ALBERT

Description: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure, despite intensive therapy. Chimeric Antigen Receptors (CARs) 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. Our main hypothesis is that modification of hematopoietic stem cells (HSCs) with CARs will lead to persistent in vivo production of target-specific immune cells in multiple lineages, enhancing graft-versus-tumor activity and development of immunological memory. Using CD19 as target, we have generated first-generation and CD28- and 4-1BB-containing-second-generation CAR lentiviral constructs for modification of human HSCs, for assessment in vitro and in vivo. Gene modification with anti-CD19 CAR of CD34+cells isolated from human umbilical cord blood (UCB) did not impair normal differentiation and proliferation, with fully functional CAR-expressing cell progeny. Transduction with lentiviral vectors consistently achieved 40-50% efficiency at the clinically relevant vector copy number of 1-2 copies/cell. While first- and second-generation CARs triggered antigen-dependent cytotoxicity by myeloid and T cells in a similar fashion, only second-generation constructs successfully activated NK cells for antigen-dependent elimination of cell targets. In vivo studies using humanized NSG engrafted with CAR-modified human UCB CD34+ cells demonstrated similar levels of engraftment of human cells as compared to non-modified UCB CD34+ cells, with CAR-expressing cells in multiple lineages (myeloid, NK, T) successfully engrafted into bone marrow, spleen, peripheral blood and thymus detectable by flow cytometry and qPCR, in stable levels up to 35 weeks of life, with gene modification with first- or second-generation anti-CD19 CARs. No animals engrafted with CAR-modified HSCs presented signs of autoimmunity or chronic inflammation. Cells presented ex vivo antigen-dependent cytotoxicity against cell targets. Mice successfully engrafted with CAR-modified HSCs harbored decreased CD19+populations, and only HSCs modified with second-generation CARs successfully led to tumor growth inhibition and survival advantage at tumor challenge. CAR-modified HSCs led to development of T cell effector memory and T cell central memory subsets, confirming the expectation of development of long-lasting phenotypes due to directed antigen specificity. Longer survival of mice with developing tumors was also significantly correlated to higher number of CAR-expressing cells infiltrating subcutaneous tumors. Our results demonstrate feasibility of CAR modification of human HSCs for cancer immunotherapy. This approach can be applied to different cancers just by adjusting the target specificity. Furthermore, it could be easily employed in the context of HSC transplantation to augment the anti-leukemic activity, with CAR-expressing myeloid and NK cells to ensure tumor-specific immunity until de novo production of T cells from CAR-modified HSCs. It also bears the possibility of decreased morbidity and mortality, being desirable for vulnerable populations such as children and elderly patients, and offers alternative treatment for patients with no available HLA-matched sources for bone marrow transplantation, benefiting ethnic minorities. Disclosures Larson: Millenium Pharmaceuticals, Inc.: Speakers Bureau.

Description: Background Although significant improvements have been made, patients with relapsed or refractory B-cell malignancies continue to have unfavorable clinical outcomes. We hypothesize that transduction of hematopoietic stem cells (HSCs) with an anti-CD19 Chimeric Antigen Receptor (CAR) will produce a multi-lineage, persistent immunotherapy that can be controlled by the HSVsr39TK suicide gene. Methods First generation anti-CD19 CAR lentiviral constructs containing the HSVsr39TK suicide gene were developed to compare vectors containing the human elongation factor alpha short (EFS) or myeloproliferative sarcoma virus U3 (MNDU3) promoters for transduction efficiency, antigen-specific cytotoxicity and ganciclovir (GCV)-induced cell death in primary human T-cells. The CD28 costimulatory domain was added to the selected construct, and high titer lentiviral vectors were generated to evaluate transduction of human umbilical cord blood (UCB) HSCs for in vitro and in vivo assays. In vitro assays were performed after culture under myeloid differentiation conditions, followed by assessment of phenotype, transduction efficiency, cytotoxic function and GCV-induced cell death. In vivo assays were conducted through transplantation of gene-modified human HSCs into irradiated NSG pups, compared to humanized NSG injected with non-modified human HSCs. Once engraftment was identified, mice from each cohort were further separated into GCV treated and untreated groups. Following GCV administration, mice were harvested to evaluate the presence of human and CAR-modified cells in the bone marrow, spleen and peripheral blood. Results In human primary T cells, the MNDU3 promoter resulted in higher percentage of CAR expressing cells and mean fluorescence intensity compared to the EFS promoter. Cytotoxicity by the transduced T cells against the huCD19+Raji cell line showed similar target cell specific lysis among the constructs. Treatment with GCV effectively decreased the in vitro survival of the cells containing the HSVsr39TK gene compared to the non-transduced and control vector. The construct with MNDU3 promoter was then used with a CD28-containing second-generation anti-CD19 CAR (CCL-MND-αCD19/z/28-sr39). Once transduction efficiency and CAR function were validated in primary human T cells, this vector was used to transduce human UCB CD34+ cells. Following transduction, these cells were evaluated in vitro and in vivo. The cells used for the in vitro studies were cultured under myeloid differentiation conditions. The average number of CAR expressing cells was 45% at the clinically relevant vector copy number of 0.5-1 copies/cell. The myeloid cells transduced with the CCL-MND-αCD19/z/28-sr39 vector demonstrated CD19-specific killing and were eliminated by GCV. In vivo studies demonstrated successful engraftment of transduced HSC with CAR-expressing cells in the different hematopoietic lineages (T, NK, myeloid) detected among human cells in the bone marrow (1.2-15.4%, mean 7.6%), spleen (0.3-15.4%, mean 5.6%), and peripheral blood (0.5-30%, mean 9.2%). Mice engrafted with anti-CD19 CAR-modified HSCs exhibited decreased huCD19+ populations, compared to the mice engrafted with non-modified HSCs. Treatment with GCV resulted in significant decrease in CAR-expressing cells only in the mice transplanted with CD34+ cells transduced with the HSVsr39TK-containing vector. Discussion Here we demonstrate that HSCs can be effectively transduced with an anti-CD19 CAR linked to the HSVsr39TK suicide gene. The CAR was detected in human cells in the bone marrow, spleen and peripheral blood and resulted in decreased B-lineage populations as an index of antigen-specific cytotoxicity; the HSVsr39TK gene conferred sensitivity to ganciclovir which eliminated transduced cells. These results provide pre-clinical support for the use of a CD19 targeted CAR in HSCs for the treatment of B-cell malignancies. Disclosures: Larson: Millenium: Speakers Bureau.

