Murine Chimeric Antigen Receptor Research Articles

Abstract LB068: Repurposing chimeric antigen receptor regulatory T cells for treating cancer

Abstract Solid tumors are the deadliest form of cancer. Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has led to remarkable regression rates in leukemia and lymphoma. However, solid tumors have remained refractory to CAR T-cell therapy, with CAR T cells either failing to penetrate the solid tumor microenvironment (TME) or becoming exhausted once in it. Mechanisms behind these properties of the TME include nutrient scarcity, aberrant vascularization, stromal cell deposition, expression of inhibitory immune receptors by cancer cells, and the presence of immunosuppressive cells. Interestingly, there is a subset of T cells found in the TME - regulatory T cells (Tregs). Dedicated to suppressing immune responses, Tregs constitute a barrier to anticancer immunity by migrating to and heavily accumulating in solid tumors, inhibiting local immunity. Tregs can be redirected towards any cell surface antigen using a CAR and have been shown to be cytotoxic towards target antigen presenting cells. Can Tregs be converted from tumor protectors to anti-tumor effector cells? To test the anti-tumor potential of CAR Tregs, we modified human Tregs with an anti-CD19 CD28-CD3z CAR. Tregs activated via the CAR or endogenous TCR/CD28 were evaluated at the functional and gene expression levels. CAR and endogenous TCR/CD28 activation resulted in similar cell expansion and Treg stability (FOXP3 and HELIOS expression). However, CAR-mediated activation markedly increased Treg proinflammatory cytokine production. Moreover, compared to their TCR/CD28-activated counterparts, CAR Tregs displayed increased cytotoxic activity towards tumor cells and a diminished capacity to modulate antigen-presenting cells and suppress T cell proliferation, suggesting a global shift from suppression to inflammation. Strikingly, CAR Tregs suppressed tumor growth in immunodeficient mice inoculated with antigen-expressing B-cell leukemia, myeloid leukemia, and lung epithelial carcinoma. Single-cell RNA sequencing identified a cell cluster enriched in CAR Tregs characterized by high expression of effector T cell genes, such as IFNG, GZMB, and CD40LG, revealing that CAR activation creates a highly active population of proinflammatory Tregs. Multiplex cytokine analysis, whole-transcriptome RNA-seq, and intracellular cytokine staining confirmed that IFNG production by FOXP3+ Tregs is induced specifically by CAR activation. Of note, murine CAR Tregs are also cytotoxic towards tumor cells. Current experiments are focused on pinpointing and validating transcription factors and surface markers characteristic of proinflammatory CAR Tregs and further assessing CAR Treg antitumor efficacy in vivo. In conclusion, CAR-mediated activation induces a novel proinflammatory Treg phenotype that can be exploited in the next generation of engineered immune cell therapies against solid tumors. Citation Format: Leonardo M. Ferreira, Russell W. Cochrane, Rob A. Robino, Bryan Granger, Eva Allen, Vishnu Gudivada, Aguirre A. de Cubas, Stefano Berto. Repurposing chimeric antigen receptor regulatory T cells for treating cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB068.

Mutation-specific CAR T cells as precision therapy for IGLV3-21R110 expressing high-risk chronic lymphocytic leukemia.

The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21R110) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells.

Mucin-1-Targeted Chimeric Antigen Receptor T Cells Are Effective and Safe in Controlling Solid Tumors in Immunocompetent Host.

The chimeric antigen receptor (CAR) T-cell therapy in solid epithelial tumors has been explored, however, with limited success. As much of the preclinical work has relied on xenograft models in immunocompromised animals, the immune-related efficacies and toxicities may have been missed. In this study, we engineered syngeneic murine CAR T cells targeting the tumor form of human mucin-1 (tMUC1) and tested the MUC1 CAR T cells' efficacy and toxicity in the immunocompetent human MUC1-expressing mouse models. The MUC1 CAR T cells significantly eliminated murine pancreatic and breast cancer cell lines in vitro. In vivo, MUC1 CAR T cells significantly slowed the mammary gland tumor progression in the spontaneous PyVMT×MUC1.Tg (MMT) mice, prevented lung metastasis, and prolonged survival. Most importantly, there was minimal short or long-term toxicity with acceptable levels of transient liver toxicity but no kidney toxicity. In addition, the mice did not show any signs of weight loss or other behavioral changes with the treatment. We also report that a single dose of MUC1 CAR T-cell treatment modestly reduced the pancreatic tumor burden in a syngeneic orthotopic model of pancreatic ductal adenocarcinoma given at late stage of an established tumor. Taken together, these findings suggested the further development of tMUC1-targeted CAR T cells as an effective and relatively safe treatment modality for various tMUC1-expressing solid tumors.

