Presence of Endogenous TCR Antigen in Vivo Attenuates Efficacy of Anti-CD19 Targeted CAR T Cell Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3721-3721
Author(s):  
Yinmeng Yang ◽  
Christopher Daniel Chien ◽  
Elad Jacoby ◽  
Haiying Qin ◽  
Waleed Haso ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Ifn Gamma ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Adoptive therapy using T cells genetically engineered to express chimeric antigen receptors (CAR) has proven extremely effective against acute lymphoblastic leukemia (ALL) in clinical trials with the use of anti-CD19 CAR T cells. Most CAR T cell protocols use autologous T cells, which are then activated, transduced with the anti-CD19 CAR, and expanded ex-vivo before infusion back into the patient. This approach minimizes the risk of graft-versus-host disease (GVHD) even in allogeneic transplant recipients, due to tolerization of the donor T cell repertoire in the recipient. However, many patients have heavy disease burden and lymphopenia due to previous treatments, which makes the isolation of healthy T cells difficult. Thus, centers are exploring the potential of allogeneic T cell donors and the possibility of universal T cell donors for CAR-based therapy including the use of virus-specific T cells. In these cases, in addition to the chimeric receptor specificity, the transduced T cell population will also have reactivity against target antigens through the endogenous TCR. However, little is known about the impact of signaling of the endogenous TCR on CAR T cell activity, particularly in vivo. To test this, we used a syngeneic transplantable ALL murine model, E2aPBx, in which CD19 CAR T cells can effectively eradicate ALL. CD4 (Marilyn) and CD8 (Matahari) T cells from syngeneic HY-TCR transgenic donors specific for the minor histocompatibility male antigen, HY, were used as CAR T cell donors to control for endogenous TCR reactivity. Splenic T cells isolated from Matahari, Marilyn, or B6 mice were activated ex-vivo using anti-CD3/anti-CD28 beads, with the addition of IL2 and IL7. T cells were transduced with a retroviral vector expressing a murine CAR composed of anti-CD19 scfv/CD28/CD3ζ on days two and three. CAR T cells are evaluated in vitro by CD107a degranulation assay and INF gamma ELISA. In response to HY peptide alone or HY+CD19- line M39M, transduced CD8 HY (Matahari) cells produced IFN gamma and expressed CD107a whereas transduced CD4 HY (Marilyn) cells only produced IFN gamma. Interestingly, in response to CD19+HY- ALL, both Matahari and Marilyn expressed CD107a and produced IFN gamma indicating that CD4 T cells can acquire CD8-like lytic activity when stimulated through a CAR receptor. When CD19 CAR transduced Marilyns and Mataharis were stimulated in the presence of HY and CD19, CD8 Mataharis had an attenuated effect against CD19, suggesting that the presence of antigen activated TCR adversely affects the potency of the CAR receptor. Efficacy of the HY and polyclonal CAR T cells were next tested in-vivo in male and female B6 mice. Mice were given 1E6 E2aPBx ALL leukemia cells on day 1, and received 500 rads sub-lethal total body irradiation on day 4 as a lymphodepleting regimen. On day 5, mice were given a low (1E5) or high (5E6) dose of CAR T cells. There was a statistically significant (p=0.0177) improvement in the survival of female versus male mice after treatment with the CD4+ HY specific anti-CD19 CAR T cells, and female mice that received HY anti-CD19 CAR T cells survived longer than untreated control females (p=0.01). Remarkably, the survival of male mice that received HY anti-CD19 CAR T cells was statistically worse than untreated control males (p=0.008). This suggests that the presence of TCR antigen negatively impacts the function of CAR T cells. Furthermore, in a separate experiment using an equally mixed population of Marilyn (CD4+) and Matahari (CD8+) HY specific T cells, males has a statistically significantly (p=0.0116) worse survival compared to females after receiving 5E5 HY specific T cells. In conclusion, simultaneous stimulation through both CAR and TCR results in attenuated cytokine production and degranulation by CD8 T cells. In vivo, in the presence of the endogenous TCR antigen, both CD4 and CD8 CAR T cells are less potent at eradicating leukemia. These have implications for the development of universal donors for CAR T cell therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals Targeting glycosylation of PD-1 to enhance CAR-T cell cytotoxicity

