ARTICLE Non Hodgkin Lymphoma Ferrata Storti Foundation Daratumumab displays in vitro and in vivo anti-tumor activity in models of B-cell non-Hodgkin lymphoma and improves responses to standard chemo-immunotherapy regimens Anna Vidal-Crespo,1* Alba Matas-Céspedes,1,2*,a Vanina Rodriguez,1 Cédric Rossi,3 Juan G. Valero,1,2 Neus Serrat,1,2 Alejandra Sanjuan-Pla,4 Pablo Menéndez,2,4,5 Gaël Roué,6 Armando López-Guillermo,2,7 Eva Giné,2,7 Haematologica 2020 Volume 105(4):1032-1041 Elías Campo,2,8,9 Dolors Colomer,2,8 Christine Bezombes,10 Jeroen Lammerts van Bueren,11,b Christopher Chiu,12 Parul Doshi12,c and Patricia Pérez-Galán1,2 a Current affiliation: Grifols, Barcelona, Spain; bCurrent affiliation: Merus, Utrecht, The Netherlands; cCurrent affiliation: Bristol Myers Squibb, Lawrenceville, NJ, USA 1 Department of Hematology-Oncology, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 2Centro de Investigación Biomédica en Red-Oncología (CIBERONC), Barcelona, Spain; 3Department of Hematology, Dijon University Hospital, Dijon, France; 4Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; 5Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; 6 Laboratory of Experimental Hematology, Department of Hematology, Vall d'Hebron Institute of Oncology, Vall d’Hebron University Hospital, Barcelona, Spain; 7Department of Hematology, Hospital Clínic-IDIBAPS, Barcelona, Spain; 8Hematopathology Unit, Department of Pathology, Hospital Clínic-IDIBAPS, Barcelona, Spain; 9Faculty of Medicine, University of Barcelona, Barcelona Spain; 10Centre de Recherches en Cancérologie de Toulouse (CRCT), UMR1037 INSERM, Université Toulouse III: Paul-Sabatier, ERL5294 CNRS, Université de Toulouse, Toulouse, France; 11 Genmab, Utrecht, the Netherlands and 12Janssen R&D, Spring House, PA, USA * AV-C and AM-C contributed equally to this work. Correspondence: ABSTRACT PATRICIA PÉREZ-GALÁN C D38 is expressed in several types of non-Hodgkin lymphoma (NHL) pperez@clinic.cat and constitutes a promising target for antibody-based therapy. Daratumumab (Darzalex) is a first-in-class anti-CD38 antibody Received: November 13, 2018. approved for the treatment of relapsed/refractory (R/R) multiple myeloma Accepted: July 9, 2019. (MM). It has also demonstrated clinical activity in Waldenström macroglob- ulinaemia and amyloidosis. Here, we have evaluated the activity and mech- Pre-published: July 11, 2019. anism of action of daratumumab in preclinical in vitro and in vivo models of mantle cell lymphoma (MCL), follicular lymphoma (FL) and diffuse large doi:10.3324/haematol.2018.211904 B-cell lymphoma (DLBCL), as monotherapy or in combination with stan- dard chemo-immunotherapy. In vitro, daratumumab engages Fc-mediated Check the online version for the most updated cytotoxicity by antibody-dependent cell cytotoxicity and antibody-depen- information on this article, online supplements, dent cell phagocytosis in all lymphoma subtypes. In the presence of human and information on authorship & disclosures: www.haematologica.org/content/105/4/1032 serum, complement-dependent cell cytotoxicity was marginally engaged. We demonstrated by Selective Plane Illumination Microscopy that daratu- mumab fully penetrated a three-dimensional (3D) lymphoma organoid and ©2020 Ferrata Storti Foundation decreased organoid volume. In vivo, daratumumab completely prevents Material published in Haematologica is covered by copyright. tumor outgrowth in models of MCL and FL, and shows comparable activity All rights are reserved to the Ferrata Storti Foundation. Use of to rituximab in a disseminated in vivo model of blastic MCL. Moreover, published material is allowed under the following terms and conditions: daratumumab improves overall survival (OS) in a mouse model of trans- https://creativecommons.org/licenses/by-nc/4.0/legalcode. formed CD20dim FL, where rituximab showed limited activity. Copies of published material are allowed for personal or inter- Daratumumab potentiates the antitumor activity of CHOP and R-CHOP in nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: MCL and FL xenografts. Furthermore, in a patient-derived DLBCL https://creativecommons.org/licenses/by-nc/4.0/legalcode, xenograft model, daratumumab anti-tumor activity was comparable to R- sect. 3. Reproducing and sharing published material for com- CHOP and the addition of daratumumab to either CHOP or R-CHOP led mercial purposes is not allowed without permission in writing from the publisher. to full tumor regression. In summary, daratumumab constitutes a novel therapeutic opportunity in certain scenarios and these results warrant fur- ther clinical development. 1032 haematologica | 2020; 105(4) Daratumumab in B-NHL Introduction mumab has been approved in combination with dexam- ethasone plus either lenalidomide or bortezomib, or poma- B-cell non-Hodgkin’s lymphoma (B-NHL) constitutes 4- lidomide for the treatment of relapsed MM patients.24 5% of all hematologic neoplasia with increasing incidence Daratumumab has a broad-spectrum killing activity in MM in western countries.1 DLBCL and FL represent the most fre- engaging complement-dependent cytotoxicity (CDC), anti- quent aggressive and indolent NHL, accounting for approx- body-dependent cellular cytotoxicity (ADCC),25 antibody- imately 35% and 20% of all lymphomas, respectively.2,3 dependent cellular phagocytosis (ADCP),26 and apoptosis.27 Moreover, roughly one third of FL patients develop a histo- Moreover, daratumumab modulates the enzymatic activity logic transformation (tFL) to DLBCL leading to a dismal of CD3828 and induces an immunomodulatory role in MM prognosis.4 Both entities are currently treated with chemo- by depleting CD38+ immune suppressive cells29 contributing immunotherapy including a rituximab backbone.5,6 FL to its antitumor activity. In chronic lymphocytic leukemia responses are usually high, although recurrence occurs in (CLL), we have demonstrated that daratumumab induces the majority of the cases.7 In DLBCL, currently classified cytotoxic activity in vitro via ADCC and ADCP in