Plasma Cell Disorders ARTICLE Preclinical development of a humanized Ferrata Storti Foundation chimeric antigen receptor against B-cell maturation antigen for multiple myeloma Lorena Perez-Amill,1 Guillermo Suñe,1 Asier Antoñana-Vildosola,1 Maria Castella,1 Amer Najjar,2 Jaume Bonet,3 Narcis Fernández-Fuentes,4 Susana Inogés,5 Ascensión López,5 Clara Bueno,6 Manel Juan,7 Alvaro Urbano- Ispizua1,8,9 and Beatriz Martín-Antonio1,8 1 Department of Hematology, Hospital Clinic, IDIBAPS, Barcelona, Spain; 2Department of Pediatrics - Research, The University of Texas M. D. Anderson Cancer Center, Houston, TX, Haematologica 2021 USA; 3Laboratory of Protein Design and Immunoengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 4Department of Biosciences, Faculty of Volume 106(1):173-184 Sciences and Technology, Universitat de Vic - Universitat Central de Catalunya, Vic, Spain; 5 Department of Hematology and Cell Therapy Area and Department of Immunology and Immunotherapy, Clinic University of Navarra, Navarra, Spain; 6Josep Carreras Leukemia Research Institute and Cell Therapy Program of the School of Medicine, University of Barcelona. Barcelona, Spain; 7Department of Immunotherapy, Hospital Clinic, IDIBAPS, Barcelona, Spain; 8Josep Carreras Leukemia Research Institute, Barcelona, Spain and 9 Department of Hematology, University of Barcelona, Barcelona, Spain ABSTRACT M ultiple myeloma is a prevalent and incurable disease, despite the development of new and effective drugs. The recent development of chimeric antigen receptor (CAR)T cells has shown impressive results in the treatment of patients with relapsed or refractory hematologic B-cell malignancies. In recent years, B-cell maturation antigen (BCMA) has appeared as a promising antigen to target using a variety of immunotherapy treatments, including CART cells, for patients with multiple myeloma. To this end, we generated clinical-grade murine CART cells directed against BCMA, named ARI2m cells. Having demonstrated its efficacy, and in an attempt to avoid the immune rejection of CART cells by the patient, the single chain variable fragment was humanized, creating ARI2h cells. ARI2h cells showed comparable in vitro and in vivo efficacy to that of ARI2m cells, and superiority in cases of high tumor burden disease. In terms of inflam- Correspondence: matory response, ARI2h cells produced less tumor necrosis factor-α and BEATRIZ MARTIN-ANTONIO were associated with a milder in vivo toxicity profile. Large-scale expansion bmartina@clinic.cat of both ARI2m and ARI2h cells was efficiently conducted following Good Manufacturing Practice guidelines, obtaining the target CART-cell dose required for treatment of multiple myeloma patients. Moreover, we Received: June 3, 2019. demonstrated that soluble BCMA and BCMA released in vesicles both Accepted: January 3, 2020. affect CAR-BCMA activity. In summary, this study sets the bases for the Pre-published: January 9, 2020. implementation of a clinical trial (EudraCT code: 2019-001472-11) to study the efficacy of ARI2h-cell treatment for patients with multiple myeloma. https://doi.org/10.3324/haematol.2019.228577 Introduction ©2021 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. Multiple myeloma (MM) remains an incurable hematologic malignancy respon- All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and sible for 15-20% of all blood cancers1,2 and new cases have been increasing, on aver- conditions: age, by 0.8% each year over the past decade.3 The natural history of MM is relapse https://creativecommons.org/licenses/by-nc/4.0/legalcode. until refractory disease without reaching a plateau of survival, with less than 10% Copies of published material are allowed for personal or inter- of patients achieving sustained complete remission beyond 5-10 years after autol- nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: ogous stem-cell transplantation.4 Moreover, patients are rarely cured after high- https://creativecommons.org/licenses/by-nc/4.0/legalcode, dose chemotherapy followed by autologous stem-cell transplantation, indicating sect. 3. Reproducing and sharing published material for com- that novel strategies are required to improve the survival of patients with mercial purposes is not allowed without permission in writing relapsed/refractory MM. from the publisher. In recent years, chimeric antigen receptor (CAR)T-cell immunotherapy, based on the infusion of autologous T cells genetically modified to recognize an antigen haematologica | 2021; 106(1) 173 L. Perez-Amill et al. expressed on the tumor cell, has changed the modality of affinities of every 9-mer peptides from both scFv that are treatment for certain hematologic malignancies. not encoded by the human genome were evaluated for 12 Specifically, in acute lymphoblastic leukemia and lym- HLA-I alleles (5 type A and 7 type B) to predict binding phomas targeting CD19 outstanding responses have been affinity. As humanized ARI2h scFv contains human frame- achieved from the use of CART cells.5-8 In MM, B-cell mat- work