Modulation of the Btla-HVEM Axis to Enhance CAR T Cell Immunotherapy Against Cancer

Introduction: The efficacy of adoptive T cell immunotherapies against cancer, such as chimeric antigen receptor (CAR) T cells, is severely blunted by the immunosuppressive tumor microenvironment (TME). We sought to investigate the role of the TME in cancer resistance to effector T cells in order to define actionable targets to enhance CAR T cell immunotherapies. We initially used Hodgkin lymphoma (HL) as an ideal tumor model since it is characterized by a TME that is profoundly infiltrated by immunosuppressive cells, and then we expanded our findings to multiple cancer models. We first sought to identify the dominant interactions of immunosuppressive cellular compartments and effector T cells by analyzing single-cell RNA sequencing data on a total of 26 (4 exploratory + 22 validation) HL patient tumor biopsies. Using the CellPhoneDB algorithm, we inferred that the ligand B- and T-lymphocyte attenuator (BTLA) on effector T cells and the receptor Herpesvirus entry mediator (HVEM, TNFRSF14) on immunosuppressive cells (e.g., regulatory T cells, monocytes) strongly interact in the TME and promote T cell dysfunction ( Fig 1a). Akin to the canonical checkpoint PD1, BTLA recruits two potent tyrosine phosphatases, SHP-1 and SHP-2, to disable early T cell activation. Thus, we rationalized that BTLA expression on T cells might reduce their anti-tumor function. We hypothesized that deleting BTLA in CAR T cells would abolish BTLA-HVEM trans interactions at the immunological synapse and unleash the cytotoxic potential of CAR T cells. Methods and Results: We first generated BTLA KO anti-CD30 CAR T (CART30) cells against HL. To test their function in vivo, 15x10 6 HDLM-2 (CD30+HVEM+ HL) cells were subcutaneously implanted into NSG mice on day 0, and on day 62, 3x10 5 CAR30+ T cells were infused intravenously. Our results demonstrated that BTLA KO significantly enhances the function of anti-CD30 CAR T cells in HVEM+ HL ( Fig 1b), as assessed via tumor size (caliper) and CART30 expansion in the peripheral blood (flow cytometry). Additionally, we generated BTLA KO 4-1BBζ CART19 cells, which showed greater in vivo anti-tumor function in a subcutaneous tumor model of DLBCL (CD19+ HVEM+ OCI-Ly18). Serum collected from OCI-Ly18-bearing NSG mice infused with BTLA KO CART19 was enriched in effector cytokines (e.g., TNF, IFNγ, IL-2) as measured by Luminex. We then extended these findings into HVEM+ solid tumor models. BTLA KO improved tumor control in vitro in short-term killing experiments (Incucyte SX5) for both CAR and TCR T cells, respectively directed against prostate cancer (HER2+ PC-3) and melanoma (GP-100+ DM-6). Importantly, we demonstrated that BTLA KO in primary BALB/c-derived murine CAR T cells enhances tumor control in A20 murine lymphoma. In this model, BTLA KO tumor-infiltrating CAR T cells showed substantially reduced exhaustion in the TME relative to wild-type CAR T cells. Mechanistically, we showed that targeted mutations in BTLA intracellular tyrosine motifs effectively reduce the recruitment of SHP-1/2 and preserve the docking domain of the pro-stimulatory Grb2, thus maintaining high NFAT signaling and increased persistence in vivo. These effects were dependent on the expression of HVEM on tumor cells. Finally, we found that high BTLA RNA expression in tisagenlecleucel infusion products correlates with poor response to treatment in patients with DLBCL and FL (NCT02030834). Conclusion: Our results reveal a critical role of the BTLA-HVEM axis in inhibiting CAR T cell function, and demonstrate that CRISPR-Cas9 deletion of BTLA leads to enhanced anti-tumor efficacy in multiple models of cancer. The key mechanism is the reduction of SHP-1/2 recruitment and the consequent increase in CAR T cell activation. The results of this study will be translated into a first in human clinical trial of BTLA-deficient CAR T cells for relapsed or refractory cancer.

Bcl11b-Edited Lymphoid Progenitors for the Generation of CAR-Engineered Natural Killer-like Cells with Potent Anti-Leukemic Activity

BACKGROUND: The transcription factor B cell CLL/lymphoma 11B (BCL11B) is indispensable for T lineage development of lymphoid progenitors. Tonic signaling of chimeric antigen receptors (CARs) during early phases of in vitro generation of hematopoietic stem cell (HSC)-derived lymphoid progenitors leads to suppressed T cell development and acquisition of NK cell-like properties (CAR-induced killer cells, termed CARiK cells) by suppression of BCL11B. Co-transplantation of CARiK cells into hematopoietic stem cell recipients mediates strong antigen-specific anti-leukemic effects. Here we investigated whether CRISPR/Cas9-based editing of Bcl11b would allow the generation of CARiK cells using clinically more relevant CARs w/o tonic signaling activity. METHODS: Antigen binders against murine CD123 were identified using phage display technology. High affinity binders were cloned into a 2nd generation murine CAR backbone (CD28CD3zeta configuration) and comparatively assessed for expression strength, functionality, specificity, and the lack of tonic signaling activity. Aiming for an all in one vector approach, a sgRNA sequence against Bcl11b was added to the construct for CRISPR-mediated gene disruption. Transduced murine LSK cells were then differentiated into lymphoid CARiK progenitors using the OP9-DLI feeder layer system. Double edited progenitors (8 x 106) and respective controls were co-transplanted into leukemia-bearing murine stem cell recipients. BCL11B suppression was assessed by Western-blotting, correlated with the appearance of a CARiK phenotype, and finally tested for in vivo functionality. RESULTS: A suitable CD123 CAR of high expression strength, specificity, and devoid of tonic signaling was generated. Despite the lack of tonic signaling, early stimulation via CD123 in culture was sufficient for some degree of NK-directed developmental shift. The degree of fratricide was limited. Early Bcl11b disruption allowed for further lymphoid cell differentiation. Bcl11b editing only let to complete BCL11B suppression as did the combined all in one vector construct (Figure 1A). CAR transduction w/o Bcl11b editing resulted in incomplete BCL11B suppression. After transduction with the lentiviral all in one construct, the strongest developmental NK cell shift was observed (Figure 1B). After co-transplantation of CARiK cells up to 100% leukemia free survival was achieved. Low expression strength of CD123 on physiologic hematopoiesis allowed for undisturbed recovery of myelopoiesis as compared to controls. CONCLUSION:Bcl11b editing of hematopoietic stem cell-derived CAR-engineered lymphoid progenitors allows the generation of a NK cell-like cell product with considerable anti-tumor activity. Genetic stability remains to be determined. Figure 1: Natural killer-like cell differentiation of HSC-derived mCD123 CAR-engineered lymphoid progenitors can be enforced by Bcl11b editing. (A) Partial suppression of BCL11B by early CAR-signaling and finally complete disruption by Bcl11b editing as shown by Western Blotting (iTom = neg. control). (B) The extent of BCL11B suppression correlates positively with the amount of NKp46+ cells within the CAR-expressing gate in flow cytometry analysis (n=3). (B) Statistics was performed using Student's t test (2 tailed). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fully murine CD105-targeted CAR-T cells provide an immunocompetent model for CAR-T cell biology

