scholarly journals 958 BNT211: a phase I/II trial to evaluate safety and efficacy of CLDN6 CAR-T cells and vaccine-mediated in vivo expansion in patients with CLDN6-positive advanced solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A1008-A1008
Author(s):  
Andreas Mackensen ◽  
Christian Koenecke ◽  
John Haanen ◽  
Winfried Alsdorf ◽  
Alexander Desuki ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Specific Antigen ◽  
Unknown Primary ◽  
Car T Cells ◽  
Car T Cell ◽  
Flat Dose ◽  
Car T

BackgroundBNT211 is a chimeric antigen receptor (CAR)-T cell product candidate that targets the tumor specific antigen Claudin-6 (CLDN6). Preclinical studies demonstrated that combining these engineered cells with a CAR-T cell Amplifying RNA Vaccine (CARVac) leads to in vivo expansion of adoptively transferred CAR-T cells, resulting in their improved persistence and functionality.MethodsThis first-in-human, open label, multi-center trial involves a bifurcated 3+3 design with separate CLDN6 CAR-T cell dose escalations (single flat-dose) for monotherapy (part 1) and the combination with CARVac (part 2) based on 3 dose levels (DL). In part 2, CARVac is applied every 3 weeks starting at day 4 post transplantation including a one-step intra-patient dose escalation. Patients with CLDN6-positive relapsed or refractory solid tumors without further standard treatment options and ECOG 0 or 1 are eligible for recruitment.ResultsAs of July 23rd 2021, 8 patients have been treated. DL1 of part 1 has been completed, while dosing of part 1 DL2 and part 2 DL1 is ongoing. One patient with cancer of unknown primary was treated with a dose below DL1 in combination with CARVac; the underlying diseases of the other 7 treated patients were testicular, ovarian and endometrial cancer as well as soft-tissue sarcoma. No acute or dose-limiting toxicities and no serious adverse events related to the drug product have been reported. Manageable cytokine release syndrome (CRS, grade 1-2, the latter managed with Tocilizumab) without any signs of neurotoxicity have been observed in both patients of part 1 DL2. Only transient and moderate elevations of IL-6 and CRP serum levels occurred in remaining patients. Notably, administration of CARVac resulted in transient flu-like symptoms resolving within 24h. Analysis of CAR-T cell frequency in peripheral blood revealed robust engraftment followed by decline after day 17. Further expansion was noted in two patients with liver metastases accompanied by elevated levels of ALT, AST and AP, while total bilirubin was not affected. First tumor assessment 6 weeks after transplantation available for 5/8 patients revealed 4 SD (3 transitioned into PD after an additional 6-18 weeks) and 1 PD. Strikingly, three patients showed initial tumor shrinkage according to RECIST1.1 (reduction of target sum: -18%, -21% and -27%).ConclusionsCLDN6 CAR-T cells +/- CARVac show a favorable safety profile at doses tested and encouraging signs of efficacy. Updated data from open cohorts and especially for combination with CARVac will be presented.AcknowledgementsBNT211-01 is funded by BioNTech Cell & Gene Therapies GmbH.Trial RegistrationClinicaltrialsgov: NCT04503278ReferencesN/A Ethics ApprovalEthics & Institutional Review Board approvals were obtained from the respective participating countries prior to initiation of the trial.ConsentN/A

scholarly journals 137 Development of anti-Mucin1 CAR-T against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A146-A146
Author(s):  
Jihyun Lee ◽  
Areum Park ◽  
Jungwon Choi ◽  
Dae Gwan Yi ◽  
Hee Jung Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity

BackgroundChimeric antigen receptor (CAR) -T cell therapies have proven to be effective against various liquid tumors. However, the development of CAR-T against solid tumors has been challenging due to insufficient efficacy and potential on-target off-tumor toxicities caused by low expression of tumor antigens on normal tissues. Testing various affinities of CARs has demonstrated that lower affinity CARs maintain its anti-tumor effect while minimizing safety concerns (1). In order to develop a CAR-T against solid tumors expressing Mucin1, we have screened for Mucin1 binding antibodies and tested their anti-tumor effect in vitro and in vivo. The potential of on-target off-tumor toxicity was also measured in vitro.MethodsAnti-Mucin1 human single chain variable fragments (scFv) were obtained via screening against a scFv display library. Anti-Mucin1 scFvs were incorporated into CARs and in vitro, in vivo functions against various tumor cells expressing Mucin1 were tested. For in vivo studies, tumor bearing NOG mice (HCC1954 cells) received anti-Mucin1 CAR-T cells. Therapeutic efficacy was evaluated by measuring tumor volumes. Potential on-target off-tumor toxicity against Mucin1 on normal cells was tested by investigating the killing effect of anti-Mucin1 CAR-T against cancer cell line (HCC70) and non-tumorigenic breast epithelial cell line (MCF-10A) in co-culture systemsResultsIn vitro activity of anti-Mucin1 CAR-T cells that displayed a range of affinities for Mucin1 (27nM to 320nM) showed similar cytokine secretion levels and cytotoxicity against Mucin-1 expressing tumor cell lines (HCC70 and T47D). Robust anti-tumor activity was also demonstrated in vivo against large tumors (400~500 mm3) with relatively small numbers of CAR-T cells (0.5 x 106 CAR-T cells per mouse). In vivo expansion of CAR-T cells were observed in all scFv-CAR-T cases and accompanied by close to complete regression of tumors within 25 days post CAR-T cell injection. Of the 4 scFv CAR-Ts, 2H08 (with a Kd of 94nM) was tested for activity against normal breast epithelial cells. When 2H08-CAR-T was cocultured with a mixture of HCC70 and MCF-10A cells, they preferentially killed only the Mucin1 overexpressing HCC70 cells leaving MCF-10 cells intact.ConclusionsOur study demonstrates anti-tumor activity of a novel scFv-derived CAR-T recognizing Mucin1 and its effectiveness in large pre-established tumors in vivo. We also demonstrate that 2H08-CAR-T can distinguish between target overexpressing cancer cells and normal epithelial cells, which suggests that by toning down the affinity of CAR against antigen one can improve the safety profile of solid tumor antigen targeting CAR-T cell therapies.ReferenceCastellarin M, Sands C, Da T, Scholler J, Graham K, Buza E, Fraietta J, Zhao Y, June C. A rational mouse model to detect on-target, off-tumor CAR T cell toxicity. JCI Insight 2020; 5:e136012Ethics ApprovalAll experiments were done under protocols approved by the Institutional Animal Care and Use Committee (IACUC) (Study#LGME21-011).ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

139 Establishment of canine CAR T cells treatment model for solid tumor immunotherapy development

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A152-A153
Author(s):  
Shihong Zhang ◽  
Karan Kohli ◽  
R Graeme Black ◽  
Brian Hayes ◽  
Cassandra Miller ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Solid Tumor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Signaling Domain

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed therapy for hematological malignancies but has not yet been established as standard of care for any solid tumors. One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant, immunocompetent animal models. In this study, we sought to establish CAR T cells for naturally occurring canine sarcomas in client owned animals as a model for human CAR T cell therapy.MethodsArchived FFPE, freshly isolated canine solid tumor samples as well as tumor lines were tested for B7H3 expression by immunohistochemistry (IHC) and flow cytometry analysis. We designed CARs using the scFv from the human B7H3-specific antibody MGA271 and confirmed the cross-reactivity to canine B7H3 (construct information see figure 1A). A truncated EGFR (tEGFR) was included in the construct to allow for IHC and flow cytometry testing for the presence of CAR T cells. Killing efficiency was evaluated using 3D tumor spheroid killing assays to monitor dynamics. Safety of the CAR products following lymphodepletion was confirmed in two healthy dogs (figure 1B).ResultsCanine solid tumors were confirmed to be B7H3 positive in almost all cases. Using the GALV-pseudotyped retrovirus system, transduction was efficient with up to 70% CAR+ cells. Post-transduction expansion was over 100 folds. B7H3 CAR transduced canine T cells were able to eliminate B7H3+ canine tumor spheroids effectively (figure 2). Safety of the CAR T cells (dose: 1 × 109/m2) were confirmed in both healthy animals following cyclophosphamide lymphodepletion. After week 6, cetuximab was given to the subjects to deplete EGFR+ cells. Subject 2 experienced fever after CAR T cell administration. Both dogs showed elevated serum ALP and ALT levels and returned to normal (figure 3). No other treatment-related adverse events were observed. Information of the CAR T cell products can be found in table 1.Abstract 139 Figure 1Construct information and safety trial design(A) Four 2nd generation CAR constructs were generated. Two B7H3 CARs were candidates for the treatment, and two HER2 CARs served as controls, as they have been shown to kill canine cancer cells. The CARs are consisted of a single chain variable fragment (scFv, either B7H3-specific MGA271 or HER2-specific FRP5), a short hinge, a transmembrane domain (tm), a canine costimulatory signaling domain (either canine CD28 or 4-1BB) and canine CD3? signaling domain. Truncated EGFR is added in the construct for CAR+ T cell detection and facilitate the depletion of CAR T cells in vivo as a safety measure. (B) Blood from the subjects were drawn 3 weeks prior to the treatment for CAR T cell production. Cyclophosphamide (Cy, 400 mg/m2) and Fludarabine (Flu, 10 mg/m2) were given to the subjects for 2 days for lymphodepletion. CAR T cells (1 × 109/m2) and cetuximab (200 mg/m2) were given to the subjects as indicated. Blood, lymph node (LN) and bone marrow (BM) aspirates were collected for CAR T cell homing and persistence analysisAbstract 139 Figure 2Killing of canine OSA spheroids by canine CAR T ce(A) Scheme of tumor cell spheroid forming and killing. The loss of GFP can be measured for cytotoxicity readout (B) FRP5 and MGA271 CAR T cells can effectively kill canine cancer spheroids. Experiments were done in triplicates and error bars indicate SDAbstract 139 Figure 3Dynamics of peripheral lymphocytes, serum ALP and Current treatment regimen effectively decreased peripheral lymphocytes number after cyclophosphamide and fludarabine administration (D-4 and D-3) and increased serum ALP and ALT level after CAR T cell infusion (D0). Dashed line in both graphs show the upper limit of ALP and ALT levels, which are both 68U/LAbstract 139 Table 1Infused CAR T cell product informationBoth subjects are adult male beagle mixConclusionsWe demonstrated that, similar to human cancers, B7H3 is a target in canine solid tumors. We successfully generated canine B7H3 specific CAR T cell products that are highly efficient at killing canine 3D tumor spheroids using a production protocol that closely models human CAR T cell production procedure and confirmed the safety in vivo. We plan to test and optimize various approaches to enhance CAR T cell efficacy for solid tumor treatment both in vitro and in canine sarcoma patients.Ethics ApprovalThe study was approved by Fred Hutchinson Cancer Research Center‘s Institutional Animal Care and Use Committee (IACUC), approval number PROTO201900860

