scholarly journals 186 Development of KSQ-001, an engineered TIL (eTIL) therapy for solid tumors through CRISPR/Cas9-mediated editing of SOCS1

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A198-A198
Author(s):  
Karrie Wong ◽  
Sharon Lin ◽  
Christopher Wrocklage ◽  
Katri Sofjan ◽  
Leila Williams ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Memory Formation ◽  
Tumor Models ◽  
Syngeneic Tumor ◽  
Genome Wide

BackgroundAdoptive cell therapy with ex vivo expanded tumor infiltrating lymphocytes (TIL) offers a potentially curative treatment for cancer. However, the immunosuppressive tumor microenvironment limits the effectiveness of TIL therapy. To address this medical need, we used our Immune-CRISPRomics® Platform to perform a series of genome-wide CRISPR/Cas9 screens to identify targets enhancing the ability of T cells to infiltrate and kill solid tumors in an in vivo setting. These screens identified SOCS1 as a top target that restrains T cell anti-tumor immunity. Based on these findings, we developed KSQ-001, an engineered TIL (eTIL) therapy created via CRISPR/Cas9-mediated editing of SOCS1 for the treatment of solid tumors.MethodsGenome-wide CRISPR/Cas9 screens were conducted in in vitro primary human T cells and TIL cultures and in in vivo primary mouse OT1 and PMEL-TCR-Tg T cells in syngeneic tumor models. The efficacy of surrogate murine KSQ-001 (mKSQ-001), in which the SOCS1 gene is inactivated by CRISPR/Cas9 in OT1 or PMEL-TCR-Tg T cells, was evaluated in both the B16-Ova and CRC-gp100 syngeneic tumor models, with memory formation and efficacy evaluated both in the presence and absence of cyclophosphamide-mediated lymphodepletion. KSQ-001 was manufactured from human TIL using SOCS1-targeting sgRNAs selected for therapeutic use based on potency and selectivity, with KSQ-001 characterized for in vitro function and in vivo anti-tumor efficacy.ResultsUpon adoptive transfer of a single dose into solid tumor-bearing hosts, mKSQ-001 was found to robustly enhance anti-tumor efficacy and eradicate tumors in 7/10 mice in the PD1-sensitive OT1/B16-Ova model and to drive responses in the PD-1 refractory PMEL/CRC-gp100 syngeneic tumor model. mKSQ-001 also showed a ten-fold increase in anti-tumor potency in vivo compared to unengineered T-cell product and established durable anti-tumor memory by persisting in the form of T central memory cells detectable at high frequency in the peripheral blood of complete responder mice. In the setting of lymphodepletion, mKSQ-001 also displayed heightened anti-tumor potency, accumulation, and memory formation in comparison to inactivation of PD-1. Importantly, human KSQ-001 displayed a transcriptional signature indicative of increased anti-tumor function, produced increased amounts of pro-inflammatory cytokines, exhibited a hypersensitivity to IL-12 signaling, and demonstrated increased anti-tumor function both in vitro and in vivo solid tumor models.ConclusionsBased on insights from our Immune-CRISPRomics® platform and demonstrated efficacy across multiple preclinical tumor models, we have developed KSQ-001, a novel eTIL therapy. These preclinical data support clinical testing of KSQ-001 in a variety of solid tumor indications.

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.

scholarly journals BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved antitumor activity

2021 ◽  
Author(s):  
Taylor L Hickman ◽  
Eugene Choi ◽  
Kathleen R Whiteman ◽  
Sujatha Muralidharan ◽  
Tapasya Pai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Solid Tumor ◽  
Cell Function ◽  
Cell Metabolism ◽  
Car T Cells ◽  
Car T

Purpose: The solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models. Experimental Design: We evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo. Results: Expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions. Conclusions: Together, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).

scholarly journals 714 PD1 x TGFβR2 bispecifics selectively block TGFβR2 on PD1-positive T cells, promote T cell activation, and elicit an anti-tumor response in solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A756-A756
Author(s):  
Gregory Moore ◽  
Suzanne Schubbert ◽  
Christine Bonzon ◽  
Kendra Avery ◽  
Rumana Rashid ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
T Cell Activation ◽  
Tumor Response ◽  
Nsg Mice ◽  
Pd1 Blockade ◽  
Anti Tumor Activity