primary into germinal center type (GCB) or activated B-cell type CLL cells and cell lines. In vivo, daratumumab significantly (ABC),8 treatment is not guided by subtype, and responses prolongs OS of animals in systemic CLL murine models. to chemo-immunotherapy are normally higher in the GCB Daratumumab also affects tumor-microenvironment inter- subtype. Nevertheless, a portion of DLBCL (20%) do not actions by blocking CLL homing and dissemination to sec- respond to this regimen.9 Several second-generation anti- ondary lymphoid organs in vitro and in vivo.30 CD20 antibodies, such as the Food and Drug In the present study, we aimed to investigate in vitro and Administration-approved obinutuzumab have been clini- in vivo activity of daratumumab on MCL, FL and DLBCL cally tested to overcome these limitations.10,11 However, an cells as monotherapy and in combination with standard alternative evolving therapeutic approach is to target a dif- therapies. ferent antigen. In this regard, both FL and DLBCL originate in the germinal center (GC) and consequently express high levels of CD38, making this molecule an attractive thera- Methods peutic target.12 MCL is a rare NHL (6% of all NHL) with an aggressive Therapeutic drugs evolution and clinically challenging.13,14 Its frontline therapy, Daratumumab (Darzalex, anti-CD38mAb, IgG1) and the iso- although heterogeneous, typically consists of rituximab- type control mAb (CNTO 3930, IgG1) were provided by Janssen. based chemo-immunotherapy followed by autologous- Rituximab (Mabthera, anti-CD20 mAb, IgG1) and the chemother- stem cell transplantation and/or rituximab maintenance. apy regimen CHOP (cyclophosphamide, doxorubicin, vincristine, Even with intensive therapy, MCL patients ultimately and prednisone) were obtained from the department of pharmacy relapse.15 Novel targeted therapies currently approved for of the Hospital Clínic of Barcelona. R/R MCL include14 the mTOR inhibitor temsirolimus, the immunomodulatory agent lenalidomide, the proteasome Subcutaneous pre-emptive mouse models inhibitor bortezomib,16 also approved in front-line, and the SCID mice (Janvier Laboratories) were subcutaneously (sc) BTK inhibitor ibrutinib that achieves the highest response injected with 10x106 RL-luc cells in the FL model or 10x106 REC-1 rates.17 However, MCL patients failing ibrutinib treatment cells in the MCL model, respectively, following a protocol have very limited therapeutic options.18 In this situation, approved by the Animal Testing Ethic committee of the University where virtually all MCL cases express some level of CD38, of Barcelona and Generalitat de Catalunya (Protocol # 9971). Mice this antigen represents a potential alternative target to be were randomly assigned into cohorts of six mice per group and explored. Moreover, CD38 is associated with nodal disease received one intraperitoneal (ip) injection of 10 mg/kg of daratu- and poorer survival19,20 and high CD38 expression correlates mumab or isotype control every other week, starting the day of with poor in vivo response to bortezomib.21 Thus, targeting cell inoculation. CD38 could hold promise as a strategy for MCL, also in bortezomib resistant tumors. Patient-derived DLBCL xenograft model CD38 is present at high levels in bone marrow (BM) pre- ST1361 is a DLBCL patient-derived xenograft model developed cursor cells and it is downregulated in resting normal B cells. using a DLBCL tumor originated in a metastatic site from a The molecule is re-expressed at high density once naïve B 58-year-old chemotherapy-naïve Hispanic male. ST1361 was lymphocytes are activated, and peaks when B cells enter GCB subtype transformed from an Epstein-Barr virus negative FL the GC. Terminally differentiated plasma cells and their tumor. pathological counterparts express the highest surface densi- The DLBCL tumor was homogeneously chopped into frag- ty among human cells, while it is completely absent in ments of similar size and injected sc into SCID mice. Treatment memory B cells.22 CD38 behaves simultaneously as an was initiated when mean tumor volume was approximately enzyme and as a receptor. The extracellular domain of 150-250 mm3. Daratumumab 20 mg/kg was administered weekly CD38 contains an enzymatic site that can generate cyclic for three weeks alone or in combination with CHOP (20 mg/kg ADP ribose (cADPR) and ADPR from nicotine adenine din- cyclophosphamide, 1.25 mg/kg doxorubicin, 0.2 mg/kg vincristine ucleotide (NAD+). This control of adenosine synthesis by [intravenously on Day 0], and 0.15 mg/kg prednisone [Days 0-4]; CD38 may be important in the context of the characteristic once a day (QD) for five days) or R-CHOP (10 mg/kg rituximab immunosuppressive tumor microenvironment. [intraperitoneally on Day 0]+ CHOP as before). Daratumumab (Darzalex) is a first-in-class, human IgG1κ monoclonal antibody (mAB) that targets the CD38 epitope. Statistical analysis It was approved by the Food and Drug Administration in Unpaired and paired t-tests were used to assess statistical differ- 2015 as a monotherapy for patients with MM, who have ences between two groups using GraphPad Prism software 4.0. received at least three prior therapies.23 Currently, daratu- For Kaplan-Meier survival curves, SPSS19 software was used. haematologica | 2020; 105(4) 1033 A. Vidal-Crespo et al. Table 1. B-cell non-Hodgkin’s lymphoma cell lines characterization and daratumumab activity. Cell line NHL subtype1 CD38 sABC2 %CD381 MFIR CD383 %CD201 MFIR CD203 %CD461 %CD551 %CD591 %ADCC4 %ADCP4 %CDC4 Jeko MCL 16253 84 22 100 46 92 98 61 58 41 7 REC-1 MCL 106234 100 155 100 77 99 99 88 60 46 18 HBL2 MCL 25317 100 92 100 99 98 96 98 28 21 0 Mino MCL 127458 100 51 100 42 97 100 96 32 35 0 UPN1 MCL 54089 99 97 97 8 98 99 63 36 73 0 Z138 MCL 186239 100 120 100 28 100 75 69 53 89 0 DOHH2 FL 22369 87 20 99 31 100 76 98 25 38 0 SC-1 FL 71726 100 40 32 4 95 96 83 41 81 1 RL FL 132684 96 76 93 49 100 94 97 52 45 6 WSU-FSCCL FL 89707 97 354 8 2 82 81 94 67 92 7 Toledo DLBCL 123539 99 186 89 13 94 99 97 34 50 14 WSU-DLCL2 