ALBERT

Description: Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+T cells with preserved TSCMpotential using theSleeping Beautyplatform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

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: Adoptive transfer of T cells expressing chimeric antigen receptor (CAR) has demonstrated clinical effectiveness in early phase clinical trials, with persistence of effector cells typically leading to improved outcomes. Most CARs directly dock with cell-surface antigens, but this limits the number of tumor-derived targets. Thus, we have adapted two technologies to target intracellular antigens and improve survival of infused T cells. This was accomplished by expressing a CAR on T effector cells that functions as a mimetic of T-cell receptor (TCR) to recognize NY-ESO-1 in the context of HLA A2 and adapting HLA-A2+ T cells to serve as antigen presenting cells (T-APC) by expressing NY-ESO-1 antigen. NY-ESO-1 is a desirable target for T-cell therapy of high risk multiple myeloma (MM) with efficacy in trials infusing T cells expressing TCR recognizing this antigen. We hypothesized combined immunotherapy with NY-ESO-1-specific CAR+ T cells and an NY-ESO-1+ T-APC vaccine will lead to enhanced anti-myeloma efficacy due to improved persistence of the CAR+ T effector cells. An NY-ESO-1-specific CAR and control TCR were expressed on primary T cells using the Sleeping Beauty (SB) transposon/transposase system. T-APC was generated by electro-transfer of DNA plasmids from SB system coding for NY-ESO-1 and membrane-bound IL-15 (mbIL15). The tethered cytokine functions as co-stimulatory molecule to improve the potency of the vaccine. In vitro studies confirmed the NY-ESO-1-specific CAR+ (and TCR+) T cells could be numerically expanded upon co-culture with T-APC. A mouse model of NY-ESO-1+HLA-A2+(CD19neg) multiple myeloma was used to compare tumor growth for CAR+ T effector cells with and without T-APC. The NY-ESO-1-specific CAR+ T effector cells displayed anti-tumor effect that was superior to control mice without T cells and mice receiving CD19-specific control CAR+ T cells. Mice receiving both NY-ESO-1-specific CAR+T effector cells and T-APC exhibited further improvement in anti-myeloma activity. This group demonstrated superior persistence of T effector cells with recovered cells exhibiting a memory phenotype. In summary, T cells can target intracellular NY-ESO-1 using a TCR mimetic CAR. Improved anti-tumor effect attributed to better persistence can be achieved by co-infusion of T-APC vaccine. These data provide the foundation to assess T cells targeting NY-ESO-1 in a clinical trial. Disclosures Patel: Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Olivares:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Singh:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Hurton:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Huls:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Employment, Equity Ownership, Patents & Royalties. Cooper:City of Hope: Patents & Royalties; Intrexon: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria.

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: Abstract 3030 Poster Board II-1006 Introduction NK cells have therapeutic potential for a wide variety of human malignancies. The major obstacle for adoptive NK cell immunotherapy is obtaining sufficient cell numbers, as these cells represent a small fraction of peripheral white blood cells, expand poorly ex vivo, and have limited life spans in vivo. Common gamma-chain cytokines are important in NK cell activation, maturation, and proliferation. Others have described improved ex vivo expansion of NK cells using soluble cytokines, when cocultured with stimulated peripheral blood mononuclear cells (PBMC) or Epstein Barr Virus (EBV) lymphoblastioid cell lines, or with artificial antigen presenting cells (aAPC) engineered with costimulatory molecules and/or membrane-bound IL-15 (mIL-15). Expansion of NK cells by these methods has been limited by senescence from telomere shortening. To generate clinical-grade T cells for adoptive transfer, our group developed aAPC derived from K562 retrovirally transduced to express the costimulatory molecules CD86 and CD137L. These aAPC were produced as a master cell bank and further genetically modified to express membrane-bound cytokines. Since IL-21 signals via STAT3, and STAT3 is a known activator of telomerase transcription, we investigated whether NK cell expansion with mIL-21 would provide a sustained proliferative advantage over