disclosure include saCas9 (Staphylococcus aureus Cas9), cjCas9 (Camphylobacter jejuni Cas9), NmeCas9 (Neisseria meningitidis Cas9), and spCas9 (Streptococcus pyrogenes Cas 9). An example of a saCas9 suitable for delivery by an AAV vector is provided in Ann Ran, F. et al. “In vivo genome editing using Staphylococcus aureus Cas9”, Nature, 9: 186-91, DOI: 10.1038 / naturel4299. gRNA

[0351] As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because Eukaryotic systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type 21 promoter U6). Synthetic gRNAs are slightly over 100 bp at the minimum length and contain a spacer portion that targets the 20 protospacer nucleotides immediately preceding the PAM sequence (e.g., NGG or AGG); gRNAs do not contain a PAM sequence. In some embodiments, a gRNA of the disclosure contains a spacer having the following nucleotide sequence: GUCUCAGGAGCGGUGUCUGU. In some embodiments, the Cas9 polypeptide has specificity for an AGG PAM sequence.

[0352] Antibodies

[0353] As reported herein, antibodies (e.g., Trastuzumab), or functional fragments thereof, that specifically bind a an HER2 antigen are useful in the methods of the disclosure, including therapeutic methods. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. As used herein, the term “antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies, such as cam elid antibodies (Riechmann, 1999, lournal of Immunological Methods 231 :25-38), composed of either a VL or a VH domain which exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments.

[0354] The antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab') 2 , as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). For example, F(ab')2, and Fab fragments that lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). Thus, the antibodies of the disclosure comprise, without limitation, whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab’, single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.

[0355] Unconventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062,1995), single domain antibodies, single chain antibodies, and antibodies having multiple valencies (e.g., diabodies, tribodies, tetrabodies, and pentabodies). Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies have the affinity and specificity of conventional antibodies although they are only half of the size of a single chain Fv fragment. The consequence of this unique structure, combined with their extreme stability and a high degree of homology with human antibody frameworks, is that nanobodies can bind therapeutic targets not accessible to conventional antibodies. Recombinant antibody fragments with multiple valencies provide high binding avidity and unique targeting specificity to cancer cells. These multimeric scFvs (e.g., diabodies, tetrabodies) offer an improvement over the parent antibody since small molecules of ~60-100kDa in size provide faster blood clearance and rapid tissue uptake See Power et al., (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting, 4, 47-58, 1999).

[0356] Various techniques for making and using unconventional antibodies have been described. Bispecific antibodies produced using leucine zippers are described by Kostelny et al. (J. Immunol. 148(5): 1547-1553, 1992). Diabody technology is described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993). Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners is described by Gruber et al. (J. Immunol. 152:5368, 1994). Trispecific antibodies are described by Tutt et al. (J. Immunol. 147:60, 1991). Single chain Fv polypeptide antibodies include a covalently linked VH::VL heterodimer which can be expressed from a nucleic acid including VH- and Vr-encoding sequences either joined directly or joined by a peptide-encoding linker as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.

[0357] In various embodiments, an antibody is monoclonal. Alternatively, the antibody is a polyclonal antibody. The preparation and use of polyclonal antibodies are also known the skilled artisan. The disclosure also encompasses hybrid antibodies, in which one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids may also be formed using humanized heavy and light chains. Such antibodies are often referred to as “chimeric” antibodies.

[0358] In general, intact antibodies are said to contain “Fc” and “Fab” regions. The Fc regions are involved in complement activation and are not involved in antigen binding. An antibody from which the Fc’ region has been enzymatically cleaved, or which has been produced without the Fc’ region, designated an “F(ab’)2” fragment, retains both of the antigen binding sites of the intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an “Fab1” fragment, retains one of the antigen binding sites of the intact antibody. Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted “Fd.” The Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.

[0359] Methods of preparing antibodies are well known to those of ordinary skill in the science of immunology. Antibodies can be made by any of the methods known in the art utilizing a soluble polypeptide, or immunogenic fragment thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding polypeptides or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the polypeptide thereby generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding human polypeptides or immunogenic fragments thereof, can be expressed in cells in vitro, followed by isolation of the polypeptide and administration of the polypeptide to a suitable host in which antibodies are raised.

[0360] Alternatively, antibodies may, if desired, be derived from an antibody phage display library. A bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins. Phage display is the process by which the phage is made to 'display' the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.

[0361] Antibodies made by any method known in the art can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.

[0362] Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotol erant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g, Pristane).

[0363] Antibodies of the disclosure can be “humanized” by methods known in the art. “Humanized” antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. Techniques to humanize antibodies are particularly useful when non-human animal (e.g, murine) antibodies are generated. Examples of methods for humanizing a murine antibody are provided in U.S. patents 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

[0364] Polynucleotide Delivery

[0365] Viral vectors are used to genetically alter cells of the present disclosure and their progeny. Viral vectors are used, as are the physical methods described below, to deliver one or more target genes, polynucleotides, antisense molecules, HDR cassettes, or ribozyme sequences, for example, into the cells.

[0366] Viral vectors and methods for using them to deliver DNA to cells are well known to those of skill in the art. Transducing viral vectors (e.g., retroviral vectors (e.g., lentiviral vectors), alphaviral vectors (e.g., Sindbis vectors), adenoviral vectors, herpes virus vectors, and adeno-associated viral vectors) can be used for introducing a polynucleotide to a cell, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a polynucleotide can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990;

[0367] Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988;

[0368] Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61, 1990; Sharp, The Lancet 337: 1277- 1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No.5,399,346). In some embodiments, the AAV vector is an AAV6 vector or an AAV- DJ vector (see, e.g., Lerch, et al. “Structure of AAV-DJ, a retargeted gene therapy vector: Cryo- Electron Microscopy at 4.5A resolution,” Structure, 20: 1310-1320 (2012)).

[0369] Calcium phosphate transfection can be used to introduce plasmid DNA containing a target gene or polynucleotide into a B cell and is a standard method of DNA transfer to those of skill in the art. DEAE-dextran transfection, which is also known to those of skill in the art, may be preferred over calcium phosphate transfection where transient transfection is desired, as it is often more efficient. Since the cells of the present disclosure are isolated cells, microinjection can be particularly effective for transferring genetic material into the cells. This method is advantageous because it provides delivery of the desired genetic material directly to the nucleus, avoiding both cytoplasmic and lysosomal degradation of the injected polynucleotide. Cells of the present disclosure can also be genetically modified using electroporation.

[0370] Liposomal delivery of DNA or RNA to genetically modify the cells can be performed using cationic liposomes, which form a stable complex with the polynucleotide. For stabilization of the liposome complex, dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPQ) can be added. Commercially available reagents for liposomal transfer include Lipofectin (Life Technologies). Lipofectin, for example, is a mixture of the cationic lipid N-[l-(2, 3-dioleyloxy)propyl]-N-N-N- trimethyl ammonia chloride and DOPE. Liposomes can carry larger pieces of DNA, can generally protect the polynucleotide from degradation, and can be targeted to specific cells or tissues. Cationic lipid- mediated gene transfer efficiency can be enhanced by incorporating purified viral or cellular envelope components, such as the purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G). Gene transfer techniques which have been shown effective for delivery of DNA into primary and established mammalian cell lines using lipopolyamine-coated DNA can be used to introduce target DNA into the lymphatic endothelial progenitor cells described herein.

[0371] Naked plasmid DNA can be injected directly into B cells. This technique has been shown to be effective in transferring plasmid DNA to skeletal muscle tissue, where expression in mouse skeletal muscle has been observed for more than 19 months following a single intramuscular injection. More rapidly dividing cells take up naked plasmid DNA more efficiently. Therefore, it is advantageous to stimulate cell division prior to treatment with plasmid DNA. Microprojectile gene transfer can also be used to transfer genes into cells either in vitro or in vivo. The basic procedure for microprojectile gene transfer was described by J. Wolff in Gene Therapeutics (1994), page 195. Similarly, microparticle injection techniques have been described previously, and methods are known to those of skill in the art. Signal peptides can be also attached to plasmid DNA to direct the DNA to the nucleus for more efficient expression.

[0372] Peptide or protein transfection is another method that can be used to genetically alter lymphatic endothelial progenitor cells of the disclosure and their progeny. Peptides such as Pep- 1 (commercially available as ChariotO), as well as other protein transduction domains, can quickly and efficiently transport biologically active proteins, peptides, antibodies, and nucleic acids directly into cells, with an efficiency of about 60% to about 95% (Morris, M.C. et al, (2001) Nat. Biotech. 19: 1173-1176).

[0373] Compositions

[0374] Compositions comprising at least one engineered B cell of the disclosure, or a polynucleotide encoding at least one polypeptide capable of binding an HER2 antigen, as described herein are provided. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.

[0375] Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are vehicles commonly used to formulate pharmaceutical compositions for animal or human administration The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include, without limitation, distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. A pharmaceutical composition or formulation of the present disclosure can further include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[0376] Further examples of formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249: 1527- 1533 (1990).

[0377] Methods for Treatment

[0378] In some embodiments, the methods herein include administering to a subject (including a human subject identified as in need of such treatment) an effective amount of a engineered B cells of the disclosure. The treatment methods are suitably administered to a subject, particularly a human, suffering from, susceptible to, or at risk of having an HER2+ cancer, such as a breast cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the subject is administered a single dose of the engineered B cells. In various embodiments, the engineered B cells are autologous to the subject to which they are administered.

[0379] Identifying a subject in need of such treatment can be based on the judgment of the subject or of a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). Briefly, the determination of those subjects who are in need of treatment or who are “at risk” or “susceptible” can be made by any objective or subjective determination by a diagnostic test (e.g., blood sample, biopsy, genetic test, enzyme, or protein marker assay), marker analysis, family history, and the like, including an opinion of the subject or a health care provider. A subject undergoing treatment can be a non-human mammal, such as a veterinary subject, or a human subject (also referred to as a “patient”).

[0380] In another embodiment, a method of monitoring the progress of treatment of an HER2+ cancer using engineered B cells of the disclosure. The method includes a diagnostic measurement (e.g., CT scan, screening assay or detection assay) in a subject having an HER2+ cancer and having been administered engineered B cells of the disclosure. The diagnostic measurement in the method can be compared to samples from healthy, normal controls; in a pre- disease sample of the subject; or in other afflicted / diseased patients to establish the treated subject’s disease status. For monitoring, a second diagnostic measurement may be obtained from the subject at a time point later than the determination of the first diagnostic measurement, and the two measurements can be compared to monitor the course of disease or the efficacy of the therapy / treatment. In certain embodiments, a pre-treatment measurement in the subject (e.g., in a sample or biopsy obtained from the subject or CT scan) is determined prior to beginning treatment as described; this measurement can then be compared to a measurement in the subject after the treatment commences and / or during the course of treatment to determine the efficacy of (monitor the efficacy of) the disease treatment.

[0381] The engineered B cells and / or chemotherapeutic agents of the disclosure may be administered to a subject by any of the routes normally used for introducing a cell or chemotherapeutic agent into a subject. Routes and methods of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intrathecal, parenteral, such as intravenous (IV) or subcutaneous (SC), vaginal, rectal, intranasal, inhalation, intraocular, intracranial, or oral. Parenteral administration, such as subcutaneous, intravenous, or intramuscular administration, is generally achieved by injection (immunization). Injectables can be prepared in conventional forms and formulations, either as liquid solutions or suspensions, solid forms (e.g., lyophilized forms) suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local.

[0382] The engineered B cells, chemotherapeutic agents, or compositions thereof, can be administered in any suitable manner, such as with pharmaceutically acceptable carriers, diluents, or excipients as described supra. Pharmaceutically acceptable carriers are determined in part by the agents being administered, as well as by the particular method used to administer the composition. Accordingly, a pharmaceutical composition comprising the engineered B cells or compositions thereof, can be prepared using a wide variety of suitable and physiologically and pharmaceutically acceptable formulations.

[0383] Administration of the engineered B cells of the disclosure, chemotherapeutic agents, or compositions thereof, can be accomplished by single or multiple doses. It may be advantageous to administer only a single dose of engineered B cells to a subject. The dose administered to a subject should be sufficient to induce a beneficial therapeutic response in a subject over time, such as to reduce tumor load in a subject by killing tumor cells in the subject. The dose required will vary from subject to subject depending on the species, age, weight, and general condition of the subject, by the severity of the cancer being treated, by the particular composition being used and by the mode of administration. An appropriate dose can be determined by a person skilled in the art, such as a clinician or medical practitioner, using only routine experimentation. One of skill in the art is capable of determining therapeutically effective amounts of engineered B cells and / or chemotherapeutic agents that provide a therapeutic effect or protection against diseases caused by HER2+ neoplastic cells.

[0384] In various embodiments, the engineered B cells administered to the subject are detectable in the subject after about or after at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 3 months, 4 months, 5 months 6 months, or a year post-administration. In some embodiments, the engineered B cells administered to the subject comprise about or at least about 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or more of the bone marrow B cells or splenic cells in the subject before or after about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 3 months, 4 months, 5 months 6 months, or a year post-administration.

[0385] In some cases, administration of the engineered B cells to the subject is associated with an increase in T cell proliferation in a solid tumor or the spleen of the subject relative to a reference subject. The administration of the engineered B cells may be associated with an increase in anti -turm or response in the subject relative to a reference subject. For example, the administration may result in a decrease in tumor volume or mass by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference.

[0386] Combination Therapies

[0387] Optionally, engineered B cells of the disclosure may be administered in combination with any other standard anti-pathogen or anti -neoplasia therapy; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin. In some embodiments, the B cells of the disclosure are administered in combination with a chemotherapy regimen. The chemotherapy regimen may be a low-intensity regimen and / or may involve administering one or more chemotherapeutic agents to a subject. Non-limiting examples of chemotherapeutic agents suitable for use in methods of the disclosure include cyclophosphamide and doxorubicin. Non-limiting examples of chemotherapeutic agents include lympho / myeloid depleting agents. In some cases, the chemotherapeutic agent contains an anti-PDl monoclonal antibody.

[0388] Kits

[0389] The disclosure also provides kits for use in preparation of engineered B cells of the disclosure and / or for use in treating a neoplasia. Kits of the instant disclosure may include one or more containers comprising an agent of the disclosure, such as an engineered B cell or a HDR cassette of the disclosure. In some embodiments, the kits further include instructions for use in accordance with the methods of this disclosure. In some embodiments, these instructions comprise a description of use of the agent to characterize a neoplasia and / or use of the agent for treatment of an HER2+ neoplasia or for preparation of an engineered B cell.

[0390] Instructions supplied in the kits of the instant disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions may be provided for practicing any of the methods described herein.

[0391] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

[0392] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for specific embodiments will be discussed in the sections that follow.

[0393] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention.

[0394] EXAMPLES Example 1: B cell receptor gene editing of primary murine B-cells for adoptive immunotherapy of breast cancer

[0395] To prepare B cells expressing B cell receptors targeting a HER2 antigen, a culture and editing regimen was first developed for murine B-cells (as depicted in FIG. 1A). Initially, CD43- negative resting B-cells were isolated from the spleen (sSP) of donor mice, and these cells were maintained in culture for two days prior to editing. The editing was conducted using the CRISPR-Cas9 method, with the donor template DNA delivered via the using a AAV-DJ vector. A crucial factor influencing the repair mechanism, favoring Homology-Directed Repair (HDR) over Non-Homologous End Joining (NHEJ), is the replicative state of the cells, where actively proliferating cells in the S2 phase exhibit greater efficiency in gene insertion compared to quiescent cells.

[0396] In a quest to enhance HDR and protein translation, different culture media were used to promote the proliferation of B-cells in culture. The first of these media was designed to mimic T- cell-dependent stimulation and incorporated IL4 (referred to as IL-4-M), while the second replicated a TLR-like stimulation, employing CpG / IL2 (referred to as CpG-M), as shown in FIG. 1A. It's worth noting that B-cells exhibited healthy growth in both types of media (illustrated in FIG. IB), but the resulting phenotypes were markedly distinct.

[0397] Upon analyzing the cultured B-cells at the end of the 6-day culture period, IL-4-M (media containing IL-4) induced a germinal center (GC)-like phenotype, marked by GL+ CD95+ cells, and a lack of plasma-cell differentiation (CD 138+ cells) (FIG. 1C). In contrast, CpG-M (media containing CpG) induced a shift toward a Marginal Zone (MZ) phenotype, accompanied by a higher percentage of plasma cells, notably expressing high levels of TACI, a characteristic marker of cells involved in T-cell independent responses, but without class switching (FIG. 1C).

[0398] Moreover, continuous monitoring of the cultured cells over the course of the culture period revealed that IL4-M-cultured cells retained a Follicular (FO) phenotype (as shown in FIG. ID) and transitioned into a GC-like state as early as day 2 of culture (illustrated in FIG. IE). Conversely, CpG-M induced a switch to MZ-cells as early as day 2. This phenotypic difference was also reflected in the surface expression of immunoglobulins (Ig); Ig G and IgE class-switching in cells cultured in IL4-M commenced as early as day 4 of culture (as depicted in FIG. IF). Additionally, an increased frequency of plasma cells was observed in cells cultured in CpG-M (illustrated in FIG. IG).

[0399] Notably, without intending to be bound by theory, IL4-M elevated the expression of activation markers on B-cells, including CD80, MHCII, CXCR4, ICOSL, and, at later time points, CD86 and PDL1, possibly rendering these cells more receptive to T-cell interactions (as depicted in FIG. 1H). To assess the antigen (Ag) presentation capabilities of the B cells, the unique characteristics of transgenic B6.Cg- Tg(TcraTcrb)425Cbn / J mice, referred to as OT-II, whose CD4+ T cells recognize chicken ovalbumin peptide residues 323-339 when presented by MHC class II molecules were used. B-cells, cultured in both media, were loaded with OVA protein and co-cultured for 3 days with CFSE-labeled OT- II CD4 cells. Following the co- culture, heightened proliferation was observed, specifically in cell cycle generations 5 and above, in the T cells co-cultured with B-cells activated in the CD40L-IL4 media (see FIG. II). Without intending to be bound by theory, this indicates improved antigen processing and antigen- presenting cell (APC) functions, suggesting that this particular phenotype may be the most suitable for anti- tumor applications.

[0400] A CRISPR-based editing approach was developed to introduce a Trastuzumab-derived Fragment-antigen binding (Fab) sequence into the endogenous IGH locus of primary murine B- cells. The Fab comprised the variable heavy-chain regions (VDJH) fused with a complete light- chain (murine CL+VJL), connected by a short linker featuring a 3x Strep-Tag motif for easy detection and isolation of edited cells. The linker also served to prevent mispairing between edited and endogenous antibody chains, thereby avoiding the formation of B-cell receptors with unpredictable and potentially autoreactive specificities.

[0401] To ensure a consistent editing approach across various antibody variants, a non- repeated intronic site situated between the final J-fragment and the initial switching region was targeted. This particular intronic region plays a pivotal role in regulating the class switching of the constant chain, housing the essential Ep enhancer. This enhancer becomes active following VDJ recombination rearrangement, a process that brings it into close proximity with each V- fragmenf s promoter. To leverage this natural process, a minimal IgH promoter (IgHV14-3) was produced at the start of the donor cassette. After target integration, this promoter became activated due to its proximity to the Ep enhancer, effectively disrupting the expression of the endogenous VDJ and mimicking the physiological expression of the B-cell receptor (FIG. 2A). A splice donor at the end of the donor cassette ensures splicing with the endogenous heavy chain. This approach enabled the simultaneous expression of the transgene in both membrane- bound and secreted forms, without being affected by VDJ or class-switching recombination events. The donor cassette was delivered using an AAV6 vector to resting primary murine B- cells, which were then cultured for 2 days in IL4 media and subjected to electroporation with CRISPR Cas9 / sgRNA ribonucleoparticles (RNP). The efficiency of editing was assessed 48 hours post-electroporation via FACS analysis using fluorescence-labeled soluble hHER2 and an anti-StrepTag antibody recognizing the StrepTagged linker of the HDR cassette. The results demonstrated a significant portion of cells were edited and capable of binding the soluble hHER2 protein (FIG. 2B). To further enhance editing efficiency, primary B-cells were also edited at different culture time points, and the highest percentages of edited cells were observed on day 2 and day 3, reaching values of 20% and 22%, respectively (FIG. 2C), while editing was minimal on day 0 due to the lack of stimulation and cell proliferation.