DLBCL 14926 94 46 99 137 95 95 98 45 15 1 SU-DHL-6 DLBCL 174033 92 383 98 172 97 44 31 64 56 5 SU-DHL-4 DLBCL 96623 96 108 100 45 93 45 95 61 34 8 Daudi BL 415667 100 276 99 30 88 17 9 63 79 96 Percentage of positive cells for CD38, CD20, CD46, CD55 and CD59 determined by flow cytometry, referred to isotype control; 2sABC: number of surface antibodies bound per 1 cell evaluated by QuantiBRITETM CD38-PE; 3MFIR: mean fluorescence ratio referred to isotype control; 4Percentage of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) induction at 1 mg/mL daratumumab. Percentage of CDC induction at 10 mg/mL daratumumab. MCL: mantle cell lymphoma, FL: follicular lymphoma; DLBCL: diffuse large B-cell lymphoma; NHL: non-Hodgkin lymphoma; BL: Burkitt lymphoma. Additional methodologic details are described in the Online S2), in a process that may resemble trogocytosis as previ- Supplementary Materials and Methods. ously described in MM studies.31,32 We next evaluated the CDC activity and observed that, similar to that reported for CLL cells,30 daratumumab Results induced low to marginal cell death in the presence of nor- mal human serum (NHS 10%) (Table 1). Very similar Daratumumab induces cell killing in the presence of results were obtained in these CDC experiments when external effectors in B-NHL 50% NHS was applied. The high expression of the com- We first assessed by calcein-AM release assay, the abili- plement regulatory proteins (CRP) CD46, CD55 and ty of daratumumab to induce ADCC, ADCP and CDC in CD59, and an insufficient number of CD38 molecules per MCL, FL and DLBCL cell lines. Using peripheral blood cell on NHL cells may explain the low CDC activity (Table mononuclear cells (PBMC) from healthy donors as exter- 1). nal effectors we demonstrated that daratumumab engages Collectively, daratumumab effectively kills CD38+ NHL ADCC in a panel of B-NHL cell lines (Table 1). cells through engagement of ADCC and ADCP but not Daratumumab induced a significant dose-dependent cell CDC, independently of the expression levels of CD38. lysis reaching its maximum antitumor activity at 1 mg/mL on MCL cell lines (mean ± standard deviation Daratumumab enters and distributes homogeneously [SD]=45±14%) (Figure 1A), at 0.1 mg/mL on FL cell lines within a 3D lymphoma model (mean ± SD=48±18%) (Figure 1B), and at 0.1 mg/mL on To model the compact aggregates of lymphoma cells DLBCL cell lines (mean ± SD=49±11%) (Figure 1C). The growing in the patients’ lymph nodes, 3D spheroids were degree of ADCC induction did not correlate with the generated by the hanging drop method,33 using RL-GFP number of CD38 molecules per NHL cell specific antibody cells previously reported as an useful tool for antibody binding capacity (sABC) (r2=0.1988; Online Supplementary therapy studies.34 We first sought to determine the degree Figure S1A and Table 1). A phagocytosis assay using mouse to which daratumumab can penetrate these lymphoma macrophages as effector cells was set up to determine aggregates depending on dose (1 and 10 mg/mL mAb) and daratumumab-mediated ADCP. Daratumumab induced time of exposure (4, 24 and 48 hours). First, we measured significant ADCP at 1 mg/mL in MCL cell lines (mean ± the percentage of mAb diffusion in the spheroid, repre- SD=54±24; Figure 1D), FL cell lines (mean ± SD=64±26%; senting the total amount of the mAb in the spheroid. The Figure 1E) and in DLBCL cell lines (mean ± SD=39±18%; 3D reconstruction images obtained by SPIM (Figure 2A) Figure 1F). The degree of ADCP induction did not corre- revealed a maximum diffusion of daratumumab at 1 late with the number of CD38 sABC on NHL cell lines mg/mL after 48 hours of treatment (Figure 2B). Next, max- (r2=0.2270; Online Supplementary Figure S1B and Table 1). imum depth of daratumumab in the spheroids was deter- Interestingly, the daratumumab-opsonized cells that mined under the same conditions and represented as per- were exposed to macrophages and not phagocytosed, suf- centage of mAb penetration in the spheroid. As observed fered a lost in CD38 surface expression, irrespective of the for the diffusion, a maximum penetration of daratumum- degree of ADCP induction (Online Supplementary Figure ab at 1 µg/mL after 48 hours of treatment was achieved 1034 haematologica | 2020; 105(4) Daratumumab in B-NHL A B C D E F Figure 1. Daratumumab induces antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in the presence of external effectors in non-Hodgkin lymphoma. (A) Mantle cell lymphoma (MCL), (B) follicular lymphoma (FL), and (C) diffuse large B-cell lymphoma (DLBCL) cell lines were treated with increasing daratumumab doses (0.0001-1 mg/mL) in the presence of peripheral blood mononuclear cell (PBMC) from healthy donors at a E:T ratio of 50:1 for four hours. Viability was then evaluated by calcein release assay. The Burkitt lymphoma (BL) cell line, Daudi, was included as positive control. (D) MCL, (E) FL, and (F) DLBCL cell lines were labeled with calcein and incubated for 4 hours with the mΦ at an E:T ratio of 1:1 in the presence of a fixed daratumumab concentration of 1 mg/mL, followed by flow cytometry analysis in triplicates. Antibody-dependent cellular phagocytosis (ADCP) was calculated as the percentage of CD19+ calcein+F4/80- cells after daratumumab treatment referred to isotype-treated cells. Daudi cells were included as positive control. ADCC: antibody-dependent cellular cytotoxicity (Figure 2C). Moreover, a significant dose-dependent day of tumor injection. Subsequently, animals received reduction of the spheroid volume was observed after dara- antibody injections every other week (10 mg/kg) for a tumumab treatment compared to isotype control (mean ± total of three doses and were sacrificed at day 30 (REC-1) SD=15±6%, 20±7%, respectively at 1 and 10 mg/mL) (Figure 3A) or day 34 (RL) (Figure 3B). Daratumumab pre- (Figure 2D) showing a direct effect of daratumumab on vented tumor formation in all mice injected with REC-1, tumor growth in 3D. This observation was validated in and in 5 of 6 mice injected with RL