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaojuan Shi ◽  
Daiqun Zhang ◽  
Feng Li ◽  
Zhen Zhang ◽  
Shumin Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inhibitory Effects ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractAsparagine-linked (N-linked) glycosylation is ubiquitous and can stabilize immune inhibitory PD-1 protein. Reducing N-linked glycosylation of PD-1 may decrease PD-1 expression and relieve its inhibitory effects on CAR-T cells. Considering that the codon of Asparagine is aac or aat, we wondered if the adenine base editor (ABE), which induces a·t to g·c conversion at specific site, could be used to reduce PD-1 suppression by changing the glycosylated residue in CAR-T cells. Our results showed ABE editing altered the coding sequence of N74 residue of PDCD1 and downregulated PD-1 expression in CAR-T cells. Further analysis showed ABE-edited CAR-T cells had enhanced cytotoxic functions in vitro and in vivo. Our study suggested that the single base editors can be used to augment CAR-T cell therapy.

Quality Assessment of Pre-Clinical Studies of Chimeric Antigen Receptor T-Cell Therapy Products: A Point of Focus On Safety

2021 ◽  
Vol 16 ◽  
Author(s):  
Vikas Maharshi ◽  
Diksha Diksha ◽  
Pooja Gupta
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Background: Serious adverse reactions have been reported with the use of chimeric antigen receptor (CAR) T-cell therapy in clinical setting despite the success of these products in pre-clinical stages of development. Objective: We evaluated the quality of available pre-clinical safety data of CAR T-cell therapy products. Methods: A 21 items safety-checklist was designed specifically for CAR T-cell. Literature was searched using search/MeSH terms in PubMed (October 2019 – February 2020). Studies were screened from title and abstract. Original pre-clinical researches related to CAR T-cell anti-cancer therapy were included. Results: Of the search results, 152 studies (3 in vivo, 39 in vitro, and 110 combined) were included. Only 7.9% studies were specifically designed to evaluate/ improve product safety. Eleven studies included target antigen(s) and no study included co-stimulatory molecule(s) expressed exclusively by tumor tissue and/or CAR T-cells. One study used CRISPR-Cas9 for CAR gene insertion. The use of switch-off mechanism and purity assessment of CAR T-cell products were reported in 13.2% and 8.6% studies respectively. Of the 149 studies with in vivo component, immuno-competent animal models were used in 24.8%. Measurement of blood pressure, temperature, body weight and serum cytokines were reported in 0, 2.7, 29.2 and 27.4% studies respectively. The tissue distribution and CAR T-cells persistence were reported in 26.5% studies. Conclusion: Majority of the checklist parameters were not reported in the pre-clinical publications to be adequately predictive of the safety of CAR T-cells in a clinical setting.

scholarly journals Use of AXL-specific CAR T cells to treat non-small-cell lung cancer

2021 ◽  
Author(s):  
Zhenfeng Zhang ◽  
Bihui Cao ◽  
Manting Liu ◽  
Yubo Zhou ◽  
Qi Zhao ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
T Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
T Cell Infiltration ◽  
Car T Cell Therapy ◽  
Car T

Abstract The application of Chimeric antigen receptor (CAR) T cells in solid tumors is hindered by lack of tumor specific targets and inefficient T cell infiltration in tumor. It has been postulated that AXL may be an ideal immunotherapy target for non-small-cell lung cancer (NSCLC). Here, we screened 208 non-tumor samples from 22 types of human organs or tissues and 90 tumor samples from NSCLC patients by immunohistochemistry or Western Blotting and identified that AXL was rarely expressed in normal tissues but highly expressed in 69% NSCLC samples, suggesting AXL is an ideal target for CAR T cell therapy for lung cancer. We generated low-, mediate-, high-affinity AXL-CARs and evaluated their killing effect on NSCLC. Our data demonstrated antitumor effects of AXL-CAR T cell therapy for various NSCLC models both in vitro and in vivo. AXL-CAR T cells alone exerted strong antitumor effect in subcutaneous lung cancer cell derived xenograft (CDX), pulmonary metastases CDX, and intraperitoneal CDX models. Intraperitoneal delivery of CAR T cells resulted in superior tumor killing effects compared with systemic infusions for the intraperitoneal CDX tumor models. AXL-CAR T combined with microwave ablation (MWA) or EGFR-TKI resulted in enhanced killing effect and CAR-T cell infiltration in vivo. Together, our current study suggests that systemic or regional infusion of AXL-CAR T cell alone or combination with other therapies might have potential translatable value for the treatment of NSCLC in clinical situation.

