Gritti, G

Ibrahim, L

Maciocia, PM

Pule, MA

Wawrzyniecka, PA

English

UCL Discovery

LETTER OPENIMMUNOTHERAPYChimeric antigen receptor T cells for gamma–delta T cellmalignanciesP. A. Wawrzyniecka1, L. Ibrahim1, G. Gritti 2, M. A. Pule 1 and P. M. Maciocia 1✉© The Author(s) 2021Leukemia; https://doi.org/10.1038/s41375-021-01385-0TO THE EDITOR:Cancers derived from the malignant transformation of gamma–delta(γδ) T cells carry very poor prognosis. The major pathologiesrecognised are γδ T acute lymphoblastic leukaemia (γδ T-ALL), andtwo lymphoma subtypes: hepatosplenic T cell lymphoma (HSTL) andprimary cutaneous γδ T cell lymphoma (PCγδ-TCL) [1]. γδ T-ALLrepresents approximately 10% of cases of T-ALL and is associatedwith high rates of induction failure, relapse and excess mortality [2].HSTL is a rare (approximately 3% of cases of T cell lymphoma [1]) buthighly aggressive disorder, which typically presents in males in the2nd or 3rd decade of life, often in association with immunosuppres-sive therapy [3]. It carries the worst prognosis of all lymphomasubtypes, with a median survival of only 6–8 months [4] and onlyisolated cases of long-term survival [5]. PCγδ-TCL is also rare(approximately 1% of skin lymphomas [1]) and presents withcutaneous involvement, typically associated with visceral and/orbone marrow disease. Again, outcomes are poor, with 75% 1-yearmortality in the largest published case series [6].Treatment for γδ malignancies is with cytotoxic chemotherapy,with no tumour-specific therapies currently available. By contrast,in analogous B-cell malignancies, highly effective immunothera-pies, including monoclonal antibodies, bispecific T cell engagersand chimeric antigen receptor (CAR)-T cells [7] are available.These therapies have revolutionised the treatment and outcomeof advanced B-cell malignancies. CAR-T cells against CD19in particular have demonstrated the potential to induce long-lasting complete remissions even in patients with advanced andrefractory cancers [7].For γδ malignancies, the defining immunophenotypic charac-teristic is expression of the γδ T cell receptor (TCR), present in>95% of cases of HSTL and in all PCγδ-TCL and γδ T-ALL [3].Importantly, in normal tissues expression is limited to γδ T cells,where it functions as the antigen recognition receptor. Here, wedeveloped CAR-T cells targeting the γδ TCR and demonstratein vitro and in vivo efficacy against γδ T cell malignancies. Ourdata offers proof-of-concept for γδ TCR-targeting with CAR-T cellsas a potential approach to bring highly potent immunotherapy tothe treatment of γδ malignancies.Primary αβ T cells were retrovirally transduced to expressanti-γδ TCR CAR or control anti-CD19 CAR (Fig. 1a). Followingtransduction with anti-CD19 CAR, a small proportion of γδ T cellspersisted in the culture, including some which expressed anti-CD19 CAR. By contrast, for anti-γδ TCR CAR, no γδ T cells weredetected in the culture, suggesting ‘purging’ of these cells by thetransduced population (Fig. 1b). CAR-T cells were then co-culturedwith T cell lines which natively express (Loucy – Vγ9Vδ2, BE13 –Vγ8Vδ1, MOLT13 – Vγ3Vδ1 [8]) or are negative for surface γδ TCR(SupT1-CD19). While control anti-CD19 CAR lysed only SupT1-CD19 cells, anti-γδ TCR CAR-T lysed only γδ TCR-positive cell lines(Fig. 1c). In addition, anti-γδ TCR CAR-T cells demonstrated specificsecretion of cytokines including interferon-γ, IL-2, IL-13 and TNF-α(Fig. 1d). Next, we co-cultured anti-CD19 or anti-γδ CAR-T cellswith normal autologous γδ T cells. At a high E:T ratio (1:1), targetnormal γδ T cells were