Accepted Article Preview: Published ahead of advance online publication Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes M Pfeifer, B Zheng, T Erdmann, H Koeppen, R McCord, M Grau, A Staiger, A Chai, T Sandmann, H Madle, B Do¨rken, Y-W Chu, A I Chen, D Lebovic, G A Salles, M S Czuczman, M C Palanca-Wessels, O W Press, R Advani, F Morschhauser, B D Cheson, P Lenz, G Ott, A G Polson, K E Mundt, G Lenz
Cite this article as: M Pfeifer, B Zheng, T Erdmann, H Koeppen, R McCord, M Grau, A Staiger, A Chai, T Sandmann, H Madle, B Do¨rken, Y-W Chu, A I Chen, D Lebovic, G A Salles, M S Czuczman, M C Palanca-Wessels, O W Press, R Advani, F Morschhauser, B D Cheson, P Lenz, G Ott, A G Polson, K E Mundt, G Lenz, Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes, Leukemia accepted article preview 24 February 2015; doi: 10.1038/leu.2015.48. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 25 August 2014; revised 8 January 2015; accepted 13 February 2015; Accepted article preview online 24 February 2015
©
2015 Macmillan Publishers Limited. All rights reserved.
1
Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes
Matthias Pfeifer1, Bing Zheng2, Tabea Erdmann3,4, Hartmut Koeppen2, Ron McCord2, Michael Grau5, Annette Staiger6, Akiko Chai2, Thomas Sandmann2, Hannelore Madle3,4, Bernd Dörken1, Yu-Waye Chu2, Andy I. Chen7, Daniel Lebovic8, Gilles Andre Salles9, Myron S. Czuczman10, Maria Corinna Palanca-Wessels11,12, Oliver W. Press11, Ranjana Advani13, Franck Morschhauser14, Bruce D. Cheson15, Peter Lenz5, German Ott6, Andrew G. Polson2, Kirsten E. Mundt2,#, and Georg Lenz3,4,# 1
Department of Hematology, Oncology and Tumor Immunology, Charité Universitätsmedizin Berlin, Germany 2 Genentech, Inc., 1 DNA Way, South San Francisco, CA, USA 3 Translational Oncology, Department of Medicine A, Albert-Schweitzer-Campus 1, University Hospital Münster, Münster, Germany 4 Cluster of Excellence EXC 1003, Cells in Motion, 48149 Münster, Germany 5 Department of Physics, Philipps-University, Marburg, Germany 6 Department of Clinical Pathology, Robert-Bosch-Krankenhaus and Dr. Margarete FischerBosch-Institute of Clinical Pharmacology, Stuttgart, Germany 7 Oregon Health & Science University, Portland, OR, USA 8 University of Michigan, Ann Arbor, MI, USA 9 Hospices Civils de Lyon - Université de Lyon, Pierre-Bénite, France 10 Departments of Medicine and Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA 11 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 12 Seattle Genetics, Inc., Bothell, WA, USA 13 Stanford University Medical Center, Stanford University, Stanford, CA, USA 14 Hematology Department, EA 4481 GRIIOT, Centre Hospitalier Régional Universitaire de Lille, Lille, France 15 Lombardi Comprehensive Cancer Center, Georgetown University Hospital, Washington DC, USA #Corresponding authors
Corresponding authors: Georg Lenz, M.D. University Hospital Münster Translational Oncology Medical Department A Albert-Schweitzer-Campus 1 48149 Münster, Germany Phone: +49 251 83 56229 Fax: +49 251 83 52673 e-mail: [email protected]
©
Kirsten E. Mundt, PhD Genentech Inc. DNA Way 1 South San Francisco CA 94080, USA Phone: +1 650 225 5462 Fax: +1 650 467 7571 e-mail: [email protected]
2015 Macmillan Publishers Limited. All rights reserved.
2
Disclosure of Conflicts of Interest B.Z., H.K., R.M., A.C., T.S., Y.W.C., A.G.P., and K.E.M. are employees of Genentech. M.C.P.W. is an employee of Seattle Genetics. G.L. received research funding from Genentech.
©
2015 Macmillan Publishers Limited. All rights reserved.
3
Abstract Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubuledisrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment due to low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.
