Engineering Precision Medicine to Increase Graft-Versus-Lymphoma Activity: Hematopoietic Stem Cells Modified with Chimeric Antigen Receptors

Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure. Trials using autologous T-cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the cells is transient, limiting efficacy. Our hypothesis is modification of hematopoietic stem cells (HSC) with CAR will lead to persistent production of target-specific immune cells in multiple lineages, enhancing graft-versus-tumor activity and development of immunological memory.

Design/Methods: Using CD19 as target, we generated second-generation CD28- and 4-1BB-costimulated CAR constructs for modification of human HSC for assessment in vitro and in vivo, using third-generation lentiviral vectors. Additionally, co-delivery of suicide gene systems was tested to allow ablation of gene-modified cells.

Results: Gene modification of HSC with anti-CD19 CAR using lentiviral vectors did not impair differentiation or proliferation, and led to functional CAR-expressing cell progeny, at 40-50% transduction efficiency and engineered antigen-dependent cytotoxicity in myeloid, NK and T-cells. In vivo studies using humanized NSG engrafted with CAR-modified HSC demonstrated similar levels of humanization to non-modified HSC, with multilineage CAR-expressing cells present in bone marrow, spleen, blood and thymus in stable levels up to 44 weeks of life. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. Ex vivo cells presented antigen-dependent cytotoxicity against targets. Mice engrafted with CAR-modified HSC had decreased CD19+ populations and successfully presented tumor growth inhibition and survival advantage at tumor challenge (55-60%). CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues.

Conclusions: CAR modification of HSC for cancer immunotherapy is feasible. This approach can be applied to different cancers by adjusting target specificity and could be employed in the context of HSC transplantation to augment the anti-lymphoma activity. It also bears the possibility of decreased morbidity and mortality and offers alternative treatment for patients with no available sources for bone marrow transplantation, benefiting ethnic minorities. These results also give proof of principle for CAR-modified HSC regulated by suicide gene; further studies are needed to enable clinical translation.