ABSTRACT The modeling of chimeric antigen receptor (CAR) T cell therapies has been mostly focused on immunodeficient models. However, there are many advantages in studying CAR-T cell biology in an immunocompetent setting. We generated a fully murine CAR targeting CD105 (endoglin), a component of the TGFβ receptor expressed on the surface of certain solid tumors and acute leukemias. CD105-targeted CAR-T cells can be grown from various murine backgrounds, tracked in vivo by congenic marks, and be activated by CD105 in isolation or expressed by tumor cells. CD105-targeted CAR-T cells were toxic at higher doses but proved safe in lower doses and modestly effective in treating wild-type B16 melanoma-bearing mice. CAR-T cells infiltrating the tumor expressed high levels of exhaustion markers and exhibited metabolic insufficiencies. We also generated a human CD105 CAR, which was efficacious in treating human melanoma and acute myeloid leukemia in vivo. Our work details a new murine model of CAR-T cell therapy that can be used from immunologists to further our understanding of CAR-T cell biology. We also set the foundation for further exploration of CD105 as a possible human CAR-T cell target.

Murine allogeneic CAR T cells integrated before or early after posttransplant cyclophosphamide exert antitumor effects

AbstractRelapse limits the therapeutic efficacy both of chimeric antigen receptor (CAR) T cells and allogeneic hematopoietic cell transplantation (allo-HCT). Patients may undergo these therapies sequentially to prevent or treat relapsed malignancy. However, direct integration of the 2 therapies has been avoided over concerns for potential induction of graft-versus-host disease (GVHD) by allogeneic CAR T cells. We have shown in murine T-cell-replete MHC-haploidentical allo-HCT that suppressive mechanisms induced immediately after posttransplant cyclophosphamide (PTCy), given on days +3/+4, prevent GVHD induction by alloreactive T cells infused as early as day +5. Therefore, we hypothesized that allogeneic CAR T cells given in a similarly integrated manner in our murine MHC-haploidentical allo-HCT model may safely exert antitumor effects. Indeed, allogeneic anti-CD19 CAR T cells given early after (day +5) PTCy or even prior to (day 0) PTCy cleared leukemia without exacerbating the cytokine release syndrome occurring from the MHC-haploidentical allo-HCT or interfering with PTCy-mediated GVHD prevention. Meanwhile, CAR T-cell treatment on day +9 or day +14 was safe but less effective, suggesting a limited therapeutic window. CAR T cells infused before PTCy were not eliminated, but surviving CAR T cells continued to proliferate highly and expand despite PTCy. In comparison with infusion on day +5, CAR T-cell infusion on day 0 demonstrated superior clinical efficacy associated with earlier CAR T-cell expansion, higher phenotypic CAR T-cell activation, less CD4+CD25+Foxp3+ CAR T-cell recovery, and transcriptional changes suggesting increased activation of CD4+ CAR T cells and more cytotoxic CD8+ CAR T cells. This study provides mechanistic insight into PTCy’s impact on graft-versus-tumor immunity and describes novel approaches to integrate CAR T cells and allo-HCT that may compensate for deficiencies of each individual approach.

A long-acting interleukin-7, rhIL-7-hyFc, enhances CAR T cell expansion, persistence, and anti-tumor activity

Chimeric antigen receptor (CAR) T cell therapy is routinely used to treat patients with refractory hematologic malignancies. However, a significant proportion of patients experience suboptimal CAR T cell cytotoxicity and persistence that can permit tumor cell escape and disease relapse. Here we show that a prototype pro-lymphoid growth factor is able to enhance CAR T cell efficacy. We demonstrate that a long-acting form of recombinant human interleukin-7 (IL-7) fused with hybrid Fc (rhIL-7-hyFc) promotes proliferation, persistence and cytotoxicity of human CAR T cells in xenogeneic mouse models, and murine CAR T cells in syngeneic mouse models, resulting in long-term tumor-free survival. Thus, rhIL-7-hyFc represents a tunable clinic-ready adjuvant for improving suboptimal CAR T cell activity.

