CD19-specific Chimeric Antigen Receptor Research Articles

8 - Differing co-stimulatory, linker and spacer domains produce variations in CD4 and CD8 cell composition and cytotoxic potential in CD19-specific chimeric antigen receptor (CAR19) T cells generated with the piggyBac transposase

8 - Differing co-stimulatory, linker and spacer domains produce variations in CD4 and CD8 cell composition and cytotoxic potential in CD19-specific chimeric antigen receptor (CAR19) T cells generated with the piggyBac transposase

6 - Direct comparison of in vivo fate of second and third-generation CD19-specific chimeric antigen receptor (CAR)-T cells in patients with b cell non-hodgkin lymphoma (B-NHL): Reversal of toxicity from tonic signaling

6 - Direct comparison of in vivo fate of second and third-generation CD19-specific chimeric antigen receptor (CAR)-T cells in patients with b cell non-hodgkin lymphoma (B-NHL): Reversal of toxicity from tonic signaling

Abstract PR10: Enhancing the efficacy of T cell-based immunotherapies using miR-155 engineered tumor-specific CD8+ T cells

Abstract Lymphodepleting regimens are employed prior to adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic cytokine sinks. These conditioning modalities, however, are often associated with severe toxicities. We found that miR-155 enabled B16 melanoma-specific CD8+ T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T cell responsiveness to limited amounts of homeostatic γc cytokines by restraining the expression of the Akt inhibitor Ship1 and multiple negative regulators of Stat5, such as Socs1 and Ptpn2, resulting in delayed cellular contraction and sustained cytokine production. To translate these findings into new clinical trials, we tested whether the overexpression of miR-155 in human T cells would enhance the antitumor efficacy of CD19-specific chimeric antigen receptor (CD19-CAR) cells against systemic acute lymphoblastic leukemia xenografts. As a safety measure, the suicide gene, inducible caspase 9, was included in the miR-155 construct. We found that miR-155 also augmented the antitumor efficacy of T cells in this xenograft tumor model. Recapitulating our findings in mouse cells, human T lymphocytes overexpressing miR-155 showed increased Stat5 signaling and enhanced survival. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be employed to increase the effectiveness of adoptive immunotherapies in a cell intrinsic manner without the need of life-threatening, lymphodepleting maneuvers. This abstract is also being presented as Poster A82. Citation Format: Yun Ji, James Hocker, Jinhui Hu, Sanjivan Gautam, Neal Lacey, Luca Gattinoni. Enhancing the efficacy of T cell-based immunotherapies using miR-155 engineered tumor-specific CD8+ T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR10.

30 - Direct Comparison of in Vivo Fate of Second and Third-Generation CD19-Specific Chimeric Antigen Receptor (CAR)-T Cells in Patients with B-Cell Non-Hodgkin Lymphoma (B-NHL): Reversal of Toxicity From Tonic Signaling

30 - Direct Comparison of in Vivo Fate of Second and Third-Generation CD19-Specific Chimeric Antigen Receptor (CAR)-T Cells in Patients with B-Cell Non-Hodgkin Lymphoma (B-NHL): Reversal of Toxicity From Tonic Signaling

Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.

Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection. The most successful CARs used to date are those targeting CD19 (ref. 2), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies. CARs are typically transduced into the T cells of a patient using γ-retroviral vectors or other randomly integrating vectors, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific interventions in human cells, including targeted gene delivery to the CCR5 and AAVS1 loci. Here we show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.

Pre-clinical development of gene modification of haematopoietic stem cells with chimeric antigen receptors for cancer immunotherapy

ABSTRACTPatients with refractory or recurrent B-lineage hematologic malignancies have less than 50% of chance of cure despite intensive therapy and innovative approaches are needed. We hypothesize that gene modification of haematopoietic stem cells (HSC) with an anti-CD19 chimeric antigen receptor (CAR) will produce a multi-lineage, persistent immunotherapy against B-lineage malignancies that can be controlled by the HSVsr39TK suicide gene. High-titer third-generation self-inactivating lentiviral constructs were developed to deliver a second-generation CD19-specific CAR and the herpes simplex virus thymidine kinase HSVsr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC were transduced with such lentiviral vectors and evaluated for function of both CAR and HSVsr39TK. Satisfactory transduction efficiency was achieved; the addition of the suicide gene did not impair CAR expression or antigen-specific cytotoxicity, and determined marked cytotoxicity to ganciclovir. NSG mice transplanted with gene-modified human HSC showed CAR expression not significantly different between transduced cells with or without HSVsr39TK, and expression of anti-CD19 CAR conferred anti-tumor survival advantage. Treatment with ganciclovir led to significant ablation of gene-modified cells in mouse tissues. Haematopoietic stem cell transplantation is frequently part of the standard of care for patients with relapsed and refractory B cell malignancies; following HSC collection, a portion of the cells could be modified to express the CD19-specific CAR and give rise to a persistent, multi-cell lineage, HLA-independent immunotherapy, enhancing the graft-versus-malignancy activity.

Construction and Testing of Two Distinct Humanized CD19-Specific Chimeric Antigen Receptors (CARs) for the Treatment of B-Cell Malignancies