cells. In this latter additional lymphoma cell lines. Daratumumab moderate- model, a 20 mm3 RL tumor was found in one daratumum- ly but significantly (P<0.0001) reduced the spheroid vol- ab-treated mouse at the endpoint (day 34). As the RL cells ume in all the cell lines analyzed, albeit in a different inoculated expressed the luciferase gene, bioluminescence degree (Online Supplementary Figure S3A-B), that was analysis at different time points further supported these accompanied in most of cases by a concomitant decrease results (Figure 3B). All animals receiving isotype control in the cell number. However, this observation did not antibody formed tumors of at least 100 mm3 within a apply to HBL-2 and WSU-DLCL2 cell lines, where daratu- mean of 18 days (REC-1) and 21 days (RL) and were bigger mumab may just generate a tighter spheroid (Online than 1,000 mm3 at the end of the experiment. Supplementary Figure S3C). These data demonstrate that daratumumab efficiently Daratumumab prolongs OS and reduces tumor penetrates a lymphoma 3D structure and moderately infiltration in systemic xenograft models of MCL and FL reduces the growth of spheroids in the absence of external Next, we generated a model for blastic MCL by inocu- effectors. lating Z-138 cells intravenously into SCID mice, and one week later mice were treated weekly with either daratu- Daratumumab prevents tumor outgrowth in MCL and mumab (D), rituximab (R) or isotype (I) control FL xenografts (20/10/10/10 mg/kg). Seven of ten isotype-treated mice As a first test of daratumumab activity in vivo in NHL had to be sacrificed between days 57 - 89 due to systemic models, we inoculated SCID mice sc with two cell lines signs of disease (Figure 4). Daratumumab significantly that we had previously set up: RL cells to model tFL,35 and improved OS (I vs. D: ***P<0.001), with 90% of the mice REC-1 cells to model MCL.36 SCID mice have active natu- treated with daratumumab surviving for over 107 days, ral killer (NK) cells and macrophages that can act as effec- (Figure 4A) compared to just 10 % alive in the controls. OS tor cells to engage daratumumab activity, as we have for the daratumumab group was slightly superior but did shown using in vitro models. Mice were treated with dara- not reach statistical significance (D vs. R: ns, P=0.2907), tumumab or an isotype control (20 mg/kg) starting on the compared to that observed in the rituximab-treated mice haematologica | 2020; 105(4) 1035 A. Vidal-Crespo et al. (I vs. R: **P<0.01), where 70% of the mice were alive at ab. By the end of the experiment (day 90), 30% of mice day 107 (Figure 4). Mean survival was 73 days for the con- treated with daratumumab were cured (Figure 4). On trol group, 97 for rituximab and 103 for daratumumab autopsy, WSU-FSCCL cells showed systemic dissemina- groups. On autopsy, the disease had spread to the brain, tion of disease in the brain, BM and spleen (identified as BM, spleen and lungs (Online Supplementary Figure S4A). huCD45+/CD19+CD10+; Online Supplementary Figure S4C). Daratumumab treatment reduced the disease burden in Daratumumab robustly decreased dissemination to the the brain, BM, and spleen by 93%, 63%, and 48%, respec- brain (90%, P< 0.01) compared to the control group, while tively (Online Supplementary Figure S4B). Rituximab-treat- rituximab was not effective (Online Supplementary Figure ed mice showed a similar reduction in tumor load to that S4D). Homing of WSU-FSCCL cells to the spleen and BM achieved by daratumumab in the brain (73%) and BM was less prominent. There were no significant differences (69%) compared to that observed with the isotype con- in the reduction of dissemination to the spleen and BM by trol, while no effect was observed in spleen infiltration. daratumumab compared to the rituximab-treated group Finally, daratumumab did not diminish lung infiltration, (Online Supplementary Figure S4C). while a moderate reduction was seen in rituximab-treated Altogether, these data confirm that daratumumab mice (18%) compared to isotype control mice (Online shows comparable to superior activity with respect to rit- Supplementary Figure S4B), suggesting an organ-dependent uximab in systemic models of MCL and FL. differential immunotherapeutic response. In the FL systemic model, CD20low WSU-FSCCL cells Daratumumab synergizes with R-CHOP to reduce tumor (Table 1) were intravenously inoculated in SCID mice and burden in pre-clinical models of MCL and FL one week later mice were treated weekly with daratu- The synergy between daratumumab and rituximab mumab, rituximab or isotype control (20/10/10/10 and/or chemotherapy regimens was evaluated in MCL mg/kg). All control-treated and rituximab-treated mice (REC-1) and tFL (RL) xenograft heterotopic models. SCID rapidly succumbed to the disease and died or had to be mice were inoculated subcutaneously with NHL cells and euthanized due to severe disease signs by day 40 (control when tumors became palpable, mice were randomly group) and day 62 (rituximab group), respectively (Figure assigned into the following groups: i) Isotype ii) daratu- 4). In contrast, in the daratumumab-treated group, OS was mumab iii) CHOP iv) D-CHOP v) R-CHOP and vi) R-D- significantly prolonged (I vs. D: ***P<0.001; I vs. R: CHOP. Tumor volume was assessed twice a week and **P<0.01; D vs. R *P< 0.05). Mean survival was 35 days for tumor weights at sacrifice. In the MCL model (Figure 5A), the control group, 41 for rituximab and 60 for daratumum- daratumumab induced significant anti-tumor activity A B C D Figure 2. Daratumumab effect in a three-dimensional model of follicular lymphoma. Three-dimesional (3D) spheroids of RL-GFP cells were obtained after three days of culture in hanging drop plates or 96-well ultra-low attachment plates, and then treated with 10 mg/mL of Isotype control (IgG1), or 1 or 10 mg/mL of daratumumab at different times. (A) 3D reconstruction images produced by SPIM; fluorescence was measured at a lex of 488nm (GFP) and 561nm (monoclonal antibody [mAb] labeling). (B) Percentage of mAb diffusion, representing quantity of the mAb in the spheroid. (C) Percentage of mAb penetration, representing maximum depth of the mAb in the spheroid. (D) Spheroid volume (mm3) time course (n=number of spheroids per treatment). Statistical differences between groups were assessed by unpaired t-test (**P<0.01; ***P<0.001). 