©
2015 Macmillan Publishers Limited. All rights reserved.
4
Introduction Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category (1). This heterogeneity can partially be explained by the existence of different molecular subtypes identified by gene expression profiling (2-5). Applying the cell of origin classification, at least two major molecular subtypes can be distinguished. The germinal center B-cell-like (GCB) DLBCLs are derived from germinal center B-cells, whereas activated B-cell-like (ABC) DLBCLs originate from activated B-cells (2, 3). GCB and ABC DLBCLs depend on different oncogenic pathways that are frequently activated by somatic mutations (1). Importantly, the existence of molecular subtypes, as well as specific mutations appear predictive of response to pathway inhibitors such as ibrutinib, immunomodulatory agents such as lenalidomide or proteasome inhibitors (6-8). Thus, the assignment of DLBCL patients into molecular subtypes is becoming increasingly important to ensure that patients are selected for the most efficacious treatment regimens. Finally, ABC and GCB DLBCLs also show significant differences in outcome when treated with immunochemotherapy (9). The introduction of the anti-CD20 monoclonal antibody (mAB) rituximab into the therapy of DLBCL has significantly improved prognosis (1012). Nevertheless, a substantial number of patients relapse after first-line therapy. These relapsed or refractory DLBCL patients are characterized by dismal outcome (13). A novel therapeutic approach is the use of antibody drug conjugates (ADCs) in which cytotoxic drugs are attached to antibodies that are directed against antigens expressed on tumor cells (14). Recently this strategy has been shown to be very effective in the treatment of malignant lymphoma patients. The anti-CD30 ADC brentuximab vedotin achieved high response rates in patients with relapsed or
©
2015 Macmillan Publishers Limited. All rights reserved.
5
refractory CD30-positive Hodgkin’s lymphoma (HL) and anaplastic large-cell lymphoma (ALCL) (15-17). CD22 and CD79B are physiologically expressed in the vast majority of B-cells and therefore represent promising targets for ADCs. Preclinical data for an antiCD79B and an anti-CD22 antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) indicated efficacy in B-cell lymphomas including Burkitt lymphoma, mantle cell lymphoma, follicular lymphoma and DLBCL (18-20). However, these studies did not investigate whether ABC or GCB DLBCL or lymphomas characterized by specific mutations respond differentially to these agents. Of particular interest in this respect are mutations affecting CD79B that have been reported to impact B-cell receptor internalization in mouse B-cells (21). To this end, we analyzed the efficacy of anti-CD22-MMAE and anti-CD79B-MMAE in a large panel of cell lines derived from DLBCL patients of both molecular subtypes including cell lines characterized by CD79B mutations. To validate our in vitro findings, we investigated responses observed in DLBCL patients treated in two recent phase-I studies.
Material and Methods
Antibody drug conjugates The following ADCs were used in the present study. As previously described, pinatuzumab vedotin is an anti-CD22-MMAE ADC (DCDT2980S), whereas polatuzumab vedotin (DCDS4501A) represents an anti-CD79B-MMAE ADC (18, 22). Pinatuzumab vedotin and polatuzumab vedotin are the drug candidates used in two phase-I clinical trials in relapsed and refractory lymphoma patients (23, 24).
©
2015 Macmillan Publishers Limited. All rights reserved.
6
Cell lines Human DLBCL, ALCL and multiple myeloma (MM) cell lines were cultured as previously described in RPMI (Life Technologies, Carlsbad, CA, USA) with 10% FCS (Sigma-Aldrich, St. Louis, MO, USA), except for OCI-Ly1, OCI-Ly2, OCI-Ly3, OCILy4, OCI-Ly7, OCI-Ly10, OCI-Ly19, and TMD8 that were cultured in Isocove’s modified Dulbecco medium (Life Technologies) supplemented with 10% FCS (25, 26).
Viability assay, sub-G1 and cell cycle analysis Detailed protocols are available in the Supplemental Material and Methods.
Determination of CD22 and CD79B surface expression by flow cytometry Detailed protocols are available in the Supplemental Material and Methods.
CD79B antibody internalization assay Cells were pre-incubated with 10 g/ml of mouse anti-human CD79B antibody (clone SN8) (27) on ice for 30 minutes and washed once to remove excess unbound antibodies. Cells were subsequently incubated at 37°C allowing for internalization. To terminate internalization, cells were washed twice and stained with PE conjugated goat anti-mouse IgG antibody. Surface CD79B expression was measured by flow cytometry. Percent of internalization was calculated as % internalization (Tn) = (gMFI(T0)-gMFI(Tn))/gMFI(T0)*100 with gMFI = geometric mean fluorescence intensity.