Introduction: Modificiation of T cells using CD19-specific chimeric antigen receptor (CAR) therapy has produced dramatic responses against a number of hematologic malignancies in multiple clinical trials. To date, most of the CARs studied in clinical trials are derived from mouse single chain fragment variable (scFv), which can elicit an immune response when infused into human patients and thereby can limit the persistency of CAR-T cells. Indeed, a subset of patients with limited persistency of infused CAR-Ts has been observed in clinical trials. However, this can be overcome by utilizing the humanized scFv in CAR design. Here, we constructed two new CD19-specific CARs, which are derived from the scFv of two distinct humanized CD19 antibody clones and compared them with the widely used CD19-CAR derived from a mouse scFv (FMC63).Methods: Lentiviral constructs encoding FMC63-BBZ CAR was generated based on the published sequence of FMC63 scFV and cloned in to a lentiviral vector driven by an EF1α promoter. The FMC63-BBZ CAR sequence was fused in frame with T2A ribosomal skip sequence followed by a puromycin resistance gene (PAC). Humanized CD19-Specific CARs were generated by replacing the FMC63 sequence with two humanized CD19-specific scFv sequences that were derived from two distinct humanized monoclonal antibodies. 293FT cells were used to make lentiviruses and the concentrated viruses were used to infect T-cells from healthy donors, NK92MI and Jurkat-NFAT-GFP (JNG) cells. Surface expressions of CARs were detected by protein-L staining using flow cytometry. Cytolytic activity of CAR-engineered T and NK cells was measured using a 4 hour co-culture assay. Cytokines (IL-2, IFN-γ and TNF-α) were measured from the co-cultured supernatants by ELISA.Results: We were able to infect primary-T cells, NK92MI and JNG cells using the FMC63 and humanized CAR constructs, and the surface expression of CARs was comparable between them. Co-culture of JNG cells stably expressing the FMC63 or humanized CARs with CD19+ve Raji (Burkitt Lymphoma), Nalm6 (Pre- B-cell acute lymphoblastic leukemia) and BV173 (Chronic Myeloid Leukemia) cancer cells lead to robust expression of GFP (indicative of activated NFAT signaling), CD69 (an activation marker for cytotoxic cells), and a rapid increase in the secretion of IL-2. None of these effects were observed in unmodified JNG cells or with the co-culture of JNG cells stably expressing FMC63 or humanized CARs with CD19-ve HL60 (Acute Promyelocytic Leukemia) and U937 (Histiocytic Lymphoma) cancer cells. Next, we made stable NK92MI cells expressing FMC63, humanized CD19-specific CARs, and a control CAR, followed by cellular cytotoxicity assay. Both FMC63-CAR and humanized CD19-CARs expressing NK92MI cells readily eliminated CD19+ve cancer cells, whereas they had no discernible effect on CD19-ve cancer cells. Furthermore, parental or a control CAR expressing NK92MI cells are without any effect, either with CD19+ve or CD19-ve cancer cells, thereby strictly highlighting the comparable specificity of our humanized CD19-CARs with FMC63-CAR against CD19+ve cells. More importantly, co-culture of NK92MI cells stably expressing FMC63-CAR and humanized CD19-CARs with direct patient samples [CD19+ve B-ALLs and CD19-ve Acute Myelogenous Leukemia’s (AMLs)] eliminated only the patient B-ALLs, but without any effect on patient AMLs. Also, increased surface expression of CD69 and robust secretion of cytokines (IFN-γ and TNF-α) were observed only when FMC63 or humanized CD19-CARs expressing NK92MI cells were incubated with CD19+ve patient B-ALLs. Finally, expression of FMC63and humanized CD19-CARs in primary donor T-cells also resulted in the elimination CD19+ve cancer cells along with a significant increase in the secretion of cytokines. Most importantly, both humanized CD19-specific CARs significantly increased the survival of CD19+ve cancer cells bearing NSG mice, whereas the unmodified T-cells or the T-cells expressing a non-specific CAR were without any effect.Conclusion: Our extensive pre-clinical study clearly shows that humanized CD19-specific CAR-T or NK cells exhibit comparable potency with the widely used FMC63-CAR, which provides the basis for the clinical testing of these humanized CARs in CD19+ve malignancies, thereby allaying the possible concern on immune mediated rejection of mouse scFv based CD19-CAR therapy. DisclosuresNo relevant conflicts of interest to declare.

Inducible MyD88/CD40 (iMC) Costimulation Provides Ligand-Dependent Tumor Eradication By CD123-Specific Chimeric Antigen Receptor T Cells

Introduction: Promising clinical results with CD19-specific chimeric antigen receptor (CAR)-directed T cells for the treatment of B cell leukemia and lymphoma suggest that CARs may be effective in other hematological malignancies, such as acute myeloid leukemia (AML). CD123/IL-3Rα is an attractive CAR-T cell target due to its high expression on both AML blasts and leukemic stem cells (AML-LSCs). However, the antigen is also expressed at lower levels on normal stem cell progenitors presenting a major toxicity concern should CD123-specific CAR-T cells show long-term persistence. Here, we describe a CAR platform, "GoCAR-T", which uses a proliferation-deficient, first generation, CD123-specific CAR together with a ligand (rimiducid (Rim))-dependent costimulatory switch (inducible MyD88/CD40 (iMC)) to provide physician-controlled eradication of CD123+ tumor cells and regulate long-term CAR-T cell engraftment.Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a bicistronic retrovirus encoding tandem Rim-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and first generation CAR targeting CD123 (SFG-iMC-CD123.ζ). The effects ofiMC costimulation on CD123-targeted CARs were assessed in coculture assays with CD123+, EGFPluciferase (EGFPluc)-modified leukemic cell lines (KG1, THP-1 and MOLM-13) with and without Rim using the IncuCyte live cell imaging system. IL-2 production was examined by ELISA from coculture supernatants. In vivo efficacy of iMC-CD123.ζ-modified T cells was assessed using an immune-deficient NSG tumor xenograft model. One million EGFPluc-expressing CD123+ THP-1 tumor cells were injected i.v. into the animals, followed by a single i.v. injection on day 7 with 2.5x106 non-transduced or iMC-CD123.ζ-modified T cells. Groups receiving CAR-T cells subsequently received i.p. injections of Rim (1 mg/kg) or vehicle only on days 0 and 15 post-T cell injection. Animals were evaluated for THP-1-EGFPluc tumor burden and weight change on a weekly basis using IVIS bioluminescent imaging (BLI) and for T cell persistence by flow cytometry and qPCR at day 30 post-T cell injection.Results: SFG-iMC-CD123.ζ efficiently transduced activated T cells (66±8%) and showed antigen-specific cytolytic function against CD123+ leukemic cell lines. However, in coculture assays both CAR antigen recognition and Rim-dependent iMC costimulation were required for IL-2 production (285±41 versus 2,541±255 pg/ml for control and 1 nM Rim, respectively), robust CAR-T cell proliferation (87-fold increase with Rim stimulation) and enhanced tumor cell killing. In NSG mice engrafted with CD123+ THP-1-EGFPluc tumor cells, only animals were treated with iMC-CD123.ζ-modified T cells and 1 mg/kg Rim on days 0 and 15 post-T cell injection controlled tumor growth, showing a 2-log (average radiance: NT = 3.0x108; CAR + Vehicle = 2.4x108; CAR + Rim=8.4x105 p/sec/cm2/sr) reduction in tumor burden compared to mice receiving CAR-T cells and vehicle only (Figure 1). Approximately two weeks after the second Rim injection (day 30), CAR-T cells were infrequent (<1.0%) in the spleen and bone marrow of both CAR groups suggesting that active costimulation is required for CAR-T persistence.Summary: GoCAR-T, a platform comprising a ligand-dependent activation switch and a proliferation-deficient first generation CAR, efficiently eradicates CD123+ leukemic cells when costimulation is provided by systemic rimiducid administration. Deprivation of iMC costimulation results in reduction of CAR-T levels, providing a user-controlled system for managing persistence and safety of CD123-specific CAR-T cells. DisclosuresFoster:Bellicum Pharmaceuticals: Employment. Duong:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Direct Comparison of In Vivo Fate of Second and Third-Generation CD19-Specific Chimeric Antigen Receptor (CAR)-T Cells in Patients with B-Cell Lymphoma: Reversal of Toxicity from Tonic Signaling