or in combination with mIL-15. Methods K562 aAPC were retrovirally transduced to express CD64, CD86, CD137L, CD19 (Clone 9), and mIL-15 (Clone 4). These clones were further modified by Sleeping Beauty integration of mIL-21 (Clone 9+IL-21 and Clone 4+IL-21). Freshly isolated PBMC from 5 donors were co-cultured with irradiated K562 aAPC (Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21) at a ratio of 2:1 (aAPC:PBMC) in the presence of 50 IU/ml of rhIL-2. Half of the media was changed every two days and cells were re-stimulated with aAPC every seven days at ratio of 2:1. Cells were counted and phenotyped on day 0, 7, 14, and 21 for CD3, CD16, CD56, NKG2D, KIR (2DL1, 2DL2/3, and 3DL1), and NCR (NKp30, NKp44, NKp46). A preclinical SOP to expand PBMC from a 20 mL blood draw was established and additional donors of known HLA type were expanded with Clone 9+mIL-21 for up to 7 weeks. Cytotoxicity function against K562, 721.221, Raji, and AML targets was measured using the Calcien-AM assay (Invitrogen). Telomere length of expanded and fresh NK cells was measured with the FlouFish assay using the telomere specific FITC conjugated (C3TA2)3 PNA probe. Results By day 14, aAPCs bearing mIL-21 induced greater total cell expansion than those with mIL-15 alone (188, 2900, and 2281-fold for Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21, respectively). However, PBMC cultured without mIL-15 contained far fewer co-expanding T cells. Exponential expansion continued for up to 7 weeks without evidence of senescence when mIL-21 was present, reaching a mean of 91,566-fold expansion of the CD3−CD16/56+ population at 4 weeks. NK cells expanded with mIL-21 had increased expression of KIR and NCR, and expressed very high CD16 and NKG2D levels. These NK cells showed much higher cytotoxicity against all targets than fresh NK cells, retained KIR inhibition, and demonstrated enhanced killing via ADCC. Furthermore, telomere lengths of NK cells expanded with Clone 9+mIL-21 were longer than that of fresh NK cells or those expanded without mIL-21, perhaps explaining the continued expansion without senescence. Thus, NK cell expansion is improved using aAPCs expressing mIL-21 rather than mIL-15. We are currently establishing a GMP-grade working cell bank of Clone 9+mIL-21 for use in clinical trials. Funding: Brenda and Howard Johnson Fund, UT MD Anderson Physician Scientist Program Disclosures No relevant conflicts of interest to declare.

Description: The safety and feasibility of adoptive immunotherapy using ex vivo-expanded differentiated human effector T cells that express tumor-specific chimeric receptors are being evaluated in clinical trials. Typically, these T cells are CCR7neg and bear a T-cell receptor of unknown specificity. To improve the therapeutic potential of genetically engineered T cells in general, and CD19-specific T cells in particular, strategies are needed to improve their ability traffic to sites of residual/macroscopic disease where infused T cells can be specifically activated for proliferation, cytokine secretion, and tumor-lysis. To accomplish these goals we have generated a selection process that uses genetically modified T cells, expressing influenza A matrix protein 1 (MP1) or CMV pp65, to act as antigen presenting cells (T-APC) in order to expand autologous viral-specific T cells in vitro and in vivo. The viral-specific effector T cells can then be genetically modified with a CD19-specific chimeric immunoreceptor (CD19R), which recognizes CD19 on malignant B cells, independent of MHC. By using these viral-specific T cells as a platform for the introduction of CD19R, we now demonstrate that bi-specific T cells express the chemokine receptor CCR7, which is implicated in the trafficking of T cells to lymph nodes. We demonstrate that this chemokine receptor functions to directionally chemotax the genetically modified bi-specific T-cells along concentration gradients of CCL19 or CCL21. We further demonstrate that both the endogenous and introduced chimeric immunoreceptor continue to function in CCR7+ bi-specific T cells. Indeed, the bi-specific T cells are capable of augmented cytokine production and proliferation upon docking with both CD19 and MP1 antigens, compared with these same T cells interacting with either CD19 or MP1 alone. This enhanced activation is an explanation for the enhanced in vivo anti-tumor activity demonstrated by bi-specific T-cells when stimulated with MP1+ T-APC in treating CD19+ lymphoma in NOD/scid mice. An advantage of this methodology is that the CCR7+ bi-specific T cells and T-APC can be genetically modified and expanded in compliance with current good manufacturing practice (cGMP) for 2nd generation Phase I/II clinical trials to test their ability to traffic to sites of lymphoma providing potent regional/local T-cell activation. Legend: (A) CCR7+ viral- and CD19-bi-specific T cells migrate along recombinant CCL19 and CCL21 concentration gradients, whereas CCR7neg CD19-specific T cells do not. (B) Stimulation of both introduced chimeric immunoreceptor and endogenous T-cell receptor on CD19- and MP1- bi-specific T-cells, using artificial APC, results in augmented cytokine production. Figure Figure

