IL-12-Secreting CD19-Targeted Cord Blood-Derived T Cells for the Immunotherapy of B-Cell Acute Lymphoblastic Leukemia

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

HJ Pegram1, TJ Purdon1, DG van Leeuwen1, KJ Curran2, SA Giralt1,3,4, JN Barker1,3,4 and RJ Brentjens1,5,6

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 4150-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.

Leukemia (2015) 29, 415–422; doi:10.1038/leu.2014.215

INTRODUCTION after allogeneic hematopoietic stem cell transplantation without 13 Allogeneic hematopoietic stem cell transplantation is the standard evidence of graft-versus-host disease. of care in therapy for many patients with high risk or relapsed Adoptive T-cell transfer therapy is also an attractive option in B-cell acute lymphoblastic leukemia (ALL). Unfortunately, many UCBT recipients to further augment graft-versus-leukemia effects, patients do not have suitable human leukocyte antigen-matched but is technically challenging due to the difficulty in generating and readily available adult donors. umbilical cord blood (UCB) sufficient numbers of effector T cells. Herein, we describe a novel transplantation (UCBT) is a viable treatment alternative for these technique to expand and genetically modify clinically meaningful patients with the advantages of rapid graft availability, flexibility of numbers of UCB-derived CAR T cells. Expansion of UCB T cells in scheduling and a reduced stringency of required (human the context of interleukin (IL)-15 and IL-12 led to over 150-fold leukocyte antigen) match.1,2 UCBT has also been associated with expansion and unique expression of both central memory markers relatively low rates of leukemic relapse likely related to a robust and effector proteins, an ideal phenotype for adoptive cell transfer graft-versus-leukemia effect.2–5 However, relapse remains a therapy. We then used retroviral transduction to generate UCB significant cause of treatment failure. In the adult series reported T cells expressing a CD19-specific CAR and an IL-12 transgene. This by Marks et al.,6 for example, the 3-year relapse incidence was 22% study demonstrates the feasibility of generating clinically relevant in 116 ALL UCBT recipients transplanted in first or second numbers of CAR UCB T cells that have effective antitumor efficacy complete remission. Moreover, the relapse risk is considerably in preclinical murine models. Our study supports the clinical increased in patients transplanted with residual disease and in translation of UCB-derived CAR T cells to further augment the recipients of reduced-intensity conditioning. For these reasons graft-versus-leukemia potency of UCBT for B-cell ALL. there is a need to enhance the antileukemic efficacy of UCBT for B-cell ALL. Expression of a tumor-targeted chimeric antigen receptor (CAR) MATERIALS AND METHODS allows for the generation of tumor-targeted T cells that are capable of non-major histocompatibility-restricted tumor recogni- Cell lines ’ tion and eradication. T cells modified to express CD19-specific Raji (Burkitt s lymphoma cell line) and Nalm6 (pre-B-cell ALL cell line) tumor CARs can mediate effective antitumor responses both in vitro and cells were maintained in RPMI 1640 (Invitrogen, Grand Island, NY, USA) 7,8 supplemented with 0.1 mM non-essential amino acids, 1 mM sodium in vivo in multiple murine models. In addition, early clinical trials − 2 fi fi pyruvate, 5 × 10 mM 2ME (Invitrogen). Retroviral producer cell lines utilizing T cells modi ed with a CD19-speci c CAR have (293 Glv9) producing 1928z, 1928z/IL-12, 4H1128z or 4H1128z/IL-12 demonstrated significant antitumor efficacy in patients with encoding virus were maintained in DMEM (Invitrogen). All media were + 9–12 CD19 B-cell malignancies. Adoptive transfer of donor- supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml derived CD19-specific CAR T cells can eradicate tumor persisting penicillin and streptomycin, and 2 mM L-glutamine.

