T-cell Antigen-presenting Cells Research Articles

Novel CD19t T-Antigen Presenting Cells Expand CD19 CAR T Cells In Vivo

Background: While immunotherapy withCD19 specific CAR T cells for relapsed/refractory (R/R) B-ALL achieves MRD negative remission in nearly all patients, relapse occurs in approximately half of patients and is frequently associated with early loss of CAR T cell persistence. Low CD19 antigen burden in the bone marrow prior to lymphodepletion and rapid contraction of CAR T cells in the blood after engraftment are predictive of early loss of CAR T cell persistence. We hypothesize that episodic antigen exposure using CD19t T cell antigen presenting cells (T-APCs) can trigger CD19 CAR T cell proliferation and re-activation in vivo, resulting in more durable CAR T cell persistence and diminished risk of CD19+ relapse. Here we report our experience to date using T-APCs following CD19 CAR T cell therapy for children and young adults with R/R B-ALL on clinical trial PLAT-03 (NCT03186118). Methods: Patient-derived T-APC products were manufactured from cryopreserved CD4/CD8 selected T cells. Cells were then activated with anti-CD3/CD28 beads, transduced with a lentiviral vector to express a truncated human CD19 (CD19t), and expanded in culture for 10 days. Subjects <25kg received 10x106 T-APCs/kg/dose and those ≥25kg received a flat dose of 5x108 T-APCs/dose, for up to a total of six-monthly doses of T-APCs. Adverse events were graded according to CTCAEv4. Results: Fourteen subjects (8-26 yrs) have been enrolled; 8 with low CD19 antigen burden, 5 with rapid CAR T cell contraction, and 1 subject with early loss of CAR T cell persistence on a predecessor study who received a re-infusion of CD19 CAR T cells on this study followed by T-APCs. T-APCs were successfully manufactured in 14/14 subjects. Two subjects lost CAR T cell persistence prior to T-APC infusion and were ineligible to receive T-APCs. To date, 11 subjects have received at least one dose of T-APCs. One of 11 subjects experienced a grade 3 febrile infusion reaction within hours of the 2nd dose of T-APCs, prohibiting further dosing. There were no other related adverse events (AEs) > grade 2 among the 11 subjects, and no cytokine release syndrome or neurotoxicity has been observed. An increase in detectable CD19 CAR T cells occurred in all subjects following T-APCs, and T-APCs can be transiently detected following infusion (Figure 1). Of the 10 treated subjects with low CD19 antigen burden or rapid T cell contraction, 8/10 had CAR T cell persistence beyond Day 63, as evidenced by B cell aplasia. At last follow-up, 5/10 have ongoing B cell aplasia, with a median follow up of 8.8 months (range, 2-18.5 months). The estimated 1-year leukemia free survival (LFS) is 69.2%. Conclusion: This first-in-human study of CD19t T-APCs demonstrates the ability to successfully manufacture T-APCs from stored apheresis products collected for CAR T cell production. In 11 subjects receiving at least one T-APC dose to date, there has been one T-APC infusion reaction and no other significant associated toxicity. Early evidence of efficacy demonstrated by secondary expansion of CAR T cells suggests the potential of CD19t T-APCs to enhance durable CD19 CAR T cell persistence. Figure 1 Disclosures Gardner: Novartis: Honoraria. Jensen:Bluebird Bio: Research Funding; Juno Therapeutics, a Celgene Company: Research Funding.

Novel CD19t T-Antigen Presenting Cells Designed to Re-Activate and Expand CD19 CAR T Cells In Vivo: Early Demonstration of Feasibility and Safety

