Expansion Of Chimeric Antigen Receptor Research Articles

SSTR2 as an anatomical imaging marker and a safety switch to monitor and manage CAR T cell toxicity

The ability to image adoptively transferred T cells in the body and to eliminate them to avoid toxicity will be vital for chimeric antigen receptor (CAR) T cell therapy, particularly against solid tumors with higher risk of off-tumor toxicity. Previously, we have demonstrated the utility of somatostatin receptor 2 (SSTR2) for CAR T cell imaging, illustrating the expansion and contraction of CAR T cells in tumor as well as off-tumor expansion. Using intercellular adhesion molecule 1 (ICAM-1)-specific CAR T cells that secrete interleukin (IL)-12 as a model, herein we examined the potential of SSTR2 as a safety switch when combined with the SSTR2-specific maytansine-octreotate conjugate PEN-221. Constitutive secretion of IL-12 led to continuous expansion of CAR T cells after rapid elimination of tumors, causing systemic toxicity in mice with intact MHC expression. Treatment with PEN-221 rapidly reduced the abundance of CAR T cells, decreasing the severity of xenogeneic graft-versus-host disease (GvHD), and prolonged survival. Our study supports the development of SSTR2 as a single genetic marker for CAR T cells that is readily applicable to humans both for anatomical detection of T cell distribution and an image-guided safety switch for rapid elimination of CAR T cells.

Intercellular Mitochondrial Transfer Enhances Metabolic Fitness and Anti-Tumor Effects of CAR T Cells

[Introduction] Chimeric antigen receptor (CAR) T-cell therapy is a potentially curative treatment for patients with relapsed or refractory (r/r) hematopoietic malignancies. However, roughly half of patients with r/r B-cell lymphoma experience treatment failure after CAR T-cell therapy possibly due to poor expansion, limited persistence, and exhaustion of CAR T cells. Given that metabolic fitness has a central role in regulating T-cell functions, manipulating the mitochondrial function is a promising approach to enhance anti-tumor effects of CAR T cells. In the current study, we tested if transfer of mitochondria isolated from HeLa cells using a novel intact mitochondrial isolation technology (Q mitochondria; WO2021015298 A1) into T cells could enhance metabolic fitness and anti-tumor effects of CAR T cells. [Methods] Purified T cells from naïve mice were stimulated with plate-bound anti-CD3/CD28 antibodies in the presence or absence of Q (50-100 µg/ml). Culture medium and Q were renewed every 24 or 48 hours. Mitochondrial respiratory function was evaluated using the XFp Flux Analyzer. For CAR T-cell generation, purified T cells were stimulated with anti-CD3/-CD28 antibodies for 48 hrs and then incubated with retroviral vector encoding anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hrs. [Results] First, we confirmed that mitochondrial structure and ATP synthesis of Q mitochondria were much better maintained compared to those of mitochondria prepared by the conventional homogenization method. When purified T cells were incubated with fluorescent-labeled Q mitochondria for 24 hrs during TCR stimulation, approx. 95% of T cells endocytosed Q mitochondria, and mitochondrial mass per cell was significantly increased. As expected, oxidative phosphorylation (OXPHOS) was markedly enhanced in Q-treated T cells compared to diluent-treated controls after TCR stimulation. The levels of reactive oxygen species (ROS) in diluent-treated T cells were increased after 72-hrs TCR stimulation, while Q mitochondria significantly mitigated accumulation of ROS in activated T cells. Q mitochondria increased TCF-1+PD-1+Tim3- precursor exhausted T cells and enhanced production of IFN-g and TNF-a after 72-hrs TCR stimulation, and improved survival after 192-hrs chronic TCR stimulation. Based on these findings, we next investigated whether Q mitochondria could affect CAR T-cell functions. CAR T cells were generated with purified T cells from naïve BALB/c mice and Q mitochondria was added from the beginning of TCR stimulation. Q-treated CAR T cells demonstrated significantly enhanced OXPHOS (Fig. 1) and cytokine production compared to diluent-treated CAR T cells. Q-treated CAR T cells also showed significantly improved proliferative capacity and in vitro cytotoxicity against syngeneic B-cell lymphoma cells (A20). Next, we examined in vivo antitumor effects of Q-treated CAR T cells. Naïve BALB/c mice were subcutaneously injected with 1.5 x 107 A20 cells on the right flank and intravenously injected with 1 x 106 Q-treated or control CAR T cells, or naïve BALB/c T cells 14 days after tumor inoculation. Although control CAR T cells significantly suppressed tumor growth compared to naïve T cells, all mice died due to tumor growth by day 31 after tumor inoculation. Importantly, tumor growth was significantly delayed (Fig. 2) in the mice injected with Q-treated CAR T cells leading to significantly prolonged survival compared to those treated with control CAR-T cells. [Conclusions] We for the first time found transfer of Q mitochondria prepared with the intact mitochondrial isolation technology into CAR T cells improved metabolic fitness, leading to enhanced proliferative capacity and cytokine production, and in vitro and in vivo anti-tumor effects. Transfer of Q mitochondria could be also useful as an adjunctive method in combination with various other genetical and pharmacological approaches to enhance effectiveness of CAR T-cell therapy. Fig. 1. Oxygen consumption rate (OCR) of Q-treated and control CAR T cells. *, P < 0.05; **, P < 0.01. Fig. 2. Tumor growth curves in the mice injected with Q-treated CAR T cells, control CAR T cells, and nai¨ve T cells on day 14 after tumor inoculation. *, P < 0.05; **, P < 0.01. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oligoclonal T-cell expansion in a patient with bone marrow failure after CD19 CAR-T therapy for Richter-transformed DLBCL

