Chimeric antigen receptors and related methods and compositions for the treatment of cancer
Aspects of the disclosure relate to novel scFv molecules that are useful for incorporation into novel chimeric antigen receptors with enhance anti-tumor activity. Further aspects relate to a polypeptide comprising a CAR comprising, in order from amino proximal to carboxy proximal end, a scFv, a transmembrane domain, a torsional linker, and a cytoplasmic region comprising a primary intracellular signaling domain, wherein the torsional linker comprises 1-12 alanine residues. Also described are nucleic acids comprising a sequence encoding a polypeptide of the disclosure, vectors, such as lentiviral vectors comprising the nucleic acids of the disclosure, cells comprising and/or expressing nucleic acids and/or polypeptides of the disclosure, and populations of cells comprising the cell embodiments of the disclosure. Also provided are methods of making cells that express a polypeptide and methods of treating patients with the polypeptides and cell compositions of the disclosure.
1 . A method of treating a patient with brain cancer, comprising administering to the patient a population of cells that express a polypeptide comprising an anti-CD20 single chain variable fragment (scFv) comprising:
a light chain variable region (VL) comprising in order from amino-proximal to carboxy-proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region I (LCDR1), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); and
a heavy chain variable region (V-H) comprising in order from amino-proximal to carboxy-proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity-determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity-determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity-determining region 3 (HCDR3), and heavy chain framework region 4 (HFR4);
wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOS:29, 30, 31, 22, 12, 13, and 14, respectively; and wherein HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOS:26, 27, 28, 18, 9, 10, and 11, respectively.
2 . The method of claim 1 , wherein the light chain variable region comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5 and a heavy chain variable region comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO:6.
3 . The method of claim 1 , wherein the VII is amino proximal to the VL.
4 . The method of claim 1 , wherein the VH is carboxy proximal to the VL.
5 . The method of claim 1 , wherein the polypeptide comprises a chimeric antigen receptor (CAR) comprising the scFv, a transmembrane domain and a cytoplasmic region comprising a primary intracellular signaling domain.
6 . The method of claim 5 , wherein the CAR further comprises an extracellular spacer between the transmembrane domain and the scFv.
7 . The method of claim 5 , wherein the transmembrane domain is an alpha or beta chain of the T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154 transmembrane domain.
8 . The method of claim 5 , wherein the cytoplasmic region further comprises one or more costimulatory domains.
9 . The method of claim 8 , wherein the one or more costimulatory domain(s) comprise a costimulatory domain from one or more of 4-1BB (CD137), CD28, IL-15Ra, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), and/or ICOS (CD278).
10 . The method of claim 1 , wherein the polypeptide comprises SEQ ID NO:33 or 42.
11 . The method of claim 5 , wherein the polypeptide further comprises a torsional linker between the transmembrane domain and the cytoplasmic region.
12 . The method of claim 11 , wherein the torsional linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alanine residues.
13 . A method of treating a patient with brain cancer, comprising administering to the patient a population of cells that express a polypeptide comprising a CAR comprising, in order from amino proximal to carboxy proximal end, an anti-CD20 scFv, a CD28 transmembrane domain, a torsional linker, and a cytoplasmic region comprising a CD28 costimulatory domain and a CD3-zeta intracellular signaling domain, wherein the torsional linker comprises 1-12 alanine residues, wherein the anti-CD20 scFv comprises
a light chain variable region (VL) comprising in order from amino-proximal to carboxy-proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); and
a heavy chain variable region (VH) comprising in order from amino-proximal to carboxy-proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity-determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity-determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity-determining region 3 (HCDR3), and heavy chain framework region 4 (HFR4);
wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOS:29, 30, 31, 22, 12, 13, and 14, respectively; and wherein HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOS:26, 27, 28, 18, 9, 10, and 11, respectively.
14 . The method of claim 1 , wherein the cells are T cells, natural killer cells, stem cells, bone marrow cells, fetal liver cells, or cord blood cells.
15 . The method of claim 13 , wherein the cells are T cells, natural killer cells, stem cells, bone marrow cells, fetal liver cells, or cord blood cells.
16 . The method of claim 14 , herein the T cells are naïve memory T cells, natural killer T cells, or invariant natural killer T cells.
17 . The method of claim 14 , wherein the stem cells are embryonic stem cells, hematopoietic stem or progenitor, or induced pluripotent stem cells.
18 . The method of claim 15 , wherein the T cells are naïve memory T cells, natural killer T cells, or invariant natural killer T cells.
19 . The method of claim 15 , wherein the stem cells are embryonic stem cells, hematopoietic stem or progenitor cells, or induced pluripotent stem cells.