partially lysed (Fig. 1e), with concomitantexpansion of anti-γδ CAR-T cells (Fig. 1f). A marked down-regulation of γδ TCR expression was noted on surviving γδ T cells(Fig. 1g). Interestingly, by contrast, at lower E:T ratios (1:2 and 1:4),paradoxical γδ T cell expansion was instead observed (Fig. 1e),associated with reduction in numbers of anti-γδ CAR-T cells(Fig. 1f). This suggests lysis of anti-γδ CAR-T by target normal γδT cells.To assay the in vivo potency of anti-γδ TCR CAR-T cells, we utilisedthe Loucy murine model of disseminated γδ TCR-positive leukaemia(Fig. 2a, d). NSG mice were intravenously injected on CAR D-12 with4 × 106 Loucy cells, engineered to stably express Firefly luciferase.Tumour engraftment was confirmed by bioluminescence imaging(BLI) at D-1 (data not shown), then mice were treated on D0 with 8 ×105 anti-γδ TCR or control anti-CD19 CAR-T cells. Mice receiving anti-γδ TCR CAR demonstrated reduction of tumour burden, as assessedby flow cytometry of bone marrow and spleen at necropsy on D14(Fig. 2b, c, Supplementary Fig. 1), BLI (Fig. 2e, f) and bleed at D30(Fig. 2g). Prolonged survival (Fig. 2h) was seen in anti-γδ TCR CARrecipients compared to CD19 CAR-treated animals, although allanimals eventually died of progressive γδ TCR-positive disease, withno evidence of antigen downregulation. In common with other NSGmodels, CAR-T cell persistence was limited, with no detectable cells inthe peripheral blood at D30 (data not shown).Despite success in B-cell malignancies, a lack of acceptabletargets means targeted immunotherapy is rarely applied toT cell malignancies. The anti-CD30 antibody-drug conjugateReceived: 7 April 2021 Revised: 6 July 2021 Accepted: 4 August 20211Department of Haematology, Cancer Institute, University College London, London, UK. 2Hematology Department, Azienda Socio Sanitario Territoriale Papa Giovanni XXIIIHospital, Bergamo, Italy. ✉email: p.maciocia@ucl.ac.ukwww.nature.com/leuLeukemiaFig. 1 In vitro testing of anti-γδ TCR CAR. a Schematic of anti-γδ TCR CAR, with 2nd generation architecture. b Example flow plot of γδ-TCRstaining on anti-CD19 or anti-γδ TCR CAR-T cells following transduction c co-cultures of anti-γδ TCR or control anti-CD19 CAR-T cells withCD19+ (SupT1-CD19) or γδ TCR+ cell lines (Loucy, MOLT13, BE13) (c) cytotoxicity at 72 h, as measured by bioluminescence-based assay (d) cytokinesecretion at 48 h. e–g 120-h co-culture of control or anti-γδ TCR CAR-T cells with autologous normal γδ T cells, n= 3. e Residual γδ T cells asproportion of starting cells f Example γδ TCR staining on normal γδ T cells after co-culture with anti-γδ TCR CAR-T or anti-CD19 CAR-T cells g residualanti-γδ TCR CAR-T or anti-CD19 CAR-T following co-culture, as proportion of starting cells. **p < 0.001, ***p< 0.0001.Fig. 2 In vivo assessment of anti-γδ TCR CAR. a Schematic of Loucy murine model (n= 6/group) b Quantification of tumour in (b) marrowand (c) spleen at D14 following CAR-T injection. d Schematic of Loucy murine model (n= 9/group) e bioluminescence (BLI) imaging at D21following CAR-T infusion. f Quantification of BLI signal at D21 g quantification of tumour burden in blood at D30 h survival curves of mice(median survival CAR19 50 days v CARγδ 69 days, HR 12.4, p= 0.0003, comparison by log-rank method). All other comparisons byMann–Whitney U test.P.A. Wawrzyniecka et al.2Leukemia1234567890();,:brentuximab vedotin is effective in anaplastic large cell lymphoma[9], however, CD30 is not typically expressed in γδ cancers [2].Suggested approaches to targeting T cell malignancies includetargeting pan-T