Generation of autologous tumor-specific T cells for adoptive transfer based on vaccination, in vitro restimulation and CD3/CD28 dynabead-induced T cell expansion

Abstract

Adoptive cell transfer (ACT) of in vitro expanded autologous tumor-infiltrating lymphocytes (TIL) has been shown to exert therapeutic efficacy in melanoma patients. We aimed to develop an ACT protocol based on tumor-specific T cells isolated from peripheral blood and in vitro expanded by Dynabeads® ClinExVivo™CD3/CD28. We show here that the addition of an in vitro restimulation step with relevant peptides prior to bead expansion dramatically increased the proportion of tumor-specific T cells in PBMC-cultures. Importantly, peptide-pulsed dendritic cells (DCs) as well as allogeneic tumor lysate-pulsed DCs from the DC vaccine preparation could be used with comparable efficiency to peptides for in vitro restimulation, to increase the tumor-specific T-cell response. Furthermore, we tested the use of different ratios and different types of Dynabeads® CD3/CD28 and CD3/CD28/CD137 T-cell expander, for optimized expansion of tumor-specific T cells. A ratio of 1:3 of Dynabeads® CD3/CD28 T-cell expander to T cells resulted in the maximum number of tumor-specific T cells. The addition of CD137 did not improve functionality or fold expansion. Both T-cell expansion systems could generate tumor-specific T cells that were both cytotoxic and effective cytokine producers upon antigen recognition. Dynabeads®-expanded T-cell cultures shows phenotypical characteristics of memory T cells with potential to migrate and expand in vivo. In addition, they possess longer telomeres compared to TIL cultures. Taken together, we demonstrate that in vitro restimulation of tumor-specific T cells prior to bead expansion is necessary to achieve high numbers of tumor-specific T cells. This is effective and easily applicable in combination with DC vaccination, by use of vaccine-generated DCs, either pulsed with peptide or tumor-lysate.

Fig. 1

Enhancement of tumor- and virus-specific T cell by in vitro restimulation and expansion by CD3/CD28 T-cell expander PBMCs from a patient with renal cell carcinoma who previously received vaccination with peptide-pulsed autologous DCs (hTERTp988 response) or a melanoma patient prior to inclusion in DC vaccination trial (Sur1M2 and CMV) were either directly stained with relevant tetramers, bead expanded for 12 days and stained with tetramers, restimulated for 14 days with peptide and stained with tetramers or restimulated with peptide for 14 days + bead expanded for 12 days and stained with tetramer. Shown is percentage of tetramer-specific T cells out of CD8 T cells on a directly ex vivo stained cells, b directly bead-expanded cells, c restimulated cells and d restimulated and bead-expanded cells. Depicted HIV controls are from the melanoma patient. Similar HIV controls were performed on cells from the renal cell carcinoma patient (data not shown)

Fig. 2

Varying the time of restimulation and bead to T-cell ratio in dynabead expansion cultures PBMCs from a patient with renal cell carcinoma who previously received vaccination with peptide-pulsed autologous DCs was restimulated with hTERTp988 peptide for 7 and 14 days and afterward bead expanded for 12 days with Dynabeads^® CD3/CD28 T-cell expander at different DB:Tc ratio. a Percentage of hTERTp988 tetramer-specific T cells out of CD8 T cells after 7 day and 14 days of restimulation culture. b Percentage of hTERTp988 tetramer-specific T cells out of CD8 T cells after bead expansion of restimulation cultures (7 and 14 days) with DB:Tc ratio of 1:1, 1:3 and 1:10. c Total number of expanded cells in restimulated and bead-expanded cultures. d Total number of hTERTp988-specific T cells after restimulation and bead expansion

Fig. 3

DCs can be used in restimulation cultures instead of peptides a PBMCs from a patient with melanoma who previously received vaccination with peptide-pulsed autologous DCs was restimulated with either peptide-pulsed autologous DCs or hTERTp30 peptide for 14 days. Shown are the hTERTp30 tetramer-specific T cells as percentage of CD8 T cells in cultures with peptide-pulsed autologous DCs and hTERTp30 peptide, respectively. b Percentage of Sur1M2, p53 K9V, and hTERTp30 out of CD8 T in one 14 days restimulation culture using peptide-pulsed autologous DCs. c PBMCs from a patient with melanoma who previously received vaccination with allogeneic tumor cell lysate-pulsed autologous DCs was restimulated with the same DCs for 14 days. Shown is the ex vivo percentage of A2 Bcl2, A2 MAGE, A2 TRP2_180–188, A2 TRP2_185–192, A2 PRDX5, and A2 p53 K9V out of CD8 T cells analyzed by tetramers and the allo-lysate-pulsed DCs restimulated percentage of A2 Bcl2, A2 MAGE, A2 TRP2_180–188, A2 TRP2_185–192, A2 PRDX5, A2 p53 K9V, and A2 HIV-1 Pol out of CD8 T cells in two individual experiments analyzed by tetramers. NA, not analyzed

Fig. 4

Tumor-specific T cells expanded by Dynabeads^® CD3/CD28 T-cell expander are functional dynabead-expanded T cells containing high percentage of hTERTp30-specific T cells were analyzed for ability to secrete IFNγ upon stimulation with hTERTp30 peptide. a Percentage of IFNγ producing hTERTp30 tetramer-specific T cells in cultures expanded by either Dynabeads^® CD3/CD28 T-cell expander or Dynabeads^® CD3/CD28/CD137 T-cell expander. One representative experiment out of 2 is shown. b Specific lysis of peptide-pulsed T2 cells by expansion cultures (Dynabeads^® CD3/CD28 T-cell expander or Dynabeads^® CD3/CD28/CD137 T-cell expander) containing high percentage of hTERTp30-specific T cells. One representative experiment out of 2 is shown. c Simultaneous production of IFNγ, TNFα and IL-2 in a Dynabeads^® CD3/CD28 expanded culture stimulated with hTERTp988 peptide. Cells stimulated with HIV control peptide did not produce cytokines. One representative experiment out of 2 is shown

Fig. 5

Phenotype and telomere length of Dynabead^® CD3/CD28-expanded T cells. a Cells were restimulated for 14 days with either peptide only (hTERTp988; 2 individual cultures from the same renal cell carcinoma patient) or peptide-pulsed DCs (hTERTp30, Sur1M2, and K9V; 3 individual cultures from the same melanoma patient) and analyzed for expression of phenotypic markers on CD8 T cell (open circles) and tetramer-specific T cells (filled circles). b Cells were restimulated for 14 days as described above and were afterward expanded by Dynabead^® CD3/CD28 T-cell expander for 12 days. Cells were analyzed for expression of phenotypic markers on CD8 T cells (open circles) and tetramer-specific T cells (filled circles). c PBMCs ± bead expansion and TIL cultures before and after rapid expansion were analyzed for telomere length as described in “Materials and methods”. Shown are “bead CD8” (3 samples before and 5 after bead expansion), “bead CD8tet” (2 samples before and 4 after bead expansion), and “CD8 TIL” (4 samples before and 2 after rapid expansion). Results are depicted as mean + range