Publications by authors named "Elisa Landoni"

9 Publications

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Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Cancer Immunol Res 2021 Feb 5. Epub 2021 Feb 5.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0451DOI Listing
February 2021

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

Clin Cancer Res 2020 Dec 8;26(23):6321-6334. Epub 2020 Sep 8.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Turin, Italy.

Purpose: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes and in immunodeficient mice.

Experimental Design: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored , in two- and three-dimensional STS cultures, and in three STS xenograft models.

Results: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK.

Conclusions: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-0357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710537PMC
December 2020

Enhancing Chimeric Antigen Receptor T-Cell Efficacy in Solid Tumors.

Clin Cancer Res 2020 06 3;26(11):2444-2451. Epub 2020 Feb 3.

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina.

Chimeric antigen receptor (CAR) T-cell therapy has been acclaimed as a revolution in cancer treatment following the impressive results in hematologic malignancies. Unfortunately, in patients with solid tumors, objectives responses to CAR T cells are still anecdotal, and important issues are driven by on-target but off-tumor activity of CAR T cells and by the extremely complex biology of solid tumors. Here, we will review the recent attempts to challenge the therapeutic impediments to CAR T-cell therapy in solid tumors. We will focus on the most promising strategies of antigen targeting to improve tumor specificity and address the tumor heterogeneity, efforts to circumvent the physical barriers of the tumor architecture such as subverted tumor vasculature, impediments of CAR T-cell trafficking and immune suppressive microenvironment.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269829PMC
June 2020

A High-Avidity T-cell Receptor Redirects Natural Killer T-cell Specificity and Outcompetes the Endogenous Invariant T-cell Receptor.

Cancer Immunol Res 2020 01 12;8(1):57-69. Epub 2019 Nov 12.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

T-cell receptor (TCR) gene transfer redirects T cells to target intracellular antigens. However, the potential autoreactivity generated by TCR mispairing and occurrence of graft-versus-host disease in the allogenic setting due to the retention of native TCRs remain major concerns. Natural killer T cells (NKT) have shown promise as a platform for adoptive T-cell therapy in cancer patients. Here, we showed their utility for TCR gene transfer. We successfully engineered and expanded NKTs expressing a functional TCR (TCR NKTs), showing HLA-restricted antitumor activity in xenogeneic mouse models in the absence of graft-versus-mouse reactions. We found that TCR NKTs downregulated the invariant TCR (iTCR), leading to iTCRTCR and iTCRTCR populations. In-depth analyses of these subsets revealed that in iTCRTCR NKTs, the iTCR, although expressed at the mRNA and protein levels, was retained in the cytoplasm. This effect resulted from a competition for binding to CD3 molecules for cell-surface expression by the transgenic TCR. Overall, our results highlight the feasibility and advantages of using NKTs for TCR expression for adoptive cell immunotherapies. NKT-low intrinsic alloreactivity that associated with the observed iTCR displacement by the engineered TCR represents ideal characteristics for "off-the-shelf" products without further TCR gene editing.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684559PMC
January 2020

Machine-Learning Prediction of Tumor Antigen Immunogenicity in the Selection of Therapeutic Epitopes.

Cancer Immunol Res 2019 10 12;7(10):1591-1604. Epub 2019 Sep 12.

Department of Microbiology and Immunology, UNC School of Medicine, Chapel Hill, North Carolina.

Current tumor neoantigen calling algorithms primarily rely on epitope/major histocompatibility complex (MHC) binding affinity predictions to rank and select for potential epitope targets. These algorithms do not predict for epitope immunogenicity using approaches modeled from tumor-specific antigen data. Here, we describe peptide-intrinsic biochemical features associated with neoantigen and minor histocompatibility mismatch antigen immunogenicity and present a gradient boosting algorithm for predicting tumor antigen immunogenicity. This algorithm was validated in two murine tumor models and demonstrated the capacity to select for therapeutically active antigens. Immune correlates of neoantigen immunogenicity were studied in a pan-cancer data set from The Cancer Genome Atlas and demonstrated an association between expression of immunogenic neoantigens and immunity in colon and lung adenocarcinomas. Lastly, we present evidence for expression of an out-of-frame neoantigen that was capable of driving antitumor cytotoxic T-cell responses. With the growing clinical importance of tumor vaccine therapies, our approach may allow for better selection of therapeutically relevant tumor-specific antigens, including nonclassic out-of-frame antigens capable of driving antitumor immunity.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774822PMC
October 2019

In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab.

