scholarly journals Modeling predicts differences in CAR T cell signaling due to biological variability

2022 ◽  
Author(s):  
Vardges Tserunyan ◽  
Stacey D Finley

In recent decades, chimeric antigen receptors (CARs) have been successfully used to generate engineered T cells capable of recognizing and eliminating cancer cells. The structure of CARs frequently includes costimulatory domains, which enhance the T cell response upon antigen encounter. However, it is not fully known how the CAR co-stimulatory domains influence T cell activation in the presence of biological variability. In this work, we used mathematical modeling to elucidate how the inclusion of one such co-stimulatory molecule, CD28, impacts the response of a population of engineered T cells under different sources of variability. Particularly, our simulations demonstrate that CD28-bearing CARs mediate a faster and more consistent population response under both target antigen variability and kinetic rate variability. We identify kinetic parameters that have the most impact on mediating cell activation. Finally, based on our findings, we propose that enhancing the catalytic activity of lymphocyte-specific protein tyrosine kinase (LCK) can result in drastically reduced and more consistent response times among heterogeneous CAR T cell populations.

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A23-A23
Author(s):  
D Lainšček ◽  
V Mikolič ◽  
Š Malenšek ◽  
A Verbič ◽  
R Jerala

BackgroundCD19 CAR T- cells (Chimeric antigen receptor T cells that recognize CD19) present a therapeutic option for various malignant diseases based on their ability to specifically recognize the selected tumour surface markers, triggering immune cell activation and cytokine production that results in killing cancerous cell expressing specific surface markers recognized by the CAR. The main therapeutic effect of CAR is a specific T cell activation of adequate cell number with sequential destruction of tumorous cells in a safe therapeutic manner. In order to increase T cell activation, different activation domains were introduced into CAR. CAR T-cells are highly efficient in tumour cell destruction, but may cause serious side effects that can also result in patient death so their activity needs to be carefully controlled.1 Several attempts were made to influence the CAR T cell proliferation and their activation by adding T cell growth factors, such as IL-2, into patients, however this approach of increasing the number of activating T cells with no external control over their number can again lead to non-optimal therapeutic effects. Different improvements were made by designing synthetic receptors or small molecule-inducible systems etc., which influence regulated expansion and survival of CAR T cells.2Material and MethodsIn order to regulate CD19 CAR-T cell activity, different NFAT2 based artificial transcription factors were prepared. The full length NFAT2, one of the main players in T cell IL2 production, a key cytokine for T cell activation and proliferation was truncated by deletion of its own activation domain. Next, we joined via Gibson assembly tNFAT21-593 coding sequence with domains of different heterodimerization systems that interact upon adding the inductor of heterodimerization. The interaction counterparts were fused to a strong tripartite transcriptional activator domain VPR and/or strong repressor domain KRAB resulting in formation of an engineered NFAT artificial transcription (NFAT-TF) factors with external control. To determine the activity of NFAT-TF HEK293, Jurkat or human T cells were used.ResultsBased on luciferase assay, carried out on NFAT-TF transfected HEK293 cells we first established that upon adding the external inductor of heterodimerization, efficient gene regulation occurs, according to VPR or KRAB domain appropriate functions. Findings were then transferred to Jurkat cells that were electroporated with appropriate DNA constructs, coding for NFAT-TF and CD19 CAR. After Raji:Jurkat co-culture ELISA measurements revealed that IL2 production and therefore CD19 CAR-T cell activity can be controlled by the action of NFAT-TF. The same regulation over the activity and subsequent proliferation status was also observed in retrovirally transduced human T-cells.ConclusionWe developed a regulatory system for therapeutic effect of CD19 CAR-T cells, a unique mechanism to control T cell activation and proliferation based on the engineered NFAT2 artificial transcription factor.ReferencesBonifant CL, et al. Toxicity and management in CAR T-cell therapy. Mol Ther Oncolytics 2016;3:16011.Wu C-Y, et al. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science 2015;80:350.Disclosure InformationD. Lainšček: None. V. Mikolič: None. Š. Malenšek: None. A. Verbič: None. R. Jerala: None.


2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3041-3041
Author(s):  
Jenny Mu ◽  
Justin Edwards ◽  
Liubov Zaritskaya ◽  
Jeffrey Swers ◽  
Ankit Gupta ◽  
...  