Patient population
©
2015 Macmillan Publishers Limited. All rights reserved.
7
Both DCT4862g and DCS4968g were phase-I dose finding studies for anti-CD22MMAE or anti-CD79B-MMAE respectively, in patients diagnosed with relapsed or refractory B-cell lymphoma. These studies are registered at ClinicalTrials.gov under NCT01209130 and NCT01290549 respectively (23, 24). All patients gave informed written consent to participate in the clinical trials and to provide tissue for biologic studies. The respective institutional ethics review boards approved both trails. To correlate pre-clinical data with clinical results, we report in this manuscript CD22 and CD79B expression data as well as the correlation of expression and efficacy in DLBCL patients who were treated at doses of 1.8 mg/kg and above. The recommended phase-II dose was determined as 2.4 mg/kg for both CD22 ADC and CD79B ADC as single agents. The complete patient characteristics and overall results of these studies will be published in a separate manuscript.
CD22 and CD79B immunohistochemistry in primary DLBCL patient samples and DLBCL cell lines CD22 and CD79B immunohistochemical stainings were performed on the Ventana Discovery XT platform (Ventana, Tucson, AZ, USA) using the Ventana CC1 standard digestion pretreatment for antigen retrieval. The anti-CD22-mAB SP104 (Ventana) was used according to the manufacturer’s instructions. The anti-CD79B-mAB AT107-2 (0.25 g/ml; AbD Serotec, Oxford, UK) was incubated for 60 minutes at 37oC. Human tonsils were used as positive controls. The staining results are reported as Hscore as previously described that includes both numbers of positive tumor cells as well as the staining intensities (28, 29). The H-score was calculated for staining of tumor cells using the following formula: H-Score = (% at 0) * 0 + (% at 1+) * 1 + (% at 2+) * 2 + (% at 3+) * 3. Thus, this score produces a continuous variable that ranges from 0 to 300 (28, 29). A modified H©
2015 Macmillan Publishers Limited. All rights reserved.
8
score was used in the cell lines that displayed a rather uniform intensity of protein expression in a given case: % cells positive * predominant expression intensity (03).
Determination of molecular DLBCL subtypes Classification into molecular DLBCL subtypes was performed analogously to Wright et al. (3). The detailed algorithm is described in Supplemental Material and Methods and the data are summarized in Supplemental Figure 1.
Determination of IC50 concentrations and correlation analyses The IC50 concentrations were calculated as described in detail in the Supplemental Material and Methods.
Mutation analysis of CD79B in primary DLBCL patient samples Detailed protocols are available in the Supplemental Material and Methods. The primer sequences are summarized in Supplemental Table 1.
Western blotting Western blotting was performed as previously described (30, 31).
Results
CD22 and CD79B expression in DLBCL To assess the expression pattern of the targets of anti-CD22-MMAE and anti-CD79BMMAE, we measured CD22 and CD79B cell surface expression of DLBCL derived
©
2015 Macmillan Publishers Limited. All rights reserved.
9
cell lines by flow cytometry. Cell lines derived from MM and ALCL that do not express either CD22 or CD79B were used as negative controls (Supplemental Figure 2A and 2B). As expected, virtually all DLBCL cell lines displayed high CD22 and CD79B surface expression compared to MM (p=2.2x10-7 for CD22 and p=8.6x10-6 for CD79B) and ALCL cell lines (p=2.2x10-7 for CD22 and p=1.3x10-5 for CD79B) albeit in a dynamic range (Supplemental Figure 2A and 2B and Supplemental Table 2). To analyze CD22 and CD79B cell surface expression in primary DLBCL, we performed immunohistochemical staining on 32 patient samples for CD22 and on 28 patient samples for CD79B obtained from patients treated in two recently completed phase-I trials investigating the efficacy of anti-CD22-MMAE and anti-CD79B-MMAE, respectively (NCT01209130 and NCT01290549). All specimens expressed CD79B, consistent with its role as a signal transducing subunit of the essential B-cell receptor (BCR; median H-score 250) (Figure 1A, 1B, and 1E and Supplemental Table 3). In contrast, the expression levels of CD22 determined by immunohistochemistry were more variable and unexpectedly low in several specimens (median H-score 90; Figure 1C, 1D, and 1E and Supplemental Table 4). These results are surprising, especially as previous assessment of CD22 surface expression in primary DLBCL samples using flow cytometry indicated CD22 expression on virtually all cases (20).
