High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity

R C Lynn; Y Feng; K Schutsky; M Poussin; A Kalota; D S Dimitrov; D J Powell Jr

doi:10.1038/leu.2016.35

Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections

Human Gene Therapy ◽

10.1089/hum.2019.149 ◽

2020 ◽

Vol 31 (7-8) ◽

pp. 423-439 ◽

Cited By ~ 1

Author(s):

Henning Olbrich ◽

Sebastian J. Theobald ◽

Constanze Slabik ◽

Laura Gerasch ◽

Andreas Schneider ◽

...

Keyword(s):

T Cells ◽

Cord Blood ◽

Human Cytomegalovirus ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Cytomegalovirus Infections

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0112 ◽

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.

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Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000896 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000896

Author(s):

Talia Velasco-Hernandez ◽

Samanta Romina Zanetti ◽

Heleia Roca-Ho ◽

Francisco Gutierrez-Aguera ◽

Paolo Petazzi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Therapy ◽

High Affinity ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundThere are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19– either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19– B-ALL relapses and CD19– preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.MethodsHere, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.ResultsConformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.ConclusionsWe report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22–CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

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High-affinity mutant Interleukin-13 targeted CAR T cells enhance delivery of clickable biodegradable fluorescent nanoparticles to glioblastoma

Bioactive Materials ◽

10.1016/j.bioactmat.2020.04.011 ◽

2020 ◽

Vol 5 (3) ◽

pp. 624-635 ◽

Cited By ~ 3

Author(s):

Gloria B. Kim ◽

Virginia Aragon-Sanabria ◽

Lauren Randolph ◽

Hali Jiang ◽

Joshua A. Reynolds ◽

...

Keyword(s):

T Cells ◽

Fluorescent Nanoparticles ◽

Interleukin 13 ◽

High Affinity ◽

Car T Cells ◽

Car T

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High-Affinity GD2-Specific CAR T Cells Induce Fatal Encephalitis in a Preclinical Neuroblastoma Model

Cancer Immunology Research ◽

10.1158/2326-6066.cir-17-0211 ◽

2017 ◽

Vol 6 (1) ◽

pp. 36-46 ◽

Cited By ~ 68

Author(s):

Sarah A. Richman ◽

Selene Nunez-Cruz ◽

Babak Moghimi ◽

Lucy Z. Li ◽

Zachary T. Gershenson ◽

...

Keyword(s):

T Cells ◽

High Affinity ◽

Car T Cells ◽

Car T

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CAR T Cells Targeted with a High Affinity Scfv Against the HCMV Glycoprotein Gb As Adoptive T Cell Therapy after Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.5721.5721 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5721-5721 ◽

Cited By ~ 2

Author(s):

Renata Stripecke ◽

Laura Gerasch ◽

Sebastian Theobald ◽

Bala Sai Sundarasetty ◽

Maksim Mamonkin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Target Cells ◽

Hematopoietic Stem ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Infected Cells

Abstract Introduction: Reactivation of human cytomegalovirus (HCMV) in immune compromised patients after hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality, particularly after cord blood transplantation (CBT). Adoptive transfer of T cells expanded in vitro is currently used as therapy for drug-refractory HCMV disease. A major limitation of this approach is the requirement of HLA-restricted HCMV-specific memory T cells. An alternative approach exploring HLA-independent T cell recognition was sought. Because the HCMV envelope glycoprotein B (gB) is highly expressed during lytic infection and in latently infected cells, we hypothesized that T cells can be redirected to recognize and kill HCMV-specific cells by means of a gB-specific chimeric antigen receptor (CAR). We have synthesized and tested a gB-specific CAR derived from the SM5-1 monoclonal antibody which binds with high affinity (KD 5.7x1011) to a conserved antigenic and non-glycosylated domain of gB. Methods: We generated two codon-optimized SM5-derived scFvs (VH->VL and VL->VH) and fused with an existing CAR backbone containing an IgG Fc spacer and intracellular signaling domains. CARs containing either CD28.zeta or 4-1BB.zeta were synthesized and expressed in T cells following a standard retroviral transduction protocol yielding 80-90% transduction rate. Expression of the CARs on T cells was confirmed by flow cytometry using goat anti-human immunoglobulin reactive against the IgG Fc region. 293T cells co-expressing gB and dTomato were used for in vitro cytotoxicity assays. Results: T cells expressing gB-CAR/CD28.zeta were cytotoxic against gB+ target cells producing 90% killing of 293T/gB-dTom cells compared with control CD19 CAR/CD28.zeta cells at an effector-to-target ratio 3:1 for 48 h (parental 293T cells were not killed). The cytolytic activity correlated with expansion of CAR T cells and concomitant loss of gB-dTom expression in the remaining viable 293T cells. Sequential co-culture of these gB-CAR T cells with freshly seeded 293T/gB-dTom resulted into further elimination of target cells. We are currently evaluating the effects of different gB-CAR T cell designs in the killing of HCMV-infected cell lines and primary cells using HCMV laboratory strains expressing the GFP and Gaussia Luciferase reporter genes. Pilot experiments indicated that gB-CAR/CD28.zeta cells with the scFv in the VL->VH orientation resulted into more clustering and killing of HepG2 cells infected with HCMV-GFP after 24h of co-culture than a control CD19 CAR/CD28.zeta. Humanized mice transplanted with cord blood CD34+ stem cells and challenged with these HCMV laboratory strains will be used to evaluate the in vivo effectivity of cord blood-derived donor-matched gB-CAR-T cells to eliminate acute and latent HCMV infections. Conclusion: These studies explore a novel approach in preventing HCMV reactivation in immunosuppressed patients by redirecting T cells expressing a high-affinity gB-CAR to eliminate HCMV-infected cells in a TCR/MHC-independent manner. Disclosures No relevant conflicts of interest to declare.

