Anti-CD19 Chimeric Antigen Receptor in Hematopoietic Stem Cells Controlled By the Suicide Gene HSV-sr39TK for Immunotherapy of B-Lineage Malignancies
Authors: Truscott Laurel Christine , Chiou Tzu Ting , De Oliveira Satiro N. 
Published: October 2019

Background: Patients with refractory or recurrent B-lineage hematologic malignancies have less than 50% of chance of cure despite intensive therapy and innovative approaches are needed to decrease their morbidity and mortality. Our previous published results demonstrate feasibility of stable gene modification of hematopoietic cells (HSC) with chimeric antigen receptor (CAR) directing antigen specificity of T-, NK- and myeloid cells that are continuously produced with effective anti-tumor activity in humanized mouse model. We hypothesize that gene modification of HSC with an anti-CD19 CAR will produce a multi-lineage, persistent immunotherapy against B-lineage hematological malignancies that can be controlled by the HSV-sr39TK suicide gene. Hematopoietic stem cell transplantation is frequently part of the standard of care for patients with relapsed and refractory B cell malignancies. Following HSC collection, a portion of these cells could be modified to express the CD19-specific CAR and give rise to a persistent, multi-cell lineage, HLA-independent immunotherapy, enhancing the graft-versus-malignancy activity. Based on current clinical trials of gene therapy, gene modification of a partial portion of HSC would be sufficient to give rise to a large number of antigen-specific immune cells, and ablation of such gene-modified cells would not cause myeloablation. The generation of NK and myeloid cells bearing CAR would allow earlier directed anti-tumor activity until post-transplant thymopoiesis can take place, enhancing the graft-versus-cancer activity.

Methods: High-titer third-generation self-inactivating lentiviral constructs were developed to deliver second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered the hyper-active herpes simplex virus thymidine kinase HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Such vector constructs were thoroughly evaluated using Jurkat cell line and primary human T-cells. Human HSC isolated from umbilical cord blood were transduced with such lentiviral vectors and evaluated for function of both CAR and HSV-sr39TK. Gene-modified human HSC were also injected into NSG pups after irradiation for in vivo evaluation of engraftment, anti-tumor activity and suicide gene activation. In vivo ablation of gene-modified cells was performed by treatment with intraperitoneal ganciclovir 50mg/kg/day over 5 days. Persistence of gene-modified cells was assessed by flow cytometry and ddPCR of animal tissues.

Results: Two promoters were compared in such vector constructs, the human elongating factor-1 short (EFS) and the retrovirus-derived MNDU3, regarding transduction efficiency of primary cells. Those vector constructs with MNDU3 as the promoter showed improved CAR expression (p=0.003) and antigen-specific cytotoxicity, despite similar integrated vector copy numbers (VCN) per cell. The addition of HSV-sr39TK to the construct did not impair CAR expression or antigen-specific cytotoxicity, and determined marked cytotoxicity to ganciclovir. Transduction of human HSC with high-titer lentiviral vectors led to satisfactory transduction efficiency with second-generation CAR and HSV-sr39TK. When tested in vivo, mice transplanted with gene-modified human HSC showed similar humanization in bone marrow and spleen (as defined by CD45+ cells) to non-modified HSC. CAR expression by flow cytometry and VCN were not significantly different between transduced cells with and without HSV-sr39TK, and expression of anti-CD19 CAR conferred anti-tumor survival advantage, with 100% of survival of mice engrafted with HSC modified with CAR and HSV-sr39TK for up to 120 days post-tumor challenge. When treated with ganciclovir, the flow cytometry detection of gene-modified cells was decreased in bone marrows (p=0.07) and spleens (p=0.05) to levels similar to background, and with significant decrease of ddPCR detection of gene-modified cells in both bone marrows (p=0.05) and spleens (p=0.01).

Conclusions: The modification of HSC with second generation anti-CD19 CAR has provided specific and persistent anti-lymphoma protection. The addition of the suicide gene system HSV-sr39TK to the construct did not affect transduction efficiency, and has determined significant ablation of gene-modified cells in mouse tissues.

Disclosures

No relevant conflicts of interest to declare.