scholarly journals 131 Genetic disruption of negative immune regulators to enhance CAR-T efficacy against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A140-A141
Author(s):  
David Mai ◽  
Omar Johnson ◽  
Carl June
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Transduction Efficiency ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundCAR-T cell therapy has demonstrated remarkable success in hematological malignancies but displays limited efficacy in solid tumors, which comprise most cancer cases. Recent studies suggest that CAR-T cell failure via T cell exhaustion is characterized by decreased surface CAR expression, cytotoxicity, and Th1 cytokine production, leading to reduced antitumor functionality.1 2 3 To address these issues, studies have turned to genetically knocking out or overexpressing targets associated with an exhaustion or effector phenotype, such as PD-1 knockout (KO) and c-Jun overexpression, among other candidates that are typically receptors or transcription factors.4 5 6 However, there are other underexplored factors that mediate various aspects of immune regulation. While genome-wide CRISPR screens may discover such factors, they are unlikely to reveal phenotypes for genes whose function is partially redundant, therefore promising candidates may be missed. Such candidates include post-transcriptional regulators (PTRs) that coordinate immune responses, which are less well-studied in the context of CAR-T cell function. We hypothesized that KO of these PTRs may increase CAR-T cell cytokine activity, phenotype, and persistence, potentially under long-term or exhaustion-inducing conditions, leading to increased tumor control. Ultimately, disruption of negative immune regulators could produce CAR-T cells with enhanced activity and persistence, narrowing the gap between efficacy in hematological and solid tumors.MethodsTo explore whether the disruption of two target PTRs improves solid tumor efficacy, we used CRISPR-Cas9 to genetically delete one or both PTRs in mesothelin-targeting human CAR-T cells and assayed their function in vitro and in vivo in NSG mice.ResultsWe show successful genetic deletion of these genes in post-thymic human T cells and that their disruption does not affect primary expansion (figure 1) or transduction efficiency (figure 2). These KO CAR-T cells display increased expression of co-stimulatory receptors and various cytokines (figure 3). While KO CAR-T cells are functionally similar to WT CAR-T cells in in vitro assays (figure 4), KO CAR-T cells demonstrate superior activity in vivo and can clear large, established tumors compared to WT CAR-T cells at low dose (figure 5).Abstract 131 Figure 1Expansion kinetics of KO CAR-T cellsAbstract 131 Figure 2Transduction efficiency and baseline phenotype of KO CAR-T cellsAbstract 131 Figure 3Costimulatory receptor and cytokine expression of KO CAR-T cellsAbstract 131 Figure 4In vitro cytotoxicity of KO CAR-T cellsAbstract 131 Figure 5In vivo activity of KO CAR-T cellsConclusionsThese results indicate that KO of our target PTRs may improve the potency of CAR-T cells in solid tumors and may have important implications on the development of effective solid-tumor cell therapies.ReferencesJE Wherry and M Kurachi, Molecular and cellular insights into T cell exhaustion, Nature Reviews Immunology 2015;15:486–499.EW Weber, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 2021;372:6537.S Kuramitsu et al. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment. Cell, in revision.BD Choi et al, CRISPR-Cas9 disruption of PD-1 enhances activity of university EGFRvIII CAR T cells in a preclinical model of human glioblastoma. Journal for ImmunoTherapy of Cancer 2019;7:304.RC Lynn et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019;576:293–300.LJ Rupp et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Scientific Reports 2017;7:737.