1036 haematologica | 2020; 105(4) Daratumumab in B-NHL compared to the isotype control (42%, P< 0.01). CHOP Moreover, tissue sections of selected mice of each group alone marginally reduced tumor growth (P=0.182) and no were analyzed by immunohistochemistry for the prolifer- difference was observed between daratumumab and D- ation marker pH3 and the angiogenesis marker CD31, CHOP groups (P=0.451). Importantly, adding rituximab further confirming these results (Online Supplementary to D-CHOP treatment (R-D-CHOP) induced a significant Figure S5C). reduction in tumor volume compared to the isotype con- In conclusion, these data demonstrate the value of the trol (86%, P<0.001) and also compared to R-CHOP (73%, combination R-D-CHOP as a new therapeutic strategy in P<0.001) and D-CHOP (51%, P< 0.05) alone. Similarly, in MCL and tFL superior to the standard-of-care (R-CHOP). the tFL model (Figure 5C-D), daratumumab treatment inhibited tumor growth compared to isotype control Daratumumab induces tumor growth inhibition and (36%, P<0.001). On the other hand, the combination D- prolongs survival in a patient-derived DLBCL xenograft CHOP inhibited tumor growth significantly better than model in combination with R-CHOP daratumumab as a single agent (57%, P<0.001), resem- To further evaluate the activity of daratumumab alone bling that achieved by R-CHOP (67%, P<0.001). In addi- or in combination with the standard-of-care therapy we tion, the combination regimen of R-D-CHOP had a used a clinically more relevant patient-derived DLBCL remarkable effect in impeding tumor growth (84% inhi- xenograft model (ST1361) with detectable CD38 expres- bition, P<0.001), which was superior than that achieved sion by immunohistochemistry (Online Supplementary by each combination regimen separately (P<0.001). The Figure S6). Identical to the MCL and tFL models, all indi- results of the RL-luc model were further validated by bio- vidual treatments were capable of reducing tumor volume luminescence and quantification of the signal captured at over time (P<0.001, Figure 6A); however, daratumumab the endpoint (Online Supplementary Figure S5A-B). alone was strikingly efficient with an anti-tumor activity A B Figure 3. Daratumumab efficacy in pre-emptive models of mantle cell lymphoma and transfomed follicular lymphoma. 10x106 REC-1 cells (A) or RL-luc cells (B) were mixed with matrigel (1:1) and subcutaneously injected in SCID mice (n=6 per group). Animals received one dose every other week (10 mg/kg of isotype control [IgG1] or dartumumab) starting the day of cell inoculation. Tumor growth curves over time clearly show total regression was achieved in both models. As RL cell line expressed the luciferase gene, sequential bioluminescence images were captured at different time points (B). Statistical differences between groups were assessed by unpaired t-test (***P<0.001). MCL: mantle cell lymphoma; tFL: transfomed follicular lymphoma. Figure 4. Daratumumab monotherapy in a systemic model of mantle cell lymphoma and follicular lymphoma compared to rituximab. 10x106 Z-138 mantle celle lymphoma (MCL) and WSU-FSCCL follicular lymphoma (FL) cells were intravenously injected in SCID mice (n=10 per group). Treatment (isotype control [IgG1]/ dara- tumumab/ rituximab) started one week after inoculation and went on weekly for four weeks (20/10/10/10 mg/kg), as indicated by the red arrows. Mice were mon- itored twice weekly for any sign of disease and were euthanized when body weight decreased 15-20%. Survival curves are represented. Statistical differences between groups were assessed by log-rank test. Z-138: Overall significance***P<0.001; Isotype; control vs. daratumumab ***P<0.01; daratumumab vs. rituximab not signficant (ns) P=0.2907. WSU-FSCCL: Overall significance ***P<0.001; isotype control vs. daratumumab ***P≤0.001; daratumumab vs rituximab *P=0.045. haematologica | 2020; 105(4) 1037 A. Vidal-Crespo et al. A B C D Figure 5. Daratumumab combined with R-CHOP in mantle cell lymphoma and transformed follicular lymphoma. 10x106 REC-1 (A-B) and RL-luc (C-D) cells were mixed with matrigel (1:1) and subcutaneously injected in SCID mice (REC-1: n=5-7 per group; RL: n=7-10 per group). Treatment as monotherapy (isotype control [IgG1]/ daratumumab/ rituximab/ CHOP) or the combination regimen DARA ± RITUX ± CHOP started one week after inoculation and went on weekly for three weeks for the monoclonal antbodies (mAb) (20/10/10 mg/kg) in the REC model, and four weeks (20/10/10/10 mg/kg) for the RL model. CHOP was given as an initial unique dose the first day of treatment in both models. Tumor growth curves over time are represented for each cohort (A and C). Tumor weight for each treatment was averaged and represented at endpoint for REC-1 (B) and RL models (D). Statistical differences between groups were assessed by unpaired t-test (**P<0.01; ***P<0.001). comparable to R-CHOP (P<0.001, Figure 6A). daratumumab-induced ADCC and ADCP in CLL cells.30 Furthermore, the addition of daratumumab to either The capacity of daratumumab to induce ADCC and CHOP or R-CHOP led to full tumor regression (P<0.001, phagocytosis may to some extent be related to other fac- Figure 6A). Importantly, tumor volume negatively corre- tors, such as the expression of different types of FcgR, lated very well with the OS of xenograft-bearing mice, KIRs and Natural Cytotoxic Receptors (NCR) on NK regardless the treatment group (Figure 6B). Thus, mice cells.37 Moreover, the expression of the so-called ‘don’t eat treated with either daratumumab alone or R-CHOP dis- me’ signals, such as CD47, on tumor cells plays an impor- played identical OS and, in line with the full tumor regres- tant role in regulating phagocytosis. In