regions, only sub-peptides from complementarity- uration antigen (BCMA)9-11 has appeared as the most determining regions and Vernier zone were considered. The promising target for CART-cell immunotherapy. affinity threshold to select only strong binders was <100 Clinical studies in patients with relapsed/refractory MM nM. To determine binding affinities, structural models receiving CART-BCMA cells have documented excellent derived for each antibody-BCMA pair were built with responses.12 Unfortunately, however, patients end up relaps- M4T22 using the crystal structure of the J22.9xi- ing.12,13 Interestingly, in comparison to CART19, a higher BCMAhuman complex (4ZFO)18 from the Protein Data CART-cell dose is required to achieve responses, 150x106 Bank23 as a template. The quality and stereochemistry of the CART cells being the the lowest dose required to obtain model was assessed using Prosa-II24 and PROCHECK,25 response in patients who have relapsed after or are refracto- respectively. For each structural complex antibody-antigen ry to a median of seven lines of treatment.12,14 Moreover, it model a total of 100 minimization trajectories were per- has been shown that a deepening of the response is formed, followed by an estimation of the binding affinity as obtained over time.12 In addition, the use of humanized or the energy difference between the complex and its separat- human CAR instead of murine CAR is emerging as the cur- ed components (ddG). Minimization and ddG analysis rent trend in CART-cell immunotherapy.15,16 were carried out with Rosetta Design Suite.26,27 The quality Here, using the same structure developed for CAR19 of the binding affinity model was very high28 because of the (ARI-0001), a CAR that has already been used in a phase I high level of sequence identity between target sequences clinical trial (NCT03144583) for B-cell malignancies at our and the template showing similar protein-protein interfaces institution,17 we generated a murine CAR against BCMA (Online Supplementary Figure S1D). (ARI2m) and a humanized version (ARI2h) for academic use. The efficacy and inflammatory response of both Clinical-grade production of ARI2 cells ARI2m and ARI2h cells were compared. Both CART cells Lentiviral particles were produced inside a clean room showed comparable anti-MM activity. However, a greater facility following Good Manufacturing Practice guidelines efficacy was observed for ARI2h cells in cases of high as previously described.17. Clinical-grade ARI2h/ARI2m tumor burden. The feasibility of clinical-grade expansion cells were produced using CliniMACS Prodigy (Miltenyi, was tested in parallel in two different institutions and suc- Biotec) as described elsewhere.17 cessfully achieved for both CAR. Finally, the impact of sol- Further details of the methods are provided in the Online uble BCMA (sBCMA) on ARI2m cell activity was ana- Supplementary Methods. lyzed, demonstrating how sBCMA can negatively affect CAR-BCMA activity. Overall, the results of this study have set the bases for a multicenter clinical trial on the use Results of ARI2h cells in MM patients in Spain (EudraCT code: 2019-001472-11). ARI2m cells demonstrate potent anti-myeloma activity The ARI2m sequence (Online Supplementary Figure S1A) contains the scFv sequence of the anti-BCMA antibody Methods J22.9,18 which has been shown to be effective against MM,29 and human CD8a, 4-1BB and CD3ζ as hinge, transmem- Ethics statement brane, co-stimulatory and signaling domains (Figure 1A). Research involving human materials was approved by CART-cell transfection efficiencies varied between 30-60% the Clinical Research Ethical Committee (Hospital Clinic, in different experiments. CAR expression was retained after Barcelona, Spain). Peripheral blood T cells were obtained cryopreservation (Online Supplementary Figure S1B). The from healthy donors after informed consent. All animal efficacy of ARI2m cells was tested against ARP1 and U266 work was performed with approval from the Animal MM cells by co-culturing T cells and MM cells at an effec- Research Ethical Committee (Hospital Clínic, Barcelona, tor:target (E:T) ratio of 1:1 over 4 days. ARI2m cells effi- Spain). ciently eliminated MM cells in comparison to untransduced (UT) T cells (Figure 1B) while no cytotoxicity was observed Cloning and humanization strategy against a BCMA-negative cell line (K562) demonstrating the The anti-BCMA single chain variant fragment (scFv) was specificity of ARI2m cells (Figure 1B). In addition, limiting designed from the J22.9 antibody.18 Human CD8a domains, dilution cytotoxicity assays with E:T ratios from 1:1 to 4-1BB and CD3 domains were obtained from the CART19 1:0.125 demonstrated a high efficacy of ARI2m cells at used at our Institution.17 Anti-CD19 scFv was substituted eliminating MM cells at a low E:T ratio at 36 h (Figure 1C), for the anti-BCMA scFv of the J22.9 antibody. In order to and the effect continued to increase to 72 h (Online obtain ARI2h, the scFv sequence of the J22.9 antibody was Supplementary