Next-Generation Manufacturing Protocols Enriching TSCM CAR T Cells Can Overcome Disease-Specific T Cell Defects in Cancer Patients

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, 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 Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

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Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Blood ◽

10.1182/blood-2019-129998 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3931-3931

Author(s):

Martina Fontaine ◽

Benjamin Demoulin ◽

Simon Bornschein ◽

Susanna Raitano ◽

Steve Lenger ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Next Generation ◽

Activated T Cells ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Anti Tumor Activity ◽

Nkg2d Receptor

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

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Visualization of human T lymphocyte-mediated eradication of cancer cells in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009092117 ◽

2020 ◽

Vol 117 (37) ◽

pp. 22910-22919

Author(s):

Xingkang He ◽

Xin Yin ◽

Jing Wu ◽

Stina L. Wickström ◽

Yanhong Duo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cancer Cells ◽

Cancer Patients ◽

Metastatic Cancer ◽

Car T Cells ◽

Human T Cell ◽

Car T ◽

Metastatic Cancer Cells

Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.

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ATA3219: A Potent Next-Generation Allogeneic Off-the-Shelf CD19-CAR T Therapy without the Need for Gene-Editing

Blood ◽

10.1182/blood-2020-141262 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-9

Author(s):

C Pham ◽

T Spindler ◽

E Hwang ◽

A Brito ◽

Y Bulliard ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Gene Editing ◽

Next Generation ◽

Publicly Traded ◽

Car T Cells ◽

Car T ◽

Signaling Domain

INTRODUCTION The development of allogeneic CD19 chimeric antigen receptor (CAR) T cells from healthy donors is a significant focus in cell therapy and is anticipated to overcome the technical and logistical challenges associated with autologous CAR-T cells. Unlike gene-edited approaches, which require inactivation of the endogenous αβ T cell receptor to reduce the risk of Graft-versus-Host Disease (GvHD), allogeneic Epstein-Barr Virus (EBV)-targeted T cells represent a clinically-advanced treatment modality that, to-date, has demonstrated a favorable safety profile with limited risks of GvHD or cytokine release syndrome [Prockop et al. JCI, 2020; Prockop et al. Blood, 2019] . As an allogeneic CAR T cell platform, EBV T cells represent a unique composition that retains critical transducibility and function, and minimizes risks for GvHD and other host interactions, without requiring complex gene editing or other cell engineering approaches to facilitate use in the allogeneic setting. Recent clinical experience with allogeneic CD19 CAR-modified EBV T cells have further supported safe and effective clinical experience in the context of B cell malignancies [Curran KJ et al. TCT 2020]. Recent advances in next-generation stimulatory domains also represent potential for improvement on current CAR-T therapies. Specifically, a modified CD3ζ domain retaining signaling capacity in 1 of 3 immune-receptor-tyrosine-based-activation-motif (ITAM) regions (referred to as 1XX) is designed to extend functional persistence without compromising potency via calibration of antigen induced CAR signaling intensity to more physiologic levels [Feucht et al. Science Trans Med 2018]. Here, we describe the first preclinical evaluation of ATA3219, a next-generation allogeneic CD19 CAR T cell therapy, combining a non-edited allogeneic EBV T cell approach with a CAR signaling domain designed to improve upon the currently clinically validated CD19 targeted CAR therapies. METHODS and RESULTS We generated EBV T cells engineered with a CD19-targeted CAR containing a modified CD3ζ signaling domain, 1XX (CD19-1XX CAR+ EBV T cells). CD19-1XX CAR+ EBV T cells demonstrate high CAR expression, polyfunctionality, expansion and in vitro potency through HLA-independent killing of CD19+ targets. Furthermore, CD19-1XX CAR+ EBV T cells demonstrate highly potent antitumor activity in an established disseminated tumor model of acute lymphoblastic leukemia and is associated with long-term persistence of the product. No treatment-related toxicities were observed in this animal model. CONCLUSIONS This preclinical dataset for CD19-1XX CAR+ EBV T cells demonstrate, persistence, polyfunctional phenotype and efficient targeting of CD19-expressing tumor cells, both in vitro and in vivo, with limited allocytoxicity against antigen-negative, HLA-mismatched targets. These findings support advancing ATA3219 to clinical evaluation. Disclosures Pham: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Spindler:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Hwang:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Brito:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Bulliard:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Aftab:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.136 ◽

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.