Successful Treatment with CAR- T Cells in a Patient with Immune Thrombocytopenia Associated with Castleman Disease

Multicentric Castleman disease (MCD), a rare lymphoproliferative disorder of incompletely understood etiology, is manifested as enlarged multiple lymph node and multiple organs involvement. The best therapeutic option for this disease remains unknown. A 21-year-old male patient of MCD with swilling lymph nodes received combination chemotherapy treatment with unsatisfactory efficacy. He developed thrombocytopenia during disease progression and failed steroid treatment. His platelet increased to normal level on day 27 of receiving infusion of humanised anti-CD19 chimeric antigen receptor (CAR) T cells (1 × 10⁶ cells/kg) and murine anti-BCMA CAR T cells (0.6 × 10⁶ cells/kg). He has a stable platelet count and no evidence of progression of lymphadenopathy for 45 months at this writing. This case report reminds clinicians to be aware of the potential clinical value of CAR T cells therapy in MCD.

Secondary Humanized CAR T Cells Salvage Therapy Improves Survival of R/R DLBCL Patients Failed to Murine CD19-CAR T Cells Therapy

Background: The Chimeric antigen receptor (CAR) T-cell therapy has shown very promising future in relapsed/refractory diffused large B cell lymphoma (R/R DLBCL) patients resisted to second-line chemotherapy and failed to auto hematopoietic cell transplantation. Trials with murine CD19-CAR T-cell therapy have shown only 40%-54% complete remission (CR) rate in patients with R/R DLBCL. However, 30%-60% of patients with previous CR relapse within 1 year. It is critical to develop new strategies to improve survival of R/R DLBCL patients failed to murine CD19-CAR T cells therapy. Forty-two R/R DLBCL patients failed to murine CD19-CAR T cells therapy who accepted secondary humanized CAR T cells salvage therapy in our center were recruited to this studyAim: 1. Investigate the effect and safety of Secondary humanized CAR T cells salvage therapy in R/R DLBCL patients failed to murine CD19-CAR T cells therapy. 2. Analyze the influence factor of curative effect of secondary humanized CAR T cells, to predict the outcome of treatment.Method: We retrospectively analyzed 42 cases treated with secondary humanized CAR T cells salvage therapy after failing to murine CD19-CAR T cells therapy in our center, enrolled from 05/01/2018 to 05/01/2020. According to the new result of tumor immunohistochemical analysis, five patients still chose to target CD19, twenty-one targeted CD20, and sixteen targeted CD22. Pretreatment regimen before infusion of CAR T cells were chemotherapy comprising fludarabine (three doses of 30 mg/m2 daily) and cyclophosphamide (three doses of 300mg/m2 daily). The T-cells with specific CAR expression in peripheral blood were dynamically detected by flow cytometry. After CAR T-cells infusion, PET-CT were performed every 3 months to evaluate the state of disease. Patients were followed up till 06/15/2021Results: The incidence of cytokine release syndrome (CRS) was 84%, of which 22% was severe CRS (≥grade 3), as the incidence of immune effector cells associated neurologic toxicity (ICANS) was 7.14%, of which 2.83% was severe ICANS. The incidence of target organ damage was 12%. The rate of treatment-related mortality was 7%. In 3 months, complete remission rate was 26.2%, partial remission rate (PR) was 33.3% and the overall response rate (ORR) was 59.5%. Ten patients (26.2%) remained in complete remission at the cutoff date. The median of progression-free survival (PFS) length was 4.42 months (95%CI: 1.87-6.02). The median of overall survival (OS) length was 9.24 months (95%CI: 4.44-~). The 1-year overall survival rate was 38.1%. We also found high level of LDH, heavy tumor burden, no less than 4 IPI score, and double expression of MYC and BCL2 were risk factors of OS, PFS and CR. The patients who had achieved CR or PR after murine CD19-CAR T cells therapy had a favorable OS and PFS. The longer the patients had PFS during murine CD19-CAR T cells therapy, the longer PFS and OS during humanized CAR T cells therapy.Conclusion: 1. Secondary humanized CAR T cells salvage therapy improves survival of R/R DLBCL patients failed to murine CD19-CAR T cells therapy. 2. The incidence of severe CRS and treatment-related causality were relatively low. 3. The patients who got better curative effect during murine CAR T cells therapy prone to a longer survival time.Keywords: Secondary humanized CAR T cells therapy, murine CAR T cells therapy, R/R DLBCL, CRS, ICANS. DisclosuresNo relevant conflicts of interest to declare.

