Clinical & Translational Immunology 2021; e1268. doi: 10.1002/cti2.1268 www.wileyonlinelibrary.com/journal/cti ORIGINAL ARTICLE Memory stem T cells modified with a redesigned CD30-chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma Carmen Alvarez-Ferna ndez1,2,3†, Laura Escriba -Garcia1,2,3†, AC Caballero1,2,3,4, Eva Escudero-Lo pez 1,2,3,4 , Cristina Ujaldo n-Miro 1,2,3,4, Rosanna Montserrat-Torres1,2,3, Paula Pujol-Fernandez 1,2,3 , Jorge Sierra1,3,4 & Javier Briones1,2,3,4 1 Hematology Service, Hospital de la Santa Creu y Sant Pau, Barcelona, Spain 2 Laboratory of Experimental Hematology-IIB, Institut Recerca Hospital de la Santa Creu y Sant Pau, Barcelona, Spain 3  Carreras Leukemia Research Institute, Barcelona, Spain Jose 4 Autonomous University of Barcelona, Barcelona, Spain Correspondence Abstract J Briones or C Alvarez-Fernandez, Hematology Service, Hospital Santa Creu y Objectives. Adoptive cell therapy (ACT) with mature T cells Sant Pau, Mas Casanovas 90, 08041 modified with a chimeric antigen receptor has demonstrated Barcelona, Spain. improved outcome for B-cell malignancies. However, its E-mails: jbriones@santpau.cat (JB) or application for others such as Hodgkin lymphoma remains a calvarezf@santpau.cat (CAF) clinical challenge. CD30 antigen, expressed in Hodgkin lymphoma † Equal contributors. cells, is absent in most healthy tissues, representing an ideal target of ACT for this disease. Despite that, efficacy of CD30-chimeric Received 4 June 2020; antigen receptor (CAR) T cells for Hodgkin lymphoma remains Revised 18 December and modest. Here, we have developed and tested a novel CD30-CAR T 4 March 2020; to improve efficacy of CD30-CAR therapy, using a targeting Accepted 4 March 2021 epitope within the non-cleavable part of CD30 receptor, and memory stem T cells (TSCM) to improve engraftment, persistence doi: 10.1002/cti2.1268 and antitumor activity. Methods. TSCM-like cultures were generated Clinical & Translational Immunology and expanded ex vivo and transduced at day 1 or 2 with a 2021; 10: e1268 lentiviral vector encoding the CD30-CAR. Therapeutic in vivo experiments were performed using NSG mice injected with L540 (sc) or L428 (iv) and treated with CD30-CAR T cells when the tumor was established. Results. CD30-CAR TSCM-like cells generated and expanded ex vivo, despite CD30 expression and fratricide killing of CD30+ CAR T cells, were not impaired by soluble CD30 and completely eradicated Hodgkin lymphoma in vivo, showing high persistence and long-lasting immunity. In addition, highly enriched CD30-CAR TSCM-like products confer a survival advantage in vivo, in contrast to more differentiated CAR T cells, with higher tumor infiltration and enhanced antitumor effect. Conclusion. This study supports the use of a refined CD30-CAR T cells with highly enriched TSCM-like products to improve clinical efficacy of CAR T for Hodgkin lymphoma. Keywords: chimeric antigen receptor, immunotherapy, memory stem T cells ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 1 CD30-CAR memory stem T cells for treating Hodgkin lymphoma C Alvarez-Fern andez et al. na€ıve, central memory (TCM) and the recently INTRODUCTION described memory stem T cells (TSCM),13 are T cells expressing chimeric antigen receptors capable to generate the complete T-cell (CARs) have revolutionised the field of ACT for repertoire, displaying in vivo long persistence, cancer in the last few years.1 CAR technology greater proliferative capacity and enhanced allows engineered T cells to recognise tumor- antitumor efficacy than TEFF. TSCM, a particular associated antigens (TAA) with high specificity in subpopulation characterised by the high a MHC-independent manner, improving T-cell expression of CD45RA, CD62L, CCR7 and CD95, proliferation and antitumor efficacy.2,3 A number had brought attention through its association of clinical trials with CD19-CAR-modified T cells with clinical efficacy of CAR therapy.13–15 (CAR19) have developed in the last years for B-cell The clinical significance of this T-cell subset has malignancies with high rates of complete been shown recently in patients receiving CAR19 remission and a significant proportion of patients cells in whom in vivo expansion of CAR T cells and being long-term progression-free.4–7 However, the clinical responses have been correlated with the application of CAR T-cell therapy to other frequency, within the infused product, of a T-cell haematological tumors such as Hodgkin subset with a phenotype closely related to TSCM.16,17 lymphoma (HL) resulted in lower remission rates, Here, we have developed a novel CAR targeting and thus, it remains a clinical challenge. CD30 with the aim of improving the antitumor CD30 protein, a member of the tumor necrosis efficacy of CD30-CAR therapy. Design novelties factor receptor family, is highly expressed by HL include the use of a scFv targeting a membrane- malignant cells,8 while its expression is highly proximal epitope to maximise the CAR therapy restricted in normal cells (i.e. expressed in antitumor effect and the interaction of the CD30- eosinophils and some subtypes of activated T and CAR with a protein domain localised in the stalk, B cells), making this receptor a very convenient non-cleavable region of the CD30 molecule, to target for Hodgkin and other CD30+ lymphomas. avoid the potential blockade by the soluble CD30 Two reported clinical trials using CD30-CAR T protein.18,19 In addition, we generated highly cells against HL have demonstrated that CAR T- enriched modified CD30-CAR TSCM-like products to cell infusion is well tolerated, with no relevant take advantage of their intrinsic potential for toxicities.9,10 However, although clinical results in durable engraftment, in vivo proliferation and those heavily treated patients are promising, tumor homing in order to improve the CAR responses are modest, and in contrast to what is therapeutic efficacy.10,13,14 Our work demonstrates seen in patients treated with a CAR19, very few that CD30-CAR-modified TSCM-like cells can be patients achieve long-term remission.10 efficiently transduced and expanded ex vivo Although the mechanisms explaining this despite CD30 protein expression. Importantly, we modest clinical efficacy are not well understood, show that, despite the presence of soluble CD30 previous studies suggest that factors such as protein, our CD30-CAR TSCM-like cells show membrane location of the targeted epitope and enhanced antitumor activity against HL, tumor persistence of CAR T cells in treated patients are homing and persistence in vivo, compared with critical points for a successful CAR T-cell cell products with more differentiated CD30-CAR therapy.9,10 In addition, the CD30 single-chain T cells. These data may significantly contribute to variable fragment (scFv) used in those studies the design of improved CD30-CAR cell therapies could be blocked by soluble CD30 protein (found for patients with HL and other CD30+ lymphoid at high concentrations in most of HL patients), malignancies, which may translate into better which may significantly limit CD30-CAR efficacy. clinical outcomes. In addition, clinical trials with CAR T cells have used mostly T-cell products with a differentiated phenotype [effector memory T cells (TEM) and RESULTS