US 12,753,437 B2 · Methods of isolating T cells and T-cell receptors from peripheral blood by single-cell analysis for immunotherapy

Abstract

Provided are methods of preparing an enriched population of T cells having antigenic specificity for a target antigen. The method may comprise isolating T cells from a blood sample of a patient; selecting the isolated T cells which have a gene expression profile; and separating the selected T cells from the unselected cells. The separated selected T cells provide an enriched population of T cells having antigenic specificity for the target antigen. Methods of isolating a TCR, preparing a population of cells that express a TCR, isolated TCRs, isolated populations of cells, pharmaceutical compositions, and methods of treating or preventing a condition in a mammal are also provided.

A method of preparing an enriched population of T cells having antigenic specificity for a target antigen, the method comprising:

isolating T cells from a blood sample of a patient;
selecting the isolated T cells which have a gene expression profile;
and
separating the selected T cells from the unselected cells, wherein the separated selected T cells provide an enriched population of T cells having antigenic specificity for the target antigen,
wherein the target antigen is a neoantigen encoded by a cancer-specific mutation, a cancer antigen, or a cancer-associated viral antigen, and wherein:
(a) the gene expression profile comprises all of ACTG1+, AES+, ANXA2+, ANXA5+, ARPC2+, ARPC3+, CD3D+, CD52+, CD7+, CD62L+, CD99+, CORO1A+, COTL1+, CRIP1+, CXCL13+, EMP3+, FLNA+, FTL+, FYB1+, GAPDH+, H2AFV+, HMGB2+, IL32+, ITGB1+, LSP1+, LTB+, PPIA+, S100A10+, S100A4+, S100A6+, SLC25A5+, SUB1+, TIGIT+, TMSB10+, VIM+, ACTB−, B2M−, BTG1−, CCL4−, CCL4L2−, CCL5−, CD74−, EEF1A1−, FTH1−, GZMK−, HLA-DRA−, MTRNR2L12−, PNRC1−, RPL10−, RPL13−, RPL3−, RPL30−, RPL32−, RPL34−, RPLP1−, RPL39−, RPL5−, RPLP0−, RPS12−, RPS14−, RPS18−, RPS19−, RPS21−, RPS23−, RPS24−, RPS3−, RPS3A−, RPS4X−, RPS6−, and ZC3HAV1−;
(b) the gene expression profile comprises all of CARS+, CD39+ (ENTPD1)+, CD62L+, CD70+, CD82+, CXCL13+, HLA-DRB1+, ITGAE+, LGALS3+, SA100A4+, TIGIT+, and TOX+;
(c) the gene expression profile comprises CD8+ and all of ALOX5AP+, ANXA2+, ANXA5+, CARS+, CD82+, CDC25B+, CHN1+, CLECL1+, COTL1+, CYTOR+, FLNA+, GATA3+, HLA-DPA1+, HLA-DQA2+, HLA-DQB1+, HLA-DRA+, HLA-DRB1+, HLA-DRB5+, ITGB1+, ITM2A+, LGALS3+, LIME1+, MYO1G+, P2RY8+, PASK+, RBPJ+, S100A11+, TIGIT+, TPM4+, TRADD+, UBXN11+, CCL4−, CCL5−, CCR7−, CYTIP−, EEF1G−, GZMH−, IMPDH2−, LINCO2446−, LYAR−, MYC−, NKG7−, NUCB2−, PITPNC1−, PLAC8−, and TCF7−;
(d) the gene expression profile comprises CD8+ and all of ALOX5AP+, ANXA2+, ANXA5+, CARS+, CD82+, CDC25B+, CHN1+, CLECL1+, COTL1+, FLNA+, HLA-DPA1+, HLA-DQA2+, HLA-DQB1+, HLA-DRA+, HLA-DRB1+, HLA-DRB5+, ITGB1+, ITM2A+, LGALS3+, LIME1+, MYO1G+, PASK+, S100A11+, TIGIT+, and UBXN11+;
(e) the gene expression profile comprises CD8+ and all of CD45RO+, CD45RA−, HLA-DR+, CD39+, and CD103+;
