767 Interferon gamma reduces CAR-T exhaustion and toxicity without compromising therapeutic efficacy in hematologic malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A815-A815
Author(s):  
Stefanie Bailey ◽  
Sonika Vatsa ◽  
Amanda Bouffard ◽  
Rebecca Larson ◽  
Irene Scarfo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Hematologic Malignancies ◽  
Car T Cells ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, ultimately leading to its FDA approval for relapsed/refractory acute lymphoblastic leukemia and large cell lymphomas in 2017. Despite the success of CAR T cells in the clinic, toxicities such as cytokine release syndrome (CRS) can be severe. Attempts to mitigate these effects have primarily focused on the blockade of macrophage-derived cytokines, such as IL-6 and IL-1B. Herein, we show that the pharmaceutical blockade or genetic deletion of interferon gamma (IFNg, a CAR-T-derived cytokine that strongly correlates with CRS in the clinic, appears to be a viable target for the reduction of CAR-T-associated toxicities.MethodsPharmacologic (blocking antibody) and genetic (CRISPR/Cas9) approaches were used to block IFNg signaling and/or production by CAR T cells. In vitro CAR-T function and cytotoxicity was tested using ELISA, flow cytometry and short-/long-term killing assays prior to their assessment in vivo. NSG mice were injected with Nalm6 or JeKo-1 cancer cells prior to treatment with IFNg-modified CAR-T and tumor size and IFNg production were measured. To determine how the loss of IFNg might affect innate immune cells, CAR-T, macrophages and tumor cells were co-cultured and assessed by flow cytometry, immunofluorescence, Luminex and RNA sequencing.ResultsIFNg could be blocked using an anti-IFNg antibody or CRISPR/Cas9 editing of the CAR T cells without affecting T cell activation, proliferation or cytokine production (IL-2, TNFa, GM-CSF). Successful blockade of the IFNg signaling pathway was confirmed by reduced phosphorylation of JAK1, JAK2 and STAT1, even in the presence of exogenous IFNg. Loss of IFNg did not reduce the cytotoxic potential or persistence of CAR-T against hematologic malignancies in vitro or in vivo. When cultured with macrophages and cancer cells, IFNg knockout (IFNgKO) CAR-T yielded decreased levels of IL-1B, IL-6, IL-13, MCP1 and CXCL10, indicating a reduction in macrophage activation induced by CAR-T in the absence of IFNg. Serum from tumor-bearing mice treated with IFNgKO CAR-T elicited lower activation of macrophages in vitro compared to those treated with IFNg-producing CAR-T cells. Furthermore, IFNgKO CAR T cells co-cultured with tumor cells and macrophages demonstrated less exhaustion as shown by reduced expression of PD1, Tim3 and Lag3 and increased IFNgKO CAR-T expansion.ConclusionsCollectively, these data suggest that IFNg is not required for the efficacy of CAR-T in hematologic malignancies and can potentially be targeted to reduce toxicity and enhance CAR-T efficacy and persistence in the clinic.

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).

Preclinical evaluation of anti-ROR1 CAR T cells employing a ROR1 binding SCFV derived from the clinical stage mab cirmtuzumab.

2020 ◽  
Vol 38 (5_suppl) ◽  
pp. 41-41
Author(s):  
Charles E. Prussak ◽  
Christopher Oh ◽  
Juliana Velez Lujan ◽  
Sharon Lam ◽  
Jieyu Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
In Vivo Studies ◽  
Tumor Escape ◽  
Cancer Stemness ◽  
2Nd Generation ◽  
Car T Cells ◽  
Car T