1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 2Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 3Adult Bone Marrow Transplantation Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 4Weill Cornell Medical College, New York, NY, USA; 5Center for Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, USA and 6Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Correspondence: Dr RJ Brentjens, Department of Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, NY, USA. E-mail: [email protected] Received 15 April 2014; revised 12 June 2014; accepted 18 June 2014; accepted article preview online 9 July 2014; advance online publication, 1 August 2014 IL-12-secreting cord blood-derived CAR T cells HJ Pegram et al 416 Isolation and expansion of T cells from UCB units mice received one systemic infusion of 5 × 106 CAR+ UCB-derived T cells. Fresh UCB units not meeting cell dose criteria for public banking were Mice were monitored for survival and were euthanized when showing obtained from the Cleveland Cord Blood Center (Cleveland, OH, USA), and signs of distress. All experiments were performed in accordance with an mononuclear cells were separated using density gradient centrifugation Institutional Animal Care and Use Committee-approved protocol (protocol with Accu-prep (axis-Shield PoC AS, Oslo, Norway). T cells were isolated, 00-05-065). Bioluminescent imaging was achieved using the Xenogen IVIS activated and expanded with Dynabeads ClinExVivo CD3/CD28 magnetic imaging system and analyzed with Living Image software (Xenogeny) as 14 beads (Invitrogen), according to the manufacturer's instructions. UCB- previously described. Briefly, tumor-bearing mice were injected intraper- derived T cells were cultured in RPMI 1640 supplemented with 10% fetal itoneally with D-Luciferin (150 mg/kg) and after 10 min were imaged under fl fi calf serum , 100 U/ml penicillin and streptomycin, and 2 mM L-glutamine, in iso uorane anesthesia. Image acquisition was achieved using a 25 cm eld the presence of 100 IU/ml recombinant human IL-2 (Proleukin, Novartis, of view, medium binning level and 60-s exposure time. Basel, Switzerland), 10 ng/ml recombinant human IL-12, 10 ng/ml recombinant human IL-7 or 10 ng/ml recombinant human IL-15 (all from R&D Statistical analyses Systems, Minneapolis, MN, USA). Viable cells were enumerated using Survival data were assessed using a log-rank test. Flow cytometry and Trypan blue (Invitrogen) exclusion. Secondary stimulation was achieved cytokine secretion data was analyzed using a Mann–Whitney test using with ClinExVivo CD3/CD28 beads at a bead to T-cell ratio of 1:2. Prism 5.0 (Graphpad Software, La Jolla, CA, USA).

Retroviral transduction of UCB T cells Activated UCB T cells were retrovirally transduced as previously RESULTS described.14 Briefly, UCB T cells were spinoculated three times with UCB T-cell culture in IL-12 and IL-15 results in over 150-fold retroviral supernatant on retronectin (Takara, Otsu, Shiga, Japan) coated expansion and a unique memory/effector phenotype plates. Transduction efficiency was assessed by flow cytometry using goat T lymphocytes can be successfully isolated from UCB units using anti-mouse antibody conjugated to phycoerythrin (PE, Invitrogen) to 16 detect 1928z or an Alexa-Fluor647 conjugated hamster antibody that CD3/CD28 beads. UCB T cells must be expanded a several fold specifically binds the 4H1128z CAR (Monoclonal Antibody Facility, for clinical relevance and adoptive cell transfer therapy. To Memorial Sloan Kettering Cancer Center). investigate the optimal culture conditions for maximal expansion of CB derived T cells, we cultured activated UCB T cells in Flow cytometric analysis combinations of 10 ng/ml IL-12, IL-15, IL-7, and/or 100 IU/ml IL-2. By enumerating viable cells, we determined that culture in the Expanded UCB T cells were analysed by flow cytometry after staining with the following antibodies according to the manufacturers instructions context of IL-15 is important for the expansion of UCB T cells. (clone numbers are shown): PE-conjugated antibodies specific for human Culture of UCB T cells in IL-12+IL-15, IL-15+IL-7, IL-15+IL-2 or IL-15 CCR7 (3D12), Granzyme B (GB11), fluorescein isothiocyanate-conjugated alone resulted in over 120-fold expansion (Figure 1) in a 12day antibodies specific for perforin (dG9), CD28 (CD28.2), CD107a (eBioH4A3), period. Flow cytometry was used to determine the CD4:CD8 T-cell PD-1 (eBioJ105), CTLA-4 (14D3) and allophycocyanin-conjugated anti- ratio and we demonstrated that UCB T cells expanded in these bodies specific for CD25 (BC96) and CD107a (ebio1D48), and PE-cyanine cytokine combinations had equivalent percentages of CD4 and dye 7-conjugated antibody specific for IFNγ (4S.B3) obtained from CD8 T cells (Figure 2a). We then investigated the expression of eBioscience (San Diego, CA, USA). PE-conjugated antibodies specific for memory markers on these cells, particularly CD62L and CCR7. IL-12 CD4 (S3.5) or CD45RA (MEM-56) and allophycocyanin-conjugated anti- fi and IL-15 expanded UCB T cells had a higher expression of these bodies speci c for human CD8 (3B5) and CD62L (Dreg-56) were all markers compared to cells expanded in IL-15+IL-7, IL-15+IL-2 or obtained from Invitrogen. Intracellular staining (for cytokines and CTLA-4) was achieved using the BD Cytofix/Cytoperm Plus Fixation/Permeabiliza- tion kit with BD GolgiPlug (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions, in combination with a fixable viability dye (efluor450, eBioscience). Proliferation of UCB T cells was achieved by labeling T cells with CellVue Claret Far Red Fluorescent cell linker mini kit (Sigma, St Louis, MO, USA), according to the manufacturer's instructions. Labeled T cells were then incubated with Nalm6 tumor cells at 1:1 ratio overnight before flow cytometric analysis. Tumor cells were excluded from analysis by staining with allophycocyanin-conjugated antibody specific for CD19 (SJ25-C1, Invitrogen).