Although adoptive transfer of T cells with second-generation CD19-specific CARs containing CD28 or 4-1BB costimulatory endodomains shows remarkable clinical efficacy against B cell malignancies, the optimal choice of costimulatory domains in these and other CARs remains controversial. Depending on the precise CAR structure and specificity, individual endodomains may be associated with deleterious ligand-independent tonic signaling in the transduced T cell. Long et al. (Nat Med 2015) established the CD28 co-stimulatory endodomain can have a toxic tonic signaling effect, but it is unclear if tonic 4-1BB signaling may have deleterious consequences as well, and if such effects can be reversed.We therefore modeled tonic CAR signaling in T cells by transducing them with gammaretroviral vectors expressing 2nd-generation CD19.CAR constructs containing either the CD28 or 4-1BB costimulatory endodomain (in addition to the CD3-ζ chain endodomain). Compared to CAR-T cells with the CD28 endodomain alone, those with 4-1BB alone expanded 70% more slowly following transduction. Impaired expansion of 4-1BB CD19.CAR-T cells was coupled with a 4-fold increase in apoptosis and a gradual downregulation of CAR expression, and was a consequence of 4-1BB-associated tonic TRAF2-dependent signaling, leading to activation of NF-κB, upregulation of Fas and augmented Fas-dependent activation-induced T cell death (AICD). Moreover, expression of 4-1BB CAR from a gammaretroviral vector increased tonic signaling through a self-amplifying/positive feedback effect on the retroviral LTR promoter.Because of the toxicity of 4-1BB in our gammaretroviral CAR.CD19 construct (manifest by delayed expansion and increased apoptosis) we could not directly compare the in vivo fate of T cells expressing CAR.CD19 4-1BB with that of co-administered CAR.CD19 CD28 T cells in patients with lymphoma. We found, however, that the adverse effects of tonic 4-1BB costimulation could be overcome in a 3rd-generation CAR.CD19 vector, containing both CD28 and 4-1BB costimulatory molecules in tandem.We thus compared the fate of a 3rd-generation vector containing both CD28 and 4-1BB costimulatory domains with that of a 2nd-generation vector containing CD28 alone. Six patients with refractory/relapsed diffuse large B-cell lymphoma received 2 cell populations, one expressing 2nd and one expressing 3rd generation vectors. To determine whether CD28 alone was optimal (which would suggest 4-1BB is antagonistic) or whether 4-1BB had an additive or synergistic effect contributing to superior persistence and expansion of the CD28-41BB combination, patients were simultaneously infused with 1-20×106 of both 2nd and 3rd generation CAR+ T cells/m2 48-72 hours after lymphodepletion with cyclophosphamide (500 mg/m2/d) and fludarabine (30 mg/m2/d) × 3. Persistence of infused T cells was assessed in blood by CD19.CAR qPCR assays specific for each CAR. Molecular signals peaked approximately 2 weeks post infusion, remaining detectable for up to 6 months. The 3rd-generation CAR-T cells had a mean 23-fold (range 1.1 to 109-fold) higher expansion than 2nd-generation CAR-T cells and correspondingly longer persistence. Two patients had grade 2 cytokine release syndrome, with elevation of proinflammatory cytokines, including IL-6, at the time of peak expansion of T cells. Of the 5 patients evaluable for response, 2 entered complete remission (the longest ongoing for 9 months), 1 has had continued complete remission after autologous stem cell transplantation, 1 had a partial response, and 1 progressed.In conclusion, our data indicate that infusion of T cells carrying a CD19.CAR containing CD28 and 4-1BB endodomains is safe and can have efficacy at every dose level tested. Additionally, in a side-by-side comparison, the 3rdgeneration vector produced greater in vivo expansion and persistence than an otherwise identical CAR-T cell population with CD28 alone. DisclosuresRooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Viracyte: Equity Ownership. Heslop:Celgene: Patents & Royalties, Research Funding; Chimerix: Other: Endpoint adjudication committee; Viracyte: Equity Ownership; Cell Medica: Patents & Royalties: Licensing agreement EBV-specific T cells.

CD19 CAR-T Cells Are Highly Effective in Ibrutinib-Refractory Chronic Lymphocytic Leukemia

BACKGROUND:Ibrutinib, a Bruton Tyrosine Kinase (BTK) inhibitor causes partial responses (PR) in a majority of patients (pts) with chronic lymphocytic leukemia (CLL). However, complete responses (CR) are rare and high-risk pts who progress on ibrutinib have short survival. Lymphodepletion chemotherapy followed by infusion of CD19-specific chimeric antigen receptor (CAR)-modified T cells has produced encouraging responses in CLL in phase 1 clinical trials, but the majority of pts in those studies had not previously received or failed ibrutinib.METHODS:We treated 18 adults with CLL who had previously received ibrutinib with anti-CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets obtained by immunomagnetic selection of leukapheresis products, formulated in a final 1:1 ratio of CD8+:CD4+ CAR+ T cells, and infused at 1 of 3 dose levels (2x105, 2x106 or 2x107 CAR-T cells/kg) after lymphodepletion chemotherapy.RESULTS:Eighteen pts (median age 60; range 40-73) with a median of 5 previous therapies (range 3-9), including 3 pts that failed prior allogeneic stem cell transplant were enrolled and treated on the study. All pts were refractory to or had relapsed after receiving a regimen containing fludarabine and rituximab, and all pts had previously received ibrutinib; 11 were ibrutinib-refractory, 3 were ibrutinib-intolerant, and 4 were refractory to venetoclax. Twelve pts had a complex karyotype and 11 pts had 17p deletion. The median percentage of abnormal B cells in marrow was 77% (range 0.4 Ð 96). All pts had extramedullary disease and 2 had CNS disease. Lymphodepletion chemotherapy consisted of cyclophosphamide (Cy) 30-60 mg/kg x 1 and fludarabine (Flu) 25 mg/m2/day x 3 days (n=15); Flu 25 mg/m2/day x 3 days alone (n=2); and Cy 60 mg/kg alone (n=1). CAR-T cells were manufactured for all pts and 16/18 received CD4+ and CD8+ CAR-T cells in the defined 1:1 ratio. Four pts received 2x105, 13 received 2x106 and 1 received 2x107 CAR-T cells/kg.Seventeen pts have completed response and toxicity assessment. Analysis of all pts with B cell malignancies treated with Cy/Flu and CAR-T cells on our trial showed that the highest dose level (2x107 CAR-T cells/kg) was too toxic for an initial CAR-T cell infusion, and identified a maximum tolerated first dose of 2x106 CAR-T cells/kg. Cytokine release syndrome (CRS) was graded according to Lee et al (Blood, 2014). After a single cycle of lymphodepletion chemotherapy and CAR-T cell infusion, 8 pts developed grade (gr) 0-1, 5 had gr 2, 3 had gr 3, and 1 had gr 4 CRS. Four pts developed gr ³3 neurotoxicity (NT). No gr 5 events were observed, no pts were intubated, and only 1 pt required pressors. Three pts received tocilizumab and dexamethasone to treat CRS and/or NT. Four pts received a second cycle of lymphodepletion chemotherapy and CAR-T cells at a 10-fold higher dose than the 1st infusion for persistent disease. CRS and NT (gr3) was observed in 3 of 4 pts after the second cycle of therapy.Restaging was performed 4 weeks after the last CAR-T cell infusion. The ORR was 76% (8 PR and 5 CR). Two of the pts with PR by lymph node size criteria (IWCLL 2008) had negative PET scans after therapy. Among ibrutinib-refractory (n=10) or intolerant pts (n=3), the ORR was 77% (7 PR and 3 CR). In venetoclax refractory pts, 2 of 4 responded (PR). Only 1 of the 3 pts who did not receive Cy/Flu lymphodepletion responded. At day 28, 11 of 13 (85%) pts who received Cy/Flu lymphodepletion and a CAR-T cell infusion at ²2x106 CAR-T cells/kg had complete elimination of marrow disease by flow cytometry; 10/13 (77%) with nodal disease had a PR or CR at restaging, 1 had a mixed response, and 2 had progressive disease (PD).No malignant IGH sequences were detected in marrow of 4/4 pts in CR who underwent IGH deep sequencing. Pts with CR had a higher peak percentage of CD8+ (p=0.006), but not CD4+ CAR-T cells in blood. Robust CAR-T cell expansion was seen in some non-responders, which in conjunction with the lower response rate in nodal sites compared to blood, suggests that factors in the malignant lymph node environment may inhibit CAR-T cell activity. No pt in CR has relapsed or died with a median follow-up of 8.4 months. For the 13 pts that received Cy/Flu lymphodepletion and ≤ 2 x 106 CAR-T cells/kg, OS is 100% and PFS is shown in Fig. 1.CONCLUSION:CD19 CAR-T cells of defined CD4:CD8 ratio are highly active in CLL and can induce high response rates and durable CRs in poor prognosis pts who have previously failed ibrutinib. [Display omitted] DisclosuresTurtle:Juno Therapeutics: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria. Li:Juno Therapeutics: Employment, Equity Ownership. Riddell:Adaptive Biotechnologies: Consultancy, Honoraria; Cell Medica: Consultancy, Honoraria; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Maloney:Juno Therapeutics: Research Funding; Genentech/Roche: Honoraria.

