scholarly journals Large-Scale Manufacturing of CMV-CD19CAR T Cells and Characterization of Their Biologic and Immunologic Properties

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3247-3247
Author(s):  
Xiuli Wang ◽  
Miriam Walter ◽  
Ryan Urak ◽  
Laura Lim ◽  
Vibhuti Vyas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Large Scale ◽  
Ex Vivo ◽  
Early Stage ◽  
Car T Cells ◽  
Car T ◽  
In Vitro Expansion

We have successfully established a clinical platform for CD19 chimeric antigen receptor (CAR) T cells and evaluated its safety and efficacy in a series of pilot clinical trials following autologous hematopoietic cell transplantation (HCT) for treatment of high-risk non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). However, the full potential of this therapy has been limited by the lack of engraftment and persistence of CAR T cells in patients, most likely due to the inadequate antigen drive to stimulate robust expansion and long-term persistence of the infused CAR T cells. Additionally, CD19+ NHL seems less responsive to current CAR T cell technology than does CD19+ ALL that might be explained by lower engraftment and persistence of CAR T cells in NHL. To improve the efficacy and durability of CAR T cells in these disease settings, we sought to examine a novel approach based on properties of cytomegalovirus (CMV)-specific T cells. Specifically, we select CMV pp65-specific T cells for ex vivo modification with a CAR targeting CD19. After in vitro expansion, CMV-CD19 bi-specific T cells will be infused into the patient. A second round of expansion will be done in vivo, using a CMV vaccine (Triplex), developed and clinically evaluated multiple clinical sites including City of Hope. Triplex is a multi-antigen recombinant modified vaccinia Ankara (MVA) virus vaccine expressing pp65, IE1 and IE2, that has proven safe and immunogenic in Phase I trials in healthy volunteers and transplant patients and was highly tolerable and efficacious in a completed Phase II vaccine trial in allogeneic HCT recipients. We hypothesis that shorter ex vivo expansion time will prevent CAR T cell exhaustion that results in better in vivo expansion, especially after Triplex vaccination. Thus far, we have performed six large-scale manufacturing process development (PD) runs of CMV-CD19 CAR T cells using T cells from healthy donors. Briefly, peripheral blood mononuclear cells (PBMCs) were collected and processed in the CliniMACS Prodigy® system, in which PBMCs were first stimulated with a GMP-grade PepTivator® overlapping CMV pp65 peptide pool, then enriched for CMV-responsive IFNɣ+ T cells using the IFNγ Catchmatrix reagent (Miltenyi Biotec Inc.). CMV-responsive IFNɣ+ T cells were next transduced with a lentiviral vector encoding EGFRt/CAR, and finally expanded for approximately 15 days in vitro. As summarized in Table 1, we consistently recovered 4.8%+/-1.4 ×106 CMV specific T cells with 78.3%±2.9 purity from 1×109 PBMC input, which is the maximum input number suggested by the CCS program associated with the Prodigy system (Miltenyi Biotec Inc.). During the early stage PD runs, we noticed that red blood cell contamination in the PBMC layer after ficoll separation could negatively impact the yield of CMV-specific T cells. Thus, we optimized the separation process by performing a second ficoll whenever a red buffy coat was observed, and increased the yield of CMV-specific T cells from 2.8 ×106 to 9.25×106 (Table 1, HD 187.2 PBMCs underwent 2 subsequent ficolls). Phenotypic analysis demonstrated that freshly isolated CMV-specific T cells consisted of four different memory subsets (TEMRA, Tscm, Tcm and effector memory T cells). After in vitro expansion for 15 days, CMV-CAR bi-specific T cell expansion was significantly improved from 60×106 cells in the early stage runs to 200×106 in the late stage optimized runs (Figure 1). The CMV-CD19CAR T cells expressed CD62L, though not exhaustion markers such as PD1. Bispecific T cells exhibited bi-functionality upon stimulation with CD19+ tumor cells or CMVpp65 antigen, as indicated by secretion of IFNγ. Interestingly, we detected stronger effector function against CD19+ tumor cells from CMV-CD19CAR T cells compared to conventional CD19CAR T cells that were derived from the same donor (Figure 2). To validate our manufacturing process, we are currently conducting IND-enabling studies using patient-derived PBMC as the starting population and will initiate our first clinical trial in early 2020 for patients with intermediate/advanced-grade B cell NHL post lymphodepletion or autologous/allogeneic HCT followed by Triplex vaccination 28 days after T cell infusion for in vivo expansion of bi-specific CAR T cells. The primary objectives of these two trials are to examine safety and persistence/expansion of CMV-CD19CAR T cell before and after Triplex vaccine boost. Disclosures Nakamura: Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: support for an academic seminar in a university in Japan; Alexion: Other: support to a lecture at a Japan Society of Transfusion/Cellular Therapy meeting ; Kirin Kyowa: Other: support for an academic seminar in a university in Japan.