scholarly journals Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma

2019 ◽  
Vol 21 (11) ◽  
pp. 1436-1446 ◽  
Author(s):  
Jing Cui ◽  
Qi Zhang ◽  
Qi Song ◽  
Herui Wang ◽  
Pauline Dmitriev ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Intratumoral Injection ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. Methods First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. Results We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. Conclusions By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

scholarly journals 136 ATA3271: An armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A145-A145
Author(s):  
Xianhui Chen ◽  
Jiangyue Liu ◽  
Shuai Yang ◽  
Amogh Oke ◽  
Sarah Davies ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Next Generation ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMesothelin (MSLN) is a GPI-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma and is an attractive target antigen for tumor surface antigen-targeting therapies. Regional administration of autologous, 2nd generation MSLN-targeted CAR-T cells for malignant pleural mesothelioma has shown promise in early clinical evaluation.1 2 More recently, a next-generation MSLN-targeted, autologous CAR T therapy leveraging 1XX CAR signaling and PD1DNR is currently under investigation for advanced mesothelioma [NCT04577326]. Although autologous MSLN CAR-T holds promise, an allogeneic approach may have more widespread application. EBV T-cells represent a unique, non-gene edited approach for allogeneic T-cell therapy. EBV-specific T-cells are currently in a phase 3 trial for EBV-positive post-transplant lymphoproliferative disease [NCT03394365] and, to-date, have demonstrated a favorable safety profile with no evidence for GvHD and cytokine release syndrome attributable to EBV T-cells. Clinical proof-of-principle studies for CAR transduced CD19-targeted allogeneic EBV T-cell therapies have shown acceptable safety and durable response.3 The first preclinical evaluation of ATA3271 was reported last year.4 Here, we describe updated preclinical data for this potential off-the-shelf, allogeneic cell therapy.MethodsWe engineered MSLN CAR+ EBV T-cells (ATA3271) with a novel 1XX signaling domain that is associated with strong effector function and favorable persistence, as well as armored with PD1DNR to provide intrinsic checkpoint blockade.5 Anti-tumor effect of ATA3271 was assessed using a MSTO-211H-derived tumor cell line overexpressing MSLN and PDL1.ResultsUpon MSLN engagement, ATA3271 showed proliferation, efficient tumor cell lysis in the presence of high-level cell-surface PD-L1 expression and secretion of effector cytokines [IL-2, TNF-α, granzyme B]. In a 16-day serial stimulation assay, with PD-L1+ tumor cells added every 2–3 days, ATA3271 expanded 4 to 45-fold without the need for external cytokines, and retained comparable antitumor function as CD3/CD28-stimulated ‘autologous’ CAR-T cells. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrated anti-tumor efficacy without toxicities. Memory markers [CD62L, CCR7] play a key role for T-cell persistence in vivo. We identified donor-to-donor variability in memory marker expression on ATA3271 and optimized our process to maximize their expression. Memory marker expression impact on ATA3271 potency, both in vitro and in vivo, will be presented.ConclusionsOverall, these in vitro and in vivo data show potent anti-tumor activity without evidence of toxicity, suggesting that ATA3271 may be a promising approach for the treatment of MSLN-positive cancers that warrants further clinical investigation.ReferencesAdusumilli Prasad S, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Res 2019;79(13 Suppl):Abstract CT036.Adusumilli Prasad S, et al. A phase I trial of regional mesothelin-targeted CAR T-cell therapy in patients with malignant pleural disease, in combination with the anti-PD-1 agent pembrolizumab. Cancer Discov 2021.Curran Kevin J, et al. Durable remission following ‘off-the-shelf’ chimeric antigen receptor (CAR) T-cells in patients with relapse/refractory (R/R) B-cell malignancies. Biol Blood Marrow Transplant 2020;26.3: S89.Liu Jiangyue, et al. 98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors. JITC 2020;8.Feucht Judith, et al. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nat Med 2019;25.1: 82–88.

