T cell receptors and methods of use thereof
The present disclosure is directed recombinant T cell receptors capable of binding a gp100 epitope and nucleic acid molecules encoding the same. In some embodiments, the nucleic acid molecules further comprise a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR. Other aspects of the disclosure are directed to vectors comprising the nucleic acid molecule and cells comprising the recombinant TCR, the nucleic acid molecule, or the vector. Still other aspects of the disclosure are directed to methods of using the same. In some embodiments, the methods comprise treating a cancer in a subject in need thereof.
1 . A nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds human gp100 (“anti-gp100 TCR”); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR,
wherein the anti-gp100 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and
wherein the beta chain comprises variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3;
wherein:
(i) the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 10;
(ii) the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 9;
(iii) the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 6;
(iv) the alpha chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 7;
(v) the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 8; and
(vi) the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 5
wherein the anti-gp100 TCR cross competes for binding to human gp100 with a reference TCR, which comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2.
2 . The nucleic acid molecule of claim 1 , wherein the anti-gp100 TCR binds to an epitope of gp100 consisting of an amino acid sequence as set forth in SEQ ID NO: 13.
3 . The nucleic acid molecule of claim 2 , wherein the epitope is complexed with an HLA class I molecule selected from an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G allele.
4 . The nucleic acid molecule of claim 3 , wherein the HLA class I molecule is an HLA-B*40 allele, optionally selected from an HLA-B*40:01 allele, HLA-B*40:02 allele, an HLA-B*40:03 allele, an HLA-B*40:04 allele, an HLA-B*40:05 allele, and an HLA-B*40:06 allele.
5 . The nucleic acid molecule of claim 1 , wherein
(i) the alpha chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 1;
(ii) the beta chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 2; or
(iii) both (i) and (ii).
6 . The nucleic acid molecule of claim 1 , wherein:
(a) the alpha chain of the anti-gp100 TCR further comprises a constant region, wherein the constant region is different from endogenous constant region of the alpha chain, and wherein
(i) the alpha chain constant region comprises an amino acid sequence having at least about 85% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 1; or
(ii) the alpha chain constant region comprises an amino acid sequence comprising at least 1 amino acid substitution relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 1;
(b) the beta chain of the anti-gp100 TCR further comprises a constant region, wherein the constant region is different from endogenous constant regions of the beta chain, and wherein
(i) the beta chain constant region comprises an amino acid sequence having at least about 85% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 2; or
(ii) the beta chain constant region comprises an amino acid sequence comprising at least 1 amino acid substitution relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 2; or
(c) both (a) and (b).
7 . The nucleic acid molecule of claim 1 , wherein
(i) the alpha chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 1;
(ii) the beta chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 2; or
(iii) both (i) and (ii).
8 . The nucleic acid molecule of claim 1 , wherein the second nucleotide sequence
(i) is one or more siRNAs that reduce the expression of endogenous TCRs, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the endogenous TCRs;
(ii) encodes Cas9; or
(iii) both (i) and (ii).
9 . The nucleic acid molecule of claim 1 , wherein the anti-gp100 TCR comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprises an amino acid sequence having at least 1 substitution within the target sequence relative to the corresponding amino acid sequence of an endogenous TCR.
10 . A vector comprising the nucleic acid molecule of claim 1 .
11 . A T cell receptor (TCR) or an antigen binding portion thereof comprising the alpha chain variable domain and the beta chain variable domain of the anti-gp100 TCR of claim 1 .
12 . A bispecific TCR comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises the TCR or an antigen-binding portion thereof of claim 11 .
13 . A cell comprising the nucleic acid molecule of claim 1 .
14 . A method of treating a cancer in a subject in need thereof, comprising administering to the subject the cell of claim 13 .
15 . A method of engineering an antigen-targeting cell, comprising transducing a cell collected from a subject in need of a T cell therapy with the nucleic acid molecule of claim 1 .