fact, CD47 expres- sion achieved, daratumumab combined with either sion has been found to be increased on MCL, FL and CHOP or R-CHOP resulted in 100% survival at the end of DLBCL cells compared to normal cells, conferring a worse the experiment (P<0.05 after Bonferroni multiplicity cor- clinical prognosis.38 Moreover, in agreement with previous rection, Figure 6A). Remarkably, full tumor regression and results in MM,32 the daratumumab-opsonized cells that 100% survival at day 60 were observed even when dara- were exposed to macrophages and not phagocytosed, suf- tumumab treatment was ceased as early as 14 days (total fered a loss in CD38 surface expression. This event may of three doses) after tumor cell inoculation. Collectively, reduce the ability of daratumumab to kill lymphoma cells these results indicate that tumor cells were completely via CDC and ADCC, compromising the therapeutic effi- eliminated within a short time and that no tumor escape cacy of daratumumab. However, MM studies demonstrat- occurred in this DLBCL patient-derived model. ed that this phenomenon occurs early in the treatment irrespective of their clinical responses.32 Therefore, CD38 reduction via trogocytosis should not necessarily be con- Discussion sidered as an escape mechanism from daratumumab treat- ment. On the contrary, trogocytosis may be beneficial and In this study we have demonstrated that daratumumab represent a novel mechanism of action of daratumumab, engages Fc-mediated cell killing of NHL cells by ADCC as there is also a transfer of tumor cell membrane frag- and ADCP. Variation in the extent of ADCC and ADCP ments containing important signaling molecules such as could not be explained by the differences in CD38 expres- CD56 CD49d and CD138 in this process. The decreased sion levels. Similar observations were also reported for expression of these adhesion molecules in tumor cells may 1038 haematologica | 2020; 105(4) Daratumumab in B-NHL A B Figure 6. Daratumumab combined with R-CHOP in a patient-derived diffuse large B-cell lymphoma xenograft model. Fragments from a patient-derived diffuse large B-cell lymphoma (DLBCL) were injected subcutaneously into SCID mice (n=8-10) and staged to approximately 150-250 mm3 mean tumor volume to measure (A) tumor growth and (B) overall survival (OS). 20 mg/kg daratumumab was administered weekly alone or in combination with CHOP (20 mg/kg cyclophosphamide, 1.25 mg/kg doxorubicin, 0.2 mg/kg vincristine, and 0.15 mg/kg prednisone) or R-CHOP (10 mg/kg rituximab+ CHOP) for a total of three weeks. compromise their interaction with the tumor microenvi- rapid reduction of CD20 in lymphoma patients treated ronment as we reported in a previous work.30 with rituximab, which has been linked to the development Daratumumab did not induce CDC in FL, or DLBCL or of acquired resistance.42,43 Thus, daratumumab treatment MCL cell lines. Flow cytometry analysis demonstrated may be considered in these scenarios where anti-CD20 that MCL and FL cells show medium to high expression of resistance has developed due to antigen shaving.44 In addi- CD38 while DLBCL cells tend to express higher levels of tion, our results indicate that daratumumab significantly CD38. However, the number of molecules per cell in these improves long-term survival when used as a single agent, NHL cell lines was lower than that found on the CDC- indicating that it may be also an alternative to rituximab in sensitive Daudi Burkitt lymphoma cell line, suggesting a NHL CD20high, evidenced by the comparable activity of threshold for CD38-targeted CDC lysis. In addition, this both antibodies in the blastic MCL model using Z-138 low induction of CDC was also associated with high cells. We have also observed a remarkable effect of dara- expression of the CRP CD55 and CD59 that were lower tumumab in the tumor cell dissemination to the brain in FL in Daudi cells. These observations are in line with our pre- and MCL systemic models. This may be related to the vious data obtained for CLL cell lines and primary cells.30 capacity of daratumumab to cross the hemato-encephalic Overall, the baseline expression of CD38/CD55/CD59 barrier. In fact, daratumumab has shown efficacy in CNS appears to be associated with the response to daratumum- plasmocytoma45 and extramedullary myeloma.46 ab-induced CDC in vitro in FL, DLBCL and MCL cells. Finally, we have examined the combination of daratu- These results mirrored those obtain with the anti-CD20 mumab with the standard-of-care therapy R-CHOP. Our rituximab in CLL cells.39 data suggest that daratumumab significantly potentiates We have shown for the first time that daratumumab, at R-CHOP activity, inducing higher rates of tumor regres- clinically achievable doses, effectively penetrates a lym- sion in MCL and FL. Moreover, we analyzed the efficacy phoma organoid in vitro model. A moderate but significant of daratumumab in a DLBCL patient derived mouse reduction of the sphere volume was observed with daratu- xenograft, generated from a transformed FL, and usually mumab in 3D lymphoma models, even though no signifi- less responsive to chemotherapy, as shown in our results cant effect was observed in a 2D model (data not shown). where indeed the CHOP regimen displayed limited activ- These results agree with daratumumab efficacy against ity. In this model, daratumumab in combination only with subcutaneous tumors in vivo (Figure 5 and Figure 6). CHOP, in the absence of rituximab leads to a complete In vivo results support the ability of daratumumab to pre- abrogation of tumor growth. This strong synergy may be vent the outgrowth of MCL and FL cells, when adminis- explained by the induction of stress-related cytokines by tered in a prophylactic setting, prior to tumor develop- chemotherapeutic agents that can effectively target cancer ment. These results open a window of opportunity for cells for removal by the innate immune system through daratumumab as an alternative to rituximab maintenance macrophage infiltration and phagocytic activity,47 