IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia

Abstract

Disease relapse or progression is a major cause of death following umbilical cord blood (UCB) transplantation (UCBT) in patients with high-risk, relapsed or refractory acute lymphoblastic leukemia (ALL). Adoptive transfer of donor-derived T cells modified to express a tumor-targeted chimeric antigen receptor (CAR) may eradicate persistent disease after transplantation. Such therapy has not been available to UCBT recipients, however, due to the low numbers of available UCB T cells and the limited capacity for ex vivo expansion of cytolytic cells. We have developed a novel strategy to expand UCB T cells to clinically relevant numbers in the context of exogenous cytokines. UCB-derived T cells cultured with interleukin (IL)-12 and IL-15 generated >150-fold expansion with a unique central memory/effector phenotype. Moreover, UCB T cells were modified to both express the CD19-specific CAR, 1928z, and secrete IL-12. 1928z/IL-12 UCB T cells retained a central memory-effector phenotype and had increased antitumor efficacy in vitro. Furthermore, adoptive transfer of 1928z/IL-12 UCB T cells resulted in significantly enhanced survival of CD19(+) tumor-bearing SCID-Beige mice. Clinical translation of CAR-modified UCB T cells could augment the graft-versus-leukemia effect after UCBT and thus further improve disease-free survival of transplant patients with B-cell ALL.

Figure 1

Optimal expansion of CD3/CD28-activated UCB-derived T cells. UCB T cells were isolated and expanded with CD3/CD28 beads and cultured in the context of stimulatory cytokines IL-2, IL-7, IL-12, IL-15 or combinations thereof. Viable cells were enumerated and fold expansion of T cells was calculated. Results shown are the average fold expansion from 7–10 UCB units in independent experiments.

Figure 2

Phenotype of IL-12 and IL-15-expanded UCB-derived T cells. The phenotype of UCB-derived T cells cultured in IL-15, IL-15+IL-2, IL-15 + IL-7 and IL-15+IL-12 was compared using flow cytometry. Representative plots show that UCB T cells expanded in IL-12 and IL-15 have similar ratios of CD4:CD8 T cells (a) and increased expression of CCR7 and CD62L (b), CD28 (c) and Granzyme B in both CD8 and CD4 T cells (d). There is no difference in expression of CTLA-4 (e) or PD-1 (f) in UCB T cells expanded in IL-15, IL-15+IL-2, IL-15+ IL-7 or IL-15+IL-12. Mean fluorescence intensity of staining is from 3–4 independent experiments, *P < 0.05.

Figure 3

Increased function of IL-12 and IL-15-expanded UCB-derived T cells. The function of UCB T cells expanded in IL-15, IL-15+IL-2, IL-15 +IL-7 or IL-15+IL-12 was determined by multiparameter flow cytometry following restimulation with CD3/CD28 beads. Representative plots show that UCB T cells cultured in IL-12 and IL-15 have increased production of IFNγ and a trend toward increased CD107a expression, a marker of degranulation. Data shown are representative of four independent experiments, *P < 0.05.

Figure 4

Expression of CD19-targeted CAR and IL-12 in UCB-derived T cells. UCB T cells cultured in IL-12 and IL-15 were modified to express the 1928z or control 4H1128z CAR with or without IL-12. (a) Schematic of constructs depicting the 5’ and 3’ long terminal repeats (LTR), splice donor (SD), splice acceptor (SA), packaging element (ψ), CD8 leader sequence (CD8), variable heavy (V_H) and variable light (V_L) chains of the single-chain variable fragment (scFv), transmembrane domain (TM), human CD28 signaling domain (hCD28), human zeta chain signaling domain (hζ chain) and flexi human IL-12 (hIL-12f). (b) Transduced UCB T cells expressed either 1928z or 4H1128z CAR following retroviral transduction, as determined by flow cytometry. Average transduction efficiency ± s.e.m. from seven experiments is shown, **P>0.05 (c) 1928z/IL-12 UCB T cells showed increased expression of CD62L, equivalent CCR7 and decreased CD25 when compared to cells transduced with 1928z CAR alone, as determined by flow cytometry. (d) Expression of 1928z/IL-12 resulted in increased expression of Perforin (Pfp) and Granzyme B (GzmB) when compared to cells transduced with 1928z CAR alone, as determined by flow cytometry. Data shown are representative of five independent experiments.

Figure 5

UCB T cells modified to express both the 1928z CAR and IL-12 have increased proliferation and IFNγ in response to Nalm6 tumor cells. The ability of modified UCB T cells to secrete IL-12p70, IFNγ and IL-2 in response to Nalm6 or Raji tumor cells was assessed using a luminex assay following coculture. 1928z/IL12 T cells secrete more IL-12p70 and IFNγ, and less IL-2 than 1928z T cells, *P < 0.05. (a) Proliferative capacity of modified UCB T cells was investigated by labeling T cells with membrane dye prior to coculture with Nalm6 tumor cells. Flow cytometry was utilized to detect dilution of membrane dye (proliferation of T cells). (b) 1928z/IL-12 T cells showed increased proliferation compared to 1928z T cells. Data shown are representative of two independent experiments. (c) The ability of modified UCB T cells to lyse Raji or Nalm6 tumor cells was assessed using a 4-h ⁵¹Cr assay. Data shown are representative of three independent experiments.

Figure 6

1928z/IL-12 UCB T cells enhance the survival of tumor-bearing mice. (a) Mice inoculated with 1 × 10⁶ Nalm6 tumor cells i.v. were treated with a systemic infusion of 5 × 10⁶ CAR⁺ UCB T cells. Survival of Nalm6 tumor-bearing mice was significantly enhanced by treatment with 1928z/IL-12 UCB T cells compared to CAR alone and control cells, *P < 0.05. (b) Bioluminescent imaging revealed decreased tumor growth in mice treated with 1928z/IL-12 UCB T cells compared to 1928z, 4H1128z, 4H1128z/IL-12 UCB T cells or untreated mice. Data shown are from UCB T cells from two units.