Description: Optimization of transgene expression is paramount for successful gene modification of primary cells for clinical applications, and careful selection of the viral vector construct is a critical part of this process. Viral promoters based on the U3 region of the Moloney murine leukemia virus (such as MNDU3 and MSCV) are currently the most commonly used for gene transfer in human primary cells. These viral promoter-containing vectors, however, can activate nearby genes, potentially causing toxicity and/or neoplastic transformation. EF1alpha (or its short, intron-less form, EFS) is a promoter that has been recently used in many clinical trials. It is a cellular-derived enhancer/promoter with decreased cross-activation of nearby promoters, therefore hypothetically decreasing the risk of genotoxicity. We have produced vector constructs carrying the internal enhancer/promoters MNDU3, MSCV, or EFS driving clinically relevant transgenes for modification of primary human T lymphocytes and hematopoietic stem cells. Lentiviral vectors containing either the MNDU3 or EFS promoters driving the EGFP reporter gene were used to transduce Jurkat cells and primary human T cells. In Jurkat cells, MNDU3-driven vectors provided 2-3 times higher vector copy integrations with a corresponding higher percentage of EGFP expression, across a wide range of multiplicity of infection (MOI). In primary T cells, however, there was no significant increase in vector copy numbers per cell, but a significant increase in transduction efficiency and geometric mean fluorescence intensity of EGFP expression in cells transduced with MNDU3-driven vectors at all MOI studied, even when corrected for vector copy number. Lentiviral vectors containing either a MNDU3 or EFS promoter driving a first-generation anti-CD19 chimeric antigen receptor (CAR) were used to transduce primary human T cells. We found that integrated vector copy numbers per cell were 0.8 with MNDU3 and 0.5 with EFS, and resultant transgene expression in the transduced populations was 45% with MNDU3 and 22% with EFS. Primary human T cells were also transduced with a lentivirus carrying MSCV or EFS driving a codon-optimized MART-1-specific T cell receptor (TCR) and then analyzed by tetramer staining. MSCV promoter-driven vectors resulted in 33.76%, 33.1%, and 29% higher transgene expression at 5 ng, 10 ng, and 25 ng p24 equivalents compared with T cells transduced with vectors driven by the EFS promoter using the same amount of p24. After correction for integrated vector copy numbers, T cells had more than 2-fold increase in transgene expression when using the MSCV promoter. CD34+ hematopoietic stem cells isolated from human cord blood were transduced using the same high-titer MSCV- or EFS-driven MART-1-specific TCR expression vectors; MSCV-driven lentiviral vectors provided an average vector copy number of 0.5 copies per cell compared to 0.7 copies per cell with the similar EFS-containing vectors. These gene-modified cells were then injected into NOD-scid-IL2rγnull mice, with peripheral blood analyzed by flow cytometry after 8 weeks. HuCD45+/huCD3+/huCD4+ and huCD45+/huCD3+/huCD8+cells had mean transgene expression of 18% and 16% in the MSCV group, compared to 0% and 0% in the EFS group. Together, these results demonstrate more efficient transgene expression is conveyed by the virally-derived MSCV and MNDU3 promoters versus the cellular EFS promoter in gene-modified primary human hematopoietic cells. Higher transgene expression relative to integrated vector copies is consistent with higher promoter function, and transgene expression may be significantly decreased when using the EFS promoter in lentiviral vectors for clinical applications. Further studies are needed to carefully evaluate genotoxic effects of the MNDU3 and MSCV promoters in comparison to the EFS promoter for safe and efficient clinical translation. Disclosures Larson: Millenium Pharmaceuticals, Inc.: Speakers Bureau.