Pre-Clinical Development of Gene Modification of Hematopoietic Stem Cells with Chimeric Antigen Receptors for Cancer Immunotherapy
Authors: Truscott Laurel Christine , Larson Sarah , Patel Amie , Kao Roy L. , De Oliveira Satiro N. 
Published: October 2019

Abstract
Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure, despite intensive therapy. Innovative approaches are needed to decrease their morbidity and mortality. Chimeric Antigen Receptors (CAR) successfully engineer antigen specificity in immune cells, with clinical trials currently being conducted using ex vivo expanded gene-modified mature T cells. Results from preclinical studies and clinical trials show that effector cells usually have transient in vivo persistence that could significantly limit clinical efficacy and allow tumor recurrence. Building upon the standard of care and seeking an approach that could foster persistence of the CAR-modified cells, we have published studies using anti-CD19 CAR-modified human hematopoietic stem cells (HSC) engrafted in NSG immunodeficient mice. We hypothesize that modification of HSC with CD19-specific CAR will generate persistent multi-lineage anti-tumor activity for immunotherapy of CD19+ hematological malignancies. To increase the safety of the modification of HSC, a suicide gene can be inserted into the vector to eradicate the modified cells in the setting of toxicity. Thorough evaluation of this approach in relevant study models is required for advancement to clinical trials.
Significance: This approach is untested in clinical translation to this date, and implies harnessing a patient's own HSC to create a whole self-renewing immune system directed to destroy cancer, a concept that can be applied to different cancers just by adjusting the target specificity. The prospect of modifying autologous cells to enhance graft-versus-cancer activity bears the possibility of decreased morbidity and mortality, being desirable for specifically vulnerable populations, as children and elderly patients, and offering alternative therapy for those without cell sources available for allogeneic HSC transplantation, benefiting patients from ethnic minorities. This approach can be easily adopted in the clinical setting for patients planned to receive autologous HSC transplant as their standard therapy, enhancing graft-versus-cancer activity with anti-CD19 specificity.
Methods: High-titer third-generation lentiviral constructs were produced carrying second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC isolated from umbilical cord blood and G-CSF-mobilized apheresed peripheral blood stem cells (PBSC) were transduced with such lentiviral vectors and injected into NSG pups after irradiation for in vivo evaluation of engraftment, function and suicide gene activation.
Results: We have consistently achieved engraftment of human cells in about 95% of study mice, with engraftment of CAR+ cells in about 80% of the animals. Human HSC were successfully transduced with lentiviral vectors carrying anti-CD19 CAR with no impairment of differentiation or proliferation in vitro and in vivo. Immune cells differentiated in vivo from CAR+ HSC had antigen-specific cytotoxicity directed by CAR. CAR+ human cells were detected in BM, spleen, blood and thymus of injected mice. CAR+ T cells were stably detected in the blood of engrafted mice up to 40 weeks post-injection, demonstrating lymphopoiesis of CAR+ T cells successfully escaping thymic deletion and persisting throughout murine lifetime. As a surrogate of the antigen specificity and efficacy, CD19+ cells were significantly decreased in all mice engrafted with anti-CD19 CAR demonstrating that CAR+ immune cells were not inactivated or developed tolerance. Mice humanized with at least 4% of CAR+ cells in blood had significant protection against challenge with CD19+ tumor cell line, with inhibition or elimination of tumor development and consequent survival advantage. Activation of HSV-sr39TK suicide gene by ganciclovir treatment successfully led to ablation of gene-modified cells in vitro and in vivo.
Conclusions: Our results demonstrate feasibility of CAR modification of human HSC for cancer immunotherapy. It could be easily employed in the context of HSC transplantation to augment the anti-cancer activity, with CAR-expressing myeloid and NK cells to ensure tumor-specific immunity until de novo production of T cells from CAR-modified HSC.

Disclosures
Larson: BMS: Consultancy.