Abbreviated T-Cell Activation on the Automated Clinimacs Prodigy Device Enhances Bispecific CD19/22 Chimeric Antigen Receptor T-Cell Viability and Fold Expansion, Reducing Total Culture Duration

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.

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P07.02 Regulation of CD19 CAR T- cell activation based on engineered Nuclear factor of activated T cells artificial transcription factors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.43 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A23-A23

Author(s):

D Lainš&ccaron;ek ◽

V Mikoli&ccaron; ◽

Š Malenšek ◽

A Verbi&ccaron; ◽

R Jerala

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cell Activity ◽

External Control ◽

Car T Cells ◽

Car T Cell ◽

Artificial Transcription Factors ◽

Car T

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. Lainš&ccaron;ek: None. V. Mikoli&ccaron;: None. Š. Malenšek: None. A. Verbi&ccaron;: None. R. Jerala: None.

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Generating Chimeric Antigen Receptors Utilizing Novel Anti-CD3 Nanobeads

Blood ◽

10.1182/blood.v124.21.5949.5949 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5949-5949

Author(s):

Liora M. Schultz ◽

Debra K Czerwinski ◽

Aihua Fu ◽

Shoshana Levy ◽

Ronald Levy

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Antigen Receptors ◽

Chimeric Antigen Receptors ◽

Car T Cell ◽

Car T ◽

Naïve T Cell ◽

Naive T Cell

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.

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Mutation of the CD28 Costimulatory Domain Confers Decreased CAR T Cell Exhaustion

Blood ◽

10.1182/blood-2018-99-110645 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 966-966 ◽

Cited By ~ 2

Author(s):

Justin C. Boucher ◽

Gongbo Li ◽

Bishwas Shrestha ◽

Maria Cabral ◽

Dylan Morrissey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Car T Cells ◽

Car T Cell ◽

Car T

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.

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

10.1101/2022.01.14.476364 ◽

2022 ◽

Author(s):

Vardges Tserunyan ◽

Stacey D Finley

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Response ◽

Target Antigen ◽

Biological Variability ◽

Car T Cell ◽

Car T ◽

Engineered T Cells

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.

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Inducible MyD88/CD40 Allows Rimiducid-Dependent Activation to Control Proliferation and Survival of Chimeric Antigen Receptor-Modified T Cells

Blood ◽

10.1182/blood.v126.23.4295.4295 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4295-4295 ◽

Cited By ~ 1

Author(s):

Aaron Foster ◽

Aruna Mahendravada ◽

Nicholas P Shinners ◽

Peter Chang ◽

An Lu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Cell Activation ◽

First Generation ◽

Car T Cell ◽

Car T ◽

Equity Ownership

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.

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IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.675179 ◽

2021 ◽

Vol 8 ◽

Author(s):

Li Du ◽

Yaru Nai ◽

Meiying Shen ◽

Tingting Li ◽

Jingjing Huang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Transfection Efficiency ◽

Cytotoxic Activities ◽

Cell Preparation ◽

Car T Cells ◽

Car T Cell ◽

Car T

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.

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A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization

Frontiers in Immunology ◽

10.3389/fimmu.2021.689697 ◽

2021 ◽

Vol 12 ◽

Author(s):

Laura Grunewald ◽

Tobias Lam ◽

Lena Andersch ◽

Anika Klaus ◽

Silke Schwiebert ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Three Dimensional ◽

Human Tumor ◽

Analysis Tool ◽

Car T Cells ◽

Car T Cell ◽

Car T

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.

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Engineering γδT cells limits tonic signaling associated with chimeric antigen receptors

Science Signaling ◽

10.1126/scisignal.aax1872 ◽

2019 ◽

Vol 12 (598) ◽

pp. eaax1872 ◽

Cited By ~ 5

Author(s):

