Gengwen Tian

Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy

Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant natural killer T (NKT) cells can build on the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKT cells were engineered to express a CAR against the GD2 ganglioside (CAR.GD2), which is highly expressed by neuroblastoma (NB). We compared CAR.GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB costimulatory endodomains. CAR.GD2 expression rendered NKT cells highly cytotoxic against NB cells without affecting their CD1d-dependent reactivity. We observed a striking T helper 1-like polarization of NKT cells by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR.GD2 enhanced in vivo persistence of NKT cells. These CAR.GD2 NKT cells effectively localized to the tumor site had potent antitumor activity, and repeat injections significantly improved the long-term survival of mice with metastatic NB. Unlike T cells, CAR.GD2 NKT cells did not induce graft-versus-host disease. These results establish the potential of NKT cells to serve as a safe and effective platform for CAR-directed cancer immunotherapy.

CD62L+ NKT cells have prolonged persistence and antitumor activity in vivo

Vα24-invariant natural killer T cells (NKTs) localize to tumors and have inherent antitumor properties, making them attractive chimeric antigen receptor (CAR) carriers for redirected cancer immunotherapy. However, clinical application of CAR-NKTs has been impeded, as mechanisms responsible for NKT expansion and the in vivo persistence of these cells are unknown. Here, we demonstrated that antigen-induced expansion of primary NKTs in vitro associates with the accumulation of a CD62L+ subset and exhaustion of CD62L- cells. Only CD62L+ NKTs survived and proliferated in response to secondary stimulation. When transferred to immune-deficient NSG mice, CD62L+ NKTs persisted 5 times longer than CD62L- NKTs. Moreover, CD62L+ cells transduced with a CD19-specific CAR achieved sustained tumor regression in a B cell lymphoma model. Proliferating CD62L+ cells downregulated or maintained CD62L expression when activated via T cell receptor alone or in combination with costimulatory receptors. We generated HLAnull K562 cell clones that were engineered to express CD1d and costimulatory ligands. Clone B-8-2 (HLAnullCD1dmedCD86high4-1BBLmedOX40Lhigh) induced the highest rates of NKT expansion and CD62L expression. B-8-2-expanded CAR-NKTs exhibited prolonged in vivo persistence and superior therapeutic activities in models of lymphoma and neuroblastoma. Therefore, we have identified CD62L as a marker of a distinct NKT subset endowed with high proliferative potential and have developed artificial antigen-presenting cells that generate CD62L-enriched NKTs for effective cancer immunotherapy.

Medulloblastoma expresses CD1d and can be targeted for immunotherapy with NKT cells

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Current therapies are toxic and not always curative that necessitates development of targeted immunotherapy. However, little is known about immunobiology of this tumor. In this study, we show that MB cells in 9 of 20 primary tumors express CD1d, an antigen-presenting molecule for Natural Killer T cells (NKTs). Quantitative RT-PCR analysis of 61 primary tumors revealed an elevated level of CD1d mRNA expression in a molecular subgroup characterized by an overactivation of Sonic Hedgehog (SHH) oncogene compared with Group 4. CD1d-positive MB cells cross-presented glycolipid antigens to activate NKT-cell cytotoxicity. Intracranial injection of NKTs resulted in regression of orthotopic MB xenografts in NOD/SCID mice. Importantly, the numbers and function of peripheral blood type-I NKTs were preserved in MB patients. Therefore, CD1d is expressed on tumor cells in a subset of MB patients and represents a novel target for immunotherapy.