41 Background: Chimeric antigen receptor (CAR)-modified T cells (CAR-T) were generated targeting cells expressing ROR1, which is present on many malignant cancers and has been associated with cancer stemness and chemo-resistance. The ROR1 CAR utilizes the humanized single-chain fragment variable (scFv) binding domain of UC-961 (cirmtuzumab), which exhibits high affinity and specificity for human ROR1 and has demonstrated an excellent safety profile in Phase 1 studies. Methods: CAR constructs with varying spacer regions and intracellular co-stimulatory domains, using the scFV of cirmtuzumab, were constructed and used to generate CAR-T cells from healthy donors. These ROR1 CAR-T cells were tested for cytotoxicity against lymphoid cancer cells in vitro and in vivo studies that employed immune-deficient mice engrafted with labeled human leukemia cells MEC1 or MEC1-ROR1, which had been transfected to stably express ROR1. Results: The 2nd generation and 3rd generation CAR-T-cells with analogous spacer regions were comparably potent and selectively cytotoxic for cells bearing the ROR1 target antigen. But the 2nd generation CARs demonstrated greater potency in vitro even at low effector to target ratios. For the in vivo studies, mice received a single injection of ROR1 CAR-T cells or activated T cells from the same donor as a control. The ROR1 CAR-T cells rapidly cleared the leukemic cells from the animals, whereas animals receiving control T cells or no therapy quickly succumbed to progressive disease within 3 weeks. The administered CAR-T products remained highly active following administration and could be detected for ≥ 3 months without evidence for T cell exhaustion. Conclusions: The generated CAR-T cells utilizing constructs with the Fv of cirmtuzumab, a humanized mAb highly specific for ROR1, onco-embryonic surface antigen, effectively and selectively killed neoplastic cells bearing ROR1 both in vitro and in vivo. As ROR1 expression and signaling has been associated with cancer stemness and chemo-resistance utilizing ROR1 CAR-T therapy to target cancer cells might mitigate tumor escape. These data strongly support the rationale for continued development of our ROR1 CAR-T.

scholarly journals Engineering light-controllable CAR T cells for cancer immunotherapy

2020 ◽  
Vol 6 (8) ◽  
pp. eaay9209 ◽  
Author(s):  
Ziliang Huang ◽  
Yiqian Wu ◽  
Molly E. Allen ◽  
Yijia Pan ◽  
Phillip Kyriakakis ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
Cancer Immunotherapy ◽  
Tumor Antigens ◽  
Nuclear Translocation ◽  
Car T Cells ◽  
Car T ◽  
Target Cancer

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.

scholarly journals 105 4–1BB and optimized CD28 co-stimulation enhances function of human mono- and bi-specific third-generation CAR T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A115-A116
Author(s):  
Emiliano Roselli ◽  
Justin Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Kristen Spitler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
The Other ◽  
Target Cells ◽  
Third Generation ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to NF-κB signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with 4-1BB or mut06 together with the combination of both (BB06). We evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed LCK recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NSG mice. Additionally, we engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased polyfunctionality and LCK recruitment to the CAR (figure 1A), after repeated-antigen stimulation these cells expanded significantly better than second-generation CAR T cells (figure 1B). A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers (figure 1C). Bi-specific CAR T cells exhibited improved in vivo anti-tumor activity with increased persistence and decreased exhaustion (figure 1D).Abstract 105 Figure 1A. pLCK is increased in h19BB06z CAR T cells and associated with the CAR. CAR T cells were stimulated with irradiated 3T3-hCD19 cells at a 10:1 E:T ratio for 24hr. Cells were lysed and CAR bound and unbound fractions were western blotted. A single-cell measure of polyfunctional strength index (PSI) of CAR T cells. B. h19BB06z CAR T cells have the highest proliferation after repeated antigen stimulations. 5x105 CAR T cells were stimulated with 1x105 irradiated 3T3-hCD19 cells. After 1 week, half of the cells were enumerated by flow cytometry and the other half was re-stimulated with 1x105 fresh irradiated 3T3-hCD19 cells. This was repeated for a total of 4 weeks. C. 5x105 CAR T cells were co-cultured with 5x105 target cells (Raji-CD19High). After 1 week half the cells were harvested enumerated and stained by flow cytometry while the other half was re-stimulated with 5x105 fresh target cells (indicated by arrows). This was repeated for a total of 4 weeks. Frequency of PD1+CD39+ cells within CD8+ CAR T cells. D. Raji-FFLuc-bearing NSG mice were treated with 1x106 CAR T cells 5 days after initial tumor cell injection. Tumor burden (average luminescence) of mice treated with bi-specific or monospecific CAR T cells, UT and tumor control. Each line represents an individual mouse. (n = 7 mice per group).ConclusionsThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono- and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