[0402] Notably, the use of the AAV-DJ vector (see, Lerch, et al. “Structure of AAV-DJ, a retargeted gene therapy vector: Cryo-Electron Microscopy at 4.5Å resolution,” Structure, 20:1310-1320 (2012)), yielded a twofold increase in editing efficiency when combined with the editing strategy, as measured by FACS and ddPCR (FIG. 2D), in comparison to the same multiplicity of infection (MOI) of AAV6. Remarkably, a 1.5-fold increase in efficiency was achieved even with a 10 times lower MOI.

[0403] When using the AAV-DJ vector alongside optimal editing timing, an approximate 90% rate of edited cells in samples examined was observed at four days post-editing (FIG. 2E). Edited cells exhibited a growth rate comparable to their unedited counterparts when cultured, with a reduced growth rate only in the days immediately following the editing procedure (FIG. 2F).

[0404] Additionally, an ELISA assay conducted on the supernatant of edited cells revealed the production of both IgM and IgG anti-hHER2 antibodies in the culture supernatant of edited samples (FIG. 2G). Throughout the culture, edited cells maintained a phenotype similar to their unedited counterparts (FIGs. 2H and 21).

[0405] In order to investigate the anti -tumor activity of HER2-specific edited B cells in a fully immunocompetent recipient, the MMTV-rtTA / tetO-HER2 mouse model (HER2) was used, an FVB background transgenic mouse. This mouse model employs an inducible Tet-on system that, upon doxycycline administration, triggers the overexpression of hHER2 exclusively in the mouse mammary glands, leading to the development of hHER2-driven tumor masses.

[0406] In the experimental protocol (FIG. 3A), 8-weeks old female HER2 mice received a doxycycline- containing diet to induce tumor growth. When the tumor mass was established (at about 16 weeks of age), mice were treated with edited and unedited B-cells. During the experiment, mice were kept under Doxycycline administration to sustain HER2 expression and tumor growth after the B cell administration. By following the tumor growth over time, no statistically significant difference was observed between mice treated with edited or unedited B- cells. Variability in tumor growth among the mice was large (FIG. 3B). Not intending to be bound by theory, this variability may be attributed to the tumor model itself, which induces hHER2 expression throughout the mammary fat pad tissue, leading to the formation of various tumor masses in various regions of the mouse's body (FIG. 3C). This inherent variability in tumor location and growth made it technically challenging to consistently monitor tumor progression over time. Despite the efforts to synchronize doxycycline administration, the variable tumor growth in each mouse posed difficulties in precisely coordinating the timing of B cell treatment and assessing its anti-tumor effects. Consequently, the decision was made to move to a more uniform setting, easier to standardize. Accordingly, an orthotopic model of murine breast cancer in C57BL6 mice was developed by taking advantage of the CD45.1 / CD45.2 mismatch system to easily detect donor B-cells.

[0407] To establish the orthotopic breast cancer cell model in C57BL6 mice, murine breast cancer cells EO771 engineered to express the human-HER2 antigen were used. Two different bidirectional lentiviral vectors (LVV) were cloned and produced to express the hHER2 antigen in combination with either GFP or OVA / Luciferase (OVA) (FIG. 4A). In both constructs, the hHER2 gene was under a minimal CMV promoter in antisense; GFP was under the control of a PGK promoter, as well as the OVA protein and luciferase protein linked by a P2A self-cleaving peptide to the GFP. After LVV transduction, EO771 cells underwent two cycles of selection by fluorescence-activated cell sorting (FACS) to isolate cells expressing the transgene. The sorted cells successfully expressed the transgenes.

[0408] These two bulk populations of cells (EO771-HER2-GFP and EO771-HER2-OVA-Luc) were expanded and transplanted into the mammary fat pad of recipient C57BL / 6J mice. Notably, the hHER2-GFP EO771 model exhibited a tumor growth rate similar to that of the WT EO771 cells, while the hHER2-0VA-LUC EO771 model showed delayed tumor growth (FIG. 4A).

[0409] Following an initial proof-of-efficacy assessment of the B-cell therapy in which both Edited and Unedited B-cells into hHER2-GFP EO771 were transplanted into tumor-bearing mice and a reduction in tumor burden was not observed (FIG. 4B), an effort was embarked upon to augment the anti-tumor potential of the B-cells by complementing their administration with low- intensity chemotherapy regimens. Not intending to be bound by theory, low-intensity chemotherapy regimen could potentially enhance B-cell engraftment in a fully immunocompetent host and elevate antigen availability. Cyclophosphamide (CPA) and doxorubicin (Doxo), both of which are recognized as lympho / myeloid-depleting agents frequently utilized as adjuvant or neoadjuvant treatments in breast cancer patients undergoing resection, were chosen for use in the chemotherapy regimens.

[0410] To determine the dose to use in combination with the B-cell treatment, experiments were conducted using hHER2-GFP and hHER2-0VA-Luc EO771 models. The results indicated that mice receiving either 3 or 4 doses of 5mg / kg of Doxo, the clinically effective dosages in mice, (FIG. 4C) or 2 doses of 116 mg / kg of CPA (FIG. 4D) exhibited complete tumor eradication. Similar outcomes were observed in mice subjected to a combination of both drugs (FIG. 4E).

[0411] The effects of CPA and Doxo, both independently and in combination, were also evaluated with or without the addition of the anti-PDl Monoclonal Antibody (MoAb). Among these variations, the combined treatment involving a single dose of 2.5 mg / kg of Doxo administered alongside a single injection of 116 mg / kg of CPA demonstrated an effective anti- tumor response, while also offering potential synergies for the B-cell therapy to further enhance treatment outcomes (FIG. 4F). This approach was selected for further testing.

[0412] Recipient CD45.2 C57BL6 mice received transplants of 0.5*10A6 hHER2-GFP or hHER- Ova-Luc EO771 cells into the mammary fat pad on day 0. Two weeks later, these mice were randomly assigned and sorted into experimental groups. On day 14 (and day 21 when reported), varying quantities of Edited or Unedited CD45.1 donor B-cells were transplanted into the recipient mice, with this transplantation taking place two days after the administration of chemotherapy (see FIG. 4G). Through longitudinal blood collection, a reduction was observed in the White Blood Cell (WBC) count in mice that had undergone chemotherapy treatment, a reduction that was subsequently reversed by the injection of B cells (FIG. 4H). Notably, chemotherapy primarily depleted the Lymphoid (LY) compartment, resulting in a temporary increase in neutrophil and monocyte counts (FIG. 41).

[0413] In order to capture the immunological changes happening at an early time point after the B-cell injection, mice were sacrificed four days after the B-cell injection and spleen (SP), tumor (TM), inguinal lymph nodes (ILN), and tumor-draining lymph nodes (TDLN) were collected and analyzed by FACS. CD45.1 donor B cells were retrieved in the TLN and ILN of mice that received edited B cells, suggesting their recruitment to lymphoid tissues after the initial injection (FIG. 5A upper panel). In those mice that received Edited B-cells in combination with chemotherapy, the frequency of donor cells within the CD19+ cells (i.e., B-cells) in both TDLN and ILN was even higher, suggesting a combinatory effect of the two treatments (FIG. 5A-lower panel right). Furthermore, an increase was seen in the GC+ B-cells in the TLN and ILN in mice receiving B cells + chemo (FIG. 5A-lower panel left), suggesting an active immune response, likely favored by the TAA release due to the chemotherapy administration.

[0414] To investigate the biodistribution and trafficking of donor B cells after injection and their possible recruitment at the tumor site, Luc-expressing B cells, both edited and unedited, were produced and transplanted into mice bearing EO771-HER2+GFP+ tumors. BLI scans were performed to follow the donor cells in vivo. Luc+ B-cells were isolated from the spleen of donor mice previously engrafted with Hematopoietic Stem and Progenitor Cells (HSPCs) transduced with Luciferase- expressing LVV. BLI signal as average radiance (p / s / cm2 / sr) was measured at 24h, 48h and 72h after B-cells injection to monitor the biodistribution of the donor B-cells, and scans at 144h (1 weeks) and 360h (2 weeks) were also performed to monitor the long-term engraftment and persistence of the cells.

[0415] No major differences were observed in the initial three days in mice receiving either Edited or Unedited B-cells alone (FIG. 5B). However, the signal escalated in mice that received edited cells combined with chemotherapy. Remarkably, by the conclusion of the two-week period following injection, the BLI signal regressed to background levels for the majority of treated mice. Interestingly, recruitment of B-cells to the tumor site was observed, which was enhanced by the chemotherapy regimen.

[0416] Low-intensity chemotherapy with CPA+Doxo was used in combination with one (FIG. 5C) or two injections of B cells (FIG. 5E), edited or unedited at day 14 and day 21 in those mice receiving a double injection. Untreated tumor-bearing mice were used as a control group.

[0417] By monitoring the tumor burden, it was observed that mice who received a single injection of Edited B-cell (up to 10*7 cells / mouse) outperformed compared to the other groups and showed a significant three-fold reduction in tumor volume compared to groups receiving chemotherapy alone or in combination with unedited B cells (FIG. 5C). By analyzing SP, TM and TDLN at the time of sacrifice, no peculiar immunological differences were observed among the groups apart from an increase frequency of GC within the resident B-cells in mice that received Edited B- cells in combination with chemo (FIG. 5D). Noteworthy, doubling the total transplanted B cells and performing a second injection resulted in a diminished antitumor effect of edited B cells, aligning their efficacy with the group treated solely with chemotherapy (FIG. 5E).

[0418] When tumors were harvested and digested at the sacrifice, uniform hHER2 transgene expression levels were found, mirroring the transgene expression in the initial EO771 bulk population (FIG. 5F).

[0419] Anti -tumor efficacy of B-cells was also tested in hHER2-OVA-Luc EO771 tumor - bearing mice. Low-intensity chemotherapy with CPA+Doxo was used in combination with two injections of B cells, edited or unedited at day 14 and day 21. Untreated tumor-bearing mice were used as a control group. All groups of mice treated with chemotherapy exhibited a reduction in tumor growth over time compared to the untreated groups (FIG. 6A), and the injection of unedited B-cells did not add any significant additional effect on tumor growth. When tumors were harvested and digested at the sacrifice, the frequency of hHER2 -positive cancer cells was low (ranging from 2% to 45%) compared to the bulk population at the time of injection (FIG. 6B), possibly explaining the observed low efficacy observed for the Edited B-cells. Notably, it was found that the percentage of hHER2-positive cells was not correlated with the treatments, but it was correlated with the tumor volume at the time of sacrifice, as shown in FIG. 6C. Larger tumors exhibited a higher percentage of hHER2 -positive cells, while smaller tumors showed lower expression of hHER2 -positive cells.

[0420] Upon examination at the time of sacrifice, a reduction in host T-cell infiltration in the TME of all the mice treated with chemotherapy was observed, along with a trend toward increased resident B- cell infiltration when donor B-cells were administered (FIG. 6D). This heightened B-cell infiltration was accompanied by an elevated frequency of germinal center (GC)-like resident B-cells in mice treated with Edited B-cells and chemotherapy, which might play a role in the formation of tertiary lymphoid structures (TLS) (FIG. 6E). Mice receiving Unedited B-cells and chemotherapy did not exhibit any TLS-like B-cells. Additionally, an increase in the expression of CD80 on infiltrating resident B-cells was confirmed in mice treated with chemotherapy compared to the control group.

[0421] Furthermore, an upsurge in the induction of GC responses in the TDLN of mice receiving Edited B-cells in conjunction with chemotherapy (FIG. 6F) was observed, along with a more pronounced overall activation of these B-cells (as evidenced by increased levels of CD80, CD86, and MHCII) when mice received chemotherapy treatment (FIG. 6G).

[0422] To maximize the possible anti-tumor effect of the B-cells observed in the EO771 hHER2- OVA- Luc model, and overcome the loss of antigen, single cloning was performed on the EO771 hHER2-0VA-Luc bulk population. After initial sorting and expansion, 3 different clones were transplanted into C57BL6 recipient mice in parallel with the bulk population. Two (Clone C3 and Clone Cl 8) of the three clones engrafted and grew comparably to the bulk population (FIG. 7 A) and also maintained hHER2 expression. Clone Cl 8 was selected for further testing.

[0423] One potential strategy to enhance the anti-tumor efficacy of Edited B-cells in the EO771 hHER2- OVA-Luc model, where two highly immunogenic antigens, hHER2 and OVA, are present, involves leveraging the capacity of B-cells to present multiple antigens through epitope spreading. This mechanism can subsequently trigger a T-cell mediated response, particularly involving OVA- specific T-cells. To explore this avenue, preliminary experiments were conducted in hHER2-0VA- Luc bearing mice by introducing CD4 OT-II cells and CD8 OT-I cells, which have T-cell receptors (TCRs) restricted to OVA (FIG. 7B). These experiments were conducted with or without the pre-inj ection of Edited B-cells.

[0424] In TDLN, increased proliferation and a distinct cell cycle distribution among the donor CD8 OT-I cells was observed, coupled with a higher frequency of Effector Memory CD8 T-cells (defined by CD44 high and CD62L low expression) and greater activation, as indicated by the percentage of CD69+ cells, in mice that received pre-inj ections of Edited B-cells (FIGs. 7C-7E). However, no significant proliferation or phenotypic variations were observed in the CD4 OT-II compartment. This absence of changes in the CD4 OT-II compartment may be attributed to a timing issue during the analysis.

[0425] Building upon the findings showing that hHER2-specific engineered B cells exhibited an antitumor effect in the EO771-hHER2-GFP model, it was decided to investigate whether this effect could be reproduced in other tumor models. To this end, an orthotopic subcutaneous (SQ) melanoma model was established by utilizing the B16F100, a cancer cell line derived from C57BL6 melanoma. B16 hHER2-GFP cell line was generated by following the same experimental procedure described for the EO771 in FIG. 4A. SQ injection of the cell line showed that it was able to engraft and grow melanoma masses into the recipient mice as the WT Bl 6, showing even a lightly higher faster growth kinetics (see FIG. 8A).

[0426] Due to the fast kinetic of the B16F100 models, donor edited or unedited B-cells were injected into recipient tumor-bearing mice already 4 days after tumor transplant without the use of any chemotherapy regimen (FIG. 8A). Conversely to what was seen on the EO771 model, by following the tumor burden over time, no anti -tumor effect of B-cells in B16 hHER2-GFP tumor-bearing mice (FIG. 8B) was observed, suggesting that the B-cell treatment was not sufficient to overcome the notably immune cold-phenotype of the melanoma. Notably, a very low tumor infiltration was noticed in all groups of mice independently of the treatment (FIG. 8C). When tumors were harvested and digested at the sacrifice, hHER2 transgene expression was found to be maintained (FIG. 8D).

[0427] In order to increase the immunogenicity of the melanoma model, B16F100 was engineered to express OVA-Luc antigens (Ags) as described in FIG. 4A for the EO771 model. B16 hHER2-0VA-Luc were able to engraft and grow as the B 16 WT (FIG. 9A).

[0428] Conversely to what was previously observed in the B16-hHER2-GFP, treating B 16 hHER2-0VA- Luc tumor-bearing mice with various amounts of B-cells (15-45*10A6 cells / mouse) significantly reduced the tumor burden independently of the B-cell antigen specificity (FIG. 9B). In contrast to what was previously noted in the B16-hHER2-GFP model, the proportion of host immune cells infiltrating the TME was higher, as depicted in FIG. 9C. At the time of sacrifice, an escalation in the frequency of germinal center (GC)-like resident B-cells in the SP, TDLN, and the TME of mice who received Edited B-cells was observed, as illustrated in FIG. 9D, 9E, 9F, and 9G, respectively. This increase in GC-like B-cells was followed by augmented B-cell infiltration into the TME. However, no significant disparities were observed in the T-cell phenotype within the SP, TDLN, or the tumor microenvironment, as indicated in FIG. 9H, 91, and 9J

[0429] When tumors were harvested and digested at the sacrifice, hHER2 transgene expression was found to have almost disappeared in those mice (FIG. 9K). The loss of transgene expression over time was also seen in the B 16 hHER2-0VA-Luc bulk population used for the transplant when these cells are kept in culture for two weeks. Conversely to what was shown in the EO771 model, this loss of expression was not correlated with either the treatment or the tumor volume.

[0430] To maximize the anti-tumor effect of the B-cells observed in the B 16 hHER2-0VA-Luc murine melanoma model, and overcome the loss of antigen, single cloning was performed on the B16 hHER2-0VA-Luc bulk population (FIG. 10A). After initial sorting and expansion, 3 different clones were transplanted into C57BL6 recipient mice in parallel with the bulk population. Two (Clone 8 and Clone 4) of the three clones engrafted and grew comparably to the bulk population (FIG. 10B) and also maintained hHER2 expression (FIG. IOC). Clone 8 was selected for further testing. By repeating the same experimental setting as depicted in FIG. 10A and following the tumor burden over time, a reduction of the tumor burden was observed in mice treated with B-cells independently from their antigen specificity (FIG. 10D), and despite no antigen loss being observed in vivo (FIG. 10E). An inverse correlation was observed between hHER2+ expression and tumor volume in B-cell treated mice, but not in untreated mice (FIG.10F). Notably, these results were achieved by transplanting a lower dose of B-cells (5*10A6 cells / mouse), which induced a very robust resident immune cells infiltration in the TME (FIG. 10G)

[0431] Relying on the fast growth kinetics of the B 16 model, the hypothesis that an early treatment could maximize the B-cell efficacy was tested. When B-cells transplanted intravenously (I.V.) at the same time as the EO771-hHER2-OVA-Luc, a significant reduction of the tumor masses was observed only in mice treated with Edited B-cells (FIG. 10H). In this experimental setting, unedited B cells were not efficacious in reducing the tumor burden.

[0432] Anti-HER2 antibody production was observed in vivo in C57BL6 mice with various tumor types. Mice with EO771 tumors, either expressing GFP or OVA-Luc, exhibited detectable levels of anti- HER2 IgM in their plasma when they received Edited B-cells in combination with a low-intensity chemotherapy regimen (FIG. 11A-B; left panels). In contrast, mice that received either Unedited B cells, and Control mice did not show any detectable levels of anti-HER2 IgM. This pattern was also observed in B16-HER2-Ova-Luc tumor-bearing mice (FIG. 11C; left panel). Conversely, the production of anti-HER2 IgG appeared to be independent of the B-cell transplant, with mice developing anti-HER2 IgG a few weeks after tumor transplantation, regardless of the treatment received. This phenomenon was more pronounced when the mice had Ova+ tumors (FIG. 11A-B-C; right panels).

[0433] To circumvent the immune response against human HER2, two novel EO771 cell lines were generated expressing a murinized version of hHER2. The same construct used for EO771 HER2 OVA-Luc was employed, but human HER2 was replaced with murine HER2. In one construct, the construct encoded the full region bound by Trastuzumab, while in the other, only the amino acids that make up the Trastuzumab epitope were encoded. These engineered EO771 cells maintained their binding affinity with Trastuzumab antibody but lost their binding capability with anti-hHER2 antibodies (FIG. 11D).

[0434] To elucidate the underlying rationale for employing edited B cells as anti-tumor agents, a lung metastatic model was established. To discern the optimal intervention timeframe, a series of timing tests were conducted (FIG. 12A). As evidenced by the orthotopic model growth, the melanoma tumor exhibited a notably accelerated growth rate in comparison to the breast cancer model. Consequently, the time points of 2 weeks post-injection and 3 weeks post-injection were selected as study endpoints for the B 16 hHER2-0VA-Luc and EO771-hHER2-OVA-Luc models, respectively.

[0435] Comprehensive flow cytometry analyses were executed on the dissociated metastatic tissues (FIG. 12B-C), focusing on classical markers for B cells (i.e., CD19+ and B220+), as well as distinguishing between inflammatory Ly6Chigh and anti-inflammatory Ly6Clow monocytes, granulocytes (Ly6G+), and CD4+ or CD8+ T cells. Considering the percentage of CD19+ / B220+ cells in the tissues, it was concluded that the lung metastasis model could be used to evaluate the efficacy of the editing of the B cells as anti-tumor agents.

[0436] What follows is a brief summary of advancements represented by the results presented in Example 1.

[0437] Optimization of primary murine B-cell culture and editing yielded efficiencies exceeding 90%, marking a significant milestone in the field.

[0438] Two distinct culture protocols were tested and refined to obtain GC-like or MZ-like B- cells in vitro by modulating cytokine compositions.

[0439] Human-HER2+ cell lines, in combination with Ovalbumin or GFP control, were developed as bulk populations or single clones. Orthotopic models of breast cancer and melanoma in C57Black6 mice were established. Engraftment of edited cells and their migration to the tumor site was successfully demonstrated in vivo.

[0440] In vivo production of anti-HER2 antibodies by edited B-cells was validated.