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First Results from the Dose Escalation Segment of the Phase I Clinical Study Evaluating Cyad-02, an Optimized Non Gene-Edited Engineered NKG2D CAR T-Cell Product, in Relapsed or Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients

Blood ◽

10.1182/blood-2020-139667 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36 ◽

Cited By ~ 1

Author(s):

Dries Deeren ◽

Johan A. Maertens ◽

Tara Lin ◽

Yves Beguin ◽

Benjamin Demoulin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Phase I ◽

Research Funding ◽

Next Generation ◽

Cell Product ◽

Car T Cells ◽

Car T Cell ◽

Car T

Background T-cells engineered to express a chimeric antigen receptor (CAR) based on the NKG2D receptor (NKG2D CAR) targeting the 8 NKG2D ligands (MICA/B, ULBP1-6) over-expressed by a large variety of malignancies have been developped to treat patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Previously, CYAD-01, the first generation of NKG2D CAR T-cell products, was evaluated in several Phase I clinical trials and showed initial signals of objective clinical responses in patients with r/r AML and MDS, albeit with short durability. Preclinical data have shown that NKG2D ligands MICA and MICB are transiently upregulated on activated CAR T-cells, and target-dependent killing of CAR T-cells post-infusion can potentially occur, leading to short in vivo persistence. In an effort to increase the persistence and potency of the NKG2D CAR T-cells, CYAD-02 was developed as a next-generation product using a non-gene editing approach to silence the expression of MICA and MICB. Aim MICA and MICB were down-regulated by inserting a single optimized short hairpin RNA (shRNA) targeting both MICA and MICB within the NKG2D CAR construct. This next-generation NKG2D CAR T-cell product is manufactured with the OptimAb process, resulting in CAR T-cells with a higher frequency of early memory T-cells secreting high levels of cytokines upon activation, and is referred to as CYAD-02. Results As compared to CYAD-01, CYAD-02 cell expansion in vitro was 3-fold increased. In an in vivo AML model, CYAD-02 showed 10-fold higher engraftment 1 week after injection and improved anti-tumor activity as compared to CYAD-01 manufactured with the initial mAb process. This led to a 2.6-fold increase of mouse survival as compared to CYAD-01 in a stress-test aggressive AML model where the dose of CYAD-01 was titrated down for minimal activity (figure). The first-in-human study evaluating CYAD-02, the CYCLE-1 study (NCT04167696), has been initiated in early 2020 in patients with r/r AML/MDS. The study evaluates three dose-levels of CYAD-02 (1x108, 3x108 and 1x109 cells/infusion), administered as a single infusion after non-myeloablative preconditioning chemotherapy (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days, CyFlu) according to a classical Fibonacci 3+3 design. As of August 2020, 6 patients have been treated with CYAD-02 at the dose of 1x108 or 3x108 cells/infusion. To date, the results demonstrate the safety and tolerability for CYAD-02 in patients with r/r AML and MDS with no dose-limiting toxicity observed. The study is currently enrolling at 1x109 cells/infusion. The CYAD-02 safety profile and preliminary clinical activity data together with the pharmacokinetics evaluation from the complete dose escalation segment will be provided at the time of presentation. Conclusion/summary The CYAD-02 is the first autologous CAR T-cell product based on the non-gene edited shRNA technology used to treat patients. This next generation NKG2D CAR T-cell product is currently investigated in the CYCLE-1 Phase I study in r/r AML/MDS patient population, a difficult to target disease due in part to the absence of truly AML-specific surface antigens, its rapid clinical progression and the absence of disease control by the CyFlu preconditioning. Both the anti-MICA and MICB shRNA hairpin and the OptimAb manufacturing process for CYAD-02 aim to improve CAR T-cell persistence and clinical responses. Figure Disclosures Lin: Mateon Therapeutics: Research Funding; Aptevo: Research Funding; Abbvie: Research Funding; Ono Pharmaceutical: Research Funding; Incyte: Research Funding; Gilead Sciences: Research Funding; Jazz: Research Funding; Astellas Pharma: Research Funding; Bio-Path Holdings: Research Funding; Celgene: Research Funding; Celyad: Research Funding; Genetech-Roche: Research Funding; Seattle Genetics: Research Funding; Tolero Pharmaceuticals: Research Funding; Trovagene: Research Funding; Prescient Therapeutics: Research Funding; Pfizer: Research Funding. Demoulin:Celyad Oncology: Current Employment. Fontaine:Celyad Oncology: Current Employment. Sotiropoulou:Celyad Oncology: Current Employment. Alcantar-Orozco:Celyad Oncology: Current Employment. Breman:Celyad Oncology: Current Employment. Dheur:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

