scholarly journals Brain capillary obstruction as a novel mechanism of anti-CD19 CAR T cell neurotoxicity

2021 ◽  
Author(s):  
Lila D Faulhaber ◽  
Kendra Jae Hartsuyker ◽  
Anthea Q Phuong ◽  
Yeheun Cho ◽  
Katie K Mand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Therapeutic Interventions ◽  
High Dose ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T Cell ◽  
Syngeneic Mouse Model ◽  
Car T

Immunotherapy for hematologic malignancies with CD19-directed CAR T cells is associated with neurotoxicity in about 40% of patients. Systemic cytokine release syndrome, endothelial activation, and disruption of endothelial integrity have all been associated with neurotoxicity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We developed a syngeneic mouse model which manifests systemic cytokine release and behavioral abnormalities within 3-5 days after infusion of high-dose murine CD19-CAR T cells. Histologic examination revealed widespread brain hemorrhages, diffuse extravascular IgG deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. In vivo two-photon imaging of blood flow revealed plugging of >10% of capillaries by leukocytes, associated with regions of localized hypoxia. These data reveal capillary obstruction and associated brain hypoxia and microvascular decline as a potential basis for neurotoxicity during CD19-CAR T cell treatment in humans, which may be amenable to therapeutic interventions.

scholarly journals Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1934-1934 ◽  
Author(s):  
Eduardo Huarte ◽  
Roddy S O'Connor ◽  
Melissa Parker ◽  
Taisheng Huang ◽  
Michael C. Milone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

scholarly journals Overcoming CD19 Antigen Loss in B-Cell Malignancies with CAR T Cells Targeting BAFF-R

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3871-3871
Author(s):  
Hong Qin ◽  
Zhenyuan Dong ◽  
Xiuli Wang ◽  
Wesley Cheng ◽  
Diane Lynne Smith ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
High Dose ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership ◽  
Antigen Loss

Background: The current success in treating hematological malignancies with CD19 targeted chimeric antigen receptor (CAR) T cells is diminished by tumor relapse from target antigen loss. The B-cell activating factor-receptor (BAFF-R), a tumor necrosis factor receptor superfamily protein (TNFRSF13C), is a particularly interesting alternative target in CD19 relapsed disease and in B cell malignancies in general. BAFF-R null mice exhibit greatly reduced normal B-cell numbers and mouse strains expressing a mutant BAFF-R exhibited decreased B-cell life spans and a dramatically reduced peripheral B-cell compartment. Thus, BAFF-R signaling is a driver of B-cell survival, which may limit the capacity of clonal B-cell tumors to escape therapy by down-regulation of antigen expression. We have exploited the potential of the BAFF/BAFF-R axis and developed a proprietary BAFF-R targeting CAR T cell using a single-chain variable fragment (scFv) of a humanized anti-human BAFF-R antibody engineered into a second generation CAR construct containing 4-1BB costimulatory and CD3ζ intracellular signaling domains (Qin et al. Sci. Transl. Med., In Press). BAFF-R CAR T cells were cytotoxic against a panel of human leukemia and lymphoma lines. Adoptively transferred BAFF-R-CAR T cells eradicated 10-day pre-established tumor xenografts after a single treatment and retained efficacy against xenografts deficient in CD19 expression, including CD19-negative variants within a background of CD19-positive lymphoma cells. BAFF-R-, but not CD19-CAR T cells, also demonstrated antitumor effects against an additional CD19 antigen loss primary patient-derived xenograft (PDX) in vivo. Methods and Results: A first-in-human clinical trial of this BAFF-R CAR T cell in B-ALL is planned. To this end, we cloned the BAFF-R CAR construct into a previously FDA approved clinical lentiviral vector (Figure, part A), and produced BAFF-R CAR T cells using both the research prototype and clinical vectors for head-to-head analysis. We observed comparable cytotoxic T lymphocyte activity against previously established leukemia and lymphoma models in vitro. In vivo, NSG mice challenged with Nalm-6 human B-ALL tumors were randomized (n=5/group) for treatment with either BAFF-R CAR produced from the research prototype or clinical vectors. The two experimental groups were indistinguishable in their antitumor efficacy (data not shown). Further, to advance IND enabling studies we also compared clinical CAR T cell manufacturing strategies. We used an early stage TN/MEM subset for CAR T cell production, as used in previous CAR T cell trials at our institution, and using TN/MEM isolated from healthy donor PBMCs achieved 36% transduction efficiency, comparable to clinical experience at City of Hope. Finally, we assessed the therapeutic effects of BAFF-R CAR TN/MEM cells in vivo. We challenged NSG mice on day 0 with 1 x 105 Nalm-6-CD19 deficient (Nalm-6-CD19KO) human ALL tumor cells and randomized them (n=5/group) to receive a single infusion of either low dose (1 x 106) or high dose (2 x 106) BAFF-R-CAR TN/MEM cells on day 10. Because transduction efficiency was 36%, 2.8 or 5.6 x 106 total T cells were infused to yield 1 or 2 x106 BAFF-R-CAR TN/MEM, respectively. Non-transduced T cells from the same donor were used as allogeneic controls (non-CAR). Tumors were monitored by bioluminescent imaging on the days indicated. Both the low and the high dose BAFF-R CAR T cell groups demonstrated complete Nalm-6 CD19KO tumor regressions, compared with controls (Figure, part B). Conclusions: Our studies support the clinical development of BAFF-R CAR T cells and a pending clinical trial of BAFF-R CAR T cell therapy in relapsed/refractory B-ALL patients who have failed prior CD19-targeted immunotherapy. Targeting BAFF-R may thereby add to existing alternative strategies to overcome relapse from CD19 antigen loss. While BAFF-R antigen loss by tumor cells is unlikely, because its expression is critically required for normal B-cell survival, this hypothesis can only be established by clinical testing. Disclosures Qin: InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership. Aldoss:AUTO1: Consultancy; Helocyte: Consultancy, Honoraria, Other: travel/accommodation/expenses; Jazz Pharmaceuticals: Honoraria, Other: travel/accommodation/expenses, Speakers Bureau; Agios: Consultancy, Honoraria. Kwak:Pepromene Bio: Consultancy, Equity Ownership, Research Funding; InnoLifes: Consultancy, Equity Ownership; Xeme BioPharma, Inc: Consultancy, Equity Ownership; Enzychem LifeSciences: Consultancy; Celltrion, Inc.: Consultancy; Celltrion Healthcare: Consultancy.