scholarly journals Preservation of T-Cell Stemness with a Novel Expansionless CAR-T Manufacturing Process, Which Reduces Manufacturing Time to Less Than Two Days, Drives Enhanced CAR-T Cell Efficacy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2848-2848
Author(s):  
Boris Engels ◽  
Xu Zhu ◽  
Jennifer Yang ◽  
Andrew Price ◽  
Akash Sohoni ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antitumor Efficacy ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background: Extended T-cell culture periods in vitro deplete the CAR-T final product of naive and stem cell memory T-cell (T scm) subpopulations that are associated with improved antitumor efficacy. YTB323 is an autologous CD19-directed CAR-T cell therapy with dramatically simplified manufacturing, which eliminates complexities such as long culture periods. This improved T-Charge™ process preserves T-cell stemness, an important characteristic closely tied to therapeutic potential, which leads to enhanced expansion ability and greater antitumor activity of CAR-T cells. Methods: The new T-Charge TM manufacturing platform, which reduces ex vivo culture time to about 24 hours and takes <2 days to manufacture the final product, was evaluated in a preclinical setting. T cells were enriched from healthy donor leukapheresis, followed by activation and transduction with a lentiviral vector encoding for the same CAR used for tisagenlecleucel. After ≈24 hours of culture, cells were harvested, washed, and formulated (YTB323). In parallel, CAR-T cells (CTL*019) were generated using a traditional ex vivo expansion CAR-T manufacturing protocol (TM process) from the same healthy donor T cells and identical lentiviral vector. Post manufacturing, CAR-T products were assessed in T-cell functional assays in vitro and in vivo, in immunodeficient NSG mice (NOD-scid IL2Rg-null) inoculated with a pre-B-ALL cell line (NALM6) or a DLBCL cell line (TMD-8) to evaluate antitumor activity and CAR-T expansion. Initial data from the dose escalation portion of the Phase 1 study will be reported separately. Results: YTB323 CAR-T products, generated via this novel expansionless manufacturing process, retained the immunophenotype of the input leukapheresis; specifically, naive/T scm cells (CD45RO -/CCR7 +) were retained as shown by flow cytometry. In contrast, the TM process with ex vivo expansion generated a final product consisting mainly of central memory T cells (T cm) (CD45RO +/CCR7 +) (Fig A). Further evidence to support the preservation of the initial phenotype is illustrated by bulk and single-cell RNA sequencing experiments, comparing leukapheresis and final products from CAR-Ts generated using the T-Charge™ and TM protocols. YTB323 CAR-T cell potency was assessed in vitro using a cytokine secretion assay and a tumor repeat stimulation assay, designed to test the persistence and exhaustion of the cell product. YTB323 T cells exhibited 10- to 17-fold higher levels of IL-2 and IFN-γ secretion upon CD19-specific activation compared with CTL*019. Moreover, YTB323 cells were able to control the tumor at a 30-fold lower Effector:Tumor cell ratio and for a minimum of 7 more stimulations in the repeat stimulation assay. Both assays clearly demonstrated enhanced potency of the YTB323 CAR-T cells in vitro. The ultimate preclinical assessment of the YTB323 cell potency was through comparison with CTL*019 regarding in vivo expansion and antitumor efficacy against B-cell tumors in immunodeficient NSG mouse models at multiple doses. Expansion of CD3+/CAR+ T-cells in blood was analyzed weekly by flow cytometry for up to 4 weeks postinfusion. Dose-dependent expansion (C max and AUC 0-21d) was observed for both YTB323 and CTL*019. C max was ≈40-times higher and AUC 0-21d was ≈33-times higher for YTB323 compared with CTL*019 across multiple doses. Delayed peak expansion (T max) of YTB323 by at least 1 week compared with CTL*019 was observed, supporting that increased expansion was driven by the less differentiated T-cell phenotype of YTB323. YTB323 controlled NALM6 B-ALL tumor growth at a lower dose of 0.1×10 6 CAR+ cells compared to 0.5×10 6 CAR+ cells required for CTL*019 (Fig B). In the DLBCL model TMD-8, only YTB323 was able to control the tumors while CTL*019 led to tumor progression at the respective dose groups. This ability of YTB323 cells to control the tumor at lower doses confirms their robustness and potency. Conclusions: The novel manufacturing platform T-Charge™ used for YTB323 is simplified, shortened, and expansionless. It thereby preserves T-cell stemness, associated with improved in vivo CAR-T expansion and antitumor efficacy. Compared to approved CAR-T therapies, YTB323 has the potential to achieve higher clinical efficacy at its respective lower doses. T-Charge™ is aiming to substantially revolutionize CAR-T manufacturing, with concomitant higher likelihood of long-term deep responses. Figure 1 Figure 1. Disclosures Engels: Novartis: Current Employment, Current equity holder in publicly-traded company. Zhu: Novartis: Current Employment, Current equity holder in publicly-traded company. Yang: Novartis: Current Employment, Patents & Royalties. Price: Novartis: Current Employment. Sohoni: Novartis: Current Employment. Stein: Novartis: Current Employment. Parent: Novartis: Ended employment in the past 24 months; iVexSol, Inc: Current Employment. Greene: iVexSol, Inc: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Niederst: Novartis: Current Employment, Current equity holder in publicly-traded company. Whalen: Novartis: Current Employment. Orlando: Novartis: Current Employment. Treanor: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: no royalties as company-held patents. Brogdon: Novartis Institutes for Biomedical Research: Current Employment.