scholarly journals 147 Memory phenotype in allogeneic anti-BCMA CAR-T cell therapy (P-BCMA-ALLO1) correlates with in vivo tumor control

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A155-A155
Author(s):  
Hubert Tseng ◽  
Yan Zhang ◽  
Stacey Cranert ◽  
Maximilian Richter ◽  
Karl Marquez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Memory Phenotype ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundThe emergence of CAR-T cell therapy has transformed the treatment of refractory/relapsed multiple myeloma (MM). Yet, autologous CAR-T cells suffer from many manufacturing challenges including mainly consistency, toxicity, and cost. To address these issues, we engineered a fully allogeneic anti-BCMA CAR-T cell candidate for MM from healthy donors (P-BCMA-ALLO1). Herein, we demonstrate that this therapy maintains a stem cell memory T cell (TSCM) phenotype through editing which correlates with in vivo antitumor efficacy.MethodsUsing Poseida’s non-viral piggyBac® (PB) DNA Delivery System in combination with the high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System and a proprietary ‘booster molecule’, we generated P-BCMA-ALLO1 from healthy donor T cells. We used CC to eliminate surface expression of both the TCR and MHC class I to make fully allogeneic CAR-T cells. In addition to the CAR molecule, PB enables the delivery of a selectable marker allowing the generation of a final cell product that is >95% CAR-positive. The inclusion of the ‘booster molecule’ in the manufacturing process improves the expansion of gene-edited cells without compromising memory phenotype or function. This process can produce up to hundreds of patient doses from a single manufacturing run which significantly reduces manufacturing cost per dose. We characterized the memory phenotype of P-BCMA-ALLO1 by assessing the mRNA and protein expression profiles of rested and activated CAR-T cells by flow cytometry and Nanostring analysis. We also assessed the antitumor capabilities of these cells using cytotoxicity assays and performed serial in vitro restimulation to assess the ability of P-BCMA-ALLO1 to undergo multiple rounds of activation and expansion. We then evaluated the relationship of these characteristics with in vivo efficacy, as defined by control of tumor in an immunodeficient RPMI-8226 subcutaneous murine tumor model.ResultsP-BCMA-ALLO1 is comprised of a high frequency of TSCM. It has potent in vivo antitumor activity, which is comparable to non-edited autologous anti-BCMA CAR-T cell therapy. Expression of memory markers at both mRNA and protein levels across individual lots significantly correlates with in vivo tumor control. Conversely, suboptimal research products with worse in vivo outcomes expressed an exhausted gene expression profile. Moreover, CAR-T products that are more effective in vivo are also more viable, cytotoxic, and proliferative following multiple rounds of restimulation in vitro.ConclusionsP-BCMA-ALLO1 is a highly potent and safe allogeneic anti-BCMA CAR with a manufacturing process that consistently maintains a TSCM phenotype, which correlates with antitumor efficacy. P-BCMA-ALLO1 is advancing rapidly towards the clinic (NCT04960579).

scholarly journals Enhancing the Antigen-Sensitivity of the CD22 CAR through Modulation of the Affinity and Linker-Length of the Single-Chain Fragment Variable

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-42
Author(s):  
M. Eric Kohler ◽  
Zachary Walsh ◽  
Kole Degolier ◽  
Terry J. Fry
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

