112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A124-A124
Author(s):  
Letizia Giardino ◽  
Ryan Gilbreth ◽  
Cui Chen ◽  
Erin Sult ◽  
Noel Monks ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Single Chain ◽  
High Affinity ◽  
Car T Cells ◽  
Car T ◽  
Animal Care ◽  
Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

CD19-Redirected Chimeric Antigen Receptor T (CART19) Cells Induce a Cytokine Release Syndrome (CRS) and Induction of Treatable Macrophage Activation Syndrome (MAS) That Can Be Managed by the IL-6 Antagonist Tocilizumab (toc).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2604-2604 ◽  
Author(s):  
Stephan A. Grupp ◽  
David L Porter ◽  
David T Teachey ◽  
David M. Barrett ◽  
Anne Chew ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Tumor Lysis Syndrome ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Abstract Abstract 2604 We previously reported on CART19 cells expressing a chimeric antigen receptor (CAR) with intracellular activation and costimulatory domains. Infusion of these cells results in 100 to 100,000× in vivo proliferation, tumor lysis syndrome followed by durable antitumor activity, and prolonged persistence in pts with B cell tumors. Here we report that in vivo proliferation of CART19 cells and potent anti-tumor activity is associated with CRS, leading to hemophagocytic lymphohistiocytosis (HLH), also termed MAS. We propose that MAS/HLH is a unique biomarker that is associated with and may be required for potent anti-tumor activity. Autologous T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3-zeta, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into ALL or CLL pts with persistent disease after 2–8 prior treatments. CART19 anti ALL activity was also modeled in a xenograft mouse model with high level of human ALL/human T cell engraftment and simultaneous detection of CAR T cells and ALL using 2-color bioluminescent imaging. We describe updated results of 10 pts who received CART19 cells elsewhere at ASH (Porter, et al), including 9 pts with CLL and 1 pediatric pt with relapsed refractory ALL. 6/9 evaluable pts had a CR or PR, including 4 sustained CRs. While there was no acute infusional toxicity, all responding pts also developed CRS. All had high fevers, as well as grade 3 or 4 hypotension/hypoxia. CRS preceded peak blood expression of CART19 cells, and then increased in intensity until the CART19 cell peak (D10–31 after infusion). The ALL pt experienced the most significant toxicity, with grade 4 hypotension and respiratory failure. Steroid therapy on D6 resulted in no improvement. On D9, noting high levels of TNFa and IL-6 (peak increases above baseline: IFNg at 6040x; IL-6 at 988x; IL-2R at 56x, IL-2 at 163× and TNFa at 17x), we administered TNFa and IL-6 antagonists entanercept and toc. This resulted in resolution of fever and hypotension within 12hr and a rapid wean from ventilator support to room air. These interventions had no apparent impact on CART19 cell expansion or efficacy: peak of CAR T cells (2539 CAR+ cells/uL; 77% of CD3 cells by flow) occurred on D11, and D23 bone marrow showed CR with negative MRD, compared to her initial on-study marrow which showed 65% blasts. Although she had no history of CNS ALL, spinal fluid showed detectable CART19 cells (21 lymphs/mcL; 78% CAR+). At 4mo post infusion, this pt remains in CR, with 17 CART19 cells/uL in the blood and 31% CAR+ CD3 cells in the marrow. Clinical assessment of subsequent responding patients shows all had evidence of MAS/HLH including dramatic elevations of ferritin and histologic evidence of HLH. Peak ferritin levels range from 44,000 to 605,000, preceding and continuing with peak T cell proliferation. Other consistent findings include rapid onset hepatosplenomegaly unrelated to disease and moderate DIC. Subsequently, 3 CLL patients have also been treated with toc, also with prompt and striking resolution of high fevers, hypotension and hypoxia. 1 received toc on D10 and achieved a CR accompanied by CART19 expansion. 1 had rapid resolution of CRS following toc administration on day 9 and follow up for response is too short. A 3rd CLL pt received toc on D3 for early fevers and had no CART-19 proliferation and no response. To model the timing of cytokine blockade, xenografts using bioluminescent primary pediatric ALL were established and then treated with extra cells from the clinical manufacture. The CART19 cells proliferated and resulted in prolonged survival. Cytokine blockade prior to T cell infusion with toc and/or etanercept abrogated disease control with less in vivo proliferation of infused CART19 cells, confirming the result seen in the one pt given early toc (D3). The optimal time and threshold to trigger cytokine blockade is currently being tested in these models. CART19 T cells can produce massive in-vivo expansion, long-term persistence, and anti-tumor efficacy, but can also induce significant CRS with features suggestive of MAS/HLH that responds rapidly to cytokine blockade. Given prior to initiation of significant CART19 proliferation, blockade of TNFa and/or IL-6 may interfere with proliferation and effector function, but if given at a point where cell proliferation is underway, toc may ameliorate the symptoms that we have observed correlate with robust clinical responses. Disclosures: Off Label Use: tocilizumab for cell therapy toxicity. Levine:University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties; TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kalos:University of Pennsylvania: Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