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DDRE-20. GENERATION OF A HIGH-AFFINITY EGFR/EGFRvIII-TARGETED D2C7 CAR-T CELL

Neuro-Oncology ◽

10.1093/neuonc/noaa215.265 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii65-ii66

Author(s):

Katherine Ryan ◽

Daniel Wilkinson ◽

Daniel Landi ◽

Peter Fecci

Keyword(s):

T Cells ◽

T Cell ◽

Murine Models ◽

Wild Type ◽

High Affinity ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract BACKGROUND The intent of this investigation is to validate a novel, high-affinity epidermal growth factor receptor (EGFR) chimeric antigen receptor (CAR) T-cell product for adoptive immunotherapy of EGFR-expressing malignancies of the central nervous system (CNS). Wild type EGFR is ubiquitously expressed on glial tumors in both children and adults, and the majority of solid tumors that metastasize to the CNS but is not expressed on healthy CNS tissues. EGFR and its isoform mutant, EGFRvIII, are hyperactivated or overexpressed receptor tyrosine kinases described in many human cancers. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) that binds to both wild type EGFR and EGFRvIII. We hypothesize that D2C7 adapted as a CAR on primary human T cells will generate potent, cytotoxic activity against EGFR-expressing tumors in vivo. METHODS In vitro interferon gamma release assays comparing the D2C7 CAR to the EGFRvIII CAR have proven that D2C7 CAR-T cells have high specificity and potent cytotoxicity against established murine glioblastoma (GBM) tumor cell lines, expressing both EGFR and EGFRvIII. Next steps include pre-clinical in vivo testing of the D2C7 CAR-T cells in murine models of CNS tumors expressing EGFR/EGFRvIII such as GBM, non-small cell lung carcinoma (NSCLC), breast carcinoma, and melanoma. CONCLUSIONS To improve and validate the effectiveness of CAR T-cell therapy for EGFR-expressing CNS tumors, our team has designed a high-affinity CAR-T cell that targets both EGFRvIII and wild-type EGFR, known as a D2C7 CAR-T cell. This investigation has established pre-clinical anti-tumor activity of D2C7-CAR T cells in vitro and we plan to present more mature data regarding efficacy in orthotopic murine models of GBM, NSCLC, breast carcinoma, and melanoma at the meeting this Fall.

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Enhanced functionality of low-affinity CD19 CAR T-cells is associated with activation priming and a polyfunctional cytokine phenotype

10.1101/2020.09.22.291831 ◽

2020 ◽

Author(s):

Ilaria M. Michelozzi ◽

Eduardo Gomez Castaneda ◽

Ruben V.C. Pohle ◽

Ferran Cardoso Rodriguez ◽

Jahangir Sufi ◽

...

Keyword(s):

T Cells ◽

Molecular Mechanisms ◽

Protein Profile ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Clinical Models

AbstractWe recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR, CAT) that showed enhanced expansion, cytotoxicity, and anti-tumour efficacy compared to the high-affinity (FMC63 based) CAR used in tisagenlecleucel, in pre-clinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a Phase I clinical study. To understand the molecular mechanisms behind the improved properties of CAT CAR T-cells, we performed a systematic in vitro characterization of the transcriptomic (RNA-seq) and protein (CyTOF) changes occurring in T-cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. We demonstrate that CAT CAR T-cells show enhanced activation to CD19 stimulation and a distinct transcriptomic/protein profile with increased cytokine polyfunctionality post-stimulation compared with FMC63 CAR T-cells. Our results suggest that the enhanced functionality of low-affinity CAR T-cells may be sustained by the establishment of a self-reinforcing circuit activated through cytokines polyfunctional crosstalk.

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