terminally differentiated effector T cells (TEMRA)], Generation of TSCM-like cells modified with a which leads to a poor persistence of T cells novel CD30-CAR in vivo.6,9 Previous studies of ACT in mice11 and non-human primates12 showed that effector T To generate CD30-CAR TSCM-like enriched cells, cells (TEFF) have a robust cytotoxic capacity, but isolated CD8+ and CD4+ na€ıve T cells from healthy only less differentiated T-cell subsets, such as donors were cultured with low doses of IL-7, IL-15 2021 | Vol. 10 | e1268 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 2 Australian and New Zealand Society for Immunology, Inc. C Alvarez-Fern andez et al. CD30-CAR memory stem T cells for treating Hodgkin lymphoma and IL-21, and activated with a short CD3/CD28 T cells, CD30-CAR T cells and UN cells, respectively) costimulation. TSCM-like cells were the most that further decrease at the end of the culture prevalent T-cell subset at day 10 of culture (3.9  0.89% vs 0.18  0.27 in CD4+ T cells; and (62.4  6.49% CD4+ T cells, 67.29  8.62% of CD8+ 6.65  2.26% vs 0.20  0.26% in CD8+ T cells, T cells; Figure 1a) and were efficiently transduced CD30-CAR T cells and UN cells, respectively; with a third-generation CD30-CAR lentiviral vector, Figure 1c). Interestingly, the decrease in the showing a CD30-CAR expression of 87.03  1.69% proportion of CD30+ T cells in transduced T-cell in bulk T cells (Figure 1b) and 94.3  1.34% in culture was correlated with an increase in T-cell CD4+ and 75.3  2.14% in CD8+ TSCM-like cells, viability from 78.95  0.83% at day 4 to respectively (Supplementary figure 1). 88.55  0.38% at the end of the culture A concern with a CAR targeting CD30 protein is (Figure 1d), while UN T-cell viability was not the potential elimination of activated CD30+ T affected during cell culture (Figure 1d), cells by CD30-CAR T cells (i.e. ‘fratricide killing’). In suggesting a possible elimination of CD30+ T cells this regard, we detected a significant increase in by CD30-CAR T cells. the proportion of CD30+ T cells during the culture Moreover, the fold expansion of transduced T peaking at day 4 compared with untransduced cells is significantly lower than that of non- (UN) T cells (38.86  1.92% vs 6.8  3.24 in CD4+ transduced T cells, which could be also related to T cells; and 40.92  3.69% vs 11.92  4.87 in CD8+ fratricide killing. Figure 1. TSCM-like are highly enriched, CD30-CAR-transduced and expanded in vitro despite CD30 expression. Na€ıve T cells from healthy donors (n = 7) were cultured with CD3/CD28 costimulation in the presence of IL-7, IL-15 and IL-21 during 10 days. (a) Frequencies of CD4+ and CD8+ T-cell subpopulations at the end of culture. TSCM-like cells were the most prevalent T-cell population (mean  SD). (b) Representative plot of T cells transduced at day 2 with CD30-CAR-encoding lentivirus and CD30-CAR expression and analysed by flow cytometry using an anti-CD3 and anti-EGFRt antibodies. (c) Expression of CD30 receptor on CD4+ and CD8+ T-cell subpopulations in untransduced cells (white bars) and transduced cells (black bars) during the culture (days 4, 7 and 10; mean  SD). (d) Percentage of T-cell viability in untransduced cells (grey line) and transduced cells (black line) at days 4, 7 and 10 of culture (mean  SD). (e) Fold expansion of CD4+ and CD8+ T cells during 10-day culture in untransduced cells (grey line) and transduced cells (black line; mean  SD). Data are representative of seven independently repeated experiments. ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 3 CD30-CAR memory stem T cells for treating Hodgkin lymphoma C Alvarez-Fern andez et al. Despite the transient CD30 protein expression for Raji). Interestingly, low amounts of secreted and a significant decrease in fold expansion IL-6 (178.45  82.1 pg mL 1 for L540 and compared with UN cells, CD30-CAR TSCM-like cells 565.17  156.58 pg mL 1 for L428 vs 130.38  could be efficiently expanded ex vivo (day 10: 13.6 pg mL 1 for Raji) and IL-10 (134.71  63.16  25.66 vs 289.5  120.81 fold expansion of 34.95 pg mL 1 for L540 and 122.18  20.47 CD4+ T cells; and 101.98  50.69 vs 632.52  125.85 pg mL 1 for L428 vs 5.99  2.11 pg mL 1 for Raji) fold expansion of CD8+ T cells, CD30-CAR T cells were detected in these same conditions (Figure 2c). and UN cells, respectively; Figure 1e). It has been reported that the ability of CAR T cells to maintain functionality in the context of high tumor burden and repeated stimulation is Enriched CD30-CAR TSCM-like cells show critical for their therapeutic efficacy. To evaluate in vitro antitumor efficacy and persistence functional persistence of CD30-CAR, TSCM-like cells against HL that is not blocked by soluble were repeatedly stimulated with CD30+ tumor CD30 cells and different functional aspects were Next, we evaluated the antitumor capacity of analysed. CD30-CAR TSCM-like enriched T cells using two Upon re-exposure to CD30+ target cells, CD30- different HL tumor cell lines, L428 and L540. CAR TSCM-like enriched T cells maintained their CD30-CAR TSCM-like enriched cells showed a potent functionality with a potent and specific cytolytic and specific cytolytic activity against both CD30+ activity against L540 (tumor cell death at 5:1 E:T HL lines (tumor cell death at 5:1 E:T ratio; ratio; 88.75% after three antigen exposition vs 89.92  5.95% vs 0% with control TSCM-like cells 0% with UN cells; Figure 3a). Interestingly, a in L540, and 71.11  8.91% vs 0% with control significant increase in CAR expression was TSCM-like cells in L428), while CD30 target cells observed within 24 h of co-incubation with CD30+ were not killed (Figure 2a). target cells (2959.5  702.1 vs 5982  871.6 MFI, Hodgkin lymphoma patients present high prior exposure and after 24 h, respectively). amounts of soluble CD30 protein (sCD30) in However, this increase declined 72 h after antigen serum, which can potentially abrogate the exposition, reaching similar MFI levels to pre- antitumor activity of CAR T cells because of CAR exposition, which were stable after subsequent blockade.10 We have observed that both HL lines antigen encounter (Figure 3b). Although an secrete high amounts of CD30 protein to increase in PD1+TIM3+ cells was observed after the extracellular environment (1790  50.2 pg mL 1 first co-culture with CD30+ target cells (Figure 3c; for L428 and 2388.75  137.53 pg mL 1 for L540; 15.35  1.48% vs 32.75  5.58% of PD1+-TIM3+ Supplementary figure 2). cells, pre-exposition and 24 h post-exposition, To simulate a scenario with comparable respectively), a significant decrease was observed amounts of sCD30 protein found in advanced HL after 72 h. The low frequency of PD1+TIM3+ cells patients,10 a cytotoxicity assay was performed in and the maintenance of CAR functionality after the presence of saturating concentrations of successive re-expositions suggest that the increase recombinant sCD30 protein (20 µg). In spite of in PD1+-TIM3+ cells observed after the first that, we observed that the efficacy of our CD30- antigen encounter was related to activation CAR T cells was not compromised and killed rather than exhaustion (Figure 3c; 32.75  5.58% CD30+ tumor cells effectively (Figure 2b). vs 3.01  2.11% of PD1+-TIM3+ cells, P < 0.006, 24 Moreover, we measured a number of cytokines and 72 h