Jonathan Fisher ◽

Roshan Sharma ◽

Dilu Wisidagamage Don ◽

Marta Barisa ◽

Marina Olle Hurtado ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

T Cell Activation ◽

Time Course ◽

Cell Activation ◽

Leukemic Cells ◽

Γδt Cells ◽

Car T Cell ◽

Car T

Despite the benefits of chimeric antigen receptor (CAR)–T cell therapies against lymphoid malignancies, responses in solid tumors have been more limited and off-target toxicities have been more marked. Among the possible design limitations of CAR-T cells for cancer are unwanted tonic (antigen-independent) signaling and off-target activation. Efforts to overcome these hurdles have been blunted by a lack of mechanistic understanding. Here, we showed that single-cell analysis with time course mass cytometry provided a rapid means of assessing CAR-T cell activation. We compared signal transduction in expanded T cells to that in T cells transduced to express second-generation CARs and found that cell expansion enhanced the response to stimulation. However, expansion also induced tonic signaling and reduced network plasticity, which were associated with expression of the T cell exhaustion markers PD-1 and TIM-3. Because this was most evident in pathways downstream of CD3ζ, we performed similar analyses on γδT cells that expressed chimeric costimulatory receptors (CCRs) lacking CD3ζ but containing DAP10 stimulatory domains. These CCR-γδT cells did not exhibit tonic signaling but were efficiently activated and mounted cytotoxic responses in the presence of CCR-specific stimuli or cognate leukemic cells. Single-cell signaling analysis enabled detailed characterization of CAR-T and CCR-T cell activation to better understand their functional activities. Furthermore, we demonstrated that CCR-γδT cells may offer the potential to avoid on-target, off-tumor toxicity and allo-reactivity in the context of myeloid malignancies.

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CAR-T Cells Are Serial Killers of Tumor Cells

Blood ◽

10.1182/blood.v126.23.3088.3088 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3088-3088

Author(s):

Misty Jenkins ◽

Alex Davenport ◽

Ryan Cross ◽

Carmen Yong ◽

David S. Ritchie ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Cell Activation ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Kinetics Of ◽

I Cells

Abstract Chimeric antigen receptor (CAR) T cells have shown clinical efficacy in refractory B cell malignancies. Despite these exciting clinical results, our fundamental understanding of CAR-T cell biology is limited, possibly limiting the broader application of CAR-T cells to additional haemopoetic and solid cancers. To date, the mechanism of CAR-T immunological synapse formation with tumor cells and kinetics of subsequent serial killing by CAR-T cells has not been explored. Here, we investigated the kinetics of CAR-T cell activation and cytotoxicity, including immune synapse formation, and kinetics of tumor cell killing, closely comparing to activation and cytotoxicity via the T cell receptor (TCR). To address this, we developed a mouse model in which the CD8+ T cells (termed CAR.OT-I cells) co-expressed two antigen receptors, the clonogenic OT-I TCR, and a second generation CAR comprising a scFV to human HER2, CD28 and CD3ζ signaling domains. Effector CAR.OT-I cells were activated via their antigen receptors using either SIINFEKL-pulsed or HER-2 expressing tumor cells, the interactions between the effector CAR.OT-I cells and tumor cells were then assessed by time lapse live microscopy. CAR.OT-I cell activation via the endogenous TCR or the CAR did not affect tumor killing kinetics, except the time taken from CAR.OT-I activation to detachment (from the dying tumor cell) was significantly slower when the endogenous TCR was engaged. Subsequently, we showed for the first time, that CAR.OT-I cells have serial killing capacity, which is important to consider when therapeutic numbers of CAR-T cells are likely to be outnumbered by tumor targets. Individual CAR.OT-I cells killed multiple tumor cells, whether activated via the endogenous TCR or the CAR. We further explored whether these findings have implications for killing of tumor cells using low effector:target cell ratio in short versus long-term killing assays, chromium release and xCELLigence killing assays respectively. We observed, no matter which antigen receptor was activated, the effector CAR.OT-I cells were equivalent killers of tumor cells in short term assays (4-8 hours). However, over a period of 50 hours, CAR.OT-I cells activated via the CAR killed tumor cells at a lower rate than when activated via the TCR. This was due to CAR.OT-I CAR expression down-regulation from 20-50 hours. This study highlights that fundamental differences occur in the way CAR-T cells kill tumor cells, depending on how the effector CAR-T cell is activated. Furthermore, the study provides important insights for CAR-T cell activation in vivo with implications for single- or dual-receptor-focused CAR-T cell therapy and improved clinical benefit. Disclosures No relevant conflicts of interest to declare.

