Systems and methods to produce B cells genetically modified to express selected antibodies

1. A method of genetically engineering B cells to express a selected antibody, the method comprising

delivering into B cells:

a gene editing agent that cuts a targeted genomic region in the B cells; and

a genetic construct comprising from 5′ to 3′:

(i) a nucleotide sequence of a heavy chain promoter;

(ii) a nucleotide sequence encoding a signal peptide;

(iii) a nucleotide sequence encoding the light chain of the selected antibody;

(iv) a nucleotide sequence encoding a flexible linker or a self-cleaving peptide or a nucleotide sequence of a skipping element;

(v) a nucleotide sequence encoding the variable region of the heavy chain of the selected antibody; and

(vi) a nucleotide sequence comprising a splice junction,

wherein the genomic region comprises a sequence as set forth in:

(A) SEQ ID NO: 1;

(B) SEQ ID NO: 2;

(C) SEQ ID NO: 3; or

(D) SEQ ID NO: 4,

and wherein the genetic construct is inserted at the genomic region of (A), (B), (C), or (D) after the cutting by the gene editing agent, thereby genetically engineering the B cells to express the selected antibody.

2. The method of claim 1 , wherein the B cells' endogenous variable heavy chain encoding genome is not excised.

3. The method of claim 1 , wherein the gene editing agent comprises a nuclease associated with a guide RNA (gRNA) having a sequence as set forth in:

SEQ ID NO: 88, 89, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, or 307 when the genomic region is (A);

SEQ ID NO: 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, or 327 when the genomic region is (B);

SEQ ID NO: 87, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, or 346 when the genomic region is (C); or

SEQ ID NO: 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, or 366 when the genomic region is (D).

4. The method of claim 1 , wherein the delivering is through electroporation, a nanoparticle, or viral-mediated delivery.

5. The method of claim 3 , wherein the gRNA and nuclease are delivered through electroporation and the genetic construct is delivered as part of an adeno-associated viral vector.

6. The method of claim 3 , wherein the nuclease is Cas9 or Cpf1.

7. The method of claim 1 , wherein the heavy chain promoter has a sequence as set forth in SEQ ID NO: 111 when the genomic region is (A) or (B); or has a sequence as set forth in SEQ ID NO: 128 when the genomic region is (C) or (D).

8. The method of claim 1 , wherein the signal peptide

has an amino acid sequence as set forth in SEQ ID NO: 185, 186, 187, 188, 189, 190, 191, or 192;

has an amino acid sequence as set forth in SEQ ID NO: 193 or 194;

has an amino acid sequence as set forth in SEQ ID NO: 118 when the genomic region is (A) or (B); or

has an amino acid sequence as set forth in SEQ ID NO: 134 when the genomic region is (C) or (D).

9. The method of claim 1 , wherein the flexible linker

is encoded by a nucleotide sequence as set forth in SEQ ID NO: 116;

has an amino acid sequence as set forth in SEQ ID NOs: 122, 180, 181, 182, 183, or 184; and/or

is a Gly-Ser linker comprising 50-80 amino acids.

10. The method of claim 1 , wherein the self-cleaving peptide has a sequence as set forth in SEQ ID NO: 176, 177, 178, or 179 or wherein the skipping element is an internal ribosome entry site (IRES).

11. The method of claim 1 , wherein the nucleotide sequence comprising the splice junction is as set forth in SEQ ID NO: 124 or 151 when the genomic region is (A) or (B); or is as set forth in SEQ ID NO: 139 when the genomic region is (C) or (D).

12. The method of claim 1 , wherein the genetic construct further encodes a tag having a sequence as set forth in SEQ ID NO: 195, 196, 197, 198, 199, 200, 201, 202, 203, or 204.

13. The method of claim 1 , wherein the selected antibody comprises an anti-Respiratory Syncytial Virus (RSV) antibody comprising

(a) a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 138 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 136;

(b) a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 138 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 205;

(c) a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 123 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 120; or

(d) a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 123 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 206;

an anti-human immunodeficiency virus (HIV) antibody comprising a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 150 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 149;

an anti-pertussis antibody comprising a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 235 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 236;

an anti-influenza antibody comprising a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 159 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 158;

an anti-Epstein Barr virus (EBV) antibody comprising variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 168 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 166; or

an anti-tumor necrosis factor (TNF) antibody comprising a variable heavy chain having the amino acid sequence as set forth in SEQ ID NO: 254 and a variable light chain having the amino acid sequence as set forth in SEQ ID NO: 255.