post-exposure, respectively). released by CD30-CAR TSCM-like enriched cells after 24-h co-culture with CD30+ HL lines and the Enriched CD30-CAR TSCM-like cells exhibited a CD30 Raji cell line. After exposure to CD30 potent antitumor effect in vivo against HL, antigen, CD30-CAR TSCM-like enriched cells secreted enhanced persistence and long-term high amounts of INF-c (48 437.5  1654.63 pg immunity mL 1 for L540 and 9052.39  1889.16 pg mL 1 for L428 vs 1193.75  8.84 pg mL 1 for Raji), TNF-a After showing that CD30-CAR TSCM-like enriched (1709.16  34.17 pg mL 1 for L540 and 1353.33  cells have significant in vitro cytotoxicity against 580.31 pg mL 1 for L428) and IL-2 (2833.67  HL, we evaluated their therapeutic potential 367.98 pg mL 1 for L540 and 2232.78  in vivo using two different HL models (iv L428 and 977.33 pg mL 1 for L428 vs 315  162.63 pg mL 1 sc L540). Mice (n = 4 per group) were treated with 2021 | Vol. 10 | e1268 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 4 Australian and New Zealand Society for Immunology, Inc. C Alvarez-Fern andez et al. CD30-CAR memory stem T cells for treating Hodgkin lymphoma Figure 2. CD30-CAR TSCM-like enriched culture efficiently eradicates HL in vitro. (a) TSCM-like enriched cells expressing CD30-CAR (n = 7) were exposed to CD30+ target cells (L540 or L428 tumor cells) and control CD30 cell line (Raji) at different effector:target (E:T) ratios. Specific cytolytic activity was measured at 24 h by bioluminescence assay. Untransduced TSCM-like cells were used as negative control (mean  SD). (b) Cytolytic capacity of untransduced and CD30-CAR TSCM-like cells (n = 3) was measured against CD30+ target cells (L540 and L428) at 24 h, in the presence or absence of saturated concentration of soluble CD30 protein (sCD30; 20 lg), at different E:T ratios. Raji cell line (CD30 ) was used as negative target control (mean  SD). (c) Cytokine secretion of CD30-CAR TSCM-like enriched culture (n = 3) 24 h after L540, L428 and Raji co- culture. Untransduced TSCM-like cells were used as negative control. Cytokine levels of IL-2, IL-6, IL-10, IFN-c and TNF-a were measured by cytometry-based multiplex analysis (mean  SD). Data are representative of seven (a) or three (b, c) independently repeated experiments. For each donor, technical duplicate was used (c). two different iv doses of CD30-CAR TSCM-like respectively), being 25 and 37 days after enriched cells (low dose: 5 9 106 and high dose: treatment administration the time needed to 10 9 106). The treatment with the higher dose achieve complete tumor clearance, respectively. induced a complete response in all mice bearing However, mice receiving the lower dose of CD30- either L540 or L428 tumors (Figure 4a and b, CAR TSCM-like enriched cells showed a decreased ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 5 CD30-CAR memory stem T cells for treating Hodgkin lymphoma C Alvarez-Fern andez et al. Figure 3. CD30-CAR TSCM-like function persists after CD30+ antigen re-exposition. (a) Representative graphic of untransduced and CD30-CAR TSCM-like enriched cells exposed to CD30+ target cells (L540) at different effector:target (E:T) ratios every 72 h after each antigen re-exposition (total stimulations: 3). Specific cytolytic activity was measured after 24 h of each antigen stimulation by bioluminescence. (b) Mean fluorescence intensity (MFI) of CD30-CAR expression and (c) percentage of PD-1-TIM-3+ T cells during antigen re-exposures (mean  SD). *P < 0.05; **P < 0.01. Data are representative of three independently repeated experiments. antitumor efficacy in both tumor models (50% immunity against HL, animals that survived after and 75% of tumor clearance in L540 and L428, systemic L428 tumor injection (n = 4/4) were respectively; Figure 4a and b). Tumor-bearing challenged again with the same tumor dose at mice that received the highest dose (i.e. 10 9 106) day 79 with no further CD30-CAR T cells. All mice of non-transduced T cells were not able to remained tumor-free 81 days after tumor eliminate the tumor. rechallenge, whereas all age-matched control Furthermore, we took advantage of the mice died of tumor progression (Figure 5a). subcutaneously implanted tumor (L540) to analyse Next, since CAR T persistence is an important tumor-infiltrating T lymphocytes (TILs) in those attribute correlated with antitumor efficacy, we mice in which the lower dose of CD30-CAR T cells look for T cells expressing CD30-CAR+ in lymphoid was not effective. Remarkably, a significant organs of mice that survived after L428 tumor increase in the proportion of PD1+TIM3+ CD4+ and rechallenge. We detected the presence of CD30- CD8+ T cells was detected in the tumor at day 48 CAR+ T cells in bone marrow and lymph nodes compared with the infused CD30-CAR T cells (72.55  1.34% and 75.45  3.6% of CAR+ T cells (CD4+: 4.41  5.12% vs 33.6  10.6%, P < 0.036; from the entire T-cell population, respectively; CD8+: 0.43  0.35% vs 13.05  1.62%, P < 0.0086; Figure 5b). Remarkably, TSCM-like CD30-CAR cells Figure 4c), suggesting a long-term exhaustion of represent the predominant T-cell population these T cells. detected in both bone marrow and lymph nodes To elucidate whether treatment with CD30-CAR (43.75  0.07% and 41.8  7.21%, respectively; TSCM-like enriched cells could induce long-term Figure 5c). 2021 | Vol. 10 | e1268 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 6 Australian and New Zealand Society for Immunology, Inc. C Alvarez-Fern andez et al. CD30-CAR memory stem T cells for treating Hodgkin lymphoma Figure 4. Therapeutic treatment with CD30-CAR TSCM-like enriched cell products induces a potent antitumor response against HL in vivo. NSG mice (n = 4 for both experimental and control groups) were injected with (a) 2.5 9 106 L540 tumor cells (sc) on day 0 and treated 3 days later with 5 or 10 9 106 CD30-CAR TSCM-like enriched cells (iv), or (b) 2 9 106 L428 tumor cells (iv) on day 0 and treated with 5 or 10 9 106 CD30- CAR TSCM-like enriched cells (iv) 22 days after tumor challenge. In both models, control mice received 10 9 106 untransduced (UN) TSCM-like enriched cells. Mice were monitored every other day for survival, and tumor growth was measured by in vivo bioluminescence. (c) Expression of exhaustion markers (PD1 and TIM3) in CD4+ and CD8+ tumor-infiltrating T cells found in L540-bearing mice treated with 5 9 106 CD30-CAR TSCM-like enriched cells, 48 days after tumor challenge, compared with CD4+ and CD8+ pre-infused T cells (mean  SD). *P < 0.05; **P < 0.01. Data are representative of two independent experiments. Collectively, these data show that CD30-CAR compared with more differentiated T cells, we TSCM-like enriched cells have long-term persistence studied the in vivo therapeutic antitumor activity after a single infusion and provide the of two different cell products, highly enriched establishment of long-lasting antitumor immunity. TSCM-like (TSCM-likeH; 70.95  0.06 in CD4+ and In addition, the proportion of T cells expressing 82.25  10.1% in CD8+) and lowly enriched TSCM-like PD1+, TIM3+ and LAG-3+ were analysed in T cells (TSCM-likeL; 28.45  12.1 in CD4+ and 18.11  11.14% from bone marrow and lymph nodes. We in CD8+) T cells (Figure 6a). observed a low proportion of T cells expressing To prove potential