scholarly journals MDSC Suppression of CAR T Cells Can be Reduced By Targeted Signaling Disruption

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4438-4438 ◽  
Author(s):  
Estelle V Cervantes ◽  
Justin C. Boucher ◽  
Sae Bom Lee ◽  
Kristen Spitler ◽  
Kayla Reid ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Research Funding ◽  
Target Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Counts ◽  
Car T

CAR T cells are genetically modified with an extracellular scFv, transmembrane domain, and intracellular costimulatory and CD3ζ domains. Two treatments received the approval from the FDA for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma. However, CAR T cell persistence remains a problem. A reason for this may be that myeloid cells such as myeloid derived suppressor cells (MDSCs) may be contributing to the reduced persistence of CAR T cells. MDSCs originate from myeloid cells and have been implicated in the suppression of the immune system in the tumor microenvironment. To determine what effect MDSCs might have during CAR T cell production we co-cultured MDSCs during CAR transduction. We found gene transfer was lower for m19z (38.7% vs 46.8%), m1928z (24.1% vs 39.1%), and m19hBBz (35.8% vs 46.2%) CAR T cells co-cultured with MDSCs compared to those that were not (Fig 1A). There was also a reduction in total T cell counts for m19z (58%), m1928z (88%), and m19hBBz (65%) after MDSC co-culture. This data suggests MDSCs present during CAR T cell production can alter gene transfer and total T cell counts. We also investigated the effect MDSCs can potentially have on CAR T cells when present during CAR T cell antigen stimulation. CAR T cells co-cultured with MDSCs in vitro had significant reductions in m19z, m1928z, and m19hBBz CAR T cell mediated killing against target cells (Fig 1B). We also found significantly lower production of IFNγ in m19z, m1928z, and m19hBBz CAR T cells cultured with MDSCs compared to cells cultured with total BM (Fig 1C). After 24hr stimulation with MDSCs and target cells, there was lower expression of activation markers PD1 and LAG3 by CAR T cells compared to culture without MDSCs. This suggests that MDSCs reduce CAR T cell activation, killing, and cytokine production. We also looked at how CAR proliferation after antigen stimulation is affected by MDSC co-culture and found MDSCs significantly reduced CAR proliferation in vitro. To evaluate if we could create a more resistant CAR T cell to MDSC suppression null mutations were incorporated into a CD28 CAR. We mutated the YMNM and PRRP subdomains of CD28 which signal through PI3K and ITK respectively leaving only PYAP active (mut06). When MDSCs were co-cultured with mut06 T cells during production mut06 had a smaller reduction in gene transfer (21% vs 38%) and T cell counts (80% vs 88%) compared to m1928z. Mut06 also had a significantly higher expression of PD1 and TIM3 compared to m1928z after production with MDSCs. In vitro when mut06 was co-cultured with MDSCs it had the same killing ability as m1928z without MDSCs and was significantly better at killing compared to m1928z co-cultured with MDSCs (Fig 1B). To examine the effect of MDSCs on CAR T cells in vivo we injected C57BL6 mice with CAR T cells followed by MDSCs a week later. In these ongoing experiments we found mut06 had significantly higher numbers of CAR T cells in the blood compared to m1928z (Fig1D). Overall our data shows that MDSCs can suppress CAR T cell function when present during production as well as CAR stimulation. It also suggests that by optimizing CD28 CAR signaling using mut06 that we were able to generate a CAR T cell that is more resistant to MDSCs. Furthermore, we may be able to recapitulate the effect of mut06 with targeted inhibitors. Figure 1 Disclosures Davila: GlaxoSmithKline: Consultancy; Novartis: Research Funding; Adaptive: Consultancy; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; Anixa: Consultancy; Atara: Research Funding.

scholarly journals Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2776-2776
Author(s):  
Salvatore Fiorenza ◽  
George S. Laszlo ◽  
Tinh-Doan Phi ◽  
Margaret C. Lunn ◽  
Delaney R. Kirchmeier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Set Domain ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Privately Held

Abstract Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 PAN antibodies"). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone. Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants. Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion. Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. Disclosures Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

scholarly journals Foamy Virus Based Vectors for CAR-T Cell Development

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4646-4646
Author(s):  
Emmanouil Simantirakis ◽  
Vassilis Atsaves ◽  
Ioannis Tsironis ◽  
Margarita Gkyzi ◽  
Kostas Konstantopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Ifn Γ