Int J Cancer 2020 01 12;146(1):236-247. Epub 2019 Oct 12.

Department of Biomedical Sciences, Institute of Translational Pharmacology, CNR, Rome, Italy.

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A -chimeric receptor (CR), and those engineered with the low-affinity CD16 -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A -CR T cells was 74 ± 10%, whereas the percentage of CD16 -CR T cells was 46 ± 15%. Only CD32A -CR T cells bound panitumumab. CD32A -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16 -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116 cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A -CR on T cells by cetuximab or panitumumab and CD16 -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR.
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http://dx.doi.org/10.1002/ijc.32663DOI Listing
January 2020

Eradication of Neuroblastoma by T Cells Redirected with an Optimized GD2-Specific Chimeric Antigen Receptor and Interleukin-15.

Clin Cancer Res 2019 05 7;25(9):2915-2924. Epub 2019 Jan 7.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Purpose: A delay in encountering the cognate antigen while in the circulation, and the suboptimal costimulation received at the tumor site are key reasons for the limited activity of chimeric antigen receptor-redirected T cells (CAR-T) in solid tumors. We have explored the benefits of incorporating the IL15 cytokine within the CAR cassette to provide both a survival signal before antigen encounter, and an additional cytokine signaling at the tumor site using a neuroblastoma tumor model.

Experimental Design: We optimized the construct for the CAR specific for the NB-antigen GD2 without (GD2.CAR) or with IL15 (GD2.CAR.15). We then compared the expansion, phenotype, and antitumor activity of T cells transduced with these constructs against an array of neuroblastoma cell lines and using a xenogeneic metastatic model of neuroblastoma.

Results: We observed that optimized GD2.CAR.15-Ts have reduced expression of the PD-1 receptor, are enriched in stem cell-like cells, and have superior antitumor activity upon repetitive tumor exposures and as compared with GD2.CAR-Ts. Tumor rechallenge experiments further highlighted the role of IL15 in promoting enhanced CAR-T antitumor activity and survival, both in the peripheral blood and tissues. Finally, the inclusion of the inducible caspase-9 gene (iC9) safety switch warranted effective on demand elimination of the engineered GD2.CAR.15-Ts.

Conclusions: Our results guide new therapeutic options for GD2.CAR-Ts in patients with neuroblastoma, and CAR-T development for a broad range of solid tumors.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1811DOI Listing
May 2019

Treating hematological malignancies with cell therapy: where are we now?

Expert Opin Biol Ther 2018 01 5;18(1):65-75. Epub 2017 Oct 5.

a Lineberger Comprehensive Cancer Center , University of North Carolina , Chapel Hill , NC , USA.

Introduction: Adoptive cell therapy (ACT) is becoming an increasingly successful and widespread form of treatment for different types of cancer. Compared to chemotherapy or monoclonal antibodies, ACT is an active biological strategy, with infused immune cells featuring dynamic migration, expansion and long-term persistence properties. ACT in hematological malignancies offered the initial proof of principle of the feasibility for this innovative 'live-drug'.

Areas Covered: In this review, the authors summarize the clinical results achieved with two specific strategies in hematological malignancies: chimeric antigen receptor (CAR) and T cell receptor (transgenic TCR) redirected T cells. Moreover, they discuss the recent pre-clinical studies aimed at increasing the feasibility, safety and efficacy of ACT.

Expert Opinion: ACT can promote cancer regression in patients with leukemia, lymphoma and multiple myeloma. Nevertheless, more precise targeting of tumor cells and containment of side effects are needed. Overcoming tumor-associated immunosuppressive mechanisms and preventing tumor escape are also emerging as critical barriers. Finally, simplification in the manufacturing procedures should promote wider application of these technologies outside academic centers. Although the enthusiasm for ACT-based therapies is high, comprehensive and systematic clinical studies are required to advance the field.
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http://dx.doi.org/10.1080/14712598.2018.1384810DOI Listing
January 2018

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease.

Blood 2017 08 21;130(5):606-618. Epub 2017 Jun 21.

Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, Program in Immunology and Bio-immunotherapy of Cancer.

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1 targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.
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http://dx.doi.org/10.1182/blood-2016-08-732636DOI Listing
August 2017