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G-CSF/Plerixafor Dual-Mobilized Donor Derived CD33CAR T-Cells As Potent and Effective AML Therapy in Pre-Clinical Models

Blood ◽

10.1182/blood-2021-153868 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1716-1716

Author(s):

Giacomo Canesin ◽

Hillary Hoyt ◽

Reid Williams ◽

Mariana Silva ◽

Melissa Chng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Immune Cell ◽

Ex Vivo ◽

Publicly Traded ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract There are currently no known acute myeloid leukemia (AML) specific antigens. Genetic ablation of CD33 using CRISPR/Cas9 engineering of the hematopoietic stem cell (HSC) transplant (VOR33) represents a synthetic biology approach to generating a leukemia-specific antigen in the transplant recipient. VOR33 enables anti-CD33 CAR-T cell killing of AML cells while sparing normal myeloid lineage development and function, thereby potentially avoiding myelosuppression and increasing the therapeutic index of anti-CD33 CAR-T therapy. Mobilized leukapheresis product represents an attractive starting material for the generation of both a CD33 null HSC transplant and a complementary CD33CAR T-cell product. In this study, we sought to determine the impact of dual mobilization with Granulocyte-Colony Stimulating Factor (G-CSF) and plerixafor (mozobil) on immune cell composition, T cell phenotype, and the functionality of these T cells to control AML tumor growth upon chimeric antigen receptor (CAR) transduction. Mobilized (mob) peripheral blood mononuclear cells (PBMCs) were collected from healthy donors injected with G-CSF (10µg/kg/day, 5 consecutive days) and plerixafor (240µg/kg, on day 4 and 5). Non-mobilized (non-mob) PBMCs, collected from the same donors, were used as controls. Cells were analyzed by flow cytometry for immunophenotyping and T cell characterization including differentiation and bone marrow homing markers, as well as responses to T cell activation with anti-CD3 (OKT3) and IL-2. Non-/mob PBMC populations were also analyzed by single-cell next generation sequencing (CITEseq) using 127 immune cell phenotypic markers in combination with extensive transcriptome and T cell receptor repertoire analysis. In addition, lentiviral transduction of anti-CD33 CAR constructs enabled functional comparisons of mob- and non-mob-CAR T-cells in AML cell co-cultures as well as AML mouse models. Ex vivo immunophenotyping of PBMC from a total of over 30 healthy donor samples showed that mobilization decreases the overall percentage of CD3 + T cells but increases that of naïve T cells (CD45RA +/CCR7 +), at the expense of T effector-memory (CD45RA -/CCR7 -) and central-memory (CD45RA -/CCR7 +) populations. Bone marrow homing factors (e.g.: CXCR4) were increased in mob compared to non-mob T cell samples. As expected, higher percentages of monocytes (CD14 +) were detected in mob compared to non-mob donor samples, but this difference disappeared after culture under T cell activation conditions. T cell activation also led to similar increases in CD25, CD69 and CD137 expression, and a decrease in CD62L expression. Single cell sequencing analyses confirmed mobilization-induced increases in naïve T cells as well as changes in monocytes/macrophages, CD4 + T cells and NK cells percentages. Notably, functional in vitro cytotoxic assays demonstrated that mob-CD33-CAR T-cells are as effective as non-mob-CD33-CAR T-cells in killing CD33 + AML cells, with reduced 'bystander' activation of non-transduced T cells. Furthermore, results from in vivo AML mouse models indicate that mob-CD33-CAR T-cells are equally effective in clearing CD33 + tumors as non-mob-CD33-CAR T-cells. Our analysis showed phenotypical ex vivo differences between mob and non-mob PBMCs, which disappeared upon activation, indicating similar responses to T cell-specific stimulation. These findings are corroborated by similar in vitro cytotoxicity profiles of non-/mob-CAR T-cells. Non-transduced T cells in the mob-CAR T-cell population showed limited 'bystander' activation, indicating a potentially favorable clinical toxicity profile. Additional in vivo assessment of mob-CAR T-cell function shows effective tumor clearance, which supports further efforts towards their clinical use in combination with engineered HSCs for the treatment of AML patients. Disclosures Canesin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hoyt: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Williams: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Silva: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chng: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ung: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Qiu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Shin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Scherer: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Kassim: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

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