Figure S1C). humanized using two predictive models (Blast and The production of pro-inflammatory cytokines by Germline). Selected amino acids (excluding complementar- ARI2m cells was analyzed after co-culturing ARI2m cells ity-determining regions and Vernier zone) were substituted and MM cells at different E:T ratios, at 24 and 48 h. A high for their homologous sequence in humans. interferon (IFN)γ production was observed at 24 h which increased at 48 h (Figure 1D). Some IFNγ production was Predictive in silico models detected for UT T cells, as expected, since UT T cells are Immunogenicity against MHC-I was predicted with activated during in vitro expansion. Minimum levels of inter- NetMHC-4.019-21 as previously described.15 In detail, binding leukin (IL)6 were detected at 24 h, with the levels increasing 174 haematologica | 2021; 106(1) CAR-BCMA for multiple myeloma at 48 h (Figure 1D). Tumor necrosis factor (TNF)α produc- (Figure 1E). ARI2m cells prevented disease progression and tion decreased at 48 h in comparison to that at 24 h, sug- performed better than UT T cells (Figure 1E, F). As expect- gesting that TNFα is produced early during CART-cell acti- ed, this translated into increased survival (Figure 1G). vation (Figure 1D). Furthermore, analysis of mice tissues at the experimental The in vivo efficacy of ARI2m cells was analyzed in a endpoint showed an absence of MM cells in the bone mar- murine model in which NSG mice received 1x106 ARP1 row and spleen (Figure 1H). T cells were mainly in the MM cells. Mice were treated 6 days later with either 10x106 spleen (Figure 1I), whereas CART cells proliferated mainly UT cells or 10x106 T cells containing 2x106 ARI2m cells in bone marrow, as indicated by a higher percentage of A B C D E F G H I J Figure 1. ARI2m cells demonstrate potent in vitro and in vivo anti-myeloma activity. (A) Design of ARI2m. (B) Cytotoxicity assays of ARI2m against ARP1 and U266 (multiple myeloma cell lines) and non-myeloma K562 cells performed from 24-96 h. (C) Limiting dilution cytotoxicity assay against ARP1 and U266 cells performed at ratios from 1:1 to 0.125:1 (T cell: tumor cell line) at 36 h. (D) Cytokine profile of interferon γ, interleukin 6 and tumor necrosis factor α after 24 h and 48 h of co- culturing T cells and ARP1 cells. (E-J) In vivo efficacy of ARI2m cells. (E, F) Diagram of experimental design and quantification of disease progression by weekly bio- luminescence imaging and overall survival of the different groups of mice (G). (H) Flow cytometry of bone marrow and spleen of mice at the end of the experiment. (I) Percentage of total T cells and chimeric antigen receptor T cells in bone marrow and spleen ofn mice treated with ARI2m. (J). Soluble B-cell maturation antigen from mice serum after being treated with ARI2m or UT T cells. *P<0.05. LTR: long terminal repeat; UT: untransduced; IFNγ: interferon γ; IL6: interleukin 6; TNFα: tumor necrosis factor α; MM: multiple myeloma; BM: bone marrow; GFP: green fluorescent protein; sBCMA: soluble B-cell maturation antigen. haematologica | 2021; 106(1) 175 L. Perez-Amill et al. CART cells from the whole T-cell population in bone mar- global higher number of T cells proliferating in the ARI2m- row than in the spleen (Figure 1I). Moreover, sBCMA was treated group, as previously observed.30 In the advanced dis- analyzed as an additional marker for MM progression in ease model, whereas ARI2m abrogated disease progression mice serum. As expected, a high amount of sBCMA was completely, minimal levels of MM disease were detected in detected in mice treated with UT T cells while no sBCMA the ARI2h-treated group at certain time points (Figure 3B), was found in mice treated with ARI2m cells (Figure 1J). although this difference was not statistically significant (Figure 3C). Interestingly, mice treated with ARI2h cells Humanization of ARI2m does not change affinity again showed increased survival, consistent with lower tox- binding against human B-cell maturation antigen icity, in comparison with that of mice treated with ARI2m and enhances cytotoxicity against highly advanced cells (Figure 3D). Accordingly, in both disease models, the myeloma disease global number of T cells was higher for ARI2m than for Early disappearance of CART cells15 is associated with ARI2h cells (Figure 3E), which might explain the greater xenorecognition of the murine component of the CAR scFV xeno-graft-versus-host disease observed in the ARI2m-treat- by the human immune system. To circumvent this problem, ed group. Importantly, the majority of T cells in bone mar- we designed a humanized sequence of the scFv of ARI2m row corresponded to CART cells, for both ARI2m and (Online Supplementary Figure S1A). Two different humanized ARI2h cells, and in both disease models (Figure 3E). Lastly, variants were created based on two different predictive algo- analysis of mice serum showed that both CAR secreted rithms (Blast and Germline) by substitution of amino acids large amounts of IFNγ. However, and in agreement with of the