Biomarkers of Cytokine Release Syndrome and Neurotoxicity after CD19 CAR-T Cells and Mitigation of Toxicity By Cell Dose

BACKGROUND:Lymphodepletion chemotherapy with infusion of CD19-specific chimeric antigen receptor (CAR)-modified T cells has produced impressive antitumor responses in CD19+ malignancies in phase 1 clinical trials, but can be associated with cytokine release syndrome (CRS) and neurotoxicity (NT). Our understanding of CRS and NT continues to evolve, and identification of predictive biomarkers and strategies to mitigate these toxicities will facilitate management of patients (pts) undergoing CD19 CAR-T cell therapy in multicenter phase 2 trials.METHODS:We treated 127 adults with ALL, NHL or CLL with anti-CD19 CAR-T cells manufactured from defined CD4+ and CD8+ T cell subsets, formulated in a 1:1 ratio of CD8+:CD4+ CAR+ T cells, and infused at 1 of 3 dose levels (2x105, 2x106 or 2x107 CAR-T cells/kg) following lymphodepletion chemotherapy. The incidence and grade (gr) of CRS and NT, and correlative biomarkers were analyzed through 28 days after infusion.RESULTS:One hundred and nine pts (45 ALL, 47 NHL, 17 CLL) have completed toxicity and response assessment. CRS was graded according to Lee et al (Blood, 2014) and developed in 71% of pts (44% gr 1-2, 22% gr 3-4, 5% gr 5). Pressors were only required in 21% of the pts who developed gr 3-4 CRS. Gr ³3 NT (CTCAE v 4.03) developed in 25% of pts, all of whom developed fever before NT. The median duration of all-cause hospitalization from the start of lymphodepletion was 7, 6, and 10 days for ALL, NHL and CLL pts, respectively.Because we observed short CAR-T cell persistence and an anti-CAR transgene immune response in an initial cohort of pts who received cyclophosphamide (Cy) lymphodepletion, fludarabine (Flu) was added to Cy. This regimen abrogated immune CAR-T cell rejection and dramatically increased early CAR-T cell expansion. We found that infusion of 2x107 CAR-T cells/kg after Cy/Flu was excessively toxic (5/9 developed gr 4-5 CRS), and identified 2x106 CAR-T cells/kg as the maximum tolerated dose in NHL and CLL. Of 26 NHL pts (22 with aggressive histology) treated with Cy/Flu and 2x106 CAR-T cells/kg, the ORR was 73% and the CR rate 46%. No pts had gr 5 CRS or required pressors, and only 12% of pts experienced either gr 3-4 CRS and/or gr ³3 NT. Of 13 CLL pts treated with Cy/Flu and ²2x106 CAR-T cells/kg, the ORR (CT+/-PET) was 85%. In 85%, of pts no marrow disease was detected by flow cytometry. Overall, 38% of pts achieved CR, including 1 pt with residual CLL after the first infusion who achieved CR after a second CAR-T cell infusion. No pts had gr 4-5 CRS or required pressors, and 23% had either gr 3 CRS and/or gr 3 NT.In ALL pts, the incidence of CRS and NT correlated with the percentage of marrow blasts and CAR-T cell dose, and further dose modification was required for pts with ³ 5% marrow blasts, in whom 2x106 CAR-T cells/kg was excessively toxic (56% gr 4-5 CRS; 56% gr 4-5 NT; n=9). CRS and NT was mitigated in pts with ³ 5% blasts by administering 2x105 CAR-T cells/kg resulting in 6% gr 4 CRS, 17% gr 3; and 17% gr 3-4 NT; n=18). Efficacy was not compromised with the T cell dose reduction for the high tumor burden cohort with 89% of pts achieving a bone marrow CR by high-resolution flow cytometry.Detailed characterization of early biomarkers of CRS and NT may provide an opportunity to develop data-driven toxicity grading to facilitate mitigation strategies that could be applied across different centers. Compared to pts with gr 0-2 CRS, those with gr 3-5 CRS had significantly higher peak levels of IL-15, IL-6, IL-2, IFN-g, C-reactive protein, and ferritin in both ALL and NHL cohorts. Importantly, in univariate analysis, the levels of IL-15, IL-6, IL-8, IL-10, soluble TNF receptor type 1, and IFN-g were significantly higher on day 1 after CAR-T cells in pts that developed gr 3-5 CRS, and might be used to identify pts to test early intervention strategies to reduce later severe toxicity. We made similar observations in pts with and without NT. Multivariate analysis of biomarkers including clinical and laboratory parameters (Figure 1) is ongoing.CONCLUSION:CD19 CAR-T cell immunotherapy can be associated with severe CRS and NT. The use of CAR-T cell products with a prescribed 1:1 CD4/CD8 composition identified CAR-T cell doses associated with a reduced incidence and severity of these complications without impairing efficacy. Additional study of correlative biomarkers to inform rational strategies for early intervention will facilitate the safe and effective clinical application of CAR-T cell therapy. [Display omitted] DisclosuresTurtle:Seattle Genetics: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Research Funding. Li:Juno Therapeutics: Employment, Equity Ownership. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria. Maloney:Juno Therapeutics: Research Funding; Genentech/Roche: Consultancy, Honoraria.