An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors

Science ◽  
2020 ◽  
Vol 367 (6476) ◽  
pp. 446-453 ◽  
Author(s):  
Katharina Reinhard ◽  
Benjamin Rengstl ◽  
Petra Oehm ◽  
Kristina Michel ◽  
Arne Billmeier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Developmentally Regulated ◽  
Car T Cells ◽  
Car T Cell ◽  
Junction Protein ◽  
Car T ◽  
Rna Vaccine

Chimeric antigen receptor (CAR)–T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses.

scholarly journals Combination of AAV-CCL19 and GPC3 CAR-T Cells in the Treatment of Hepatocellular Carcinoma

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Min Meng ◽  
Yi-chen Wu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Tissue ◽  
Malignant Tumors ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background. Chimeric antigen receptor-modified T cell (CAR-T) therapy has great potential for treating malignant tumors, especially hematological malignancies. However, the therapeutic effect of solid tumors is limited. One of the most important factors is the homing of CAR-T cells to tumor tissues in vivo. Method. a recombinant adeno-associated virus 2 (AAV2) subtype carrying the CCL19 gene was used to pretreat the tumor before the Glypican-3 (GPC3) CAR-T treatment. The tumor tissue continuously expressed CCL19 and analyzed the tumor-suppressive effect of AAV-CCL19 on GPC3 CAR-T by in vitro and in vivo experiments. Result. Under the chemotaxis of CCL19, CAR-T cells had a significant increase in the degree of tumor tissue infiltration; also, the antitumor effect in vitro was significantly enhanced. AAV-CCL19 combined with GPC3 CAR-T significantly increased the survival time of mice. The aforementioned results showed that the combination of AAV-CCL19 and GPC3 CAR-T cells effectively increased the ability of CAR-T cells to go home into the tumor tissue, making the CAR-T cell treatment more effective. Conclusion. This study is expected to solve the dilemma in treating CAR-T cell solid tumors and achieve better clinical results.

scholarly journals 120 Chemokine receptor engineering enhances trafficking and homing of primary and iPSC-derived CAR-T cells to solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A129-A129
Author(s):  
Martin Hosking ◽  
Soheila Shirinbak ◽  
Joy Grant ◽  
Yijia Pan ◽  
Angela Gentile ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Chemokine Receptor ◽  
Dependent Manner ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptor (CAR)-T cells for solid tumors have shown modest effectiveness as compared to hematologic malignancies, a consequence of antigen heterogeneity, the immuno-suppressive tumor microenvironment (TME), limited cell persistence, and perhaps most notably, the trafficking of the CAR-T cell to the tumor itself. Early detection of CAR-T cells within a solid tumor has been associated with better outcomes across several clinical trials in diverse tumor settings, suggesting that strategies focused on enhancing CAR-T cell homing to and infiltration into the tumor can yield therapeutic benefit.MethodsHere, we demonstrate that following irradiation or exposure to common chemotherapy drugs, selected tumor cell lines (breast, ovarian, and prostate) specifically upregulate several chemokines, notably the CXCR2 ligand, interleukin (IL)-8, up to 4-fold over baseline control (e.g. 24ng/ml increased to 79ng/ml for SKOV3; 2.9ng/ml increased to 12.5ng/ml for MDA-MB-231). To leverage the upregulation of IL-8 as a mechanism of directing CAR-T cells to the tumor site, we initially engineered primary CAR-T cells to express CXCR2 and demonstrated functional migration, in a dose-dependent manner, to recombinant IL-8 in an in vitro transwell chemotaxis assay; maximal migration of approximately 2-fold over baseline was observed with 10ng/ml of rhIL-8. Similarly, supernatant from pre-conditioned tumor lines also elicited functional enhancements in migration (up to 4-fold specific migration). In addition, ovarian tumors were sub-optimally treated with paclitaxel in vivo, which promoted infiltration of CXCR2+ CAR-T cells and demonstrated enhanced tumor control.ResultsWe then incorporated these findings into our off-the-shelf, iPSC-derived CAR-T cell product platform. Induced pluripotent stem cells (iPSCs) were precisely engineered to co-express CAR and CXCR2 and subsequently differentiated to T cells to generate iPSC-derived CAR-T cells (CAR-iT cells). Like their primary CAR-T cell counterparts, functional chemotaxis of CXCR2+ CAR-iT cells was also observed in response to recombinant IL-8 and preconditioned tumor media. Importantly, CXCR2 expression did not limit CAR-dependent cytolytic function and the specificity of CAR-iT cells, underscoring the compatibility of this approach. Further in vitro and in vivo studies are ongoing and will be presented.ConclusionsCollectively, these data demonstrate that rational engineering of unique chemokine receptors to deliver the ideal chemokine/chemokine receptor match between tumors and effector cells can be leveraged to enhance tumor targeting and trafficking of CAR-iT cells for more effective treatment of solid tumors.Ethics ApprovalThese studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

