Rapid Expansion of Highly Functional Antigen-Specific T Cells from Patients with Melanoma by Nanoscale Artificial Antigen-Presenting Cells

Abstract

Purpose: Generation of antigen-specific T cells from patients with cancer employs large numbers of peripheral blood cells and/or tumor-infiltrating cells to generate antigen-presenting and effector cells commonly requiring multiple rounds of restimulation ex vivo. We used a novel paramagnetic, nanoparticle-based artificial antigen-presenting cell (nano-aAPC) that combines anti-CD28 costimulatory and human MHC class I molecules that are loaded with antigenic peptides to rapidly expand tumor antigen-specific T cells from patients with melanoma.

Experimental design: Nano-aAPC-expressing HLA-A*0201 molecules and costimulatory anti-CD28 antibody and HLA-A*0201 molecules loaded with MART-1 or gp100 class I-restricted peptides were used to stimulate CD8 T cells purified from the peripheral blood of treatment-naïve or PD-1 antibody-treated patients with stage IV melanoma. Expanded cells were restimulated with fresh peptide-pulsed nano-aAPC at day 7. Phenotype analysis and functional assays including cytokine release, cytolysis, and measurement of avidity were conducted.

Results: MART-1-specific CD8 T cells rapidly expanded up to 1,000-fold by day 14 after exposure to peptide-pulsed nano-aAPC. Expanded T cells had a predominantly stem cell memory CD45RA⁺/CD62L⁺/CD95⁺ phenotype; expressed ICOS, PD-1, Tim3, and LAG3; and lacked CD28. Cells from patients with melanoma were polyfunctional; highly avid; expressed IL2, IFNγ, and TNFα; and exhibited cytolytic activity against tumor cell lines. They expanded 2- to 3-fold after exposure to PD-1 antibody in vivo, and expressed a highly diverse T-cell receptor V beta repertoire.

Conclusions: Peptide-pulsed nano-aAPC rapidly expanded polyfunctional antigen-specific CD8 T cells with high avidity, potent lytic function, and a stem cell memory phenotype from patients with melanoma.

Figure 1.. Frequency of MART-1 Tet⁺ CD8⁺ T-cells expanded by nano-aAPC or autologous DCs.

(A) Schematic diagram of Enrichment + Expansion using nano-aAPC. Isolated CD8⁺ T-cells were incubated with nano-aAPC loaded with MART-1 peptide, and then the cell-particle mixture was passed through a magnetic column. After enrichment, magnet-bound fractions of enriched cells and nano-aAPC were eluted and cultured in vitro. (B) Representative data of frequency of MART-1 Tet⁺ CD8⁺ T-cells expanded by CD3/CD28 Dynabeads, mature DCs or nano-aAPC at day 0 (pre-expansion), day 7 and day 14. (C) Summary of frequency, cell number and fold change of MART-1 Tet⁺ CD8⁺ T-cells from the same melanoma patients (n=8). (D) Representative data of frequency of MART-1 Tet⁺ CD8⁺ T-cells expanded by nano-aAPC with Enrichment or Non-enrichment method at day 0 (pre-expansion), day 7 and day 14. (E) Summary of frequency and fold change of MART-1 Tet⁺ CD8⁺ T-cells from the same melanoma patients (n=5). (F) Representative data of frequency of MART-1 Tet⁺ CD8⁺ T-cells expanded by nano-aAPC in the presence of IL mix (IL) or cytokine mix (CM) at day 0 (pre-expansion), day 7 and day 14. (G) Summary of frequency and fold change of MART-1 Tet⁺ CD8⁺ T-cells from the same melanoma patients (n=8). Success rates were 51/53 for E + E with nano-aAPC, 16/16 for DCs, 8/10 for Dynabeads from stage IV melanoma patients. Significance was assessed by Student’s two-tailed paired t test.

Figure 2.. Comparison of nano-aAPC T cell expansion from melanoma patients and healthy donors.

(A) The frequency of MART-1 Tet⁺ CD8⁺ T-cells by nano-aAPC expansion on day 0, 7 and 14 from healthy individuals and melanoma patients. Representative flow data of one melanoma patient and one healthy donor were shown. (B) Summary of frequency, fold change and cell number of MART-1 Tet⁺ CD8⁺ T-cells expanded by nano-aAPC in 8 healthy donors (HD) and 8 melanoma patients (Melanoma). Significance was assessed by Student’s two-tailed t test. (C) Representative staining of MART-1 Tet⁺ CD8⁺ T-cells from melanoma patients expanded by DCs or nano-aAPC for CD45RA, CD62L (L-selectin) and CD95 at day 14. The staining pattern of T_SCM and T_Naïve was shown. (D) Summary of phenotypes on MART-1 Tet⁺ CD8⁺ T-cells expanded by DCs or nano-aAPC from the same melanoma patients (n=6). Phenotypes of T-cells were categorized based on CD45RA, CD62L and CD95 expression into central memory CD45RA⁻, CD62L⁺, (T_CM), effector memory CD45RA⁻, CD62L⁻ (T_EM), stem cell memory CD45RA⁺, CD62L⁺, CD95⁺ (T_SCM), naïve CD45RA⁺, CD62L⁺, CD95⁻ (T_N), and effector CD45RA⁺, CD62L⁻ (T_E) populations. (E) Expression of KLRG1 on T_{Effector Memory} and T_Effector cells of MART-1 Tet⁺ CD8⁺ T-cells was analyzed (n=6). (F) Relative telomere length from 6 melanoma patients was analyzed based on those of 1301 cells which has long telomere length. Error bars indicate mean ± SD. Significance was assessed by Student’s two-tailed paired t test.