The advent of chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape of refractory B-cell malignancies. Real-world evidence has highlighted the high incidence of hematological toxicity, including prolonged and profound cytopenia. Here, we present a case of bone marrow (BM) failure in a 57-year old man with DLBCL-type Richter transformation receiving tisagenlecleucel in a standard-of-care setting. His clinical course was notable for underlying BM infiltration, severe cytokine release syndrome, and multiple infectious complications in the setting of prolonged, profound neutropenia. Cytokine profiling revealed a signature consistent with acquired aplastic anemia - including downregulation of CD40-L and EGF. Flow cytometric analyses demonstrated migration of CAR T-cells to the BM, as well as peripheral blood expansion of both CAR and non-CAR bearing CD8+CD57+ T-cells, an immunophenotype linked to oligoclonality in aplastic anemia. From single-cell level transcriptomics, this cytotoxic T-cell population was characterized by T-cell receptor Vβ oligoclonal expansion and post-CAR clonal drift. Gene expression profiling revealed upregulation of exhaustion markers on (CAR) T-cells and decreased expression of genes involved in STAT signalling and inflammatory response. In conclusion, this case highlights the complex nature of CAR-T-related hematological toxicity and introduces oligoclonal (CAR) T-cell expansion as a potential contributing pathophysiologic mechanism.

Oncolytic virus-mediated expansion of dual-specific CAR T cells improves efficacy against solid tumors in mice.

Oncolytic viruses (OVs) encoding a variety of transgenes have been evaluated as therapeutic tools to increase the efficacy of chimeric antigen receptor (CAR)-modified T cells in the solid tumor microenvironment (TME). Here, using systemically delivered OVs and CAR T cells in immunocompetent mouse models, we have defined a mechanism by which OVs can potentiate CAR T cell efficacy against solid tumor models of melanoma and glioma. We show that stimulation of the native T cell receptor (TCR) with viral or virally encoded epitopes gives rise to enhanced proliferation, CAR-directed antitumor function, and distinct memory phenotypes. In vivo expansion of dual-specific (DS) CAR T cells was leveraged by in vitro preloading with oncolytic vesicular stomatitis virus (VSV) or reovirus, allowing for a further in vivo expansion and reactivation of T cells by homologous boosting. This treatment led to prolonged survival of mice with subcutaneous melanoma and intracranial glioma tumors. Human CD19 CAR T cells could also be expanded in vitro with TCR reactivity against viral or virally encoded antigens and was associated with greater CAR-directed cytokine production. Our data highlight the utility of combining OV and CAR T cell therapy and show that stimulation of the native TCR can be exploited to enhance CAR T cell activity and efficacy in mice.

Overview of approved CAR-T therapies, ongoing clinical trials, and its impact on clinical practice.

In recent years, we have seen rapid expansion of chimeric antigen receptor T‐cell (CAR‐T) therapies in multiple malignancies. CAR‐T therapy has profoundly altered the treatment landscape of non‐Hodgkin lymphoma, B‐cell acute lymphoblastic leukemia, and multiple myeloma. Currently available CD19 and B‐cell maturation antigen‐directed CAR‐T therapies have shown high overall response rate and durable remissions in patients who have failed standard therapies. Multiple studies are underway exploring the role of CAR‐T‐cell therapy as earlier line of treatment. In high‐grade B‐cell lymphoma, CD19 CAR‐T therapy may replace autologous hematopoietic cell transplantation as second line therapy in near future. CAR‐T‐cell therapy targeting novel tumor‐associated antigens will help expand utility of this treatment modality in other hematological malignancies. It may also help overcome limitations of currently approved CAR‐T‐cell therapies. In this review, we have provided an overview of currently approved CAR‐T therapies and upcoming clinical trials which may potentially impact the clinical practice.

Dexamethasone Enhanced CAR T Cell Persistence and Function through Upregulation of Interleukin 7 Receptor