14. The method of claim 1 , wherein the selected antibody comprises a CDRH1 sequence as set forth in SEQ ID NO: 207, a CDRH2 sequence as set forth in SEQ ID NO: 208, a CDRH3 sequence as set forth in SEQ ID NO: 209; a CDRL1 sequence as set forth in SEQ ID NO: 210, a CDRL2 sequence as set forth in SEQ ID NO: 211, and a CDRL3 sequence as set forth in SEQ ID NO: 212;

a CDRH1 sequence as set forth in SEQ ID NO: 213, a CDRH2 sequence as set forth in SEQ ID NO: 214, a CDRH3 sequence as set forth in SEQ ID NO: 215, a CDRL1 sequence as set forth in SEQ ID NO: 216, a CDRL2 sequence as set forth in SEQ ID NO: 217, and a CDRL3 sequence as set forth in SEQ ID NO: 218;

a CDRH1 sequence as set forth in SEQ ID NO: 219, a CDRH2 sequence as set forth in SEQ ID NO: 220, a CDRH3 sequence as set forth in SEQ ID NO: 221, a CDRL1 of QYGS, a CDRL2 sequence as set forth in SGS, and a CDRL3 sequence as set forth in SEQ ID NO: 222;

a CDRH1 sequence as set forth in SEQ ID NO: 223, a CDRH2 sequence as set forth in SEQ ID NO: 224, a CDRH3 sequence as set forth in SEQ ID NO: 225; a CDRL1 sequence as set forth in SEQ ID NO: 226, a CDRL2 sequence as set forth in SEQ ID NO: 227, and a CDRKL3 as set forth in SEQ ID NO: 228;

a CDRH1 sequence as set forth in SEQ ID NO: 229, a CDRH2 sequence as set forth in SEQ ID NO: 230, a CDRH3 sequence as set forth in SEQ ID NO: 231, a CDRL1 sequence as set forth in SEQ ID NO: 232, a CDRL2 sequence as set forth in SEQ ID NO: 233, and a CDRL3 sequence as set forth in SEQ ID NO: 234;

a CDRH1 sequence as set forth in SEQ ID NO: 237, a CDRH2 sequence as set forth in SEQ ID NO: 238, a CDRH3 sequence as set forth in SEQ ID NO: 239, a CDRL1 sequence as set forth in SEQ ID NO: 240, a CDRL2 sequence as set forth in SEQ ID NO: 241, and a CDRL3 sequence as set forth in SEQ ID NO: 242;

a CDRH1 sequence as set forth in SEQ ID NO: 243, a CDRH2 sequence as set forth in SEQ ID NO: 244, a CDRH3 sequence as set forth in SEQ ID NO: 245, a CDRL1 sequence as set forth in SEQ ID NO: 246, a CDRL2 of KTS, and a CDRL3 sequence as set forth in SEQ ID NO: 247;

a CDRH1 sequence as set forth in SEQ ID NO: 248, a CDRH2 sequence as set forth in SEQ ID NO: 249, a CDRH3 sequence as set forth in SEQ ID NO: 250, a CDRL1 sequence as set forth in SEQ ID NO: 251, a CDRL2 sequence as set forth in SEQ ID NO: 252, and a CDRL3 sequence as set forth in SEQ ID NO: 253;

a CDRH1 sequence as set forth in SEQ ID NO: 256, a CDRH2 sequence as set forth in SEQ ID NO: 257, and a CDRH3 sequence as set forth in SEQ ID NO: 258; a CDRL1 sequence as set forth in SEQ ID NO: 259, a CDRL2 sequence as set forth in SEQ ID NO: 260, and a CDRL3 sequence as set forth in SEQ ID NO: 261;

a CDRH1 sequence as set forth in SEQ ID NO: 262, a CDRH2 sequence as set forth in SEQ ID NO: 263, and a CDRH3 sequence as set forth in SEQ ID NO: 264; a CDRL1 sequence as set forth in SEQ ID NO: 265, a CDRL2 sequence as set forth in SEQ ID NO: 266, and a CDRL3 sequence as set forth in SEQ ID NO: 267; or

a CDRH1 sequence as set forth in SEQ ID NO: 268, a CDRH2 sequence as set forth in SEQ ID NO: 269, and a CDRH3 sequence as set forth in SEQ ID NO: 270; or a CDRL1 sequence as set forth in SEQ ID NO: 271, a CDRL2 sequence as set forth in SEQ ID NO: 272, and a CDRL3 sequence as set forth in SEQ ID NO: 273,

each according to Kabat numbering.