Figure 3.. Analysis of activation, maturation, checkpoint, proliferation and senescence markers on MART-1 Tet⁺ CD8⁺ T-cells expanded by DCs or nano-aAPC.

(A-E) Expression of activation (A, CD69, 4–1BB, ICOS and OX-40), maturity (B, CD27 and CD28), checkpoint (C, PD-1, Tim3 and LAG3), proliferation (D, Ki67) and senescence molecules (E, KLRG1) on MART-1 Tet⁺ CD8⁺ T-cells expanded by DCs or nano-aAPC from the same melanoma patients (n=6). Significance was assessed by Student’s two-tailed paired t test.

Figure 4.. Nano-aAPC expanded MART-1 Tet⁺ CD8⁺ T-cells are polyfunctional.

(A) Representative staining pattern of MART-1 Tet⁺ CD8⁺ T-cells for CD107a and indicated cytokines. Expanded CD8⁺ T-cells were co-cultured with T2 cells pulsed with the MART-1_{26–35 A27L} peptide. (B) Summary of expression of CD107a, intracellular TNFα, IFNγ, IL-2 from 6 melanoma patients. (C) Summary of perforin and granzyme B expression from 8 melanoma patients. (D) Polyfunctional analysis of MART-1 Tet⁺ CD8⁺ T-cells from 6 melanoma patients. All combinations of responses were shown. The data were summarized in a pie chart. (E) MART-1 Tet⁺ CD8⁺ T-cells expressing 2 or more functions were compared with those expressing 0 and 1 function. (F) The frequency of MART-1 Tet⁺ CD8⁺ T-cells that expressed each cytokine (from 1⁺ to 4⁺) and no cytokines (No exp.). (G) Cytotoxic activity of MART-1 Tet⁺ CD8⁺ T-cells at various E:T ratios (n=3). (H and I) Cytotoxic activity of MART-1 Tet⁺ CD8⁺ T-cells from 4 melanoma patients. The percent specific lysis against T2 cells which were pulsed with MART-1 or gp100 peptide (10⁻⁸ (M) respectively). (J) Specific lysis of MART-1 Tet⁺ CD8⁺ T-cells expanded by nano-aAPC (ET=1) or DCs (ET=1 and 10) from the same melanoma patient. (K) The functional avidity of MART-1 Tet⁺ CD8⁺ T-cells expanded by nano-aAPC or DCs from same patients (n=5). Error bars indicate mean ± SD. Significance was assessed by Student’s two-tailed t test.

Figure 5.. Cytotoxic activity of nano-aAPC expanded CD8⁺ T-cells against melanoma cell lines.

(A) Specific lysis of CD8⁺ T-cells were analyzed. CD8⁺ T-cells were co-cultured with melanoma cell lines SK-MEL-28 (HLA-A*0201⁻, MART-1⁺), A375 (HLA-A*0201⁺, MART-1⁻), MeWo (HLA-A*0201⁺, MART-1⁺), Malme-3M (HLA-A*0201⁺, MART-1⁺) at various ET ratios. Significance was assessed by Student’s two-tailed t test at ET=10. (B) Specific lysis of nano-aAPC expanded CD8⁺ T-cells against MeWo at ET=10 comparing with unexpanded CD8⁺ T-cells from 5 melanoma patients. Error bars indicate mean ± SD. Significance was assessed by Student’s two-tailed paired t test.

Figure 6.. MART-1 TCR Repertoires from healthy donors and melanoma patients post expansion with nano-aAPCs.

(A) Productive frequency of clones in CD8⁺ T-cell samples of two patients, with blue representing the sum of all clones with frequencies lower than 0.001%. (B) Vβ gene usage of CD8 samples in two patients prior to DCs or nano-aAPC expansion. (C) Frequency of individual MART-1-specific T cell clones ordered from highest to lowest frequency for healthy donor (HD) vs. melanoma patients. (D) Frequency of each Vβ for MART-1-expanded samples from healthy donors and melanoma patients. (E) Productive Entropy of CD8 samples from two patients compared to their corresponding MART-1 Tet⁺ CD8⁺ T-cells expanded by DCs or nano-aAPC. (F) Productive clonality of CD8 samples from two samples compared to their corresponding MART-1 Tet⁺ CD8⁺ T-cells expanded by DCs or nano-aAPC. (G) Summary of expansion by nano-aAPC or DCs.