MATERIALS AND METHODS FOR ENGINEERING CELLS AND USES THEREOF IN IMMUNO-ONCOLOGY

• US 20190314414A1
• Filed: 06/07/2019
• Published: 10/17/2019
• Est. Priority Date: 05/12/2017
• Status: Active Grant

• Current Assignee: CRISPR Therapeutics AG
• Original Assignee: CRISPR Therapeutics AG
• Inventors: TERRETT, Jonathan Alexander, KALAITZIDIS, Demetrios, KLEIN, Lawrence
• Granted Patent: US 10,729,725 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2239/26 Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 2239/56 Kidney
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/464411 Immunoglobulin superfamily
• CPC Class Codes: A61K 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/464414 CD74, Ii, MHC class II invariant chain or MHC class II gamma chain
• CPC Class Codes: A61K 39/464417 Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
• CPC Class Codes: A61K 39/464438 Tumor necrosis factors [TNF], CD70
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0066 Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70517 CD8
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 14/70578 : NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/02 : containing a signal sequence
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C12N 15/102 : Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1138 : against receptors or cell surface proteins
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/63 : Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/86 : Viral vectors
• CPC Class Codes: C12N 15/907 : in mammalian cells
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2750/14143 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 5/0634 : Cells from the blood or the immune system
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 2521/301 : Endonuclease

Abstract

Materials and methods for producing genome-edited cells engineered to express a chimeric antigen receptor (CAR) construct on the cell surface, and materials and methods for genome editing to modulate the expression, function, or activity of one or more immuno-oncology related genes in a cell, and materials and methods for treating a patient using the genome-edited engineered cells.

Claims

1-186. -186. (canceled)

187. A population of cells comprising engineered human T cells, wherein the engineered human T cells comprise:

(a) a disrupted T cell receptor alpha chain constant region (TRAC) gene comprising a deletion of the nucleotide sequence of SEQ ID NO;

76, and comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising (i) an ectodomain that comprises an anti-BCMA single-chain variable fragment (scFv), wherein the anti-BCMA scFv comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO;

1524, 1530, or 1589, and a variable light chain comprising the amino acid sequence of SEQ ID NO;

1526, 1527, 1531, or 1590, (ii) a CD8 transmembrane domain, and (iii) an endodomain that comprises a CD28 or 41BB co-stimulatory domain and a CD3z co-stimulatory domain; and

(b) a disrupted beta-2-microglobulin (B2M) gene.

188. The population of cells of claim 187, wherein the anti-BCMA scFv comprises:

(i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO;

1524 and a variable light chain comprising the amino acid sequence of SEQ ID NO;

1526;

(ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO;

1524 and a variable light chain comprising the amino acid sequence of SEQ ID NO;

1527;

(iii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO;

1530 and a variable light chain comprising the amino acid sequence of SEQ ID NO;

1531; and

(iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO;

1589 and a variable light chain comprising the amino acid sequence of SEQ ID NO;

1590.

189. The population of cells of claim 188, wherein the anti-BCMA scFv comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:

1589 and a variable light chain comprising the amino acid sequence of SEQ ID NO;

1590.

190. The population of cells of claim 189, wherein the CAR comprises an endodomain comprising a 41BB co-stimulatory domain and a CD3z co-stimulatory domain.

191. The population of cells of claim 187, wherein (i) at least 70% of the engineered human T cells do not express a detectable level of TCR surface protein;

(ii) at least 50% of the engineered human T cells do not express a detectable level of B2M surface protein; and

/or (iii) at least 50% of the engineered human T cells express a detectable level of the CAR.

192. The population of cells of claim 187, wherein (i) co-culture of the engineered human T cells with BCMA+ B cells results in lysis of at least 50% of the BCMA+ cells;

(ii) the engineered human T cells produce interferon gamma in the presence of BCMA+ cells; and

/or (iii) the engineered human T cells do not proliferate in the absence of cytokine stimulation, growth factor stimulation, or antigen stimulation.

193. The population of cells of claim 187, wherein the disrupted B2M gene comprises an insertion of at least one nucleotide base pair and/or a deletion of at least one nucleotide base pair.

194. The population of cells of claim 193, wherein the disrupted B2M gene comprises a nucleotide sequence selected from the group consisting of:

SEQ ID NO;

1560;

SEQ ID NO;

1561;

SEQ ID NO;

1562;

SEQ ID NO;

1563;

SEQ ID NO;

1564; and

SEQ ID NO;

1565.

213. A composition comprising the population of cells of claim 187, and a pharmaceutically acceptable carrier.

195. A population of cells comprising engineered human T cells, wherein the engineered human T cells comprise:

(a) a disrupted TRAC gene comprising a deletion of the nucleotide sequence of SEQ ID NO;

76, and comprising a nucleic acid encoding a CAR comprising (i) an ectodomain that comprises an anti-BCMA scFv, wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO;

1503, 1504, 1505, 1506, 1509, 1511, 1513, or 1514, (ii) a CD8 transmembrane domain, and (iii) an endodomain that comprises a CD28 or 41BB co-stimulatory domain and a CD3z co-stimulatory domain; and

(b) a disrupted B2M gene.