3041 Background: Conventional chimeric antigen receptor T cell (CAR-T) therapies have achieved limited clinical success in the treatment of solid tumors, in part due to the challenges of identifying tumor antigen(s) that are uniquely expressed on tumor cells. The dearth of such targets requires that current CAR-T therapies be re-engineered to preferentially target tumor cells thereby mitigating potential on-target off-tumor toxicity to normal cells. Herein we describe a novel cell therapy platform comprising Antigen Receptor Complex T (ARC-T) cells that are readily activated, silenced, and reprogrammed in vivo by administration of a novel tumor-targeting soluble protein antigen-receptor X-linker (sparX). The formation of the ARC-T, sparX, and tumor complex is required for the ARC-T to kill the tumor. Because ARC-T activity is entirely dependent on the dose of sparX administered, therapeutic doses of sparX may be defined that preferentially target cells over-expressing a target antigen and thus limit coincident kill of normal cells expressing lower levels of target antigen. Methods: We have created a library of sparX that bind different cell surface antigens, including HER2. The HER2 sparX was tested as both monovalent and bivalent constructs in vitro by assessing ARC-T cell activation, cytokine release and target cell cytotoxicity. In vivo efficacy models utilized NSG mice and incorporated tumor volume measurements and histopathologic assessments to evaluate tumor clearance. Results: In vitro studies demonstrate that co-culture of ARC-T cells, sparX-HER2 and HER2-expressing target cells drives T cell activation, expansion, cytokine secretion and cytotoxicity of target cells in a dose-dependent manner. Furthermore, by affinity tuning the HER2 binding domain and bivalent formatting of sparX-HER2, we achieved selective killing of HER2-overexpressing breast cancer cells with minimal effect on cells expressing HER2 levels representative of normal tissues. In vivo proof-of-principal studies with ARC-T/sparX-HER2 similarly demonstrate complete eradication of HER2-overexpressing solid tumor cells. Conclusions: These results demonstrate that a single intravenous dose of ARC-T cells can traffic to a solid tumor site and induce tumor eradication upon systemic administration and co-localization of tumor-targeting sparX in a mouse model. Bivalent formatting of sparX-HER2 further enabled ARC-T sensitivity to target antigen density to avoid the on-target off-tumor toxicity that has hindered conventional monovalent CAR-T treatments.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5949-5949
Author(s):  
Liora M. Schultz ◽  
Debra K Czerwinski ◽  
Aihua Fu ◽  
Shoshana Levy ◽  
Ronald Levy

Abstract The processes of ex vivo transduction of T cells to express chimeric antigen receptors (CARs) and of CAR+ T cell expansion influence the phenotype, function and ultimate fate of the final CAR+T cell product infused into patients. CAR constructs, despite expression of endogenous activation signals, require exogenous T cell activation during CAR transduction to allow optimal lenti-viral or retroviral-mediated integration of the CAR gene of interest into T cells. Clinical CAR therapy trials utilize anti-CD3 antibody-mediated activation or combined CD3 and CD28 stimulation using CD3, CD28 specific magnetic beads. We introduce novel magnetic nanoparticle beads generated from iron oxide nanoparticles conjugated to streptavidin and bound to biotinylated T cell activating antibodies for the purpose of CAR transduction. The small size of these nanobeads confers the advantage of decreased steric hindrance and enhanced capability of bead surface antibodies to access T cell surface antigen for binding and stimulation. We achieve efficient CAR transduction using anti-CD3 nanobead-mediated T cell stimulation and demonstrate CD19 specific CAR-mediated cytotoxicity of CD19+ tumor using an annexin V and 7AAD cytotoxicity assay. Evaluation of T cell phenotype following anti-CD3 nanobead-mediated T cell activation demonstrates preferential activation of naïve T cells as compared to central and effector memory cells. Addition of anti-CD28 costimulation is not necessary to achieving or inhibiting this preferential naïve T cell activation. Naïve T cells exhibit greater replicative capacity and anti-tumor function as compared to both effector and central memory T cells for adoptive transfer. We anticipate that preferential generation of naïve T cell derived CAR+ T cells achieved by introducing anti-CD3 nanobead stimulation can further improve the outcomes of clinical trials using CAR therapy. Disclosures Fu: NVIGEN Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 966-966 ◽  
Author(s):  
Justin C. Boucher ◽  
Gongbo Li ◽  
Bishwas Shrestha ◽  
Maria Cabral ◽  
Dylan Morrissey ◽  
...  