Cytotoxicity assays The cytotoxic capacity of UCB-derived gene-modified T cells was assessed using a standard 51chromium (51Cr) assay, described elsewhere.15 Briefly, 51Cr-radiolabeled tumor cells were incubated with transduced UCB T cells at varying CAR+ effector T cell to target ratios. The amount of 51Cr released in the supernatant was measured using a Perkin Elmer Top Count NXT (Perkin Elmer, Irvine, CA, USA) and correlated with percentage-specific lysis.

Cytokine detection assays UCB-derived T cells were co-cultured with Raji or Nalm6 tumor cells at a 1:1 CAR+ T-cell:tumor-cell ratio for 24 h. Supernatant was collected and cytokines were detected using a multiplex human cytokine detection assay (Millipore Corp., Billerica, MA, USA), according to the manufacturer's instructions. Data were analyzed using the Luminex IS100 system and IS2.3 software. Figure 1. Optimal expansion of CD3/CD28-activated UCB-derived T cells. UCB T cells were isolated and expanded with CD3/CD28 In vivo SCID-Beige murine studies beads and cultured in the context of stimulatory cytokines IL-2, IL-7, Fox Chase CB17 (CB17.Cg-PrkdcscidLystbg-J/Crl, SCID-Beige mice; Charles IL-12, IL-15 or combinations thereof. Viable cells were enumerated River Laboratories, Wilmington, MA, USA) of 6–8 weeks of age were and fold expansion of T cells was calculated. Results shown are the inoculated intravenously with 1 × 106 Nalm6 tumor cells, expressing a GFP- average fold expansion from 7–10 UCB units in independent ﬁreﬂy luciferase fusion protein (Nalm6-eGFP-FFluc). The following day, 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, *Po0.05.

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, *Po0.05.

