No abstract

Key Points GD2-specific CAR renders NKT cells cytotoxic against NB cells and results in potent in vivo antitumor activity without graft-versus-host disease. The 4-1BB-containing CAR constructs induce T helper 1–like polarization in NKT cells.

No abstract

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but are associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL-7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Co-expressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and anti-tumor activity during repeated exposure to tumor cells, without T cell dysfunction or autonomous T cell growth. Furthermore, C7R co-expressing CAR-T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer.

Vα24-invariant NKT cells inhibit tumor growth by targeting tumor-associated macrophages (TAMs).

Purpose: Va24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs.Experimental Design: Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma.Results: Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activationinduced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2. CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround in vitro tumor cell killing. Following transfer into mice bearing neuroblastoma xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared with GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathologic analysis.Conclusions: Our results informed selection of the CD28based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).

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 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.

One of the major limitations of modern cancer vaccine vectors is that, unlike infectious pathogens, to which the immune system has evolved to respond, they are not sufficiently effective in delivering tumor-associated antigens (TAAs) in an immunogenic form to intact professional antigen-presenting cells (APCs) at their anatomic location. To overcome this challenge, we exploited Salmonella Pathogenicity Island 2 (SPI2) and its type III secretion system (T3SS) to deliver a TAA of choice into the cytosol of APCs in situ. We have systematically compared candidate genes from the SPI2 locus of Salmonella typhimurium in the vaccine design, using model antigens and a codon-optimized human TAA, survivin (coSVN). In a screen of 20 SPI2 promoter/effector combinations, the PsifB::sseJ pair demonstrated the maximal potency for antigen translocation in the APC cytosol, presentation to CD8 T cells, and immunogenicity in mice. Therapeutic vaccination with the PsifB::sseJ-coSVN construct (p8032) resulted in CXCR3-dependent tumor infiltration with CD8 T cells, reversal of the CD8:Treg ratio at the tumor site, and potent anti-tumor activity in a CT26 colon carcinoma model. The vaccine’s immunogenicity and anti-tumor potency were further enhanced by co-administration of an NKT-cell ligand, 7WD8-5, which strongly enhanced production of IL-12 and IFNγ in vaccinated mice. Furthermore, therapeutic vaccination with p8032/7WD8-5 resulted in complete tumor regression in an A20 lymphoma model, with the generation of protective memory. Thus, oral antigen delivery via SPI2-encoded T3SS of Salmonella may be the foundation for an effective cancer vaccine platform.