[0441] Significant tumor reduction was observed in mice treated with edited B-cells compared to those receiving unmodified counterparts.

[0442] Activation of the endogenous B-cell pool to form germinal centers and induction of T- cell responses against HER2 was demonstrated in vivo.

[0443] Example 2: B cell receptor gene editing of primary human B-cells for adoptive immunotherapy of breast cancer

[0444] In a manner akin to the murine model described in Example 1, a CRISPR / Cas9 strategy was developed to redirect the B cell specificity of primary human B-cells by inserting a Fab derived from Trastuzumab into the IGH locus. Initial attempts using previously published protocols yielded minimal cell growth. Therefore, a commercially available CD40L-based B-cell growth medium was used to maximize cell proliferation and, subsequently, improve homology- directed repair (HDR) efficiency. The experiments involved the use of primary CD43- resting human B-cells isolated from healthy donor-derived Peripheral Blood Mononuclear Cells (PBMCs). These B-cells were edited following four days of pre-stimulation using ribonucleoparticles (RNPs) and delivery of donor vectors through AAV6 (as shown in FIGs. 13A and 22A). The selected culture medium allowed the primary B-cells to proliferate in culture for up to 14 days (as depicted in FIG. 13B) and induced the expression of various activation markers, notably CD80 and CD86, which play a crucial role in B-T cell interaction (see FIG. 13C). These cultured B-cells retained their differentiation capacity over an extended culture period. Their differentiation was monitored over time using flow cytometry markers, such as CD27, CD38, and CD 138, observing transitions from transitional (CD38+CD27-), naive (CD27- CD38-), memory (CD27+ CD38-), plasmablasts (PB: CD27+CD38+CD138-), and plasmacells (PC: CD27+CD38+CD138+) (as shown in FIG. 13D-left and in FIG. 22B). As the cells underwent isotype switching and differentiated into surface Ig- plasmacells, IgM surface expression was progressively reduced while IgG expression and Ignegcells increased with time.

[0445] The donor vector and the targeted genomic region used for engineering the primary B- cells followed the same principle illustrated in FIG. 14A for the murine model. The donor Fab contained the variable heavy-chain regions (VDJH) fused with a complete light-chain (human CL+VJL), connected by a short linker featuring a 3x Strep-Tag motif for easy detection and isolation of edited cells. A signal peptide and a minimal promoter (IGHV1-69) were inserted at the 5 ’-end, and a splice donor site (SD) at the 3 ’-end (see FIG. 13E).

[0446] The selected sgRNA (IgH296) was initially tested on the K562 cell line, and its efficiency was compared to the well-established AAVS1 safe-harbor sgRNA. The percentage of non-homologous end joining (NHEJ) was estimated using the Interference of CRISPR Edits (ICE) bioinformatics tool by Synthego, yielding comparable estimates for both guides (FIG. 13F-left) Similar NHEJ efficiency was observed when testing the IgH296 sgRNA on different cell lines (FIG. 13F-right). Notably, when tested on primary human B-cells, the efficiency of NHEJ with IgH296 was even higher than the control sgRNA (FIG. 13G).

[0447] The IgH296 sgRNA was chosen for further HDR testing in combination with the AAV6 vector delivering the donor cassette. B-cells undergoing the editing process exhibited a slower proliferation rate compared to unedited cells (FIG. 13H). Through the optimized culture and editing procedure, efficient editing of primary human B-cells was achieved. This was confirmed by flow cytometry using soluble fluorescent-HER2 protein and an anti-Tag antibody, which showed up to 60% of edited cells (FIG. 131) and 30% of edited alleles measured by droplet digital PCR (ddPCR), suggesting monoallelic integration of the transgene (FIG. 131). Analysis of the cellular phenotype at the end of six days in culture revealed a slower differentiation in the edited samples, with an increased fraction of naive cells at day 6 of culture (FIG. 13J). Importantly, within the edited sample, there was no difference in terms of phenotype between cells that successfully underwent HDR (Tag+HER2+) and those that did not integrate the donor vector (Tag-HER2-) during the editing procedure (FIG. 13K-left). However, Tag-HER2- cells exhibited a higher frequency of Ig-negative cells compared to Tag+HER2+ or unedited cells, suggesting that NHEJ events in those cells undergoing the editing procedure without successful HDR might lead to BCR silencing (FIG. 13K- right).

[0448] To assess the effectiveness of different in vitro culture media in promoting robust B lymphocyte expansion, B-cell growth, B-cell phenotype, and editing efficiency were measured in different culture media. B-cells were cultured using six different CD40L-based stimulation media, including those based on commercial xeno-free and animal component-free supplements (referred to as “SC supplement”) and custom- made cytokine mixtures (referred to as “stimulation cit”). Four different media (IMDM, ExCellerate, ImmunocultXF-T, and ImmunocultXF-B) served as base media. Media composition significantly influenced the relative distribution of B cell subsets, particularly at later time points (FIG. 13L). The increased differentiation of cells cultured with custom-made cytokines was not aligned with the goal of transplanting less differentiated cells. These cultures also exhibited lower proliferation and significantly reduced editing efficiency compared to standard media. Notably, ImmunocultXF-B base media, despite promoting even higher B-cell proliferation than the standard ImmunocultXF- T base media, resulted in lower editing efficiency (FIGs. 13M and 13N).

[0449] As a result, the previously-tested ImmunocultXF-T + SC medium was deemed the best choice to proceed with.

[0450] To assess the functionality of the edited B-cell receptor (BCR) membrane, flow cytometric analysis was conducted to detect the intracellular phosphorylation of downstream signaling proteins, such as ERK, upon binding to the soluble HER2 ligand. The edited BCR's ability to transduce intracellular signals was confirmed by observing an increase in ERK phosphorylation in Tag+ cells compared to Tag- cells (as depicted in FIG. 14A).

[0451] Furthermore, ELISA assays carried out on the culture media confirmed the secretion of the soluble forms of IgM and IgG anti-HER2 antibodies (FIG. 14B), and this secretion increased over time, in line with the PC differentiation in culture.

[0452] Taking into consideration that B cells have the capacity to direct the killing of cells expressing their target antigen through various mechanisms (such as Granzyme B, TRAIL, and Fas / FasL), an in vitro killing assay was developed. In this assay, edited or unedited B cells were co-cultured with CFSE-stained HER2+ SK-OV-3 (ovarian cancer cell line) or HER2+ MDA- MB-453 (breast cancer cell line) cells at different target-to-effector ratios. A statistically significant increase in the percentages of live target cells (7-AADnegAnnexin Vllcg) was observed for tumor cells co-cultured with mock-electroporated or untreated B cells relative to percentages observed for co- culture with HER2-specific edited B cells (FIG. 14C).

[0453] Without wishing to be bound by theory, the presence of B cells alone in the co-culture induced partial death of the target cells, suggesting that BCR recognition and engagement played a partial role in target recognition and B-cell-mediated direct killing activity.

[0454] To investigate the potential anti -tumor effects of B cells on breast cancer progression, NOD-SCID IL2rgnull (NSG™) mice were orthotopically implanted with human HER2+ breast cancer cells from the MDA-MB-453 cell line and subsequently treated with either HER2-edited or unedited B cells. First, to track the in vivo biodistribution and potential tumor homing and infiltration, Luc+-edited or Luc-unedited B cells were transplanted into tumor-bearing mice.

[0455] Baboon-pseudotyped (BaERLess) lentiviral vectors (LVV) encoding Luciferase were used to efficiently transduce B cells, allowing for real-time monitoring via bioluminescence imaging (BLI) over time. BLI signals, quantified as average radiance (p / s / cm2 / sr), were assessed at 24 hours, 48 hours, and 72 hours post-B cell injection to observe the biodistribution of the donor B cells. Subsequent scans at 1 and 2 weeks were performed to monitor the long-term engraftment and persistence of the cells (FIG. 15A). Notably, there were no significant differences observed in mice receiving either Edited or Unedited B cells (FIG. 15B). Remarkably, despite an initial decline in the first four days after injection, the BLI signal remained above background levels for the majority of treated mice by the end of the two-week period. The recruitment of B cells to the tumor site was observed in all experimental groups.

[0456] As illustrated in FIG. 15C, to assess the anti -tumor efficacy of B cells, both edited and unedited B cells were transplanted 14 days after tumor establishment. Given that NSG™ mice have limited support for B cell engraftment, and the absence of T cells can influence the anti- tumor effectiveness of the approach, peripheral blood mononuclear cells (PBMC) were co- transplanted with syngeneic edited or unedited B cells (FIG. 15C). Additionally, a group of mice received injections of edited B cells only.

[0457] The phenotypic composition of the infused B cells was found to be similar between the unedited and edited samples. The editing efficiency of the infused B cells was approximately 50% as determined by fluorescence-activated cell sorting (FACS), corresponding to approximately 25% of HDR-edited alleles as confirmed by ddPCR ( FIG. 15D).

[0458] This editing efficiency was consistent across the main subpopulations (i.e., naive and memory cells) at the time of transplantation (FIG. 15E). The syngeneic PBMC population was primarily composed of T cells (FIGs. 15F and 15G).

[0459] Through longitudinal flow cytometric blood analyses, a robust expansion of human cells was observed starting from day 12 post-injection (FIG. 15H). Importantly, these cells were predominantly T cells (FIG. 151), and this phenomenon was observed specifically in mice co- transplanted with B cells and PBMC, leading to the in vivo production of anti-HER2 IgM and IgG antibodies in mice receiving Edited B cells along with PBMC (FIG. 15J). Notably, the production of antibodies was absent in mice that received only Edited B cells without PBMC, and the IgM antibody titer showed a direct correlation with human engraftment, which was also absent in mice not receiving supporting PBMC (FIG. 15K).

[0460] Through continuous monitoring of tumor volume over time, a significant reduction in tumor burden in mice treated with Edited B cells at early time points (FIG. 16A) was observed. Extended follow- up demonstrated sustained anti-tumor efficacy primarily in mice receiving Edited B cells along with PBMC (FIG. 16A). This was further confirmed by measuring tumor weight at the time of sacrifice (FIG. 16B), emphasizing the critical role of T-cell-mediated anti- tumor cellular responses in controlling tumor growth.

[0461] At the time of sacrifice, human engraftment in the spleen, bone marrow, and tumor tissues was assessed. Analyzing the composition of the tumor mass, a notably higher proportion of mice transplanted with Edited B cells + PBMC showing human cell infiltrates in the tumor microenvironment (TME) (FIG. 16C) was observed. These infiltrates were primarily composed of CD4 T-cells (FIG. 16D). A trend was also observed toward a higher frequency of human CD45+ cells in both the spleen and bone marrow of mice receiving Edited B cells + PBMC compared to those receiving Unedited B cells + PBMC (FIG. 16E and 16G). Importantly, no human engraftment was detected in mice that received only Edited B cells. No differences were found in the composition of human infiltrates in the spleen or bone marrow (FIG. 16F and 16H)

[0462] The contribution of human T cells activated by B cells in the anti -tumoral response was assessed through an enzyme-linked immunospot (ELISPOT) assay. This assay allowed for the detection and enumeration of individual cells secreting a specific protein, which, in this case, was interferon-gamma (IFN- y), which was released by T cells upon antigen-specific immunity development. Human T cells from the spleens of transplanted mice were expanded ex vivo for 12 days with a-CD3 / CD28 beads and subsequently co-cultured overnight with MDA MB-453 cells. Human T cells isolated from the spleen of mice treated with Edited B cells + PBMC exhibited enhanced growth in culture (FIG. 161) and a higher proportion of CD8 Effector Memory (TEM) cells at the end of the culture (FIG. 16J). Analysis of the IFN-y positive areas demonstrated a modest increase in specific T cell activation in mice treated with engineered B cells compared to those receiving untreated B cells (FIG. 16K). Without intending to be bound by theory, these findings suggested that human T cells were activated and proliferated primarily in the presence of HER2-edited B cells, and this expansion enhanced the fraction of tumor-specific T cells.

[0463] To discern the specific roles of cellular-mediated and humoral immune responses in the tumor model, NSG™ mice bearing tumors were treated with Trastuzumab Monoclonal Antibody (MoAb). In the initial experiment, the clinically effective dose of the MoAb was administered (FIG. 17A), which resulted in complete tumor eradication in mice that received either Trastuzumab alone or Trastuzumab in combination with PBMC (FIG. 17B). Notably, all mice that received the MoAb coupled with PBMC injections developed secondary tumors five months after the injection of the primary tumor (FIG. 17B).

[0464] Throughout the experiment, blood samples were longitudinally collected from the mice and an ELISA assay was conducted on the plasma to assess the levels of the MoAb in the bloodstream.

[0465] The concentration of anti-HER2 IgG found in the treated mice's blood was substantial, reaching peaks of almost 20 pg / ml (FIG. 17C) 12 days after the first injection. Since these values greatly exceeded the actual blood levels of anti-HER2 IgG found in the mice treated with Edited B-cells + PBMC (refer to FIG. 15K), the decision was made to replicate the latter scenario by administering a lower dose of Trastuzumab (FIG. 17D). Using a reduced Trastuzumab dose, comparable to the amount produced in vivo by the transplanted Edited B- cells, no effect on tumor growth was observed (FIG. 17E). Not intending to be bound by theory, this result suggested that the anti-tumor effect seen in mice treated with Edited B-cells + PBMC was primarily due to cellular-mediated immune responses and T-cell stimulation by B-cells, rather than the production of soluble antibodies against HER2.

[0466] To further corroborate this discovery, a membrane-bound form of Trastuzumab (FIG. 17F) was cloned, allowing one to distinguish the effects of T-cell-mediated anti-tumor immunity from the humoral functions of Trastuzumab. Specifically, a Trastuzumab Fab was cloned containing the variable heavy-chain regions (VDJH) fused with a complete light-chain (human CL+VJL) connected by a short linker featuring a 3x Strep-Tag motif. This construct was placed downstream of a signal peptide and a minimal promoter. In frame with the Fab, a human IgM constant region was incorporated, including the transmembrane domain, followed by a polyA sequence. This donor cassette was initially inserted between the IGH296 sgRNA homology arm and tested on primary human B-cells. Unfortunately, the editing efficiency was lower than expected (FIG. 17G), possibly due to rearrangement phenomena occurring in the BCR locus, potentially favored by homology between the endogenous IgM constant chain gene and the donor cassette.

[0467] B cells edited in the AAVS1 control locus were used to establish that the anti-tumor effect exerted by BCR-edited B cells was not an artifact resulting from the cellular modifications induced by the editing procedure but rather a consequence of BCR specificity. These control B cells underwent the exact same procedure as the BCR-edited B cells. Mice bearing MDA-MBA- 453 tumors were treated with AAVSl-edited B cells along with PBMC, following the experimental protocol outlined above (refer to FIG. 15C). Through continuous monitoring of tumor growth, no reduction was observed in tumor burden over time in mice receiving AAVS1- Edited B-cells (FIG. 18A). This outcome was further confirmed by evaluating tumor weight at the time of sacrifice (FIG. 18B).

[0468] These results conclusively demonstrated that the anti-tumor efficacy observed in the NSG™ model is indeed driven by the specificity of the BCR.

[0469] To enhance the B-cell response in the NSG™ model, the potential benefits of using a low-chemotherapy regimen in the xenotransplantation model was explored. Since NSG™ mice do not require lymphodepletion, the decision was made to avoid Cyclophosphamide (CPA) and to instead use Doxorubicin alone. The goal was to increase inflammation and improve antigen release and presentation.

[0470] A single low-dose injection of Doxorubicin was administered to the NSG™ mice. It's important to note that this drug can be highly toxic in this model due to impaired DNA damage repair mechanisms. Three different doses of Doxorubicin (1 mg / kg, 1.5 mg / kg, and 2 mg / kg) were evaluated as a single injection on day 14 after tumor implantation in HER2+ MDA-MB- 453 tumor-bearing mice (as shown in FIG. 19A).

[0471] The results showed that all doses of Doxorubicin were well- tolerated by the mice and partially reduced tumor burden (FIG. 19A and 19B), although with a subtherapeutic result compared to other models. The next step involved combining the Doxorubicin treatment with B- cell injection to maximize the impact of the treatment approach.

[0472] To replicate a metastatic model that mimics natural cancer metastasis and allows for examination of the impact of B-cells on HER2+ metastatic cells, tumor cells were administered intravenously (TV.) to recipient NSG™ mice. Specifically, the HER2+ human epithelial ovarian cancer cell line, SK-OV3, known for its higher metastatic potential compared to HER2+ DA- MB-453 and greater susceptibility to Trastuzumab treatment was utilized.

[0473] In the initial experiment, 1 million SK-OV3 cells, which had been transduced with a bidirectional LVV expressing the Luc and NGFR tracking genes, were injected intravenously into recipient NSG™ mice. One week later, Edited or Unedited B-cells were introduced in conjunction with syngeneic PBMCs into the tumor-bearing mice (FIG. 20A). Subsequently, the metastatic tumors were monitored through weekly BLI scans (FIG. 20B). It was observed that, instead of growing over time, the tumor burden was decreasing in both treated groups (FIG. 20C). This trend was consistent with a high human CD45 engraftment in the blood of the treated mice (FIG. 20D), potentially indicating that PBMCs were mediating tumor rejection.

[0474] In an attempt to address this issue, the experimental approach was modified. First, mice were transplanted with PBMCs, and one week later they received intravenous injections of tumor cells. The following week, either Edited or Unedited PBMCs, or in some cases, no B- cells were administered (FIG. 20E). Subsequent monitoring through weekly BLI scans (FIG. 20F) revealed an increase in tumor burden over time, with a slower rate of increase in mice receiving Edited B-cells (FIG. 20G). It's important to note that in this setup, the engraftment of human cells was significantly lower (FIG. 20H) and was only detectable in mice receiving Edited B- cells.

[0475] The initial experimental approach aimed to utilize humanized NSG™ mice pre-engrafted with CD34+ HSPCs isolated from G-CSF mobilized apheresis and subsequently treated with syngeneic B cells from the same apheresis to prevent alloreactive responses. The plan involved isolating the CD34+ cell fraction for immediate transplantation into sub-lethally irradiated NSG™ female mice, while CD34- PBMCs, initially frozen, would be thawed, B cells isolated, edited, and transplanted after hematopoietic reconstitution (8 weeks). However, this plan proved to be quite challenging due to the high mortality of frozen PBMCs, which made achieving a satisfactory editing efficiency impossible.

[0476] As a result, a different strategy was chosen. In this new strategy, CD34+ cells were edited in either the BCR locus or the AAVS1 control locus and then engrafted into lethally irradiated NSG™ mice. Cord blood-derived CD34+ cells were chosen, which are known for their superior engraftment capability in NSG™ mice. After the mice displayed adequate peripheral reconstitution, they were orthotopically transplanted with HER2+ MDA-MB-453 cells (FIG. 21A). Through longitudinal blood collection and FACS analysis, it was observed that the engraftment of human cells, monitored as the percentage of human-CD45+ cells in the blood, was lower than expected in both groups but increased after tumor implantation (FIG. 21B). Notably, mice engrafted with AAVSl-edited or BCR-edited CD34+ cells exhibited distinct immunological reconstitution patterns (FIG. 21C and FIG. 21D). B-cell reconstitution began to decline approximately 6 weeks after tumor implantation in favor of T-cell expansion in mice receiving BCR-edited CD34+ cells (FIG. 21C). Conversely, the majority of engrafted cells remained as B-cells until the end of the experiment in mice receiving AAVS1- edited CD34+ cells (FIG. 21D). Gene marking was comparable in both groups of mice within the B- cell compartment in the blood (FIG. 21E) and aligned with the in vitro editing efficiency of CD34+ cells (VCN 0.05 = 5% editing efficiency).

[0477] Interestingly, while monitoring tumor burden over time, a slight reduction in tumor volume was observed in mice engrafted with BCR-edited CD34+ cells (FIG. 21F). However, this effect was significantly less pronounced compared to when MDA-MB-453-bearing mice were treated with mature edited B-cells. One of the most noteworthy findings was the presence of edited B-cells in the organs of mice engrafted with BCR-edited CD34+ cells (FIG. 21G).

[0478] What follows is a brief summary of advancements represented by the results presented in Example 2.

[0479] A significant advancement in the optimization of primary human B-cell culture and BCR editing, achieving efficiencies of up to 60%, was achieved. The method allows for the culture of B cells for up to 14 days while preserving their differentiation capacity. These edited B cells expressed the edited BCR on their surface and demonstrated the production of soluble anti- HER2 IgM and IgG in culture, marking the highest reported efficiency in the field to date.