cell antigens such as CD5 [10] or CD7 [11].However, such strategies deplete the entire normal T cellcompartment and may induce profound immunosuppression[11], potentially requiring rescue by allogeneic hematopoieticstem cell transplant.More refined approaches that target clonal elements of the TCR,such as selective targeting of TRBC1 and TRBC2 in αβ T cellmalignancies, allow depletion of only part of the normal T cellcompartment [12]. While analogous approaches are potentiallypossible in γδ TCR malignancies, simple targeting of the γδ TCRmay be feasible. This approach could concomitantly depletenormal γδ T cells. These constitute <5% of peripheral blood T cells,are more abundant in tissues and have a range of proposedancillary immunological functions [13]. Importantly, geneticallyγδ-deficient mice display a very mild phenotype [14], and there isno known human pathology associated with γδ T cell deficiency.This suggests that depletion of the γδ T cell compartment maybe clinically tolerable, although initially clinical exploration ofanti-γδ TCR CAR-T should proceed cautiously: for instance withco-expression of a suicide gene [15], availability of back-upcryopreserved peripheral blood mononuclear cells, and closemonitoring for development of atypical infections.Indeed, it is unclear if anti-γδ CAR-T treatment would lead to γδT cell aplasia. An interesting observation in our study was that, whileanti-γδ CAR-T expanded when cultured with normal γδ T cells at highE:T ratio, the reverse was observed when normal γδ T was in excess.Thus, anti-γδ CAR-T were themselves depleted from the culture, withexpansion of normal γδ T cells. The probable explanation is that CARbinding to the TCR of γδ T cells induced CAR signalling, but alsosignalling via the TCR of the γδ T cell, leading to a 2-way synapse witheach cell potentially both target and effector. When normal γδ Toutnumbered anti-γδ CAR-T, the balance of cytotoxicity resulted inanti-γδ CAR-T cell lysis and expansion of the normal cells. Thepotential clinical consequences for anti-γδ TCR CAR therapy areunclear and would be difficult to ascertain pre-clinically due to a lackof relevant immunocompetent models. However, in patients receivinganti-γδ CAR-T, it is likely that the CAR-T: normal γδ T cell ratio at thetumour site would be high in the critical initial CAR-T expansionphase, following lymphodepleting chemotherapy.Here, we have demonstrated the feasibility of engineeringnormal αβ T cells to express anti-γδ TCR CAR and have shown thatanti-γδ TCR CAR-T cells can specifically kill malignant γδ cells bothin vitro and in vivo. Our approach offers the first proposedstrategy to bring highly potent CAR-T cells to the treatment ofγδ T cell malignancies, where there is a major unmet needfor effective therapies. Clinical assessment of this approach iswarranted.REFERENCES1. Swerdlow SH, Campo E, Harris NL. WHO classification of tumours of haemato-poietic and lymphoid tissues. Lyon; IARC; 2008.2. Matos DM, Rizzatti EG, Fernandes M, Buccheri V, Falcão RP. Gammadelta andalphabeta T cell acute lymphoblastic leukemia: comparison of their clinical andimmunophenotypic features. Haematologica. 2005;90:264–6.3. Tripodo C, Iannitto E, Florena AM, Pucillo CE, Piccaluga PP, Franco V, et al.Gamma-delta T cell lymphomas. Nat Rev Clin Oncol. 2009;6:707–17.4. Vose J, Armitage J, Weisenburger D. International T Cell Lymphoma Project.International peripheral T cell and natural killer/T cell lymphoma study: pathologyfindings and clinical outcomes. J Clin Oncol. 