The advent of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL). CD19 directed CAR T cells have demonstrated the ability to induce complete remissions in up to 90% of r/r ALL patients. Despite this remarkable upfront success, relapse after CAR T cell therapy remains a major obstacle to long term remissions. A major mechanism for relapse after CD19-directed CAR T cell therapy is the recurrence of antigen-negative ALL cells. In recent years, CD22 CAR T cell therapy has emerged as an effective salvage therapy for patients with CD19-negative ALL. In a phase I clinical trial, CD22 CAR T cells were able to induce remission in up to 80% of patients with CD19-negative ALL. Patients achieving remission, who did not undergo a consolidative hematopoietic stem cell transplant, were found to be at high risk of relapse due to downregulation of the CD22 antigen below the threshold required for effective CD22 CAR T cell activity. Thus, strategies to increase the antigen-sensitivity of CD22 CAR T cells have the potential to enhance the induction and duration of remission in ALL patients. As the properties of a CAR that influence sensitivity to antigen are not well defined, we began by testing the impact of increasing the affinity of the single-chain fragment variable (scFv) for the CD22 antigen. T cells from healthy donors were activated and transduced with a second-generation, 4-1BB CAR containing either the standard affinity (SA)-m971 scFv used in the prior clinical trial, or a high affinity (HA) scFv generated by affinity maturation of the m971 scFv. SA- and HA-CD22 CAR T cells were evaluated in vitro and in vivo against clones of the pre-B ALL cell line, NALM6, which express CD22 at wild type levels (CD22WT), sub-physiologic levels (CD22Lo), supra-physiologic levels (CD22Hi) or in which CD22 was deleted (CD22Neg). We found that the amount of CD22 expressed on the leukemia cells resulted in dose-dependent expression of activation markers, such as CD69 and CD25 (p<0.05) on CD22 CAR T cells. Similarly, CAR T cell functions, such as the secretion of interferon-gamma (IFNg, p<0.0001) and interleukin-2 (IL-2, p<0.0001) as well as cytotoxic degranulation (p<0.0001) were all significantly impacted by the amount of CD22 on the surface of NALM6. A similar pattern of antigenic dose-response was seen in the signaling of CAR T cells, with phosphorylation of ERK reflecting the level of CD22 antigen (p<0.001) and correlating with the increased in vivo efficacy of the CAR T cells against CD22WT NALM6, relative to CD22Lo NALM6. Increasing the affinity of the CD22 CAR did not impact the in vivo efficacy against CD22WT NALM6 at either a therapeutic or subtherapeutic dose, however, HA-CD22 CAR T cells significantly prolonged the survival of NSG mice with CD22Lo NALM6, relative to SA-CD22 CAR T cells (p<0.01). The enhanced activity of HA-CD22 CAR T cells against CD22Lo leukemia did not correlate with improved in vitro functionality, as the HA-CD22 CAR T cells surprisingly demonstrated lower IL-2 secretion (p<0.01), lower proliferation (p<0.05) and diminished in vitro lysis of CD22Lo NALM6 (p<0.05), relative to SA-CD22 CAR T cells. ERK phosphorylation, however, was significantly increased in HA-CD22 CAR T cells (p<0.01) and was the only in vitro marker which correlated with the enhanced in vivo activity seen with the affinity-matured CAR. Previous clinical experience has demonstrated the importance of using a short linker (consisting of a single G4S sequence) between the heavy and light chains of the m971 scFv, therefore we next evaluated the impact of linker length on the activity of the HA-CD22 CAR. HA-CD22 CARs were generated with either a short- or long-linker (G4S x1 vs G4S x3, respectively) and evaluated in vitro and in vivo. While the short linker improved proliferation in vitro, there was no significant impact of linker length on cytokine production or lysis of CD22Lo NALM6. In a xenograft model, HA-CD22 CAR T cells with the long-linker demonstrated slower progression of CD22Lo leukemia and significantly prolonged survival of NSG mice with CD22WT leukemia relative to HA-CD22 CAR T cells with the short-linker (p<0.01). Taken together, these studies suggest that increasing the affinity of a scFv is a promising strategy for enhancing CAR sensitivity to low levels of target antigen, with the potential to decrease post-CAR T cell relapses due to antigen downregulation. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD19/BAFF-R Dual-Targeted CAR T Cells for the Treatment of Mixed Antigen-Negative Variants of Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2783-2783
Author(s):  
Xiuli Wang ◽  
Zhenyuan Dong ◽  
Wen-Chung Chang ◽  
Wesley Cheng ◽  
Vibhuti Vyas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Dual Targeting ◽  
Car T Cell Therapy ◽  
Car T