Murine Donor 1928z CAR T Cells Exert Potent Graft-Versus-Lymphoma Activity without Graft-Versus-Host-Disease

Chimeric antigen receptors (CARs) promote tumor cell target recognition of adoptively transferred T cellsand clinical studies have shown that autologous T cells transduced with CARs targeting CD19, expressed on normal B cells and several B cell malignancies, are effective against CD19+ malignancies. The potential of donor-derived CD19-CAR T cells to treat relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) has been relatively unexplored due to concerns for graft-versus-host disease (GVHD) and other toxicities. However, recent clinical trials of donor-derived CD19-CAR T cells in allo-HSCT have noted a surprisingly low incidence of GVHD. We studied the GVHD and anti-tumor potential of donor-derived mouse CD19-specific CAR 1928z (m1928z) T cells in preclinical allo-HSCT and lymphoma models.We constructed a panel of retroviral vectors targeting mouse CD19 (Figure 1A) to test various aspects of CAR stimulation and co-stimulation and generated T cells expressing these constructs. We then used these constructs in mouse allo-HSCT models comprising BALB/c recipients and B6 donors (Figure 1B).A20 lymphoma-inoculated BALB/c recipients of B6 BM treated with donor B6 m1928z T cells had significantly higher tumor-free survival and improved survival compared to m19delta treated recipients (Figure 1C, P < 0.0001). Adoptive transfer of B6 m1928z T cells caused significantly less GVHD compared to B6 m19delta T cells (P < 0.001) in BALB/c recipients of B6 BM confirming that m1928z T cells cause less GVHD. We explored the requirement of CAR signaling for attenuated GVHD using hum1928z (P < 0.001) (anti-human CD19-CAR, non reactive in mice) T cells and noted significantly worse survival and GVHD scores in mice as compared to with m1928z indicating that CAR signaling was necessary for this protection (Figure 1D). We also examined the importance of co-stimulation and noted increased GVHD mediated by m19z T cells (P < 0.05) (CAR with no co-stimulation) (Figure 1E) and m19BBz (P < 0.01) (CAR with 4-1BB co-stimulation) (Figure 1F) indicating that CD28 co-stimulation was also vital for attenuated GVHD. To study the effect of concurrent TCR and CAR signaling in vitro, we used T cells engineered to co-express 1928z CAR and OT-1 TCR in a cold-target inhibition killing assay and noted that activation of the T cells through 1928z can impair lytic activity mediated by TCR. To elucidate the consequence of concurrent TCR and CAR activation, we studied the TCR signaling pathway and found increased phosphorylated PKCa, pERK1/2, pS6, pSTAT1, pSTAT3, and pSTAT5 in donor m1928z T cells compared to m19delta T cells, indicating a hyperactivated state. In order to further study the effect of concurrent stimulation of alloreactive TCR and m1928z CAR, we utilized B6 ABM-RAG1 knockout (ABM) T cells, which express monoclonal TCR specific to I-Abm12 expressed on B6.C-H2bm12 (BM12) mice. In accordance with our previous findings, we observed significantly less GVHD in allo-HSCT BM12 recipients of ABM m1928z T cells versus m19delta T cells (P < 0.0001). We performed global transcriptional analysis of ABM m1928z T cells and m19delta harvested from adoptively transferred BM12 recipients of B6 BM and differential upregulation of Bim, Bid, FasL and Apaf1 in m1928z T cells. Phenotypic analysis of these cells also showed increased PD-1 and Fas expression, consistent with post-stimulatory programmed cell deathIn conclusion, we show that donor m1928z T cells mediate strong GVT effects with decreased GVHD. Following adoptive transfer, alloreactive m1928z T cells (but not m19BBz) undergo progressive loss of effector function and proliferative potential, and eventual deletion, which significantly decreases graft-versus-host activity. The non-alloreactive m1928z T cells present in bulk donor T cell populations retain significant anti-lymphoma activity. Our findings support the administration of donor 1928z T cells to prevent relapse of CD19+ malignancies after allo-HSCT and provide mechanistic insight into recent clinical trial results demonstrating that allogeneic CD19-specific 1928z CAR T cells promote anti-lymphoma activity while causing minimal GVHD. [Display omitted] DisclosuresDavila:Adaptive Biotechnologies: Membership on an entity’s Board of Directors or advisory committees; Celyad: Consultancy; Precision Biosciences: Membership on an entity’s Board of Directors or advisory committees; Kite Pharma: Membership on an entity’s Board of Directors or advisory committees; Servier: Membership on an entity’s Board of Directors or advisory committees. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

Anti-CD19 Chimeric Antigen Receptor in Hematopoietic Stem Cells Controlled By the Suicide Gene HSV-sr39TK for Immunotherapy of B-Lineage Malignancies

Background: Patients with refractory or recurrent B-lineage hematologic malignancies have less than 50% of chance of cure despite intensive therapy and innovative approaches are needed to decrease their morbidity and mortality. Our previous published results demonstrate feasibility of stable gene modification of hematopoietic cells (HSC) with chimeric antigen receptor (CAR) directing antigen specificity of T-, NK- and myeloid cells that are continuously produced with effective anti-tumor activity in humanized mouse model. We hypothesize that gene modification of HSC with an anti-CD19 CAR will produce a multi-lineage, persistent immunotherapy against B-lineage hematological malignancies that can be controlled by the HSV-sr39TK suicide gene. Hematopoietic stem cell transplantation is frequently part of the standard of care for patients with relapsed and refractory B cell malignancies. Following HSC collection, a portion of these cells could be modified to express the CD19-specific CAR and give rise to a persistent, multi-cell lineage, HLA-independent immunotherapy, enhancing the graft-versus-malignancy activity. Based on current clinical trials of gene therapy, gene modification of a partial portion of HSC would be sufficient to give rise to a large number of antigen-specific immune cells, and ablation of such gene-modified cells would not cause myeloablation. The generation of NK and myeloid cells bearing CAR would allow earlier directed anti-tumor activity until post-transplant thymopoiesis can take place, enhancing the graft-versus-cancer activity.Methods: High-titer third-generation self-inactivating lentiviral constructs were developed to deliver second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered the hyper-active herpes simplex virus thymidine kinase HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Such vector constructs were thoroughly evaluated using Jurkat cell line and primary human T-cells. Human HSC isolated from umbilical cord blood were transduced with such lentiviral vectors and evaluated for function of both CAR and HSV-sr39TK. Gene-modified human HSC were also injected into NSG pups after irradiation for in vivo evaluation of engraftment, anti-tumor activity and suicide gene activation. In vivo ablation of gene-modified cells was performed by treatment with intraperitoneal ganciclovir 50mg/kg/day over 5 days. Persistence of gene-modified cells was assessed by flow cytometry and ddPCR of animal tissues.Results: Two promoters were compared in such vector constructs, the human elongating factor-1 short (EFS) and the retrovirus-derived MNDU3, regarding transduction efficiency of primary cells. Those vector constructs with MNDU3 as the promoter showed improved CAR expression (p=0.003) and antigen-specific cytotoxicity, despite similar integrated vector copy numbers (VCN) per cell. The addition of HSV-sr39TK to the construct did not impair CAR expression or antigen-specific cytotoxicity, and determined marked cytotoxicity to ganciclovir. Transduction of human HSC with high-titer lentiviral vectors led to satisfactory transduction efficiency with second-generation CAR and HSV-sr39TK. When tested in vivo, mice transplanted with gene-modified human HSC showed similar humanization in bone marrow and spleen (as defined by CD45+ cells) to non-modified HSC. CAR expression by flow cytometry and VCN were not significantly different between transduced cells with and without HSV-sr39TK, and expression of anti-CD19 CAR conferred anti-tumor survival advantage, with 100% of survival of mice engrafted with HSC modified with CAR and HSV-sr39TK for up to 120 days post-tumor challenge. When treated with ganciclovir, the flow cytometry detection of gene-modified cells was decreased in bone marrows (p=0.07) and spleens (p=0.05) to levels similar to background, and with significant decrease of ddPCR detection of gene-modified cells in both bone marrows (p=0.05) and spleens (p=0.01).Conclusions: The modification of HSC with second generation anti-CD19 CAR has provided specific and persistent anti-lymphoma protection. The addition of the suicide gene system HSV-sr39TK to the construct did not affect transduction efficiency, and has determined significant ablation of gene-modified cells in mouse tissues. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of Humanized CD19-Targeted Chimeric Antigen Receptor (CAR)-Modified T Cells in Children and Young Adults with Relapsed/Refractory Acute Lymphoblastic Leukemia