IL-15 alone (Figure 2b). In addition, increased expression of CD28 both Granzyme B and perforin compared to cells expressing the in these cells was observed (Figure 2c), and cells expanded in IL-12 CAR alone (Figure 4d). and IL-15 displayed increased expression of the cytotoxic effector We then sought to determine the in vitro antitumor function of protein, Granzyme B, in both CD4+ and CD8+ T cells (Figure 2d). transduced UCB T cells. We utilized Luminex technology to We also determined the expression of CTLA-4 and PD-1 on measure UCB T-cell cytokine release following a 24-h coculture expanded UCB T cells to ensure that expansion did not result in with Nalm6 or Raji tumor cells. 1928z/IL-12 UCB T cells produced anergy/exhaustion. There is no significant difference in expression significant levels of IL-12 compared to control UCB T cells of PD-1 or CTLA-4 on UCB T cells expanded in IL-15, IL-15+IL-2, expressing the CAR alone (Figure 5a). In addition, 1928z/IL-12 IL-15+IL-7 or IL-15+IL-12 (Figures 2e and f). IL-12- and IL-15- UCB T cells secreted increased levels of IFNγ in response to Raji expanded UCB T cells had a unique phenotype with increased and Nalm6 tumor cells, compared to 1928z UCB T cells (Figure 5a). expression of central memory markers which was seen alongside Consistent with previous findings in murine T cells,17 we found expression of cytotoxic effector proteins thereby combining 1928z/IL-12 UCB T cells secreted decreased levels of IL-2 central memory and effector T-cell phenotypes. compared to 1928z UCB T cells following coculture with tumor We then assessed the functional capacity of UCB T cells using (Figure 5a). We investigated T-cell proliferation using membrane- intracellular staining and flow cytometry. UCB T cells expanded in dye dilution assay and a 24 h coculture with Nalm6 tumor cells. IL-12 and IL-15 demonstrated increased production of IFN-γ Flow cytometry analysis revealed that 1928z/IL-12 UCB T cells following secondary stimulation compared to UCB T cells exhibited enhanced proliferation compared to 1928z UCB T cells expanded in IL-15+IL-7, IL-15+IL-2 or IL-15 alone (Figure 3). These (Figure 5b). To determine the cytolytic capacity of transduced UCB cells also trended toward increased CD107a expression, indicating T cells, we performed a 51Cr release assay and demonstrated that increased cytotoxic capacity, although this was not statistically 1928z/IL-12 UCB T cells mediated increased lysis of Raji and Nalm6 significant (Figure 3). From these studies, we conclude that culture tumor cells compared to 1928z UCB T cells (Figure 5c). of UCB T cells in the context of exogenous IL-12 and IL-15 Finally, we investigated the in vivo antitumor capacity of supports maximal expansion of UCB T cells and a combined transduced UCB-derived T cells using a preclinical SCID-Beige central memory/effector T-cell phenotype, ideal for adoptive cell murine model. Initial experiments utilized Raji tumors and we therapy. showed that 1928z UCB T cells can cure mice without the need for irradiation or IL-2 support (Supplementary Figure 1). Therefore, to UCB-derived T cells can be effectively targeted to tumor with a determine the effect of IL-12 transgene on the antitumor efficacy CD19-specific CAR and IL-12 transgene of these cells we used a more aggressive model employing Nalm6 6 Meaningful adoptive therapy with UCB T cells requires improved tumor cells. Treatment of Nalm6 tumor-bearing mice with 5 × 10 + antitumor efficacy. To address this challenge, we targeted UCB CAR 1928z/IL-12 UCB-derived T cells resulted in significantly T cells to tumors with a CAR. The CAR consisted of an antibody enhanced survival compared to mice treated with 1928z, 4H1128z recognition domain (specific for CD19 for 1928z or the control or 4H1128zIL-12 or untreated mice (Figures 6a and b). Given that ovarian carcinoma antigen, MUC-16, for 4H11) linked to the CD28 1928z UCB T cells were cultured in exogenous IL-12 prior to and the CD3 zeta signaling domains (Figure 4a), as previously adoptive transfer and these cells did not inhibit tumor progres- reported.7 Given the favorable effects of IL-12 on UCB T-cell sion, the requirement for continued autocrine IL-12 stimulation phenotype and function described above, and previous studies in for the antitumor efficacy of 1928z/IL-12 UCB T cells was our laboratory demonstrating that IL-12 enhances T-cell mediated demonstrated. antitumor effects,17 we also modified UCB T cells with a vector encoding both the CAR and a flexi human IL-12 fusion protein.17 DISCUSSION Following retroviral transduction of expanded UCB T cells, flow cytometry was used to detect CAR expression. UCB T cells Tumor-targeted CAR UCB T cells could greatly augment the transduced with 1928z, 1928z/IL-12, 4H1128z or 4H1128z/IL-12 antileukemic potency of UCBT. This has significant therapeutic expressed the chimeric receptor at reproducible levels (Figure 4b). implications for patients with high-risk or refractory disease. The mean transduction from seven experiments (SEM) for UCB However, given the correlation between high cell dose and T cells transduced with 1928z was 67.24% (9.90), with 1928z/IL-12 improved UCB transplant outcome, only a small percentage of the was 47.7% (6.72), with 4H1128z was 75.52% (12.86) and with UCB unit would be available for T-cell isolation and generation of 4H1128z/IL-12 was 62.66% (10.49). Consistent with previous CAR UCB T cells. Therefore, maximal expansion of CAR UCB T cells finding in murine T cells,17 expression of the CAR and IL-12 is required for therapeutic application.18 Herein, we describe a resulted in maintaining high CD62L and low CD25 expression novel protocol for expansion and genetic modification of UCB compared to 1928z UCB T cells. However these cell types had T cells that yields a clinically relevant dose of functional antitumor equivalent expression of CCR7 (Figure 4c), as determined by flow UCB T cells. Specifically, we demonstrate that ex vivo culture of cytometry.17 1928z/IL-12 UCB T cells had increased production of UCB T cells with exogenous IL-12 and IL-15 resulted in optimal

