ALBERT

Description: Human endogenous retroviruses (HERVs) are ancient viruses forming 8% of human genome. One subset of HERVs, the HERV-K has recently been found to be expressed on tumor cells including melanoma, breast cancer and lymphoma but not on normal body cells. Thus, targeting HERV-K protein as a tumor associated antigen (TAA) may be a potential treatment strategy for tumors that are resistant to conventional therapies. One approach to improve therapeutic outcome is by infusing T cells rendered specific for such TAAs preferentially expressed on tumor cells. Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a chimeric antigen receptor (CAR) on T cells using gene therapy. This approach is currently being used in our clinical trials adoptively transferring CD19-specific CAR+ T cells into patients with B-lineage malignancies. Preliminary analysis of HERV-K env protein expression in 268 melanoma samples and 139 normal organ donor tissues using immunohistochemistry demonstrated antigen expression in tumor cells and absence of expression in normal organ tissues. The scFv region from a mouse monoclonal antibody to target HERV-K env was used to generate a CAR and cloned into Sleeping Beauty (SB) plasmid for stable expression in T cells. HERV-K-specific CAR+T cells were selectively propagated ex vivo on artificial antigen presenting cells (aAPC) using an approach already in our clinical trials. Indeed, after genetic modification of T cells and selection on HERV-K+ aAPC, over 95% of propagated T cells stably expressed the introduced HERV-K-specific CAR and exhibited redirected specificity for HERV-K+ melanoma (Figure 1). Further, the adoptive transfer of HERV-K-specific CAR+T cells killed metastatic melanoma in a mouse xenograph model. While we have chosen melanoma as our tumor model, this study has the potential to be applied to other malignancies, including lymphoma and myeloma due to restricted expression of HERV-K envelope (env) protein on these tumor cells. These data demonstrate that it is feasible to generate T cells expressing a HERV-K-specific CAR using a clinically-appealing approach as a treatment strategy for HERV-K env+ tumors. Disclosures: No relevant conflicts of interest to declare.

Description: Background T cells can be genetically modified ex vivo to redirect specificity upon enforced expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in autologous and allogeneic T cells manufactured in compliance with current good manufacturing practice (cGMP) for Phase I/II trials. Methods T cells were electroporated using a Nucleofector device to synchronously introduce DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. Results To date we have enrolled and manufactured product for 25 patients with multiply-relapsed ALL (n=12) or B-cell lymphoma (n=13) on three investigator-initiated trials at MD Anderson Cancer Center to administer thawed patient- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=7), allogeneic adult (n=14) or umbilical cord (n=4) hematopoietic stem-cell transplantation (HSCT). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing under CLIA. Currently, five patients have been infused with the CAR+ T cells following allogeneic HSCT, including one patient with cord blood-derived T cells (ALL, n=4; NHL, n=1), beginning at a dose of 106 and escalating to 107 modified T cells/m2. Three patients treated at the first dose level of 106 T cells/m2 have progressed; the patient treated at the next dose level with 107 T cells/m2 remains in remission at 5 months following HSCT. Assessment for response too early for patient treated with UCB T cells. Four patients with non-Hodgkin’s lymphoma have been treated with patient-derived modified T cells following autologous HSCT at a dose of 5x107 T cells/m2, and all patients remain in remission at 3 months following HSCT. No acute or late toxicities have been noted to date. PCR testing for persistence of CAR-modified T cells is underway. Conclusion We report the first human application of the SB and aAPC systems to genetically modify clinical-grade cells. Importantly, infusing CD19-specific CAR+ T cells in the adjuvant HSCT setting and thus targeting minimal residual disease is feasible and safe, and may provide an effective approach for maintaining remission in patients with high risk, CD19+ lymphoid malignancies. Clinical data is accruing and will be updated at the meeting. This nimble manufacturing approach can be readily modified in a cost-effective manner to improve the availability, persistence and therapeutic potential of genetically modified T cells, as well as target tumor–associated antigens other than CD19. Disclosures: No relevant conflicts of interest to declare.