variable regions of heavy and light chains. In vitro com- previous observations of slower kinetics for ARI2h cells, parison of the efficacy of both variants demonstrated that IFNγ production was slower in the ARI2h group. Thus, in the Germline variant had slightly higher anti-MM activity the early model, IFNγ could not be detected in the ARI2h (Figure 2A) and equal specificity, since neither of them elim- group 3 days after CART-cell infusion but its levels was inated K562 cells (Figure 2A). Therefore, the Germline vari- higher at day 31 (Figure 3F). Similarly, in the advanced dis- ant, which was termed ARI2h, was selected for additional ease model, no IFNγ was detected at day 5 for ARI2h cells, characterization. Predictive in silico models for the immuno- but levels increased at day 21 (Figure 3F). genicity of both scFv demonstrated higher immunogenicity These results suggested a faster activity of ARI2m than of for ARI2m than for ARI2h (Figure 2B). Binding affinity pre- ARI2h cells, which in cases of high tumor burden, might diction of both scFv against murine and human BCMA lead to faster exhaustion of CART cells and their disappear- showed that both scFv bind to human BCMA with similar ance. To test this hypothesis, a third in vivo experiment with affinity while being unable to bind murine BCMA (Figure a lower CART-cell dose (3x106) was performed. Disease bur- 2C). A structural comparison between the two scFv showed den was higher at the time of CART-cell injection than in the that most structural changes during the humanization were previous experiments (Figure 3B vs. Figure 4A). In this concentrated in the heavy chain of the antibody. model, neither ARI2m nor ARI2h cells could prevent disease Complementarity-determining regions on the antibody progression (Figure 4B). However, the performance of included no mutations and presented almost no structural ARI2h cells at slowing disease progression was better than drift (Figure 2D), reinforcing the idea that both antibodies that of ARI2m cells (Figure 4B). At the last time-point, when have an almost identical binding surface. Subsequently, we mice were euthanized due to disease progression and not to analyzed whether T-cell transfection with either ARI2m or xeno-graft-versus-host disease, T cells were almost unde- ARI2h CAR constructs would lead to different phenotypes tectable (Figure 4C). However, mice treated with ARI2h cells in ARI2 cells. We did not observe any difference in either the had higher numbers of ARI2h cells in the bone marrow proportion of ARI2+CD4 and ARI2+CD8 cell populations (Figure 4C). These data suggest that slower CART-cell pro- (Figure 2E) or the proportion of memory T-cell subsets liferation could lead to longer CART-cell persistence and (Online Supplementary Figure S1E). superior antitumor activity in cases of high tumor burden. Moreover, as it was noted that the in vitro efficacy of To further support these findings, we exposed both ARI2m ARI2h was slightly lower than that of ARI2m cells (Figure and ARI2h cells to consecutive in vitro challenges with MM 2A), a long-term cytotoxicity assay was performed in which (Figure 4D). In agreement with the lower ARI2m efficacy tumor and ARI2 cells were cultured together at a low E:T observed in animals with a high tumor burden, these exper- ratio (0.125:1). This assay showed slower killing kinetics for iments demonstrated more durable persistence for CD4 and ARI2h cells, but an equal anti-MM activity at later time- CD8 ARI2h cells, which was not consistent for ARI2m cells points (Figure 2F). Accordingly, T-cell proliferation upon (Figure 4E). ARI2h cells induce less tumor necrosis factor α CAR-antigen binding was slower for ARI2h cells (Figure 2G). In addition, whereas the same level of IFNγ was pro- duced by both CART cells, lower TNFα and similar IL6 pro- production than do ARI2m cells duction was detected for ARI2h cells (Figure 2H). Previous studies have shown that macrophages, after The anti-MM activity of ARI2h and ARI2m cells was fur- being activated by CART cells, are the main producers of ther evaluated using two different in vivo models of MM IL6, IL1b and TNFα31,32 which lead to the development of (early and advanced disease models). Mice received MM cytokine release syndrome (CRS) in patients. To further ana- cells on day 0 and were treated with 5x106 CART cells on lyze the pro-inflammatory profile of both CAR, we there- day 6 or 14 to create an early and advanced model of dis- fore established an in vitro system adding macrophages. ease, respectively (Figure 3A, B). In the early disease model, Thus, effector and target cells were co-culture in the pres- ARI2h and ARI2m cells prevented MM progression equally ence of macrophages (Figure 5A). The addition of (Figure 3A, C). Around day 50, mice started to show signs of macrophages did not affect CART-cell cytotoxicity (Figure toxicity, which were more severe in the ARI2m group and 5B). The level of IFNγ was only slightly increased but very translated into a lower survival rate for this