[0480] In vitro experiments have showcased the ability of edited B -cells to effectively target and eliminate HER2+ cancer cells.

[0481] Utilizing a xenotransplantation model in NSG™ mice, HER2+ MDA-MB-453 cancer cell lines were implanted into the mammary fat pad. Subsequent treatment with edited or unedited cells in combination with syngeneic PBMC facilitated enhanced B-cell engraftment. These findings demonstrated successful engraftment of edited cells in NSG™ recipients, showcasing migration to the tumor site.

[0482] In vivo studies further revealed the production of anti-HER2 IgM and IgG antibodies by edited B-cells. Significant tumor reduction was observed in mice treated with edited B-cells compared to those receiving unmodified counterparts.

[0483] Importantly, alongside tumor reduction, induction of in vivo T-cell responses against HER2 was observed, indicating a substantial contribution of T-cell immunity to tumor suppression, rather than solely relying on antibody production against HER2. This is the first report of the use of Edited human B-cells as efficacious anti -tumor agents.

[0484] Example 3: Anti-tumor effect of engineered B cells was mediated by antigen-specific T cell activation

[0485] Experiments were undertaken to demonstrate that human T cells were activated / primed in vivo in the presence of HER2-specific B cells, and that this expansion enhanced the fraction of tumor-specific T cells. The tumor reduction measured in a mouse xenotransplantation model did not correlate with the serologic levels of anti-HER2 IgG or IgM (FIG. 24A). Conversely, the tumor size at the endpoint inversely correlated with human cell expansion (FIG. 24B) in spleen and tumor, mostly composed of CD3 T cells (FIG. 24C), particularly CD8 T cells (FIG. 24D). Importantly, both these T-cells / tumor correlations were not significant in the mice injected with unedited B cells and PBMCs (FIG. 24E), suggesting a qualitative difference in the T cells that expand and infiltrate the tumors in the two groups of mice.

[0486] Overall, these data demonstrated that tumor-specific human B cells could induce a T-cell- dependent anti-tumor response and trigger enhanced priming and / or activation of tumor-specific T cells. Example 4: CD4 T cells were central orchestrators of B and CD8 T cell responses in T Cell- dependent tumor control

[0487] Having established the importance of B cell specificity in triggering an anti -tumor response and inducing a T cell-specific activation, experiments were undertaken to determine which T cell subset was primarily responsible for the observed anti-tumor effect in vivo, and to what extent. Mouse orthotopic models of HER2+ breast cancer were transplanted with B cell receptor (BCR) edited or unmodified B cells along with sorted CD4 and CD8 T cells, either separately or combined, to reproduce a physiological CD4:CD8 ratio (approximately 2: 1) observed in PBMCs used in prior experiments (FIG. 25A). The BCR-edited cells expressed a B cell receptor modified to target an HER2 antigen.

[0488] When the BCR-edited B cells were transplanted with CD8 T cells alone, no evidence of human cell engraftment or infiltration was found in the mice, either during weekly blood sampling or at endpoint organ analysis (FIG. 25B). In line with the lack of long-term engraftment of both B and T cells, no tumor reduction was observed in mice treated with BCR- edited B cells and CD8 T cells compared to controls (FIG. 25C). In contrast, when tumor- bearing mice were injected with CD4 T cells, high human CD45 (hCD45) engraftment was detected in both blood and organs at the endpoint (FIG. 25D). By comparing the mean frequency of hCD45 in blood, spleen, and tumors, a 2 to 3-fold increase in hCD45 engraftment was observed compared to mice treated with unedited or BCR edited B cells in combination with peripheral blood mononuclear cells (PBMCs’), regardless of B cell specificity. A significant tumor reduction was observed in mice treated with BCR-edited B cells and CD4 T cells, compared to those treated with unmodified B cells and CD4 (FIG. 25E). When CD4 and CD8 T cells were combined in a 2: 1 ratio, maintaining the same total number of transplanted T cells, thus reducing the total number of CD4 T cells in the pool, a 1.5 to 2-fold increase in hCD45 engraftment in the blood and organs, compared to mice treated with unedited or BCR edited B cells in combination with peripheral blood mononuclear cells (PBMCs’) (FIG. 25F). This suggested that CD4 T cells played a critical role in driving overall engraftment and supporting CD8 T cell engraftment, which did not occur without CD4 help. This combination also reduced tumor growth in mice treated with BCR-edited B cells and CD4 / CD8 T cells compared to those receiving unmodified B cells (FIG. 25E). While both CD4 alone and the CD4 / CD8 combination led to tumor reduction, highlighting the T cell-dependent mechanism of action of cancer-specific B cells, the underlying mechanisms appear distinct. In general, transplanting purified T cells alongside with engineered B cells, thus removing engraftment competition from the B cells present in the PBMC fraction, resulted in higher engraftment of the in vitro activated B cells. Flow cytometry revealed the presence of Tag+ (BCR-edited) B cells five weeks post-injection (FIG. 25G), with around 10-20% of total splenic B cells and 5% of bone marrow B cells being Tag+ (FIG. 25H). Notably, the frequency of Tag+ B cells, even without competition from PBMC-derived B cells, was lower than the in vitro editing efficiency of about 50%, likely due to the selective advantage of unedited B cells, or the in vivo differentiation of B cells into plasmablasts and plasma cells after transplantation, where BCR expression is no longer present on the cell surface. This latter limitation could be addressed by engineering B cells to express the edited BCR along with a fluorescent or selection marker that is expressed independently of differentiation. Nonetheless, increased B cell engraftment was observed when the proportion of CD4 T cells was higher, suggesting a supportive role of CD4 T cells in B cell survival and engraftment (FIG. 25H).

[0489] Consistent with the higher engraftment of ex vivo modified B cells, increased production of specific anti-HER2 IgM and IgG antibodies was observed in mice transplanted with either CD4 alone or the CD4 / CD8 combination (FIGs. 251 and 25 J). However, despite a 10-fold increase in anti-HER2 IgM and IgG titers, no additional anti-tumor activity was observed compared to mice treated with BCR-edited B cells plus PBMCs, underscoring again that antibody production was not the main mechanism of action in this setting (FIGs. 25K and 25L).

[0490] Interestingly, CD8 T cells generated a stronger anti-tumor microenvironment (TM) IFN-y response despite not being the limiting factor in anti-tumor efficacy and relying on CD4 T helper cells. Although mice were transplanted with a 2: 1 CD4:CD8 ratio, the final engraftment in tumors, spleens, and bone marrow skewed toward CD4 T cells, reaching a 3-4: 1 ratio (FIG. 25M). Ex vivo testing of T cells from transplanted mice against parental HER2+MDA-MB-453 cells revealed a higher frequency of IFN-y enzyme linked immunosorbent spot (ELISPOT) responders when CD8 T cells were paired with CD4 T cells in the mice (FIG. 25N). Additionally, the frequency of IFN-y-positive ELLISPOT responders in mice administered both CD8 T cells and CD4 T cells was three times larger than that observed in mice administered only CD8 T cells (FIG. 250).

[0491] In conclusion, the above experiments demonstrated that CD4 T cells played a central role in driving the engraftment of both B cells and CD8 T cells, and in enhancing the anti -tumor effect of BCR-edited B cells. The interaction between CD4 T cells and BCR-edited B cells appeared critical for both B cell survival and function. CD4 T cells not only promoted B cell engraftment but also supported the engagement of CD8 T cells, which, in the absence of CD4 T cells, failed to engraft effectively. While antibody production (IgM / IgG) was elevated in mice administered BCR-edited B cells in combination with CD4, it did not appear to be a primary mechanism behind tumor reduction. Instead, the anti-tumor response was likely driven by T cell activation, in which CD4 T cells acted as key orchestrators of both B cell and CD8 T cell responses, ultimately leading to enhanced tumor control.

[0492] The following materials and methods were employed in Example 1.

[0493] Mice

[0494] Six to eight weeks old female C57BL / 6J, FVB, B6.Cg-Tg(TcraTcrb)425Cbn / J (OT-II), B6.SJL-Ptprca Pepcb / BoyJ (CD45.1) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). MMTV-rtTA / tetO-HER2 mice were tumors were induced by Doxycycline administration as Nutritionally complete grain-based rodent diet (200 mg / kg) when female mice reach 8 weeks of age.

[0495] Cell lines

[0496] B16F10 mouse melanoma cells and EO771 murine breast cancer cells were obtained from the American Type Culture Collection (ATCC; CRL-6475 and CRL-3461 respectively). B16F10 cells were kept in culture in RPMI-1640 (Corning, Coming, NY, USA) supplemented with 10% fetal bovine serum (FBS, GeminiBio, West Sacramento, CA, USA), 1% Penicillin / Streptomycin (P / S, Thermo Fisher Scientific Inc., Waltham, MA, USA), lx NEAA (Thermo Fisher Scientific Inc., Waltham, MA, USA), 5 mM 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1 mM sodium pyruvate (Corning, Corning, NY, USA). EO771 were kept in culture in Dulbecco’s Modified Eagle’s Medium (DMEM; Coming, Coming, NY, USA) supplemented with 10% FBS, 1% P / S and 20 mM HEPES.

[0497] Engineered cell lines were transduced with a bidirectional lentiviral vector (LVV) expressing HER2-GFP or HER2-OVA-LUC (see below for details sequences). Single clones of the hHER2- OVA-Luc B16 and hHER2-OVA-Luc EO771 were produced by limiting dilution of the HER2+ sorted bulk population.

[0498] All cells were cultured in a humidified, 5% CO2 incubator at 37°C.

[0499] Flow cytometry

[0500] Flow cytometry analyses were performed on LSRFortessa (BD Bioscience, Franklin Lakes, NJ, USA) instrument. All Antibodies were purchased from Biolegend (San Diego, CA, USA) or BD Biosciences (Franklin Lakes, NJ, USA), unless stated otherwise. Staining was performed in 50 pL of FACS buffer consisting PBS with 1% Bovine Serum Albumin (BSA, Sigma- Aldrich Inc., St. Louis, MO, USA) and 0.1% sodium azide (Sigma-Aldrich Inc., St. Louis, MO, USA).

[0501] For surface stainings of the B-cells, cells were labeled with a cocktail including combinations of the following antibodies: CD138 BV650 (281-2, Rat, BioLegend), TACI BV421 (8F10, Rat, Biolegend), CD19 BV785 (6d5, Rat, BioLegend), CDld PE (53-7.3, Rat, BioLegend), CD5 PE- Cy5 (53-7.3, Rat Biolegend), CD21 AF700 (7E9, Rat, BioLegend), CD23 BV605 (B3B4, Rat, BioLegend), CD274 (PD-L1) BV650 (10F.9G2, Rat, BioLegend), CD73 AF700 (TY / 11.8, Hamster, BD), CD80 BV421 (16-10A1, Rat, BD), CD86 AF700 (GL-1, Hamster, BD), CD95 (Fas) PE (Jo2, Rat, BioLegend), CXCR5 PE / Dazzle 594 (L138D7, Rat, BioLegend), GL7 PE-Cy7 (GL7, Rat, BioLegend), ICOSL PE (HK5.3, Goat, BioLegend), IgD BV605 (1 l-26c.2A, Rat, BioLegend), IgE PE (RME-1, Rat, BD), IgGl FITC (A85-1, Rat, BD), IgM BV510 (RMM1, Rat, BioLegend), MHC-

[0502] II (IA / IE) BV510 (M5 / 114, Rat, BioLegend), TIM1 PE (RMT1-4, BioLegend), PD1 BV786 (29F.1A12, Rat, BioLegend), Strep-Tactin PE (6-5000-001, IBA), Soluble HER2 FITC (Aero Biosystems). T cell phenotype was assessed by means of CD4, CD8, CD44, CD62L, CD69. T- cell proliferation was assessed by means of Carboxyfluoresceinsuccinimidylester (CFSE) (Thermo Fisher Scientific Inc., Waltham, MA, USA) labelling following manufacturer’s instructions.

[0503] Isolation of primary murine B-cells

[0504] Spleen was collected from B6.SJL-PtprcaPepcb / BoyJ (B6 CD45.1) mice (Jackson Laboratory; Bar Harbor, ME, USA). Splenocyte single cell suspension was obtained after mashing and filtering the spleen with a 40 pm strainer (Corning, Coming, NY, USA). Single cells were washed with cold MACS buffer (Lonza, Basel, Switzerland) and red blood cells were lysed by incubating with ammonium-chloride-potassium (ACK, StemCell Technologies, Vancouver, Canada) for 2 minutes at Room Temperature (RT). CD43- Resting B cell lymphocytes were isolated by negative selection using the Mouse B Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) containing a cocktail of biotin-conjugated monoclonal antibodies against CD43, CD4 and Ter-119, according to the manufacturer’s instructions.

[0505] Culture of primary murine B-cells

[0506] Isolated B cells were cultured in two different CD40L-based stimulation media; B-cells were kept in culture in ExCellerate B cell media (R&D Systems, Minneapolis, MN, USA) supplemented with 10 mM HEPES, 100 ng / mL recombinant mouse CD40 Ligand / TNFSF5 (HA- tag) Protein (CD40L, R&D Systems, Minneapolis, MN, USA), 100 ng / mL HA-Tag antibody (R&D Systems, Minneapolis, MN, USA), 55 pM β-Mercaptoethanol (Thermo Fisher Scientific Inc., Waltham, MA, USA). Two different cytokines, either 7 ng / mL recombinant mouse IL-4 Protein (R&D Systems, Minneapolis, MN, USA) (CD40L-IL-4) or 50 ng / mL recombinant mouse IL-2 Protein (StemCell Technologies, Vancouver, Canada) in combination with 1 pg / mL CpG (ODN1826; Miltenyi Biotec, Bergisch Gladbach, Germany) (CD40-CpG / IL-2) were used to stimulate B-cell growth and differentiation.

[0507] B-cells were cultured at a concentration of 2.5x105 / mL, counted and expanded every second day, unless stated otherwise, until the day of the analysis.

[0508] CRISPR / Cas9 editing

[0509] B cells were electroporated after two days of pre- stimulation, unless stated otherwise, by using a 4D-Nucleofector device (Lonza, Basel, Switzerland). Chemically synthetized single- guide RNAs (sgRNAs) (muIgH367 sgRNA: UUAUACAGUAUCCGAUGCAU targeting a sequence next to an AGG PAM; Synthego, Redwood City, CA, USA) were complexed in a 2: 1 molar ratio with 50 pmolCas9 (Alt-R™ S.p. Cas9 Nuclease V3, IDT, Newark, NJ, USA) for 15 minutes at room temperature (RT). The resulting ribonucleoproteins (RNPs) were combined with murine B cells in P4 Primary Cell Nucleofector Solution (Lonza, Basel, Switzerland). The electroporation was performed in 20 pL Nucleocuvette Strips or 100 pL Nucleocuvette Vessels on the Lonza electroporation device according to the manufacturer’s instructions (program DI 100). Pre- warmed cell culture medium was added to the cells immediately after electroporation and donor vector was delivered after 20 minutes by means of an AAV2 / DJ vector (Boston children’s Hospital Viral Core Facility, Boston, MA) at a multiplicity of infection (MOI) of 2.17xl05. Cells were transduced overnight at 37°C at a concentration of 1.5xl06 / ml, and later expanded at 2.5xlO5 / mL for a total of 6 days in culture. Untreated (UT) cells were used as control.

[0510] Editing efficiency was assessed with the following antibodies: CD19, Streptavidin-Tag, human HER-2.

[0511] Plasmid cloning

[0512] Plasmid cloning was performed by using standard molecular biology techniques. Briefly, plasmid was digested using different restriction enzymes (New England BioLabs, Ipswich, MA, USA), fragments were separated by agarose gel electrophoresis and the correct ones inserted into a dephosphorylated linearized AAV6 backbone with Quick Ligase (New England BioLabs, Ipswich, MA, USA) after purification with Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) according to manufacturer’s instructions. After ligation, TOPIO chemically competent

[0513] E. coli bacteria (Thermo Fisher Scientific Inc., Waltham, MA, USA) were transformed according to the manufacturer’s instructions and plated on LB agar plates containing Carbenicillin for selection. Plasmid DNA was extracted from single bacterial colonies and purified with Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA) and EndoFree Plasmid Maxi Kit (QIAGEN, Hilden, Germany). Colonies were screened with control digestions and sequenced.

[0514] HDR donor vector

[0515] The HDR cassette consisted of the J5558H10 heavy chain promoter region, followed by a Trastuzumab-derived Fragment-antigen binding (Fab) sequence and a splice donor site. The Fab is a fusion of the codon-optimized variable heavy-chain regions (VDJH) and a complete light- chain (murine CKappaL+VJL) connected with a 57 bp linker that includes a 3x Strep-Tag II motif. The splice junction is followed by 60 base pairs derived from the mouse IGHJ3 gene segment. The entire cassette is cloned in between a 503-base pair upstream homology arm and a 968-base pair downstream homology arm flanking the sgRNA TTATACAGTATCCGATGCATAGG cutting site.

[0516] The donor cassette in between the homology arms were cloned into an AAV2 / DJ backbone plasmid.

[0517] LVV vectors

[0518] Both vectors were used to transduce Bl 6F 10 and EO771 cell lines. In both vectors hHER2 gene was under a minimal CMV promoter in antisense; GFP was under the control of a PGK promoter, as was the OVA protein and luciferase protein linked to one another by a P2A self-cleaving peptide. WPRE sequence was introduced following Luc or GFP. Both vectors were cloned into a LVV backbone plasmid. For the murinized HER2 vectors, an identical construct to the one containing hHER2-OVA-Luc was employed, with the replacement of hHER2 with murine HER2. In one construct, the full domain bound by Trastuzumab was preserved, while in the other, only the amino acids constituting the Trastuzumab epitope were included. Both vectors were subsequently utilized to transduce the EO771 cell lines. NHEJ evaluation

[0519] NHEJ efficiency was evaluated on genomic DNA (gDNA). DNA was purified using QuickExtract DNA Extraction Solution (Biosearch Technologies, Hoddesdon, UK); 2 pL of DNA were combined with a master mix containing 2.5 pL 10 mM Primer, 25 pL Taq- Polymerase mix (New England Biolabs, Ipswich, USA) and 18 pL ddH2O. A 1 kb ladder (New England Biolabs, Ipswich, USA) was used to identify the size of the DNA bands.

[0520] Primer sequence:

[0521] PCR program:

[0522] The PCR product was separated on an 1% agarose gel. To visualize the DNA using UV- light, SYBR Safe (NEB, Thermo Fisher Scientific Inc., Waltham, MA, USA) (1 : 10.000) was added to the agarose gel. The electrophoresis was running at 120 V for 30 min.

[0523] The amplified band was cut and extracted from the by means of Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) following the manufacturer’s instructions. Sanger sequence results were obtained (Genewiz from Azenta Life Sciences ; South Plainfield, NJ, USA) and the percentage of NHEJ was estimated using the Interference of CRISPR Edits (ICE) bioinformatics tool by Synthego.

[0524] Droplet digital PCR (ddPCR) for HDR efficiency evaluation

[0525] Total gDNA was isolated from cell pellet with DNeasy Blood&Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. ddPCR was run according to the manufacturer’s instruction by using the following:

[0526] Primer sequence:

[0527] PCR program:

[0528] The plate was read in a BioRad QX200 Droplet Reader (Bio-Rad, Hercules, CA, USA) and data analyzed with QuantaSoft software.

[0529] Untreated (UT) B -cells and H2O were included as negative controls. The frequency of edited alleles was determined as: edited alleles (%) = (positive droplets for target locus) / (positive droplets for RPP30) *100.

[0530] B and T cells Co-culture

[0531] B cells were isolated as previously described. After two days of pre-stimulation in culture (CD40L-IL-4 or CpG / IL-2 media), B lymphocytes were collected.

[0532] On the same day, CD4 T cells were isolated from the spleen of OT-II mice by using a CD4+T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) which contains biotin- conjugated monoclonal antibodies against CD8a, CD1 lb, CD11c, CD 19, CD45R (B220), CD49b (DX5), CD105, Anti-MHC Class II, Ter-119 and TCRγ / δ. After the isolation, CD4 T cells were labeled with Carboxyfluoresceinsuccinimidylester (CFSE) (Thermo Fisher Scientific Inc., Waltham, MA, USA) following manufacturer’s instructions.