Description: Relapse of B-lineage (CD19+) acute lymphoblastic leukemia (ALL) remains a major impediment to the therapeutic success of allogeneic umbilical cord blood transplant (UCBT). The adoptive transfer of donor-derived tumor-specific T-cells is a conceptually attractive means to improve the graft-versus-leukemia-effect at the time of minimal residual disease to improve relapse-rates without exacerbating graft-versus-host-disease. However, adoptive immunotherapy after banked UCBT has been limited by the functional naïveté of neonatal T cells and difficulty obtaining T cells from the unrelated donor. These hurdles can now be overcome by genetically rendering cord blood-derived T cells to be specific for CD19 and expanding the T cells ex vivo from small numbers of cord blood cells, in compliance with current good manufacturing practices for phase I/II trials. To generate T cells that target CD19+ malignant cells, we have used non-viral gene transfer to introduce a DNA plasmid to express a CD19-specific chimeric immunoreceptor, designated CD19R, which binds to cell-surface CD19 via an scFv, independent of MHC, and triggers T-cell activation through CD3- ζ. To safeguard the safety of recipients of adoptive immunotherapy, the DNA plasmid co-expresses the bi-functional hygromycin phosphotransferase/HSV-1 thymidine kinase (HyTK) selection/suicide gene. To assess in vivo the fate of adoptively transferred T cells in mice; a novel tri-functional gene linking firefly luciferase (ffLuc) with HyTK (ffLucHyTK) was generated. The process ex vivo to expand cord blood-derived T cells, which is currently employed at COH in human trials, uses reiterative 14-day additions of OKT3, rhIL-2, cytocidal concentrations of hygromycin, and irradiated peripheral blood mononuclear cells (PBMC) and LCL as feeder cells. The expanded genetically manipulated cord-blood derived T cells express cell-surface markers of differentiated effector cells, similar to the phenotype of CD19-specific T cells derived from PBMC. In vitro the CD19R+HyTK+ cord blood-derived T cells are activated for cytolysis and cytokine production by CD19+ tumor cells. In vivo these genetically modified T cells can be used to eradicate established CD19+ tumors and undergo ganciclovir-mediated ablation, as demonstrated by non-invasive serial imaging of luciferase-mediated bioluminescence (see Figure). These data support a clinical trial to test the safety and feasibility of adoptive transfer of CD19-specific umbilical cord-blood derived T-cells for patients with high risk B-lineage ALL undergoing UCBT. Legend: Non-invasive in vivo biophotonic imaging demonstrates that (A) CD19+ tumor expressing ffLuc gene are eliminated by CD19R+HyTK+ cord-blood derived T cells, and (B) CD19R+ffLuc+HyTK+ cord-blood derived T cells are ablated by ganciclovir. Top row: prior to adoptive immunotherapy or ganciclovir treatment. Bottom row: after adoptive immunotherapy or ganciclovir treatment. Two representative mice are shown. Figure Figure

Description: Abstract 4097 Poster Board III-1032 CD19-specific chimeric antigen receptors (CARs) based on first and second generation designs (Figure) expressed by genetically manipulated T cells are being evaluated in clinical trials and it is apparent that the long term in vivo survival of the infused T cells will be critical to achieving therapeutic successes. We have previously demonstrated that the first-generation CAR can be modified to include and provide an improved T-cell survival signal through addition of a chimeric CD28 endodomain (designated second generation CAR, CD19RCD28). However, it is recognized that optimal activation of T cells through CD28 for sustained proliferation may require co-stimulation through other signaling molecules. To address this issue we have altered the second generation CAR to include additional intracellular signal transduction domains from CD137 (4-1BB) or, CD134 (OX40) to