scholarly journals Therapeutic Targeting of Mesothelin in Acute Myeloid Leukemia with Chimeric Antigen Receptor T Cell Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11 ◽  
Author(s):  
Quy Le ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Anisha Loeb ◽  
Amanda R. Leonti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cell Surface ◽  
Myeloid Leukemia ◽  
Car T Cells ◽  
Car T ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is one of the most highly refractory hematologic malignancies despite intensive combination chemotherapy and bone marrow stem cell transplantation. Lack of curative treatments is in large part due to our poor understanding of the disease biology and paucity of therapeutic targets. In an effort to identify actionable targets, we recently completed the largest genome, epigenome and transcriptome profiling of AML in nearly 3000 children and young adults. This discovery effort has led to the identification of a library of novel AML-restricted targets (high expression in AML, minimal-to-no expression in normal hematopoiesis) for therapeutic development. One such target was MSLN which encodes for mesothelin, a cell surface adhesion molecule that is highly expressed in 30-50% of AML cases in pediatric (Children Oncology Group) and adult (MD Anderson) cohorts and is entirely absent in normal bone marrow and peripheral blood CD34+ cells. MSLN expression in normal tissues is confined to mesothelial cells lining the pleura, pericardium, and peritoneum. Previous studies targeting MSLN in solid tumors have demonstrated clinical efficacy with minimal toxicities. Given that T cells genetically modified to express chimeric antigen receptors (CARs) are extremely effective at eradicating relapsed and refractory malignancy, we developed MSLN-directed CAR T cells for pre-clinical evaluation in AML. Methods: From primary patient samples, we verified MSLN expression by RT-PCR and confirmed mesothelin surface protein expression on leukemic blasts by flow-cytometry as well as detected soluble mesothelin in the plasma by ELISA. The VH and VL sequences from Amatuximab were used to create the scFv domain of the standard CAR (41-BB and CD3Zeta). For in vivo CAR T study, Nomo-1 cells, which express endogenous level of MSLN, and Kasumi-1 cells engineered to express MSLN with a lentivirus construct (Kasumi-1 MSLN+) were transplanted into NSG mice. Mock transduced MSLN-directed CAR T cells were infused 1 week (Nomo-1) and 2 weeks (Kasumi-1 MSLN+) following leukemic cell injection. Leukemic burden was measured by bioluminescence IVIS imaging weekly. For in vitro study, Nomo-1 cells were treated with GM6001 (50uM), a metalloprotease inhibitor, or DMSO control for 48 hr prior to evaluation of surface mesothelin by flow cytometry and soluble mesothelin in the culture supernatant by ELISA. Results: In vivo cytotoxicity of CAR T cells against Nomo-1 and Kasumi-1MSLN+ AML models demonstrated potent, target-dependent tumor killing. After 1- and 2-weeks post CAR T infusion, leukemic cells were eradicated in both Nomo-1 (p<0.0005, week 2, Figure 1A) and Kasumi-1 MSLN+ xenografts (p<0.005 at week 2, Figure 1B). Mesothelin undergoes shedding at the cell membrane as a result of ADAM17-mediated cleavage. Blocking ADAM17 activity with GM6001 in Nomo-1 cells led to increased cell surface mesothelin (Figure 1C) with a corresponding reduction in the shed form (Figure 1D), suggesting that GM6001 treatment stabilizes mesothelin on the cell surface. Furthermore, GM6001 treatment during co-culture of Nomo-1 and CAR T cells enhanced cytolytic activity of CAR T cells (Figure 1E). GM6001 treatment did not significantly impact cell viability of Nomo-1 cells in the absence of CAR T cells (data not shown). Conclusion: In this study, we demonstrate that mesothelin is a viable therapeutic target and a potential diagnostic biomarker in AML. We show that MSLN CAR T cells were highly effective in eliminating MSLN-positive AML cells in vitro and in vivo. Shedding contributes to the loss of mesothelin antigen and provides a source of soluble mesothelin that may interfere with antibody-based therapies, including CAR T cells. Modulating MSLN shedding by inhibiting ADAM17-mediated cleavage resulted in stabilized mesothelin and improved CAR T cell functionality. This work warrants further evaluation of MSLN CAR T cells to be tested in clinical trials for AML and demonstrates that inhibiting MSLN shedding is a promising approach to improve CAR T efficacy. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD72 Nanobody-Based CAR-T Cells Have Potent Anti-Tumor Efficacy in B Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1717-1717
Author(s):  
Matthew A Nix ◽  
William C Temple ◽  
William Karlon ◽  
Donghui Wang ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
In Vitro Cytotoxicity ◽  
B Cell Malignancies ◽  
Cytotoxicity Assays ◽  
Car T Cells ◽  
Car T