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Chimeric Antigen Receptor-Engineered T-Cells - A New Way and Era for Lymphoma Treatment

Recent Patents on Anti-Cancer Drug Discovery ◽

10.2174/1574892814666191022164641 ◽

2020 ◽

Vol 14 (4) ◽

pp. 312-323

Author(s):

Romeo G. Mihăilă

Keyword(s):

T Cells ◽

T Cell ◽

Response Rate ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Car T Cells ◽

Future Developments ◽

Car T Cell ◽

Large B Cell Lymphoma ◽

Car T

Background: Patients with refractory or relapsed diffuse large B-cell lymphoma have a poor prognosis with the current standard of care. Objective: Chimeric Antigen Receptor T-cells (CAR T-cells) are functionally reprogrammed lymphocytes, which are able to recognize and kill tumor cells. The aim of this study is to make progress in this area. Method: A mini-review was achieved using the articles published in Web of Science and PubMed in the last year and the new patents were made in this field. Results: The responses to CAR T-cell products axicabtagene ciloleucel and tisagenlecleucel are promising; the objective response rate can reach up to 83%, and the complete response rate ranges between 40 and 58%. About half of the patients may have serious side effects, such as cytokine release syndrome and neurotoxicity. Current and future developments include the improvement of CAR T-cell expansion and polyfunctionality, the combined use of CAR T-cells with a fusion protein between interferon and an anti-CD20 monoclonal antibody, with checkpoint inhibitors or small molecule sensitizers that have apoptotic-regulatory effects. Furthermore, the use of IL-12-expressing CAR T-cells, an improved technology for the production of CAR T-cells based on targeted nucleases, the widespread use of allogeneic CAR T-cells or universal CAR T-cells obtained from genetically engineered healthy donor T-cells are future developments actively considered. Conclusion: CAR T-cell therapy significantly improved the outcome of patients with relapsed or refractory diffuse large B-cell lymphoma. The advances in CAR T-cells production technology will improve the results and enable the expansion of this new immunotherapy.

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Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals

International Journal of Molecular Sciences ◽

10.3390/ijms22052476 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2476

Author(s):

Kento Fujiwara ◽

Masaki Kitaura ◽

Ayaka Tsunei ◽

Hotaka Kusabuka ◽

Erika Ogaki ◽

...

Keyword(s):

Signal Transduction ◽

T Cells ◽

T Cell ◽

Second Generation ◽

Independent Manner ◽

Cell Functions ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

The Impact

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.

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Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

Biomarker Research ◽

10.1186/s40364-021-00264-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Laura Castelletti ◽

Dannel Yeo ◽

Nico van Zandwijk ◽

John E. J. Rasko

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Malignant Mesothelioma ◽

Oncolytic Viruses ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

AbstractMalignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.

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Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001877 ◽

2021 ◽

Vol 9 (3) ◽

pp. e001877

Author(s):

Irfan N Bandey ◽

Jay R T Adolacion ◽

Gabrielle Romain ◽

Melisa Martinez Paniagua ◽

Xingyue An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Adoptive Cell Therapy ◽

Population Heterogeneity ◽

Serial Killer ◽

Car T Cells ◽

Car T ◽

Single Cell Profiling ◽

Killer T Cells

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

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