clinically meaningful either PD1+, TIM3+ or LAG-3+ in lymph nodes differences in antitumor efficacy between both (5.84  2.22% of PD1+; 4.72  4.1% of TIM3+; cell products, mice were treated with a single 0.3  0.28% of LAG-3+) and bone marrow infusion of a suboptimal dose of CD30-CAR T cells (18.45  3.32% of PD1+; 3.06  1.91% of TIM3+; (5 9 106 iv) in both cases, and a delay in the 3.62  3.05% of LAG-3+; Figure 5d). treatment administration time regarding the tumor challenge. Under this stringent therapeutic condition, mice treated with TSCM-likeH CD30-CAR Highly enriched CD30-CAR TSCM-like cells showed a significant reduced tumor growth (TSCM-likeH) cells confer superior antitumor throughout the entire study, compared with those capacity compared with lowly enriched mice treated with TSCM-likeL CD30-CAR cells CD30-CAR TSCM-like (TSCM-likeL) cells (Figure 6b; P < 0.001). That lower tumor growth To analyse whether a high proportion of less translates into a significant superior survival of differentiated CD30-CAR T cells within the infused mice treated with TSCM-likeH CD30-CAR compared product could enhance the antitumor efficacy with those treated with TSCM-likeL CD30-CAR cells ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 7 CD30-CAR memory stem T cells for treating Hodgkin lymphoma C Alvarez-Fern andez et al. Figure 5. TSCM-like enriched cells expressing CD30-CAR have high persistence in vivo and confer long-lasting immunity against HL. (a) NSG mice surviving from L428 tumor administration (n = 4) were rechallenged again with the same tumor dose (2 9 106 cells per mouse, iv), 79 days (arrow) after the first tumor challenge (grey discontinued line). An age-matched mouse group (n = 4) was injected with 2 9 106 L428 tumor cells (iv) as tumor control (pointed line). Black line represents control mice receiving 10 9 106 untransduced (UN) TSCM-like enriched cells. Mice were followed every other day for survival, and tumor growth control was done by in vivo bioluminescence. (b) Bone marrow and lymph nodes from surviving CD30-CAR-treated mice were analysed for CAR T cell by flow cytometry (mean  SD). (c) T-cell subpopulations were analysed in bone marrow and lymph nodes by flow cytometry (mean  SD). (d) Expression of exhaustion markers (PD1, TIM3 and LAG-3) was analysed by flow cytometry in T cells found in lymph nodes and bone marrow (mean  SD). Data are representative of two independent experiments. (median survival: 51 vs 37 days post-challenge, P = 0.0006; Figure 6e and Supplementary respectively, P = 0.006; Figure 6c). figure 3). Importantly, although the frequency of tumor- infiltrating T cells founded in tumors of TSCM-likeH- DISCUSSION and TSCM-likeL-treated mice was similar (29  4% and 23.5  2.5% from total tumor mass, Most HL patients may be cured with current respectively; data not shown), the proportion of chemotherapy treatments; however, about 20% CD30-CAR+ T cells within the tumor-infiltrating T have a relapsing/refractory (R/R) disease, with very cells was significantly higher in mice treated with poor outcome.20 Immunotherapy targeting CD30 TSCM-likeH than in TSCM-likeL mice (78.78  10.64% (i.e. brentuximab vedotin) or immune checkpoint vs 53.75  3.43%, respectively, P = 0.002; molecules for these R/R patients may yield Figure 6d), suggesting a higher trafficking of complete responses, but long-term control of the CD30-CAR TSCM-likeH cells to the tumor site. disease is infrequent.21,22 Despite that, responses Moreover, we detect a higher amount of CD30- observed with these agents emphasise the CAR T cells in blood in those mice treated with sensitivity of HL to immunotherapy strategies. TSCM-likeH cells than in mice treated with TSCM-likeL Chimeric antigen receptor T-cell therapy cells (740  106.22 CAR+ T cell mL 1 vs redirected to CD30 has been tested in R/R HL 137.66  18.45 CAR+ T cell mL 1, respectively, patients.9,10 Although results are promising, 2021 | Vol. 10 | e1268 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 8 Australian and New Zealand Society for Immunology, Inc. C Alvarez-Fern andez et al. CD30-CAR memory stem T cells for treating Hodgkin lymphoma Figure 6. CD30-CAR TSCM-likeH products have higher antitumor efficacy and T-cell persistence than CD30-CAR TSCM-likeL products. (a) Composition of TSCM-likeHand TSCM-likeL cultures at the day of treatment (mean  SEM). (b, c) NSG mice (N = 4/group) were challenged with 2.5 9 106 L540 tumor cells (sc) on day 0 and treated when the tumor was well established (day 5) with 5 9 106 CD30-CAR TSCM-likeH (pointed line and ▲) or TSCM-likeL (discontinued line and ■) cells (iv). A group of mice were injected with 2.5 9 106 L540 tumor cells (sc) as tumor control (black line and ●). Mice were monitored every other day for (B) tumor growth and (c) survival, measured by in vivo bioluminescence (mean  SD). (d) Percentage of CD30-CAR expression in tumor-infiltrating T cells was analysed in TSCM-likeH and TSCM-likeL treated mice by flow cytometry (e) CD30-CAR T-cell detection in blood from mice treated with TSCM-likeH or TSCM-likeL cultures. (mean  SD). *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two independent experiments. clinical efficacy is limited, in contrast to data from could lead to CAR blockade by CD30 present in a B-cell tumors treated with CAR19.23 Although soluble form in the plasma of HL patients with CD30 remains an excellent target for CAR T advanced/aggressive disease, significantly therapy of HL, a number of obstacles should be diminishing its antitumor effect.26,27 Here, we solved to clinically improve their efficacy, such as show that the effectiveness of our CD30-CAR was persistence of CAR T cells, trafficking to tumor not blocked by the presence of high amounts of and increase of tumor cytotoxicity. soluble CD30 protein (20 lg, equivalent to Here, we have evaluated the efficacy of a CD30- 12 000 units mL 1). In addition, epitope location CAR targeting a membrane-proximal epitope on appears to be a major determinant of CAR T the CD30 molecule, to maximise its antitumor efficacy, with membrane-proximal epitopes being effect and to avoid the inhibition with soluble better than those located on a distal region.28 In CD30. Furthermore, we have explored the use of line with this, our CD30-CAR could be CAR T-cell products highly enriched in TSCM-like advantageous over others since not only it is not cells to foster engraftment, persistence and affected by sCD30, but also it may have an trafficking into HL tumors. enhanced cytolytic activity because of the Although the effectiveness of CD30-CAR proximal location of the targeted epitope. targeting a distal epitope on the CD30 molecule It has become evident that long-term in previous studies was apparently not affected by persistence, high engraftment, expansion and soluble CD30 molecule,24,25 recent clinical trials survival of adoptively transferred T cells are have shown a correlation between sCD30 levels critical parameters for a good antitumor and the limited antitumor effect with CD30-CAR T efficacy.6,9,10,13,16 Thus, recent studies have been cells.10 The affinity of the CD30-specific scFv used focused on the study of the most appropriate T- to construct the CAR that recognises a distal cell subsets for ACT and CAR T-cell therapy, and