scholarly journals Monocytes Are Required for Both Optimal Anti-Leukemic Efficacy and the Cytokine Release Syndrome By CAR-T Cells: Lessons from an Innovative Xenotolerant Mouse Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 997-997
Author(s):  
Margherita Norelli ◽  
Monica Casucci ◽  
Barbara Camisa ◽  
Laura Falcone ◽  
Catia Traversari ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Secondary Transfer

Abstract Background: Chimeric antigen-receptor (CAR)-engineered T cells promise to cure chronic and acute leukemias refractory to standard treatments. Before this promise is fulfilled, however, two crucial issues need to be solved: i) how to circumvent the emergence of secondary resistance (e.g. due totarget-antigen loss; leukemic lineage switch); ii) how to manage associated toxicities (e.g. the cytokine release syndrome, CRS; lineage aplasias). Unfortunately, all these issues cannot be addressed pre-clinically in currently available NSG mouse models, because they lack human hematopoiesis and, furthermore, ultimately develop xenograft-versus-host disease (X-GVHD), preventing the evaluation of long-term effects. Methods: We have developed an innovative xenotolerant model by transplanting human hematopoietic stem cells (HSCs) intraliver in newborn NSG mice triple transgenic for human SCF, GM-SCF and IL-3 (SGM3). Differently from "classical" NSG, SGM3 mice reconstituted high levels of human T cells (>1000 cells per microL at week 8), which, once transferred in secondary recipients, persisted up to 200d without causing X-GVHD, even after irradiation. Robust and specific xenotolerance was confirmed by in vitrohyporesponsiveness to NSG, bot not to C57/Bl6 antigens (irradiated splenocytes) or human HLAs (PBMCs). Secondary transfer experiments in leukemic and/or HSC-humanized SGM-3 mice have been then designed for studying the determinants of CAR-T cell efficacy and associated toxicities in the absence of confounding xenoreactivity. Results: SGM3-derived T cells were transduced ex vivo with either a CD19 or a CD44v6 CAR (both having a CD28 2G design) after activation with CD3/CD28-beads and IL-7/IL-15, resulting in a preferential and functional CD45RA+/CD62L+/CD95+ stem memory T cell (TSCM) phenotype. Once transferred in secondary recipients previously engrafted with a CD19+/CD44v6 leukemic cell line, CD19 or CD44v6 CAR-T cells equally mediated rapid tumor clearance both in low and high tumor-burden settings, in the absence of malaise or elevated human IL-6 levels in vivo. At later time points (after 100d), however, approximately 50% of responding mice relapsed despite significant CAR-T cell persistence in vivo (>50 cells per microL). A significant fraction of leukemia relapses were characterized by post-transcriptional down-regulation of CD44v6 expression or CD19 loss, respectively. Conversely, secondary transfer of SGM3-derived CAR-T cells in leukemic SGM3 mice that had been previously humanized with HSCs resulted in the development of a clinical syndrome similar to the CRS observed in clinical trials (high fevers, elevated IL-6, TNF-alpha and serum amyloid A levels - mouse analog of C-reactive protein in humans), resulting in 30% lethality. This CRS was anticipated and shortened for CD44v6 compared with CD19 CAR-T cells and worse in the case of 4-1BB compared with the original CD28 2G CAR designs. Strikingly, mice recovering from the CRS benefited from durable leukemic remissions, yet experienced long-lasting CD19+ B-cell or CD44v6+ monocyte aplasias. Deepness of remission was confirmed in "tertiary" recipients, which did not develop leukemia after the infusion of bone-marrow cells from mice in remission 150d since CAR-T cell infusion. Interestingly, in this model, tocilizumab administration at the time of either CD19 or CD44v6 CAR-T cell infusion efficiently prevented the CRS, but did not interfere with their comparable and long-term anti-leukemic effects. Conversely, depleting monocytes/macrophages before therapeutic CAR-T cell infusion by either lyposomal clodronate or by the prophylactic CD44v6 CAR-T cells inhibited CRS development, but also resulted in significantly worse leukemia-free survival (at 250d, 0% vs 80%, P<0.0001). Conclusions: A number of lessons can be learned from this innovative xenotolerant mouse model of CAR-T cell immunotherapy: monocytes are required for both i) optimal anti-leukemic efficacy, and ii) the occurrence of CRS; iii) tocilizumab prevents the CRS without interfering with efficacy; iv) monocyte aplasia induced by CD44v6 CAR-T cells does not impact on their efficacy, at least in the theraeputic setting, and may ameliorate CRS toxicity. As for CD44v6 CAR-T cells, this model could be used for effectively predicting the efficacy and associated toxicities of new CAR-T cell therapies, speeding up their clinical development. Disclosures Traversari: MolMed SpA: Employment. Bordignon:MolMed SpA: Employment. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Bondanza:Formula Pharmaceuticals: Honoraria; TxCell: Research Funding; MolMed SpA: Research Funding.