IL-21 Selectively Protects CD62L+ NKT Cells and Enhances Their Effector Functions for Adoptive Immunotherapy

T cells expressing CD19-specific chimeric Ag receptors (CARs) produce high remission rates in B cell lymphoma, but frequent disease recurrence and challenges in generating sufficient numbers of autologous CAR T cells necessitate the development of alternative therapeutic effectors. Vα24-invariant NKTs have intrinsic antitumor properties and are not alloreactive, allowing for off-the-shelf use of CAR-NKTs from healthy donors. We recently reported that CD62L+ NKTs persist longer and have more potent antilymphoma activity than CD62L− cells. However, the conditions governing preservation of CD62L+ cells during NKT cell expansion remain largely unknown. In this study, we demonstrate that IL-21 preserves this crucial central memory–like NKT subset and enhances its antitumor effector functionality. We found that following antigenic stimulation with α-galactosylceramide, CD62L+ NKTs both expressed IL-21R and secreted IL-21, each at significantly higher levels than CD62L− cells. Although IL-21 alone failed to expand stimulated NKTs, combined IL-2/IL-21 treatment produced more NKTs and increased the frequency of CD62L+ cells versus IL-2 alone. Gene expression analysis comparing CD62L+ and CD62L− cells treated with IL-2 alone or IL-2/IL-21 revealed that the latter condition downregulated the proapoptotic protein BIM selectively in CD62L+ NKTs, protecting them from activation-induced cell death. Moreover, IL-2/IL-21–expanded NKTs upregulated granzyme B expression and produced more TH1 cytokines, leading to enhanced in vitro cytotoxicity of nontransduced and anti–CD19-CAR–transduced NKTs against CD1d+ and CD19+ lymphoma cells, respectively. Further, IL-2/IL-21–expanded CAR-NKTs dramatically increased the survival of lymphoma-bearing NSG mice compared with IL-2–expanded CAR-NKTs. These findings have immediate translational implications for the development of NKT cell–based immunotherapies targeting lymphoma and other malignancies.

512. Tumor-Associated Macrophages Via Up-Regulation of PD1 Ligands Protect Neuroblastoma from Immunotherapy With NKT Cells Expressing GD2-Specific Chimeric Antigen Receptor

Immunotherapy with Natural Killer T cells engineered to express a GD2-specific chimeric antigen receptor (CAR.GD2 NKTs) demonstrated potent anti-tumor activity in a metastatic model of neuroblastoma in humanized (hu)NSG mice. However, tumors eventually progressed without the loss of GD2 expression suggesting that tumor escape is due to inhibition of effector function of CAR. GD2 NKTs. Since NKTs co-localize and specifically interact with M2-like tumor-associated macrophages (TAMs), we examined whether TAMs inhibit effector functions of NKT and CAR.GD2 NKT cells. In vitro experiments revealed a reciprocal inhibition of NKTs and M2-polarized macrophages. We found that NKTs selectively kill M2-polarized macrophages or induce their differentiation toward M1-like cells. However, both M2- and M1-macrophages suppressed NKT-cell proliferation and IFNγ production in response to TCR or CAR stimulation. This was associated with rapid up-regulation of PD-L1 and PD-L2 on both M2 and M1 macrophages upon addition of supernatant from activated NKT cells. Moreover, M1 macrophages constitutively expressed PD-L1 and were resistant to NKT-cell cytotoxicity. The analysis of tumor tissues in hu-NSG mice revealed strong up-regulation of PD-L1 expression exclusively on TAMs and not on neuroblastoma cells after treatment with CAR. GD2 NKTs. Neither neuroblastoma cells nor TAMs expressed PD1 ligands in untreated mice or in primary NB tissues from patients with stage 4 disease. Importantly, a combination of CAR.GD2 NKT cell immunotherapy with anti-PD1 blocking mAb produced durable tumor regression. The results provide a rationale for a combined use of CAR-redirected NKT cells with PD1-blocking therapeutics for immunotherapy of neuroblastoma and possibly other solid tumors.