Abstract The results of clinical trials evaluating CD19-targeting chimeric antigen receptor (CAR) T cells are impressive, with overall response rates of up to 90% in B cell acute lymphoblastic leukemia (ALL) and 50-80% in lymphoma. Despite initial responses, antigen-negative relapse is common following treatment with CD19-targeted therapies and is estimated to occur in up to 39% of patients. One approach of addressing this problem is to utilize dual-targeting CAR T cells, a strategy that has recently been applied to CD19/CD22 for ALL, CS1/BCMA, and BCMA/GPRC5D for multiple myeloma. Dual-targeting CARs can simultaneously target two tumor antigens and, therefore, potentially eradicate heterogeneous tumors. The initial response to dual-targeted CAR T cells is expected to provide greater tumor coverage compared to single-targeted therapy, and can potentially circumvent antigen escape. Moreover, if one tumor antigen becomes downregulated during treatment, the second targeting domain will continue to be reactive to tumors. Therefore, it is critical to identify novel targets that can be combined with CD19 in a dual-targeted immunotherapeutic platform. To expand the potential for dual targeting of ALL, we developed a CAR T cell therapy against a novel target, B-cell activating factor receptor (BAFF-R), based on the remarkable specificity of anti-BAFF-R antibodies that we previously generated. BAFF-R is expressed almost exclusively on B cells, including in patients with CD19-negative relapse, making it an ideal immunotherapeutic target. Studies demonstrate that the role of BAFF-R in B cell function and survival is conclusive, an important feature that may mitigate the tumor's ability to escape therapy through antigen loss, particularly if a non-redundant role for BAFF-R is confirmed. BAFF-R-CAR T cells demonstrate in vitro effector function and in vivo therapeutic efficacy in CD19-negative models, including patient-derived xenograft models, and are currently being evaluated clinically for the treatment of ALL (NCT04690595). We hypothesized that simultaneous targeting of CD19 and BAFF-R in a bispecific CAR platform could confer a therapeutic advantage and avoid the challenges of sequential administration of CD19 and BAFF-R monospecific CAR T cells. We leveraged our experience with CD19- and BAFF-R-CAR T cells to develop a dual-targeting, bispecific CAR with a 41BB costimulatory domain (CD19/BAFF-R dual CAR). Here, we identified the optimal orientation of the single-chain variable fragment (loop scFv) domains within the dual construct and tested the CD19/BAFF-R dual CAR T cells for their in vitro effector function and in vivo anti-leukemia activity. To evaluate the specific targeting to CD19 and BAFFR, we developed Nalm-6-BAFF-R-knockout (KO) and Nalm-6-CD19-KO cell lines. CD19/BAFF-R dual CAR T cells specifically released IFN-g following incubation with Nalm-6 CD19-/- or BAFF-R-/- cells (P<0.001) compared with un-transduced mock T cells. Both CD4+ and CD8+ CAR T cell populations exhibited effector function. To evaluate the antigen-dependent targeting of the CD19/BAFF-R dual CAR T cells in vivo, we utilized a mixed B-cell leukemia model that simulates clinical tumor heterogeneity. NOD-scid IL2Rgammanull (NSG) mice were inoculated with 2-3x10 5 of mixed Nalm-6 BAFF-R-/- and CD19-/- cells at a 1:1 ratio with a single injection. 1x10 6 CD19/BAFF-R dual CAR, CD19, or BAFF-R single CAR-T cells were administered intravenously 9-10 days later. Tumor growth was monitored by bioluminescent imaging weekly. We observed superior tumor eradication (P<0.01) and survival (P<0.01) (Figure 1) by CD19/BAFF-R dual CAR T cells compared to either single-targeting CAR and mock T cells. The adoptively transferred CD19/BAFF-R dual CAR T cells were able to persist in vivo. Our unique CD19/BAFF-R dual-targeting CAR T cells will be the first to target this combination of tumor-associated antigens. Our study demonstrated the reliability of bispecific CD19/BAFF-R dual CAR T cell therapy in inducing remission in ALL consisting of CD19-/- and BAFF-R-/- tumors. We hypothesize that simultaneous immunotherapy targeting of heterogeneous leukemic cell populations may diminish the likelihood of antigen escape and may have a significant impact on leukemia treatment by improving the therapeutic benefits of CAR T cell therapy. Figure 1 Figure 1. Disclosures Wang: Pepromene Bio, Inc.: Consultancy. Forman: Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company. Kwak: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company. Qin: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company.

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

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1715-1715
Author(s):  
Ryan Urak ◽  
Ashlie Munoz ◽  
Hui-Ju Hsieh ◽  
Ellie Taus ◽  
Stephen J Forman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Interleukin 7 ◽  
Car T Cell Therapy ◽  
Car T

Abstract 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. Figure 1 Figure 1. Disclosures Forman: 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.

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