Background: Immunotherapy with CD19 specific chimeric antigen receptor (CAR) T cells for relapsed/refractory acute lymphoblastic leukemia (ALL) demonstrated a minimal residual disease (MRD) negative remission rate of 93% on the phase 1 portion of Pediatric Leukemia Adoptive Therapy (PLAT)-02. However, the 1-year event free survival (EFS) was 50.8%, largely due to recurrence of disease which was frequently associated with early loss of CAR T cell persistence. Subjects on PLAT-02 with <15% CD19 antigen burden in the bone marrow prior to lymphodepletion had a decreased magnitude of CAR T cell expansion and were less likely to have durable persistence of CAR T cells. Also, subjects with a rapid contraction of CAR T cells in the blood between Day +10 and Day+14, as measured by flow cytometry, had inferior long term persistence. These findings led to the hypothesis that loss of persistence may be due to decreased antigen stimulation and subsequent quiescence of CAR+ effector T cells. Episodic target exposure using T cell antigen presenting cells (T-APCs) expressing a truncated human CD19 (CD19t) could trigger CD19 CAR T cell proliferation and re-activation in vivo, resulting in more durable CAR T cell persistence and diminished risk of CD19+ relapse.Methods: Eligible subjects for PLAT-03 (NCT03186118), a pilot study of CD19t T-APCs, must have previously enrolled on the phase 2 portion of PLAT-02 and have a stored apheresis product available. Cohorts include subjects that have an identified predictive factor to lose CAR T cell persistence early, or have already experienced early loss of CAR T cell persistence before 6 months and can receive a reinfusion of CD19 CAR T cells prior to planned T-APCs. Patient-derived T-APC products are manufactured with selected and cryopreserved CD4 and CD8 cells from the subject's original apheresis product. The T cells are activated and transduced with a lentiviral vector to express the CD19t transgene. Transduced cells are expanded in culture for 10-14 days, cryopreserved and release-tested. Subjects <25kg receive 10x106 T-APCs/kg and those ≥25kg receive a flat dose of 5x108 T-APCs. Subjects receive at least one, and up to six total doses of T-APCs at monthly intervals.Results: Seven subjects (ages 9-23 years) have enrolled on PLAT-03; 5 enrolled based on low CD19 antigen burden at the time of lymphodepletion, and 2 based on rapid CAR T cell contraction between Days 10 and 14. T-APC products were successfully manufactured in 5/5 subjects, with two products pending manufacture. One of 5 subjects was unable to receive his planned T-APCs, as he lost CAR T cell persistence prior to T-APC infusion and failed to engraft a second infusion of CD19 CAR T cells. Of the 4 subjects that have received at least one dose of T-APCs, there have been no related adverse events (AEs) > grade 2, and no fever or cytokine release syndrome (CRS) has been observed. Grade 1-2 AEs that are at least possibly related to T-APCs have included hypogammaglobulinemia, nausea, vomiting and headache. Two subjects have completed T-APC treatment; one received all 5 available T-APC doses (subject S001), and one experienced loss of CAR T cell persistence after two T-APC doses and therefore was not eligible for further doses. Data is shown for subject S001, a 20 year old man with multiply relapsed B-ALL. He enrolled on PLAT-03 due to a low CD19 antigen burden of 4.79% by flow cytometry (inclusive of both normal and malignant CD19+ cells) just prior to lymphodepletion. He had minimal toxicity (grade 1 CRS) following CD19 CAR T cell infusion, and subsequently received 5 monthly doses of T-APCs. An increase in the quantity of detectable CAR T cells (EGFRt+ cells) was observed following T-APCs as shown below, and the subject has ongoing CAR T cell persistence at 8 months, as evidenced by B cell aplasia.Conclusion: The first-in-human, pilot study PLAT-03 introduces CD19t T-APCs in an attempt to enhance durable CAR T cell persistence and leukemia remission following CD19 CAR T cell immunotherapy. T-APCs have been successfully manufactured from stored apheresis products collected for CAR T cell production. Four subjects have been successfully treated with at least one T-APC infusion to date, without any significant toxicity and with early evidence of efficacy. Accrual of subjects is ongoing to further assess the safety and feasibility of administering CD19t T-APCs, and to examine the impact of T-APCs on CD19 CAR T cell efficacy. [Display omitted] DisclosuresPark:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

T-Cell Therapy for Multiple Myeloma Using NY-ESO-1+ T-Cell Antigen Presenting Cells (T-APC) Combined with Adoptive Cellular Transfer (ACT) to Augment Immunotherapy

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. [Display omitted] [Display omitted] DisclosuresCooper:InCellerate: Equity Ownership; Sangamo: Patents & Royalties; Targazyme: Consultancy; GE Healthcare: Consultancy; Ferring Pharmaceuticals: Consultancy; Fate Therapeutics: Consultancy; Janssen Pharma: Consultancy; BMS: Consultancy; Miltenyi: Honoraria.

