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M. Guercio et al.
of exhaustion and limited in vivo persistence. Moreover, in the case of CAR.CD30, the low CD30 expression on CD3+ T cells could also cause chronic antigen stimulation and the occurrence of cellular fratricide.31
Our experimental data indicate that the inclusion of an AC10-derived scFv in the CAR construct, independently of the co-stimulatory domains, was not associated with exhaustion, even after multiple CD30+ lymphoma-cell stimulations. Furthermore, we did not observe fratricidal activity among CAR.CD30 T-cell populations. With respect to co-stimulatory domains, we comparatively eval- uated two III-CAR.CD30, incorporating either CD28.OX40 or CD28.4-1BB. The clinical impact of com- bining co-stimulatory domains has not yet been clearly established, and it may be construct- and disease- specific.32,33 However, in the setting of repetitive antigen engagement, driving T-cell maturation to terminally differ- entiated cells associated with loss of CCR7, the combined CD28ζ-OX40 signaling CAR rescued CCR7– T cells from apoptosis resulting, in turn, in more efficient antitumor efficacy.34 For early clinical development, trial designs com- paring two CAR T cells simultaneously administered to the same patient have provided invaluable evidence of the pharmacokinetic effect of co-stimulatory domains.35-37 Ultimately, experimental evidence38,39 suggests that the positioning of co-stimulatory domains within the endodomain of a CAR can influence CAR T-cell activity, and no algorithm, up to now, has been able to predict which co-stimulatory combination is optimal for a specific CAR construct, making the search for optimized activity strictly dependent on experimental conditions. We recently showed the advantage of using the CD28.4-1BB co-stimu- latory domain to optimize CAR T-cell therapy targeting GD2+ neuroblastoma.40 In the current study, however, we observed an in vivo superiority of the CAR.CD30 construct incorporating the CD28.OX40 co-stimulatory domains, in terms of both anti-lymphoma activity and CAR T-cell per- sistence. We also confirmed the in vitro data in an in vivo model, showing greater stability of the CAR.CD30.CD28.OX40 T-cell population, both in terms of CAR+ cell percentage and CAR expression (MFI), even after repeated/prolonged exposures to tumor. CAR.CD30.CD28.OX40 T-cell activation profile correlates with efficient tumor control, stable expression of CAR molecule on the cell membrane and high production of IFN-γ, TNF-α and IL2 cytokines (Th1 profile). Evaluation of the cytokine activation profile is especially relevant in the context of lymphoma. Indeed, HL malignant cells express high levels of PDL1 and produce the immunosuppressive IL10, TGFβ, galectin 1 and prostaglandin E2 molecules, which inhibit T-cell effector functions and induce apopto- sis of activated Th1 and CD8+ T cells.41,42 We demonstrated that CAR.CD30.CD28.OX40 T cells and, in particular, the CD8+ fraction, exert significant and prolonged anti-lym- phoma activity even in this strongly immune-modulating environment.
Importantly, we confirmed that the manufacturing process based on IL7/IL15 is crucial for optimizing CAR T-cell activity also in the lymphoma setting. In particular, we proved, both in vitro and in vivo, that CAR.CD30 T
cells (IL7/IL15) systematically exposed to CD30+ lym- phoma cells were significantly enriched in CM and EM subpopulations, in both CD4+ and CD8+ T-cell subsets. Moreover, in a NHL xenograft mouse model, in which we mimic lymphoma relapse through tumor re-challenge, CAR.CD30.CD28.OX40 T cells were able to re-expand significantly and exert tumor control. We also showed that the number of CAR.CD30.CD28.OX40 T cells declined in peripheral blood upon lymphoma eradication. Thorough characterization of mouse tissues after long- term in vivo experiments (day +240) revealed the presence of CAR.CD30.CD28.OX40 T cells in several organs, including bone marrow, lymph nodes, kidney, liver, spleen and thyroid.
The inclusion of the inducible “safety switch” iCasp943 is crucial to render the therapeutic approach safer, con- trolling potential unwanted side effects in the context of CAR T cells. We showed both in vitro and in vivo that AP1903 is able to significantly reduce iCasp9.CAR.CD30 cells. However, the persistence, albeit at very low levels, of genetically modified T cells with a low expression of CAR.CD30 after AP1903 treatment cannot be excluded.
Overall, the significant in vivo reactivity, the high poten- cy, the negligible toxicity in animals and the long persist- ence of CAR.CD30.CD28.OX40 T cells contribute to the value of this CAR design, which will be tested in a clinical trial for patients with relapsed/refractory HL and anaplas- tic large-cell lymphoma.
Disclosures
No conflicts of interest to disclose. A patent application has been made (n. 102018000003464).
Contributions
MG, CQ, BDA and FL designed experimental studies, supervised the conduction of the project, analyzed the data and wrote the manuscript. MG, DO, SDC, MS, SC, IB, ZA, AC, BC, KB, IC, CDS, MP, EG, MS, SM, RC and RDV per- formed the in vitro experiments. CDS and MP performed immunohistochemistry assays. MG, IB, BDA and CQ per- formed the in vivo experiments. DO, MG and BDA cloned the retroviral vector. MG, EG, MS and MS performed FACS analysis. AR, FDB, PM, LV, KG, RDV, LM and FL provided patients’ samples, medical advice and expertise in pediatric lym- phoma. SB, ACi and MT analyzed data. All authors read and approved the final version of the manuscript.
Acknowledgments
We are grateful to Bellicum Pharmaceuticals for kindly pro- viding the AP1903 dimerizing drug.
Funding
The experimental work was supported by grants awarded by
Ricerca Finalizzata GR-2016-02364546 (to BDA), Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC)- Special Project 5×1000 n. 9962 (to FL), AIRC IG 2018 id. 21724 (to FL), Ricerca Finalizzata GR-2013-02359212 (to CQ), Ricerca Corrente (to CQ and BDA). Progetto Ministeriale CAR-T (to FL), and Associazione “Raffaele Passarelli” Onlus (to BDA).
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haematologica | 2021; 106(4)