BackgroundTGFβ production by solid tumors and their microenvironment is a major mechanism used by tumors to avoid immunosurveillance. Blockade of TGFβ has been shown to promote an anti-tumor response; however, systemic blockade of TGFβ has also been associated with toxicity. We hypothesized that a PD1 x TGFβR2 bispecific antibody could selectively block the suppressive activity of TGFβ on tumor T cells and enhance their anti-tumor activity while avoiding the toxicity associated with systemic blockade.MethodsWe engineered bispecific antibodies that simultaneously engage PD1 and TGFβR2 using Xencor’s XmAb platform. The anti-TGFβR2 arm was tuned for optimal activity by introducing affinity-modulating amino acid substitutions. The activity of PD1 x TGFβR2 bispecifics was evaluated in vitro using a signaling assay to measure phosphorylated SMAD (pSMAD) by flow cytometry with exogenous TGFβ in unactivated and activated PBMC. In vivo activity was evaluated by monitoring the engraftment of human PBMC in NSG mice (huPBMC-NSG). Anti-tumor activity was assessed in huPBMC-NSG mice engrafted with established human cancer cell lines. Antibodies against other T cell targets were also incorporated into TGFβR2 bispecifics, and similarly evaluated in vitro and in vivo.ResultsPD1 x TGFβR2 bispecifics were confirmed to bind PD1 and block binding of TGFβ to TGFβR2. In vitro, we found that T cells from activated, serum-deprived PBMC exhibited robust induction of pSMAD in response to TGFβ, and PD1 x TGFβR2 bispecifics selectively inhibited pSMAD induction in PD1-positive T cells as demonstrated by over a 100-fold potency increase compared to an untargeted anti-TGFβR2 control. Additionally, we saw an enhancement of potency when evaluating blocking activity in activated (PD1-high) vs. unactivated (PD1-low) T cells. Similar selectivity was measured when comparing inhibition of pSMAD induction for activated T cells versus other PD1-negative, TGFβ-responsive immune cells. Intriguingly, TGFβR2 bispecifics incorporating antibodies against other T cell targets allowed for the targeting of a broader population of T cells while still conferring potent selectivity against target-negative cells. In vivo, treatment of huPBMC-NSG mice with TGFβR2 bispecifics promoted superior T cell engraftment and combined additively with PD1 blockade. Furthermore, TGFβR2 bispecific treatment of huPBMC-NSG mice containing established MDA-MB-231 triple-negative breast cancer tumors promoted an anti-tumor response that was also augmented with PD1 blockade.ConclusionsMultiple PD1 x TGFβR2 bispecifics were engineered to selectively block TGFβR2 on PD1-positive T cells and evaluated in vitro and in vivo. Compelling activity, including additivity with PD1 blockade, suggests that clinical development is warranted for the treatment of human malignancies.

scholarly journals Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors

2021 ◽  
Vol 9 (4) ◽  
pp. e002173
Author(s):  
Guanmeng Wang ◽  
Xin Zhou ◽  
Giovanni Fucà ◽  
Elena Dukhovlinova ◽  
Peishun Shou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Antigen Expression ◽  
Human Antibody ◽  
Single Chain ◽  
Car T Cells ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs.MethodsWe used Humabody VH domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) VH and mesothelin (MSLN) VH sequences and redirect T cell with VH based-CAR. The antitumor activity and mode of action of PSMA VH and MSLN VH were evaluated in vitro and in vivo compared with the traditional scFv-based CARs.ResultsHuman VH domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. VH modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression.ConclusionsFully human antibody VH domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, VH domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.

scholarly journals 126 Regional administration of IL-12 endowed CAR T cells effectively targets systemic disease