Dexamethasone (Dex) has been a mainstay for the treatment of inflammatory pathologies, such as in autoimmunity and cytokine release syndrome (CRS) from immunotherapies. However, its effects on chimeric antigen receptor (CAR) T cells, during CRS events have not been interrogated. In this study, we treated the CAR T cells with different concentrations of Dex (0, 1, and 10µM) single or multiple times in vitro and expanded them for one week. We demonstrated that Dex treatment did not inhibit CAR T cell growth and functionality even with concentrations higher than what would be used in the clinic. Interestingly, we observed that the Dex treatment significantly upregulated endogenous gamma chain cytokine receptor-interleukin 7 receptor alpha (IL7Rα) at the mRNA and protein levels (P=0.0005) (Fig 1a). These effects are not T cell subset dependent because we observed upregulation of IL7Rα on PBMC and enriched naïve and memory T cell-derived CAR T cells. Furthermore, un-transduced T cells also exhibited IL7Rα increase, which suggests that the upregulation of IL7Rα is the general mechanism of Dex for T cells. IL7Rα is well accepted as a key element to CAR T cell persistence and memory T cell formation. However, the IL7R-IL-7 signaling pathway is limited due to the downregulation of high-affinity IL7Rα during the activation and expansion of CAR T cells. We found out that Dex can upregulate endogenous IL7Rα in a reversible manner, which is an important factor for safety in clinical application. We showed that ex vivo upregulation of IL7Ra by a single Dex treatment subsequently enhanced CAR T cell persistence and anti-tumor efficacy in vivo in the presence of IL-7. To further confirm the positive effects of Dex on CAR T cell therapy, we performed a combinatorial therapy by delivering CD19 CAR T cells to acute lymphoid leukemia (ALL) tumor-bearing NOD-scid IL2Rgammanull (NSG) mice and then administering Dex (1mg/kg) and IL-7-expressing CHO cells. Consistently, we observed a complete cure of tumor-bearing mice only in the CD19 CAR T cell group that was given both Dex and IL-7, but not CAR alone and Dex only groups (Fig 1b-c) (P=0.0006). Mice survived, tumor-free, over 150 days. Supportively, we observed CAR T cell persistence only in the CAR T cells combined with Dex and IL7 group but not in the CAR group. To determine if Dex influenced CAR T cells beyond IL7Rα, we performed gene analysis and demonstrated that IL7Rα, but not other γ chain cytokines was selectively upregulated by Dex, which supports previous reports from Lee et al. that Dex and glucocorticoid receptors (GR) complex binds upstream of the IL-7rα promoter preferentially regulating IL7Rα. Furthermore, we utilized Nanostring technology to analyze CAR T cells with and without Dex treatment for mRNA signatures that related to signaling pathways. We observed pathways related to activation, migration, persistence, and chemokine production were upregulated, while pathways related to apoptosis and TCR diversity were downregulated after Dex treatment. The results indicated that Dex may regulate multiple functions of CAR T cells.Overall, our studies in both in vitro and in vivo treatment support that Dex does not have negative effects on CAR T cell potency but provides insight into an unforeseen strategy to improve CAR T cell therapies through upregulation of IL-7Rα and improving T cell activation, trafficking, and persistence. We believe our observations could extend beyond hematological malignancies to a potentially potent and durable therapy for solid tumors, as Dex is not only an immunosuppressive agent but also an anti-cancer drug used against a multitude of tumors to prevent tumor growth as well as modulate the microenvironment. Our data also provided rationale on starting CAR T cell therapy without the necessity of tapering off the ongoing steroid treatment. [Display omitted] DisclosuresForman: Allogene: Consultancy; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company. Wang: Pepromene Bio, Inc.: Consultancy.

Understanding the CD8+ CAR T Cell Response to Antigen-Low Leukemias

Chimeric antigen receptor (CAR) T cell therapy has emerged as a highly efficacious treatment for B-lineage acute lymphoblastic leukemias (B-ALL). However, downregulation of the CAR-targeted antigen on leukemia cells, predicted to reduce cellular avidity, is associated with post-CAR T cell leukemic relapse following CD22 CAR treatment (Fry et al., Nat. Med., 2017). We have observed reduced function of human CAR T cells against low target antigen site density (Ag Lo) human leukemia in immunodeficient mouse models, relative to CARs responding to high-antigen expressing leukemia. Thus, a better understanding of CAR responses to Ag Lo leukemia could help to increase the durability of remissions. We set out to develop a model system in which we could further interrogate the consequences of low-avidity interactions on CAR immunobiology, generating variants of a murine B-ALL driven by the E2A-PBX fusion protein (E2A) with different levels of target antigen to use in an immunocompetent syngeneic mouse model.We observed impaired expansion (p<0.0001) and tumor clearance (p<0.001) of CAR T cells responding to low-antigen variants of E2A (E2A-Ag Lo) as compared to wildtype E2A expressing high levels of antigen (E2A-WT). While CD8+ CAR T cell (CAR8) transcription factor (TF) expression in response to E2A-Ag Lo versus E2A-WT was largely similar early after CAR infusion, by day 9 post-CAR, CAR8s responding to E2A-Ag Lo exhibited decreased expression of multiple TFs, with Eomes (p<0.01), Irf4 (p<0.001) and Blimp1 (p<0.01) showing the largest magnitude change relative to CAR8s responding to E2A-WT. Additionally, CAR8s from mice bearing E2A-Ag Lo became enriched for cells of a “terminally exhausted” phenotype (Eomes+/PD1 Hi/TOX Hi) by day 11 post-CAR, and negatively-enriched for the “progenitor exhausted” (Tcf1+/PD1 Int) phenotype which can be functionally rescued by anti-PD1 therapy (p<0.0001, p<0.01). These data suggest that continual stimulation by low density antigen leads to a gradual reduction in the ability of CAR8s to mount an effector response, and eventually to T cell states with sub-optimal anti-tumor efficacy.Following in vitro stimulation of human CD22 CARs across a range of leukemic antigen densities, we saw that the percentage of CAR+ cells capable of producing IFNγ and IL2 corresponded to target antigen density (p<0.01, p<0.001). As human CARs are commonly manufactured from heterogenous bulk donor T cells, we hypothesized that antigen sensitivity is impacted by the prior antigen-experience of a given T cell. We predicted that T cells which had encountered cognate antigen through their TCR prior to CAR manufacturing (CAR8 AgEx) would have enhanced capacity to respond to low-avidity stimulation compared to CARs manufactured from naïve CD8+ T cells (CAR8 Naïve). We used a well-characterized ovalbumin vaccination model with OT-I TCR-transgenic T cells, allowing defined control of T cell antigen experience, to generate CAR8 AgEx. We found that CAR8 AgEx were highly antigen-sensitive relative to CAR8 Naïve, showing almost no reduction in numbers of cells capable of producing IFNγ and TNFα in vitro against E2A-Ag Lo as compared to E2A-WT. In vivo, CAR8 AgEx showed near complete depletion of E2A-Ag Lo in bone marrow by day 11 post-CAR, while mice treated with CAR8 Naïve maintained a substantial tumor burden (p<0.01). To test our hypothesis in human cells, we manufactured CD22 CAR T cells from naïve (CD45RO-) versus non-naïve (CD45RO+) starting T cell populations, and again found that CAR AgEx outperformed CAR Naïve against Ag Lo leukemia in production of IFNγ and IL2 in vitro (p<0.001, p<0.01) and in early leukemic clearance in vivo (p<0.0001, day 13).In conclusion, we have established a model to study the immunobiology of the CAR T cell response to Ag Lo B-ALL in an intact host. Preliminary findings indicate impaired expansion and tumor clearance of Ag Lo leukemia, associated with altered CAR T cell transcriptional profiles and features of T cell exhaustion. Furthermore, T cell history prior to CAR manufacturing has a drastic impact on the capacity to respond to Ag Lo leukemia. Future studies with this model will expand our characterization of CAR T cells responding to Ag Lo leukemia, with the goal of optimizing antigen sensitivity.We expect that advancing our understanding on the interplay of antigen density and CAR differentiation status will prove useful in developing more effective iterations of this therapy. DisclosuresFry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