Overexpression of Rorγt in CAR T Cells Improves Persistence and Reduces Exhaustion

Although T cells modified with a chimeric antigen receptor (CAR) are highly effective, durability of remissions may be limited by the poor persistence of adoptively transferred CAR T cells in patients. Growing evidence supports the idea that the pre-infusion phenotype of CAR T cells affects the efficacy of CAR T cell therapies, in part due to the increased ability of certain subsets of CAR T cells to persist long-term. Studies using conventional T cells have suggested that adoptive transfer of T cells programmed to have Th17/Tc17 effector functions can increase the anti-tumor efficacy and persistence of transferred cells when compared to adoptive transfer of non-Th17/Tc17 cells. However, standard CAR formats currently in clinical use that contain either a CD28 or 4-1BB costimulatory domain fail to generate substantial numbers of Th17/Tc17-like cells. Thus, few studies have investigated whether Th17/Tc17 CAR T cells could enhance the efficacy of CAR T cell therapies.We generated a multi-cistronic lentiviral construct encoding ROR-related Orphan Receptor gamma (RORγt), a key lineage-specifying transcription factor involved in Th17/Tc17 differentiation, followed by a murine CAR targeting CD19 that contains a CD28 costimulatory domain (1928z). RORγt expression did not hinder degranulation in response to CAR antigen upon co-culture with E2aPbx, a murine acute lymphoblastic leukemia cell line. Significantly higher numbers of RORγt-1928z CAR T cells produce IL-17 compared to 1928z CAR T cells (p<0.0001) suggesting that RORγt expression is sufficient to program type-17 effector functions in CAR T cells.To determine the impact of RORγt overexpression on CAR T cell phenotype and persistence in vivo, we adoptively transferred 1x10 6 1928z or RORγt-1928z CAR T cells into leukemia-bearing C57Bl6/J recipients and analyzed CAR T cells from the bone marrow at days 4, 11, and 31. As early as day 4 post-transfer, RORγt-1928z CAR T cells were present at significantly higher numbers than 1928z CAR T cells, and remained higher through Day 31 (0.16% of the bone marrow for 1928z CAR T cells vs 0.84% of the bone marrow for RORγt-1928z CARs; p= 0.0012, n=5 mice per group. The RORγt-1928z CAR T cells were enriched for an effector/effector memory phenotype (CD62L-) compared to 1928z CAR T cells, which were mostly of a central memory phenotype (CD62L+). Interestingly, the RORγt-1928z CAR T cells were enriched for IL-7Rα+ cells and expressed more IL7Rα than their 1928z CAR T cell counterparts (p=0.0012), suggesting an increased capacity for self-renewal in RORγt-1928z CAR T cells.Previous work from our lab has demonstrated that CAR T cells that persist after leukemia clearance have an exhausted phenotype, with increased expression of PD1 and a reduced functionality 1. After leukemia clearance, RORγt-1928z CAR T cells expressed lower levels of PD1 than their 1928z counterparts (p<0.0001). Finally, 1928z CAR T cells form a population of Eomes hiPD1 hi cells, associated with terminal exhaustion, that is absent in the RORγt-1928z CAR T cells.Taken together, our preliminary data demonstrates that overexpression of RORγt is sufficient to program type-17 effector functions in CAR T cells and suggests that RORγt overexpression can enhance the ability of CAR T cells to persist and self-renew, and prevent terminal exhaustion. RORγt overexpression may enhance persistence of CAR T cells and durability of remissions in hematologic malignancies. Furthermore, RORγt overexpression may also be a strategy to enhance CAR T cell efficacy against solid tumors, where an immunosuppressive microenvironment contributes to T cell dysfunction.1.Yinmeng Yang, Mark Eric Kohler, Terry J. Fry; Effect of Chronic Endogenous Antigen Stimulation on CAR T Cell Persistence and Memory Formation. Blood2017; 130 (Supplement 1): 166. doi: https://doi.org/10.1182/blood.V130.Suppl_1.166.16 DisclosuresFry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

Allogeneic CAR Invariant Natural Killer T Cells Exert Potent Antitumor Effects through Host CD8 T-Cell Cross-Priming.

The development of allogeneic chimeric antigen receptor (CAR) T-cell therapies for off-the-shelf use is a major goal that faces two main immunologic challenges, namely the risk of graft-versus-host disease (GvHD) induction by the transferred cells and the rejection by the host immune system limiting their persistence. In this work we assessed the direct and indirect antitumor effect of allogeneic CAR-engineered invariant natural killer T (iNKT) cells, a cell population without GvHD-induction potential that displays immunomodulatory properties. After assessing murine CAR iNKT cells direct antitumor effects in vitro and in vivo, we employed an immunocompetent mouse model of B-cell lymphoma to assess the interaction between allogeneic CAR iNKT cells and endogenous immune cells. We demonstrate that allogeneic CAR iNKT cells exerted potent direct and indirect antitumor activity when administered across major MHC barriers by inducing tumor-specific antitumor immunity through host CD8 T-cell cross-priming. In addition to their known direct cytotoxic effect, allogeneic CAR iNKT cells induce host CD8 T-cell antitumor responses, resulting in a potent antitumor effect lasting longer than the physical persistence of the allogeneic cells. The utilization of off-the-shelf allogeneic CAR iNKT cells could meet significant unmet needs in the clinic.