group (Figure significant increases were detected for IL6, TNFα and IL1b, 3D). These results suggested that toxicity was related to a the last one not being detected in the absence of 176 haematologica | 2021; 106(1) CAR-BCMA for multiple myeloma Figure 2. Humanization of ARI2m into ARI2h and com- parison of the affinity and immunogenicity of ARI2m vs ARI2h. (see also Online Supplementary Figure S1). (A) A Limiting dilution cytotoxicity assay of ARI2m versus both humanized versions (Blast and Germline). (B) Predicted affinities of 9-mer peptides derived from either the ARI2m or ARI2h single chain variant fragment (scFv) against HLA-I. The total number of sites with predicted affinity <100 nM is shown on the left, and specific inter- actions of HLA-I alleles with each 9-mer peptide of the scFv is shown on the right. (C) Estimation of the binding affinity of both scFv against human and murine B-cell maturation antigen (BCMA). The difference in Gibbs free energy between the complex and its separated compo- nents for each protein pair highlights the inability of any of the antibodies to target murine BCMA. (D). Local struc- tural comparison between the two scFv performed through RMSD. Positions of the complementarity-deter- mining regions are highlighted in green while sequence B differences between the two antibodies are mapped in gray under the curve. The resolution of the template structure of the antibody is represented as a dashed line. The graphic shows low structural drift on the anti- bodies’ complementarity-determining regions between the two antibody variants. (E). Phenotype characteriza- tion of T cells transduced with either ARI2m or ARI2h chimeric antigen receptor construct and after being expanded for 7 days (n=3). (F) Long-term cytotoxic assay comparing ARI2m versus ARI2h cells against ARP1 (mul- tiple myeloma cells) and K562 (non-myeloma cells) for proliferation (G) and interferon γ, tumor necrosis factor α and Interleukin 6 production (H). *P<0.05. UT: untrans- duced; ddg: difference in Gibbs free energy; RMSD: root mean square deviation; E:T; effector to target cell ratio; CSFE: carboxyfluorescein succinimidyl ester; IFNγ: inter- feron γ; TNFα: tumor necrosis factor α; IL6: interleukin 6. C D E F G H haematologica | 2021; 106(1) 177 L. Perez-Amill et al. A B C D Figure 3. In vivo efficacy of ARI2m cells and ARI2h cells. Mice received multi- ple myeloma cells on day 0, and were treated at the indicated days. Disease progression was followed by weekly bioluminescence E in the early disease model (A) and advanced disease model (B) and quantified (C). (D) Kaplan-Meier curve representing the overall survival of the different groups of mice. (E) Total CD3+ T cells and percent- age of ARI2 cells from the CD3+ T-cell populations found in the bone marrow and spleen in both the early and advanced disease models. (F) Enzyme-linked F immunosorbent assay of interferon γ from murine serum at days 3 and 31 for the early disease model and at days 5 and 21 for the advanced disease model. *P<0.05. MM: mul- tiple myeloma; CAR: chimeric antigen receptor; BM: bone marrow; IFNγ: interferon γ; CART: chimeric antigen receptor T-cell treatment. 178 CAR-BCMA for multiple myeloma macrophages (Figure 5C). Next, we compared the pro- ARI2m and ARI2h cells were efficiently expanded and the inflammatory activity of ARI2m cells and ARI2h cells over 2 required CART-cell dose (>150x106 CART cells)12 was days using this setting. We observed similar IFNγ, IL6 and achieved in all cases (Figure 6A, B). Anti-MM activity for IL1b production for both CAR (Figure 5D) but TNFα pro- both ARI2h and ARI2m cells was also demonstrated (Figure duction was lower for ARI2h cells, consistent with the find- 6C). In both institutions, all productions achieved the mini- ings of the long-term in vitro assay previously performed mum threshold required for product release (Figure 6D, E). (Figure 2H) and the lesser in vivo toxicity of ARI2h compared to ARI2m cells. Soluble and released B-cell maturation antigen affects ARI2 cells activity Efficient clinical production and activity of ARI2 cells Clinical studies with CART19 in acute lymphoblastic Pre-clinical data presented here support the development leukemia have shown that 1x106 CART cells/kg are suffi- of a phase I multicenter clinical trial for MM patients cient to achieve complete remission.5 In MM however a (EudraCT code: 2019-001472-11) to evaluate the efficacy of higher dose is needed (>150x106).12 BCMA is cleaved and treatment with ARI2 cells. The feasibility of large-scale, clin- released as sBCMA into the extracellular milieu.33 We ical-grade production was tested both for ARI2m and ARI2h hypothesized that sBCMA can bind to CAR-BCMA, partial- cells using T cells from healthy donors. A CART-cell produc- ly hampering its anti-MM activity, thereby explaining the tion system has already been established at our institution high CAR-BCMA dose required to achieve complete remis- and is being used in an ongoing phase I clinical trial with a sion in MM