Primary Analysis of ZUMA-12: A Phase 2 Study of Axicabtagene Ciloleucel (Axi-Cel) As First-Line Therapy in Patients with High-Risk Large B-Cell Lymphoma (LBCL)

Primary Analysis of ZUMA-12: A Phase 2 Study of Axicabtagene Ciloleucel (Axi-Cel) As First-Line Therapy in Patients with High-Risk Large B-Cell Lymphoma (LBCL)

BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells.

The transcription factors NFAT and AP-1 (Fos-Jun) cooperate to promote the effector functions of T cells, but NFAT in the absence of AP-1 imposes a negative feedback program of T cell hyporesponsiveness (“exhaustion”). Here we show that BATF and IRF4 cooperate to counter T cell exhaustion in mouse tumor models. Overexpression of BATF in CD8+ T cells expressing a chimeric antigen receptor (CAR) promoted the survival and expansion of tumor-infiltrating CAR T cells, increased the production of effector cytokines, decreased the expression of inhibitory receptors and the exhaustion-associated transcription factor TOX, and supported the generation of long-lived memory T cells that controlled tumor recurrence. These responses were dependent on BATF-IRF interaction, since cells expressing a BATF mutant unable to interact with IRF4 did not survive in tumors and did not effectively delay tumor growth. BATF may improve the anti-tumor responses of CAR T cells by skewing their phenotypes and transcriptional profiles away from exhaustion and towards increased effector function.

Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing

With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin’s lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

Intensification of Lymphodepletion Optimizes CAR Re-Treatment Efficacy

▪Background: Post chimeric antigen receptor (CAR) therapy relapse has generally occurred via two mechanisms; emergence of antigen-negative leukemia, or via loss of CAR T cells. Anecdotally, CAR re-treatment has had limited success, potentially due to immune-mediated rejection of CAR T cells, or from immunogenicity of epitopes on murine constructs-which may be less applicable to humanized constructs. After several re-infusion attempts on our phase I dose-escalation study of CD22 CAR, in an effort to improve response, we incorporated use of an intensified lymphodepletion regimen prior to CAR re-infusion and report on our findings. Furthermore, preclinical models suggest that modulation of antigen expression, despite persistent positivity, may also impact the CAR response. Therefore, in this re-treatment analysis, we also explore the role of antigen expression on treatment response.Design: Children and young adults with relapsed/refractory CD22+ acute lymphoblastic leukemia (ALL) enrolled on our phase I dose escalation anti-CD22 CAR therapy protocol (NCT02315612) who met eligibility criteria for a second infusion were evaluated. All patients had bone marrow evaluations by morphology and flow cytometry prior to second infusion to demonstrate persistent CD22+ ALL and loss of CD22 CAR T cells, and subsequently had restaging at day 28 (+/- 4 days) post second CAR infusion. Additionally, lymphodepleting chemotherapy, either with the original dosing schema (Fludarabine 25 mg/m2 x 3 days and Cyclophosphamide 900 mg/m2 x 1 day) or at intensified dosing (Fludarabine 30 mg/m2 x 4 days and Cyclophosphamide 600 mg/m2 x 2 days) was utilized at the discretion of the treating team. All patients were re-infused with their original cell dose; 3 x 105 CAR T cells/kg (n=1), 1 X 106 CAR T cells/kg (n=4), or 3 X 106 CAR T cells/kg (n=1). Flow cytometry was utilized to assess CD22 antigen-binding capacity (ABC) of leukemia cells pre and post infusion. Patients were analyzed for response and expansion of CAR T cells.Results: From June 2015 to July 2017, 6 patients with ALL were eligible for a second infusion of CD22 CAR T cells. This included 4 patients who relapsed with CD22+ disease after attaining a complete remission and 2 patients who had stable disease with limited CAR expansion and evidence of clinical benefit following the first infusion. The median time to relapse after a first infusion was 5 months (range: 3-11 months) with a median time to re-infusion of 4 months (range: 2-11 months) from the first infusion. Prior to re-infusion, all patients had <5% circulating CAR T cells, and all had significant disease burden, > M2 marrow (>5% blasts). Our initial re-infusion experience with 4 subjects, which utilized the identical lymphodepletion regimen as with the first infusion, demonstrated CAR expansion in only 2 patients. Following this, the pre-infusion lymphodepleting chemotherapy was intensified, with 2/2 patients having CAR expansion-including one patient who had a minimal expansion with the initial infusion, but robust expansion following the intensified lymphodepletion prior to second infusion. Both patients tolerated the intensified regimen well without any significant chemotherapy related toxicities. Following re-infusion, 4/6 patients had complete clearance of CD22+ leukemia. Notably, in this cohort, 4/6 patients were found to have a drop in their CD22 ABC, between the time from first to second infusion, with a median drop in site density of 3,141 sites/cell (range: 703-6460) with 2 of the re-infusion non-responders having approximately 50% decrease in the ABC from the CAR pre-treatment value.Conclusion: Our preliminary experience with CAR re-infusions demonstrates that in the presence of preserved antigen expression, re-infusions can be effective, and that an intensified lymphodepletion regimen may optimize re-treatment response, both in patients with limited CAR expansion following a first infusion or with post-CAR relapse. Despite antigen positivity, decreased antigen density post-relapse may be one potential mechanism contributing to lack of CAR efficacy with second infusions, and higher level of antigen density may be needed to optimize anti-tumor response. Future directions will include incorporating an intensified lymphodepletion regimen for all second infusions and exploring methods to enhance antigen expression at the time of re-infusion. DisclosuresNo relevant conflicts of interest to declare.