B-Cell Maturation Antigen (BCMA)-Specific, CentyrinTM-Based, PiggyBacTM-Transposed CAR-T Memory Stem Cells Are Effective Against p53-/- and Patient-Derived Multiple Myeloma Tumors

B-Cell Maturation Antigen (BCMA)-Specific, CentyrinTM-Based, PiggyBacTM-Transposed CAR-T Memory Stem Cells Are Effective Against p53-/- and Patient-Derived Multiple Myeloma Tumors

Co-Transplantation of CAR-Gene Engineered Precursor T Cells Leads to Suppressed T Cell Development and Increased NK Cell-Based CAR-Mediated Anti-Leukemic Activity

BACKGROUND: Production of chimeric antigen receptor (CAR) -engineered T cells is currently done for each patient individually and relies on a patient-derived T cell product of heterogeneous quality. On target/off tumor effects represent a major obstacle for a broader application of CARs. Broader application of this powerful therapeutic tool is hampered by limited production capacity and the non-desired sequelae from CAR-derived on target/off tumor effects. Here we investigated whether precursor T cells (preTs) engineered to express a clinically relevant CAR can potentially address some of these limitations.METHODS: Murine LSK cells were transduced with a 2nd generation murine CAR (CD28CD3zeta configuration) against CD19 in a lentiviral backbone and consecutively differentiated into preTs using the OP9-DL1 feeder layer system. CAR-engineered preTs (8 x 106) of MHC matched (B6/NCrl) or mismatched origin (B10.A) were co-transplanted with syngeneic T-cell-depleted bone marrow into irradiated B6 recipients. Mice were challenged with a lethal dose of CD19+ murine leukemia cells 20 days after adoptive transfer and assessed for B cell recovery and survival. For in vivo NK cell depletion studies, the anti-NK1.1 antibody (clone: PK136) was used. Sorted CAR preTs previous to co-transfer and after in vivo maturation were analyzed by microarray technology.RESULTS: Afterco-transplantation of in vitro generated CAR-engineered preTs up to 60% (p<0.001) and 100% (p<0.0001) leukemia free survival was achieved in fully MHC-mismatched or MHC-matched recipients respectively (Figure 1A). No graft-versus-host disease was induced. Anti-leukemic effects entirely relied on the in vivo development of CAR-expressing natural killer (NK) cells since NK cell depletion after hematopoietic stem cell transplantation (HCT) resulted in a complete abrogation of anti-leukemic effects (Figure 1B, p<0.001). As shown by gene expressing profiling enforced CAR-expression on preTs fosters transcriptional key factors being associated with NK cell development such as Nfil3, Id2, and Eomes. CAR-expression on co-transplanted preTs blocked thymic seeding completely and prevented further T-cell development in vivo. In contrast, CAR-expression favored NK cell development with strong anti-leukemic effects when compared to signaling-defective CAR preTs early after adoptive transfer of engineered preTs (Figure 1C, p<0.001). CAR-bearing NK-cells persisted for up to 60 days post-transplant allowing for complete B-cell recovery thereafter.CONCLUSION: Co-transplantation of CAR-engineered preTs is of high clinical relevance since they can be prefabricated and frozen as a “off the shelf product”. Limited persistence of their NK-progenies after HCT makes them an attractive platform for CARs when on target/off tumor effects are of specific concern.Figure 1: Co-transplantation of mCD19 CAR-engineered preTs provides strong anti-leukemia effects that are mediated by preT-derived CAR-expressing NK cells. (A) Irradiated B6/NCrl recipients were transplanted with either 3 x 106 T cell depleted bone marrow cells (TCDBM) only (n= 10) or additionally with 8 x 106 of either syngeneic (n= 10) or allogeneic CAR-transduced preTs (n= 10). Mice were challenged with 1.2 x 106 C1498-CD19-GFP cells on day 20 after transplantation (day 0) and followed for survival (B) Mice co-transplanted with CAR-preTs were treated i.p. weekly either with a depleting anti-NK1.1 antibody (200µg) or with PBS for control (n= 10, respectively). All mice received 1.2 x 106 C1498-CD19-GFP cells on day 20 after transplantation and survival was assessed after leukemia challenge. (C) Progenies of co-transplanted CAR-, ΔCAR- (CAR defective for signaling) or Ctrl-transduced preTs were analyzed within the BM of transplanted mice by flow cytometry 14 days after transplantation. Numbers of NK1.1+NKp46+ cells within the gate are depicted (CAR, ΔCAR, Ctrl, n=4 respectively). (A, B) Survival curves were analyzed with Mantel-Cox (log-rank) test and (C) with one-way ANOVA followed by Turkey's post-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. [Display omitted] DisclosuresSauer:Neovii: Research Funding.

CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Simple SummaryEfficient trafficking and survival of CAR T cells within the hostile tumor microenvironment are important prerequisites for potent solid tumor attack that have not yet been achieved. We deployed monospecific murine instead of polyclonal human T cells for CAR T cell generation to evaluate second generation L1CAM- and HER2-specific CARs with different spacer length and either the CD28 or 4-1BB co-stimulatory domain in mouse models of neuroblastoma and ovarian carcinoma. This mouse-in-mouse approach ensured CAR T cell trafficking unhindered by species-specific discrepancies and demonstrated superior solid tumor attack by CAR T cells harboring the CD28 compared to 4-1BB co-stimulatory domain. Our approach has the potential to improve prediction and selection of promising clinical CAR candidates against solid tumors in the future.Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