▪BackgroundTargeted immunotherapy with CTL019, CD19-specific chimeric antigen receptor (CAR)-modified T cells, can produce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). However, a subset of patients has limited persistence, which can increase the risk of relapse. Most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin; therefore, anti-mouse reactivity is one potential cause of immune-mediated rejection that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19-directed CAR T cells (CTL119).DesignA pilot/phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (CTL119) enrolled children and young adults with relapsed/refractory B-ALL with or without prior exposure to a CAR T cell product. Patients previously treated with CD19-specific CAR-modified T cells were eligible if they met 1 of 3 criteria: 1) CD19+ relapse 2) no response to prior CAR T cell therapy or 3) early B cell recovery indicating poor persistence of CAR T cells. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of CD3z, 4-1BB, and humanized anti-CD19 scFv domains and activated/expanded with anti-CD3/CD28 beads. The humanized scFv domain was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Patients received lymphodepletion with cyclophosphamide and fludarabine 1 week prior to infusion with CTL119.ResultsThirty children and young adults aged 29 mo-24 yr were infused with CTL119. Eighteen patients had received prior allogeneic stem cell transplant (SCT). Eleven patients who previously received murine-derived CD19-specific CAR-modified T cells (CTL019, n=7; other, n=4) were retreated for B cell recovery (n=5), CD19+ relapse (n=5), or no response to prior CAR T cells (n=1). CNS disease or other extramedullary disease was the indication for enrollment in 6 and 3 patients, respectively.At assessment 1 month after infusion, 26/30 patients (87%) achieved a complete response (CR), defined as morphologic remission with B cell aplasia. Of 11 patients previously treated with murine CD19-specific CAR-modified T cells, 7 (64%) achieved a CR at 1 month, 4 demonstrated no response. Multiparameter flow cytometry for minimal residual disease (MRD) was negative at a detection level of 0.01% in 5/7 responding patients. Two responding patients with positive MRD progressed to CD19+ relapse at 1.6 and 3 mo.In patients with no prior exposure to a CD19 CAR T cell product, MRD-negative CR was achieved in 19/19 patients (100%). One patient relapsed with CD19+ extramedullary disease at 2.8 mo. With a median follow-up of 4.2 mo (range, 1.0-14.1 mo) for all responding patients in both cohorts, 23/26 remain in remission with 1 proceeding to SCT in remission.B cell aplasia, a functional marker of CD19-targeted CAR T cell persistence, continued for 3 months or more in 11/18 patients with adequate follow-up: 1/6 retreatment, 10/12 CAR-naïve. Cytokine release syndrome (CRS) was observed in 28/30 patients and mild in most patients (grade 1, n=6; grade 2, n=18). Three patients experienced grade 3 CRS requiring supplemental oxygen or low-dose vasopressor support and 1 experienced grade 4 CRS requiring high-dose vasopressor and ventilatory support. Severe CRS was successfully managed with the IL6R antagonist tocilizumab in 3 patients. Neurologic toxicity included encephalopathy (n=5) and seizure (n=4) and was fully reversible.ConclusionIn the first study of humanized anti-CD19 CAR T cells, CTL119 induced remissions in children and young adults with relapsed/refractory B-ALL, including 64% of patients previously treated with murine CD19-directed CAR T cells and 100% of CAR-naïve patients. Further investigation into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. DisclosuresMaude:Novartis: Consultancy. Barrett:Novartis: Research Funding. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding; Vitality Institute: Research Funding. Brogdon:Novartis: Employment. Scholler:Novartis: Patents & Royalties: Royalties, Research Funding. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Frey:Amgen: Consultancy; Novartis: Research Funding. Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Employment. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership. Grupp:Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding.

Efficacy and Safety of CTL019 in the First US Phase II Multicenter Trial in Pediatric Relapsed/Refractory Acute Lymphoblastic Leukemia: Results of an Interim Analysis

Background: CTL019 is an investigational therapy derived from autologous T-cells expressing a CD19-specific chimeric antigen receptor (CAR). A single center, phase I/IIa trial of CTL019 showed complete and durable remissions in pediatric/young adult patients (pts) with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) (Maude et al NEJM 2014); these results have yet to be reproduced in a multicenter setting. Here, we report results from a 6-month interim analysis of the first multicenter phase II trial of an engineered cell therapy in leukemia. Methods: 9 US sites participated in this single-arm phase II study in pediatric/young adult pts with R/R B-ALL. Leukapheresis products were shipped for centralized manufacturing according to the University of Pennsylvania (Penn) process in an academic-industry collaboration. T cells were transduced with a lentiviral vector encoding a CAR composed of anti-CD19 scFv, CD3ζ and 4-1BB domains. Following lymphodepletion with fludarabine and cyclophosphamide, a single dose of CTL019 cells was administered (target dose 2.0-5.0×10 6 cells/kg for ≤50 kg and 1.0-2.5×10 8 cells for >50 kg). The primary endpoint was overall remission rate (ORR = CR + CRi [CRi, complete remission with incomplete blood count recovery] maintained at 2 evaluations ≥28 days apart) as determined by an Independent Review Committee. Secondary objectives included minimal residual disease (MRD), relapse-free survival (RFS), overall survival (OS) and safety. All analyses were performed on infused patient set. Results: 29/35 pts enrolled (82.9%) were infused with CTL019; 6 withdrew prior to infusion (2 manufacturing failures [1 lack of growth, 1 contamination]; 4 deaths [median, 48 days from enrollment; 2 progressive disease, 1 multi-organ failure, 1 pneumonia]). Mean bone marrow involvement at enrollment was 68.2% (SD, 27.3%; Table 1). 2 pts did not receive lymphodepleting chemotherapy due to leukopenia. Collection, manufacturing and infusion were feasible in a multicenter setting with a median time from enrollment to infusion of 37 days. Target cell dose was met in 24/33 (72.7%) manufactured products. ORR in all infused pts was 69.0% (20/29 pts; 98.95% CI 43.6, 88.1). Of the 5 pts who received CTL019 below the target dose, 2 achieved CR/CRi. Of note, deep remission with no evidence of MRD ( Conclusions: In this first multicenter trial of CAR-modified T cell therapy, CTL019 therapy was feasible and efficacious, showing a high ORR with durable remissions in pediatric/young adult pts with R/R B-ALL. Despite the high rate of toxicity with CTL019 in the R/R setting, the rate of grade 3 or 4 CRS was comparable to the single center study, and standardized management of CRS was successful in a multicenter trial with no deaths attributable to CRS. In this highly refractory population, a vast majority of eligible pts can be successfully infused in a timely fashion and outcomes appear reproducible in a multicenter setting despite a more heterogeneous population than the single center study. The trial is continuing under Novartis manufacturing. Disclosures Maude: Novartis: Consultancy. Pulsipher: Medac: Other: Travel support for a study group; Chimerix: Consultancy, Other: Advisory Board ; Jazz Pharmaceutical: Consultancy, Other: Advisory Board; Novartis: Consultancy, Other: Advisory Board, Steering Committee for Phase II Study. Grupp: Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Davies: Novartis: Honoraria. Verneris: Bimogen: Other: Advisory Board. Schlis: Novartis: Honoraria. Driscoll: Novartis: Consultancy. June: Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership. Levine: GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Wood: Novartis Pharmaceuticals: Employment, Other: Stock. Yi: Novartis: Employment.