poten- therapy in these models, in the context of a complete tially augmenting the described ability of daratumumab to response after the induction therapy.40,41 Moreover, we engage ADCP.26 have demonstrated that daratumumab shows single agent In the phase II clinical trial CARINA, LYM2001 in R/R activity both in systemic and subcutaneous models of NHL, daratumumab monotherapy did not yield the NHL, when administered after disease onset. More impor- expected results in heavily pretreated FL and DLBCL tantly, the data from the FL model using WSU-FSCCL cells patients, while no results could be concluded in MCL point to a possible role for daratumumab as a therapeutic because of insufficient patient recruitment. However, we alternative to rituximab in NHL with reduced CD20 have identified certain NHL scenarios were daratumumab expression. Previous studies have shown a substantial and shows comparable antitumor activity as rituximab (blastic haematologica | 2020; 105(4) 1039 A. Vidal-Crespo et al. MCL) and other where exhibits superiority (transformed Acknowledgments CD20dim FL). Moreover, daratumumab potentiates the We thank Dr. Adrian Wiestner for his support with this study antitumor activity of CHOP and R-CHOP in all three and his critical revision of the manuscript. Jocabed Roldan, Laura models. Jiménez, Sandra Cabezas and Ariadna Giro for their technical In addition, as demonstrated in MM, it is likely that assistance. The authors would like to thank Dr J Rouquette and daratumumab may modulate the activity and frequency Ms L Teyssedre (Imaging facility, ITAV, Toulouse) for performing of CD38 expressing immune suppressive cell populations Daratumumab imaging within the 3D lymphoma cultures by present in NHL, an effect not exerted by rituximab, and SPIM, and Dr JJ Fournié (CRCT, Toulouse) for article comments. therefore may offer a superior overall antitumor effect.29 These may be of special relevance in FL and DLBCL where Funding infiltration of diverse T and myeloid subpopulations is This work was carried out at the Esther Koplowitz Center, specially prominent.48 The immune-compromised mouse Barcelona. Genmab and Janssen pharmaceuticals funded this models used in this study preclude the analysis of this research. Additional grants that contributed to this work included: immune-modulating activity of daratumumab. Spanish Ministry of Economy and Competitiveness & European In conclusion, our findings warrant further clinical Regional Development Fund (ERDF) “Una manera de hacer development of daratumumab in NHL providing a strong Europa” for SAF2011/29326 and SAF2014/57708R to PP-G, rationale for examining its clinical efficacy in different sce- SAF2015/31242R and SAF2015-31242-R to DC, CIBERONC narios, including maintenance therapy after induction (CB16/12/00334 and CB16/12/00225), the Integrated therapy, cases with anti-CD20 resistance, FL histologic Excellence Grant from the Instituto de Salud Carlos III (ISCIII) transformation, and as frontline in combination with the PIE1313/00033 to EC and PP-G, and finally Generalitat de standard of care (CHOP/ R-CHOP). Catalunya support for AGAUR 2017SGR1009 to DC. 10. Lim SH, Vaughan AT, Ashton-Key M, et al. 21. Pérez-Galán P, Mora-Jensen H, Weniger References Fc gamma receptor IIb on target B cells pro- MA, et al. Bortezomib resistance in mantle motes rituximab internalization and cell lymphoma is associated with plasma- 1. Chihara D, Nastoupil LJ, Williams JN, Lee reduces clinical efficacy. Blood. 2011; cytic differentiation. Blood. 2011; P, Koff JL and Fowers CR. New insights 118(9):2530-2540. 117(2):542-553. into the epidemiology of non-Hodgkin 11. Edelmann J, Gribben JG. Obinutuzumab 22. Deaglio S, Vaisitti T, Aydin S, Ferrero E, lymphoma and implications for therapy. for the treatment of indolent lymphoma. Malavasi F. In-tandem insight from basic Expert Rev Anticancer Ther. 2015; Futur Med. 2016;12(15):1769-1781. science combined with clinical research: 15(5):531-544. 12. Mantei K, Wood BL. Flow cytometric eval- CD38 as both marker and key component 2. Swerdlow SH, Campo E, Harris NL, et al. uation of CD38 expression assists in distin- of the pathogenetic network underlying WHO Classification of tumours of guishing follicular hyperplasia from follicu- chronic lymphocytic leukemia. Blood. haematopoietic and lymphoid tissues. lar lymphoma. Cytom Part B Clin Cytom. 2006;108(4):1135-1144. 2008. 2009;76(5):315-320. 23. McKeage K. Daratumumab: First Global 3. Swerdlow SH, Campo E, Pileri SA, et al. 13. Pérez-Galán P, Dreyling M, Wiestner A. Approval. Drugs. 2016;76(2):275-281. The 2016 revision of the World Health Mantle cell lymphoma: biology, pathogen- 24. https://www.accessdata.fda.gov/drugsatf- Organization (WHO) classification of lym- esis, and the molecular basis of treatment da_docs/label/2017/761036s005lbl.pdf. phoid neoplasms. Blood. 2016; in the genomic era. Blood. 2011;117(1):26- Janssen Biotech Inc. DarzalexTM (daratu- 127(20):2375-2390. 38. mumab): prescribing information 2017;1- 4. Lossos IS, Gascoyne RD. Transformation of 14. Campo E and Rule S. Mantle cell lym- 29. follicular lymphoma. Best Pr Res Clin phoma: evolving management strategies. 25. de Weers M, Tai Y-T, van der Veer MS, et Haematol. 2011;24(2):147-163. Blood. 2015;125(1):48-55. al. Daratumumab, a novel therapeutic 5. Hiddemann W, Kneba M, Dreyling M, et al. 15. Dreyling M, Campo E, Hermine O, et al. human CD38 monoclonal antibody, Frontline therapy with rituximab added to Newly diagnosed and relapsed mantle cell induces killing of multiple myeloma and the combination of cyclophosphamide, lymphoma: ESMO Clinical Practice other hematological tumors. J Immunol. doxorubicin, vincristine, and prednisone Guidelines for diagnosis, treatment and fol- 2011;186(3):1840-1848. (CHOP) significantly improves the out- low-up. Ann Oncol. 2017;28(suppl-4):iv62- 26. Overdijk MB, Verploegen S, Bögels M, et al. come for patients with advanced-stage fol- iv71. Antibody-mediated phagocytosis con- licular lymphoma compared with therapy 16. Cavalli F. Bortezomib-based therapy for tributes to the anti-tumor activity of the with CHOP alone. Blood. 