patients. We therefore measured the amount of CAR19 product (ARI-0001).17 Four expansions were con- sBCMA in serum from patients with monoclonal gam- ducted for each CAR in two different institutions. Both mopathy of undetermined significance, patients with newly A B C D E Figure 4. Humanization of ARI2m enhances cytotoxicity against highly advanced myeloma disease. (A) Schematic representation and images of an in vivo experi- ment in mice receiving ARP1 multiple myeloma cells (MM) and treated with either ARI2m or ARI2h cells. (B) Disease progression was followed by weekly biolumines- cence of the experiment in (A). (C) Total CD3+ T cells and percentage of ARI2 cells from the CD3+ T-cell population found in the bone marrow. (D) Graph showing the scheme of consecutive challenges of chimeric antigen receptor T (CART) cells to MM cells. (E) Percentage of CART cells in CD4 or CD8 T-cell subsets after each chal- lenge. *P<0.05. **P<0.0001. haematologica | 2021; 106(1) 179 L. Perez-Amill et al. diagnosed MM and MM patients at relapse. As expected, whether this effect results in enhanced ARI2m-cell activity. we observed larger amounts of sBCMA in MM patients This analysis was conducted in transwell plates, in which (Figure 7A). untouched MM cells were placed in the upper well and co- To test whether sBCMA inhibits CART-cell activity, MM cultured ARI2m and MM cells in the lower well (Figure 7F). cells were co-cultured with ARI2m cells in the presence of In this setting, MM cells in the upper well released sBCMA recombinant BCMA protein. The results confirmed that continuously. A cytotoxicity assay showed reduced ARI2m- recombinant BCMA blocks the activity of ARI2m cells, in cell cytotoxicity and IFNγ production in the presence of terms of cytotoxicity and IFNγ production (Figure 7B). MM sBCMA (with MM cells in the upper well) than in the con- patients had around 100 ng/mL of sBCMA and a titration trol without MM cells in the upper well (Figure 7G). assay with recombinant BCMA demonstrated inhibition of Moreover, the addition of DAPT to this transwell assay ARI2m-cell activity up to 32 ng/mL of BCMA (Figure 7C). enhanced the cytotoxicity and IFNγ production of ARI2m BCMA shedding is mediated by γ-secretase, which directly cells without affecting the proliferation of ARI2m cells cleaves and releases BCMA into the milieu, decreasing sur- (Figure 7G). face BCMA expression. BCMA shedding can, therefore, be In addition, using confocal fluorescence microscopy we blocked using γ-secretase inhibitors.33 To analyze the effect observed that BCMA is released from MM cells in vesicles of a γ-secretase inhibitor (DAPT), we measured membrane- (Figure 7H) through a mechanism that could also reduce bound BCMA in MM cells and the amount of sBCMA CART-cell activity. In order to confirm that these BCMA before and after treating MM cells with DAPT. As expected, vesicles could temporarily affect CAR-BCMA activity, MM DAPT treatment increased BCMA surface expression in cells overexpressing BCMA fused to green fluorescent pro- MM cells and decreased the release of sBCMA (Figure 7D, tein (MM-BCMA-GFP) were co-cultured with ARI2m cells E). The increased membrane-bound BCMA and decreased for 3 h and time-lapse in vivo imaging was performed. We sBCMA associated with DAPT treatment were also detect- confirmed that BCMA released in vesicles bind to ARI2m ed after co-culturing MM cells with UT T cells (Figure 7D, cells, distracting ARI2m cells from their target MM cells E). In the case of co-cultures of ARI2m and MM cells, the (Figure 7I and Online Supplementary Movie S1). Moreover, we addition of DAPT decreased the amount of sBCMA, as also observed that, after being in contact with MM cells, expected. However, membrane-bound BCMA was poorly ARI2m cells could acquire BCMA in their membranes from detected because of the high ARI2m-cell activity in vitro, the surface of the MM cells and, as a consequence, fratricide eliminating MM cells (Figure 7D, E). Having demonstrated was observed between ARI2m cells (Figure 7J and Online that DAPT treatment prevents BCMA shedding, we tested Supplementary Movie S2). To further confirm this event, A B C D Figure 5. ARI2h cells induce less tumor necrosis factor α production than do ARI2m cells. (A) Schematic representation of monocyte and T-cell isolation from the same buffy coat, differentiation of the monocytes into macrophages, in vitro expansion and co-culture of chimeric antigen receptor T (CART) cells with multiple myelo- ma (MM) cell lines. (B) Cytotoxicity and (C) production of the cytokines interferon γ, interleukin 6, tumor necrosis factor α and interleukin 1b by ARI2m cells co-cultured with ARP1 cells with or without macrophages. (D) Cytokine production over 48 h after co-culturing ARI2m/ARI2h with macrophages and ARP1 MM cells. Macrophages were added at a 1:3 macrophage:CART ratio. *P<0.05. **P<0.0001. M-CSF: macrophage colony-stimulating factor; UT: untransduced; Mac: macrophages; IFNγ: interferon γ; IL6: interleukin 6; TNFα: tumor necrosis factor α; IL1b: interleukin 1b. 180 haematologica | 2021; 106(1) CAR-BCMA for multiple myeloma Figure 6. Efficient clinical production A B and activities of ARI2m and ARI2h cells. (A, B) Clinical expansion of ARI2m and ARI2h cells showing the total T-cell (A) and total chimeric antigen receptor T-cell numbers (B) achieved at the end of the expansion. (C) Cytototoxicy assays against the U266 multiple myeloma cell line of both ARI2m and ARI2h cells at the end of the expansion. Results from (A-C) are the median of four clinical expansions of ARI2m and ARI2h cells. (D, E). Detailed comparison of the four clinical productions per- formed in two different institutions, C showing the percentage of each cell population achieved (D) and the speci- fied parameters for product release (E). *P<0.05. **P<0.0001. MM: multiple myeloma; UT: untransduced. D E haematologica | 2021; 106(1) 181 L. Perez-Amill et al. A B C D E F G H I J K L Figure 7. Soluble and released B-cell maturation antigen affects the activity of ARI2 cells. (A) Measurement of soluble B-cell maturation antigen (sBCMA) from patients with monoclonal gammopathy of undetermined significance, multiple myeloma (MM) at diagnosis and MM at relapse. (B) Cytotoxicity assay and interferon γ production of ARI2m cells co-cultured with ARP1 MM cells, adding recombinant BCMA protein (BCMA) at 10,000 ng/mL. (C) Cytotoxicity assays (at 24 h) of ARI2m cells with ARP1 MM cells adding recombinant BCMA at the indicated doses. (D, E) Mean fluorescence intensity of BCMA (D) and concentration of sBCMA (E) of ARP1 MM cells alone or in co-culture with either ARI2m or UT T cells with or without DAPT. (F) Design of the cytotoxic assays in transwell plates to analyze the impact of sBCMA and DAPT. ARI2m cells and ARP1 cells were co-cultured in the well, and additional ARP1 cells were added to the transwells as a source of continuous release of sBCMA. (G) Cytotoxicity, interferon γ production and T-cell proliferation in the experiment described in (F). (H) Confocal fluorescence image of MM cells stained with a cell tracker, CMAC, in which BCMA is visualized with a monoclonal anti-BCMA. (I, J) Time lapse images from two different in vivo time lapse experiments over 3 h of ARI2m cells stained with the cell tracker, CMAC, and co-cultured with either RPMI MM cells (I) or ARP1 MM cells (J) overexpressing BCMA in green fluorescent protein (GFP) (see also Online Supplementary Movies S1 and S2). (K) Co-culture assay of T cells with MM-RPMI cells overexpressing BCMA-GFP to which latrunculin A was added in parallel. Percentage of T cells acquiring BCMA-GFP on their surface is shown. (L). MM-RPMI cells and ARI2m cells were co-cultured for 2 h to allow transfer of BCMA-GFP to ARI2m cells, then ARI2m cells were sorted and added into a 48 h cytotoxicity assay against RPMI-MM cells. *P<0.05. **P<0.0001. MGUS: monoclonal gammopathy of undetermined significance; Dx: diagnosis; E:T: effector:target cell ratio; IFNγ: interferon γ; UT: untransduced; MFI: mean fluorescence intensity; DAPT; a γ-secretase inhibitor; TW: transwell; LatA: latrunculin A. 182 haematologica | 2021; 106(1) CAR-BCMA for multiple myeloma ARI2m and MM cells co-cultured in the presence of a trogo- of high tumor burden, and longer persistence after consecu- cytosis inhibitor (latrunculin A) showed a decreased percent- tive challenges to tumor cells. age of BCMA acquisition by ARI2m cells (Figure 7K). In CRS and neurotoxicity are common after the administra- addition, after acquiring BCMA in their membranes, ARI2m tion of CART cells.5,12,13,34,40-42 Although these problems are cells showed decreased anti-MM activity (Figure 7L). efficiently managed by following international guidelines,43,44 the ideal CART-cell treatment should try to minimize the development of CRS. Here, the use of ARI2h cells instead of Discussion ARI2m was further supported by the observation of lower TNFα production in vitro with the ARI2h cells. Whereas IL6 BCMA was identified in 201310 as the most promising is the effector cytokine for CRS,31,45,46 and exponentially antigen for CART-cell immunotherapy for the treatment of increases as CRS develops, other cytokines such as TNFα patients with relapsed/refractory MM, a finding which was and IL1b31,32 are the main initiators of CRS, as they are pro- confirmed in different clinical studies in MM patients12,13 and duced at early time points by monocytes and macrophages led us to develop our murine CAR-BCMA cells with 4-1BB once they are activated by IFNγ produced by CART cells. In as a co-stimulatory domain (ARI2m cells). ARI2m cells fact, TNFα, acts as an initiator cytokine orchestrating the demonstrated strong anti-MM activity which is retained in cytokine cascade in many inflammatory diseases, appearing their humanized version (ARI2h cells). Additionally, a as a therapeutic target in inflammatory diseases.46 Here, our greater efficacy of ARI2h versus ARI2m cells was observed in in vitro model with macrophages mimicking a model more vivo in mice with high tumor burden. Our results set the similar