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A135-A135
Author(s):  
Hee Jun Lee ◽  
Cody Cullen ◽  
John Murad ◽  
Jason Yang ◽  
Wen-Chung Chang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Models ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundWhile chimeric antigen receptor (CAR) T cell therapy has shown impressive clinical efficacy for hematological malignancies,1 efficacy remains limited for solid tumors due in large part to the immunosuppressive tumor microenvironment.2 Tumor-associated glycoprotein 72 (TAG72) is an aberrantly glycosylated protein overexpressed on ovarian cancer3 and is an exciting target for CAR T cell immunotherapy. Our lab previously developed a second-generation TAG72 CAR T cell product and showed its potency against TAG72-expressing ovarian tumor cells both in vitro and in preclinical mouse models.4 We report here further modification of our TAG72 CAR T cells, with incorporation of interleukin-12 (IL-12) and interleukin-15 (IL-15), and evaluate the therapeutic benefits in peritoneal ovarian tumor models.MethodsIn this preclinical study, we build upon our earlier work with in vitro and in vivo evaluation of 9 different second-generation TAG72 CAR constructs varying in single-chain variable fragment, extracellular spacer, transmembrane, and intracellular co-stimulatory domains. We then engineer CAR T cells with two types of cytokines – IL-12 and IL-15 – and put these engineered cells against challenging in vivo tumor models.ResultsThrough in vitro and in vivo studies, we identify the most optimal construct with which we aim to evaluate in a phase 1 clinical trial targeting TAG72-positive ovarian cancer in 2021. Despite thorough optimizations to the CAR backbone, CAR T cells can be additionally engineered for improved anti-tumor response. Therefore, we further engineered CAR T cells with IL-12 or IL-15 production that greatly improves the effectiveness of TAG72-CAR T cells in difficult-to-treat in vivo tumor models. We observed that modification of CAR T cells with IL-15 displayed toxicity when regionally delivered in vivo, yet introduction of IL-12 not only demonstrated safe and superior therapeutic responses, but also allowed the regional administration of CAR T cells to address systemic disease. We are now expanding these findings by evaluating these therapies using syngeneic immunocompetent mouse tumor models.ConclusionsThe tumor microenvironment (TME) harbors various factors that thwart the killing of tumor cells by CAR T cells. Thus, CAR T cells will likely require further engineering to overcome this barrier. We show that amplifying cytokine pathways is one way to overcome the TME and improve the efficacy of CAR T cell therapy for solid tumors.ReferencesMaude SL, Teachey DT, Porter DL, Grupp SA. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood 2015 Jun 25;125(26):4017–23.Priceman SJ, Forman SJ, Brown CE. Smart CARs engineered for cancer immunotherapy. Curr Opin Oncol 2015 Nov;27(6):466–74.Chauhan SC, Vinayek N, Maher DM, Bell MC, Dunham KA, Koch MD, Lio Y, Jaggi M. Combined Staining of TAG-72, MUC1, and CA125 Improves Labeling Sensitivity in Ovarian Cancer: Antigens for Multi-targeted Antibody-guided Therapy. J Histochem Cytochem 2007 Aug;55(8):867–75.Murad JP, Kozlowska AK, Lee HJ, Ramamurthy M, Chang WC, Yazaki P, Colcher D, Shively J, Cristea M, Forman SJ, Priceman SJ. Effective Targeting of TAG72+ Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells. Front Immunol 2018 Nov 19;9:2268.

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.

CAR-T cells to deliver engineered peptide antigens and reprogram antigen specific T cell responses against solid tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2530-2530
Author(s):  
Daniel Lee ◽  
Andy J Minn ◽  
Lexus R Johnson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
In Vivo Studies ◽  
Peptide Antigens ◽  
Car T Cells ◽  
Car T