Activity of anti-CD22-MMAE and anti-CD79B-MMAE in different subtypes of DLBCL in vitro and in vivo Anti-CD22 and anti-CD79B ADCs have shown promising activity in preclinical models of various subtypes of malignant lymphoma (18). To specifically investigate the effects of anti-CD22-MMAE and anti-CD79B-MMAE in different molecular DLBCL subtypes, we assessed cell viability following treatment with these agents in a large panel of 27 DLBCL cell lines, previously assigned to either ABC or GCB DLBCL (7 ©
2015 Macmillan Publishers Limited. All rights reserved.
10
ABC DLBCL and 20 GCB DLBCL cell lines). We analyzed dose-dependent sensitivity of every cell line in the range of 0-50 µg/ml ADC and calculated the corresponding IC50. To differentiate responding from non-responding cell lines, we employed a cutoff value of IC50 ≤ 5 µg/ml for responders, based on the clinical exposure at trough drug levels being above 5 µg/ml for the recommended phase-II dose of 2.4 mg/kg for DLBCL (Figure 2A and 2B and Supplemental Table 5). Using this cut-off, we observed a similar pattern of responsiveness of DLBCL cell lines to both ADCs. 22 out of 27 (81%) cell lines were sensitive to anti-CD22-MMAE and 22 out of 27 (81%) cell lines showed toxicity following anti-CD79B-MMAE treatment. The vast majority of DLBCL cell lines responded to both anti-CD22-MMAE and anti-CD79B-MMAE respectively (Figure 2A and 2B and Supplemental Table 5). In contrast, a minority of cell lines was sensitive to either anti-CD22-MMAE or anti-CD79B-MMAE treatment. Only two cell lines (SUDHL-2 and WSU-NHL) were resistant to both drugs. For both agents, we could not detect a preferential response in either ABC or GCB DLBCL cell lines (p=0.10 for anti-CD22-MMAE and p=0.47 for anti-CD79B-MMAE; one-tailed two sample t-tests). These results demonstrate that anti-CD22-MMAE and anti-CD79BMMAE are active in DLBCL models irrespective of their molecular subtype. To validate if indeed both primary ABC and GCB DLBCL patients respond equally to anti-CD22-MMAE and anti-CD79-MMAE, we determined the molecular DLBCL subtype in 52 out of 87 (60%) patients treated in the recently completed phase-I trials using these agents. 26 patients (50%) were classified as GCB, 22 (42%) as ABC, and 4 (8%) were unclassifiable DLBCL (Supplemental Figure 1). Overall the response evaluation of 28 DLBCL patients treated with anti-CD22-MMAE and 29 patients treated with anti-CD79B-MMAE at doses of ≥ 1.8 mg/kg was available (Table 1). In these 57 patients, a molecular DLBCL subtypes classification was available in 29 patients (14 patients treated with anti-CD22-MMAE and 15 ©
2015 Macmillan Publishers Limited. All rights reserved.
11
patients treated with anti-CD79B-MMAE; Table 2). For the remaining patients we could not obtain sufficient material to perform a molecular subtype classification. The in vivo patient data confirmed our in vitro observations that both antiCD22-MMAE and anti-CD79B-MMAE are active agents in DLBCL. Treatment with anti-CD22-MMAE induced either a complete (CR) or a partial remission (PR) in 12 out of 28 (43%) patients at dose levels ≥ 1.8 mg/kg (Table 1). As suggested from our cell line data we observed activity in both ABC and GCB DLBCL patients. Three out of seven ABC (2 CRs) and two out of seven GCB DLBCL patients (2 CRs) responded to anti-CD22-MMAE (dose level ≥ 1.8 mg/kg; Table 2). Anti-CD79B-MMAE was similarly effective in DLBCL patients of both molecular subtypes. Responses were observed in 16 out of 29 (55%) patients. Four out of five ABC DLBCL patients responded (1 CR), whereas five out of nine GCB DLBCL obtained a PR (Table 2). Despite the small numbers of evaluable patients, these data suggest that anti-CD22MMAE and anti-CD79B-MMAE are active in both ABC and GCB DLBCL patients (Table 2).