Enhanced antilymphoma efficacy of CD19-redirected influenza MP1–specific CTLs by cotransfer of T cells modified to present influenza MP1

AbstractTo enhance the in vivo antitumor activity of adoptively transferred, CD19-specific chimeric antigen receptor (CAR)–redirected cytotoxic T lymphocytes (CTLs), we studied the effect of restimulating CAR+ CTLs through their endogenous virus-specific T-cell antigen receptor (TcR) by the cotransfer of engineered T-cell antigen–presenting cells (T-APCs). Using influenza A matrix protein 1 (MP1) as a model antigen, we show that ex vivo–expanded CD4+ and CD8+ T-APCs expressing a hygromycin phosphotransferase-MP1 fusion protein (HyMP1) process and present MP1 to autologous human leukocyte antigen (HLA)–restricted, MP1-specific CD4+ and CD8+ CTL precursors. The MP1-specific CTLs are amenable to subsequent genetic modification to express a CD19-specific CAR, designated CD19R, and acquire HLA-unrestricted reactivity toward CD19+ leukemia and lymphoma tumor targets while maintaining HLA-restricted MP1 specificity. The restimulation of MP1×CD19 dual-specific CTLs in vivo by the adoptive transfer of irradiated HyMP1+ T-APCs resulted in the enhanced antilymphoma potency of bispecific effector cells, as measured by elimination of the biophotonic signal of established firefly luciferase–expressing Burkitt lymphoma xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/scid) animals compared with control groups restimulated by Hy+MP1neg T-APCs. Engineered T-APCs are a novel and versatile antigen-delivery system for generating antigen-specific T cells in vitro and enhancing the in vivo effector functioning of CAR-redirected antitumor effector cells.

Retrovirus-Transduced T Cell Blasts Have Not Only Antigen-Presenting Capabilities but Also Suppressor Regulatory T Cell-Inducing Capability.

EBV-associated T/NK-lymphoproliferative diseases (LPD) are relatively rare but their prognosis is very poor. In order to understand the pathogenesis and rationalize immunotherapy, it is important to know whether EBV-infected T/NK cells have antigen-presenting capacities and how they escape the EBV-specific immunity that controls virus-infected B cells and epithelial cells. As a model for T cell antigen presenting cells (T-APC), we used activated T cells expressing pp65, the immunodominant antigen of CMV, after retroviral transduction. Two to three days after activation with CD3/28, T cells were transduced with retrovirus supernatants for 24h to 48 hr on retronectin-coated plates, and then expanded for one to two weeks with IL-2 before use as APCs or target cells. As control APCs, we used LCLs transduced with a retrovirus vector expressing pp65 (pp65-LCL). Pp65-expressing T cells functioned as target cells, since they were readily killed by pp65-specific cytotoxic T cells (CTL), reactivated using Adenovirus-pp65-pulsed dendritic cells. Furthermore, pp65T-APCs could reactivate pp65-specific CD8+ CTL from autologous PBMC. However, the pp65T-APC could not sustain prolonged pp65-specific CTL expansion. The ratio of CD4+: CD8+ T cells increased in responders after the 2nd stimulation with pp65-T-APC, and after the 3rd stimulation, the CD8+ CTL had virtually disappeared so that most of live cells were CD4+ T cells. The majority of the CD4+ T cells co-expressed CD25, the glucocoticoid-induced TNF receptor (GITR) and intracellular antigen CD152, all markers of T regulatory cells. To determine if they had regulatory function, the CD4+CD25+ population was isolated and shown to inhibit the proliferation of autologous PBMCs stimulated by pp65-LCL. In contrast, the CD4+CD25− fraction did not inhibit proliferation. Transwell experiments revealed that the inhibition required cell-to-cell contact. CFSE analysis showed that the CD4+CD25+ cells expanded while inhibiting the proliferation of stimulated PBMC. Taken together, these findings indicated that T-APC could reactivate antigen specific CD8+ CTL, but at the same time they activated regulatory CD4+ T cells, which prevented the long-term expansion of the CD8+ CTL. Similar results were found when the LMP2 antigen of EBV, which is expressed in T/NK-LPD, was used as antigen. Thus the immune escape of EBV-associated T/NK-LPD might result in part from their induction of regulatory T cells which inhibit T cell-specific CTL generation and function. T cells in general may evade immune-mediated recognition of their “novel” T cell receptors in the same way.