CD28H domain-containing chimeric antigen receptors and methods of use
View Patent ↗Chimeric antigen receptors including (a) an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a first intracellular signaling domain from CD28 homolog (CD28H) and a second intracellular signaling domain are provided. In some examples, the second intracellular domain is from 2B4, TCRζ, FcεR1γ, or DAP12. Chimeric antigen receptors including (a) an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a first intracellular signaling domain from CD28H, a second intracellular signaling domain from 2B4, and a third intracellular signaling domain are also provided. In some examples, the third intracellular domain is from TCRζ, FcεR1γ, or DAP12. Nucleic acid molecules encoding the CARs and expression vectors including the nucleic acids are also provided. Isolated cells (such as T cells or natural killer cells) expressing the CARs and methods of treating a subject with cancer with the isolated cells are provided.
1 . A chimeric antigen receptor comprising:
(a) a target binding domain comprising a CD19 scFv or a CD28H extracellular domain;
(b) a transmembrane domain comprising a CD28H transmembrane domain; and
(c) an intracellular domain comprising a first intracellular signaling domain from CD28 homolog (CD28H) and a second intracellular signaling domain, wherein the first and second intracellular signaling domains can be in either order.
2 . The chimeric antigen receptor of claim 1 , wherein the CD28H intracellular signaling domain comprises an amino acid sequence with at least 90% identity to the amino acids 172-282 of SEQ ID NO: 2 or comprises the amino acid sequence of amino acids 172-282 of SEQ ID NO: 2.
3 . The chimeric antigen receptor of claim 1 , wherein the second intracellular signaling domain is from 2B4, TCRζ, FcεR1γ, or DAP12.
4 . The chimeric antigen receptor of claim 1 , wherein the second intracellular domain is from TCRζ and wherein the TCRζ intracellular signaling domain comprises an amino acid sequence with at least 90% identity to the amino acids 283-395 of SEQ ID NO: 2 or comprises the amino acid sequence of amino acids 283-395 of SEQ ID NO: 2.
5 . The chimeric antigen receptor of claim 1 , wherein the second intracellular signaling domain is from 2B4 and wherein the 2B4 intracellular signaling domain comprises an amino acid sequence with at least 90% identity to the amino acids 172-291 of SEQ ID NO: 10 or comprises the amino acid sequence of amino acids 172-291 of SEQ ID NO: 10.
6 . The chimeric antigen receptor of claim 1 , wherein the intracellular domain further comprises an intracellular region between the transmembrane domain and the first intracellular signaling domain.
7 . The chimeric antigen receptor of claim 6 , wherein the transmembrane domain and intracellular region are from CD16, NKp46, NKp30, NKp44, or KIR2DS4.
8 . A chimeric antigen receptor comprising:
(a) a target binding domain comprising a CD19 scFv or a CD28H extracellular domain;
(b) a transmembrane domain comprising a CD28H transmembrane domain; and
(c) an intracellular domain comprising a first intracellular signaling domain from CD28 homolog (CD28H), a second intracellular signaling domain from 2B4, and a third intracellular signaling domain, wherein the first, second, and third intracellular signaling domains can be in any order.
9 . The chimeric antigen receptor of claim 8 , wherein:
the CD28H intracellular signaling domain comprises an amino acid sequence with at least 90% identity to the amino acids 172-282 of SEQ ID NO: 2 or comprises the amino acid sequence of amino acids 172-282 of SEQ ID NO: 2; and/or
wherein the 2B4 intracellular signaling domain comprises an amino acid sequence with at least 90% identity to the amino acids 172-291 of SEQ ID NO: 10 or comprises the amino acid sequence of amino acids 172-291 of SEQ ID NO: 10.
10 . The chimeric antigen receptor of claim 8 , wherein the third intracellular signaling domain is from TCRζ, FcεR1γ, or DAP12.
11 . The chimeric antigen receptor of claim 1 , further comprising:
a hinge domain, wherein the hinge domain is C-terminal to the target binding domain and N-terminal to the transmembrane domain; and/or
an amino-terminal signal sequence.
12 . The chimeric antigen receptor of claim 11 , wherein the hinge region comprises a CD8α hinge region.
13 . The chimeric antigen receptor of claim 11 , wherein the signal sequence is a CD8α signal sequence.
14 . The chimeric antigen receptor of claim 1 , comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2, 6, 12, 16, and 20 or comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 12, 16, and 20.