Abstract The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, a major obstacle with continued clinical development of CAR T cells is the limited understanding of CAR T cell biology and its mechanisms of immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation causing acute toxicities, but also lead to T cell exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. We hypothesized that by incorporating null mutations of the CD28 subdomains (YMNM, PRRP, or PYAP) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ (Fig1A), IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytoxicity was enhanced with the PYAP only CAR T cells compared to non-mutated CARs (Fig1B). When we examined the PYAP (mut06) only mutant in an immune competent mouse model we found similar B cell aplasia and CAR T cell persistence compared to non-mutated CD28 CAR T cells. Additionally, PYAP only CAR T cells injected into mice had decreased (82% to 62%) expression of PD1 in the BM. Using a pre-clinical immunocompetent mouse tumor model we found the PYAP only CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells, with 100% survival of mice given PAYP only CAR T cells compared to 50% survival of mice given non-mutated CAR T cells (Fig1C). We next sought to determine what role CAR T cell exhaustion was playing using a Rag knockout mouse system. CAR T cells were given to Rag-/- mice and 1 week later mice were challenged with tumor. Studies in Rag-/- mice also showed PYAP only CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. We also found PYAP only CAR T cells had significantly less expression of PD1 compared to non-mutated CAR T cells (Fig1D). We then co-cultured CAR T cells with target cells expressing CD19 and PDL1 and found PYAP only CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This shows that PYAP only CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. Using Nur77, pAkt, and pmTOR to measure CAR signaling we found PYAP only CAR T cells had significantly reduced levels of Nur77 while still having higher expression then first generation CAR T cells. We then examined what affect the PYAP only CAR had on transcription factors. We found similar AP1 and NF-kB expression between PYAP only and non-mutated CD28 CAR T cells but a significant reduction of NFAT in the PYAP only mutants compared to non-mutated CD28 CAR T cells. This suggests reduced NFAT expression contributes to the PYAP only CAR's resistance to exhaustion. Finally, we made human CAR constructs of the PYAP only mutant. We found PYAP only human CAR T cells had increased cytoxicity and decreased exhaustion in vitro compared to non-mutated human CD28 CAR T cells. NFAT levels in human PYAP only CAR T cells were significantly reduced compared to non-mutated CAR T cells supporting our findings in mice. Our results demonstrate that CAR T cells with only a PYAP CD28 subdomain have better cytoxicity and decreased exhaustion compared to non-mutated CD28 CAR T cells. Our results suggest this is the result of decreased CAR and NFAT signaling. Additionally, we were able to validate these findings using human CAR constructs. This work allows for development of an enhanced 2nd and 3rd generation CAR T cell therapies for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities. Figure 1 Figure 1. Disclosures Davila: Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4295-4295 ◽  
Author(s):  
Aaron Foster ◽  
Aruna Mahendravada ◽  
Nicholas P Shinners ◽  
Peter Chang ◽  
An Lu ◽  
...  