(f) the gene expression profile comprises CD8+ and all of CD45RO+, CD45RA−, HLA-DR+, CD39+, and TIGIT+;
(g) the gene expression profile comprises CD8+ and all of CD45RO+, CD45RA−, HLA-DR+, CD39+, and PDCD1+;
(h) the gene expression profile comprises CD4+ and all of CD45RO+, CD45RA−, HLA-DR+, and CD39+;
(i) the gene expression profile comprises CD4+ and all of AK4+, APOBEC3G+, C12orf75+, CCL5+, CD74+, CLIC1+, COTL1+, CST7+, CXCL13+, CXCR3+, DUSP2+, EEF1A1+, F2R+, GAPDH+, GNLY+, GZMA+, GZMK+, HCST+, HLA-DPA1+, LYAR+, LYST+, MRPL10+, MYO1G+, NKG7+, PABPC1+, PDCD1+, PFN1+, PRF1+, RAB27A+, RPL10+, RPL11+, RPL13+, RPL18A+, RPL19+, RPL30+, RPL32+, RPL34+, RPL8+, RPL9+, RPLP1+, RPS12+, RPS13+, RPS23+, RPS3A+, RPS8+, SARAF+, SELL+, TC2N+, TMSB4X+, and TPT1+;
(j) the gene expression profile comprises CD4+ and all of AC004585.1+, ACTB+, ACTG1+, ALOX5AP+, ANXA1+, ANXA5+, CD52+, CD99+, CNN2+, COTL1+, FAM45A+, FTH1+, FYB1+, GAPDH+, GIMAP4+, GYPC+, IFITM1+, IFITM2+, IGFBP4+, ITGB1+, LCP1+, LIMS1+, LMO4+, MALAT1+, MIF+, MSN+, MT-ND3+, NDUFA12+, PASK+, PFN1+, PGAM1+, PPP2R5C+, RARRES3+, RILPL2+, RPL30+, RPL32+, RPL34+, RPL9+, RPS13+, RPS25+, RPS3A+, S100A11+, S1PR4+, SERF2+, SLC25A5+, SMC4+, TIMP1+, TMSB4X+, VDAC1+, and ZFP36L2+;
(k) the gene expression profile comprises all of AHNAK+, AK4+, ALOX5AP+, ANXA2+, ANXA5+, ANXA6+, ARL6IP1+, ARPC4+, ATP2B4+, BIN1+, BRI3+, C12orf75+, CALHM2+, CAPN2+, CAPNS1+, CARHSP1+, CD74+, CD81+, CDC25B+, CDCA7+, CLDND1+, CNN2+, COTL1+, CRIP1+, CXCR3+, CYTOR+, DOK2+, DYNLL1+, EIF3A+, ELOVL5+, EMB+, ESYT1+, FLNA+, GPR171+, GYG1+, GZMA+, GZMK+, H1FX+, HACD4+, HIST1H1C+, HLA-DMA+, HLA-DPA1+, HLA-DPB1+, HLA-DQB1+, HLA-DRA+, HLA-DRB1+, HLA-DRB5+, ICAM3+, IDH2+, IFI27L2+, INPP5D+, IQGAP2+, ITGAL+, ITGB1+, ITGB7+, ITM2A+, JPT1+, LAG3+, LGALS1+, LGALS3+, LIME1+, LIMS1+, MAD1L1+, MAP2K2+, MAP4K1+, MBD2+, MED15+, MIS18BP1+, MKNK2+, MXD4+, MYADM+, MYO1F+, MYO1G+, NCK2+, NDUFA7+, NFATC2+, OPTN+, OSBPL8+, P2RY8+, PAG1+, PARP1+, PASK+, PHACTR2+, PRDX3+, PREX1+, PRKCB+, PSD4+, PSMA2+, PYCARD+, RAD23B+, RASA3+, RBM38+, RBPJ+, RCSD1+, RNPEPL1+, S1PR4+, SH2D1A+, SH3KBP1+, SHMT2+, SIT1+, SLC16A3+, SLC2A4RG+, SLC4A7+, SLF1+, SPN+, STK24+, TC2N+, TEX264+, TGFB1+, TIGIT+, TLN1+, TMC8+, TMX4+, TOX+, TPM4+, TRAPPC5+, TXN+, UBXN11+, UCP2+, VOPP1+, WNK1+, YWHAE+, and YWHAQ+;
(l) the gene expression profile comprises all of ALOX5AP+, ARID5B+, CCR4+, CD55+, CDKN1B+, COTL1+, CREM+, DCXR+, DGKA+, ELOVL5+, EML4+, EZR+, GATA3+, GPR183+, ICAM2+, IL7R+, ISG20+, ITGB1+, ITM2A+, LEF1+, LEPROTL1+, LTB+, NR3C1+, P2RY10+, PASK+, PLP2+, PPP2R5C+, PRKX+, RALA+, RASA3+, RCAN3+, RHBDD2+, RNASET2+, S100A11+, S1PR1+, S1PR4+, SAMHD1+, SAMSN1+, SELL+, SESN3+, SETD2+, SMCHD1+, TMEM123+, TRAT1+, and ZFP36+;