Marked Re-Expansion of Chimeric Antigen Receptor (CAR) T Cells and Tumor Regression Following Nivolumab Treatment in a Patient Treated with Axicabtagene Ciloleucel (axi-cel; KTE-C19) for Refractory Diffuse Large B Cell Lymphoma (DLBCL)

▪Background: ZUMA-1 is a pivotal, multicenter trial of axi-cel, an autologous anti-CD19 CAR T therapy, for the treatment of patients (pts) with refractory, aggressive non-Hodgkin lymphoma. At the primary analysis, the objective response rate was 82% with a 54% complete response rate; 44% had ongoing responses (Locke et al. ASCO 2017. #7512). Grade ≥ 3 cytokine release syndrome (CRS) and neurologic events (NE) occurred in 13% and 28% of pts, respectively. Programmed cell death-1 (PD-1) is expressed on antigen-activated CAR T cells (Perez et al. ASH 2015. #2042) and checkpoint genes, including programmed cell death-ligand 1 (PD-L1), may be upregulated in the tumor tissue of pts treated with axi-cel (Galon et al. ASCO 2017. #3025). Thus, there is a strong scientific rationale and significant clinical interest in exploring the role of immune checkpoint inhibitors to augment anti-tumor responses of axi-cel. Here we report a case of a pt with rapidly progressing refractory DLBCL whose disease did not respond to axi-cel, who then went on to receive the anti-PD-1 monoclonal antibody nivolumab (nivo) off-study and experienced rapid tumor regression.Methods and Results: A 46-y-old male with germinal center DLBCL without Myc, Bcl-2, or Bcl-6 translocations and with markedly positive PD-L1 expression had lymphoma refractory to R-CHOP, R-ICE, and R-DHAP and was enrolled in ZUMA-1. After lymphodepletion (cyclophosphamide 500 mg/m2 + fludarabine 30 mg/m2 daily × 3 days) and axi-cel infusion, he developed fevers, tachycardia, hypotension, and hypoxia consistent with grade 3 CRS. He received tocilizumab on d 3 with resolution of CRS. Shortly after infusion, the pt developed signs of rapid tumor progression. Abdominal pain, serum LDH, and total bilirubin increased progressively from d 5 to d 9, and repeat CT-PET demonstrated interval progression of abdominal disease relative to pre-infusion imaging. His serum LDH increased from a nadir of 538 U/L on d 4 to 940 U/L on d 10. He developed gastric outlet and recurrent common bile duct obstruction. Due to objective evidence of progressive disease and rapid clinical decline, the pt went to the long-term follow-up portion of the study and was allowed to receive subsequent therapy.On d 11, he received nivo 3 mg/kg. Within 24 h, serum LDH increased from 934 U/L to 1856 U/L and by 3 d after nivo administration, the patient developed grade 3 CRS, grade 1 NE, and marked peripheral lymphocytosis to a peak of 7700 lymphocytes/uL from 2400 lymphocytes/uL before nivo. His abdominal pain improved and oral intake was restored. Remarkably, after 1 cycle of nivo, abdominal CT showed regression of abdominal lymphadenopathy with a 67% reduction in index lesion volume, consistent with partial response. The pt received additional doses of nivo on d 23, 35, and 51. CT scan after 2 additional cycles of nivo confirmed continued disease volume reduction, but the patient's disease progressed on d 64.To determine the contribution of axi-cel on the highly unusual rapid tumor regression following nivo, correlative blood specimens collected before and after nivo on d 7, 13, 27, 51, 65, 90, and 93 and were analyzed for CAR T cells. At day 7 following axi-cel infusion, CAR T cells had expanded to 30 cells/uL, equivalent to the median level measured on d 7 in ZUMA-1 (28 cells/uL). On day 13, two days following nivo administration, dramatic CAR T cell re-expansion was observed, reaching a peak of 1927 CAR T cells/uL, a 64-fold increase above d 7 and a > 150-fold increase over the median d 14 level in ZUMA-1. Levels were elevated at 3 months compared with ZUMA-1 (20 CAR T cells/uL vs median, 0.4 CAR T cell/uL). Additional correlative results including cytokines will be presented.Conclusions:A patient with PD-L1-expressing DLBCL with rapidly progressive disease despite axi-cel treatment experienced a marked secondary expansion of CAR T cells and brisk tumor regression upon subsequent treatment with nivo. This case highlights the possibility that immune checkpoints play a role in preventing response to axi-cel and provides evidence that checkpoint blockade may work synergistically with CAR T cells when delivered in combination with axi-cel treatment. DisclosuresRoberts:Kite Pharma: Employment, Equity Ownership. Rossi:Kite Pharma: Employment, Equity Ownership. Smith:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Morphosys: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Kite Pharma: Research Funding.

Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.

Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients

BackgroundGenetically engineered chimeric antigen receptor (CAR) T lymphocytes are promising therapeutic tools for cancer. Four CAR T cell drugs, including tisagenlecleucel (tisa-cel) and axicabtagene-ciloleucel (axi-cel), all targeting CD19, are currently approved for treating B cell malignancies. Flow cytometry (FC) remains the standard for monitoring CAR T cells using a recombinant biotinylated target protein. Nevertheless, there is a need for additional tools, and the challenge is to develop an easy, relevant, highly sensitive, reproducible, and inexpensive detection method. Molecular tools can meet this need to specifically monitor long-term persistent CAR T cells.MethodsBased on 2 experimental CAR T cell constructs, IL-1RAP and CS1, we designed 2 quantitative digital droplet (ddPCR) PCR assays. By targeting the 4.1BB/CD3z (28BBz) or 28/CD3z (28z) junction area, we demonstrated that PCR assays can be applied to approved CD19 CAR T drugs. Both 28z and 28BBz ddPCR assays allow determination of the average vector copy number (VCN) per cell. We confirmed that the VCN is dependent on the multiplicity of infection and verified that the VCN of our experimental or GMP-like IL-1RAP CAR T cells met the requirement (< 5 VCN/cell) for delivery to the clinical department, similar to approved axi-cel or tisa-cel drugs.Results28BBz and 28z ddPCR assays applied to 2 tumoral (acute myeloid leukemia (AML) or multiple myeloma (MM) xenograft humanized NSG mouse models allowed us to quantify the early expansion (up to day 30) of CAR T cells after injection. Interestingly, following initial expansion, when circulating CAR T cells were challenged with the tumor, we noted a second expansion phase. Investigation of the bone marrow, spleen and lung showed that CAR T cells disseminated more within these tissues in mice previously injected with leukemic cell lines. Finally, circulating CAR T cell ddPCR monitoring of R/R acute lymphoid leukemia or diffuse large B cell lymphoma (n = 10 for tisa-cel and n = 7 for axi-cel) patients treated with both approved CAR T cells allowed detection of early expansion, which was highly correlated with FC, as well as long-term persistence (up to 450 days), while FC failed to detect these events.ConclusionOverall, we designed and validated 2 ddPCR assays allowing routine or preclinical monitoring of early- and long-term circulating approved or experimental CAR T cells, including our own IL-1RAP CAR T cells, which will be evaluated in an upcoming phase I clinical trial.

Phase I experience with first in class TnMUC1 targeted chimeric antigen receptor T-cells in patients with advanced TnMUC1 positive solid tumors.

e14513 Background: MUC1 is a glycoprotein that is expressed in healthy tissues on the luminal surface of simple and glandular epithelium. In tumors that arise from these cells, an alternate form with aberrant glycosylation is frequently over expressed and distinguishes tumor associated TnMUC1 from normal MUC1. We have generated a novel chimeric antigen receptor (CAR) targeting the TnMUC antigen comprised of a mouse anti-human scFv derived from the monoclonal antibody 5E5 which recognizes the epitope comprising Tn glycan of MUC1, a CD8a transmembrane region and dual CD2 and CD3z intracellular signaling domains. CD2 signaling in T-cells has been demonstrated to result in delayed exhaustion. The novel incorporation of this co-stimulatory domain may lead to enhanced persistence of the CART cells which is believed to be critical for efficacy in solid-tumors. Methods: This is a multi-center first in human Phase I study to evaluate the safety and preliminary efficacy of CART-TnMUC1-Cells for the treatment of solid-tumors. Solid-tumors included in the dose-escalation phase include metastatic treatment-resistant ovarian cancer (OC), pancreatic adenocarcinoma (PC), triple-negative breast cancer (TNBC) or non-small lung cancer (NSCLC). All patients must have TnMUC1 expression as determined by immunohistochemistry. Results: As of January 2021, a total of six patients were treated. Three in Cohort 1 (no lymphodepletion; dose = 1-2 x 107 TDN CART cells; tumors = 1 OC, 1 TNBC and 1 PC) and 3 in Cohort 2 (flu/cy lymphodepletion; dose = 1-2 x 107 TDN CART cells; tumors = 1 NSCLC and 2 OC). None of the patients treated experienced DLT’s. The trial is currently enrolling to Cohort 3 (flu/cy lymphodepletion, 5-6 x 107 TDN). No CRS, neurotoxicity, serious adverse reactions and no on-target/off-tumor toxicity was observed at these dose levels. The most common AE’s were low-grade GI symptoms (e.g., nausea, abdominal pain) in 5/6 patients (83.3%), generalized disorders (e.g., chills, fatigue) in 5/6 (83.3%) and hematologic disorders (e.g., anemia, neutropenia) in 3/6 (50%) of patients. CAR expansion was demonstrated in all patients and was improved in Cohort 2 following LD chemotherapy. Preliminary efficacy assessed by RECIST v1.1 at Day +28 demonstrate SD in all patients in Cohort 2. Conclusions: This is the first report of a novel CART-TnMUC1 construct containing a CD2 co-stimulatory domain that has been used in clinical trials for the treatment of refractory solid-tumor malignancies. While the study is still early in dose-escalation having completed only 2 of 6 planned dose levels there is no evidence of safety concerns or on-target/off-tumor toxicity. Additional safety, efficacy and biomarker data is currently being reviewed and will be presented. Clinical trial information: NCT04025216.