[0533] The co-culture was performed in 96-U bottom well-plates in DMEM (Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with 10% FBS, 1.5 mM sodium pyruvate, lx NEAA, lx MEM vitamin solution (Thermo Fisher Scientific Inc., Waltham, MA, USA), lx L- Asparagine (Sigma- Aldrich Inc., St. Louis, MO, USA), L-Arginine (Sigma-Aldrich Inc., St. Louis, MO, USA), 2 mM L-Glutamine (Thermo Fisher Scientific Inc., Waltham, MA, USA), 14 pM folic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) in 0.1 M sodium hydroxide (Sigma- Aldrich Inc., St. Louis, MO, USA) and 57.2 pM β-Mercaptoethanol.

[0534] A total of 50.000 T cells were co-cultured with B-cells in a ratio of 1 : 1. A final concentration of 100 μg / mL of OVA-protein was added to the experimental conditions. T cells with unpulsed B cells, T cells only and T cells cultured with the protein only were used as negative controls. Dendritic cells (BM-DC) cultured in a ration of 1 :5 with T-cells and lOOpg / ml of OVA-protein was used as a positive control.

[0535] After 72 hours at 37°C, the cells were harvested and analyzed in the flow cytometer to investigate proliferation and phenotypical changes of the T cells.

[0536] Live / dead discrimination was performed using Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) following manufacturer’s instructions. The following antibodies were used: CD62L, CD44, CD4, CD 19.

[0537] ELISA

[0538] A 96-well ELISA microplate was pre-coated with 100 pg / mL Carrier-Free Recombinant Human ErbB2 / Her2 Chimeric Protein (R&D Systems, Minneapolis, MN, USA) overnight at 4°C, blocked with lOOpL / well of blocking buffer (PBS 3% bovine serum albumin, BSA) for 2 hours at RT and then incubated with plasma (diluted 1 :25 in blocking buffer) or culture supernatant for 2 hours at RT. After 3 washes with washing buffer (70pL / well, PBS 0.05% Tween20), the plate was inJubated at RT for 1 hour protected from direct light exposure with secondary goat anti-murine IgM (p chain specific)-Alkaline Phosphatase Conjugated Antibody (Jackson Immunoresearch, West Grove, PA, USA) or goat anti-murine IgG-Alkaline Phosphatase Conjugated Antibody (Jackson Immunoresearch, West Grove, PA, USA) diluted 1 : 1250 in blocking buffer. The plate was then washed twice in washing buffer and developing solution Alkaline Phosphate Yellow pNPP Liquid Substrate (Sigma, St. Louis, MO, USA) was added. The plate was kept in the dark and the readings performed at 405nm at the CLARIOStar microplate reader (BMG Labtech, Ortenberg, Germany) after 15 and 45 minutes. No murine isotype of Trastuzumab was commercially available to be used as a standard, therefore data are reported solely as OD.

[0539] Tumor transplants

[0540] Eight-week-old female C57BL / 6J mice were injected subcutaneously with B16F10 melanoma cancer cells. Therefore, the cells were harvested at a confluency of 80% using PBS with 1 mM EDTA. They were counted and resuspended in PBS at a concentration of 1 million cells / mL. Subsequently, 100 pL containing 5xl05cells were injected into each mouse.

[0541] To induce tumor engraftment of the breast cancer models, EO771 cancer cells were injected into the mammary fat pat of eight-week-old C57BL / 6J mice. The cells were harvested as previously described and adjusted to a concentration of 2 million cells / mL. To inject the cells, the mice were anesthetized with isoflurane and surgery was performed injecting 50 pL containing 5xl05cells into each mouse.

[0542] B cell transplant

[0543] BCR-edited B cells were injected IV into B16F10 tumor-bearing mice on Day 0 or on Day4 according to the experimental protocol, and on EO771 tumor-bearing mice on Dayl4. EO771 tumor-bearing mice received a low-intensity chemotherapy regimen with 116 pg / gr of Cyclophosphamide (CPA; Thermo Fisher Scientific Inc., Waltham, MA, USA) and 2.5 pg / gr of Doxorubicin (Doxo; Thermo Fisher Scientific Inc., Waltham, MA, USA) on Dayl2. A group of mice was treated with unedited B cells and mice which received only tumor cells were used as control. Different doses of B-cells were used.

[0544] Tumor burden was monitored 3 times / week until the day of sacrifice and tumor volume estimation was performed by digital caliper measurement and computed using the following formula: (length x width2) / 2.

[0545] In some experiments, different doses of chemo were used, such as:

[0546] 5 pg / gr of Doxo on Day 10-12-14-16

[0547] 2.5 mg / kg of Doxo on Day 10-12-14-16

[0548] 116 mg / kg CPA on Day 14-21

[0549] 2.5 mg / kg Doxo on Day 10-12-14-16 + 116mg / kg CPA on Day 10-16

[0550] - 1.25 mg / kg Doxo on Day 10-12-14-16 + 116mg / kg CPA on Day 10-16

[0551] Bioluminescence

[0552] Femura and tibiae were collected on Day 0 from CD45.1 animals and murine HSPCs were isolated from by magnetic separation with Murine Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin- cells were cultured in StemSpan™ SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 100 ng / ml of SCF, 50 ng / ml TPO, 20 ng / ml IL3 and 100 ng / ml of FLIT (all from StemCell Technologies, Vancouver, Canada).

[0553] In Vivo Bioluminescent Imaging (BLI) was performed at the Dana-Faber Cancer Institute mouse facility. D-Luciferin (7.5 mg / 250 mL PBS) is injected IP. Imaging was performed in the Xenogen IVIS 200 (Perkin Elmer). Mice were constantly kept under narcosis with 1.5-4% Isofluran at 37°C. Bioluminescence pictures were analyzed with the Living Image Software (Perkin Elmer).

[0554] Necroscopy

[0555] Depending on the tumor model, mice were euthanized after 16 (Bl 6F 10 model) or 35 (EO771 model) days from the tumor cell transplant. Tumor, spleen and tumor-draining lymph node were collected in PBS. The tumor mass was weighted and digested subsequently. EO771 tumors were digested in 10 mg / ml Collagenase type I (Sigma-Aldrich Inc., St. Louis, MO, USA), 2.278 mg / ml Hyaluronidase (Sigma-Aldrich Inc., St. Louis, MO, USA) and 7.5% BSA Fraction V, 50 pg / mL Gentamycin (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 5 pg / mL Insulin (Thermo Fisher Scientific Inc., Waltham, MA, USA) in DMEM / F12 (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C for 40 minutes.

[0556] B16F10 tumors were digested with 2.5 mg / ml of Collagenase D (Sigma-Aldrich Inc., St. Louis, MO, USA) diluted in Hanks' Balanced Salt solution (HBSS, Thermo Fisher Scientific Inc., Waltham, MA, USA) with calcium and magnesium cloride at 37°C for 20 minutes.

[0557] Where stated, lymphocytes were isolated by means of a 63%-47%-33% Percoll (Cytiva Life Sciences, Marlborough, MA) gradient stratification and centrifugation at 1800rpm for 20min without brakes.

[0558] Single cell suspension of digested tumor, spleen and lymph nodes underwent red blood cell lysis using ACK lysing reagent and were subsequently stained for cytofluorimetric analysis. For certain experiments, cancer cells derived from the tumors after digestion were cultured in the appropriate culture medium. These cancer cells were then collected on a weekly basis for a total of eight weeks to analyze HER-2 expression using flow cytometry. Live / dead discrimination was performed using 7-AAD and to detect HER-2 at the cell surface anti-HER-2 was used.

[0559] Statistical analysis

[0560] All results are expressed as means ± SDs, unless stated otherwise. All the data were analyzed with GraphPad Prism software. Normal distribution of samples was assessed by using a D’Agostino Pearson omnibus normality test. Parametric or non-parametric t-test, one-way or two-way ANOVA test was run to assess statistical significance according to the data. A P value of less than .05 was considered statistically significant. *P < .05, **P < .01, ***p < .001 and P < .0001.

[0561] The following materials and methods were employed in Example 2. Cell lines

[0562] MDA-MB-453 human HER2+ breast cancer cell line and SK-OV-3 human HER2+ ovarian cancer cell lines were cultured in McCoy’s 5 A medium (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gemini Bio Products, West Sacramento, CA, USA), 2% L-Glutamine (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA), 1% Penicillin / Streptomycin (Cytiva, Marlborough, MA, USA) and passaged every 4 or 5 days. Human embryonic kidney cells (HEK293) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Coming, Coming, NY, USA) supplemented with 10% FBS, 1% Penicillin / Streptomycin and passaged every 4 days. JEKO-1 (Mantle Cell Lymphoma) cell line was cultured at 0.25x106 cells / mL in RPMI-1640 medium (HyClone, Logan, Utah, USA) supplemented with 10% FBS, 2% L-Glutamine, 1% Penicillin / Streptomycin and passaged every 5 days.

[0563] Primary B cells Isolation

[0564] Human peripheral blood mononuclear cells (PBMCs) were purified from discarded, de- identified leukapheresis collars, a byproduct of platelet donation, using density gradient separation (Lymphoprep, StemCell Technologies, Vancouver, Canada), upon Institutional Review Board (IRB) approval of the non-human subject research study (IRB review on 02 / 23 / 2021, IRIS reference #395201). Untouched B lymphocytes were isolated by negative selection with the Human B Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) exploiting a cocktail of biotin-conjugated monoclonal antibodies against CD2, CD14, CD16, CD36, CD43 and CD235a according to manufacturer’s instructions. Purity was assessed by flow cytometry analysis as percentage of CD19+ live cells and cells were cultured at 0.25x106 cells / mL concentration in a stimulation medium for 4 days.

[0565] B cell culture

[0566] Cells were cultured in different CD40L-based stimulation media were tested using different mix and concentration of the following reagents: 10% FBS, 1% Penicillin / Streptomycin, MEGACD40L (Enzo Lifescience, Farmingdale, NY, USA), IL-2 (Biolegend, San Diego, CA, USA), IL- 10 (Biolegend, San Diego, CA, USA), IL- 15 (Biolegend, San Diego, CA, USA), IL-4 (Biolegened, San Diego, CA, USA), CpG (IDT, Newark, NJ, USA). Either IMDM (Sigma, St. Louis, MO, USA), ExCellerate B cell Media (R&D Systems, Minneapolis, MN, USA) or ImmunoCultXF T Cell Expansion Medium (StemCell Technologies, Vancouver, Canada) were used as base media. ImmunoCult™- ACF Human B Cell Expansion Supplement (StemCell Technologies, Vancouver, Canada) was used in combination with ImmunoCultXF T Cell Expansion Medium according to the manufacturer’s instruction. B cells were cultured in the above-mentioned stimulation media for 4 days unless stated otherwise. After 4 days in culture, cells were collected for genome editing and flow cytometry analysis for phenotype assessment. For long-term phenotype monitoring, flow cytometry analysis and cell counting were performed after additional 6 and 9 days and the culture volume adjusted to maintain the same cell concentration.

[0567] AAV6 production

[0568] AAV6 vectors were produced in HEK293 cells and used as donor delivery vector. Briefly, 12xl06cells were plated onto 10 x 15cm2dishes in a final volume of 20mL medium each the day before transfection. Right before transfection, cells were treated with sodium butyrate IM (Sigma, St. Louis, MO, USA) for 15 minutes to activate transcription and then transfected by polyethylenimine (PEI, Polysciences, Warrington, PA, USA) using a 6:22 ratio of donor plasmid and pDGM6 plasmid (Addgene; #110660). The volume of PEI is based on a 4: 1 ratio of PELtotal DNA. PEI diluted in Optimem (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) was dropwise added to the donor DNA diluted in Optimem and the solution was briefly vortexed before being added to the cell culture (ImL / dish). Cells were harvested 48 hours after transfection, pelleted 2000xg at 4°C for 10 minutes and AAV6 was collected with the AAVpro Purification Kit (TaKaRa Bio, Kusatsu, Shiga, Japan) following manufacturer’s instructions. For each viral preparation, viral titers were determined by digital droplet PCR (ddPCR): AAV6 vectors were lysed with QuickExtract DNA Extraction Solution (Lucigen, Middleton, WI, US) for 8 minutes at 68°C and 5 minutes at 98°C, and serial dilutions from 10-2 to 10-10 were used for absolute quantification. AAV6 vector productions were carried out by the Boston Children’s Hospital (BCH, Boston, MA, USA) Viral Core.

[0569] Genome editing CRISPR / Cas9 editing protocol

[0570] B cells were electroporated after four days of pre-stimulation, unless stated otherwise, by using a 4D-Nucleofector device (Lonza, Basel, Switzerland). Chemically synthetized single- guide RNAs (sgRNAs) (IgH296 sgRNA: GUCUCAGGAGCGGUGUCUGU targeting a sequence next to an AGG PAM or AAVS1 sgRNA: GUCACCAAUCCUGUCCCUAG targeting a sequence next to an ACC PAM; IDT, Newark, NJ, USA) were complexed in a 2:1 molar ratio with 50 pmolCas9 (Alt-R™ S.p. Cas9 Nuclease V3, IDT, Newark, NJ, USA) for 15 minutes at room temperature (RT). The resulting ribonucleoproteins (RNPs) were combined with human B cells in P3 Primary Cell Nucleofector Solution (Lonza, Basel, Switzerland). The electroporation was performed in 20 pL Nucleocuvette Strips or 100 pL Nucleocuvette Vessels on the Lonza electroporation device according to the manufacturer’s instruction (program DI100). Pre-warmed cell culture medium was added to the cells immediately after electroporation and donor vector was delivered after 20 minutes by means of an AAV6 vector (Boston children’s Hospital Viral Core Facility, Boston, MA) at a multiplicity of infection (MOI) of 2 104. Cells were transduced for 6h at 37°C at a concentration of 3.3xl06 / ml, and later expanded at 2.5xlO5 / mL for a total of 6 days in culture. Untreated (UT) cells were used as control.

[0571] Editing efficiency was assessed with the following antibodies: CD19, Streptavidin-Tag, human HER-2 or GFP expression.

[0572] Cord Blood CD34+ cells were electroporated after two days of pre-stimulation. The editing procedure is performed as described above. The following changes were made: electroporation program EOIOO; transduction ON a lxl06 / ml.

[0573] Donor construct

[0574] Plasmid cloning was performed by using standard molecular biology techniques. Briefly, plasmid was digested using different restriction enzymes (New England BioLabs, Ipswich, MA, USA), fragments were separated by agarose gel electrophoresis and the correct ones inserted into a dephosphorylated linearized AAV6 backbone with Quick Ligase (New England BioLabs, Ipswich, MA, USA) after purification with Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) according to manufacturer’s instructions. After ligation, TOPIO chemically competent E. coli bacteria (Thermo Fisher Scientific Inc., Waltham, MA, USA) were transformed according to the manufacturer’s instructions and plated on LB agar plates containing Carbenicillin for selection. Plasmid DNA was extracted from single bacterial colonies and purified with Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA) and EndoFree Plasmid Maxi Kit (QIAGEN, Hilden, Germany). Colonies were screened with control digestions and sequenced.

[0575] The BCR HDR cassette consisted of the IGHV1-69 heavy chain promoter region, followed by a human IgK signal peptide, Trastuzumab-derived Fragment-antigen binding (Fab) sequence and a splice donor site. The Fab is a fusion of the codon-optimized variable heavy- chain regions (VDJH) and a complete light-chain (human CKappaL+VJL) connected with a 57 bp linker that includes a 3x Strep-Tag II motif. The splice junction is followed by 60 base pairs derived from the mouse IGHJ3 gene segment. The entire cassette is cloned in between a 450- base pair upstream and downstream homology arm flanking the sgRNA GTCTCAGGAGCGGTGTCTGTAGG cutting site. The donor cassette in between the homology arms were cloned into an AAV6 backbone plasmid.

[0576] The AAVS1 HDR cassette consisted of a GFP cassette under the control of PGK promoter and followed by a polyA sequence. The entire cassette is cloned in between a 796-base pair upstream and a 258-bp downstream homology arm flanking the sgRNA GTCACCAATCCTGTCCCTAG ACC cutting site.

[0577] The membrane-bound BCR HDR cassette consisted of the IGHV1-69 heavy chain promoter region, followed by a human-IgK signal peptide, a Trastuzumab-derived Fragment- antigen binding (Fab) sequence and a complete human IgM constant chain, including the transmembrane region. The Fab is a fusion of the codon-optimized variable heavy-chain regions (VDJH) and a complete light-chain (human CKappaL+VJL) connected with a 57 bp linker that includes a 3x Strep-Tag II motif. The IgM constant chain is followed by beta-globin derived polyA sequence. The entire cassette is cloned in between a 450-base pair upstream and downstream homology arm flanking the sgRNA GTCTCAGGAGCGGTGTCTGTAGG cutting site. The donor cassette in between the homology arms were cloned into an AAV6 backbone plasmid.

[0578] Ex vivo cytotoxicity assay

[0579] On day 0, HER2+ SK-OV-3 or HER2+ MDA-MB-453 cancer cells were labeled with the fluorescent dye CFSE (CellTrace CFSE Cell Proliferation Kit 0.5pM for IxlO6cells; Thermo Fisher Scientific Inc., Waltham, MA, USA) following manufacturer’s instructions. Briefly, 2xl04cancer cells were seeded with 300pL of medium in a 48-well plate and cultured for 48 hours. Two days after, 200pL of medium were removed from each well, and B cells (edited or mock edited or UT - 2 days after editing, in B cell media) were added at an effector-to-target (ET) ratio of 5: 1 B cells:cancer cells, 10: 1, 15: 1 or 30: 1. Additional B cell medium was added to reach the final volume of 300pL and cells were co-cultured overnight in mixed media conditions (100 pL McCoy5A complete + 200 pL of B cell medium). The following day, all the cells were collected from each well (first cells in suspension, then adherent cells were detached with Trypsin) and stained for cytofluorimetric analysis. B cell-only and target-only controls were included. Both technical and biological replicates were included in the experiment design.

[0580] Enzyme-linked immunosorbent (ELISA) assay

[0581] 96-well ELISA microplate was pre-coated with 100 pg / mL Carrier-Free Recombinant Human ErbB2 / Her2 Chimeric Protein (R&D Systems, Minneapolis, MN, USA) overnight at 4°C, blocked with lOOpL / well of blocking buffer (PBS 3% bovine serum albumin, BSA) for 2 hours at RT and then incubated with plasma (diluted 1 : 50 in blocking buffer) or culture supernatant for 2 hours at RT. After 3 washes with washing buffer (70pL / well, PBS 0.05% Tween20), the plate was incubated at RT for 1 hour protected from direct light exposure with secondary goat anti-human IgM (p chain specific)- Alkaline Phosphatase Conjugated Antibody (Sigma, St. Louis, MO, USA) diluted 1 :5000 in blocking buffer. The plate was then washed twice in washing buffer and developing solution Alkaline Phosphate Yellow pNPP Liquid Substrate (Sigma, St. Louis, MO, USA) was added. The plate was kept in the dark and the readings performed at 405nm at the CLARIOStar microplate reader (BMG Labtech, Ortenberg, Germany) after 15 and 45 minutes. Plasma antibody quantity was determined computing the ng / pL concentration based on the OD after normalization with blank samples. No IgM isotype of Trastuzumab is commercially available to be used as a standard, therefore IgM data are reported solely as OD. Levels of anti-HER2 IgG were measured by means of a commercially available ELISA test (ImmunoGuide Trastuzumab ELISA; IBL America, Spring Lake Park, MN, USA) according to the manufacturer’s instruction.

[0582] Enzyme-linked immunospot (ELISPOT) assay

[0583] MDA-MB-453 cells were treated with Mytomicin C 0.2pg / mL for 2 hours prior co- culture with mouse splenic CD4+ and CD8+ T cells (see section 4.7.5) on pre-coated IFN-y ELISPOT plate (BD, Franklin Lakes, NJ, USA). Effector-to-target (ET) ratio was set to 2:1 and 5:1. After an overnight co-culture, the plate was washed and developed according to manufacturer’s instructions. Spot development was monitored for up to 1 hour before stopping the reaction with deionized water. The plate was read at the ELISPOT reader (via SellNet Consulting Inc, Fort Lee, NJ, USA) after at least 24 hours of air-drying protected from direct light exposure. The mitogen phytohemagglutinin (PHA; Ipg / mL; Roche Diagnostics Gmbh, Mannheim, Germany) was used as positive control; target-only and effector-only conditions were included as negative controls.