scholarly journals CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression

2021 ◽  
Author(s):  
Katherine Mueller ◽  
Nicole Piscopo ◽  
Matthew Forsberg ◽  
Louise Saraspe ◽  
Amritava Das ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Viral Vectors ◽  
Viral Vector ◽  
Tumor Regression ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) T cells traditionally harbor viral vectors that encode the CAR transgene in the genome. However, viral vector manufacturing typically is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. Here, CAR T cells were generated within nine days using recombinant SpCas9 protein and nucleic acids, without any viral vectors or animal components. In comparison to traditional retroviral CAR T cells, nonviral CRISPR CAR T cells exhibit TRAC-targeted genomic integration of the CAR transgene, higher frequency of gene expression signatures associated with a memory phenotype, low receptor signaling prior to infusion, and potent cytotoxicity against GD2+ neuroblastoma in vitro and in vivo. This proof-of-principle study eliminating viral vectors and animal components during CAR gene transfer could enable more flexible and scalable manufacturing of clinically-relevant, high-quality CAR T cells to treat cancers, including solid tumors.

scholarly journals The roles of T cell competition and stochastic extinction events in chimeric antigen receptor T cell therapy

2021 ◽  
Vol 288 (1947) ◽  
Author(s):  
Gregory J. Kimmel ◽  
Frederick L. Locke ◽  
Philipp M. Altrock
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy is a remarkably effective immunotherapy that relies on in vivo expansion of engineered CAR T cells, after lymphodepletion (LD) by chemotherapy. The quantitative laws underlying this expansion and subsequent tumour eradication remain unknown. We develop a mathematical model of T cell–tumour cell interactions and demonstrate that expansion can be explained by immune reconstitution dynamics after LD and competition among T cells. CAR T cells rapidly grow and engage tumour cells but experience an emerging growth rate disadvantage compared to normal T cells. Since tumour eradication is deterministically unstable in our model, we define cure as a stochastic event, which, even when likely, can occur at variable times. However, we show that variability in timing is largely determined by patient variability. While cure events impacted by these fluctuations occur early and are narrowly distributed, progression events occur late and are more widely distributed in time. We parameterized our model using population-level CAR T cell and tumour data over time and compare our predictions with progression-free survival rates. We find that therapy could be improved by optimizing the tumour-killing rate and the CAR T cells' ability to adapt, as quantified by their carrying capacity. Our tumour extinction model can be leveraged to examine why therapy works in some patients but not others, and to better understand the interplay of deterministic and stochastic effects on outcomes. For example, our model implies that LD before a second CAR T injection is necessary.

scholarly journals In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4736-4745 ◽  
Author(s):  
Greta Maria Paola Giordano Attianese ◽  
Virna Marin ◽  
Valentina Hoyos ◽  
Barbara Savoldo ◽  
Irene Pizzitola ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Lymphocytes ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Car T Cells ◽  
Car T

Abstract Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19+CD5+CD20dim B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR+ T cells showed specific cytotoxic activity against CD23+ tumor cell lines (average lysis 42%) and primary CD23+ CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR+ T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR+ T cells. Redirected T cells were also effective in vivo in a CLL Rag2−/−γc−/− xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR+ T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR+ T cells represent a selective immunotherapy for the elimination of CD23+ leukemic cells in patients with CLL.

scholarly journals A CAR RNA FISH assay reveals functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2020 ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Semih U. Tareen ◽  
Michael C. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer and HIV sanctuary sites. Here, we describe a CAR RNA FISH histocytometry platform combined with a dnnRRS image analysis algorithm to quantitate spatial distribution and in vivo functional ability of a CAR T cell population at a single cell resolution. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions engineered cells in situ with their target cells in tissues with high statistical power and thus, as an important tool for preclinical and potentially clinical assessment of CAR T cell effectiveness.One Sentence SummaryWe developed a CAR RNA FISH assay to study chimeric antigen receptor T cell trafficking and function in human and mouse tissue.Impact statementWe developed an imaging platform and analysis pipeline to study large populations of engineered cells on a single cell level in situ.

scholarly journals High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms

2021 ◽  
Vol 11 ◽  
Author(s):  
Radhika Thokala ◽  
Zev A. Binder ◽  
Yibo Yin ◽  
Logan Zhang ◽  
Jiasi Vicky Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood–brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

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.

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