15. The method of claim 1 , wherein the selected antibody is an anti-Respiratory Syncytial Virus (RSV) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-Dengue virus antibody, an anti- Bordatella pertussis antibody, an anti-hepatitis C antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, an anti-metapneumovirus (MPV) antibody, an anti-cytomegalovirus antibody, an anti-Epstein Barr virus antibody; an anti-herpes simplex virus antibody, an anti- Clostridium difficile bacterial toxin antibody, or an anti-tumor necrosis factor (TNF) antibody.

16. The method of claim 1 , wherein the genetic construct comprises a homology arm 5′ of the heavy chain promoter and a homology arm 3′ of the splice junction.

17. The method of claim 16 , wherein the genetic construct comprises a nucleotide sequence as set forth in SEQ ID NO: 102 comprising a coding sequence encoding an anti-RSV antibody having the amino acid sequence as set forth in SEQ ID NO: 126;

a nucleotide sequence as set forth in SEQ ID NO: 103 comprising a coding sequence encoding an anti-RSV antibody having the amino acid sequence as set forth in SEQ ID NO: 141;

a nucleotide sequence as set forth in SEQ ID NO: 104 comprising a coding sequence encoding an anti-RSV antibody having the amino acid sequence as set forth in SEQ ID NO: 141;

a nucleotide sequence as set forth in SEQ ID NO: 105 comprising a coding sequence encoding an anti-HIV antibody having the amino acid sequence as set forth in SEQ ID NO: 152;

a nucleotide sequence as set forth in SEQ ID NO: 106 comprising a coding sequence encoding an anti-influenza antibody having the amino acid sequence as set forth in SEQ ID NO: 160; or

a nucleotide sequence as set forth in SEQ ID NO: 107 comprising a coding sequence encoding an anti-EBV antibody having the amino acid sequence as set forth in SEQ ID NO: 169.

18. The method of claim 16 , wherein

when the genomic region is (A),

the 5′ homology arm is as set forth in SEQ ID NO: 110 or 153, and the 3′ homology arm is as set forth in SEQ ID NO: 125;

the 5′ homology arm is as set forth in SEQ ID NO: 92, and the 3′ homology arm is as set forth in SEQ ID NO: 93; or

the 5′ homology arm is as set forth in SEQ ID NO: 94, and the 3′ homology arm is as set forth in SEQ ID NO: 95

or

when the genomic region is (C),

the 5′ homology arm is as set forth in SEQ ID NO: 127 and the 3′ homology arm is as set forth in SEQ ID NO: 140;

the 5′ homology arm is as set forth in SEQ ID NO: 90 and the 3′ homology arm is as set forth in SEQ ID NO: 91; or

the 5′ homology arm is as set forth in SEQ ID NO: 142 and the 3′ homology arm is as set forth in SEQ ID NO: 143.

19. The method of claim 16 , wherein

when the genomic region comprises (A), the genetic construct comprises:

(i) the 5′ homology arm sequence as set forth in SEQ ID NO: 110;

(ii) the heavy chain promoter having the sequence as set forth in SEQ ID NO: 111;

(iii) a coding sequence encoding the signal peptide having the amino acid sequence as set forth in SEQ ID NO: 118;

(iv) a coding sequence encoding the light chain of the selected antibody having the amino acid sequence as set forth in SEQ ID NO: 285;

(v) a coding sequence encoding the flexible linker having an amino acid sequence as set forth in SEQ ID NO: 122;

(vi) a coding sequence encoding the variable region of the heavy chain of the selected antibody having the amino acid sequence as set forth in SEQ ID NO: 123;

(vii) the splice junction having a sequence as set forth in SEQ ID NO: 124; and

(viii) the 3′ homology arm sequence as set forth in SEQ ID NO: 125; or

when the genomic region comprises (A), the genetic construct comprises:

(i) the 5′ homology arm sequence as set forth in SEQ ID NO: 110;