generate (Figure) third generation CARs (CD19RCD28CD137 or, CD19RCD28CD134, respectively). The third generation CARs were electro-transferred into peripheral blood T cells using the Sleeping Beauty transposon/transposase system and propagated on K562-derived CD19+ artificial antigen presenting cells (aAPCs). We observed the selective outgrowth of CD19-specific T cells expressing similar percentages of expression and densities of CD19RCD28, CD19RCD28CD137, and CD19RCD28CD134 CARs with a subset of the propagated T cells displaying a central memory phenotype (CD62L+CD28+). The T cells expressing the third generation CARs exhibited redirected-killing and were able to produce IFN-g in response to CD19. Although the cytolytic ability and Tc1 cytokines released by both second and third generation CARs were similar, differences due to the presence of the CD137 and CD134 intracellular signaling endodomains were apparent using in vitro experiments designed to mimic in vivo T-cell persistence, by measuring long-term propagation in the absence of exogenous cytokines added to the T-cell culturing process. We observed an approximate 200% increase in the numeric expansion of CD19RCD28CD137+ and CD19RCD28CD134+ T cells as compared to the CD19RCD28+ T cells at the end of 14 days of co-culture on CD19+ aAPC. These data highlight the fact that CD19-dependent signaling through chimeric CD28 might be improved by the addition of signaling through chimeric CD137 or CD134 domains so as to fully maintain T-cell activation and sustain proliferation in vivo after adoptive transfer. Figure Schematic of first (CD19R), second (CD19RCD28), and two third (CD19RCD28CD137 and CD19RCD28CD134) generation CD19-specific CARs (shown as homodimers on the cell surface) expressing one, two, or three T-cell signaling chimeric endodomains. TM = transmembrane. Figure. Schematic of first (CD19R), second (CD19RCD28), and two third (CD19RCD28CD137 and CD19RCD28CD134) generation CD19-specific CARs (shown as homodimers on the cell surface) expressing one, two, or three T-cell signaling chimeric endodomains. TM = transmembrane. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3035 Poster Board II-1011 Redirecting specificity to a selected cell surface tumor-associated antigen can be accomplished by the genetic modification of T cells to express a chimeric antigen receptor (CAR). Despite systematic modifications to the CAR endodomains to provide T cells with competent signaling, CAR-dependent T-cell activation may remain incomplete resulting in inferior in vivo persistence leading to a suboptimal therapeutic response. To improve T-cell survival and therefore an anti-tumor response, investigators have co-infused soluble recombinant cytokines such as IL-2 and IL-7. IL-7 is a homeostatic cytokine for T cells and supports survival of memory T cells. To provide IL-7 mediated signaling to T cells in the tumor microenvironment and thus enhance the proliferation and survival of CAR+ T cells, we constructed a version of IL-7 as a novel membrane-bound molecule (mIL7) designed to stimulate T cells in cis and trans. The mIL7 construct was electro-transferred with a CD19-specific CAR (on day 0) into primary T cells via multiple transposition of Sleeping Beauty DNA plasmids. These genetically modified T cells could be numerically expanded ex vivo without additional soluble cytokine supplementation on CD19+ artificial antigen presenting cells (aAPC) derived from K562. This resulted in the preferential outgrowth of T cells expressing both mIL7 and CAR (Figure A and B) while CAR+ T cells receiving no soluble cytokine supplementation did not sustain proliferation (Figure B). These mIL7+CAR+ T cells exhibited redirected specific lysis of CD19+ tumor targets. Significantly, the kinetics of propagation of mIL7+CAR+ T cells in the absence of exogenous cytokine was comparable to CAR+ T cells that were numerically expanded in the presence of soluble IL-2. Signaling by IL-7 receptor signal induction in mIL7+CAR+ T cells appeared comparable to signaling by soluble IL-7 in CAR+ T cells, as assessed by phosphorylation of signal transducer and activator of transcription 5 (STAT5). These data demonstrate that mIL7 can be expressed by CAR+ tumor-redirected T cells to enhance their proliferation without the need for additional cytokine support. These results have implications for the design of clinical trials to evaluate whether mIL7+CAR+ T cells can exhibit enhanced persistence and thus improved therapeutic potential. 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.