89723 CD105-targeted CAR T cells for the treatment of acute myeloid leukemia

ABSTRACT IMPACT: Our work might lead to a new treatment for patients with acute myeloid leukemia OBJECTIVES/GOALS: Acute myeloid leukemia (AML) is a devastating hematologic malignancy, with dismal 5-year survival. Chimeric antigen receptor (CAR) T cells have been approved for B cell malignancies but not for AML. The goal of this study is to explore the safety and efficacy of CAR T cells targeting CD105 (endoglin) to treat AML. METHODS/STUDY POPULATION: We have constructed human and murine CAR T cells targeting CD105. The CARs were created by sequencing the V(D)J regions of hybridomas and designing single chain variable fragments that target CD105 which were subsequently introduced in a CAR backbone via Gibson assembly. The CAR T cells were produced via transduction using retrovirus or lentivirus. Leukemia cell lines were assessed for CD105 expression with flow cytometry. Killing assays were performed via measurement of luminescence of target cells after co-culture with CAR T cells. Activation assays were performed with co-culture of CAR T cells and target cells and measurement of activation markers with flow cytometry. To assess in vivo efficacy and safety, murine CAR T cells were infused into C57BL/6J mice carrying B16 melanoma after lymphodepletion. RESULTS/ANTICIPATED RESULTS: All human leukemia cell lines assessed (Nalm6, MOLM-14, MV4-11, Kasumi-1, THP-1) expressed some degree of endoglin apart from the T cell leukemia Jurkat. Human CD105 CAR T cells were activated by co-culture with leukemia cell lines and effectively killed leukemia cells in vitro in a CD105-specific manner. Murine CAR T cells killed efficiently both murine solid tumors (B16 melanoma) and murine leukemias (C1498) in vitro. Murine CAR T cells did not exhibit any toxicity when infused after low-dose lymphodepletion (cyclophosphamide 100mg/kg) but caused significant morbidity after higher doses (cyclophosphamide 200mg/kg). Murine CAR T cells delayed the growth of B16 melanoma in immunocompetent mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have constructed human and murine CD105 CAR T cells with excellent activity in vitro. The activity of human CD105 CAR T cells in xenografts and the biologic relevance of the toxicity of murine CD105 CAR T cells in humans needs to be further investigated. CD105 CAR T cells might prove an important therapeutic option for patients with AML.

A phase 1 study of a novel fully human BCMA-targeting CAR (CT103A) in patients with relapsed/refractory multiple myeloma

AbstractB-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies have shown efficacy in relapsed/refractory multiple myeloma (RRMM). Because the non-human originated antigen-targeting domain may limit clinical efficacy, we developed a fully human BCMA-specific CAR, CT103A, and report its safety and efficacy in a phase 1 trial. Eighteen consecutive patients with RRMM, including 4 with prior murine BCMA CAR exposures, were enrolled. CT103A was administered at 1, 3, and 6 × 106 CAR-positive T cells/kg in the dose-escalation phase, and 1 × 106 CAR-positive T cells/kg in the expansion cohort. The overall response rate was 100%, with 72.2% of the patients achieving complete response or stringent complete response. For the 4 murine BCMA CAR–exposed patients, 3 achieved stringent complete response, and 1 achieved a very good partial response. At 1 year, the progression-free survival rate was 58.3% for all cohorts and 79.1% for the patients without extramedullary myeloma. Hematologic toxicities were the most common adverse events; 70.6% of the patients experienced grade 1 or 2 cytokine release syndromes. No immune effector cell–associated neurotoxicity syndrome was observed. To the cutoff date, CAR transgenes were detectable in 77.8% of the patients. The median CAR transgene persistence was 307.5 days. Only 1 patient was positive for the anti-drug antibody. Altogether, CT103A is safe and highly active in patients with RRMM and can be developed as a promising therapy for RRMM. Patients who relapsed from prior murine BCMA CAR T-cell therapy may still benefit from CT103A. This trial was registered at http://www.chictr.org.cn as #ChiCTR1800018137.

Novel high-affinity EGFRvIII-specific chimeric antigen receptor T cells effectively eliminate human glioblastoma.

ObjectivesThe increasing success of Chimeric Antigen Receptor (CAR) T cell therapy in haematological malignancies is reinvigorating its application in many other cancer types and with renewed focus on its application to solid tumors. We present a novel CAR against glioblastoma, an aggressive, malignant glioma, with a dismal survival rate for which treatment options have remained unchanged for over a decade.MethodsWe use the human Retained Display (ReD) antibody platform (Myrio Therapeutics) to identify a novel single‐chain variable fragment (scFv) that recognises epidermal growth factor receptor mutant variant III (EGFRvIII), a common and tumor‐specific mutation found in glioblastoma. We use both in vitro functional assays and an in vivo orthotopic xenograft model of glioblastoma to examine the function of our novel CAR, called GCT02, targeted using murine CAR T cells.ResultsOur EGFRvIII‐specific scFv was found to be of much higher affinity than reported comparators reverse‐engineered from monoclonal antibodies. Despite the higher affinity, GCT02 CAR T cells kill equivalently but secrete lower amounts of cytokine. In addition, GCT02‐CAR T cells also mediate rapid and complete tumor elimination in vivo.ConclusionWe present a novel EGFRvIII‐specific CAR, with effective antitumor functions both in in vitro and in a xenograft model of human glioblastoma.

Generation of Murine Chimeric Antigen Receptor T Cells for Adoptive T Cell Therapy for Melanoma.