410. The Development of HLAnull K562 Cells Expressing CD1d and Co-Stimulatory Molecules as Artificial Antigen-Presenting Cells for Clinical Scale Expansion of NKT Cells With Superior In Vivo Persistence and Anti-Tumor Potential

CD1d-restricted type-I Natural Killer T cells (NKTs) have been shown to mediate antitumor responses in mouse models and are associated with improved outcome in several types of cancer. However, the therapeutic application of NKTs has been limited by low numbers and functional defects of these cells in patients with cancer. Herein we present an effective method for ex-vivo NKT-cell numeric expansion for adoptive immunotherapy. The method is based on engineered properties of K562 cells to function as artificial antigen-presenting cells (aAPC). K562 cells express a single endogenous HLA allele, HLA-Cw3, an NK-cell activating ligand which favors the expansion of NK cells that compete with NKTs in culture. We rendered K562 cells HLAnull by eliminating HLA-C gene from K562 cell genome using a HLA-C-specific zinc finger nuclease. We then transduced parental and HLAnull K562 cells with CD1d cDNA followed by single cell sorting and clonal expansion. The clones were pulsed with αGalactosylceramide and tested as irradiated aAPC for NKTs using CFSE proliferation assay. We found that in contrast to HLA-Cw3+ K562/CD1d, HLAnullK562/CD1d clones selectively expanded NKTs when added to primary PBMC, and clones with an intermediate level of CD1d expression induced the highest rate of NKT-cell proliferation. Next, a selected HLAnullCD1dmed clone was further modified to express CD86 alone or in combination with 4-1BBL and/or OX40L followed by single cell sorting and clonal expansion, producing 250 clones. One clone with the phenotype HLAnullCD1dmedCD86high4-1BBmedOX40Lmed (designated B-8-2) consistently induced the highest rate of NKT-cell expansion. B-8-2 aAPC were able to efficiently expand both primary NKTs as well as NKTs modified to express a chimeric antigen receptor (CAR) specific for CD19 antigen, a clinically validated therapeutic target for B-cell malignancies. CAR expression in NKTs rendered them highly cytotoxic against CD19+ Raji leukemia cells. Compared with CAR-NKTs expanded with autologous PBMC, those expanded with B-8-2 cells exhibited a Th-1-skewed cytokine profile, prolonged in vivo persistence, and superior therapeutic activity in Raji leukemia model. Gene expression analysis of NKTs expanded with B-8-2 cells vs. those expanded with autologous PBMC revealed a significant up-regulation of genes associated with T- and NK-cell memory (CCR7, CD45RA, KLRG1, KLRD1, IL-21R), a mixture of Th17- and Th-1-associated molecules (IL-17A, IL-17F, STAT1, IL12RB1, GZMA, GZMB GNLY) and downregulation of a particular pro-apoptotic gene from the BCL2 family, BCL2L11. Therefore, the engineered K562-derived B-8-2 cells can be used as a highly efficient aAPC for ex vivo propagation of fully functional primary as well as gene-modified NKTs amenable for cancer immunotherapy.

Abstract B08: Invariant NKT cells as a platform for cancer immunotherapy with chimeric antigen receptors

Advances in the design of chimeric antigen receptors (CARs) have improved the anti-tumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells and their restriction by polymorphic HLA molecules limit their therapeutic potential and are associated with toxicity, especially in allogeneic settings. We proposed that CAR expression in Vα24-invariant NKT cells (NKTs) can build upon the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKTs were engineered to express a CAR against the GD2 ganglioside, which is highly expressed by neuroblastoma. We compared CAR constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB co-stimulatory endodomains. CAR expression rendered NKTs highly cytotoxic against neuroblastoma cells without affecting their TCR-induced proliferation and CD1d-dependent killing of M2-polarized macrophages. We observed a striking Th1-like polarization of NKTs by 4-1BB-containing CARs in vitro and in mice. Expression of both CD28 and 4-1BB endodomains in the CAR maximized persistence of NKTs in NSG mice with neuroblastoma xenografts. These NKTs, expressing CAR with two co-stimulatory endodomains localized to the tumor site more effectively than T cells with the same CAR (p Citation Format: Andras Heczey, Daofeng Liu, Gengwen Tian, Ekaterina Marinova, Xiuhua Gao, Linjie Guo, John Hicks, Gianpietro Dotti, Leonid Metelitsa. Invariant NKT cells as a platform for cancer immunotherapy with chimeric antigen receptors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B08.