epitope within the cleavable part of CD30 evidences indicate that the use of less molecule9,10,19 may be a consequence of the differentiated T cells, and particularly TSCM, results limited activity of CD30-CAR. This recognition in greater persistence, cell expansion and ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 9 CD30-CAR memory stem T cells for treating Hodgkin lymphoma C Alvarez-Fern andez et al. antitumor efficacy11,13,29–32 in contrast to a chemokine secreted by HL cells, facilitating unselected bulk T cells.6,9,32 tumor infiltration of T cells,41,43 which is in line According to previous studies, the potential with our finding that CD30-CAR TSCM-like enriched elimination of activated CD30+ T cells responding products have increased infiltration into HL to other antigens and CD30-CAR T cells tumors. Importantly, the use of IL-21 within the themselves is not a limitation for TSCM-Like cell culture would contribute further to the enhanced expansion ex vivo with an optimal cell viability antitumor efficacy of our CD30-CAR T cells, and CAR expression.3,25 Moreover, we observed a promoting the generation of less differentiated correlation between the decrease in CD30+ T cells memory T cells and increasing antitumor along with the culture and an increase in cell activity.44 Besides, CD30-CAR T cells may kill viability, which is not observed in UN T cells, Hodgkin´s TME regulatory T cells, which express suggesting fratricide elimination of CD30+ T cells. high levels of CD30, thus contributing to Other mechanisms could explain the reduction in ameliorate immunosuppression. CD30+ T cells including the internalisation of the Other strategies described to improve resistance protein or the sequestration of CD30 protein in of adoptive T cells to HL-derived the cytoplasm of T cells, similar to what has been immunosuppression included the use of CD30-CAR shown with other CARs targeting molecules cells gene-modified to express CCR4,45 to increase expressed in activated T cells.33 tumor homing, and a dual targeting of HL tumor Long-lasting responses have been associated and myeloid suppressor cells by using a CD123- with the persistence of CAR T cell and potent T- redirected CART,46 all of them are being cell expansion.5,34,35 In addition, the trafficking of translated to the clinic. CAR T cells to tumors is of critical importance for According to previous studies with TSCM-like obtaining an antitumor effect. In this study, we cells,11,13,29–32 we observed a long-term persistence have generated CD30-CAR TSCM-like enriched of CD30-CAR TSCM-like cells that were able to fully products to increase persistence and trafficking eradicate the tumor after a second tumor into HL tumors. We observed that an optimal rechallenge. Moreover, CD30-CAR expression dose of CD30-CAR TSCM-like enriched products had persisted and TSCM-like remained the most frequent a potent and specific antitumor efficacy against cellular subpopulation in lymph nodes and bone HL in vitro and in vivo, clearing completely both a marrow of those mice. Importantly, CD30-CAR T systemic and a localised (simulating an extra- cells found after the second tumor challenge nodal lesion) HL tumor. Interestingly, we found display a low expression of PD1, TIM3 and LAG-3, tumor-infiltrating T cells coexpressing high levels demonstrating that these cells were not exhausted of PD1 and TIM3 in those animals treated with a and remained functional.36–39 suboptimal dose that were not able to eradicate Differentiated CAR T cells redirected to CD30 or the tumor, which is correlated with a profound T- CD19 have limited antitumor efficacy, mostly cell dysfunction.36–38 T cells expressing PD1 in because of poor proliferation and migration to combination with inhibitory markers such as TIM3 tumor sites.6,9,10,25 Importantly, in patients and LAG-3 are associated with relapses or receiving bulk CD19-CAR T cells, in vivo expansion refractory disease,38,39 and may be a mechanism of these cells has been correlated with the for decreasing the effectiveness of CAR T-cell frequency, within the infusion product, of a T-cell therapy. Targeting of TIM3 and PD1 pathways, in subset closely related to TSCM16,17,47; however, the combination with CAR T-cell therapy, could frequency of these cells is low in the majority of increase the tumor control and restore T-cell cell products currently used in clinical trials.16,34 function, a strategy that already has been proved To gain insight into the antitumor activity of in B-cell lymphoma patients treated with CAR19.40 CD30-CAR with different proportions of TSCM-like Our approach described here may represent a way cells, we established a therapeutic model with to reduce the known immunosuppressive effect of two different proportions of TSCM-like cells, namely HL tumor microenvironment (TME) on T cells.41 T ‘highly enriched’ (>50% of CD30-CAR TSCM-like cells generated under IL-7 and IL-15 show cells) and ‘lowly enriched’ (<30% of CD30-CAR increased expression of CXCR4,42 a chemokine TSCM-like cells). Remarkably, we found higher that promote T-cell migration to peripheral tissues persistence, trafficking to tumor and frequency of and tumors. In addition, under those conditions, T circulating CD30-CAR T cells in mice receiving cells also express CXCR3 that interacts with CCL5, CD30-CAR TSCM-likeH products than in those 2021 | Vol. 10 | e1268 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 10 Australian and New Zealand Society for Immunology, Inc. C Alvarez-Fern andez et al. CD30-CAR memory stem T cells for treating Hodgkin lymphoma treated with TSCM-likeL, which, importantly, at Hospital Sant Pau. All experiments and care of animals translated into a significant survival were conducted according to the European Animal Care guidelines and approved by the Ethical Committee of advantage.9,10,17 Collectively, although we did not Animal Experimentation at Hospital Sant Pau. attempt to quantify the minimum threshold of TSCM-like proportion in infused CAR T-cell products that confer maximum antitumor activity, our data Cell lines suggest that an elevated frequency of this cell HEK-293T cells (LentiX; Clontech, Mouintain View, subset expressing the CAR could significantly California, USA) were cultured in DMEM supplemented improve the outcome of CAR T therapy. with 10% heat-inactivated foetal bovine serum (FBS), penicillin (100 U mL 1) and streptomycin (100 µg mL 1; Although xenograft mouse models are Thermo Fisher Scientific, Waltham, Massachusetts, USA). extensively used to study antitumor efficacy of T- The CD30+ HL-derived cell lines, L428 and L540, were cell adoptive therapy, they have some limitations, obtained from the German Collection of Cell Cultures particularly related to the absence of relevant (DSMZ, Braunschweig, Germany). Raji cell line was obtained TME and lack of interaction with the host from the American Type Culture Collection (ATCC; Rockville, immune system.48 For T-cell therapy, some models MD, USA). Tumor cell lines were cultured in RPMI-1640 medium supplemented with 10–20 % FBS, penicillin could also fail to correctly evaluate off-tumor (100 U mL 1) and streptomycin (100 µg mL 1; Thermo toxicity, which is an important issue when T-cell Fisher Scientific). All cell cultures were performed at 37 °C therapies are translated to the clinical scenario. in a fully humidified atmosphere with 5% CO2 in air. Tumor With all these pros and cons, NSG models allow cell lines were modified with a lentiviral vector to express studying persistence and efficacy of human CAR T firefly luciferase (ffluc) and enhanced green fluorescence protein (eGFP; Addgene, Watertown, Massachusetts, USA). cells and thus representing a good option to test For in vivo experiments, ffluc-transduced tumor cells the effect of novel CARs. were thawed from a common frozen stock and grown In summary, we demonstrate that TSCM-like can be in vitro for 3 days before use. On the day of tumor efficiently transduced and ex vivo-expanded with a injection, cells were washed with complete medium and novel CD30-CAR that surpasses fratricide killing of diluted to the appropriate concentration in 0.1 mL of CD30-expressing T cells and confers potent in vivo phosphate-buffered saline (PBS) per mouse. antitumor efficacy against HL. We have shown that highly enriched CD30-CAR TSCM-like products have Construction of CD30-CAR-encoding enhanced tumor trafficking and antitumor activity lentiviral vector compared with those composed predominantly of A codon-optimised scFv comprising the variable heavy and more differentiated cell subsets. light chains of the anti-CD30 monoclonal antibody (mAb; These proof-of-concept studies support the use of clone T105),19 separated by a Whitlow peptide linker,49 was a refined CD30-CAR with highly enriched TSCM-like synthesised (GeneArt; Thermo Fisher, Regensburg, cell products for improving clinical efficacy of CAR T Germany), and cloned into the third-generation lentiviral expression vector pHIV7,50,51 where it was fused to a CD8 therapy of patients with HL. hinge, a 4.1BB-CD3f signalling module in cis with a T2A element, and the truncated human epidermal growth factor receptor (EGFRt), as previously described.52 METHODS Blood samples and peripheral blood Lentivirus production mononuclear cell isolation Lentivirus was produced in HEK-293T cells co-transfected with the pHIV7 lentiviral vector and the packaging vectors Peripheral blood samples were obtained from healthy pCHGP-2, pCMV-rev2 and pCMV-VSV-G, (Addgene) using donors (n = 7) after informed consent following the calcium phosphate (Clontech). Medium was changed 16 h protocol approved by the Ethics Committee of Hospital de after transfection, and the cells were incubated for another la Santa Creu i Sant Pau (Barcelona). Peripheral blood 36–48 h. Lentiviral supernatants were collected, centrifuged mononuclear cells were isolated by Ficoll–Hypaque density to remove cellular debris for 15 min at 2217 g and gradient centrifugation (Lymphoprep; Axis-shield, Dundee, concentrated for 2 h at 55126 g. The lentiviral pellet was UK) and frozen until use. resuspended in TBS-5 buffer, snap-frozen on dry ice and stored at 80°C. Mice Female NOD.Cg-Prkdc scid IL2rgtm1Wjl/SzJ (NSG) mice T-cell isolation and culture (6–7 weeks of age; Charles River, France) were used for in vivo experiments. Animals were housed under specific Na€ıve T cells were isolated using a Human Na€ıve Pan T Cell pathogen-free conditions at the Laboratory Animal Facility Isolation Kit (StemCell Technologies, Vancouver, British ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 11 CD30-CAR memory stem T cells for treating Hodgkin lymphoma C Alvarez-Fern andez et al. Columbia, Canada) according to the manufacturer’s In some cases, bone marrow, lymph nodes, tumor and instructions. To obtain TSCM-like highly enriched cultures, blood of treated and control mice were harvested at the purified na€ıve T cells (higher than 95 %) were cultured and end of the experiment, and CAR T cells were analysed by expanded as previously described.15 Briefly, cells were flow cytometry. Briefly, tumor and axillar lymph nodes activated during 48 h with anti-CD3/CD28 magnetic beads were disaggregated by mechanical procedures and (Life Technologies, Waltham, Massachusetts, USA) in 1:2 collected in 5 and 3 mL of complete medium, respectively, bead/T-cell ratio and then cultured with IL-7, IL-15 and IL-21 and bone marrow was flushed from the long bones (femur at 25 ng mL 1 each (Stem Cell Technologies, Vancouver, and tibia). Cellular suspensions were filtered through a 70- British Columbia, Canada). 2.5 9 105 cells mL 1 per well lm cell strainer (BC Falcon; Cultek S.L.U., Foster City, were seeded in a 24-well plate. California, USA) and centrifuged at 385 g during 5 min. In To generate products with low proportion of TSCM-like the case of bone marrow and blood, erythrocytes were cells (TSCM-likeL), T cells were isolated using a Human Pan T lysed using an ammonium chloride solution (Pharm Lyse Cell Isolation Kit (StemCell Technologies) according to the Buffer; BD Biosciences, Franklin Lakes, New Jersey, USA) manufacturer’s instructions. Purified T cells were activated during 3 min in agitation at room temperature. Finally, with anti-CD3/CD28 magnetic beads (Life Technologies) in cells were counted and maintained in 5 mL of complete 1:1 bead/T-cell ratio, and cultured with IL-2 at 50 UI mL 1 medium until use. Blood samples were incubated with Fc (Miltenyi Biotec, Bergisch Gladbach, North Rhine- Block and corresponding antibodies prior to 10-min Westphalia, Germany). 5 9 105 cells mL 1 per well were erythrocyte lysis (Pharmlyse Buffer; BD Bioscience), seeded in a 48-well plate. centrifuged at 385 g during 5 min and acquired on T cells were cultured in RPMI-1640 medium MACSQuant Analyzer 10 (Miltenyi Biotec). supplemented with 10% heat-inactivated FBS, penicillin (100 U mL 1) and streptomycin (100 µg mL 1; Thermo Fisher Scientific). All cell cultures were performed at 37°C in Functional in vitro analysis of CD30-CAR T a fully humidified atmosphere with 5% CO2 in air. cells Cytokines and medium were replaced every 3–4 days, and cells were counted every 3–4 days by trypan blue dye In vitro cytolytic activity of CD30-CAR TSCM-like cells was exclusion. analysed by a bioluminescence-based assay using ffluc- transduced CD30+ tumor cells (L540 and L428, at different effector:target (E:T) ratios)), after 24-h co-culture. T-cell transduction Persistence of CD30-CAR T cells was evaluated by continued re-exposure assays. TSCM-like CD30 CAR T cells Transduction was performed 24 h (for TSCM-likeL cultures) or were co-cultured again with CD30+ tumor cells 72 h after 48 h (for TSCM-likeH cultures) after initial stimulation by exposition, and cytolytic activity was analysed by 800 g spinoculation for 45 min at 32°C in the presence of bioluminescence-based assay using ffluc-transduced CD30+ polybrene (5 lg mL 1; Sigma-Aldrich, St. Louis, Missouri, tumor cells. Levels of IFN-c, IL-2, TNF-a, IL-6 and IL-10 were USA). CAR expression was detected by flow cytometry at analysed in supernatants obtained after a 24-h co-culture of days 6–7 of culture. CAR T cells with L540, L428 and Raji cell lines (E:T ratio 5:1). Cytokines were detected using Multi-Analyte Flow Assay (LEGENDplex; BioLegend, San Diego, California, USA) Cytometry and CAR expression analysis according to the manufacturer’s instructions. Acquisition Cells were stained for 30 min at 4°C in PBS containing 1% was performed on a MACSQuant Analyzer 10 (Miltenyi FBS and 