IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Abstract

Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

convertibleCARs: A chimeric antigen receptor system for flexible control of activity and antigen targeting

“…And lastly, as CARs have been demonstrated to persist in humans for years post-infusion 34 , the ability to recall resident convertibleCAR-Ts to attack primary or secondary malignancies (either with the original targeting MicAbody or a different one) without having to reengineer or generate a new batch of CAR cells should be highly advantageous. Unlike scenarios where CARs have been engineered to constitutively express cytokines 35,36 , delivery of cytokines exclusively to convertibleCAR-T cells can be modulated depending upon the manufacturing or clinical needs.…”

Autologous CD19-Targeted CAR T Cells in Patients with Residual CLL following Initial Purine Analog-Based Therapy

“…39 We have explored strategies to overcome CLL-mediated immune resistance in mouse models by further modifying CAR T cells to constitutively express an additional costimulatory ligand (e.g., 4-1BBL, CD40L) or to secrete immunoregulatory cytokines (e.g., IL-12, IL-18), and a phase I clinical trial of 19-28z/4-1BBL CAR T cells in patients with R/R CLL is ongoing at our institution. [40][41][42][43][44][45] Several other clinical investigations are ongoing to combine immunologic checkpoint blockade or the oral Bruton's tyrosine kinase (BTK) inhibitor ibrutinib based on preclinical and preliminary clinical evidence showing that ibrutinib can enhance T cell proliferation, decrease PD-1 and CD200 expression, and increase skewing of the central memory phenotype of CAR T cells. 12,16,32,39,46 In summary, administration of 19-28z CAR T cells to patients with residual CLL following initial chemoimmunotherapy with PCR was safe and well tolerated, and a subset of patients achieved a meaningful clinical response.…”

Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward

“…Avanzi et al, have developed CAR T cells that constitutively secrete IL-18. Their results revealed that IL-18 not only can enhance CAR T cell survival and antitumor activity both in vitro and in vivo, but also can activate cells of endogenous immune system in immunocompetent mice (37). In a similar study, Hu et al, discovered that anti-CD19 and antimesothelin CAR T cells engineered to secrete IL-18 not only can support in vivo engraftment and persistence of CAR T cells but also enhance secretion of IFN-γ and several other cytokines such as IL-2, G-CSF, GM-CSF, TNF-α, IL-17A, and IP-10 (38).…”