2008;26:4124–30.5. Rashidi A, Cashen AF. Outcomes of allogeneic stem cell transplantation inhepatosplenic T cell lymphoma. Blood Cancer J. 2015;5:e318.6. Willemze R, Jansen PM, Cerroni L, Berti E, Santucci M, Assaf C, et al. Subcutaneouspanniculitis-like T cell lymphoma: definition, classification, and prognostic factors: anEORTC Cutaneous Lymphoma Group Study of 83 cases. Blood. 2008;111:838–45.7. Grupp SA, Laetsch TW, Buechner J, Bittencourt H, Verneris MR, Boyer MW, et al.Analysis of a global registration trial of the efficacy and safety of CTL019 inpediatric and young adults with relapsed/refractory acute lymphoblastic leukemia(ALL). Blood. 2016;128:221.8. Sandberg Y, Verhaaf B, van Gastel-Mol EJ, Wolvers-Tettero ILM, de Vos J, MacLeodRAF. et al. Human T cell lines with well-defined T cell receptor gene rearrange-ments as controls for the BIOMED-2 multiplex polymerase chain reaction tubes.Leukemia. 2007;21:230–7.9. Horwitz S, O’Connor OA, Pro B, Illidge T, Fanale M, Advani R, et al. Brentuximabvedotin with chemotherapy for CD30-positive peripheral T cell lymphoma (ECHELON-2): a global, double-blind, randomised, phase 3 trial. Lancet. 2019;393:229–40.10. Mamonkin M, Rouce RH, Tashiro H, Brenner MK. A T cell-directed chimeric antigenreceptor for the selective treatment of T cell malignancies. Blood. 2015;126:983–92.11. Gomes-Silva D, Srinivasan M, Sharma S, Lee CM, Wagner DL, Davis TH, et al.CD7-edited T cells expressing a CD7-specific CAR for the therapy of T cellmalignancies. Blood. 2017;130:285–96.12. Maciocia PM, Wawrzyniecka PA, Philip B, Onuoha SC, Legut M, Sewell AK, et al.Targeting the T cell receptor β-chain constant region for immunotherapy of T cellmalignancies. Nat Med. 2017;89:3909–12.13. Vantourout P, Hayday A. Six-of-the-best: unique contributions of γδ T cells toimmunology. Nat Rev Immunol. 2013;13:88–100.14. Fujihashi K. gamma/delta T cell-deficient mice have impaired mucosalimmunoglobulin A responses. J Exp Med. 1996;183:1929–35.15. Philip B, Kokalaki E, Mekkaoui L, Thomas S, Straathof K, Flutter B, et al. A highlycompact epitope-based marker/suicide gene for easier and safer T cell therapy.Blood. 2014;124:1277–87.ACKNOWLEDGEMENTSThe authors would like to thank members of the Pule laboratory for help and advice.AUTHOR CONTRIBUTIONSPAW designed, performed and analysed the experiments and wrote the manuscript.LI assisted with in vitro experiments. GG provided reagents and samples. MAP wrotethe manuscript and provided laboratory support. PMM conceived the idea, designed,performed and analysed experiments and wrote the manuscript.COMPETING INTERESTSPAW, LI and GG disclose no conflicts of interest. MAP is founder/chief scientificofficer, and holds stocks/ equity in Autolus Ltd. PMM holds stocks in Autolus Ltd.ADDITIONAL INFORMATIONSupplementary information The online version contains supplementary materialavailable at https://doi.org/10.1038/s41375-021-01385-0.Correspondence and requests for materials should be addressed to P.M.M.Reprints and permission information is available at http://www.nature.com/reprintsPublisher’s note Springer Nature remains neutral with regard to jurisdictional claimsin published maps and institutional affiliations.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directlyfrom the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2021P.A. Wawrzyniecka et al.3Leukemia

Chimeric antigen receptor T cells for gamma-delta T cell malignancies

https://discovery.ucl.ac.uk/10133380/1/s41375-021-01385-0.pdf

Chimeric antigen receptor T cells for gamma-delta T cell malignancies

Abstract

Similar works

Full text

Available Versions

UCL Discovery