US 11,898,207 B2
Methods of isolating neoantigen-specific T cell receptor sequences
Yong-Chen Lu, Rockville, MD (US); Peter Fitzgerald, Silver Spring, MD (US); Zhili Zheng, Gaithersburg, MD (US); and Steven A. Rosenberg, Potomac, MD (US)
Assigned to The United States of America, as represented by the Secretary, Department of Health and Human Services, Bethesda, MD (US)
Appl. No. 16/495,508
Filed by The United States of America, as represented by the Secretary, Department of Health and Human Services, Bethesda, MD (US)
PCT Filed Mar. 28, 2018, PCT No. PCT/US2018/024828
§ 371(c)(1), (2) Date Sep. 19, 2019,
PCT Pub. No. WO2018/183485, PCT Pub. Date Oct. 4, 2018.
Claims priority of provisional application 62/479,398, filed on Mar. 31, 2017.
Prior Publication US 2020/0056237 A1, Feb. 20, 2020
Int. Cl. A61K 35/17 (2015.01); C12Q 1/6881 (2018.01); G16B 30/10 (2019.01); G16B 30/20 (2019.01); C07K 14/725 (2006.01); C12N 5/0783 (2010.01); C12N 15/10 (2006.01)
CPC C12Q 1/6881 (2013.01) [A61K 35/17 (2013.01); C07K 14/7051 (2013.01); C12N 5/0636 (2013.01); C12N 15/1003 (2013.01); C12N 15/1096 (2013.01); G16B 30/10 (2019.02); G16B 30/20 (2019.02); C12N 2502/30 (2013.01)] 10 Claims
 
1. A method of isolating nucleic acid(s) comprising one or more nucleotide sequences encoding a T cell receptor (TCR) comprising a TCR alpha chain polypeptide and a TCR beta chain polypeptide, or an antigen-binding portion thereof, the method comprising:
(a) isolating, from a biological sample, T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation;
(b) co-culturing the isolated T cells with antigen presenting cells (APCs) that present the mutated amino acid sequence so that the T cells express one or more T cell activation markers;
(c) sorting the co-cultured T cells into separate single T cell samples;
(d) isolating mRNA from each separate single T cell sample;
(e) sequencing the mRNA from each separate single T cell sample, wherein the sequencing comprises:
(i) producing cDNA from the mRNA and amplifying the cDNA;
(ii) producing multiple fragments of the amplified cDNA and tagging the multiple fragments;
(iii) amplifying the tagged, multiple fragments of the cDNA; and
(iv) sequencing the amplified, tagged multiple fragments of the cDNA;
wherein the sequencing identifies the sequences of each of the multiple fragments of cDNA;
(f) aligning the sequences of each of the multiple fragments of cDNA to a known sequence of the one or more T cell activation markers to identify which single T cell sample contained a single T cell which expressed the one or more T cell activation markers;
(g) aligning the sequences of each of the multiple fragments of cDNA to a reference TCR sequence database to identify TCR alpha chain variable (V) segment sequences and TCR beta chain V segment sequences of the multiple fragments of cDNA of each separate single T cell sample which was identified in (f) to express one or more T cell activation markers;
(h) identifying TCR complementarity determining region 3 (CDR3) sequences in the multiple fragments of cDNA containing the TCR alpha chain V segment sequences identified in (g) and in the multiple fragments of cDNA containing the TCR beta chain V segment sequences identified in (g);
(i) counting the number of multiple fragments of cDNA which share the same alpha chain CDR3 amino acid sequence and the number of multiple fragments of cDNA which share the same beta chain CDR3 amino acid sequence;
(j) collecting the highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, the highest number of multiple fragments of cDNA which encode the same beta chain CDR3 sequence and, optionally, the second highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, wherein the alpha chain CDR3 sequence encoded by the second highest number of multiple fragments of cDNA is different from the alpha chain CDR3 sequence encoded by the highest number of multiple fragments of cDNA
to identify the TCR alpha and beta chain CDR3 sequences;
(k) identifying the TCR alpha chain V segment sequence of the highest number of multiple fragments of cDNA collected in (j), the TCR beta chain V segment sequence of the highest number of multiple fragments of cDNA collected in (j) and, optionally, the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA collected in (j)
to identify the TCR alpha and beta chain V segment sequences; and
(l) assembling one or more nucleic acid(s) comprising nucleotide sequences encoding:
a TCR alpha chain polypeptide comprising the TCR alpha chain V segment sequence identified in (k) and the TCR alpha chain CDR3 sequence collected in (j) and
a TCR beta chain polypeptide comprising the TCR beta chain V segment sequence identified in (k) and the TCR beta chain CDR3 sequence collected in (j),
optionally assembling a second one or more nucleic acid(s) comprising nucleotide sequences encoding:
a second TCR alpha chain polypeptide comprising the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA identified in (k) and the TCR alpha chain CDR3 sequence of the second highest number of multiple fragments of cDNA collected in (j) and
the TCR beta chain polypeptide comprising the TCR beta chain V segment sequence identified in (k) and the TCR beta chain CDR3 sequence collected in (j)
to produce isolated nucleic acid(s) comprising one or more nucleotide sequences encoding the TCR comprising the TCR alpha chain polypeptide and the TCR beta chain polypeptide, or an antigen-binding portion thereof, wherein the TCR has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.