Activity of anti-CD22-MMAE and anti-CD79B-MMAE in CD79B mutated DLBCL Roughly 20% of primary ABC DLBCL patient samples harbor mutations affecting CD79B (32). Previous work suggested that these mutations might negatively affect BCR internalization in mouse models (21). Hence, we examined whether CD79B mutations would interfere with anti-CD79B-MMAE activity, as ADC internalization is crucial for its mechanism of action. To this end, we investigated the kinetics of internalization of the naked anti-CD79B antibody in the CD79B (Y196H) mutated cell line TMD8 compared to those in the CD79B wild-type cell lines SUDHL-4 and WSUDLCL2 (32). The vast majority of the anti-CD79B antibody was rapidly internalized in all three cell lines within the first hour of incubation. The internalization rate in TMD8 ©
2015 Macmillan Publishers Limited. All rights reserved.
12
and WSU-DLCL2 cells were virtually identical and higher compared to the rate in SUDHL-4 cells (Figure 2C) suggesting that the internalization rate in human CD79B mutated and wild-type lymphoma cells is comparable. This was further underscored as treatment of the two CD79B mutated cell lines HBL1 and TMD8 (32) with antiCD79B-MMAE induced toxicity in both cell lines. Both HBL1 and TMD8 were highly sensitive to anti-CD79B-MMAE treatment with IC50s of 0.02
g/ml and 0.16
g/ml,
respectively (Supplemental Table 5). This suggests that CD79B mutations do not seem to impair the efficacy of anti-CD79B-MMAE in vitro. Next, we analyzed if mutations in CD79B might impact response to antiCD79B-MMAE in primary patients. To evaluate this, we determined the CD79B mutation status in 35 primary DLBCL samples from patients treated in both trials. We identified two patient samples harboring either a CD79B Y196H or a CD79B Y196C mutation, respectively. In contrast, 33 patients had two wild-type alleles for CD79B. As the two patients with CD79B mutations were treated with anti-CD22-MMAE (both achieved a CR), we cannot evaluate the in vivo efficacy of anti-CD79B-MMAE treatment. However, our in vitro data suggest that anti-CD79B-MMAE should be active in both patients with wild-type or mutated CD79B.
Anti-CD22-MMAE and anti-CD79B-MMAE induce cell death in DLBCL To obtain a better understanding of the growth inhibitory effects of anti-CD22-MMAE and anti-CD79B-MMAE treatment observed in DLBCL cell lines, we investigated whether cell death or cell cycle arrest were induced. Treatment of the GCB DLBCL cell lines OCI-Ly7 and RL with anti-CD22-MMAE resulted in a significant increase in the sub-G1 fraction after 72 hours, indicating an increase in cell death (Figure 2D and Supplemental Figure 3A). Similarly, anti-CD79B-MMAE treatment induced cell death as measured by a sub-G1 peak increase in OCI-Ly19 and RL cells (Figure 2E and ©
2015 Macmillan Publishers Limited. All rights reserved.
13
Supplemental Figure 3B). We also observed an increase of the G2/M peak in RL and OCI-Ly7 cells treated with anti-CD22-MMAE and in RL cells treated with anti-CD79BMMAE (Supplemental Figure 3A and 3B), indicative of a mitotic arrest, consistent with the mechanism of action of a tubulin targeted agent. To assess the contribution of the respective antibody moieties to the ADC’s mechanism of action, we measured viability of DLBCL cell lines treated with the unconjugated anti-CD22 and anti-CD79B antibodies. The unconjugated antibodies did not induce any changes in cell viability or cell cycle suggesting that the toxic effect of these ADCs is mediated solely by MMAE (Figure 2D and 2E and Supplemental Figure 3A and 3B). To confirm the effect of MMAE on cell viability, we treated our panel of cell lines with MMAE in the absence of the antibody. Indeed, MMAE significantly reduced viability in all investigated cell lines (Supplemental Table 5), demonstrating the sensitivity of these cell lines to the payload drug MMAE. Co-expression of MYC and BCL2 is associated with adverse survival in DLBCL patients and expression of BCL2 family members such as MCL1 has been shown to affect response to conventional chemotherapy in DLBCL (30, 33, 34). Therefore, we analyzed if expression of either MYC and/or the BCL2 family members BCL2, BCL-XL or MCL1 is associated with either response or resistance to antiCD22 and anti-CD79B-MMAE. To this end, we determined the expression pattern of these proteins by Western blotting in cell lines that are characterized by either response to both agents or by response to only one agent or that are characterized by resistance to both drugs (Figure 3A). We detected BCL2 and MCL1 expression in only a fraction of the investigated cell lines, whereas BCL-XL and MYC were expressed in virtually all lines albeit at different levels (Figure 3A). We could not detect a preferential expression pattern in either one of the response groups (Figure 3A). ©