BATF and IRF4 cooperate to counter exhaustion in tumour-infiltrating CAR T cells

Abstract Cooperative interactions among transcription factors are essential for gene transcription. We previously showed that NFAT and AP-1 (Fos-Jun) transcription factors cooperate to promote the effector functions of T cells, but that under conditions where it is unable to cooperate with AP-1, NFAT imposes a negative feedback programme of T cell hyporesponsiveness (“exhaustion”). Here we show that BATF and IRF4 cooperate to counter T cell exhaustion. Overexpression of Batf in CD8+ T cells expressing a chimeric antigen receptor (CAR) promoted the survival and expansion of tumour-infiltrating CAR T cells, increased their production of effector cytokines, decreased their expression of inhibitory receptors and the exhaustion-associated transcription factor TOX, and led to the generation of long-lived memory T cells that controlled tumour recurrence. These responses were dependent on the BATF-IRF interaction, since cells expressing a Batf mutant unable to interact with Irf4 did not survive in tumours and did not effectively delay tumour growth. We suggest that BATF overexpression is a therapeutically viable option for improving the anti-tumour responses of CAR TILs, by skewing their phenotypes and transcriptional profiles away from exhaustion and towards increased effector function.

IL-6 trans-signaling promotes the expansion and anti-tumor activity of CAR T cells.

Chimeric antigen receptor (CAR) T cell therapies lead to high clinical response rates in B cell malignancies, and are under investigation for treatment of solid tumors. While high systemic interleukin- (IL-) 6 levels are associated with clinical cytokine release syndrome (CRS), the role of IL-6 trans-signaling within CAR T-cells has not been reported. We generated CAR T cells that constitutively express hyper IL-6 (HIL-6), a designer cytokine that activates the trans-signaling pathway. HIL-6-expressing CAR T-cells exhibited enhanced proliferation and antitumor efficacy in vitro and in xenograft models. However, HIL-6 CAR T cells caused severe graft-versus-host disease (GVHD). Transcriptomic profiling revealed that HIL-6 stimulation of CAR T cells upregulated genes associated with T cell migration, early memory differentiation, and IL-6/GP130/STAT3 signaling. Since IL-6 trans-signaling acts via surface GP130, we generated CAR T cells expressing a constitutively-active form of GP130 and found these retained improved antitumor activity without signs of GVHD in preclinical models of B-cell leukemia and solid tumors. Taken together, these results show that IL-6 trans-signaling can enhance expansion and antitumor activity of CAR T cells via the GP130/STAT3 pathway, and suggest that expression of GP130 within CAR T cells could lead to improved antitumor efficacy without systemic IL-6 trans-signaling.

A Novel Method for Molecular Enumeration of Circulating Tisa-Cel and Axi-Cel in Lymphoma Patients

Background: Chimeric antigen receptor (CAR) T-cell therapy is an effective treatment in approximately 40% of relapsed/refractory lymphomas. High rates of complete remission (CR) observed in clinical trials, led to fast-track FDA and EMA approval and commercialization of two CD19-directed CAR T-cell products (tisagenlecleucel/Tisa-cel/Kymriah and axicabtagene ciloleucel/Axi-cel/Yescarta) for refractory/relapsed diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). In these studies, CAR T-cell engraftment and in vivo expansion have a crucial impact on disease response and toxicity. It is of paramount importance to develop homogeneous standardized approaches for monitoring expansion and persistence of CAR T-cells also in the case of cells not detectable by flow cytometry (FCM) as these data can guide clinical decision making. Aims of this study were: to assess relevant time-points for FCM analysis across the different products to develop a unique quantitative PCR assay to detect both tisa-cel and axi-cel. Methods: We prospectively collected samples from 28 patients (pts) (16 DLBCL, 5 transformed follicular lymphoma and 7 PMBCL) treated with axi-cel (n=15) and tisa-cel (n=13) at the Fondazione IRCCS Istituto Nazionale Tumori between Nov 2019 and July 2020. CAR T-cells were monitored in the peripheral blood (PB) on days 0, 4, 7, 9, 14, 21, 28 and monthly post infusion by FCM. Cells were stained with CD19 CAR Detection Reagent (Miltenyi), Anti-Biotin-PE, CD3-FITC, CD4-VioGreen, CD8-APC-Vio770, CD45-VioBlue, CD14-APC and 7-AAD Staining Solution and analyzed by FCM using the MACSQuant® Analyzer (Miltenyi). To evaluate CAR T-cell persistence, we also generated a unique droplet digital PCR (ddPCR) primer-probe assay to target both tisagen-cel and axi-cel. We obtained a partial cDNA sequence of the two CAR constructs by amplification and direct sequencing of fragments generated with primers designed by educated guess to bind to the anti-CD19 murine single-chain variable fragment (scFV) and to the CD8α or CD28 trans-membrane regions of tisagen-cel and axi-cel vectors respectively. The assay was used to quantify CAR+ cells on genomic DNA extracted from PB cells collected serially after infusion. FCM and molecular data were correlated to outcome. Results: Peak expansion of CAR T-cells by FCM occurred within the first 14 days post-infusion. At the point of maximal expansion, collectively a mean of 17% of circulating CD3+ T cells were CAR+ and CAR+ cells were more abundant among CD3+ cells in pts receiving axi-cel (26% vs 8.5% for tisagen-cel, p&amp;lt;0.05). Patients achieving CR at 1 month from CAR T-cell therapy (n=10) had significantly more CAR+ cells at day 9 than non-CR pts (mean 146 vs 18 CAR+ cells/ul, p&amp;lt;0.05) and their magnitude of expansion in the first 14 days was higher than the one of non-CR pts (mean area under the curve (AUC)= 1110 vs 177; p&amp;lt;0.05). A cut-off value of CAR+ cells at day 9&amp;gt;62/ul was prognostic with sensitivity of 67% and specificity of 76% (Receiver operating characteristic analysis). At subsequent time points the number of CAR T-cells decreased, but continued to be detectable by FCM up to 6 months post infusion although at very low levels (mean 2 CAR+ cell/ul) irrespective of outcome. To evaluate whether these low counts reliably represent CAR T+ cells or are rather signals due to aspecific binding of the antibodies, a ddPCR assay was set up. The assay displayed an excellent separation between positive and negative droplets on both axi-cel and tisagen-cel products, good reproducibility and specificity with max standard deviation of 0.06 and no CAR positive signal in healthy donors. Additionally, the CAR specific signal was detectable up to the 0,01% dilution. FCM and ddPCR data had a good correlation (r=0.93, p&amp;lt;0.001): in accordance with FCM, ddPCR revealed a peak expansion of CAR+ signals at day 9 post infusion, followed by a progressive reduction of the CAR+ signal over time in all pts that becomes undetectable in case of relapse despite the presence of 1-2 cells/ul by FCM that probably represent aspecific findings. Conclusion: The combined use of FCM and the novel molecular assay facilitates the precise enumeration of commercial anti-CD19 CAR T-cells. Higher frequencies of CAR+ T cells at day 9 distinguish responders irrespectively of the product they received in our small prospective series. Updated results will be presented. Disclosures Chiappella: Servier: Honoraria; Roche: Honoraria; Takeda: Honoraria; Iqone: Honoraria; Janssen: Honoraria; Gilead-Kite: Honoraria; Celgene: Honoraria. Corradini:Kite: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Incyte: Consultancy; BMS: Other; Takeda: Consultancy, Honoraria, Other; Servier: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; F. Hoffman-La Roche Ltd: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Travel and accommodations paid by for; KiowaKirin: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel and accommodations paid by for; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Gilead: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Daiichi Sankyo: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Other: Travel and accommodations paid by for.