Abstract Background: Approximately 50% of pediatric B-ALL patients treated with clinically approved CD19-targeting CAR-T cells do not remain in remission one year after therapy. CD22-targeting CAR-T cells appear to be curative in only a small fraction of CD19-refractory patients and this therapeutic strategy is primarily used as a bridge to stem cell transplant. Additional immunotherapeutic targets thus remain urgently needed. Our laboratory recently used cell surface proteomics to identify CD72 as a B-cell specific marker especially upregulated on poor prognosis, KMT2A/MLL-rearranged B-ALL (Nix et al., Cancer Discovery (2021)). In this published work, we used a best-in-class nanobody library displayed on yeast to develop binders to CD72. We demonstrated for the first time that fully synthetic nanobodies can generate CAR-T cells that are highly potent in vitro and in vivo. While we previously focused on these "nanoCARs" in KMT2A/MLLr B-ALL, in this follow-up study we aimed to 1) further expand our nanoCAR indications to other CD72-expressing B-cell malignancies; 2) biophysically characterize our synthetic nanobodies; 3) evaluate the potential for further humanization of the nanobody binder amino acid sequence while retaining anti-tumor efficacy; and 4) characterize the potency and T-cell immunophenotypes in the context of our lead nanobody binder ("NbD4") placed on different CAR backbones. Methods: Flow cytometry of primary patient samples for CD72 was performed in a CLIA-certified laboratory. NbD4 nanobody was recombinantly expressed in E. coli and biolayer interferometry was used to determine the binding affinity to recombinantly-expressed CD72 extracellular domain. CAR-T cells were generated from peripheral blood donor CD4+ and CD8+ cells (1:1) ratio via lentiviral transduction. In vitro cytotoxicity assays were performed at a range of effector:tumor ratios. In vivo studies were performed in human cell line orthotopic xenografts in NSG mice. 1e6 luciferase-labeled Jeko cells were implanted at Day 0 followed by administration of 4e6 CAR-T cells at Day 6. Tumor burden was assessed by bioluminescence. Results: Flow cytometry on primary non-Hodgkin B-cell lymphoma obtained from fine needle aspiration biopsy (n = 5) confirmed CD72 surface expression (not shown), consistent with RNA-seq across larger cohorts. Biolayer interferometry demonstrated that NbD4 bound with surprisingly low affinity to recombinant CD72 (K D ~800 nM) (Fig. 1A), with both slow on rate (k on 8.38e4 M -1s -1) and rapid off rate (k off 6.82e-2 s -1). This affinity stands in contrast to that reported for FMC63 single chain variable fragment (scFv) used in clinically approved CD19-targeting CAR-T cells (K D 0.3-5 nM), despite similar in vitro and in vivo efficacy of both products. Our NbD4 framework region shows ~82% homology to a human IgG variable heavy domain, significantly higher than FMC63 (~59% homology). We made additional substitutions in the framework domain to increase human homology up to 89%. In vitro cytotoxicity assays with SEM B-ALL cells showed several humanized variants with similar efficacy to NbD4 (Fig. 1B). We further evaluated the impact of placing NbD4 on different CAR backbones, including combinations of CD28 or 4-1BB costimulatory domains and CD8 or IgG4-based transmembrane and hinge regions (Fig. 1C). In vivo, CD72 nanoCARs with Backbone 3 showed significantly increased potency (Fig. 1D). Indeed, tumors treated with Backbone 3 CAR-Ts showed complete tumor clearance and did not develop new tumors even after re-challenge with 1e6 Jeko cells at Day 52 (Fig. 1D). Preliminary characterization of effector and memory CAR-T cell phenotypes before exposure to tumor suggested that Backbone 3 had an increased number of naïve T cells compared to empty CAR and CD19 CAR-T cells (data not shown). Conclusions: Our results demonstrate that our fully synthetic CD72 nanoCARs can effectively eliminate CD72-expressing B-cell malignancy models despite low nanobody binding affinity. Humanized NbD4 variants may serve as clinical candidates with even further reduction in possible immunogenicity of the llama amino acid framework. Alterations to the CAR backbone have a major impact on anti-tumor efficacy and phenotypes of our synthetic nanobodies. CD72-targeting therapies may be effective therapeutics not only KMT2A/MLLr B-ALL but also across a broader spectrum of refractory B-cell malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma

2020 ◽  
Author(s):  
Liqing Kang ◽  
Jian Zhang ◽  
Minghao Li ◽  
Nan Xu ◽  
Wei Qi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

Abstract Background: Treatment with chimeric antigen receptor (CAR)-engineered T cells directed against the B-cell maturation antigen (BCMA) promoted transient recovery from multiple myeloma (MM). However, the absence of this antigen on immature plasma cells may limit the efficacy of this modality and facilitate relapse. The purpose of this study is to characterize a novel CAR that includes both a single-chain variable fragment (scFv)-BCMA and an scFv-CD19 in tandem orientation (tan-CAR) in an attempt to target both BCMA and CD19 expression on MM cells. Method: The scFv sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T-cell signaling domains to generate the tan-CAR construct. Specificity and efficacy of activated tan-CAR T cells were analyzed using in vitro proliferation, cytokine release, and cytolysis assays. We also evaluated the in vivo efficacy with a xenograft mouse model that included target tumor cells that expressed CD19 or BCMA and compared the results to those obtained with conventional CAR T cells. Results: The in vitro studies revealed specific activation of tan-CAR T cells by K562 cells that overexpressed CD19 and/or BCMA. Cell proliferation, cytokine release, and cytolytic activity were all comparable to the responses of single scFv CAR T cells. Importantly, in vivo studies of tan-CAR T cells revealed specific inhibition of tumor growth in the mouse xenograft model that included cells expressing both CD19 and BCMA. Systemic administration of tan-CAR T cells resulted in complete tumor remission, in contrast to the reduced efficacies of BCMA-CAR T and CD19-CAR T alone in this setting. Conclusion: We report the successful design and execution of novel tan-CAR T cells that promote significant anti-tumor efficacy against both CD19 and BCMA antigen-positive tumor cells in vitro and in vivo . The data from this study reveal a novel strategy that may help to reduce the rate of relapse in the treatment with single scFv-CAR T cells.

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 Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

2017 ◽  
Vol 9 (10) ◽  
pp. 867-878 ◽  
Author(s):  
Yanjing Song ◽  
Chuan Tong ◽  
Yao Wang ◽  
Yunhe Gao ◽  
Hanren Dai ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Antitumor Activity ◽  
Cancer Cells ◽  
Gastric Cancer Cells ◽  
Car T Cells ◽  
Car T

scholarly journals IMMU-20. EVALUATION OF CAR T CELLS IN EPENDYMOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii364-iii364
Author(s):  
Anandani Nellan ◽  
Andrew Donson ◽  
Jacob Calhoun ◽  
Andrea Griesinger ◽  
Terry Fry ◽  
...  
Keyword(s):  
T Cells ◽  
Brain Tumors ◽  
Tumor Cells ◽  
Second Generation ◽  
High Risk Patient ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Abstract BACKGROUND Ependymoma is the third most common pediatric brain tumor and current treatment still results in a 10-year relapse rate of over 70% in the highest risk groups. The treatment refractory nature of ependymoma to standard therapies strongly supports the development of novel interventions. Ependymoma tumor cells express HER2 and there are active clinical trials treating children with ependymoma using local delivery of second-generation HER2 CAR T cells. METHODS Two high-risk patient-derived ependymoma cell lines, MAF811 and MAF928, that display HER2 surface expression are used for testing. We tested second-generation HER2-BBz CAR T cells in vitro and in vivo. RESULTS HER2 CAR T cells effectively kill ependymoma tumor cells in culture, but this strategy cannot eradicate the same tumor cells in mice when implanted in the fourth ventricle of the brain. HER2 CAR T cells proliferate and traffic into the tumor, but this causes a dramatic influx of immune cells, tumor swelling and lethal toxicity in a subset of mice. Mice that survive this initial tumor swelling, display significant tumor shrinkage but all tumors eventually start growing again. Ependymoma tumor cells release high amounts of inflammatory chemokines that strongly attract neutrophils and monocytes to the tumor, compared to other brain tumors, and can downregulate HER2 expression to escape recognition by CAR T cells. CONCLUSION The immunosuppressive microenvironment as well as tumor heterogeneity make HER2 CAR T cells ineffective in ependymoma. Studying these two hurdles in CAR T cell therapy is critical to effectively treat brain tumors with CAR T cells.

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