Expression of chimeric antigen receptors can redirect T cell specificity and allow for MHC-independent recognition of melanoma associated antigens. Retroviral transduction is used to express chimeric antigen receptor constructs in murine CD4+ and CD8+ T cells. Here we describe the production of retroviral supernatants and the activation, transduction, expansion, and selection of murine T cells expressing the chimeric-PD1 receptor. This protocol can be modified for any murine chimeric antigen receptor construct.

Molecular Imaging of Chimeric Antigen Receptor T Cells By ICOS-Immunopet

Introduction: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Non-invasive molecular imaging of CAR T cell therapy by positron emission tomography (PET) is a promising approach providing spatial, temporal and functional information. Reported strategies for PET-based monitoring of CAR T cells rely on additional manipulation of the cell product such as the incorporation of reporter transgenes or ex vivo biolabeling, which significantly limits the wider application of CAR T cell molecular imaging. In the present study, we assessed the ability of antibody-based PET (immunoPET) to non-invasively visualize CAR T cells in vivo. Methods: For analysis of human CAR T cell activation, we analyzed publicly available RNA sequencing data (GSE136891) obtained at serial time points during in vitro culture of CD19.CD28z CAR T cells. We analyzed by mass cytometry (CyTOF) the ex vivo ICOS expression on human CD19-28z CAR T cells obtained from 31 patients receiving axicabtagene ciloleucel (Axi-cel) for relapsed/refractory diffuse large B-cell lymphoma (DLBCL). For in vivo murine experiments, CD19-expressing B-cell lymphoma A20 cells (2.5×10e5 cells) were injected by tail vein intravenously (i.v.) into sub-lethally (4.4 Gy) irradiated Thy1.2+ BALB/c mice. Seven days later, murine CD19.CD28z Luc+ Thy1.1+ CAR T cells (1×10e6) were i.v. injected. ICOS expression was analyzed by flow cytometry on CAR T cells recovered from spleen and bone marrow 5 days after injection. For imaging studies, anti-ICOS monoclonal antibody (mAb) specific for murine ICOS (clone:7E.17G9, BioXcell) was modified with the bifunctional chelator deferoxamine (DFO/p-SCN-Bn-Deferoxamine). The DFO-ICOS mAb conjugate was radiolabeled with 37 MBq (~1 mCi) of 89Zr-oxalate (final specific activity 6 µCi/µg/ml and radiochemical purity of 99%). 89Zr-DFO-ICOSmAb (45 μCi ± 3.6, 7.5 μg± 0.6) was injected i.v. 5 days post-CAR T cell administration and PET-CT imaging performed 48 hours later. Following PET-CT, mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter. Results: Analysis of RNA-sequencing data from human CAR T cells identified ICOS as an activation marker whose transcription was up-regulated and sustained during in vitro culture. ICOS was preferentially expressed on CAR+ T cells recovered at day 7 from axi-cel treated patients compared with CAR- cells (p&amp;lt;0.001; Figure 1A). Phenotypic analysis in a murine model of B cell lymphoma infiltrating the spleen and the bone marrow confirmed preferential ICOS expression on murine CAR T cells compared to resident cells in both spleen (p=0.003) and bone marrow (p=0.008). Figure 1B shows representative volume-rendered technique (VRT) PET/CT images of 89Zr-DFO-ICOS mAb-injected tumor-bearing mice either untreated (left panels) or that received mCD19.28z CAR T cells (right panels). 89Zr-DFO-ICOS mAb similarly accumulated in highly vascularized organs (heart, liver and spleen) of both untreated and CAR T cell treated mice, consistent with the biodistribution and clearance of intact antibodies. We detected pronounced 89Zr-DFO-ICOS mAb-PET signals in the bones of CAR T cell treated mice, particularly prominent in the lumbar spine, iliac bones, femur, tibia and humeral heads (Figure 1B). Region of interest analysis confirmed markedly increased radiotracer uptake in bones rich in bone marrow from CAR T treated mice compared with those of untreated mice (lumbar spine vertebrae p&amp;lt;0.001; iliac bones p=0.001; femur p=0.002; tibia p=0.002). Moreover we observed a slight, but statistically significant increase in radiotracer accumulation in the heart of CAR T cell-treated mice (p=0.004) while no significant differences were detected in spleen and liver. As expected, there was no significant signal difference in the muscle, considered background. Biodistribution analysis using gamma counting of tissues confirmed the PET results. Conclusions: We describe for the first time an immunoPET approach to monitor the in vivo dynamics of CAR T cell migration, expansion, and persistence that does not require the addition of reporter genes or ex vivo labeling, being therefore applicable to the clinical setting for the study of any commercially available and investigational CAR T cell products. Disclosures Miklos: Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents &amp; Royalties, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Lyell Immunopharma: Consultancy, Current equity holder in private company; NeoImmune Tech: Consultancy; Nektar Therapeutics: Consultancy; Apricity Health: Consultancy, Current equity holder in private company. Gambhir:CellSight Inc: Current equity holder in private company. Negrin:Amgen: Consultancy; BioEclipse Therapeutics: Current equity holder in private company; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; Biosource: Current equity holder in private company; KUUR Therapeutics: Consultancy; UpToDate: Honoraria.