Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions - PubMed (original) (raw)

. 2008 May 1;180(9):6116-31.

doi: 10.4049/jimmunol.180.9.6116.

Yong F Li, Mona El-Gamil, Yangbing Zhao, Jennifer A Wargo, Zhili Zheng, Hui Xu, Richard A Morgan, Steven A Feldman, Laura A Johnson, Alan D Bennett, Steven M Dunn, Tara M Mahon, Bent K Jakobsen, Steven A Rosenberg

Affiliations

Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions

Paul F Robbins et al. J Immunol. 2008.

Abstract

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

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Figures

FIGURE 1

FIGURE 1

Generation of AAS variants of 1G4, DMF5, and DMF4 TCRs (A). Pairs of oligonucleotide primers designated _α_1 and _α_2 or _β_1 and _β_2 were used to amplify regions of TCR _α_-or _β_-chain sequences, respectively, that were upstream of the AAS. The AAS were encoded within a primer that partially overlapped with the _α_2 or _β_2 primer, designated _α_3 or _β_3, that was used to carry out a second PCR with primer _α_4 or _β_4. The products of the first two PCR were combined and amplified using primers _α_1 and _α_4 or _β_1 and _β_4. The nucleotide and amino acid sequences of the 1G4, DMF5, and DMF4 α and β TCR chains are presented in B, C, and D, respectively, and specific primers used to generate the AAS variants detailed in Table I.

FIGURE 2

FIGURE 2

Rapid screening of NY-ESO-1/HLA-A*02-reactive TCR 1G4 CDR3_α_ variants in CD8+ and CD4+ T cells. T cells were activated with anti-CD3 Ab and isolated CD8+ (A and B) or CD4+ (C) T cells were cotransfected with RNA 1G4 TCR _α_-chain constructs encoding AAS within the CDR3 region along with a WT 1G4 TCR _β_-chain RNA construct. Transfected T cells were incubated overnight with T2 cells pulsed with the indicated concentrations of NY-ESO-1 peptide or with 1 _μ_M control peptide, gp100:154-162 (A), or with HLA-A*02+ melanoma and breast cancer target cell lines that did or did not also express the NY-ESO-1 transcript (B and C). The release of IFN-γ was measured the following day.

FIGURE 3

FIGURE 3

Peptide titration of responses of CD8+ and CD4+ T cells transfected with NY-ESO-1/HLA-A*02-reactive 1G4 WT, CDR2_β_, and CDR3_α_ TCR variants. T cells were activated with anti-CD3 Ab and isolated CD8+ (A and B) or CD4+ (C and D) T cells were cotransfected with RNA-encoding TCR constructs containing substitutions in the 1G4 _α_- or _β_-chains along with the complementary 1G4 WT TCR chain. The releases of IFN-γ in response to T2 cells pulsed with varying doses of the NY-ESO-1 peptide or with 10 nM control HLA-A*02-binding peptide from gp100 corresponding to amino acids 154-163 (KTWGQYWQV) were measured the following day.

FIGURE 4

FIGURE 4

Rapid screening of NY-ESO-1/HLA-A*02-reactive TCR 1G4 CDR2 and CDR3 β variants in CD8+ and CD4+ T cells. T cells were activated with anti-CD3 Ab and isolated CD8+ (A-C) or CD4+ (D) T cells were cotransfected with RNA 1G4 TCR _β_-chain constructs encoding AAS within the CDR2 or CDR3 regions along with a WT 1G4 TCR _α_-chain RNA construct. Transfected T cells were incubated overnight with T2 cells pulsed with the indicated concentrations of NY-ESO-1 peptide or a control peptide, gp100:154-162 (A and B), or HLA-A*02+ melanoma target cell lines that did or did not also express NY-ESO-1 transcript (C and D). The release of IFN-γ was measured the following day.

FIGURE 5

FIGURE 5

Effects of NY-ESO-1/HLA-A*02-reactive TCR 1G4 CDR3_α_ and CDR2_β_ variants on the cytolytic activity of CD8+ and CD4+ T cells. The cytolytic activities of anti-CD3 Ab stimulated CD8+ (A) and CD4+ (B) T cells cotransfected with 1G4 WT and variant TCRs to the HLA-A*02+ and NY-ESO-1+ melanoma cell lines 624.38 and A375 as well as the response to the HLA-A*02+, but the NY-ESO-1- renal cell carcinoma line 2661R was measured in standard 4-h 51Cr-release cell lysis assays.

FIGURE 6

FIGURE 6

Effects of NY-ESO-1/HLA-A*02-reactive 1G4 WT and variant TCRs on CD8+ and CD4+ T cell activity following retroviral transduction. Anti-CD3 Ab stimulated PBMC were transduced on days 2 and 3 with retroviral supernatants that coexpressed either the 1G4 WT α and β, α_95:LY and WT β, or WT_α and _β_51:AI TCR chains. Ten days following anti-CD3 Ab stimulation, CD8+ (A and C) and CD4+ (B and D) T cells were separated and were tested 4 days later for their ability to release IFN-γ (A and B) or IL-2 (C and D) in response to melanoma, lung carcinoma, and renal cell carcinoma cell lines. The results presented are representative of those obtained following the transduction of three samples of PBMC. Cell surface TCR expression was evaluated by FACS analysis of V_β_13 as well as NY-ESO-1/HLA-*02 tetramer staining 10 days following anti-CD3 Ab stimulation, and coculture assays were initiated on day 13.

FIGURE 7

FIGURE 7

Rapid screening of MART-1/HLA-A*02-reactive TCR DMF5 and DMF4 WT and CDR2_β_ variants. T cells were activated with anti-CD3 Ab and isolated CD8+ (A and C) and CD4+ (B and D) T cells were cotransfected with RNA constructs encoding the WT α, along with either the WT β_-chain or CDR2_β variant chains generated from either the DMF5 TCR (A and B) or the DMF4 TCR (C and D). Transfected T cells were evaluated the following day for their ability to generate IFN-γ in response to melanoma target cell lines.

FIGURE 8

FIGURE 8

Effects of MART-1/HLA-A*02-reactive TCR DMF4 and DMF5 CDR2_β_ variants on the cytolytic activity of CD8+ and CD4+ T cells. The cytolytic activities of CD8+ (A and B) and CD4+ (C and D) T cells transfected with RNA encoding the DMF4 and DMF5 WT and variant TCRs to the HLA-A*02+ and MART-1+ melanoma cell line 624.38 (A and C) and to the HLA-A*02+ but MART-1- tumor cell line renal cell carcinoma 2661R (B and D) were evaluated in standard 4-h 51Cr-release cell lysis assays.

FIGURE 9

FIGURE 9

Peptide titration of responses of CD8+ and CD4+ T cells transfected with MART-1/HLA-A*02-reactive DMF4 and DMF5 WT TCRs and their CDR2_β_ variants. T cells were activated with anti-CD3 Ab and isolated CD8+ (A) or CD4+ (B) cells were cotransfected with RNA encoding TCR constructs containing substitution of DMF4 or DMF5 _β_-chains along with the complementary DMF4 or DMF5 WT _α_-chains. The releases of IFN-γ in response to T2 cells pulsed with varying doses of the MART-1 peptide or with a 1 _μ_M control HLA-A*02-binding peptide corresponding to tyrosinase residues 369-377 (YMDTMSQV) were measured the following day.

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