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 (VH) and variable light (VL) 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, **P40.05 (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. expansion, and that these cells have a unique central memory and 150-fold expansion achieved in our study will permit generation of effector phenotype for effective adoptive cell transfer therapy. clinically relevant T-cell doses. For example, in a recent 128-patient In this study, we first determined optimal cytokine cultures for double unit UCBT Memorial Sloan Kettering Cancer Center series maximal expansion of UCB T cells. Early UCB T-cell expansion (median weight 68 kilograms, kg), the median infused viable CD3+ studies utilized OKT3 and IL-2 resulting in a 39-fold expansion.19 cell dose of the dominant unit was 4.27 (0.8–15.3) × 106/kg.23 An improved expansion of 4140-fold has since been demon- One-hundred fifty-fold expansion of even 5% of many UCB units strated with CD3/CD28 magnetic beads. However, these could therefore provide T-cell doses in excess of what is routinely expanded UCB T cells have an effector memory phenotype with used in donor lymphocyte infusions in adult patients. These data loss of CCR7 expression.16 By contrast, our study revealed that combined with previously published clinical data using CAR T-cell culture in the context of exogenous IL-12 and IL-15 led to over therapy, we conclude that these numbers are sufficient for 150-fold expansion of UCB T cells with expression of central effective clinical use.9,10 Use of 10% of the graft to generate CAR memory markers and cytotoxic molecules. A 14-fold expansion of T cells may be clinically feasible in smaller pediatric patients, and UCB T cells has been reported with IL-2, IL-7, IL-12 and IL-15 and pursuing this approach in the context of single unit transplant in autologous virus pulsed dendritic cells.20–22 By contrast, the children is supported by the results of the recent pediatric Blood

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, *Po0.05. (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, *Po0.05. (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.

Leukemia (2015) 415 – 422 © 2015 Macmillan Publishers Limited IL-12-secreting cord blood-derived CAR T cells HJ Pegram et al 421 and Marrow Transplant Clinical Trials Network (BMT CTN) 0501 Inclusion of the inducible caspase9 gene into the 1928z/IL-12 randomized trial of single- versus double-unit UCBT. Moreover, retroviral construct could allow eradication of modified cells in the new data concerning the ability to optimize unit selection by case of serious adverse events, thereby improving the safety of considering the pre-freeze CD34+ cell dose could facilitate the this approach. Eradication of CAR T cells with the suicide gene will safety of reserving a small percentage of the graft for adult of course eliminate antitumor efficacy, which may result in patients.23 decreased response to tumor or potential relapse. Although UCB T cells are a classically naive population, we Our findings provide strong rationale for the clinical translation demonstrated that following activation/transduction, CAR UCB of expanded CAR UCB T cells as additional therapy for UCBT T cells have high lytic capacity and can mount cytokine responses recipients in a phase I study. We propose that adoptive transfer of to tumor cells in vitro. Despite demonstrating the importance of CAR UCB T cells will augment graft-versus-leukemia effect, and IL-15 for UCB T-cell expansion, previous studies have shown that thereby promote disease-free survival after UCBT. modifying T cells with an IL-15 transgene carries high risk of malignant transformation.24,25 Our results suggest that while IL-15 is important in UCB expansion, IL-12 is important for phenotypic CONFLICT OF INTEREST changes, including increased CD62L, CD28, GzmB and IFNγ RJB is a co-founder, stockholder, and consultant for Juno Therapeutics. The remaining (Figures 2 and 3). In addition, previous studies from our laboratory authors declare no conflict of interest. show that CAR/IL-12 T cells mediate improved antitumor efficacy and may be resistant to Tregs. Therefore, we chose to use an IL-12 transgene in our retroviral CAR construct. Our expansion protocol ACKNOWLEDGEMENTS and expression of 1928z/IL-12 resulted in a maintained central HJP was supported by the National Health and Medical Research Council of Australia. memory phenotype, in contrast to other studies where UCB T cells expressing a CD19-targeted CAR (with either 4-1BB or CD28 AUTHOR CONTRIBUTIONS signaling domains) resulted in low levels of CD62L expression.26 Therefore, our studies support the use of IL-15 and IL-12 for UCB HJP designed and performed the experiments, analyzed the data and prepared T-cell expansion and modification of UCB T cells with both the CAR the manuscript. TJP and DGvL designed and performed the experiments and and IL-12 for adoptive therapy. analyzed the data. KJC, JNB and RJB designed the experiments, analyzed the The in vitro antitumor efficacy of these cells was measured by data and revised the manuscript. SAG designed the experiments, analyzed determining the cytokine release, T-cell proliferation and cyto- the data. toxicity of CAR UCB T cells. 1928z/IL-12 UCB T cells secreted significantly more IL-12 and, consequently, IFNγ, as well as decreased levels of IL-2. We have previously noted that reduced REFERENCES levels of IL-2 secretion from CAR/IL-12 T cells may relate to the 1 Eapen M, Rocha V, Sanz G, Scaradavou A, Zhang MJ, Arcese W et al. Effect of graft mechanism for potential resistance to Tregs.17 UCB T cells mediate source on unrelated donor haemopoietic stem-cell transplantation in adults with increased lysis of tumor targets when modified to express both acute leukaemia: a retrospective analysis. 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