2015 Macmillan Publishers Limited. All rights reserved.
14
CD22 and CD79B surface expression and sensitivity to MMAE as predictors of response in DLBCL Next, we investigated whether the degree of cell surface expression of CD22 and CD79B correlates with response to anti-CD22-MMAE and anti-CD79B-MMAE in DLBCL cell lines. Indeed, we detected a significant correlation between antigen surface expression and response to both anti-CD22-MMAE (r=-0.75; p=6x10-6; Figure 3B) and anti-CD79B-MMAE (r=-0.68, p=1x10-4; Figure 3C). However, several cell lines responded to treatment with anti-CD22-MMAE and anti-CD79B-MMAE despite low surface target antigen expression (e.g. OCI-Ly1 and NUDUL-1 for antiCD22-MMAE treatment and OCI-Ly10, OCI-Ly3, OCI-Ly2, OCI-Ly4, and WSUDLCL2 for anti-CD79B-MMAE treatment; Supplemental Table 2 and 5). These data suggest that using a predefined cut-off level of surface target expression might not accurately predict which patients may respond to anti-CD22-MMAE or anti-CD79BMMAE treatment. In order to explore this possibility, we evaluated whether response to treatment in primary DLBCL patients correlates with CD22 and CD79B expression levels by immunohistochemistry. Response evaluation and immunohistochemistry was available for 18 anti-CD22-MMAE treated and 18 anti-CD79B-MMAE treated patients (dose level ≥1.8 mg/kg). The response to both ADCs was not correlated to the expression of the targets CD22 and CD79B respectively (p=0.2 for CD22 and p=0.8 for CD79B; chi-square test; Figure 3D and 3E). A low H-score did not predict resistance to these agents. Thus, the in vivo data supported our hypothesis that using a predefined CD22 or CD79B expression cut-off level for treatment selection might exclude patients who may still benefit from effective ADC therapies.
©
2015 Macmillan Publishers Limited. All rights reserved.
15
To investigate if the discrepant results in cell lines and primary patient samples regarding the correlation of CD22 and CD79B expression and response to either antiCD22-MMAE or anti-CD79B-MMAE is related to the applied technique (flow cytometry vs. immunohistochemistry), we determined CD22 and CD79B expression in our panel of cell lines additionally by immunohistochemistry (Supplemental Table 6). Subsequently, we correlated response to treatment in the cell lines with the CD22 and CD79B H-score. For CD79B we could not detect a significant correlation between expression and response to anti-CD79B-MMAE treatment any more (r=0.21; p=0.3), whereas for CD22 only a weak correlation between CD22 expression and response to anti-CD22-MMAE was detectable (r=0.53; p=4x10-3; Supplemental Table 5 and 6). These data suggest that immunohistochemistry is less sensitive in accurately determining CD22 and CD79B expression levels compared to flow cytometry. Finally, we investigated whether sensitivity to MMAE correlated with sensitivity to either anti-CD22-MMAE or anti-CD79B-MMAE measured by the corresponding IC50 values in our panel of DLBCL cell lines. For anti-CD22-MMAE (r=0.25; p=0.2; Figure 3F) and for anti-CD79B-MMAE (r=0.30; p=0.1; Figure 3G) we did not detect a significant correlation, most likely due to the fact that virtually all cell lines were extremely sensitive to MMAE treatment (Supplemental Table 5).