(ii) the heavy chain promoter having a sequence as set forth in SEQ ID NO: 111;

(iii) a coding sequence encoding the signal peptide having an amino acid sequence as set forth in SEQ ID NO: 118;

(iv) a coding sequence encoding the light chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 287;

(v) a coding sequence encoding the flexible linker having an amino acid sequence as set forth in SEQ ID NO: 122;

(vi) a coding sequence encoding the variable region of the heavy chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 150;

(vii) the splice junction having a sequence as set forth in SEQ ID NO: 151; and

(viii) the 3′ homology arm sequence as set forth in SEQ ID NO: 125; or

when the genomic region comprises (A), the genetic construct comprises:

(i) the 5′ homology arm sequence as set forth in SEQ ID NO: 153;

(ii) the heavy chain promoter having a sequence as set forth in SEQ ID NO: 111;

(iii) a coding sequence encoding the signal peptide having an amino acid sequence as set forth in SEQ ID NO: 118;

(iv) a coding sequence encoding the light chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 288;

(v) a coding sequence encoding the flexible linker having an amino acid sequence as set forth in SEQ ID NO: 122;

(vi) a coding sequence encoding the variable region of the heavy chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 159;

(vii) the splice junction having a sequence as set forth in SEQ ID NO: 151; and

(viii) the 3′ homology arm sequence as set forth in SEQ ID NO: 125; or

when the genomic region comprises (A), the genetic construct comprises:

(i) the 5′ homology arm sequence as set forth in SEQ ID NO: 153;

(ii) the heavy chain promoter having a sequence as set forth in SEQ ID NO: 111;

(iii) a coding sequence encoding the signal peptide having an amino acid sequence as set forth in SEQ ID NO: 118;

(iv) a coding sequence encoding the light chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 289;

(v) a coding sequence encoding the flexible linker having an amino acid sequence as set forth in SEQ ID NO: 122;

(vi) a coding sequence encoding the variable region of the heavy chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 168;

(vii) the splice junction having a sequence as set forth in SEQ ID NO: 151; and

(viii) the 3′ homology arm sequence as set forth in SEQ ID NO: 125; or

when the genomic region comprises (C), the genetic construct comprises:

(i) the 5′ homology arm sequence as set forth in SEQ ID NO: 127;

(ii) the heavy chain promoter having a sequence as set forth in SEQ ID NO: 128;

(iii) a coding sequence encoding the signal peptide having an amino acid sequence as set forth in SEQ ID NO: 134;

(iv) a coding sequence encoding the light chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 286;

(v) a coding sequence encoding the flexible linker having an amino acid sequence as set forth in SEQ ID NO: 122;

(vi) a coding sequence encoding the variable region of the heavy chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 138;

(vii) the splice junction having a sequence as set forth in SEQ ID NO: 139; and

(viii) the 3′ homology arm sequence as set forth in SEQ ID NO: 140; or

when the genomic region comprises (C), the genetic construct comprises:

(i) the 5′ homology arm sequence as set forth in SEQ ID NO: 142;

(ii) the heavy chain promoter having a sequence as set forth in SEQ ID NO: 128;

(iii) a coding sequence encoding the signal peptide having an amino acid sequence as set forth in SEQ ID NO: 134;

(iv) a coding sequence encoding the light chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 286;

(v) a coding sequence encoding the flexible linker having an amino acid sequence as set forth in SEQ ID NO: 122;

(vi) a coding sequence encoding the variable region of the heavy chain of the selected antibody having an amino acid sequence as set forth in SEQ ID NO: 138;

(vii) the splice junction having a sequence as set forth in SEQ ID NO: 139; and

(viii) the 3′ homology arm sequence as set forth in SEQ ID NO: 143.

20. The method of claim 1 , wherein the B cell is an antibody-producing B cell, a memory B cell, a naïve B cell, a B1 B cell or a marginal zone B cell.

21. A B cell modified according to a method of claim 1 .

22. The B cell of claim 21 , wherein the B cell is an antibody-secreting B cell, a memory B cell, a naïve B cell, a B1 B cell or a marginal zone B cell.

23. A method of providing an antibody in a subject in need thereof comprising administering a therapeutically effective amount of the B cells of claim 21 to the subject, thereby providing an antibody to the subject.