The Roles of Aryl Hydrocarbon Receptor in T Cells at IDO-Positive Tumor Microenvironment

Introduction:Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor well known to generate biological responses to dioxins. A recent study indicated that kynurenine was an endogenous ligand of AhR and was associated with suppression of the antitumor response. The essential amino acid tryptophan metabolism proceeds via the enzymes IDO, which generates the immunosuppressive metabolites, such as kynurenine. Accumulation of these tryptophan derivatives block antigen-specific T-cell proliferation and induces T-cell death through the AhR. IDO is known to be produced by tumor cells and by some immune cells, such as dendritic cells and macrophages, which reside in tumor-draining lymph nodes or are recruited to tumors. IDO is overexpressed in several human cancers, including prostate, breast, brain, and hematologic malignancies.We previously showed that both IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in non-Hodgkin lymphoma patients. We also revealed that tumor IDO activity can inhibit CD19-specific chimeric antigen-receptor (CAR) T-cells (CARTs) therapy through the action of tryptophan metabolites (Blood. 2015; 125(25): 3905-3916). Therefore, we focused on the AhR in T-cells at IDO-positive tumor microenvironment.Methods and Results:We collected PBMCs from healthy blood donors, and activated the cells with anti-CD3 and anti-CD28 Abs, which mimic physiological T-cell activation. We measured the expression of AhR in activated T-cells using by flow cytometry, and found that the range of expression were from 4.5% to 38%. We then studied the effect of kynurenine on the proliferation of T-cells. The proliferation was assessed by MTS assay. We found that kynurenine significantly inhibited the proliferation of T-cells with high expression of AhR. The percentage inhibition with kynurenine 25µM was 33%. While T-cells with low expression of AhR were not inhibited with even 50µM of kynurenine. To investigate potential mechanisms of T-cell inhibition by kynurenine, we assessed the effect of kynurenine on T-cell apoptosis. We found that kynurenine was also associated with increased the apoptosis of T-cells with high AhR, as assessed by Annexin-V staining (percent Annexin-V positive and 7-AAD negative cells in 0 and 25 µM kynurenine was 11.8% and 36%, respectively). To evaluate the effect of inhibiting AhR in T-cells, we used AhR inhibitor (CH-223191). We found that CH-223191 could blocked the inhibitory effects of kynurenine on T-cells. The percent Annexin-V positive and 7-AAD negative cells in 25 µM kynurenine plus CH-223191 was 22.5%. Next, to assess the functional effects of tryptophan metabolites on the T-cells, we cocultured T-cells (1x106) with GFP-transduced wild-type Raji cells or IDO-transduced Raji cells (1x106). After 72 hours, cells were stained with CD8 antibody to distinguish tumor (GFP+ CD8-) and T-cells (GFP- CD8+). We found that the percentage of T-cells with high AhR in culture with IDO negative and positive Raji cells was 46.7% and 61.4%, respectively, while the percentage of T-cells with low AhR was 41.6% and 39.4%. These data showed that only T-cells with high AhR were inhibited at IDO-positive tumor microenvironment. Next, bone marrow (BM) samples were obtained from multiple myeloma (MM) patients at diagnosis. We could generated the activated T-cells from 10 of 12 BM samples. The expression of AhR in T-cells was ranged from 3.3% to 26.6%. We found that there was a positive correlation between the expression of AhR and the proportion of plasma cells in BM (r=0.76, P=0.04).Discussion:The expression of AhR in activated T cells play an important role in the effect of kynurenine, which is a metabolite produced by IDO. A few clinical trials are evaluating the effects of IDO inhibitors that competitively block the degradation of tryptophan to kynurenine by the enzyme. Because anti-PD-1 and anti-CTLA-4 therapies, which block directly the inhibitory signal in T-cells, have been getting some clinical benefits against such as melanoma and Hodgkin lymphoma. Therefore, AhR in T cells might be a target for IDO-positive hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

Combination Immunotherapy with NY-ESO-1-Specific CAR+ T Cells with T-Cell Vaccine Improves Anti-Myeloma Effect

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. DisclosuresPatel: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.

Development of Novel Non-Immunoglobulin Centyrin-Based Cars (CARTyrins) Targeting Human Bcma

Chimeric-antigen receptor (CAR)-T cell immunotherapy is a promising type of cancer therapy and substantial progress has been made in developing adoptive T cell approaches for B cell malignancies. B cell maturation antigen (BCMA) is an attractive target for patients with multiple myeloma (MM) due to its high level of expression on tumor cells and restricted expression on normal tissues. Traditionally, the antigen-binding domain of a CAR is a single chain variable fragment (scFv) comprised of heavy chain (HC) and light chain (LC) variable fragments joined by a flexible linker that has been derived from a non-human monoclonal Ab (mAb). However, there are a number of disadvantages to scFv-based CARs including the limited availability of scFv, their potential to elicit antibody responses, and their association with tonic signaling due, in part, to inherent instability and flexibility of the structure and the potential for both HC/LC domain swapping and multimer formation through framework region interactions. Thus, replacement with alternative binding technologies may improve CAR-T efficacy in the clinic.Centyrins are alternative scaffold molecules that bind protein targets with high affinity and specificity, similar to scFv molecules. However, unlike scFv, Centyrins are smaller, derived from human consensus tenascin FN3 domains and are predicted to have decreased immunogenicity. Additionally, a monomeric Centryin in CAR format (i.e. CARTyrin molecule) is less likely to engage in domain swapping or interact with other Centyrins at the cell surface, thereby limiting the potential for the tonic signaling that drives the functional exhaustion of CAR T cells. Centyrins can be isolated against virtually any antigen through ex vivo panning of an extensive Centyrin library, yielding many distinct binders with a range of affinities and target epitopes.Panning with soluble BCMA protein yielded a large pool of BCMA-specific Centyrins, from which 11 distinct monomeric binders and 1 non-monomeric binder were selected for further study in CAR format. In addition, we tested numerous signal peptides, linkers, transmembrane domains and signaling domains to determine optimal configuration. We then created all CARTyrins by fusing each Centyrin with a CD8a leader peptide, spacer and transmembrane domain, as well as an intracellular signaling domain derived from both 4-1BB and CD3ζ. High quality mRNA of each CARTyrin construct was produced in order to rapidly screen CARTyrin cell surface expression and functionality in human pan T cells against BCMA+ targets. We also constructed scFv-based CARs against CD19 and BCMA for comparison. Previously CD3/CD28-stimulated T cells were electroporated (EP) with mRNA encoding each of the 12 anti-BCMA CARTyrins and, the following day, analyzed for surface expression of CARTyrin and their ability to degranulate against BCMA+ tumor cells.All 12 CARTyrins were detected on the cell surface and the 11 monomeric CARTyrins imparted BCMA-specific killing capacity to T cells. Notably, in these assays, CARTyrins were functionally comparable to scFv-based CARs against BCMA or to CD19-specific scFv-based CARs in a parallel assay with CD19+ tumor cells. The 11 functional anti-BCMA CARTyrins were further characterized for functional avidity by determining their activity against a panel of target cells with titrated levels of surface BCMA expression. To create this panel, various amounts of high quality BCMA mRNA were electroporated into BCMA- K562 tumor cells. After 4 hours of co-culture with the panel of BCMA expressing cells, CARTyrin+ T cell activity was measured as a function of CD107a expression. We observed a range of activities by each CARTyrin and show that this assay can be utilized to determine the minimal effective dose of BCMA needed to induce killing by CARTyrin+ cells. Furthermore, we establish that certain BCMA-specific CARTyrins are responsive to target cells with extremely low levels of surface BCMA expression. These results confirm that Centyrins are viable replacements for scFv in the construction of functional CARs and establish their potential utility in generating novel BCMA-specific CAR molecules, as well as other novel targetable tumor antigens. DisclosuresBarnett:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