[0584] Droplet Digital PCR (ddPCR)

[0585] Total genomic DNA was extracted from 0.5 to Ix106cells 2 days after editing using the QuickExtract DNA Extraction Solution (Lucigen, Middleton, WI, USA) following manufacturer’s protocol. Fifty nanograms of genomic DNA were loaded in each well, along with 20X Her2 and TTC5 Primer and Probe and a 2X ddPCR Supermix for Probes, no dUTPs (Bio- Rad, Hercules, CA, USA). TTC5 was used as normalizer (HEX probe, Bio-Rad, Hercules, CA, USA). Genomic amplification was carried out under the following conditions: 10’ 95°C; 40 cycles of 30” 94°C, 1’ 55°C, 2’ 72°C; 10’ 98°C. Droplets were prepared on the BioRad AutoDG Automated Droplet Generator (Bio-Rad, Hercules, CA, USA), the plate was read in the BioRad QX200 Droplet Reader (Bio-Rad, Hercules, CA, USA) and data analyzed with QuantaSoft software. Untreated (UT), mock-electroporated cells and H2O were included as negative controls. The frequency of edited alleles was determined as: edited alleles (%)= positive droplets for target locus positive droplets for TTC5x 100

[0586] Vector Copy Number (VCN) was determined by calculating the ratio of the target molecule concentration to the reference molecule concentration, times the number of copies of reference species in the genome: Copy Number= concentration of target species concentration of TTC5 species x 2.

[0587] For AAV6 titering, primers and probes used were the following: GGAACCCCTAGTGATGGAGTT (forward), CGGCCTCAGTGAGCGA (reverse), FAM probe CACTCCCTCTCTGCGCGCTCG.

[0588] Genomic amplification was carried out under the following conditions: 10’ 95°C; 45 cycles of 30” 94°C, 1’ 60°C; 10’ 98°C.

[0589] PCR for non-homologous end joining (NHEJ)

[0590] One hundred nanograms of genomic DNA were amplified by PCR using GoTaq DNA polymerase (Promega, Madison, WI, USA) according to manufacturer’s instructions. Genomic amplification was carried out under the following conditions: 2’ 98°C; 40 cycles of 45” 98°C, 45” 61°C, 1’ 75°C; 5’ 72°C. The resulting PCR products were separated by agarose gel electrophoresis and the correct fragment was purified with Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and Sanger- sequenced (Genewiz, South Plainfield, NJ, USA). The frequency of indels (% ICE) in gRNA / Cas9-electroporated cells relative to untreated control cells was determined using the Inference of CRISPR Editing (ICE) algorithm (Synthego, Menlo Park, CA, USA) and then computed as: NHEJ (%)=[100— (% edited alleles by ddPCR)] x (% ICE). Untreated and mock-electroporated B cells was used as control.

[0591] Mice

[0592] Seven or eight weeks old NOD-SCID IL2rgnull (NSG™) female mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All procedures were reviewed by the local Institutional Care and Use Committee (IACUC) and approved (protocol #20-004; Dr. Pietro Genovese). All animal procedures was approved by the IACUC committee and performed according to the approved protocol (20-004) and as described in the DFCI Animal Facility Standard Operating Procedures (SOPs).

[0593] Tumor and B cell transplants

[0594] Eight weeks old female NSG™ mice were orthotopically injected with 6xl06MDA-MB- 453 cells in the mammary fat pad on the right side of the abdomen. A subcutaneous injection of Mel oxicam 2 mg / kg was performed prior to the surgery and the following day; injection site and overall health was closely monitored for the first 4 days after tumor transplant. After two weeks, mice received intravenously 5xl06B cells either edited, mock-electroporated or untreated (UT), alone or along with 2xl06syngeneic PBMCs. Mice weight and tumor burden were longitudinally monitored until the day of sacrifice, five weeks later. Tumor volume estimation was performed by digital caliper measurement and computed using the following formula: (length x width2) / 2. Mice which received just cancer cells were used as control.

[0595] Bioluminescence

[0596] Briefly, for the in-vivo B-cell tracking, 5*106Luc+ Edited or Unedited B-cells were injected on day 14 into MDA-MB-453 tumor bearing mice. In the metastatic model, l*106Luc+ SK-OV3 cells were injected IV into recipient mice.

[0597] In Vivo Bioluminescent Imaging (BLI) was performed. D-Luciferin (7.5 mg / 250 mL PBS) is injected IP. Imaging was performed in the Xenogen IVIS 200 (Perkin Elmer). Mice were constantly kept under narcosis with 1.5-4% Isofluran at 37°C. Bioluminescence pictures were analyzed with the Living Image Software (Perkin Elmer).

[0598] CD34+ cell transplant

[0599] Briefly, CD34+ cells are isolated via magnetic separation (Miltenyi Biotec, Bergisch Gladbach, Germany) from discarded and anonymized Cord Blood bags on Day 0. Cells were cultured in StemSpan™ medium (StemCell Technologies, Vancouver, Canada) supplemented with 100 ng / ml of hFLT3, 20ng / ml of hTPO, lOOng / ml of hSCF, 35nM of UM171, lOnM of SRI, 0.2 mg / ml of hIL6, 2% L-Glutamine, 1% Penicillin / Streptomycin. On day 2, cells are electroporated as described previously and cultured l*106cells / ml.

[0600] On day 3, 0.25xl06cells were transplanted IV into lethally irradiated (2x 450 Rad) NSG™ mice. Twelve weeks after CD34+ cell transplant, mice underwent MDA-MB-453 tumor implantation in the mammary fat pad. Metastatic SK-OV3 model l*106Luc+ SK-OV3 cells were injected intravenously (IV) into recipient NSG™ mice on Day 0; depending on the experimental setting, mice received 5*106Edited or Unedited B- cells in combination with 2*106of syngeneic PBMC IV at day 7 or 2*106of PBMC on Day -7 and 5*106Edited or Unedited B-cells on Day 7.

[0601] Trastuzumab administration

[0602] MDA-MB-453 tumor-bearing mice were treated with lOmg / kg or 67pg / kg of MoAb Trastuzumab (ichorbio, Wantage, UK) IP on day 14, 21 and 28. Tumor burden was monitored using calipers.

[0603] Chemotherapy regimens

[0604] MDA-MB-453 tumor-bearing mice were treated with Img / kg or 1.5mg / kg or 2mg / kg of Doxorubicin (Thermo Fisher Scientific Inc., Waltham, MA, USA) IV on day 12. Tumor burden was monitored using calipers.

[0605] Blood and plasma collection

[0606] Blood was collected in citrate phosphate dextrose (Sigma, St. Louis, MO, USA) by means of heparinized capillaries (Thermo Fisher Scientific Inc., Waltham, MA, USA) from the retro-orbital site every 7-10 days. After plasma collection (5’ centrifugation at 14000 rpm 4°C), erythrocytes were lysed with ACK Lysing Reagent (StemCelll Technologies, Vancouver, Canada), blood stained for cytofluorimetric analysis and a dry cell pellet was collected and stored at -80°C for DNA extraction for downstream molecular analysis.

[0607] Necroscopy

[0608] Five weeks from the B cell transplant, mice were euthanized, and tumor, spleen and bone marrow was collected in PBS 2% FBS. Residual tumor mass was weighed and digested with 0.2mg / ml Collagenase type IV (Worthington Biochemical Corporation, Lakewood, NJ, USA), 2mg / ml Dispase II (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) and 0.5 mg / ml DNAsel (Roche Diagnostics GmbH, Mannheim, Germany) at 37°C for 1 hour. Bone marrow and spleen were smashed on a 40pM filter and underwent red blood cell lysis with ACK Lysing Reagent (StemCelll Technologies, Vancouver, Canada). Cells were stained for cytofluorimetric and dry pellets were collected for DNA extraction for downstream molecular analysis. One million of human splenic T cells were stimulated in Iscove’s Modified Dulbecco Medium (IMDM, Sigma, St. Louis, MO, USA) supplemented with 10% FBS, 2% L- Glutamine, 1% Penicillin / Streptomycin, 5ng / pL human IL-7 (Biolegend, San Diego, CA, USA), 5ng / pL human IL-15 (Biolegend, San Diego, CA, USA) with anti-CD3 / CD28 beads (Dynabeads Human T- Activator CD3 / CD28, at 1 :3 T cell:beads ratio; Thermo Fisher Scientific Inc., Waltham, MA, USA). At day 6 beads were removed by using the MagnaRack™ Magnetic Separation Rack (Thermo Fisher Scientific Inc., Waltham, MA, USA) and T cells were cultured for additional 6 days in IMDM 10% FBS, 2% L-Glutamine, 1% Penicillin / Streptomycin, 5ng / pL human IL-7, 5ng / pL human IL-15, 200U / mL human IL-2 (Biolegend, San Diego, CA, USA).

[0609] Flow cytometry analysis

[0610] Flow cytometry analyses were performed on LSRFortessa (BD Bioscience, Franklin Lakes, NJ, USA) instrument. 0.25xl05or 0.5xl05cells incubated in 50pL of FACS buffer consisted in PBS added with 1% Bovine Serum Albumin (BSA, Sigma, St. Louis, MO, USA) and 0.1% sodium azide (Sigma, St. Louis, MO, USA). All Antibodies were purchased from Biolegend (San Diego, CA, USA), unless otherwise stated.

[0611] B cells were labeled with LIVE / DEAD™ Fixable Yellow (LDY) Dead Cell Stain Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), CD19 PerCP / Cyanine5.5 (clone HIB19), CD27 APC (clone MT-271), CD38 APC / Fire 750 (clone HB-7), CD138 Pacific Blue (clone MI15), IgM BV650 (clone MHM-88), IgG PE / Cyanine7 (clone HP6017), Her2 FITC (Aero biosystems, Newark, DE, USA), StrepTactin PE (IB A Lifesciences GmbH; Gottingen, Germany). The following gating strategy was used: FSC-A / FSC-H (singlets) > SSC / FSC (cells) > LDY / FSC (live cells) > CD19 / FSC (B cells). Infused human PBMCs were stained with antibody cocktails containing LDY, CD19 PerCP / Cyanine5.5, CD27 APC, CD38 APC / Fire 750, CD138 Pacific Blue, CD3 APC / Cyanine7 (clone OKT3) CD13 BV711 (clone WM15). The following gating strategy was used: FSC-A / FSC-H (singlets) > SSC / FSC (cells) > LDY / FSC (live cells) > CD13 / CD3 (CD3+ T cells), CD19 / CD3 (CD19+ B cells), CD13 / SSC (CD13+ myeloid cells).

[0612] For cytotoxicity assays, cells were stained with antibody cocktails containing CD 19 BV711, Fas Pacific Blue (clone DX2), FasL PE / Cyanine7 (clone NOK-1), AnnexinV PE / Dazzle 594, 7-AAD, defined volume of Precision Count Beads (Biolegend) to calculate cell numbers. The following gating strategy was used: FSC-A / FSC-H (singlets) > SSC / FSC (cells) > CD19 / CFSE (B cells and cancer cells) > 7-AAD / FSC (viable cells) > Annexin V / FSC (non- apoptotic / necrotic cells). Mouse samples were incubated with mouse and human Fc blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) before adding the antibody cocktail. T cell staining mix contained LDY, human CD45 PE / Cyanine7 (clone 2D1), murine CD45 FITC (clone 30-F11), human CD3 APC / Cyanine7 (clone 0KT3), human CD13 BV711 (clone WM15), human CD8 Pacific Blue (clone SKI). B cell staining mix contained LDY, human CD45 PE / Cyanine7, murine CD45 FITC, human CD38 APC / Fire 750, human CD 19 BV711, human CD27 APC, human CD138 Pacific Blue, StrepTactin PE (IB A Lifesciences GmbH; Gottingen, Germany). The following gating strategy was used: FSC-A / FSC-H (singlets) > SSC / FSC (cells) > LDY / FSC (live cells) > mCD45 / hCD45 (human CD45+ cells). For T cell panel: hCD13 / hCD3 (T cells) > hCD8 / hCD4. For B cell panel: hCD19 / hCD45 (B cells) > hCD27 / hCD38 > hCD138 / hCD38.

[0613] Single-stained cells or compensation beads were used as positive control, Fluorescence Minus One (FMO) controls were used in cytotoxicity assays and with murine samples. Flow cytometry data were analyzed using FlowJo software.

[0614] Statistical analysis

[0615] All results are expressed as means ± SDs, unless stated otherwise. All the data were analyzed with GraphPad Prism software. Normal distribution of samples was assessed by using a D’Agostino Pearson omnibus normality test. Parametric or non-parametric t-test, one-way or two-way ANOVA test was run to assess statistical significance according to the data. A P value of less than .05 was considered statistically significant. *P < .05, **P < .01, ***P< 001 and .0001.

[0616] Sequences

[0617] What follows are sequences relating to the HDR cassettes used in the Examples.

[0618] Nucleotide sequence of the HDR cassette used for Human B-cells: cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtc gcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttc ctgcggccgcaCGCGTatgtgacgcccggagacagaaggtctctgggtggctgggtttttgtgg ggtgaggatggacattctgccattgtgattactactactactactacatggacgtctggggcaa agggaccacggtcaccgtctcctcaggtaagaatggccactctagggcctttgttttctgctac tgcc tgtggggt ttcc tgagca t tgcaggt tggtcctcggggca tgttccgaggggacc tgggc ggactggccaggagggga tgggcactggggtgcc t tgaggatctgggagcctctgtgga ttttc cga tgc ctt tgga a a a tggga c tcagg t tggg tgcg tctga tggag taac tgagc c tgggggc t tggggagccaca t t tggacgaga tgcc tgaacaaaccaggggtct tagtga tggc tgaggaa tg tgtctcaggagcggtgtcgAATTCGTCCTGCTGGACACTCATGTAGGGTAACGAGTGGCCACCT TTTCAGTGTTACCAGTGAGCTCTGAGTGTTCCTAATGGGACCAGGATGGGTCTAGGTGCCTGCT CAATGTCAGAGACAGCAATGGTCCCACAAAAAACCCAGGTAATCTTTAGGCCAATAAAATGTGG GTTCACAGTGAGGAGTGCATCCTGGGGTTGGGGTTTGTTCTGCAGCGGGAAGAGCGCTGTGCAC AGAAAGCTTAGAAATGGGGCAAGAGATGCTTTTCCTCAGGCAGGATTTAGGGCTTGGTCTCTCA GCATCCCACACTTGTACAGCTGATGTGGCATCTGTGTTTTCTTTCTCATCCTAGATCAGGCTTT GAGCTGTGAAATACCCTGCCTCATGCATATGCAAATAACCTGAGGTCTTCTGAGATAAATATAG ATATATTGGTGCCCTGAGAGCATCACATAACAACCACATTCCTCCTCTGAAGAAGCCCCTGGGA GCACAGCTCATGCCACCATGGAAGCTCCAGCTCAGCTGCTGTTTCTGCTGCTGCTGTGGCTGCC TGATACCACCGGCGAGATCGACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTG GGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACGTGAACACAGCCGTGGCTTGGTATC AGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCAGCTTTCTGTACAGCGGCGT GCCCAGCAGATTCAGCGGCTCTAGAAGCGGCACCGACTTCACCCTGACCATAAGCAGTCTGCAG CCCGAGGACTTCGCCACCTACTACTGTCAGCAGCACTACACCACACCTCCAACCTTTGGCCAGG GCAC'CAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGA CGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAA GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAATAGCCAAGAGAGCGTGACCG AGCAGGACAGCAAGGACTCTACCTACAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTA CGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAG AGCTTCAACCGGGGCGAGTGTTGGAGCCATCCTCAGTTCGAGAAGTGGTCACACCCACAGTTTG AGAAATGGTCCCATCCGCAATTCGAAAAAGGCGGCAGCTCTGGCAGCGGCAGCGGATCTACTGG AACAAGCTCTAGCGGCACAGGCACAAGCGCTGGCACAACAGGCACATCTGCCAGCACATCTGGC TCTGGATCTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGATCAGCAGGCGGAGAAGTGCAGCTTG TTGAATCTGGCGGTGGCCTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGG CTTCAACATCAAGGACACCTACATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGG GTCGCCAGAATCTACCCCACCAACGGCTACACCAGATACGCCGACTCTGTGAAGGGCAGATTCA CCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACTCCCTGAGAGCCGAGGA CACCGCCGTGTACTACTGTTCTAGATGGGGAGGCGACGGCTTCTACGCCATGGATGTTTGGGGA CAGGGCACCCTGGTCACCGTGTCATCTTCAGGTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCA GGTGTACTGGGCCAGGCAAGGGCTTTGGATCCtgtaggactgcaagatcgctgcacagcagcga atcgtgaaatattttctttagaattatgaggtgcgctgtgtgtcaacctgcatcttaaattctt tat tggc tggaaagagaactgtcggagtgggtgaatccagccaggagggacgcgtagccccggt cttgatgagagcagggttgggggcaggggtagcccagaaacagtggctgccgtcctgacagggg cttagggaggctccaggacctcagtgccttgaagctggtttccatgagaaaaggattgtttatc ttaggaggcatgcttactgttaaaagacaggatatgtttgaagtggcttctgagaaaaatggtt aagaaaattatgacttaaaaatgtgagagattttcaagtatattaatttttttaactgtccaag tatttgaaattcttatcatttgattaacacccattcTCGACGCTAGCGTTTCCTTCCCCTGGCT ATTCTGCTCAACCTTCCTATCAGAAAAAAAGGGGAAGCGATTCTAGGGAGCAGTCTCCATGACT GTGTGTGGAGTGTTGACAAGAGTTcGGATATTTTATTCTCTACTCAGAATcGCTGCTCCCCCTC ACTCTGTTCTGTGTTGTCATTTCCTCTTTCTTTGGTAAGCTTTTAATTTCCAGTTGCATTTTAC TAAATTAATTAAGCTGGTTATTTACTTCCCATCCTGATATCAGCTTCCCCTCCTCCTTTCCTCC CAGTCCTTCTCTCTCTCCTCTCTCTTTCTCTAATCCTTTCCTTTCCCTCAGTTCATTTCTTCTT CTTTGATCTACtTTTGTTTGTCTTTTTAAATATTGCCTTGTAACTTGCTCAGAGGACAAGGAAG ATATGTCCCTGTTTCTTCTCATAGCTCTCAAGAATAGTAGCATAATTGGCTTTTATGCCAGGGT GACAGGGGAAGAATATATTTTACATATAAATTCTGTTTGACATAGGATTCTTATAATAATTTGT

[0619] CAGTAGTTTAAGGTTGCAAACCtcgagaccgagcggccgcaggaacccctagtgatggagttgg ccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgccc gggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg

[0620] (SEQ ID NO: 1)

[0621] In the above nucleotide sequence, the following regions are annotated in order from 5’ to 3’ as indicated in parenthesis:

[0622] 5’ ITR (inverted terminal repeat) (bold, lowercase)

[0623] Homology Arm (HA Left) (bold, italic, lowercase)

[0624] IgHVl-69 minimal promoter (ALL CAPS, BOLD)

[0625] Human IgK Signal peptide (ALL CAPS, UNDERLINE)

[0626] Trastuzumab derived Variable Light Chain (ALL CAPS BOLD, ITALIC)

[0627] IgK constant light chain (ALL CAPS PLAIN TEXT)

[0628] 3X StrepTag II motif (ALL CAPS. DOUBLE-UNDERLINE)

[0629] Gly / Ser linker (ALL .CAPS, DASHED UNDERL.INE)

[0630] Trastuzumab derived Variable Heavy Chain (BOLD, ITALIC, UNDERLINE, ALL CAPS)

[0631] 60 bp from IgHJl intronic sequence Homology Arm (HA Right) (lowercase, bold, underline)

[0632] Murine HBB intronic sequence (ALL CAPS. BOLD. DOUBLE UNDERLINE) (in various embodiments, this sequence may be omitted or replaced with a polynucleotide sequence of sufficient length to allow proper packaging of the HDR cassette within an AAV capsid) 3 ’ ITR (lowercase, underline)

[0633] The below polypeptide is encoded by the above polynucleotide sequence:

[0634] MEAPAQLLFLLLLWLPDTTGEIDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI

[0635] KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

[0636] STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECWSHPQFEKWSHPQFEKWSHP

[0637] QFEKGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGEVQLVESGGG

[0638] LVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSSGESAVWG.

[0639] The following table provides a nucleotide sequence for the IgHvl-69 promoter and amino acid sequences for components of polypeptides encoded by the above HDR cassette nucleotide sequence. Table 1 : Nucleotide sequence for the IgHvl-69 promoter and amino acid sequences for components of polypeptides encoded by the HDR cassette nucleotide sequence.