Discussion ADCs represent a promising novel approach for the treatment of malignant lymphomas. Recently the anti-CD30 ADC brentuximab vedotin was approved for the treatment of relapsed CD30-positive HL and ALCL by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Previous work indicated that anti-CD22-MMAE and anti-CD79B-MMAE are toxic to preclinical ©
2015 Macmillan Publishers Limited. All rights reserved.
16
models derived from various malignant B-cell lymphomas (18-20). However, these studies did not investigate whether different molecular subtypes or lymphomas with specific, somatically acquired mutations respond preferentially to these agents. In the present study we demonstrated that the viability of the vast majority of ABC and GCB DLBCL cell lines was significantly impaired following anti-CD22-MMAE and antiCD79B-MMAE treatment. This was validated in two clinical studies, as both ABC and GCB DLBCL patients responded to these agents when treated for their relapsed or refractory disease: seven out of 12 (58%) ABC and seven out of 16 (44%) GCB DLBCL patients responded to treatment with an ADC. However, one has to be very cautious in drawing definitive conclusions from our phase-I studies given the small patient numbers. Nevertheless, the observation of responses to single agent antiCD22-MMAE and anti-CD79B-MMAE suggests that both DLBCL subtypes could be responsive to these agents. Our data are encouraging, especially as relapsed and primary refractory DLBCL patients typically experience dismal outcome (13). Similarly, CD79B mutations that predominantly occur in ABC DLBCL patients that are associated with inferior survival, did not impair response to anti-CD79B-MMAE in our in vitro models. This is interesting, as previous data in mouse B-cells suggested that mutations in the CD79B ITAM tyrosine residues elevate surface BCR expression by inhibiting receptor internalization (21). In our analyses, the internalization rate of the anti-CD79B antibody was similar in CD79B mutated TMD8 cells compared to two CD79B wild-type cell lines. These results were underscored as the CD79B ITAM mutations did not decrease the efficacy of anti-CD79B-MMAE in HBL1 and TMD8 cells. However, results of future studies in patients with mutated CD79B treated with anti-CD79B-MMAE need to be awaited to confirm these data in a clinical setting. The finding that anti-CD22 and anti-CD79B ADCs can successfully be utilized therapeutically in both ABC and GCB DLBCL patients is clinically relevant. In contrast ©
2015 Macmillan Publishers Limited. All rights reserved.
17
to anti-CD22 and anti-CD79B ADCs, various novel compounds such as the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib have been implicated to be preferentially active in ABC DLBCL patients (6). Similarly, the immunomodulatory drug lenalidomide had activity predominantly in relapsed or refractory ABC DLBCL patients in a recent phase-II trial (7). Thus, for the optimal use of these agents an accurate molecular diagnosis and assignment into cell of origin subgroups presumably will become a prerequisite. However, despite recent improvements using novel techniques such as the NanoString technology, the classification into ABC and GCB DLBCL is still not routinely done in clinical practice (35, 36). Thus, having agents such as anti-CD22-MMAE and anti-CD79B-MMAE that are equally effective in the treatment of both ABC and GCB DLBCL patients and for which molecular classification is not absolutely required might be advantageous. Interestingly, the anti-CD22 and anti-CD79B antibodies alone did not induce any toxicity in the investigated DLBCL cell lines. These data indicate that the toxic effects of the anti-CD22 and anti-CD79B ADCs are caused solely by MMAE. However, sensitivity to MMAE did not correlate with sensitivity to anti-CD22-MMAE or anti-CD79B-MMAE suggesting that additional features or the combination of different factors determine sensitivity to these agents. Potentially both target gene expression as well as sensitivity to MMAE are of major importance in dictating response to antiCD22 and anti-CD79B ADCs. We detected a significant correlation between sensitivity to anti-CD22-MMAE and anti-CD79B-MMAE and CD22 and CD79B surface expression determined by flow cytometry in DLBCL cell lines. However, anti-lymphoma responses of ADC treatment were observed in DLBCL cell lines with expression levels across the entire dynamic range of surface expression. Additionally, in our panel of cell lines CD79B expression determined by immunohistochemistry and response to anti-CD79B©
2015 Macmillan Publishers Limited. All rights reserved.
18
MMAE did not correlate, whereas for CD22 only a weak correlation between expression and response to anti-CD22-MMAE was detectable. Even more importantly CD22 and CD79B expression determined by immunohistochemistry did not correlate with response to anti-CD22-MMAE and anti-CD79B-MMAE in primary DLBCL patients indicating that the target expression levels detected by immunohistochemistry may not reliably predict patient response upfront. Even patients with no detectable levels of CD22 by immunohistochemistry responded. A similar phenomenon has previously been observed in patients with various CD30positive lymphoma subtypes treated with brentuximab vedotin (37). In these patients no correlation was observable between CD30 expression and response (37). Our data suggest that the expression levels required for activity of CD22 ADC may be below the level of detection of the immunohistochemistry assay used in our studies. Thus, our collective data indicate that patients with DLBCL should not be excluded from therapy with these agents based on low CD22 or CD79B expression by immunohistochemistry. In summary, anti-CD22-MMAE and anti-CD79B-MMAE represent active agents for the therapy of different DLBCL subtypes. Despite the small number of treated patients, we detected responses in both ABC and GCB DLBCL patients recapitulating the activity seen in vitro with representative cell lines.