Very Rapid Production of CAR+ T-Cells upon Non-Viral Gene Transfer Using the Sleeping Beauty System

T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) for in vivo clinical applications. CAR-modified T cells have demonstrated redirected specificity and, in several clinical trials, potent anti-tumor activity. Manufacture, to date, is based upon gene transfer in cycling T cells followed by a period of tissue culture to achieve stable expression of introduced CARs. In contrast, we have adapted the non-viral-based Sleeping Beauty (SB) system to avoid the need for (i) T-cell activation and (ii) extended ex vivo tissue culture; thereby developing an approach whereby T cells can be both manufactured and delivered at multiple points-of-care (POC). This shortened culture decreases the time frame for manufacturing CAR+ T cells compared with current protocols for viral- or non-viral-based methodologies and is a foundation of our POC technology. Furthermore, reducing the ex vivo culture time preserves the memory and sustained persistence of CAR+ T cells by avoiding the differentiation programming induced by activation events typically required before or after gene transfer. We have previously demonstrated that co-expressing a membrane-bound version of interleukin-15 (mbIL15) significantly enhances the in vivo persistence of CAR+ T cells that are generated following 28-day culture after electro-transfer of SB derived DNA plasmids. Herein, we incorporated mbIL15 to generate POC CD19-specific CAR+ T cells. Peripheral blood mononuclear cells were genetically modified with mbIL15 and 2nd generation CAR coded from individual SB DNA plasmids and placed in culture for less than 2 days prior to adoptive transfer. NSG mice burdened by established and disseminated CD19+ leukemia were intravenously injected with just 7.5 x 105 CAR+ T cells, or an equivalent total T-cell dose of CARneg (unmodified or mock-treated) T cells. The mbIL15-CAR T-cell infusion yielded excellent disease-free survival, anti-tumor activity (Figure), and T-cell persistence. This approach to expediting the generation of genetically modified T cells enables the administration of CAR-modified naïve T cells and demonstrates that POC T cells have potent anti-tumor effects, even at a reduced CAR+ T-cell dose. This improvement to non-viral gene transfer and T-cell production reduces the requirement for tissue culture and thus time to manufacture within a GMP facility which translates to improvements in scalability and reduced costs. In summary, these data provide a translational pathway to undertake clinical trials by rapidly infusing T cells after genetic modification using the SB system. [Display omitted] DisclosuresHurton:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Singh:Immatics: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Switzer:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Mi:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Maiti:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Su:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Huls:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Employment, Equity Ownership, Patents & Royalties. Champlin:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Cooper:Immatics: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.: Equity Ownership; Sangamo BioSciences: Patents & Royalties; Intrexon: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria.

Abstract B082: Migratory T cells demonstrate superior persistence and enhanced tumor control

Abstract Adoptive cell therapy (ACT) based on the transfer of chimeric antigen receptor (CAR) T cells rendered specific for CD19 has demonstrated significant anti-tumor effects in patients with refractory CD19+ B-cell malignancies. There are however no biomarkers that can predict the potency of T-cell infusion products and thus the ability to enrich functionally superior cells is lacking. Existing clinical data supports at least two important functions of the T cells in mediating tumor regression: (a) immediate cytotoxicity to enable tumor cell killing, and (b) long-term persistence to ensure lasting and durable responses. We demonstrate here that migratory capacity of T cells can be used as a biomarker to segregate functionally superior cells that display enhanced polyfunctionality in vitro and superior tumor control in vivo. The simplicity of sorting cells based on motility and the scalability of the approach imply that these can be routinely adapted in standard GMP settings to enrich for functional T cells among any heterogeneous population. Populations of CD19-specific chimeric antigen receptor (CAR) T cells were biomanufactured using our standard clinical protocol. Timelapse Imaging In Nanowell Grids (TIMING) was used to enable the simultaneous quantification of the interaction between thousands of individual tumor-specific CD8+ T cells and multiple tumor cells. These results demonstrated that IFN-gamma secretion was the most common function elicited upon tumor cell conjugation. We found that CD8+ T cells with killing ability, especially serial killing ability, required shorter durations of target cell conjugation in comparison to IFN-gamma secreting mono-functional cells, indicating rapid synapse termination by T cells capable of killing versus cytokine secretion. Tracking the velocities of these cells by time-lapse imaging revealed that these serial killer T cells (with or without IFN-gamma secretion) had a high out-of-contact basal motility. In order to gain a better understanding of the phenotypic characteristics of the migratory T cells, we performed molecular profiling of T cells identified only by their basal motility (in the absence of tumor cells) using single-cell multiplexed transcriptional profiling, microscopy and flow cytometry, and found that the costimulatory molecules CD2 and CD244, and the chemokine receptor CXCR3 were expressed at higher levels in highly motile T cells compared to the non-motile T cells. In order to test the hypothesis that migratory capacity can serve as a biomarker T cell polyfunctionality, we next sorted motile cells using a modified migration assay, and confirmed that the migrating cells were enriched in highly motile cells. We characterized these cells phenotypically by flow-cytometry and demonstrated that T-cell differentiation markers including CCR7, CD95 and CD45RA were not differentially expressed between the motile and non-motile cells. Consistently however, the migrated cells displayed superior killing and serial killing in comparison to the non-migrated T cells. This superior in vitro polyfunctionality of migratory T cells was confirmed in a mouse xenograft model of leukemia in which motile CAR+ T cells were superior in controlling the disease in comparison to either their non-motile counterparts or the parent unsorted population. In aggregate, these results demonstrate the utility of our TIMING single cell methodology in uncovering not only the dynamic profile of T-cell behavior but also the ability to identify subpopulations of T-cell with enhanced polyfunctionality. Our studies support the use of motility as a surrogate and selective marker of higher CAR+ T cell bioactivity. These results also open up avenues to molecularly engineer T cells for an increased motility that could translate to better in vivo outcomes. Note: This abstract was not presented at the conference. Citation Format: Gabrielle Romain, Harjeet Singh, Ivan Liadi, Jay R Adolacion, Laurence J.N. Cooper, Navin Varadarajan. Migratory T cells demonstrate superior persistence and enhanced tumor control [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B082.