[0640] Nucleotide sequence of the HDR cassette used for Murine B-cells: cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtc gcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactAGGGGTTC CTGCGGCCGCACGCGT cccaggggtga ttctagtcagactctggggt ttt tgtcgggta tagag gaaaaatccactattgtgattactatgctatggactactggggtcaaggaacctcagtcaccgt ctcctcaggtaagaatggcctctccaggtctttatttttaacctttgttatggagttttctgag ca t tgcagactaatct tgga tatttgtccc tgagggagccggc tgagagaagt tgggaaa taaa ctgtctagggatctcagagcctttaggacagattatctccacatctttgaaaaactaagaatct gtgtga tggtgt tggtggagtccc tgga tga tggga tagggact t tggaggctca t t tgaagaa ga tgc taaaacaa tecta tggc tggaggga tagt tggggc tgtagt tggaga t t ttcagt tttt agaa taaaagta t tagt tgtggaa ta tacttcaggaccacctctgtgacagca ttta tacagta tccgatgACCGGTGAGACATGTTCTCTTGCTGTCATTTGTGTAATATTTTAGTATGCAACCTTT TGGAAAGGCCAATATTATTTAAATATATATGAGAGAAGATTGCTAACTCTCATAAATGTATTGGTTTTTTTTTTTTTAATTTCCAGTAAGTGTTATCCTCATTGCTACTACCACCAATCAATTTTTTCACTAAGACAAGTGAGTGTCTCAGGTTAGGATTCTATTTTAAAATTGAGATATTAGGCTTTGATA CTACATCTAAATGGTCTGCACATGTCTCGAAGAAAGTTCTTCAGACAGAGTTAGGACTTGGACC CAGGAGTTAGGACTTGGACTGACTCAGGAGCACTCTAGTTTCTTCTTCTCCAGCTGGAATGTCC TTATGTAAGAAAAGCCTTGCCTCATGAGTATGCAAATCATGTGAGACTGTGATGATTAATATAG GGAAATCCACACCAAACATCATATGAGCCCTATATTCTCTACAGACACTGAATCTCAAGGTCCT TACCATGGTGTTCACCCCTCAGATCCTGGGCCTGATGCTGTTCTGGATCAGCGCCAGCAGAGGC GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCA CCTGTAGAGCCAGCCAGGACGTGAACACAGCCGTGGCTTGGTATCAGCAGAAGCCTGGCAAGGC CCCTAAGCTGCTGATCTACTCTGCCAGCTTCCTGTACAGCGGCGTGCCCTCTAGATTCAGCGGC AGCAGATCTGGCACCGACTTCACCCTGACAATCAGCAGCCTGCAGCCTGAGGACTTCGCCACCT ACTACTGTCAGCAGCACTACACCACACCTCCAACCTTCGGCCAGGGCACCAAGGTGGAAATCAA GAGAGCTGATGCCGCTCCTACCGTGTCTATCTTCCCACCTAGCAGCGAGCAGCTGACATCTGGC GGAGCCTCTGTCGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGA TCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCTTGGACCGACCAGGACAGCAAGGACTC CACCTACAGCATGAGCAGCACCCTGACACTGACCAAGGACGAGTACGAGAGACACAACAGCTAC ACATGCGAGGCCACACACAAGACCAGCACAAGCCCCATCGTGAAGTCCTTCAACAGAAACGAGT GCTGGTCCCATCCTCAGTTCGAGAAGTGGTCACACCCACAGTTTGAAAAGTGGTCCCATCCGCA ATTTGAGAAAGGCGGCAGCTCTGGCAGCGGCTCTGGATCTACTGGCACAAGCAGTAGCGGCACA GGCACATCTGCTGGCACCACTGGAACAAGCGCCTCTACATCTGGATCTGGCTCTGGCGGAGGCG GAGGATCTGGTGGTGGTGGATCTGCAGGCGGAGAAGTGCAGCTGGTTGAAAGTGGCGGAGGACT TGTTCAGCCTGGCGGAAGCCTGAGACTGTCTTGTGCTGCCAGCGGCTTCAACATCAAGGACACC TACATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGAATCTACCCCA CCAACGGCTACACCAGATACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCGCCGACACCAG CAAGAACACCGCCTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGC TCTAGATGGGGCGGAGATGGCTTCTACGCTATGGATGTGTGGGGCCAGGGAACACTAGTCACCG TGTCATCTTCAGGTGAGTCCTAACTTCTCCCATTCTAAATGCATGTTGGGGGGATTCTGGGCCT T C AGGAC CGGAT C Ccatagggacaaagagtggagtggggcactttctttagatttgtgaggaat gttccgcactagattgtttaaaacttcatttgttggaaggagagctgtcttagtgattgagtca agggagaaaggcatctagcctcggtctcaaaagggtagttgctgtctagagaggtctggtggag cctgcaaaagtccagctttcaaaggaacacagaagtatgtgtatggaatattagaagatgttgc ttttactcttaagttggttcctaggaaaaatagttaaatactgtgactttaaaatgtgagaggg ttttcaagtactcatttttttaaatgtccaaaattcttgtcaatcagtttgaggtcttgtttgt gtagaactgatattacttaaagtttaaccgaggaatgggagtgaggctctctcataacctattc agaactgacttttaacaataataaattaagtttcaaatatttttaaatgaattgagcaatgttg agttggagtcaagatggccgatcagaaccagaacacctgcagcagctggcaggaagcaggtcat gtggcaaggctatttggggaagggaaaataaaaccactaggtaaacttgtagctgtggtttgaa gaagtggttttgaaacactctgtccagccccaccaaaccgaaagtccaggctgagcaaaacacc acctgggtaatttgcatttctaaaataagttgaggattcagccgaaactggagaggtcctcttt taacttattgagttcaaccttttaattttagcttgagtagttctagtttccccaaacttaagtt tatcgacttctaaaatgtatttagaattcattttcaaaattaggttatgtaagaaattgaagga ctttagtgtctttaatttctaatatatttagaaaacttcttaaaattactctattattcttccc tctgattattggtctccattcaGTCGACGCTAGCCCGAGCGGCCGCAGGAACCCCTagtgatgg agttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccg acgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg

[0641] 5’ ITR (inverted terminal repeat) (bold, lowercase)

[0642] Homology Arm (HA Left) (bold, italic, lowercase) IgHVl-69 minimal promoter (ALL CAPS, BOLD) Murine IgK Signal peptide (ALL CAPS, UNDERLINE) Trastuzumab derived Variable Light Chain — codon optimized for mouse (ALL CAPS

[0643] BOLD, ITALIC)

[0644] IgK murine constant light chain (ALL CAPS PLAIN TEXT) 3X StrepTag II motif (ALL CAPS. DOUBLE-UNDERLINE)

[0645] Gly / Ser linker (ALL CAPS, DASHED UNDERL.INE)

[0646] Trastuzumab derived Variable Heavy Chain - codon optimized for mouse (BOLD,

[0647] ITALIC, UNDERLINE, ALL CAPS)

[0648] Splice donor site (CAGGT) ( AL L CAPS. BOLD. DOUBLE-UNDERLINE)

[0649] 60 bp from IgHJl intronic sequence Homology Arm (HA Right) (lowercase, bold, underline)

[0650] 3 ’ ITR (lowercase, underline)

[0651] Other Embodiments

[0652] From the foregoing description, it will be apparent that variations and modifications may be made to the embodiments and aspects of the disclosure described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0653] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single emb...

Claims

CLAIMSWhat is claimed is:

1. A polynucleotide comprising an insertion region to be inserted into the genome of a cell, wherein the polynucleotide comprises from 5’ to 3’:A) a left homology arm;B) an insertion region comprising: a) a promoter; b) a sequence encoding a polypeptide comprising: i) a heavy chain variable region of an HER2 -binding antibody (VH), a kappa light chain constant region (CLK), a peptide linker, and a light chain variable region of the HER2 -binding antibody (VL); or ii) a light chain variable region of an HER2 -binding antibody (VL), a kappa light chain constant region (CLK), a peptide linker, and a heavy chain variable region of the HER2 -binding antibody (VH); and c) a splice donor site; andC) a right homology arm, wherein the right homology arm and the left homology arm are each capable of binding a polynucleotide strand in the genome of the cell between a region of the immunoglobulin heavy chain (IGH) encoding joining gene segments and an iEp intronic enhancer.

2. The polynucleotide of claim 1 further comprising an inverted terminal repeat at the 5’ end and an inverted terminal repeat at the 3’ end.

3. The polynucleotide of claim 1, wherein the VH comprises the following complementarity determining regions (CDRs):VH CDR1 : DTYIHW;VH CDR2: RIYPTNGYTRYADSVKG; andVH CDR3: WGGDGFYAMDV; and wherein the VL comprises the following CDRs:VL CDR1 : RASQDVNTA;VL CDR2: SAS FLYS; andVL CDR3: QQHYTTPPT.

4. The polynucleotide of claim 1 or claim 3, wherein the VH comprises the following framework (FR) regions:VH FR1 : EVQLVESGGGLVQPGGSLRLSCAASGFNIK;VH FR2: VRQAPGKGLEWVA;VH FR3: RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR; andVH FR4: WGQGTLVTVSS; and wherein the VL comprises the following FR regions:VL FR1 : DIQMTQSPSSLSASVGDRVTITC;VL FR2: VAWYQQKPGKAPKLLIY;VL FR3: GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC; andVL FR4: FGQGTKVEIK.

5. The polynucleotide of claim 1, wherein the VH comprises an amino acid sequence with at least about 85% identity to the following sequence:EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS; and wherein the VL comprises an amino acid sequence with at least about 85% identity to the following sequence:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSG SRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK.

6. The polynucleotide of claim 1, wherein the CLK comprises an amino acid sequence having at least about 85% identity to the following sequence:RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

7. The polynucleotide of claim 1, wherein the peptide linker is a Gly / Ser peptide linker.

8. The polynucleotide of claim 1, wherein the peptide linker comprises an amino acid sequence having at least about 85% identity to the following sequence:GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG.

9. The polynucleotide of claim 1 or claim 8, wherein the peptide linker further comprises a streptavidin tag.

10. The polynucleotide of claim 9, wherein the streptavidin tag is at the N-terminal end or the C -terminal end of the peptide linker.

11. The polynucleotide of claim 9, wherein the streptavidin tag comprises a sequence having at least about 85% identity to the following sequence: WSHPQFEKWSHPQFEKWSHPQFEK.

12. The polynucleotide of claim 1, wherein the splice donor site comprises the following nucleotide sequence, or a fragment thereof capable of mediating splicing of RNA transcribed from the polynucleotide:GTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCAGGTGTACTGGGCCAGGCAAGGGCTTTG.

13. The polynucleotide of claim 1, wherein the polynucleotide strand in the genome of the cell between the region of the IGH encoding joining gene segments and the intronic enhancer iEp comprises the following nucleotide sequence, or a fragment thereof:GGCCCTCCCCGGGCTCAGTCTGAGAGGGTCCCAGGGACTTAGCGGGGTGCCAGTTCTTGCCTGG GGTCCTGGCATTGTTGTCACAATGTGACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCAGGTGAGTCCTCACCACCCCCTCTCTGAGTCCACTTAGGGAGACTCAGCTTGCCAGGGTCTCAGGGTCAGAGTCTTGGAGGCATTTTGGAGGTCAGGAAAGAAAGCTGGGGAGAGGG ACCCTTCGAATGGGAACCCAGCCTGTCCTCCCCAAGTCCGGCCACAGATGTCGGCAGCTGGGGG GCTCCTTCGGCTGGTCTGGGGTGACCTCTCTCCGCTTCACCTGGAGCATTCTCAGGGGCTGTCGTGATGATTGCGTGGTGGGACTCTGTCCCGCTCCAAGGCACCCGCTCTCTGGGACGGGTGCCCCC CGGGGTTTTTGGACTCCTGGGGGTGACTTAGCAGCCGTCTGCTTGCAGTTGGACTTCCCAGGCC GACAGTGGTCTGGCTTCTGAGGGGTCAGGCCAGAATGTGGGGTACGTGGGAGGCCAGCAGAGGGTTCCATGAGAAGGGCAGGACAGGGCCACGGACAGTCAGCTTCCATGTGACGCCCGGAGACAGAA GGTCTCTGGGTGGCTGGGTTTTTGTGGGGTGAGGATGGACATTCTGCCATTGTGATTACTACTA CTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGCCACTCTAGGGCCTTTGTTTTCTGCTACTGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCT CGGGGCATGTTCCGAGGGGACCTGGGCGGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTG AGGATCTGGGAGCCTCTGTGGATTTTCCGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGTCTGATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCACATTTGGACGAGATGCCTGAACAAACC AGGGGTCTTAGTGATGGCTGAGGAATGTGTCTCAGGAGCGGTGTCTGTAGGACTGCAAGATCGC TGCACAGCAGCGAATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGCATCTTAAATTCTTTATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGACGCGTAGCCCCGGTCTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACGGTGGCTGCCGTCCTGACAGGGGCTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAAAGGATTGTTTATCTTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCT GAGAAAAATGGTTAAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTT TTAACTGTCCAAGTATTTGAAATTCTTATCATTTGATTAACACCCATGAGTGATATGTGTCTGG AATTGAGGCCAAAGCAAGCTCAGCTAAGAAATACTAGCACAGTGCTGTCGGCCCCGATGCGGGA CTGCGTTTTGACCATCATAAATCAAGTTTATTTTTTTAATTAATTGAGCGAAGCTGGAAGCAGA TGATGAATTAGAGTCAAGATGGCTGCATGGGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGC AGGTCACCGCGAGAGTCTATTTTAGGAAGCAAAAAAACACAATTGGTAAATTTATCACTTCTGG TTGTGAAGAGGTGGTTTTGCCCAGGCCCAGATCTGAAAGTGCTCTACTGAGCAAAACAACACCT GGACAATTTGCGTTTCTAAAATAAGGCGAGGCTGACCGAAACTGAAAAGGCTTTTTTTAACTAT CTGAATTTCATTTCCAATCTTAGCTTATCAACTGCTAGTTTGTGCAAACAGCATATCAACTTCT AAACTGCATTCATTTTTAAAGTAAGATGTTTAAGAAATTAAACAGTCTTAGGGAGAGTTTATGA CTGTATTCAAAAAGTTTTTTAAATTAGCTTGTTATCCCTTCATGTGATAATTAATCTCAAATAC TTTTTCGATACCTCAGAGCATTATTTTCATAATGACTGTGTTCACAATCTTTTTAGGTTAACTC GTTTTC.

14. The polynucleotide of claim 1, wherein the left homology arm comprises the following nucleotide sequence or a fragment thereof capable of mediating insertion of the insertion region within the genome of the cell: atgtgacgcccggagacagaaggtctctgggtggctgggtttttgtggggtgaggatggacatt ctgccattgtgattactactactactactacatggacgtctggggcaaagggaccacggtcacc gtctcctcaggtaagaatggccactctagggcctttgttttctgctactgcctgtggggtttcc tgagcattgcaggttggtcctcggggcatgttccgaggggacctgggcggactggccaggaggg gatgggcactggggtgccttgaggatctgggagcctctgtggattttccgatgcctttggaaaa tgggactcaggttgggtgcgtctgatggagtaactgagcctgggggcttggggagccacatttg gacgagatgcctgaacaaaccaggggtcttagtgatggctgaggaatgtgtctcaggagcggtg tc.

15. The polynucleotide of claim 1, wherein the right homology arm comprises the following nucleotide sequence or a fragment thereof capable of mediating insertion of the insertion region within the genome of the cell: tgtaggactgcaagatcgctgcacagcagcgaatcgtgaaatattttctttagaattatgaggt gcgctgtgtgtcaacctgcatcttaaattctttattggctggaaagagaactgtcggagtgggt gaatccagccaggagggacgcgtagccccggtcttgatgagagcagggttgggggcaggggtagcccagaaacagtggctgccgtcctgacaggggcttagggaggctccaggacctcagtgccttga agctggtttccatgagaaaaggattgtttatcttaggaggcatgcttactgttaaaagacagga tatgtttgaagtggcttctgagaaaaatggttaagaaaattatgacttaaaaatgtgagagatt ttcaagtatattaatttttttaactgtccaagtatttgaaattcttatcatttgattaacaccc at.

16. The polynucleotide of claim 1, wherein the promoter is a minimal promoter.

17. The polynucleotide of claim 16, wherein the minimal promoter is IGHV1-69.

18. The polynucleotide of claim 1, wherein the encoded polypeptide further comprises an N- terminal signal peptide.

19. The polynucleotide of claim 18, wherein the N-terminal signal peptide comprises an amino acid sequence with at least 85% identity to the following sequence, or a fragment thereof capable of mediating targeting of the encoded polypeptide to a secretory pathway and / or membrane localization: MEAPAQLLFLLLLWLPDTTGEI.

20. The polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence with at least about 85% identity to the following sequence:MEAPAQLLFLLLLWLPDTTGEIDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECWSHPQFEKWSHPQFEKWSHP QFEKGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGEVQLVESGGG LVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSSGESAVWG.

21. The polynucleotide of claim 1, wherein the polynucleotide comprises DNA.

22. A polynucleotide comprising a homology-directed repair cassette comprising the following nucleotide sequence:CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTATGTGACGCCCGGAGACAGAAGGTCTCTGGGTGGCTGGGTTTTTGTGGGGTGAGGATGGACATTCTGCCATTGTGATTACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGCCACTCTAGGGCCTTTGTTTTCTGCTACTGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCTCGGGGCATGTTCCGAGGGGACCTGGGCGGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTGAGGATCTGGGAGCCTCTGTGGATTTTCCGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGTCTGATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCACATTTGGACGAGATGCCTGAACAAACCAGGGGTCTTAGTGATGGCTGAGGAATGTGTCTCAGGAGCGGTGTCGAATTCGTCCTGCTGGACACTCATGTAGGGTAACGAGTGGCCACCTTTTCAGTGTTACCAGTGAGCTCTGAGTGTTCCTAATGGGACCAGGATGGGTCTAGGTGCCTGCTCAATGTCAGAGACAGCAATGGTCCCACAAAAAACCCAGGTAATCTTTAGGCCAATAAAATGTGGGTTCACAGTGAGGAGTGCATCCTGGGGTTGGGGTTTGTTCTGCAGCGGGAAGAGCGCTGTGCACAGAAAGCTTAGAAATGGGGCAAGAGATGCTTTTCCTCAGGCAGGATTTAGGGCTTGGTCTCTCAGCATCCCACACTTGTACAGCTGATGTGGCATCTGTGTTTTCTTTCTCATCCTAGATCAGGCTTTGAGCTGTGAAATACCCTGCCTCATGCATATGCAAATAACCTGAGGTCTTCTGAGATAAATATAGATATATTGGTGCCCTGAGAGCATCACATAACAACCACATTCCTCCTCTGAAGAAGCCCCTGGGAGCACAGCTCATGCCACCATGGAAGCTCCAGCTCAGCTGCTGTTTCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCGAGATCGACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACGTGAACACAGCCGTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCAGCTTTCTGTACAGCGGCGTGCCCAGCAGATTCAGCGGCTCTAGAAGCGGCACCGACTTCACCCTGACCATAAGCAGTCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGCACTACACCACACCTCCAACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAATAGCCAAGAGAGCGTGACCGAGCAGGACAGCAAGGACTCTACCTACAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGTTGGAGCCATCCTCAGTTCGAGAAGTGGTCACACCCACAGTTTGAGAAATGGTCCCATCCGCAATTCGAAAAAGGCGGCAGCTCTGGCAGCGGCAGCGGATCTACTGGAACAAGCTCTAGCGGCACAGGCACAAGCGCTGGCACAACAGGCACATCTGCCAGCACATCTGGCTCTGGATCTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGATCAGCAGGCGGAGAAGTGCAGCTTGTTGAATCTGGCGGTGGCCTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGAATCTACCCCACCAACGGCTACACCAGATACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTTCTAGATGGGGAGGCGACGGCTTCTACGCCATGGATGTTTGGGGACAGGGCACCCTGGTCACCGTGTCATCTTCAGGTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCAGGTGTACTGGGCCAGGCAAGGGCTTTGGATCCTGTAGGACTGCAAGATCGCTGCACAGCAGCGAATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGCATCTTAAATTCTTTATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGACGCGTAGCCCCGGTCTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACAGTGGCTGCCGTCCTGACAGGGGCTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAAAGGATTGTTTATCTTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCTGAGAAAAATGGTTAAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTTTTAACTGTCCAAGTATTTGAAATTCTTATCATTTGATTAACACCCATTCTCGACGCTAGCGTTTCCTTCCCCTGGCTATTCTGCTCAACCTTCCTATCAGAAAAAAAGGGGAAGCGATTCTAGGGAGCAGTCTCCATGACTGTGTGTGGAGTGTTGACAAGAGTTCGGATATTTTATTCTCTACTCAGAATCGCTGCTCCCCCTCACTCTGTTCTGTGTTGTCATTTCCTCTTTCTTTGGTAAGCTTTTAATTTCCAGTTGCATTTTACTAAATTAATTAAGCTGGTTATTTACTTCCCATCCTGATATCAGCTTCCCCTCCTCCTTTCCTCCCAGTCCTTCTCTCTCTCCTCTCTCTTTCTCTAATCCTTTCCTTTCCCTCAGTTCATTTCTTCTTCTTTGATCTACTTTTGTTTGTCTTTTTAAATATTGCCTTGTAACTTGCTCAGAGGACAAGGAAGATATGTCCCTGTTTCTTCTCATAGCTCTCAAGAATAGTAGCATAATTGGCTTTTATGCCAGGGTGACAGGGGAAGAATATATTTTACATATAAATTCTGTTTGACATAGGATTCTTATAATAATTTGTCAGTAGTTTAAGGTTGCAAACCTCGAGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG.