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Khadijeh Alishah ◽  
Matthias Birtel ◽  
Elham Masoumi ◽  
Leila Jafarzadeh ◽  
Hamid Reza Mirzaee ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Solid Tumors ◽  
Target Cells ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. Methods In this study, we hypothesized that knocking out the TGFβ receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFβRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFβRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. Results Our experiments demonstrated that TGFβRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFβ in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFβRII KO CAR T-cells has been recorded. TGFβRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFβRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. Conclusion The TGFβRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

scholarly journals Point-of-Care Manufacturing of CD20.19 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cells in a Standard Academic Cell Processing Facility for a Phase I Clinical Trial in Relapsed, Refractory NHL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4553-4553 ◽  
Author(s):  
Fenlu Zhu ◽  
Nirav N Shah ◽  
Dina Schneider ◽  
Huiqing Xu ◽  
Katherine Chaney ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
Target Cells ◽  
Cell Processing ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, which has led to FDA approvals for two CAR-T products. However, limitations exist with commercial CAR-T centralized production: (1) off-site manufacturing can take several weeks and requires shipping from and to the treating facility; (2) off-site manufacturing limits treatment options for progressing patients; (3) high cost of the commercial products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy device, a GMP-compliant closed system, to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR). CAR-T manufacturing was performed exclusively using the CliniMACS Prodigy device and reagents obtained from Miltenyi Biotec. Production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory, an ISO7 air handling environment. Manufacturing was set at 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) were collected by apheresis, with a collection goal of 4 blood volumes to eliminate risk of a low CD3 yield in heavily pre-treated patients. Next, MNC were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. To start the culture process, enriched T cells were suspended in TexMACS medium supplemented with 3% human AB serum and 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. The following day (day 1), lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3ζ and 4-1BB stimulatory domains was added to the stimulated cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on Day 14. Protein L staining was used to detect expression of CD20.19 CAR on the T cells. On Day 14, eligible patients received fresh CAR-T cells, while for others the product was cryopreserved and administered on a later date. To date, the MCW Cell Therapy Laboratory has successfully generated CAR-T cell products for all 6 patients enrolled thus far on the Phase 1 clinical trial with no production failures (Table 1). Three patients received cryopreserved product and 3 patients received fresh product. The enriched T cells were 94.3% CD3+ (87.8-97.4%), and average T cell recovery from the apheresis cell products was 65.2% (54.2-80.0%). Protein L staining indicated 20.8% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFN-gamma in response to the same target cells. An average yield of 5.9e+8 (4.3-7.9e+8) CAR T cells was obtained at harvest, which exceeded the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with higher expression on CD4 T cells; average CAR-T CD4:CD8 ratio on the final products was 2.8. The majority of T cells (average of 81.5%) had an effector-memory phenotype. In-process testing performed on Day 8 of manufacturing demonstrated sufficient numbers of CAR-T cells needed for patient infusions were already present, and that the CAR-T cells only expanded an additional 1.9 to 3.5-fold between Days 8 and 14. In conclusion, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. Time to production was efficient (14 days), and patient-derived CAR-T cell products were reproducibly generated in a standard cell processing laboratory within an academic medical center. A major clinical advantage of CAR-T cells generated on-site is the flexibility in treatment. Patients can receive cells either immediately (i.e., fresh) or the cells can be cryopreserved for later infusion if the patient is not able to receive fresh cells. Based on our results, we intend to decrease the cell processing time to 10 days. Disclosures Zhu: Lentigen Technology Inc., A Miltenyi Biotec Company: Research Funding. Shah:Juno Pharmaceuticals: Honoraria; Oncosec: Equity Ownership; Geron: Equity Ownership; Exelexis: Equity Ownership; Miltenyi: Other: Travel funding, Research Funding; Lentigen Technology: Research Funding. Schneider:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Keever-Taylor:Medical College of Wisconsin: Research Funding. Dropulic:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Hari:Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Kite Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding. Johnson:Miltenyi: Research Funding.

scholarly journals Mutation of the CD28 Co-Stimulatory Domain Confers Enhanced CAR T Cell Function

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 248-248 ◽  
Author(s):  
Justin C. Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Maria Cabral ◽  
Dylan Morrissey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Signaling ◽  
Research Funding ◽  
Chromatin Accessibility ◽  
Target Cells ◽  
T Cell Signaling ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

scholarly journals CRISPR/Cas9-mediated TGFβRII Disruption Enhances Anti-tumor Efficacy of Human Chimeric Antigen Receptor T Cells in Vitro

2021 ◽  
Author(s):  
Khadijeh Alishah ◽  
Matthias Birtel ◽  
Elham Masoumi ◽  
Leila Jafarzadeh ◽  
Hamid Reza Mirzaee ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Solid Tumors ◽  
Target Cells ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. Methods: In this study, we hypothesized that knocking out the TGFβ receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFβRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFβRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells.Results: Our experiments demonstrated that TGFβRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFβ in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFβRII KO CAR T-cells has been recorded. TGFβRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFβRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. Conclusion: The TGFβRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

scholarly journals Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

2021 ◽  
Vol 9 (6) ◽  
pp. e002140
Author(s):  
Giulia Pellizzari ◽  
Olivier Martinez ◽  
Silvia Crescioli ◽  
Robert Page ◽  
Ashley Di Meo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
T Cells ◽  
T Cell ◽  
Type I ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Tumor Associated Antigen ◽  
Car T

BackgroundCancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies.MethodsEmploying mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy.ResultsWe identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected.ConclusionsThese findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies.

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 Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3921-3921 ◽  
Author(s):  
Cesar Sommer ◽  
Hsin-Yuan Cheng ◽  
Yik Andy Yeung ◽  
Duy Nguyen ◽  
Janette Sutton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cells ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

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