Inducible secretion of IL-21 augments anti-tumor activity of piggyBac-manufactured chimeric antigen receptor T cells

BackgroundThe efficiency of chimeric antigen receptor (CAR) T-cell-based therapies depends on a sufficient expansion of CAR T cells in vivo and can be weakened by intra-tumoral suppression of CAR T cell functions, leading to a failure of therapy. For example, certain B-cell malignancies such as chronic lymphocytic leukemia are weakly sensitive to treatment with CAR T cells. Co-expression of proinflamatory cytokines such as IL-12 and IL-18 by CAR T cells have been shown to enhance their antitumor function. We similarly engineered CAR T cell to co-express IL-21 and studied the effects of IL-21 on CAR T cells specific to CD19 and prostate-specific membrane antigens using an in vitro co-culture model and NSG mice transplanted with B-cell tumors. ResultsIL-21 enhanced the expansion of CAR T cells after antigenic stimulation, reduced the level of apoptosis of CAR T cells during co-culture with tumor cells and prevented differentiation of CAR T cells toward late memory phenotypes. In addition, induced secretion of IL-21 by CAR T cells promoted tumor infiltration by CD19-specific CAR (CAR19) T cells in NSG mice, resulting in reduced tumor growth. By co-culturing CAR19 T cells with bone-marrow fragments infiltrated with CLL cells we demonstrate that IL-21 reduces the immunosupressive activity of CLL cells against CAR19 T cells. ConclusionsCAR19 T cells armed with IL-21 exhibited enhanced antitumor functions. IL-21 promoted their proliferation and cytotoxicity against chronic lymphocytic leukemia (CLL). The results suggest that arming CAR T cells with IL-21 could boost the effectiveness of CAR T-mediated therapies.

4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling.

Clinical response to chimeric antigen receptor (CAR) T cell therapy is correlated with CAR T cell persistence, especially for CAR T cells that target CD19+ hematologic malignancies. 4-1BB-costimulated CAR (BBζ) T cells exhibit longer persistence after adoptive transfer than do CD28-costimulated CAR (28ζ) T cells. 4-1BB signaling improves T cell persistence even in the context of 28ζ CAR activation, which indicates distinct prosurvival signals mediated by the 4-1BB cytoplasmic domain. To specifically study signal transduction by CARs, we developed a cell-free, ligand-based activation and ex vivo culture system for CD19-specific CAR T cells. We observed greater ex vivo survival and subsequent expansion of BBζ CAR T cells when compared to 28ζ CAR T cells. We showed that only BBζ CARs activated noncanonical nuclear factor κB (ncNF-κB) signaling in T cells basally and that the anti-CD19 BBζ CAR further enhanced ncNF-κB signaling after ligand engagement. Reducing ncNF-κB signaling reduced the expansion and survival of anti-CD19 BBζ T cells and was associated with a substantial increase in the abundance of the most pro-apoptotic isoforms of Bim. Although our findings do not exclude the importance of other signaling differences between BBζ and 28ζ CARs, they demonstrate the necessary and nonredundant role of ncNF-κB signaling in promoting the survival of BBζ CAR T cells, which likely underlies the engraftment persistence observed with this CAR design.