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&lt;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&lt;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&lt;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.

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

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

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

scholarly journals 206 The development of ‘chimeric CD3e fusion protein’ and ‘anti-CD3-based bispecific T cell activating element’ engineered T (CAB-T) cells for the treatment of solid malignancies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A217-A217
Author(s):  
Andy Tsun ◽  
Zhiyuan Li ◽  
Zhenqing Zhang ◽  
Weifeng Huang ◽  
Shaogang Peng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
In Vivo Studies ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

BackgroundCancer immunotherapy has achieved unprecedented success in the complete remission of hematological tumors. However, serious or even fatal clinical side-effects have been associated with CAR-T therapies to solid tumors, which mainly include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), macrophage activation syndrome, etc. Furthermore, CAR-T therapies have not yet demonstrated significant clinical efficacy for the treatment of solid tumors. Here, we present a novel T cell therapeutic platform: a Chimeric CD3e fusion protein and anti-CD3-based bispecific T cell activating element (BiTA) engineered T (CAB-T) cells, which target tumor antigens via the secretion of BiTAs that act independently of MHC interactions. Upon BiTA secretion, CAB-T cells can simultaneously achieve anti-tumor cytotoxic effects from the CAB-T cells and simultaneously activate bystander T cells.MethodsCAB-T cells were generated by co-expressing a chimeric CD3e fusion protein and an anti-CD3-based bispecific T cell activating element. The chimeric CD3e contains the extracellular domain of CD3e, a CD8 transmembrane domain, 4-1BB costimulatory domain, CD3z T cell activation domain and a FLAG tag, while the BiTA element includes a tumor antigen targeting domain fused with an anti-CD3 scFv domain and a 6x His-tag. CAR-T cells were generated as a control. Cytokine release activity, T cell activation and exhaustion markers, T cell killing activity and T cell differentiation stages were analysed. We also tested their tumor growth inhibition activity, peripheral and tumor tissue distribution, and their safety-profiles in humanized mouse models.ResultsCAB-T cells have similar or better in vitro killing activity compared with their CAR-T counterparts, with lower levels of cytokine release (IL-2 and IFNγ). CAB-T cells also showed lower levels of exhaustion markers (PD-1, LAG-3 and TIM-3), and higher ratios of naive/Tscm and Tcm T cell populations, after co-culture with their target tumor cells (48h). In in vivo studies, CAIX CAB-T and HER2 CAB-T showed superior anti-tumor efficacy and tumor tissue infiltration activity over their corresponding CAR-T cells. For CLDN18.2 CAB-T cells, similar in vivo anti-tumor efficacy was observed compared to CAR-T after T cell infusion, but blood glucose reduction and animal mortality was observed in the mice administered with CAR-T cells.ConclusionsThe advantages of CAB-T in in vitro and in vivo studies may result from TCR signal activation of both the engineered CAB-T cells and the non-engineered bystander T cells via cross-bridging by the secreted BiTA molecules, thus offering superior anti-tumor efficacy with a potential better safety-profile compared to conventional CAR-T platforms.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3931-3931
Author(s):  
Martina Fontaine ◽  
Benjamin Demoulin ◽  
Simon Bornschein ◽  
Susanna Raitano ◽  
Steve Lenger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity ◽  
Nkg2d Receptor

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

scholarly journals A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR T Cells in Vivo

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 567 ◽  
Author(s):  
Susana Pascoal ◽  
Benjamin Salzer ◽  
Eva Scheuringer ◽  
Andrea Wenninger-Weinzierl ◽  
Caterina Sturtzel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cellular Therapy ◽  
Xenograft Model ◽  
Model Systems ◽  
Preclinical Model ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.

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