0.01% NaN3 (Sigma-Aldrich; staining buffer). All Biotec), and data analysis was done using LEGENDplex antibodies were titrated before use, and corresponding Analysis software (BioLegend). isotypes for each antibody were used as controls. For T-cell population analysis, cells were labelled with fluorescent antibodies against human CD3-FITC (REA613), CD4-APC-Vio- In vivo efficacy of CD30-CAR T cells 770 (clone VIT4), CD8-VioGreen (clone BW135/80), CD45RA- PerCPVio700 (clone T6D11), CD45RO-PE (clone UCHL1), Mice (four per group) were injected intravenously (iv) with CD27-VioBlue (clone M-T271), CD95-FITC (clone DX2) and L428 tumor cells (2 9 106 cells per mouse) or CCR7-PE vio770 (clone REA108; all from Miltenyi Biotech). subcutaneously (sc) with L540 (2.5 9 106 cells per mouse). Different T-cell subpopulations were determined following When tumors were well established (22 days for L428 a previously published gating strategy.15,53 Alexa Fluor 647- tumor model and 3 days for L540), mice were treated with labelled anti-EGFRt antibody (cetuximab; Roche, Basel, a TSCM-like enriched cell suspension (5–10 9 106 CAR+ T cells Switzerland) was used to detect CAR expression. CD30 per mice, iv). Control mice received the same dose of expression was detected using anti-CD30-APC (clone Ki-2; untransduced T cells. In some experiments, mice surviving Miltenyi Biotec). In addition, anti-PD1 (clone PD1.3.1.3), to L428 lymphoma were rechallenged with a second tumor anti-TIM3 (clone F38-2E2) and anti-LAG-3 (clone REA351) dose (2 9 106 cells per mouse, iv) 79 days after the first antibodies (all from Miltenyi Biotec) were used to analyse tumor injection. An untreated age-matched mice group T-cell exhaustion. All data were acquired on a MACSQuant receiving the same tumor dose was used as control. Analyzer 10 (Miltenyi Biotec). Data from flow cytometry To analyse differences between products with a high were analysed using the FlowJo version 10 software (TSCM-likeH cultures) or low (TSCM-likeL cultures) proportion of (TreeStar, Ashland, Oregon, USA). CD30-CAR TSCM-like cells, L540 tumor-bearing mice (n = 4) 2021 | Vol. 10 | e1268 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 12 Australian and New Zealand Society for Immunology, Inc. C Alvarez-Fern andez et al. CD30-CAR memory stem T cells for treating Hodgkin lymphoma received either 5 9 106 CAR+ highly enriched or lowly 3. Ramos CA, Heslop HE, Brenner MK. CAR-T-cell therapy enriched TSCM-like cells (iv) 5 days after tumor injection. for lymphoma. Annu Rev Med 2016; 67: 165–183. Tumor growth was measured by in vivo bioluminescence 4. Davila ML, Riviere I, Wang X et al. Efficacy and toxicity imaging (IVIS Spectrum In Vivo Imaging System; management of 19–28z CAR T-cell therapy in B cell PerkinElmer, Waltham, Massachusetts, USA) in all cases and acute lymphoblastic leukemia. Sci Transl Med 2014; 6: analysed with specialised software (Living Image, 224ra25. PerkinElmer, USA). Animals were followed every other day 5. Kalos M, Levine BL, Porter DL et al. T cells with chimeric for survival and sacrificed when moribund. antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011; 3: 95ra73. Soluble CD30 protein detection 6. Brentjens RJ, Riviere I, Park JH et al. Safety and persistence of adoptively transferred autologous CD19- Soluble CD30 protein was measured in supernatants of L540 targeted T cells in patients with relapsed or and L428 cell cultures by enzyme-linked immunoabsorbent chemotherapy refractory B-cell leukemias. Blood 2011; assay (Human CD30 PicoKine ELISA Kit; Boster Biological 118: 4817–4828. Technology, Pleasanton, California, USA). 7. Kochenderfer JN, Wilson WH, Janik JE et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically Statistical analysis engineered to recognize CD19. Blood 2010; 116: 4099– Inter-donor variability was assessed using standard 4102. deviation (SD). Results are expressed as the mean  SD. The 8. van der Weyden CA, Pileri SA, Feldman AL et al. Kaplan–Meier plots were used to analyse mice survival, and Understanding CD30 biology and therapeutic targeting: the significant differences between survival curves were a historical perspective providing insight into future assessed by the log-rank test. For all other data, the t-tests directions. Blood Cancer J 2017; 7: e603. were performed to analyse the differences between groups. 9. Wang CM, Wu ZQ, Wang Y et al. Autologous T cells All statistical analysis and graphics were performed using expressing CD30 chimeric antigen receptors for relapsed GraphPad Prism 6 (GraphPad Software Inc. California, USA). or refractory hodgkin lymphoma: an open-label phase I trial. Clin Cancer Res 2017; 23: 1156–1166. 10. Ramos CA, Grover NS, Beaven AW et al. Anti-CD30 ACKNOWLEDGMENTS CAR-T-cell therapy in relapsed and refractory hodgkin lymphoma. J Clin Oncol 2020; 38: 3794–3804. This work was supported in part by grants from La Marato  11. Gattinoni L, Zhong XS, Palmer DC et al. Wnt signaling TV3 (Exp. 20130710), Deutsche Jose  Carreras Leuka € mie arrests effector T cell differentiation and generates Stiftung (DJCSL 10R/2016), Fundacio  n Cientıfica Asociacion CD8+ memory stem cells. Nat Med 2009; 15: 808–813. Espan~ ola Contra el Ca ncer (AECC-AIO2017), Instituto de 12. Berger C, Jensen MC, Lansdorp PM et al. Adoptive Salud Carlos III (PI15/1383 y PI18/01023), Fundacio  n Bancaria transfer of effector CD8+ T cells derived from central ‘La Caixa’, TerCel (SG/11/2008), Ministerio de Economıa y memory cells establishes persistent T cell memory in Competitividad (RETOS; RTC 2015-3393-1) and AGAUR primates. J Clin Invest 2008; 118: 294–305. (2017SGR1395). 13. Gattinoni L, Lugli E, Ji Y et al. A human memory T cell subset with stem cell-like properties. Nat Med 2011; 17: 1290–1297. CONFLICT OF INTEREST 14. Klebanoff CA, Gattinoni L, Restifo NP. Sorting through subsets: which T-cell populations mediate highly The authors declare no conflict of interest. effective adoptive immunotherapy? J Immunother 2012; 35: 651–660. 15. Alvarez-Fernandez C, Escriba-Garcia L, Vidal S et al. A AUTHOR CONTRIBUTIONS short CD3/CD28 costimulation combined with IL-21 Laura Escriba  Garcia: Investigation. Ana C Caballero: enhance the generation of human memory stem T cells Investigation. Eva Escudero-lo  pez: Investigation. Cristina for adoptive immunotherapy. J Transl Med 2016; 14: Ujaldo n-Miro  : Investigation. Rosanna Montserrat-Torres: 214. Investigation. Paula Pujol-Ferna  ndez: Investigation. Jorge 16. Xu Y, Zhang M, Ramos CA et al. Closely related T- Sierra: Investigation. memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. Blood 2014; 123: 3750–3759. REFERENCES 17. Fraietta JA, Lacey SF, Orlando EJ et al. Determinants of response and resistance to CD19 chimeric antigen 1. June CH, Riddell SR, Schumacher TN. Adoptive cellular receptor (CAR) T-cell therapy of chronic lymphocytic therapy: a race to the finish line. Sci Transl Med 2015; 7: leukemia. Nat Med 2018; 24: 563–571. 280ps7. 18. Nagata S, Onda M, Numata Y et al. Novel anti-CD30 2. Dai H, Wang Y, Lu X et al. Chimeric antigen receptors recombinant immunotoxins containing disulfide- modified T-cells for cancer therapy. J Natl Cancer Inst stabilized Fv fragments. Clin Cancer Res 2002; 8: 2345– 2016; 108: djv439. 2355. ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 13 CD30-CAR memory stem T cells for treating Hodgkin lymphoma C Alvarez-Fern andez et al. 19. Nagata S, Ise T, Onda M et al. Cell membrane-specific adults: a phase 1 dose-escalation trial. Lancet 2015; 385: epitopes on CD30: potentially superior targets for 517–528. immunotherapy. Proc Natl Acad Sci USA 2005; 102: 35. Kochenderfer JN, Dudley ME, Kassim SH et al. 7946–7951. Chemotherapy-refractory diffuse large B-cell lymphoma 20. Shah GL, Moskowitz CH. Transplant strategies in and indolent B-cell malignancies can be effectively relapsed/refractory Hodgkin lymphoma. Blood 2018; treated with autologous T cells expressing an anti-CD19 131: 1689–1697. chimeric antigen receptor. J Clin Oncol 2015; 33: 540– 21. Chen R, Zinzani PL, Lee HJ et al. Pembrolizumab in 549. relapsed or refractory Hodgkin lymphoma: 2-year 36. Fourcade J, Sun Z, Benallaoua M et al. Upregulation of follow-up of KEYNOTE-087. Blood 2019; 134: 1144– Tim-3 and PD-1 expression is associated with tumor 1153. antigen-specific CD8+ T cell dysfunction in melanoma 22. Chen R, Gopal AK, Smith SE et al. Five-year survival and patients. J Exp Med 2010; 207: 2175–2186. durability results of brentuximab vedotin in patients 37. Sakuishi K, Apetoh L, Sullivan JM et al. Targeting Tim-3 with relapsed or refractory Hodgkin lymphoma. Blood and PD-1 pathways to reverse T cell exhaustion and 2016; 128: 1562–1566. restore anti-tumor immunity. J Exp Med 2010; 207: 23. Jacobson CA. CD19 chimeric antigen receptor therapy 2187–2194. for refractory aggressive B-cell lymphoma. J Clin Oncol 38. Granier C, Dariane C, Combe P et al. Tim-3 expression 2019; 37: 328–335. on tumor-infiltrating PD-1+CD8+ T cells correlates with 24. Hombach A, Heuser C, Sircar R et al. An anti-CD30 poor clinical outcome in renal cell carcinoma. Cancer chimeric receptor that mediates CD3-zeta-independent Res 2017; 77: 1075–1082. T-cell activation against Hodgkin’s lymphoma cells in 39. Yang ZZ, Kim HJ, Villasboas JC et al. Expression of LAG- the presence of soluble CD30. Cancer Res 1998; 58: 3 defines exhaustion of intratumoral PD-1+ T cells and 1116–1119. correlates with poor outcome in follicular lymphoma. 25. Savoldo B, Rooney CM, Di Stasi A et al. Epstein Barr Oncotarget 2017; 8: 61425–61439. virus specific cytotoxic T lymphocytes expressing the 40. Chong EA, Melenhorst JJ, Lacey SF et al. PD-1 blockade anti-CD30f artificial chimeric T-cell receptor for modulates chimeric antigen receptor (CAR)-modified T immunotherapy of Hodgkin disease. Blood 2007; 110: cells: refueling the CAR. Blood 2017; 129: 1039–1041. 2620–2630. 41. Connors JM, Cozen W, Steidl C et al. Hodgkin 26. Pizzolo G, Vinante F, Chilosi M et al. Serum levels of lymphoma. Nat Rev Dis Primers 2020; 6: 61. soluble CD30 molecule (Ki-1 antigen) in Hodgkin’s 42. Jourdan P, Vendrell JP, Huguet MF et al. Cytokines and disease: relationship with disease activity and clinical cell surface molecules independently induce CXCR4 stage. Br J Haematol 1990; 75: 282–284. expression on CD4+ CCR7+ human memory T cells. J 27. Gause A, Pohl C, Tschiersch A et al. Clinical significance Immunol 2000; 165: 716–724. of soluble CD30 antigen in the sera of patients with 43. Bromley SK, Mempel TR, Luster AD. Orchestrating the untreated Hodgkin’s disease. Blood 1991; 77: 1983–1988. orchestrators: chemokines in control of T cell traffic. 28. Haso W, Lee DW, Shah NN et al. Anti-CD22-chimeric Nat Immunol 2008; 9: 970–980. antigen receptors targeting B-cell precursor acute 44. Singh H, Figliola MJ, Dawson MJ et al. Reprogramming lymphoblastic leukemia. Blood 2013; 121: 1165–1174. CD19-specific T cells with IL-21 signaling can improve 29. Gattinoni L, Klebanoff CA, Palmer DC et al. Acquisition adoptive immunotherapy of B-lineage malignancies. of full effector function in vitro paradoxically impairs Cancer Res 2011; 71: 3516–3527. the in vivo antitumor efficacy of adoptively transferred 45. Di Stasi A, De Angelis B, Rooney CM et al. T CD8+ T cells. J Clin Invest 2005; 115: 1616–1626. lymphocytes coexpressing CCR4 and a chimeric antigen 30. Klebanoff CA, Gattinoni L, Torabi-Parizi P et al. Central receptor targeting CD30 have improved homing and memory self/tumor-reactive CD8+ T cells confer superior antitumor activity in a Hodgkin tumor model. Blood antitumor immunity compared with effector memory T 2009; 113: 6392–6402. cells. Proc Natl Acad Sci USA 2005; 102: 9571–9576. 46. Ruella M, Kenderian SS. Next-generation chimeric 31. Hinrichs CS, Borman ZA, Cassard L et al. Adoptively antigen receptor T-cell therapy: going off the shelf. transferred effector cells derived from naive rather than BioDrugs 2017; 31: 473–481. central memory CD8+ T cells mediate superior 47. Singh N, Perazzelli J, Grupp SA et al. Early memory antitumor immunity. Proc Natl Acad Sci USA 2009; 106: phenotypes drive T cell proliferation in patients with 17469–17474. pediatric malignancies. Sci Transl Med 2016; 8: 320ra3. 32. Sommermeyer D, Hudecek M, Kosasih PL et al. Chimeric 48. Srivastava S, Riddell SR. Chimeric antigen receptor t-cell antigen receptor-modified T cells derived from defined therapy: challenges to bench-to-bedside efficacy. J CD8+ and CD4+ subsets confer superior antitumor Immunol 2018; 200: 459–468. reactivity in vivo. Leukemia 2016; 30: 492–500. 49. Jena B, Maiti S, Huls H et al. Chimeric antigen receptor 33. Gogishvili T, Danhof S, Prommersberger S et al. (CAR)-specific monoclonal antibody to detect CD19- SLAMF7-CAR T cells eliminate myeloma and confer specific T cells in clinical trials. PLoS One 2013; 8: selective fratricide of SLAMF7+ normal lymphocytes. e57838. Blood 2017; 130: 2838–2847. 50. Jetani H, Garcia-Cadenas I, Nerreter T et al. CAR T-cells 34. Lee DW, Kochenderfer JN, Stetler-Stevenson M et al. T targeting FLT3 have potent activity against FLT3-ITD+ cells expressing CD19 chimeric antigen receptors for AML and act synergistically with the FLT3-inhibitor acute lymphoblastic leukaemia in children and young crenolanib. Leukemia 2018; 32: 1168–1179. 2021 | Vol. 10 | e1268 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 14 Australian and New Zealand Society for Immunology, Inc. C Alvarez-Fern andez et al. CD30-CAR memory stem T cells for treating Hodgkin lymphoma 51. Yam P, Jensen M, Akkina R et al. Ex vivo selection Supporting Information and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 Additional supporting information may be found after HIV vector transduction: effects on lymphocytes, online in the Supporting Information section at monocytes, and CD34+ stem cells. Mol Ther 2006; 14: the end of the article. 236–244. 52. Wang X, Chang WC, Wong CW et al. A transgene- encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 2011; 118: 1255–1263. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial- 53. Lugli E, Gattinoni L, Roberto A et al. Identification, NoDerivs License, which permits use and isolation and in vitro expansion of human and distribution in any medium, provided the original nonhuman primate T stem cell memory cells. Nat Protoc work is properly cited, the use is non-commercial 2013; 8: 33–42. and no modifications or adaptations are made. ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2021 | Vol. 10 | e1268 Australian and New Zealand Society for Immunology, Inc. Page 15