Abstract Introduction: Adoptive transfer of T cells, genetically engineered to express chimeric antigen receptors (CARs) containing costimulatory domains, such as CD28 or 4-1BB, has yielded impressive clinical results in some blood cancers, but severe toxicities have been observed due to unchecked T cell activation. In contrast, CAR-T cells have demonstrated limited clinical efficacy, associated with poor engraftment, survival and proliferation of adoptively transferred cells when used to target a variety of solid tumors. Thus, technologies that can regulate T cell activation and proliferation in vivo should both mitigate toxicities and maximize anti-tumor efficacy, expanding their clinical utility to a wider range of indications. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), activated by a small molecule chemical inducer of dimerization, rimiducid, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 and transduced with a retrovirus encoding tandem rimiducid-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 signaling elements, and first generation CARs (CAR.ζ) targeting CD19 or PSCA (SFG-iMC-2A-CD19.ζ or SFG-iMC-2A-PSCA.ζ, respectively). iMC activation was measured by treating T cells with and without rimiducid and measuring cytokine production by ELISA and T cell activation markers by flow cytometry. Coactivation through iMC and CAR was tested in coculture assays with or without rimiducid using various tumor cells (CD19+, Raji and Daudi lymphoma; PSCA+, Capan-1 and HPAC pancreatic adenocarcinoma). Efficacy of iMC-modified CAR-T cells were assessed using an immune-deficient NSG mouse tumor model. For CD19-targeted CARs, 1x105 Raji tumor cells were injected i.v. followed on day 7 by a single i.v. injection at various doses of iMC-CD19.ζ-modified T cells. For PSCA-targeted CARs, 2x106 HPAC tumor cells were injected s.c. followed by iMC-PSCA.ζ-modified T cells on day 10. In both models, iMC was activated in vivo by weekly i.p. injections of rimiducid (5 mg/kg). In some experiments, iMC-CAR-modified T cells were engrafted into tumor-free mice. Tumor burden and CAR-T cell expansion in vivo was assessed using luciferase bioluminescent imaging and flow cytometry. Results: T cells transduced with either iMC-CD19.ζ or iMC-PSCA.ζ produce cytokines (e.g., IFN-γ and IL-6) in response to rimiducid; however, the key growth and survival cytokine, IL-2, was only produced when both iMC and CAR were activated simultaneously by rimiducid and tumor antigen, respectively. CD19+ Raji tumor-bearing mice treated with iMC-CD19.ζ-modified T cells with or without rimiducid administration increased survival compared to non-transduced T cells (p = 0.01). However, rimiducid treatment induced a 7.3-fold CAR-T cell expansion compared to mice infused with iMC-CD19.ζ, but untreated with dimer drug (p = 0.02). Additionally, treatment of NSG mice bearing large (>200 mm3) HPAC tumors with a single dose iMC-PSCA.ζ, resulted in complete elimination in 10/10 mice (100%) of tumors both with and without rimiducid treatment compared to mice receiving non-transduced T cells (p = 0.0003). Rimiducid administration again dramatically increased CAR-T cell levels, resulting in a 23-fold expansion of iMC-PSCA.ζ-modified T cells compared to mice not receiving rimiducid (p = 0.02), justifying ongoing experiments using larger tumors at baseline with fewer T cells. In addition, in tumor-free mice, rimiducid prolonged iMC-PSCA.ζ-modified T cell engraftment and survival for 28 days compared to those mice not treated with dimerizer (p = 0.03). Importantly, following rimiducid withdrawal, CAR-T cell numbers declined, consistent with the requirement of MC-mediated costimulation in combination with CAR activation. Summary: Inducible MyD88/CD40 represents a novel activation switch that can be used to provide a controllable costimulatory signal to T cells transduced with a first generation CAR. The separation of the cytolytic signal 1 (CD3ζ) domain from a potent, regulatable, signal 2 costimulation (iMC) in the novel platform, called "GoCAR-T", allows the expansion of T cells only in response to both rimiducid and tumor antigen, and their decrease in number by withdrawal of rimiducid-induced iMC costimulation. The "GoCAR-T" platform may allow the development of a new generation of more effective CAR-T cell therapies. Disclosures Foster: Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Morschl:Bellicum Pharmaceuticals: Employment. Shaw:Bellicum Pharmaceuticals: Employment. Saha:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.


2021 ◽  
Vol 8 ◽  
Author(s):  
Li Du ◽  
Yaru Nai ◽  
Meiying Shen ◽  
Tingting Li ◽  
Jingjing Huang ◽  
...  

Adoptive immunotherapy using CAR-T cells is a promising curative treatment strategy for hematological malignancies. Current manufacture of clinical-grade CAR-T cells based on lentiviral/retrovirus transfection of T cells followed by anti-CD3/CD28 activation supplemented with IL-2 has been associated with low transfection efficiency and usually based on the use of terminally differentiated effector T cells. Thus, improving the quality and the quantity of CAR-T cells are essential for optimizing the CAR-T cell preparation. In our study, we focus on the role of IL-21 in the γc cytokine conditions for CAR-T cell preparation. We found for the first time that the addition of IL-21 in the CAR-T preparation improved T cell transfection efficiency through the reduction of IFN-γ expression 24–48 h after T cell activation. We also confirmed that IL-21 enhanced the enrichment and expansion of less differentiated CAR-T cells. Finally, we validated that IL-21 improved the CAR-T cell cytotoxicity, which was related to increased secretion of effector cytokines. Together, these findings can be used to optimize the CAR-T cell preparation.


2021 ◽  
Vol 12 ◽  
Author(s):  
Laura Grunewald ◽  
Tobias Lam ◽  
Lena Andersch ◽  
Anika Klaus ◽  
Silke Schwiebert ◽  
...  

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4551-4551 ◽  
Author(s):  
Sandeep K Srivastava ◽  
Sandhya R. Panch ◽  
Jianjian Jin ◽  
Haneen Shalabi ◽  
Nirali N. Shah ◽  
...  