196. The population of cells of claim 195, wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO:

1509.

197. The population of cells of claim 196, wherein the CAR comprises an endodomain comprising a 41BB co-stimulatory domain and a CD3z co-stimulatory domain.

198. The population of cells of claim 195, wherein the anti-BCMA scFv is encoded by the nucleotide sequence of SEQ ID NO:

1479, 1480, 1481, 1482, 1485, 1487, 1489, or 1490.

199. The population of cells of claim 198, wherein the anti-BCMA scFv is encoded by the nucleotide sequence of SEQ ID NO:

1485.

200. The population of cells of claim 195, wherein (i) at least 70% of the engineered human T cells do not express a detectable level of TCR surface protein;

(ii) at least 50% of the engineered human T cells do not express a detectable level of B2M surface protein; and

/or (iii) at least 50% of the engineered human T cells express a detectable level of the CAR.

201. The population of cells of claim 195, wherein (i) co-culture of the engineered human T cells with BCMA+ B cells results in lysis of at least 50% of the BCMA+ B cells;

(ii) the engineered human T cells produce interferon gamma in the presence of BCMA+ cells; and

/or (iii) the engineered human T cells do not proliferate in the absence of cytokine stimulation, growth factor stimulation, or antigen stimulation.

202. The population of cells of claim 195, wherein the disrupted B2M gene comprises an insertion of at least one nucleotide base pair and/or a deletion of at least one nucleotide base pair.

203. The population of cells of claim 202, wherein the disrupted B2M gene comprises a nucleotide sequence selected from the group consisting of:

SEQ ID NO;

1560;

SEQ ID NO;

1561;

SEQ ID NO;

1562;

SEQ ID NO;

1563;

SEQ ID NO;

1564; and

SEQ ID NO;

1565.

214. A composition comprising the population of cells of claim 195, and a pharmaceutically acceptable carrier.

204. A population of cells comprising engineered human T cells, wherein the engineered human T cells comprise:

(a) a disrupted TRAC gene comprising a deletion of the nucleotide sequence of SEQ ID NO;

76, and comprising a nucleic acid encoding a CAR, wherein the CAR comprises the amino acid sequence of SEQ ID NO;

1453, 1454, 1455, 1456, 1457, 1460, 1461, 1463, 1465, or 1466; and

(b) a disrupted B2M gene.

205. The population of cells of claim 204, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

1460.

206. The population of cells of claim 204, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

1461.

207. The population of cells of claim 204, wherein the CAR is encoded by the nucleotide sequence of SEQ ID NO:

1427, 1428, 1429, 1430, 1431, 1434, 1435, 1437, 1439, or 1440.

208. The population of cells of claim 207, wherein the CAR is encoded by the nucleotide sequence of SEQ ID NO:

1435.

209. The population of cells of claim 204, wherein (i) at least 70% of the engineered human T cells do not express a detectable level of TCR surface protein;

(ii) at least 50% of the engineered human T cells do not express a detectable level of B2M surface protein; and

/or (iii) at least 50% of the engineered human T cells express a detectable level of the CAR.

210. The population of cells of claim 204, wherein (i) co-culture of the engineered human T cells with BCMA+ B cells results in lysis of at least 50% of the BCMA+ B cells;

(ii) the engineered human T cells produce interferon gamma in the presence of BCMA+ cells; and

/or (iii) the engineered human T cells do not proliferate in the absence of cytokine stimulation, growth factor stimulation, or antigen stimulation.

211. The population of cells of claim 204, wherein the disrupted B2M gene comprises an insertion of at least one nucleotide base pair and/or a deletion of at least one nucleotide base pair.

212. The population of cells of claim 211, wherein the disrupted B2M gene comprises a nucleotide sequence selected from the group consisting of:

SEQ ID NO;

1560;

SEQ ID NO;

1561;

SEQ ID NO;

1562;

SEQ ID NO;

1563;

SEQ ID NO;

1564; and

SEQ ID NO;

1565.

215. A composition comprising the population of cells of claim 204, and a pharmaceutically acceptable carrier.

216. A method for producing an engineered human T cell, the method comprising delivering to a human T cell:

(a) a RNA-guided nuclease;

(b) a gRNA targeting a TRAC gene;

(c) a gRNA targeting a B2M gene; and

(d) a vector comprising a nucleic acid comprising (i) a nucleotide sequence that comprises a left region of homology to the TRAC locus, (ii) a nucleotide sequence encoding an anti-BCMA CAR, and (iii) a nucleotide sequence that comprises a right region of homology to the TRAC gene locus,

thereby producing an engineered human T cell.