CD70 CAR-T Compositions and Methods for Cell-based Therapy

An engineered cell comprising an anti-CD70 chimeric antigen receptor (CAR), wherein the anti-CD70 CAR comprises:

• a) an antigen-binding protein, or a fragment thereof, that specifically binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementarity determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region comprising a complementarity determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein
• 1)
• i) the VH CDR1 comprises an amino acid sequence of any one of SEQ ID NOs:
• 73, 67, 70, 76, 79, and 82;
• ii) the VH CDR2 comprises an amino acid sequence of any one of SEQ ID NOs:
• 74, 68, 71, 77, 80, and 83;
• iii) the VH CDR3 comprises an amino acid sequence of any one of SEQ ID NOs:
• 75, 69, 72, 78, 81, and 84;
• iv) the VL CDR1 comprises an amino acid sequence of any one of SEQ ID NOs:
• 55, 49, 52, 58, 61, and 64;
• v) the VL CDR2 comprises an amino acid sequence of any one of SEQ ID NOs:
• 56, 50, 53, 59, 62, and 65;
• and
• vi) the VL CDR3 comprises an amino acid sequence of any one of SEQ ID NOs:
• 57, 51, 54, 60, 63, and 66;
• and/or
• 2) wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of:
• i) SEQ ID NOs:
• 73, 74, 75, 55, 56, and 57, respectively;
• ii) SEQ ID NOs:
• 67, 68, 69, 49, 50, and 51, respectively;
• iii) SEQ ID NOs:
• 70, 71, 72, 52, 53, and 54, respectively;
• iv) SEQ ID NOs:
• 76, 77, 78, 58, 59, and 60, respectively;
• v) SEQ ID NOs:
• 79, 80, 81, 61, 62, and 63, respectively;
• or
• vi) SEQ ID NOs:
• 82, 83, 84, 64, 65, and 66, respectively;
• b) a transmembrane domain;
• and
• c) an intracellular domain comprising a costimulatory domain comprising an amino acid sequence of SEQ ID NO:
• 99 or 101.

A method of making an engineered cell comprising an anti-CD70 CAR, comprising:

• 1)
• (a) contacting the cell with a first group of lipid nanoparticles (LNPs) comprising a LNP comprising a uracil glycosylase inhibitor (UGI) and at least one LNP comprising a base editor and at least one guide RNA that is cognate to the base editor and targets a genomic locus chosen from the HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 locus;
• and
• (b) contacting a cell with
• i) a second group of LNPs comprising a LNP comprising a UGI and at least one LNP comprising a base editor and at least one guide RNA that is cognate to the base editor and targets a genomic locus chosen from the HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 locus that are different from the at least one guide RNA contained in the first group of LNPs of the step (a);
• and
• ii) at least one LNP comprising a RNA-guided cleavase and at least one gRNAs that is cognate to the RNA-guided cleavase and targets the TRAC locus;
• and
• iii. iii) a nucleic acid encoding an anti-CD70 CAR for insertion into an editing site (e.g., a double strand break) at the TRAC locus;
• or
• 2)
• (a) contacting a cell with a first group of lipid nanoparticles (LNPs) comprising a first LNP comprising a base editor and a gRNA that targets the HLA-A locus;
• a second LNP comprising a base editor and comprising a gRNA that targets the HLA-B locus, a third LNP comprising a base editor and comprising a gRNA that targets the CIITA locus, and a fourth LNP comprising a UGI;
• and
• (b) contacting a cell with (i) a second group of LNPs comprising a fifth LNP comprising a base editor and comprising a gRNA that targets the TGFBR2 locus;
• a sixth LNP comprising a base editor and comprising a gRNA that targets the CD70 locus;
• (ii) a seventh LNP comprising an RNA-guided DNA cleavase and a gRNA that is cognate to the RNA-guide DNA cleavase and targets the TRAC locus, and an eight lipid LNP comprising a UGI;
• and (iii) a nucleic acid encoding an anti-CD70 CAR for insertion into an editing site (e.g., a double strand break) at the TRAC locus.

An engineered cell comprising a genetic modification in the HLA-A gene, a genetic modification in the HLA-B gene, a genetic modification in the TRAC gene, a genetic modification in the CIITA gene, a genetic modification in the TGFBR2 gene, and/or a genetic modification in the CD70 gene, wherein the genetic modification in the HLA-A gene is within the genomic coordinates chr6:

• 29942891-29942915;
• wherein the genetic modification in the HLA-B gene is within the genomic coordinates chr6:
• 31355222-31355246, chr6:
• 31355221-31355245, or chr6:
• 31355205-31355229;
• wherein the genetic modification in the TRAC gene is within the genomic coordinates chr14:
• 22547524-22547544;
• wherein the genetic modification in the CIITA gene is within the genomic coordinates chr16:
• 10907504-10907528 or chr16:
• 10906643-10906667;
• wherein the genetic modification in the TGFBR2 gene is within the genomic coordinates chr3:
• 30674205-30674229 or chr3:
• 30671941-30671961;
• and wherein the genetic modification in the CD70 gene is within the genomic coordinates chr19:
• 6590121-6590145 or chr19:
• 6590998-6591018, wherein the engineered cell comprises an anti-CD70 chimeric antigen receptor (CAR) or comprises a nucleic acid encoding an anti-CD70 CAR.