scholarly journals Physiological (TCR-like) regulated lentiviral vectors for the generation of improved CAR-T cells

2021 ◽  
Author(s):  
Maria Tristan-Manzano ◽  
Noelia Maldonado-Perez ◽  
Pedro Luis Justicia-Lirio ◽  
Pilar Munoz ◽  
Marina Cortijo-Gutierrez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Side Effects ◽  
Large Scale ◽  
Proximal Promoter ◽  
Car T Cells ◽  
Car T ◽  
Was Gene

Background. Chimeric antigen receptor (CAR) T cells directed against CD19 have achieved impressive outcomes for the treatment of relapsed/refractory B lineage lymphoid neoplasms. However, CAR-T therapy still has important limitations due to severe side effects and the lack of efficiency in 40-50% of the patients. Most CARs-T products are generated using retroviral vectors with strong promoters. However, high CAR expression levels can lead to tonic signalling, premature exhaustion and over-stimulation of CAR-T cells, reducing efficacy and increasing side effects. TCR-like expression of the CAR through genome editing resulted in enhanced anti-tumour potency, reducing tonic signalling and improving CAR-T phenotype. In this manuscript, we searched for LVs that mimic the TCR expression pattern as a closer-to-clinic alternative for the generation of improved CAR-T cells. Methods. Different LVs containing viral and human promoters were analysed to select those that closely mimic a TCR-like kinetic profile upon T-cell activation. WAS gene proximal promoter-driven LVs (AW-LVs) were selected to express a second generation 4-1BB CD19 CAR (ARI-0001) into T cells to generate AWARI αCAR-T cells. TCR-like AWARI and EF1α-driven ARI CAR T cells were analysed for in vitro and in vivo killing efficiency using leukaemia and lymphoma cellular models. Tonic signalling, exhaustion markers and phenotype were determined by flow cytometry. Large-scale automated manufacturing of AWARI CAR-T cells was performed in a CliniMACs Prodigy bioreactor. Results. Our data showed that LVs expressing the transgene through the WAS gene proximal promoter mimic very closely the TCR (CD3) expression pattern kinetic upon TCR stimulation or antigen encounter. Compared to EF1α-driven ARI CAR-T cells, AWARI CAR-T cells exhibited a higher proportion of naive/stem cell memory T cells with less exhausted phenotype after efficient killing of CD19+ cells both in vitro and in vivo. AWARI CAR-T cells also showed lower tonic signalling and reduced secretion of pro-inflammatory cytokines and were efficiently manufactured in large-scale GMP-like conditions. Conclusions. WAS-gene-promoter driven LVs can be used to generate physiological 4-1BB-CAR-T cell products with lower tonic signalling, improved phenotype and a safer profile. We propose the use of TCR-like LVs as an alternative to strong-promoter driven LVs for the generation of CAR-T products.

