Anti-MUC1 compositions and methods of use
Disclosed are antibodies against MUC1, MUC1-CAR compositions and methods for use of these antibodies and compositions to target a MUC1 protein, wherein a cell expressing the MUC1 protein may be targeted and killed by, for instance, a cytotoxic T cell.
1 . A chimeric antigen receptor (CAR) comprising
(a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one anti-MUC1 single chain variable fragment (scFv) comprising a heavy chain variable region comprising the amino acid sequence of
QVQLVQSGAEVKKPGSSVKVSCKTSGYAFSNFWMNWVRQAPGOGLEWIGQIYPGDGDTNYNAKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYFCARSYYRSAWFAYWGOGTLVTVSS (SEQ ID NO:4); and
a light chain variable region comprising the amino acid sequence of
EILLTOSPDFQSVTPKEKVTFTCRASQSIGTSIHWYQQKPNQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSLESEDIATYYCQQSNNWPLTFGOGTKLEIK (SEQ ID NO:9), wherein the scFv comprises a linker between the heavy chain variable region and the light chain variable region;
(b) a transmembrane domain, and
(c) an endodomain comprising at least one signal transduction domain.
2 . The CAR of claim 1 , wherein the linker comprises the amino acid sequence of SEQ ID NO: 59.
3 . The CAR of claim 1 , wherein the scFv comprises the amino acid sequence of SEQ ID NO: 125.
4 . The CAR of claim 1 , wherein the ectodomain further comprises a signal peptide.
5 . The CAR of claim 4 , wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 57.
6 . The CAR of claim 1 , wherein the CAR further comprises a hinge region between the antigen recognition region and the transmembrane domain.
7 . The CAR of claim 6 , wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 61.
8 . The CAR of claim 1 , wherein the transmembrane domain comprises a sequence encoding a CD8 transmembrane domain.
9 . The CAR of claim 8 , wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 63.
10 . The CAR of claim 1 , wherein the at least one signal transduction domain comprises a CD3ζ intracellular signaling domain, a 4-1BB intracellular signaling domain, or a combination thereof.
11 . The CAR of claim 1 , wherein the at least one signal transduction domain comprises a CD3ζ intracellular signaling domain and a 4-1BB intracellular signaling domain, and wherein the 4-1BB intracellular signaling domain is located between the transmembrane domain and the CD3ζ costimulatory-intracellular signaling domain.
12 . The CAR of claim 11 , wherein the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 65.
13 . The CAR of claim 11 , wherein the CD3ζ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 67.
14 . The CAR of claim 1 , wherein
the ectodomain comprises a signal peptide,
the CAR further comprises a hinge region between the antigen recognition region and the transmembrane domain,
the transmembrane domain comprises a sequence comprising a CD8 transmembrane domain; and
the at least one signal transduction domain comprises a CD3ζ intracellular signaling domain and a 4-1BB intracellular signaling domain, and wherein the 4-1BB intracellular signaling domain is located between the transmembrane domain and the CD3ζ intracellular signaling domain.
15 . The CAR of claim 14 , wherein
the scFv comprises an amino acid sequence of SEQ ID NO: 125;
wherein the signal peptide comprises SEQ ID NO: 57;
wherein the hinge region comprises SEQ ID NO: 61;
wherein the CD8 transmembrane domain comprises SEQ ID NO: 63;
wherein the 4-1BB intracellular signaling domain comprises SEQ ID NO: 65; and
wherein the CD3ζ intracellular signaling domain comprises SEQ ID NO: 67.
16 . The CAR of claim 14 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 13.
17 . The CAR of claim 14 , wherein the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 31 or SEQ ID NO: 167.
18 . The CAR of claim 17 , wherein the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 167.
19 . A polynucleotide comprising a nucleic acid sequence encoding the CAR of claim 1 .
20 . A vector comprising the polynucleotide of claim 19 .
21 . A cell comprising the CAR of claim 1 .
22 . A population of cells, wherein a plurality of the population of cells are modified to express the CAR of claim 1 .
23 . The population of cells of claim 22 , wherein the plurality of modified cells is a plurality of modified immune cells.
24 . The population of cells of claim 22 , wherein the plurality of modified cells is a plurality of modified T-cells.
25 . The population of cells of claim 22 , wherein the plurality of the population of cells comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells that express the CAR of claim 1 .
26 . A composition comprising the cell of claim 21 .
27 . A composition comprising the population of cells of claim 23 .
28 . A pharmaceutical composition comprising the composition of claim 26 and a pharmaceutically acceptable carrier.
29 . A method of treating a MUC1 positive cancer in a subject in need thereof comprising administering a therapeutically effective amount of the composition of claim 27 .
30 . The method of claim 29 , wherein the cancer is a MUC1-C positive cancer.
31 . The method of claim 29 , wherein the cancer is a lung cancer, a brain cancer, a head and neck cancer, a breast cancer, a skin cancer, a liver cancer, a pancreatic cancer, a stomach cancer, a colon cancer, a rectal cancer, a uterine cancer, a cervical cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a skin cancer or an esophageal cancer.
32 . The cell of claim 21 further comprising a a gene encoding a dihydrofolate reductase (DHFR) protein.
33 . The cell of claim 32 , wherein the gene encoding the DHFR protein comprises the sequence of SEQ ID NO: 93 or 174.
34 . The cell of claim 32 , wherein the DHFR protein comprises the sequence of SEQ ID NO: 92.