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M. Guercio et al.
large B-cell lymphoma.2,3 However, alternative targets are needed for other types of lymphoma lacking CD19 expression, including diseases such as classical Hodgkin lymphoma (HL), anaplastic large-cell lymphoma and other T-cell lymphomas.
Although most patients with HL or NHL are cured with first-line therapies, a relevant proportion of them have primary refractory disease or experience relapse after ini- tial response to treatment.4 The standard of care for patients who relapse after first-line treatment is intensive chemotherapy followed, in responders, by autologous stem cell transplantation. Although autologous transplan- tation offers the potential to cure about half of patients, the prognosis of subjects relapsing after the autograft or not eligible for transplantation is poor.5 Novel therapies are, therefore, desirable for patients with relapsed/refrac- tory lymphoma.
Despite biological differences, HL and NHL have proven to be good targets for immunotherapy: indeed, both occur in the immune-rich lymphoid tissues and are easily accessible to antibody- and cell-based immunother- apy.5 Moreover, CD30, a cell-membrane protein belong- ing to the tumor-necrosis-factor receptor superfamily 8, can be found on the cell-surface of both HL and selected NHL including anaplastic large-cell lymphoma, diffuse large B-cell lymphoma, primary mediastinal B-cell lym- phoma,6 peripheral T-cell lymphoma,7 and adult T-cell leukemia/lymphoma,8 as well as in rare solid tumors,9 including embryonal carcinomas10 and seminomas.11 Its restricted expression on a subset of normal, activated T and B cells12,13 renders CD30 an excellent candidate for immune-based therapies, with a low risk of off-tumor, on-target toxicity.
CD30 has been extensively explored as a target for anti- body-based therapy. The most remarkable results have been achieved with brentuximab-vedotin, an antibody- drug conjugate directed against CD30, shown to be well tolerated and associated with relevant activity in HL and anaplastic large-cell lymphoma.14 Although brentuximab- vedotin appears to induce excellent responses.15,16 this antibody-drug conjugate is also associated with adverse events leading to treatment discontinuation in a signifi- cant proportion of patients.17 To overcome the challenges presented by antibody-based therapy, namely limited response durability and reduced tumor penetration.18 CAR T cells have been explored.
Immunotherapeutic approaches with CAR targeting CD30 have shown efficacy in preclinical models,19,20 and these results have been translated into the clinic in two trials based on second-generation CD30.CAR T cells, including either CD28 or 4-1BB co-stimulatory domains.21,22 The clinical efficacy of these second-genera- tion CD30.CAR T cells was, however, suboptimal, as inconsistent responses were observed, most patients hav- ing either stable disease after multiple CAR T-cell infu- sions, or no response at all. Overall, lymph nodes showed better responses than extranodal lesions and CAR T cells did not persist longer than 60 days after infusion. Notably, two studies with CD30.CAR T cells supported several other clinical observations in different settings,23,24 showing a correlation between CAR T-cell persistence and patients’ outcome. We therefore sought to optimize the CAR.CD30 T-cell approach by using a novel single- chain variable fragment (scFv), as well as a novel third- generation construct.
We demonstrate that, with our CAR.CD30 T-cell approach, the use of the novel scFv, the combination of the co-stimulatory molecules CD28 and OX40, as well as a production process based on the addition of interleukin 7 (IL7) and interleukin 15 (IL15), are all relevant to drive high in vivo proliferation/expansion, long-term persistence and establishment of the immunological memory neces- sary to control lymphoma recurrence.
Methods
Generation of retroviral vectors and transduction method of T cells
The scFv for CAR.CD30 molecules is derived from the AC10 monoclonal antibody.25 The details of the constructs are provided in Figure 1A, Online Supplementary Figure S1A, Online Supplementary Materials and Online Supplementary Table S1, which report the amino-acid sequences for all the construct components. Retroviral supernatant was generated in 293T-cells19,26,27 and quan- tified using a Retro-XTM qRT-PCR Titration Kit (Takara) to be used at 109 retrovirus-copies/0.5x106 T cells. The supernatant was used to transduce primary T cells derived from peripheral blood mononuclear cells of healthy donors (Ethical Committee approval n. 969/2015 protocol n. 669LB). More details on transduction are provided in the Online Supplementary Materials.
Phenotypic analysis
The following monoclonal antibodies were used: CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CD223, CD274, CD279, and TIM3 (BD Pharmigen, USA). The expression of CAR.CD30 on T cells was evaluated using anti-CD34 antibody (R&D, USA) or the Pierce Recombinant Biotinylated Protein L.28 (Thermo Fisher Scientific, USA). The gating strategy is reported in Online Supplementary Figure S2.
In vitro anti-lymphoma activity
CAR T-cell cytotoxicity was evaluated using a 51Cr-release
assay.20 For co-culture experiments, T cells and lymphoma-cell lines (Ethical Committee approval n. 652/2018 protocol n. GR- 2016-02364546), were plated for 7 days, and residual tumor ana- lyzed by fluorescent activated cell sorting (FACS). For “stressed” co-cultures, tumor cells were added on days 0, 5, 10, 15 and 20 at an effector:target (E:T) ratio of 1:1. The residual tumor cells and persisting T cells were analyzed by FACS 5 days after each tumor addition.
Cytokine analysis
Supernatant collected from 24 h co-culture experiments was analyzed by an enzyme-linked lectin assay (ELLA) (R&D System).
In vivo experiments
In vivo experiments were approved by the Italian Health
Ministry (n. 88/2016-PR). Specifically, 6-week old NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; from Charles River) mice were engrafted intravenously (i.v.) with 0.2x106 CD30+ Karpas299-eGFP-FFLuc (NHL model) or 2x106 CD30+ L428- eGFP-FFLuc (HL model). After tumor engraftment, mice received an i.v. injection of effector T cells (10x106/mouse). Tumor growth was evaluated using an IVIS imaging system (Perkin Elmer, USA).27 For tumor re-challenge, mice of the NHL model surviving until day +140, were infused i.v. with 0.2x106 Karpas299-eGFP-FFLuc cells. Mice were followed for an addi- tional 110 days, without effector T-cell administration.
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haematologica | 2021; 106(4)