23. The polynucleotide of claim 1, wherein the cell is a human cell.

24. The polynucleotide of claim 1 or claim 23, wherein the cell is a B cell, or a progenitor thereof.

25. A vector comprising the polynucleotide of claim 1 or claim 2.

26. The vector of claim 25, wherein the vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

27. The vector of claim 26, wherein the AAV vector is an AAV6 vector.

28. The vector of claim 26, wherein the AAV vector is an AAV-DJ vector.

29. An immune cell expressing the polypeptide encoded by the polynucleotide of claim 1.

30. The immune cell of claim 29, wherein the immune cell is a B cell.

31. A method for preparing a B cell expressing an engineered B cell receptor, the method comprising:I) contacting a B cell or progenitor thereof with a polynucleotide comprising an insertion region to be inserted into the genome of the cell, wherein the polynucleotide comprises from 5’ to 3’ :A) a left homology arm;B) an insertion region comprising: a) a promoter; b) a sequence encoding a polypeptide comprising: i) a heavy chain variable region of an HER2 -binding antibody (VH), a kappa light chain constant region (CLK), a peptide linker, and a light chain variable region of the HER2 -binding antibody (VL); or ii) a light chain variable region of an HER2 -binding antibody (VL), a kappa light chain constant region (CLK), a peptide linker, and a heavy chain variable region of the HER2 -binding antibody (VH); and c) a splice donor site; andC) a right homology arm; andII) contacting the cell with a nucleic acid programmable DNA binding protein (napDNAbp) having endonuclease activity, or a polynucleotide encoding the napDNAbp, and a guide RNA (gRNA) targeting a sequence within an immunoglobulin heavy chain (IGH) locus in the genome of the cell, wherein the right homology arm and the left homology arm are each capable of binding a polynucleotide strand in the genome of the cell between the region of the IGH encoding joining gene segments and an iEp intronic enhancer, thereby inserting the insertion region within the genome of the B cell at the IGH locus.

32. The method of claim 31 further comprising differentiating the B cell progenitor cell into a B cell.

33. The method of claim 31, wherein the cell is a human cell.

34. The method of claim 31 further comprising culturing the B cell or progenitor thereof in a medium containing cluster of differentiation (CD40) ligand (CD40L).

35. The method of claim 34, wherein culturing the B cells or progenitors thereof in said medium results in a reduction or elimination of development of Bregcells relative to B cells cultured in a medium that does not contain CD40L.

36. The method of claim 31, wherein the napDNAbp is a Cas9 polypeptide or a Casl2 polypeptide.

37. The method of claim 31, wherein the gRNA comprises the following nucleotide sequence at the 5’ end: GUCUCAGGAGCGGUGUCUGU.

38. The method of claim 31, wherein the polynucleotide comprising the insertion region is contacted with the cell using a viral vector.

39. The method of claim 38, wherein the viral vector is an adeno-associated virus (AAV) vector.

40. The method of claim 39, wherein the AAV vector is an AAV6 vector or an AAV-DJ vector.

41. The method of any one of claims 36-38, wherein the polynucleotide comprising the insertion region further comprises an inverted terminal repeat at the 5’ end and an inverted terminal repeat at the 3’ end.

42. The method of claim 31, wherein the VH comprises the following complementarity determining regions (CDRs):VH CDR1 : DTYIHW;VH CDR2: RIYPTNGYTRYADSVKG; andVH CDR3: WGGDGFYAMDV; and wherein the VL comprises the following CDRs:VL CDR1 : RASQDVNTA;VL CDR2: SAS FLYS; andVL CDR3: QQHYTTPPT.

43. The method of claim 31 or claim 42, wherein the VH comprises the following framework (FR) regions:VH FR1 : EVQLVESGGGLVQPGGSLRLSCAASGFNIK;VH FR2: VRQAPGKGLEWVA;VH FR3: RFTI SADTSKNTAYLQMNSLRAEDTAVYYCSR; andVH FR4: WGQGTLVTVSS; and wherein the VL comprises the following FR regions:VL FR1 : DIQMTQSPSSLSASVGDRVTITC;VL FR2: VAWYQQKPGKAPKLLIY;VL FR3: GVPSRFSGSRSGTDFTLTI SSLQPEDFATYYC; andVL FR4: FGQGTKVEIK.

44. The method of claim 31, wherein the VH comprises an amino acid sequence with at least about 85% identity to the following sequence:EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS; and wherein the VL comprises an amino acid sequence with at least about 85% identity to the following sequence:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSG SRSGTDFTLTI SSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK.

45. The method of claim 31, wherein the CLK comprises an amino acid sequence having at least about 85% identity to the following sequence:RTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

46. The method of claim 31, wherein the peptide linker is a Gly / Ser peptide linker.

47. The method of claim 31, wherein the peptide linker comprises an amino acid sequence having at least about 85% identity to the following sequence:GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG.

48. The method of claim 31 or claim 47, wherein the peptide linker further comprises a streptavidin tag.

49. The method of claim 31, wherein the streptavidin tag is at the N-terminal end or the C- terminal end of the peptide linker.

50. The method of claim 49, wherein the streptavidin tag comprises a sequence having at least about 85% identity to the following sequence: WSHPQFEKWSHPQFEKWSHPQFEK.

51. The method of claim 31, wherein the splice donor site comprises the following nucleotide sequence, or a fragment thereof capable of mediating splicing of RNA transcribed from the polynucleotide:GTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCAGGTGTACTGGGCCAGGCAAGGGCTTTG.

52. The method of claim 31, wherein the polynucleotide strand in the genome of the cell between the region of the IGH encoding joining gene segments and the intronic enhancer iEp comprises the following nucleotide sequence, or a fragment thereof:GGCCCTCCCCGGGCTCAGTCTGAGAGGGTCCCAGGGACTTAGCGGGGTGCCAGTTCTTGCCTGGGGTCCTGGCATTGTTGTCACAATGTGACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTCCTCACCACCCCCTCTCTGAGTCCACTTAGGGAGACTCAGCTTGC CAGGGTCTCAGGGTCAGAGTCTTGGAGGCATTTTGGAGGTCAGGAAAGAAAGCTGGGGAGAGGG ACCCTTCGAATGGGAACCCAGCCTGTCCTCCCCAAGTCCGGCCACAGATGTCGGCAGCTGGGGGGCTCCTTCGGCTGGTCTGGGGTGACCTCTCTCCGCTTCACCTGGAGCATTCTCAGGGGCTGTCG TGATGATTGCGTGGTGGGACTCTGTCCCGCTCCAAGGCACCCGCTCTCTGGGACGGGTGCCCCC CGGGGTTTTTGGACTCCTGGGGGTGACTTAGCAGCCGTCTGCTTGCAGTTGGACTTCCCAGGCCGACAGTGGTCTGGCTTCTGAGGGGTCAGGCCAGAATGTGGGGTACGTGGGAGGCCAGCAGAGGG TTCCATGAGAAGGGCAGGACAGGGCCACGGACAGTCAGCTTCCATGTGACGCCCGGAGACAGAA GGTCTCTGGGTGGCTGGGTTTTTGTGGGGTGAGGATGGACATTCTGCCATTGTGATTACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGC CACTCTAGGGCCTTTGTTTTCTGCTACTGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCTCGGGGCATGTTCCGAGGGGACCTGGGCGGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTGAGGATCTGGGAGCCTCTGTGGATTTTCCGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGT CTGATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCACATTTGGACGAGATGCCTGAACAAACC AGGGGTCTTAGTGATGGCTGAGGAATGTGTCTCAGGAGCGGTGTCTGTAGGACTGCAAGATCGC TGCACAGCAGCGAATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGC ATCTTAAATTCTTTATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGAC GCGTAGCCCCGGTCTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACGGTGGCTGCC GTCCTGACAGGGGCTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAA AGGATTGTTTATCTTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCT GAGAAAAATGGTTAAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTT TTAACTGTCCAAGTATTTGAAATTCTTATCATTTGATTAACACCCATGAGTGATATGTGTCTGG AATTGAGGCCAAAGCAAGCTCAGCTAAGAAATACTAGCACAGTGCTGTCGGCCCCGATGCGGGA CTGCGTTTTGACCATCATAAATCAAGTTTATTTTTTTAATTAATTGAGCGAAGCTGGAAGCAGA TGATGAATTAGAGTCAAGATGGCTGCATGGGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGC AGGTCACCGCGAGAGTCTATTTTAGGAAGCAAAAAAACACAATTGGTAAATTTATCACTTCTGG TTGTGAAGAGGTGGTTTTGCCCAGGCCCAGATCTGAAAGTGCTCTACTGAGCAAAACAACACCT GGACAATTTGCGTTTCTAAAATAAGGCGAGGCTGACCGAAACTGAAAAGGCTTTTTTTAACTAT CTGAATTTCATTTCCAATCTTAGCTTATCAACTGCTAGTTTGTGCAAACAGCATATCAACTTCT AAACTGCATTCATTTTTAAAGTAAGATGTTTAAGAAATTAAACAGTCTTAGGGAGAGTTTATGA CTGTATTCAAAAAGTTTTTTAAATTAGCTTGTTATCCCTTCATGTGATAATTAATCTCAAATAC TTTTTCGATACCTCAGAGCATTATTTTCATAATGACTGTGTTCACAATCTTTTTAGGTTAACTC GTTTTC.

53. The method of claim 31, wherein the left homology arm comprises the following nucleotide sequence or a fragment thereof capable of mediating insertion of the insertion region within the genome of the cell: atgtgacgcccggagacagaaggtctctgggtggctgggtttttgtggggtgaggatggacatt ctgccattgtgattactactactactactacatggacgtctggggcaaagggaccacggtcacc gtctcctcaggtaagaatggccactctagggcctttgttttctgctactgcctgtggggtttcc tgagcattgcaggttggtcctcggggcatgttccgaggggacctgggcggactggccaggaggg gatgggcactggggtgccttgaggatctgggagcctctgtggattttccgatgcctttggaaaa tgggactcaggttgggtgcgtctgatggagtaactgagcctgggggcttggggagccacatttg gacgagatgcctgaacaaaccaggggtcttagtgatggctgaggaatgtgtctcaggagcggtg tc.

54. The method of claim 31, wherein the right homology arm comprises the following nucleotide sequence or a fragment thereof capable of mediating insertion of the insertion region within the genome of the cell: tgtaggactgcaagatcgctgcacagcagcgaatcgtgaaatattttctttagaattatgaggt gcgctgtgtgtcaacctgcatcttaaattctttattggctggaaagagaactgtcggagtgggt gaatccagccaggagggacgcgtagccccggtcttgatgagagcagggttgggggcaggggtag cccagaaacagtggctgccgtcctgacaggggcttagggaggctccaggacctcagtgccttga agctggtttccatgagaaaaggattgtttatcttaggaggcatgcttactgttaaaagacagga tatgtttgaagtggcttctgagaaaaatggttaagaaaattatgacttaaaaatgtgagagatt ttcaagtatattaatttttttaactgtccaagtatttgaaattcttatcatttgattaacaccc at.

55. The method of claim 31, wherein the promoter is a minimal promoter.

56. The method of claim 55, wherein the minimal promoter is IGHV1-69.

57. The method of claim 31, wherein the encoded polypeptide further comprises an N- terminal signal peptide.

58. The method of claim 57, wherein the N-terminal signal peptide comprises an amino acid sequence with at least 85% identity to the following sequence, or a fragment thereof capable of mediating targeting of the encoded polypeptide to a secretory pathway and / or membrane localization: MEAPAQLLFLLLLWLPDTTGEI.

59. The method of claim 31, wherein the encoded polypeptide comprises an amino acid sequence with at least about 85% identity to the following sequence:MEAPAQLLFLLLLWLPDTTGEIDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECWSHPQFEKWSHPQFEKWSHP QFEKGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGEVQLVESGGG LVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSSGESAVWG.

60. A method for preparing a B cell expressing an engineered B cell receptor, the method comprising:I) contacting a B cell or progenitor thereof with an adeno-associated virus (AAV) vector comprising a polynucleotide comprising the following sequence:CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTATGTGACGCCCGGAGACAGAAGGTCTCTGGGTGGCTGGGTTTTTGTGGGGTGAGGATGGACATTCTGCCATTGTGATTACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGTAAGAATGGCCACTCTAGGGCCTTTGTTTTCTGCTACTGCCTGTGGGGTTTCCTGAGCATTGCAGGTTGGTCCTCGGGGCATGTTCCGAGGGGACCTGGGCGGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTGAGGATCTGGGAGCCTCTGTGGATTTTCCGATGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGTCTGATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCACATTTGGACGAGATGCCTGAACAAACCAGGGGTCTTAGTGATGGCTGAGGAATGTGTCTCAGGAGCGGTGTCGAATTCGTCCTGCTGGACACTCATGTAGGGTAACGAGTGGCCACCTTTTCAGTGTTACCAGTGAGCTCTGAGTGTTCCTAATGGGACCAGGATGGGTCTAGGTGCCTGCTCAATGTCAGAGACAGCAATGGTCCCACAAAAAACCCAGGTAATCTTTAGGCCAATAAAATGTGGGTTCACAGTGAGGAGTGCATCCTGGGGTTGGGGTTTGTTCTGCAGCGGGAAGAGCGCTGTGCACAGAAAGCTTAGAAATGGGGCAAGAGATGCTTTTCCTCAGGCAGGATTTAGGGCTTGGTCTCTCAGCATCCCACACTTGTACAGCTGATGTGGCATCTGTGTTTTCTTTCTCATCCTAGATCAGGCTTTGAGCTGTGAAATACCCTGCCTCATGCATATGCAAATAACCTGAGGTCTTCTGAGATAAATATAGATATATTGGTGCCCTGAGAGCATCACATAACAACCACATTCCTCCTCTGAAGAAGCCCCTGGGAGCACAGCTCATGCCACCATGGAAGCTCCAGCTCAGCTGCTGTTTCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCGAGATCGACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACGTGAACACAGCCGTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCAGCTTTCTGTACAGCGGCGTGCCCAGCAGATTCAGCGGCTCTAGAAGCGGCACCGACTTCACCCTGACCATAAGCAGTCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGCACTACACCACACCTCCAACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAATAGCCAAGAGAGCGTGACCGAGCAGGACAGCAAGGACTCTACCTACAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGTTGGAGCCATCCTCAGTTCGAGAAGTGGTCACACCCACAGTTTGAGAAATGGTCCCATCCGCAATTCGAAAAAGGCGGCAGCTCTGGCAGCGGCAGCGGATCTACTGG AACAAGCTCTAGCGGCACAGGCACAAGCGCTGGCACAACAGGCACATCTGCCAGCACATCTGGCTCTGGATCTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGATCAGCAGGCGGAGAAGTGCAGCTTG TTGAATCTGGCGGTGGCCTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGG CTTCAACATCAAGGACACCTACATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGG GTCGCCAGAATCTACCCCACCAACGGCTACACCAGATACGCCGACTCTGTGAAGGGCAGATTCA CCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACTCCCTGAGAGCCGAGGA CACCGCCGTGTACTACTGTTCTAGATGGGGAGGCGACGGCTTCTACGCCATGGATGTTTGGGGA CAGGGCACCCTGGTCACCGTGTCATCTTCAGGTGAGTCTGCTGTCTGGGGATAGCGGGGAGCCA GGTGTACTGGGCCAGGCAAGGGCTTTGGATCCTGTAGGACTGCAAGATCGCTGCACAGCAGCGA ATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACCTGCATCTTAAATTCTT TATTGGCTGGAAAGAGAACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGACGCGTAGCCCCGGT CTTGATGAGAGCAGGGTTGGGGGCAGGGGTAGCCCAGAAACAGTGGCTGCCGTCCTGACAGGGG CTTAGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAAAGGATTGTTTATC TTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTTGAAGTGGCTTCTGAGAAAAATGGTT AAGAAAATTATGACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTTTTAACTGTCCAAG TATTTGAAATTCTTATCATTTGATTAACACCCATTCTCGACGCTAGCGTTTCCTTCCCCTGGCT ATTCTGCTCAACCTTCCTATCAGAAAAAAAGGGGAAGCGATTCTAGGGAGCAGTCTCCATGACT GTGTGTGGAGTGTTGACAAGAGTTCGGATATTTTATTCTCTACTCAGAATCGCTGCTCCCCCTC ACTCTGTTCTGTGTTGTCATTTCCTCTTTCTTTGGTAAGCTTTTAATTTCCAGTTGCATTTTAC TAAATTAATTAAGCTGGTTATTTACTTCCCATCCTGATATCAGCTTCCCCTCCTCCTTTCCTCC CAGTCCTTCTCTCTCTCCTCTCTCTTTCTCTAATCCTTTCCTTTCCCTCAGTTCATTTCTTCTT CTTTGATCTACTTTTGTTTGTCTTTTTAAATATTGCCTTGTAACTTGCTCAGAGGACAAGGAAG ATATGTCCCTGTTTCTTCTCATAGCTCTCAAGAATAGTAGCATAATTGGCTTTTATGCCAGGGT GACAGGGGAAGAATATATTTTACATATAAATTCTGTTTGACATAGGATTCTTATAATAATTTGT CAGTAGTTTAAGGTTGCAAACCTCGAGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGG CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG;II) contacting the cell with a Cas9 polypeptide and a guide RNA (gRNA) comprising the sequence GUCUCAGGAGCGGUGUCUGU, thereby insertion a portion of the polynucleotide sequence of I) into the genome of the cell; andIII) culturing the cell in a medium containing cluster of differentiation (CD40) ligand (CD40L).

61. The method of claim 60 further comprising differentiating the B cell progenitor into a B cell.

62. The method of claim 60, wherein the AAV vector is an AAV6 vector or an AAV-DJ vector.

63. A pharmaceutical composition comprising the B cell of claim 30 or a B cell prepared according to the method of any one of claims 31-62, and a pharmaceutically acceptable excipient.

64. A cell prepared according to the method of any one of claims 31-62.

65. The cell of claim 64, wherein the cell expresses a B cell receptor comprising the polypeptide encoded by the insertion region and / or secretes an anti-HER2 polypeptide comprising the polypeptide encoded by the insertion region.

66. A method for treating a subject in need thereof having a neoplasia surface-expressing an HER2 antigen, wherein the method comprises administering to the subject a dose of B cells prepared according to the method of any one of any one of claims 31-6267. The method of claim 66, wherein the method comprises administering to the subject only a single dose of the B cells.

68. The method of claim 66, wherein the neoplasia is a breast cancer.

69. The method of claim 66, wherein the neoplasia comprises a solid tumor.

70. The method of claim 66 further comprising administering to the subject one or more chemotherapeutic agents.

71. The method of claim 69, wherein the subject is administered doxorubicin and / or cyclophosphamide.

72. The method of claim 70 or claim 71, wherein the chemotherapeutic agent is administered at a low dose.

73. The method of any one of claims 66-72, wherein the B cells are autologous to the subject.

74. The method of claim 66, wherein the B cells remain detectable in the subject five weeks following the administering.

75. The method of claim 66, wherein at least about 5% of total splenic B cells and / or at least about 2.5% of bone marrow B cells in the subject are the B cells the engineered B cell receptor following the administering.

76. The method of claim 66, wherein the administering is associated with increased T cell proliferation in the solid tumor and / or in the spleen of the subject relative to a reference subject.

77. The method of claim 66, wherein the administering is associated with an increase in an anti -turn or immune response in the subject relative to a reference subject.

78. A kit suitable for use in the method of any one of claims 31-65 or 69-77, wherein the kit comprises the polynucleotide of any one of claims 1-24, the vector of any one of claims 25-28, the cell of any one of claims 29, 30, 64, or 65, or the pharmaceutical composition of claim 63, and a container.