to the in vivo scenario, demonstrated that ARI2h cells bases for a multicenter phase I/II clinical trial of treatment induced less TNFα production by macrophages, a relevant with ARI2h cells for patients with relapsed/refractory MM finding, as CRS in MM patients after CAR-BCMA treatment in Spain. associates with a higher peak of TNFα.12 Moreover, CAR The success of BCMA as a target for CART-cell with faster kinetics associate with higher CRS,37 suggesting immunotherapy was demonstrated for the first time in that the slower kinetics of ARI2h cells in comparison to patients with a CAR-BCMA with CD28 as the co-stimula- ARI2m cells might also explain the observed lower level of tory domain. However, this CAR displayed marked in vivo toxicity. toxicity.14 The CD28 was therefore replaced by 4-1BB: the Last, we analyzed the impact of sBCMA on CART-cell new CAR (bb2121) demonstrated manageable toxicity and activity. Even though studies with CAR-BCMA in MM have it was found that a minimum dose of 150x106 CART cells not found any correlation between sBCMA and CART-cell was required to obtain responses.12 In parallel, two other activity,10,12,34,47 we observed that the high in vitro CART activ- studies,13,34 demonstrated that a lower number of previous ity rapidly eliminating MM cells impeded a proper analysis treatments is associated with better responses, and that of the role of sBCMA in preclinical studies. Our in vitro mod- short-term CART-cell expansion is more consistent after els performed at a low CAR-BCMA:MM ratio, with the cre- lymphodepletion.34 Different factors influence CART-cell ation of an environment with continuous release of sBCMA, expansion and persistence, which enhance the long-term and the addition of a γ-secretase inhibitor confirmed the control of the disease.5,12,13,34 Work needs to be done to negative impact of sBCMA on CAR-BCMA activity. improve these aspects of CAR-BCMA therapy, because a Moreover, we observed that BCMA is also released in vesi- high number of MM patients end up relapsing.12,13 One of the cle structures, distracting CART cells from their targets, and suggested reasons for this is the limited persistence of CART that BCMA can be transferred to CART cells through a cells. In this regard, human or humanized CAR, by avoiding mechanism which appears to be trogocytosis, a finding the immunological reaction of the human immune system already made for CART19 cells,48 causing decreased anti- against the murine components of the scFv of the CAR, MM activity of the ARI2m cells. might increase the persistence of CART cells.15,16,30 Based on In conclusion, we have developed CAR-BCMA cells with previous studies, and supported by our results showing that 4-1BB as a co-stimulatory domain which have been human- both ARI2m and ARI2h cells prevented disease progression ized, retaining high efficacy and demonstrating less toxicity equally, we selected ARI2h cells, which are humanized than their murine counterparts. ARI2 cells can be efficiently CAR-BCMA cells, to be used for a clinical trial for MM expanded under Good Manufacturing Practice conditions patients (EudraCT code: 2019-001472-11). for use in a clinical trial. Moreover, we demonstrated that Long-term CART-cell persistence is also associated with sBCMA and BCMA released from MM cells can affect durability of remission.5,12,13,34 Factors influencing long-term CART-cell activity. CART-cell persistence include the exhaustion profile of CART cells29,35 and the affinity for the target antigen in com- Disclosures bination with the co-stimulatory domain of CART cells. In No conflicts of interests to disclose. this regard, studies on CART19 with CD28 and 4-1BB demonstrated that strong activation of CART cells, due to Contributions high affinity or high expression of the target antigen com- LPA and BMA designed the study, LPA performed in vitro and bined with the CD28 domain, leads to faster CART-cell pro- in vivo experiments. GS performed clonings, virus production and liferation with increased exhaustion and shorter persistence. in vivo experiments. LPA and BMA analyzed data and wrote the Contrariwise, slower CART-cell activation, due to a lower manuscript. AN designed the scFv of ARI2m and provided viral affinity to the target antigen or to the 4-1BB domain, reduces vector for GFP-Ffluc. MC and AA provided advice for experi- exhaustion, thereby improving persistence.35-39 Here, even ments. AA performed some of the CART in vitro expansions. MJ, though ARI2m and ARI2h cells had the 4-1BB co-stimulato- SI and AL performed clinical grade ARI2 production. JB and NFF ry domain and presented the same affinity against human performed predictive models for bind-ing affinity. CB performed cell BCMA, ARI2h cells had slower kinetic activity, and demon- sortings. AUI provided funding and constructive ideas. All authors strated greater efficacy than ARI2m cells in a murine model reviewed the manuscript. haematologica | 2021; 106(1) 183 L. Perez-Amill et al. 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