Ex Vivo AKT Inhibition Promotes the Generation of Potent CD19CAR T Cells for Adoptive Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3086-3086
Author(s):  
Ryan Urak ◽  
ChingLam Wong ◽  
Wen-Chung Chang ◽  
Elizabeth E. Budde ◽  
Christine Brown ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Ex Vivo ◽  
Akt Inhibitor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Abstract Insufficient persistence and effector function of Chimeric Antigen Receptor (CAR) re-directed T cells in vivo has been a challenge for adoptive T cell therapy. Generation of long-lived potent CAR T cells is an increasing demand in the field. AKT activation triggered by convergent extracellular signals evokes a transcription program that enhances effector functions. However, sustained AKT activation severely impairs T cell memory and protective immunity because AKT drives differentiation of effectors, therefore diminishing T cell potential to survive and differentiate into memory cells. We now investigate whether inhibition of AKT signaling during ex vivo expansion can prevent terminal differentiation of CD19- chimeric antigen receptor (CD19 CAR) engineered T cells and increase the number of memory CD19 CAR T cells, which would enhance the antitumor activity following adoptive therapy. CD8+ T cells from healthy donors were isolated, activated with CD3/CD28 beads, and then transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain and two mutations (L235E; N297Q) within the CH2 region on the IgG4-Fc spacers which enhances potency and persistence by blocking Fc receptor binding. In addition, the lentiviral construct also expresses a truncated human epidermal growth factor receptor (huEGFRt) which allows us to use as a selectable marker and a mechanism to ablate the CAR T cells if necessary. IL-2 (50U/mL) and AKT inhibitor (1uM/mL) were supplemented every other day. Transduced CD19CAR T cells without AKT inhibitor treatment were used as controls. The engineered CD19CAR T cells were expanded in vitro for 21 days before in vitro and in vivo analyses. We found that AKT inhibitor did not compromise the CD19CAR T cell proliferation and survival in vitro. There was a comparable CD19CAR T cell expansion after culturing in the presence or absence AKT inhibitor. Functionally, AKT inhibitor did not dampen the effector function of CD19CAR T cells as indicated by equivalent levels of interferon gamma production and CD107a expression upon CD19 antigen stimulation. Memory-like phenotype such as CD62L and CD28 expression on CAR T cells is associated with better antitumor activity in vivo. We therefore characterized the CD19CAR T cells after ex vivo expansion. We found that 40% of AKT-inhibited CD19CAR T cells expressed CD62L and co-expressed CD28. More importantly, no exhaustion markers such as KRLG and PD-1 were induced on the AKT inhibitor treated cells. In contrast, only 10% of control untreated CD19CAR T cells expressed CD62L and they were CD28 negative, indicating that AKT-inhibited CD19CAR T cells with higher levels of CD62L and CD28 expression may have superior anti-tumor activity following adoptive transfer. To test the potency of the AKT inhibitor treated CAR T cells, 0.5x106 CD19+ acute lymphoid leukemic cells (SupB15) engineered to express firefly luciferase were inoculated intravenously into NOD/Scid IL-2RgammaCnull (NSG) mice. Five days post tumor engraftment, 2x106 CD8+ CD19CAR T cells were intravenously injected into tumor bearing mice. Control mice received either no T cells, non-transduced T cells (Mock), or CD19CAR T cells that were not treated with AKT inhibitor during in vitro expansion. Tumor signals post T cell infusion were monitored by biophotonic imaging. Compared to the untreated CD19CAR T cells, which exhibited lower and transient anti-tumor activity, AKT inhibitor treated CD19CAR T cells completely eradicated the CD19+ tumor in all mice (Figure 1) 21 days post CD19CAR T cell infusion. In conclusion, our results demonstrate that inhibition of AKT signaling during the ex vivo priming and expansion gives rise to a CD19CAR T cell population that possesses superior antitumor activity. These findings suggest that ex vivo therapeutic modulation of AKT might be a strategy to augment antitumor immunity for adoptive CAR T cell therapy, which could easily be transitioned into the clinic with the availability of pharmaceutical grade AKT inhibitor. Disclosures Forman: Amgen: Consultancy; Mustang: Research Funding.

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

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

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.

scholarly journals Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽  
2021 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Base Editing ◽  
Car T Cells ◽  
Dna And Rna ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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

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

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

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

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

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

scholarly journals Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

Blood ◽  
2019 ◽  
Vol 133 (21) ◽  
pp. 2291-2304 ◽  
Author(s):  
Diego Sánchez-Martínez ◽  
Matteo L. Baroni ◽  
Francisco Gutierrez-Agüera ◽  
Heleia Roca-Ho ◽  
Oscar Blanch-Lombarte ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Antileukemic Activity ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

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

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

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

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