2530 Background: Neoantigen depleted malignancies such as colorectal cancer demonstrate primary resistance to immune checkpoint blockade, and solid tumors in general have shown resistance to chimeric antigen receptor (CAR) T cell therapy. However, CAR-T cells have been shown to be capable of delivering various therapeutic molecules in a targeted fashion to the tumor microenvironment, in some cases through extracellular vesicles (EVs). In vivo studies have shown that the presentation of foreign viral peptides by solid tumors can reprogram bystander virus-specific cytotoxic T cells (CTLs) against tumor cells. In this study, we demonstrate that CAR-T cells can deliver engineered peptide antigens to solid tumors, leading to presentation on tumor cells and anti-tumor response. Methods: Second generation CAR-T cells (41BB endodomain) targeting human CD19 (19BBz) or human mesothelin (M5BBz) were generated via retroviral and lentiviral transduction respectively. CAR-T cells were engineered to co-express peptides such as SIINFEKL of ovalbumin and NLVPMVATV of CMV pp65 among others. Peptides were isolated from EVs via ultracentrifugation. For in vivo studies, C57BL/6 or NSG mice were injected on the flank with relevant tumors and treated with peptide-CAR-T cells. In vitro studies utilized flow cytometry and xCELLigence killing assays. Results: Murine 19BBz CAR-T cells expressing the SIINFEKL peptide of ovalbumin (ova-19BBz) were found to transfer SIINFEKL peptide to tumor cells via EVs in vitro and in vivo, leading to peptide presentation on MHC-I of tumor cells. This resulted in significantly delayed tumor growth in tumor bearing mice transfused with OT-I T cells to mimic an existing antigen specific T cell pool. We expanded on these findings by isolating EVs from human M5BBz CAR-T cells expressing CMV viral peptides. Peptide-CAR-T EVs were co-cultured with human ovarian cancer cells to assess presentation to Jurkat T cells. Finally, we utilized primary human T cells from CMV+ healthy donors to assess the clinical feasibility of our peptide delivery approach. Conclusions: CAR-T cells can be engineered to deliver peptides to tumor cells for presentation and subsequent targeting by antigen specific CTLs. This represents a novel strategy for the treatment of non-immunogenic tumors.

scholarly journals Locally secreted BiTEs complement CAR T cells by enhancing solid tumor killing

2021 ◽  
Author(s):  
Yibo Yin ◽  
Jesse Rodriguez ◽  
Nannan Li ◽  
Radhika Thokala ◽  
MacLean P Nasrallah ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumor ◽  
Tumor Therapy ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Bispecific T-cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. BiTEs can be secreted by T cells through genetic engineering and perform anti-tumor activity. We hypothesized that BiTE-secreting T cells could be a valuable T cell-directed therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity and represent a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and glioma cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the scFvs to generate new BiTE molecules. Compared with 806CAR T cells and Hu08CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE secreting bivalent targeting T cells compared with bivalent targeting CAR T cells, which significantly delayed tumor growth in a glioma mouse model. In summary, BiTEs secreted by mono- or multi- valent targeting T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

scholarly journals In Vitro-Transcribed mRNA Chimeric Antigen Receptor T Cell (IVT mRNA CAR T) Therapy in Hematologic and Solid Tumor Management: A Preclinical Update

2020 ◽  
Vol 21 (18) ◽  
pp. 6514
Author(s):  
Thangavelu Soundara Rajan ◽  
Agnese Gugliandolo ◽  
Placido Bramanti ◽  
Emanuela Mazzon
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Solid Tumor ◽  
Antigen Receptor ◽  
Hematologic Malignancies ◽  
Car T ◽  
Tumor Management

Adoptive T cell immunotherapy has received considerable interest in the treatment of cancer. In recent years, chimeric antigen receptor T cell (CAR T) therapy has emerged as a promising therapy in cancer treatment. In CAR T therapy, T cells from the patients are collected, reprogrammed genetically against tumor antigens, and reintroduced into the patients to trigger an immense immune response against cancer cells. CAR T therapy is successful in hematologic malignancies; however, in solid tumors, CAR T therapy faces multiple challenges, including the on-target off-tumor phenomenon, as most of the tumor-associated antigens are expressed in normal cells as well. Consequently, a transient in vitro-transcribed anti-mRNA-based CAR T cell (IVT mRNA CAR T) approach has been investigated to produce controlled cytotoxicity for a limited duration to avoid any undesirable effects in patients. In vitro and in vivo studies demonstrated the therapeutic ability of mRNA-engineered T cells in solid tumors, including melanoma, neuroblastoma and ovarian cancer; however, very few clinical trials are registered. In the present review, we discuss the effect of IVT mRNA CAR T therapy in preclinical studies related to hematologic malignancies and solid tumor management. In addition, we discuss the clinical trial studies based on IVT mRNA CAR T therapy in cancer.

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

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