scholarly journals Adoptive Immunotherapy beyond CAR T-Cells

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 743
Author(s):  
Aleksei Titov ◽  
Ekaterina Zmievskaya ◽  
Irina Ganeeva ◽  
Aygul Valiullina ◽  
Alexey Petukhov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
Adoptive Immunotherapy ◽  
Γδ T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T

Adoptive cell immunotherapy (ACT) is a vibrant field of cancer treatment that began progressive development in the 1980s. One of the most prominent and promising examples is chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of B-cell hematologic malignancies. Despite success in the treatment of B-cell lymphomas and leukemia, CAR T-cell therapy remains mostly ineffective for solid tumors. This is due to several reasons, such as the heterogeneity of the cellular composition in solid tumors, the need for directed migration and penetration of CAR T-cells against the pressure gradient in the tumor stroma, and the immunosuppressive microenvironment. To substantially improve the clinical efficacy of ACT against solid tumors, researchers might need to look closer into recent developments in the other branches of adoptive immunotherapy, both traditional and innovative. In this review, we describe the variety of adoptive cell therapies beyond CAR T-cell technology, i.e., exploitation of alternative cell sources with a high therapeutic potential against solid tumors (e.g., CAR M-cells) or aiming to be universal allogeneic (e.g., CAR NK-cells, γδ T-cells), tumor-infiltrating lymphocytes (TILs), and transgenic T-cell receptor (TCR) T-cell immunotherapies. In addition, we discuss the strategies for selection and validation of neoantigens to achieve efficiency and safety. We provide an overview of non-conventional TCRs and CARs, and address the problem of mispairing between the cognate and transgenic TCRs. Finally, we summarize existing and emerging approaches for manufacturing of the therapeutic cell products in traditional, semi-automated and fully automated Point-of-Care (PoC) systems.

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).

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p<0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P<0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p<0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

scholarly journals AAV-Mediated In Vivo CAR Gene Therapy for Targeting Human T Cell Leukemia

2021 ◽  
Author(s):  
Waqas Nawaz ◽  
Bilian Huang ◽  
Shijie Xu ◽  
Yanlei Li ◽  
Linjing Zhu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Car T Cells ◽  
Car T Cell ◽  
Human T Cell ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cell therapy is the most active field in immuno-oncology and brings substantial benefit to patients with B cell malignancies. However, the complex procedure for CAR T cell generation hampers its widespread applications. Here, we describe a novel approach in which human CAR T cells can be generated within the host upon injecting an Adeno-associated virus (AAV)vector carrying the CAR gene, which we call AAV delivering CAR gene therapy (ACG). Upon single infusion into a humanized NCG tumor mouse model of human T cell leukemia, AAV generates sufficient numbers of potent in vivo CAR cells, resulting in tumor regression; these in vivo generated CAR cells produce antitumor immunological characteristics. This instantaneous generation of in vivo CAR T cells may bypass the need for patient lymphodepletion, as well as the ex vivo processes of traditional CAR T cell production, which may make CAR therapy simpler and less expensive. It may allow the development of intricate, individualized treatments in the form of on-demand and diverse therapies.Significance StatementAAV can generate enough CAR cells within the host. That act as a living drug, distributed throughout the body, and persist for weeks, with the ability to recognize and destroy tumor cells.

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