Description: Background The ability to transplant across HLA disparities makes allogeneic umbilical cord blood (UCB) an attractive graft source for hematopoietic stem-cell transplantation (HSCT). Disease relapse remains a limitation, and adoptive transfer of tumor-specific T cells post UCB HSCT has not been feasible due to the functionally naïve CB T cells, and the small size as well as anonymity of the donor. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific chimeric antigen receptor (CAR, designated CD19RCD28) on UCB-derived T cells manufactured in compliance with current good manufacturing practice (cGMP). Methods After thawed UCB units are washed for clinical infusion 5% to 10% of cells are used to generate CAR+ T cells. The mononuclear cells are electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR are retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. Enrolled patients on our phase I trial receive two UCB units, thus two genetically modified T-cell products are made for each patient. We infuse thawed donor-derived CD19-specific CAR+ T cells from the dominant CB unit based on peripheral blood chimerism on days 40-100 post transplant in the adjuvant setting after double UCB HSCT Results To date we have successfully manufactured 8 products for 4 patients (ALL n=3, NHL=1) enrolled on trial. The median number of T cells in the starting CB aliquot was 8.6x106 (range, 2.5x106 to 54.8x106) with final modified T cell count at median 3x109 (range,1.7x108 to 4.1x1010) at time of cryopreservation days 28-32. In the final product, the median CD19-CAR+ cell purity by flow was 88% (range, 81.9% to 95.8%). The modified T cell product consisted of median 97.3% CD3+, 2.7 CD3-/CD56+ cells. All of the products exhibited CD19-specific killing by chromium assay as illustrated (Figure). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing. One patient with ALL has been infused to date with a T cell dose of 106T cells/m2 and no toxicity has been observed. The patient remains alive and in continued molecular remission at 111 days post HSCT. Conclusion We combined the SB system and aAPC-mediated propagation of T cells to successfully manufacture disease-specific T cells from small aliquots of UCB used to restore hematopoiesis. Importantly, this approach allows us to employ adoptive therapy to enhance the graft-versus-tumor effect in UCB HSCT as an approach to improve overall survival for these recipients. Accrual to the trial continues and updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: T cells have been previously genetically manipulated to act as a cellular vaccine to present viral antigens such as MP1 from influenza A. We have now genetically modified T cells as antigen presenting cells (T-APC) to express the tumor-associated antigen (TAA) NY-ESO-1 (Figure 1). This TAA is reportedly found in the majority of patients with high risk multiple myeloma, as well as other malignancies, but expression is absent from normal tissues. Therefore, immunologic targeting of NY-ESO-1 may be a potential treatment strategy for myeloma that is resistant to conventional therapies. One approach to improve therapeutic outcome is by adoptive transfer of T cells rendered specific for a TAA preferentially expressed on tumor cells. T-cell specificity can be redirected to intracellular TAAs by enforced expression of a characterized T-cell receptor (TCR) or chimeric antigen receptor (CAR) that recognizes processed antigen in the context of restricting human leukocyte antigen (HLA). However, persistence of these infused genetically modified cells, which is directly related to a favorable clinical response, may be variable. Therefore, T-APC may have a dual function of increasing persistence of adoptively transferred CAR+ and/or TCR+ T cells as well as inducing long term immunity through direct and cross priming of endogenous immune cells. To enhance these presentation functions, a new co-stimulatory molecule that tethers IL-15 to the cell surface (membrane-bound IL-15, mIL15) was expressed on NY-ESO-1+T-APC using the Sleeping Beauty (SB) gene transfer system. NY-ESO-1-specific HLA A2+ T cells expressing TCR and CAR could be selectively propagated ex vivo on autologous mIL15+NY-ESO-1+T-APC. The T-APC were also able to selectively propagate NY-ESO-1-specific T cells from autologous peripheral blood which can be described by binding of specific pentamer (Figure 2). Our data demonstrate that it is feasible to generate T-APC that can activate T cells engineered to have a defined specificity for NY-ESO-1 as well as grow out NY-ESO-1-specific T cells. The SB system is in place for genetic modification of clinical-grade T cells to express CAR, TCR, NY-ESO-1, and mIL15. Thus, we are proceeding to a human trial to infuse T-APC with and without genetically modified T cells in patients with NY-ESO-1+ tumors such as myeloma. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Cooper: InCellerate: Equity Ownership; Sangamo: Patents & Royalties; Targazyme: Consultancy; GE Healthcare: Consultancy; Ferring Pharmaceuticals: Consultancy; Fate Therapeutics: Consultancy; Janssen Pharma: Consultancy; BMS: Consultancy; Miltenyi: Honoraria.