2005; mantle-cell lymphoma. N Engl J Med. therapeutic antibody daratumumab in lym- 106(12):3725-3732. 2015;372(23):2271. phoma and multiple myeloma. MAbs. 6. Federico M, Luminari S, Dondi A, et al. R- 17. Wang ML, Rule S, Martin P, et al. Targeting 2015;7(2):311-320. CVP versus R-CHOP versus R-FM for the BTK with ibrutinib in relapsed or refractory 27. Overdijk MB, Jansen JHM, Nederend M, et initial treatment of patients with advanced- mantle-cell lymphoma. N Engl J Med. al. The therapeutic CD38 monoclonal anti- stage follicular lymphoma: Results of the 2013;369(6):507-516. body daratumumab induces programmed FOLL05 trial conducted by the Fondazione 18. Martin P, Maddocks K, Leonard JP, et al. cell death via Fc Receptor-mediated cross- Italiana Linfomi. J Clin Oncol. 2013; Postibrutinib outcomes in patients with linking. J Immunol. 2016;197(3):807-813. 31(12):1506-1513. mantle cell lymphoma. Blood. 2016; 28. Lammerts van Bueren J, Jakobs D, 7. Kridel R, Sehn LH, Gascoyne RD. 127(12):1559-1663. Kaldenhoven N, et al. Direct in vitro com- Pathogenesis of follicular lymphoma. J Clin 19. Orchard J, Garand R, Davis Z, et al. A sub- parison of daratumumab with Surrogate Invest. 2012;122(10):3424-3431. set of t(11; 14) lymphoma with mantle cell analogs of CD38 antibodies MOR03087, 8. Alizadeh AA, Eisen MB, Davis RE, et al. features displays mutated IgV H genes and SAR650984 and Ab79 [abstract]. Blood. Distinct types of diffuse large B-cell lym- includes patients with good prognosis, 2014;124(21):Abstract 3474. phoma identified by gene expression profil- nonnodal disease. Blood. 2003; 29. Krejcik J, Casneuf T, Nijhof IS, et al. ing. Nature. 2000;403(6769):503-511. 101(12):4975-4981. Daratumumab depletes CD38+ immune- 9. Tilly H, Dreyling M. Diffuse large B-cell 20. Camacho FI, Algara P, Rodríguez A, et al. regulatory cells, promotes T-cell expansion, non-Hodgkin’s lymphoma: ESMO Clinical Molecular heterogeneity in MCL defined and skews T-cell repertoire in multiple Recommendations for diagnosis, treatment by the use of specific V H genes and the fre- myeloma. Blood. 2016;128(3):384-394. and follow-up. Ann Oncol. 2009;20 Suppl quency of somatic mutations. Blood. 2003; 30. Matas-Céspedes A, Vidal-Crespo A, 4:110-112. 101(10):4042-4046. Rodriguez V, et al. The human CD38 mon- 1040 haematologica | 2020; 105(4) Daratumumab in B-NHL oclonal antibody daratumumab shows mantle cell lymphoma. Leukemia. 2014; analysis. Br J Haematol. 2001;114(4):881- antitumor activity and hampers leukemia- 28(10):2049-2059. 883. microenvironment interactions in chronic 37. Campbell KS, Hasegawa J. Natural killer 43. Beers SA, French RR, Chan HT, et al. lymphocytic leukemia. Clin Cancer Res. cell biology: an update and future direc- Antigenic modulation limits the efficacy of 2017;23(6):1493-505. tions. J Allergy Clin Immunol. 2013; anti-CD20 antibodies: Implications for 31. Krejcik J, van de Donk NWCJ. Trogocytosis 132(3):536-544. antibody selection. Blood. 2010; represents a novel mechanism of action of 38. Chao MP, Alizadeh AA, Tang C, et al. Anti- 115(25):5191-5201. daratumumab in multiple myeloma. CD47 Antibody Synergizes with 44. Beum PV, Peek EM, Lindorfer MA, et al. Oncotarget. 2018;9(72):33621-33622. Rituximab to Promote Phagocytosis and Loss of CD20 and bound CD20 antibody 32. Krejcik J, Frerichs KA, Nijhof IS, et al. Eradicate Non-Hodgkin Lymphoma. Cell. from opsonized B cells occurs more rapidly Monocytes and granulocytes reduce CD38 2010;142(5):699-713. because of trogocytosis mediated by Fc expression levels on meloma cells in 39. Golay J, Lazzari M, Facchinetti V, et al. receptor-expressing effector cells than patients treated with daratumumab. Clin CD20 levels determine the in vitro suscep- direct internalization by the B cells. J Cancer Res. 2017;23(24):7498-7511. tibility to rituximab and complement of B- Immunol. 2011;187(6):3438-3447. 33. Eder T, Eder IE. 3D Hanging drop culture to cell chronic lymphocytic leukemia: further 45. Elhassadi E, Murphy M, Hacking D, Farrell establish prostate cancer organoids. In: regulation by CD55 and CD59. Blood. M. Durable treatment response of relapsing Koledova Z, editor. 3D Cell Culture: 2001;98(12):3383-3389. CNS plasmacytoma using intrathecal Methods and Protocols. New York, NY: 40. Vidal L, Gafter-Gvili A, Dreyling MH, et al. chemotherapy, radiotherapy, and Springer New York; p. 167-175. Maintenance therapy for Patients with Daratumumab. Clin Case Rep. 2018; 34. Decaup E, Jean C, Laurent C, et al. Anti- mantle cell Lymphoma (MCL) - a systemat- 6(4):723-728. tumor activity of obinutuzumab and ritux- ic review and meta-analysis of randomized 46. Touzeau C, Moreau P. How I treat imab in a follicular lymphoma 3D model. controlled trials (RCTs). Blood. 2016; extramedullary myeloma. Blood. 2016; Blood Cancer J. 2013;9(3):131. 128(22):1802. 127(8):971-976. 35. Matas-Céspedes A, Rodriguez V, Kalko SG, 41. Vidal L, Gafter-Gvili A, Salles G, et al. 47. Pallasch CP, Leskov I, Braun CJ, et al. et al. Disruption of follicular dendritic cells- Rituximab maintenance improves overall Sensitizing protective tumor microenviron- follicular lymphoma cross-talk by the pan- survival of patients with follicular lym- ments to antibody-mediated therapy. Cell. PI3K inhibitor BKM120 (Buparlisib). Clin phoma-Individual patient data meta-analy- 2014;156(3):590-602. Cancer Res. 2014;20(13):3458-3471. sis. Eur J Cancer. 2017;76216-76225. 48. Tosolini M, Algans C, Pont F, Ycart B 36. Moros A, Rodríguez V, Saborit-Villarroya I, 42. Foran JM, Norton AJ, Micallef IN, et al. Fournié JJ. Large-scale microarray profiling et al. Synergistic antitumor activity of Loss of CD20 expression following treat- reveals four stages of immune escape in lenalidomide with the BET bromodomain ment with rituximab (chimaeric monoclon- non-Hodgkin lymphomas. inhibitor CPI203 in bortezomib-resistant al anti-CD20): A retrospective cohort Oncoimmunology. 2016;5(7):e1188246. haematologica | 2020; 105(4) 1041