(m) the gene expression profile comprises all of ALOX5AP+, ANXA2+, ANXA5+, ARID5B+, CAPN2+, CARS+, CDC25B+, CLDND1+, COTL1+, CREM+, CRIP1+, CXCR3+, CYTOR+, DCXR+, EMB+, FBXW5+, FLNA+, GATA3+, HLA-DPA1+, HLA-DPB1+, HLA-DQB1+, HLA-DRA+, HLA-DRB1+, HLA-DRB5+, HNRNPUL1+, ICAM2+, IL10RA+, ISG15+, ISG20+, ITGB1+, ITGB7+, ITM2A+, KLF2+, LGALS3+, LIME1+, MED15+, MX1+, NDUFA12+, NR3C1+, NSMCE1+, P2RY8+, PASK+, PPP2R5C+, RHBDD2+, RNASET2+, S100A11+, S1PR4+, SAMHD1+, SAMSN1+, SELPLG+, SMCHD1+, SPN+, TIGIT+, TRADD+, and UBXN11+;
(n) the gene expression profile comprises all of ALOX5AP+, ANXA2+, ANXA5+, APOBEC3G+, ARHGEF1+, ARID5B+, BIN1+, BIN2+, C12orf75+, C4orf48+, CAMK4+, CAPN2+, CAPZB+, CARD16+, CARS+, CCNDBP1+, CD5+, CD55+, CD82+, CDC25B+, CHN1+, CLECL1+, CNN2+, CORO1B+, COTL1+, CRIP1+, CYTOR+, DCXR+, DYNLL1+, DYNLT1+, EID1+, EIF3A+, ELOVL5+, EMB+, ETHE1+, FLNA+, FYB1+, GATA3+, GNG2+, HLA-DPA1+, HLA-DPB1+, HLA-DQA2+, HLA-DQB1+, HLA-DRA+, HLA-DRB1+, HLA-DRB5+, ICAM2+, ICAM3+, IL10RA+, IRF7+, ISG15+, ISG20+, ITGAE+, ITGB1+, ITGB7+, ITM2A+, KLF2+, LGALS3+, LIME1+, LY6E+, MAD1L1+, MED15+, MFNG+, MTERF4+, MX1+, MYO1G+, NDUFA12+, NDUFB9+, NELL2+, NR3C1+, OCIAD2+, OPTN+, P2RY8+, PARP1+, PASK+, PLP2+, PPP1R7+, PPP2R5C+, PSMB2+, PSTPIP1+, PYCARD+, RBPJ+, RHBDD2+, RNASEH2B+, RNASET2+, S100A11+, S100A4+, S1PR4+, SAMSN1+, SELPLG+, SH3KBP1+, SHMT2+, SIT1+, SMCHD1+, SPN+, STK38+, SYTL1+, SYTL3+, TAGAP+, TBC1D10C+, TIGIT+, TMPO+, TMX4+, TPGS1+, TPM4+, TRADD+, TSPO+, TXN+, UBE2L6+, UBXN11+, UCP2+, and YWHAB+;
or
(o) the gene expression profile comprises all of ALOX5AP+, ANXA2+, ANXA5+, APOBEC3G+, ARHGEF1+, ARID5B+, BIN1+, BIN2+, C12orf75+, C4orf48+, CAMK4+, CAPN2+, CAPZB+, CARD16+, CARS+, CCNDBP1+, CD5+, CD55+, CD82+, CDC25B+, CHN1+, CLECL1+, CNN2+, CORO1B+, COTL1+, CRIP1+, CYTOR+, DCXR+, DYNLL1+, DYNLT1+, EID1+, EIF3A+, ELOVL5+, EMB+, ETHE1+, FBXW5+, FLNA+, FYB1+, GATA3+, GNG2+, GSTK1+, HLA-DPA1+, HLA-DPB1+, HLA-DQA2+, HLA-DQB1+, HLA-DRA+, HLA-DRB1+, HLA-DRB5+, HNRNPUL1+, ICAM2+, ICAM3+, IL10RA+, IRF7+, ISG15+, ISG20+, ITGAE+, ITGB1+, ITGB7+, ITM2A+, KLF2+, LGALS3+, LIME1+, LY6E+, MAD1L1+, MED15+, MENG+, MTERF4+, MX1+, MYO1G+, NDUFA12+, NDUFB9+, NELL2+, NR3C1+, NUDT21+, OCIAD 2+, OPTN+, P2RY8+, PARP1+, PASK+, PLP2+, PPP1R7+, PPP2R5C+, PSMB2+, PSTPIP1+, PYCARD+, RBPJ+, RHBDD2+, RNASEH2B+, RNASET2+, S100A11+, S100A4+, S1PR4+, SAMSN1+, SELPLG+, SH3KBP1+, SHMT2+, SIT1+, SMCHD1+, SPN+, STK38+, SYTL1+, SYTL3+, TAGAP+, TBC1D10C+, TGFB1+, TIGIT+, TMPO+, TMX4+, TPGS1+, TPM4+, TRADD+, TSPO+, TXN+, UBE2L6+, UBXN11+, UCP2+, YWHAB+, ANKRD12−, APMAP−, CCL4−, CCL5−, CCR7−, CD48−, CD8B−, CXCR4−, CYTIP−, DARS−, EEF1B2−, EEF1G−, GZMH−, HSP90AB1−, IMPDH2−, ISCU−, LBH−, LINCO2446−, LYAR−, MGST3−, MT-ND2−, MT-ND5−, MYC−, NDUFV2−, NFKBIA−, NKG7−, NUCB2−, PDCD4−, PITPNC1−, PLAC8−, PRF1−, PRMT2−, RPL17−, RPS17−, SNHG7−, SNHG8−, STK17A−, TCF7−, TOMM7−, WSB1−, and ZFAS1−.

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Methods of isolating T cells and T-cell receptors from peripheral blood by single-cell analysis for immunotherapy

Life of this patent

Methods of isolating T cells and T-cell receptors from peripheral blood by single-cell analysis for immunotherapy

Life of this patent

Abstract

Claims20