Abstract Introduction: As clinical applications for Chimeric Antigen Receptor (CAR) T-cell therapy expand, cell manufacturing incorporating closed-system, automated instruments are supplanting traditional open-system, labor-intensive culture methods. At our institution and others, the CliniMACS Prodigy (Miltenyi Biotec), a closed-system automated device, has demonstrated success in the production of CAR T-cells from T-cell enrichment, activation, viral transduction, and expansion to downstream harvest for cryopreservation/fresh infusion. However, the duration of T-cell activation/viral transduction, and total T-cell culture duration are variable across centers (2-5 days and 7-13 days) and merit evaluation prior to routine use. Methods: Following the opening of our clinical protocol (Clinicaltrials.gov NCT03448393), CD19/CD22 Bispecific CAR T-cell products were manufactured on the Prodigy for 4 patients (Original Method, OM) but CAR T cell manufacturing was felt to be suboptimal. Consequently, we investigated a modified processing method (Modified Method, MM), for the manufacture of clinical grade products using the Prodigy. Specifically, TransAct CD3/CD28 reagent mediated T-cell activation/stimulation and lentiviral transduction (MSCV-CAR1922-WPRE; Lentigen Inc.) was terminated with a wash step at Day 3 (instead of the wash step at Day 5, as in the OM). Overstimulation of the relatively more sensitive patient cells was proposed as a likely cause of suboptimal cell viability and expansion in the OM runs. Final cell harvest was planned between culture days 7-12. A total of 4 apheresis products were evaluated using this MM and compared with the 4 prior runs using the OM. All products were obtained from live or deceased patients with disease profiles similar to patients on the clinical trial. Other process parameters (enrichment for CD4/CD8 subsets, in-process media changes with GMP-TexMACS Medium supplemented with human IL-2 (200IU/mL) and 3% human AB serum) were kept unchanged across the 2 methods. Transduction by Protein L expression, viability and cell phenotype (CD3, CD4/CD8) were measured by flow cytometry. Results: From ~0.1x109 CD4/8 enriched T-cells placed into the Prodigy culture chamber on day 0, the mean viable Total Nucleated Cells (TNC) obtained in the final product was 1.93x109 ± 0.27x109 in the 4 MM runs. This cell dose was accomplished by culture Day 7. In contrast, in the OM runs, the mean viable TNC obtained in the final products between Days 9 and 12 was 0.8x109 ± 0.7x109 (Figure 1a). Viable CAR transduced CD3+ fold increase was calculated for days 0-7 of the MM cultures and 0-9 and 0-12 of the OM cultures depending on day of harvest and for the 4 MM products the average fold increase was 15.3 ± 4.2 by Day 7 (Figure 1b) which was ~3 fold greater than OM products harvested on day 9 or 12. Viability of transduced cells was >80% throughout MM culture. In contrast, viability was about 31% during manufacturing of one of the OM products (Figure 1c). On the day of harvest, >99% of the cells were CD3+ T-cells for all 4 MM products (Figure 1d) with no remaining CD19+CD22+ cells. The CD4/CD8 ratio was as expected and favored CD4 T-cells over CD8 T-cells (Figure 1e). Transduction efficiency based on Protein L binding was >70% for the MM products and passed clinical release criteria (Figure 1f). A head-to-head comparison of the 2 methods from the same starting fraction in one patient product (Figure 1, 3A, 3B) also confirmed all findings above. Conclusions: Our data demonstrate that the modified CD19/CD22 Bispecific CAR T-cell manufacturing method (MM) which terminated T-cell activation/transduction by culture Day 3, resulted in reproducible and robust CAR T cell production, even in the relatively more sensitive patient cells. Viability, Viable TNC recovery, CD3% and Protein L expression were consistently higher with the MM compared to the OM. All final products in the MM met product release criteria. In addition, final product dose requirements were consistently met by culture Day 7 when using the MM, augmenting process efficiency. Consequently, we have adopted the MM for the manufacture of clinical CD19/CD22 Bispecific CAR T cells. However, to determine if this change effects CAR T cell potency, studies have been initiated to compare differences in T-cell subsets, activation/exhaustion/senescence and differentiation markers, and the metabolic activity of cells manufactured by the 2 methods. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 736-736
Author(s):  
Kole Degolier ◽  
Jennifer Cimons ◽  
Michael Yarnell ◽  
Mark Eric Kohler ◽  
Terry J. Fry

Abstract 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. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.


2021 ◽  
Vol 118 (9) ◽  
pp. e2019285118
Author(s):  
Geoff P. O’Donoghue ◽  
Lukasz J. Bugaj ◽  
Warren Anderson ◽  
Kyle G. Daniels ◽  
David J. Rawlings ◽  
...  

T cells experience complex temporal patterns of stimulus via receptor–ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide–presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways.


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