Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells

Abstract

The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. We report long-term results from three clinical trials to evaluate gammaretroviral vector-engineered T cells for HIV. The vector encoded a chimeric antigen receptor (CAR) composed of CD4 linked to the CD3ζ signaling chain (CD4ζ). CAR T cells were detected in 98% of samples tested for at least 11 years after infusion at frequencies that exceeded average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells; integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells had stable levels of engraftment, with decay half-lives that exceeded 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression before T cell transfer is not required to achieve long-term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient-years of follow-up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long-term delivery of protein-based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases.

Figure 1. Persistence of CD4ζ modified CAR T-cells over 11 years post infusion

(A) Total samples tested at annual visits and the corresponding number of samples with detectable CD4-ζ. Persistence of CAR T cells for the 43 individual patients in the (B) Mitsuyasu (8), (C) Deeks (9) and (D) Aronson (clinicaltrials.gov NCT01013415) trials, at annual visits beginning at 1 year post infusion. The LOD for the assay is plotted as a dotted reference line.

Figure 2. Transcriptional activity and CAR function in persisting cells

(A) The CD4ζ RNA level (y axis) is plotted versus the number of DNA CD4ζ DNA copies per million PBMC of each tested sample. Samples from the Deeks, Mitsuyasu and Aronson studies are plotted as red, blue and green symbols, respectively. CD4ζ RNA expression was calculated from the ΔCt values for RT-PCR of CD4ζ and GAPDH mRNA. GAPDH is expressed at a high level, so that greater expression of CD4ζ results in a smaller expression difference and so a smaller ΔCt. The values are significantly correlated by linear regression analysis (p=0.0018) testing whether rho=0 or not. No RT-controls were run in parallel and all plotted CD4ζ samples were negative, confirming the signal observed is due to RNA template. Two subjects did not have detectable CD4ζ RNA. (B) Design of the proliferation assay used to interrogate function of CD4-ζ CAR in T-cells. This assay was validated prior to employing as described in Fig. S1. Functionality is measured as the relative increase in the average copy number of CD4ζ cells following anti-CD4 antibody activation over percentage of CD4ζ before stimulation. (C) Fold-increase of CD4ζ expressing cells following three 10-day rounds of anti-CD4 mAb loaded irradiated K562 artificial antigen presenting cells expressing the high affinity Fc Receptor CD64 (KT64) and 100 IU of IL-2. CD4ζ copy numbers were evaluated from the gDNA of subject PBMCs before and after activation by qPCR analysis. The final percentage of CD4ζ in each culture is indicated by the number at the top of each bar. Each bar is designated at the bottom with the subject ID and year post-infusion of the sample.

Figure 3. Integration site analysis of CD4ζ modified CAR T cells

(A) Longitudinal abundance and dynamics of CD4ζ modified T-cells. The top-left corner of each panel shows the patient number. Q-PCR measurements of total CD4ζ copy number per million PBMCs are shown longitudinally for individual subjects (blue line). The x-axis shows months post infusion, the y axis shows Q-PCR vector copy number. Stacked bar graphs are shown directly above time points where integration sites were isolated and depict the relative abundance of integration sites based on the proportion of sequence reads detected using MseI and Tsp509I. The top five abundant sites are differentially colored with all other less abundant sites colored grey. The total number of unique sites detected at a given time point is shown above the corresponding bar graph. (B) Frequency of integration near cancer-associated gene 5′ ends. Integration sites were separated into four bins with one bin for preinfusion sites and three bins for post infusion sites (x-axis). The percent of sites found within 50kb from a gene 5′ end that were also within 50kb from a cancer-associated gene’s 5′ end are shown (y-axis). The number of sites <50kb from a gene’s 5′ end are shown at the top of each bin. No significant difference between the preinfusion and post infusion bins was found in pairwise comparisons using Fisher’s exact tests.