Multiepitope CD8(+) T cell response to a NY-ESO-1 peptide vaccine results in imprecise tumor targeting

Abstract

The cancer-testis antigen NY-ESO-1 is one of the most promising candidates for generic vaccination of cancer patients. Here we analyzed the CD8(+) T cell response to a NY-ESO-1 peptide vaccine composed of the two previously defined peptides 157-165 and 157-167, administered with GM-CSF as a systemic adjuvant. The NY-ESO-1 peptide vaccine elicited a CD8(+) T cell response directed against multiple distinct epitopes in the 157-167 region, as revealed by using A2/peptide multimers incorporating overlapping A2 binding peptides in this region. However, only a minor fraction of the elicited CD8(+) T cells, namely those recognizing the peptide 157-165 with sufficiently high functional avidity, recognized the naturally processed target on NY-ESO-1(+) tumor cells. In contrast, the majority of peptide 157-165-specific CD8(+) T cells exhibited lower functional avidity and no tumor reactivity. In addition, vaccine-elicited CD8(+) T cells specific for other overlapping epitopes in the 157-167 region failed to significantly recognize NY-ESO-1-expressing tumor targets. Thus, because of the complexity of the CD8(+) T cell repertoire that can be elicited by vaccination with synthetic peptides, a precise definition of the targeted epitope, and hence, of the corresponding peptide to be used as immunogen, is required to ensure a precise tumor targeting.

Figure 1

M 9-157⁺ CD8⁺ T cell responses in sarcoma patients receiving the NY-ESO-1 peptide vaccine. Before and after vaccination PBMC samples from patient LUD 24/1 (after-vaccination sample day 169) were stimulated in vitro with peptide NY-ESO-1 9-157 or with peptide NY-ESO-1 11-157 (1 μM), cultured during 1 week, and stained with A2/9-157 multimers^PE and anti-CD8 mAb^FITC.

Figure 2

Sarcoma patients vaccinated with NY-ESO-1 peptides 157-165 and 157-167 respond to distinct overlapping A2-restricted epitopes in the 157-167 region. Polyclonal monospecific CD8⁺ multimer⁺ populations were isolated by cell sorting with the corresponding multimers and further expanded in vitro by mitogen-driven stimulation. After 2 weeks of in vitro expansion, each population was stained with multimers incorporating each of the overlapping 9-mer and 10-mer in the 157-167 region.

Figure 3

Fine specificity of antigen recognition and tumor reactivity of distinct NY-ESO-1 multimer⁺ CTL recognizing overlapping epitopes in the 157-167 region. (a) Recognition of NY-ESO-1 peptides by polyclonal monospecific multimer⁺–sorted populations as well as by the NY-ESO-1–specific tumor-reactive clone LAU 156/5 used as an internal control was assessed in a ⁵¹Cr release assay on T2 target cells in the presence of graded concentration of the indicated peptides. (b) Tumor recognition was similarly assessed on Me 275 (A2⁺ NY-ESO-1⁺) or Me 260 (A2^–) tumor target cells at the indicated E/T ratio.

Figure 4

Functional clonal heterogeneity in M 9-157 polyclonal population’s impact on tumor reactivity. (a) Tumor recognition by T cell clones derived from M 9-157⁺ CD8⁺–sorted T cells from patient LUD 24/1 was assessed on Me275 (A2⁺ NY-ESO-1⁺) or NA8 (A2⁺ NY-ESO-1^–) tumor target cells at the indicated E/T ratio. (b) Functional avidity of recognition of 9-157 T cell clones derived from M 9-157⁺ CD8⁺ sorted T cells from patient LUD 24/1 as well as by clone LAU 156/5 used as an internal control was assessed on T2 target cells in the presence of graded peptide concentrations.

Figure 5

Ex vivo analysis of vaccine-induced 9-157– and 9-159–specific CD8⁺ T cells. (a) Cryopreserved PBMCs from vaccinated patients were thawed, incubated overnight at 37°C in CTL medium, and stained with A2/9-157 or A2/9-159 multimers^PE and anti-CD8 mAb^FITC. (b) PBMCs from patient LUD 24/1 were stained as in a. M⁺CD8⁺ positive cells were directly isolated by cell sorting and cloned under limiting dilution conditions. Tumor recognition by T cell clones obtained following this procedure was assessed on Me275 (A2⁺ NY-ESO-1⁺) or NA8 (A2⁺ NY-ESO-1^–) tumor target cells at the indicated E/T ratio in the absence or in the presence of the corresponding peptide.

Figure 6

Resistance of NY-ESO-1 peptides in the 157-167 region to degradation in human serum. NY-ESO-1 peptides 9-157, 9-157 substituted analogue C165A, 9-159, 11-157, or Melan-A 27-35 (AAGIGILTV) were incubated in human serum for the indicated time periods, and antigenic activity was then evaluated in CTL assay by using as effector cells the indicated NY-ESO-1 polyclonal monospecific lines or, in the case of the Melan-A peptide, the Melan-A–specific clone LAU 203/17. Antigen-specific lysis was assessed on ⁵¹Cr-labeled T2 cells in the presence of serial dilutions of each degradation sample. (a) Peptide 9-157 tested with line M 9-157 (upper panel) and peptide 9-159 tested with line M 9-159 (lower panel). (b) Peptide 11-157 tested with line M 9-157 (upper panel) or with line M 9-159 (lower panel). (c) Peptide 9-157 C165A tested with line M 9-157 (upper panel) and peptide Melan-A_27–35 tested with clone LAU 203/17 (lower panel). The human serum used in this experiment was heat-inactivated, but similar results were obtained by using fresh serum.