Description: Objectives: T cells can be genetically modified ex vivo to redirect specificity upon expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We employ non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in patient (pt)- or donor-derived T cells for patients with advanced B-cell malignancies. Methods: T cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of designer g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. Results: To date we have successfully manufactured product for 42 pts with multiply-relapsed ALL (n=19), NHL (n=17), or CLL (n=5) on 4 investigator-initiated trials at MD Anderson Cancer Center to administer thawed pt- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=5), allogeneic (n=21) or umbilical cord (n=4) hematopoietic cell transplantation (HCT), or for the treatment of active disease (n=12). Each clinical-grade T-cell product was subjected to a battery of in-process and final release testing. Adjuvant trials: Twelve pts have been infused with donor-derived CAR+ T cells following allogeneic HCT, including 2 pts with cord blood-derived T cells (ALL, n=10; NHL, n=2), beginning at a dose of 106 and escalating to 5x107 modified T cells/m2. Three pts, all with ALL, remain alive and in remission at median 5 months following T cell infusion. Five pts with NHL have been treated with pt-derived modified T cells following autologous HCT at a dose of 5x108 T cells/m2, and 4 pts remain in remission at median 12 months following T-cell infusions. Relapse trials: Thirteen pts have been treated for active disease (ALL, n=8; NHL, n=3; CLL, n=2) with pt or donor-derived (if prior allo-HCT) modified T cells at doses 106-5x107/m2, and 3 remain alive and in remission at median 3 months following T-cell infusions. No acute or late toxicities, including excess GVHD, have been noted. Conclusion: We report the first human application of the SB and AaPC systems to genetically modify clinical-grade cells. Furthermore, infusing CD19-specific CAR+ T cells in the adjuvant HCT setting and thus targeting minimal residual disease may provide an effective and safe approach for maintaining remission in pts at high risk for relapse. Next steps: The SB system serves as a nimble and cost-effective platform for genetic engineering of T cells. We are implementing next-generation clinical T-cell trials targeting ROR1, releasing T cells for infusion within days after electro-transfer of SB DNA plasmid coding for CAR and mRNA coding for transposase, and infusing T cells modified with CAR designs with improved therapeutic potential. Figure: Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Figure:. Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1908 Chimeric antigen receptors (CARs) are employed to genetically modify T cells to redirect their specificity to target antigens on tumor cells. Typically a second generation CAR is derived by fusing an extracellular domain derived from the scFv of monoclonal antibody (CAR) specific to targeted antigen with CD3 zeta, and CD28 endodomains. CD123 (IL3RA) is expressed on 45% to 95%of acute myelogenous leukemia (AML) and B-cell lineage acute lymphoblastic leukemia (B-ALL). Expression of CD123 is high in the leukemic stem cell (LSC) population, but not in normal hematopoietic stem cells. Thus, CD123 appears to be potential target for immunotherapy in leukemias through chimeric antigen receptor (CAR). We hypothesized that the generation of CD123 specific CAR can redirect the specificity of T cells to CD123 and this was tested by cloning the scFv of CD123 mAb in our CAR construct. The sleeping beauty system was used to express the CAR and DNA plasmids were electroporated into peripheral blood mononuclear cells and cells were numerically expanded on artificial antigen presenting cells genetically modified to express co stimulatory molecules CD86, 4-1BBL, membrane-bound IL-15, and CD123 antigen in presence of IL-21 and 1L–2. CAR+ T numerically expanded to clinically relevant numbers and showed antigen specific cytotoxicity in leukemic celllines. CAR+ T cells expressed both effector and memory markers showing the potential for in vivo persistence after T cell infusion. The bonemarrow homing receptor CXCR4 was expressed by CAR T cells shows the potential to target LSC that reside in BM niches. The preliminary data suggests that mirroring an approach we are using to manufacture clinical grade CD19 specific CAR+ T cells.Figure 1:(A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.Figure 1:. (A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.CD3CD3 Disclosures: No relevant conflicts of interest to declare.