Acknowledgements
This work was supported by research grants to G.L. from Genentech and the Deutsche Krebshilfe. In addition, this work was supported by the Deutsche Forschungsgemeinschaft, DFG EXC 1003 Cells in Motion - Cluster of Excellence,
©
2015 Macmillan Publishers Limited. All rights reserved.
19
Münster, Germany, as well as by a Doctoral Scholarship to M.G. from the PhilippsUniversity Marburg.
©
2015 Macmillan Publishers Limited. All rights reserved.
20
Disclosure of Conflicts of Interest B.Z., H.K., R.M., A.C., T.S., Y.W.C., A.G.P., and K.E.M. are employees of Genentech. M.C.P.W. is an employee of Seattle Genetics. G.L. received research funding from Genentech.
Supplementary information is available at Leukemia´s website.
©
2015 Macmillan Publishers Limited. All rights reserved.
21
References 1.
Nogai H, Dorken B, Lenz G. Pathogenesis of Non-Hodgkin's Lymphoma. J Clin Oncol 2011 May 10; 29(14): 1803-1811.
2.
Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403: 503-511.
3.
Wright G, Tan B, Rosenwald A, Hurt EH, Wiestner A, Staudt LM. A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A 2003 Aug 19; 100(17): 99919996.
4.
Shipp MA, Ross KN, Tamayo P, Weng AP, Kutok JL, Aguiar RCT, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nature Medicine 2002; 8: 68-74.
5.
Monti S, Savage KJ, Kutok JL, Feuerhake F, Kurtin P, Mihm M, et al. Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood 2005 Mar 1; 105(5): 1851-1861.
6.
Wilson W, Gerecitano J, Goy A, de Vos S, Kenkre V, Barr P, et al. The Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), Has Preferential Activity in the ABC Subtype of Relapsed/Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): Interim Results of a Multicenter, Open-Label, Phase 2 Study Blood 2012; Abstract 623.
7.
Hernandez-Ilizaliturri FJ, Deeb G, Zinzani PL, Pileri SA, Malik F, Macon WR, et al. Higher response to lenalidomide in relapsed/refractory diffuse large B-
©
2015 Macmillan Publishers Limited. All rights reserved.
22
cell lymphoma in nongerminal center B-cell-like than in germinal center B-celllike phenotype. Cancer 2011 Nov 15; 117(22): 5058-5066. 8.
Dunleavy K, Pittaluga S, Czuczman MS, Dave SS, Wright G, Grant N, et al. Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma. Blood 2009 Jun 11; 113(24): 60696076.
9.
Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 2008 Nov 27; 359(22): 2313-2323.
10.
Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002; 346(4): 235242.
11.
Pfreundschuh M, Schubert J, Ziepert M, Schmits R, Mohren M, Lengfelder E, et al. Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60). Lancet Oncol 2008 Feb; 9(2): 105-116.
12.
Habermann TM, Weller EA, Morrison VA, Gascoyne RD, Cassileth PA, Cohn JB, et al. Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J Clin Oncol 2006 Jul 1; 24(19): 3121-3127.
13.
Gisselbrecht C, Glass B, Mounier N, Singh Gill D, Linch DC, Trneny M, et al. Salvage regimens with autologous transplantation for relapsed large B-cell lymphoma in the rituximab era. J Clin Oncol 2010 Sep 20; 28(27): 4184-4190.
14.
Leslie LA, Younes A. Antibody-drug conjugates in hematologic malignancies. Am Soc Clin Oncol Educ Book 2013: 108-113. ©
2015 Macmillan Publishers Limited. All rights reserved.
23
15.
Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 2004 Oct 15; 10(20): 7063-7070.
16.
Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med 2010 Nov 4; 363(19): 1812-1821.
17.
Pro B, Advani R, Brice P, Bartlett NL, Rosenblatt JD, Illidge T, et al. Brentuximab vedotin (SGN-35) in patients with relapsed or refractory systemic anaplastic large-cell lymphoma: results of a phase II study. J Clin Oncol 2012 Jun 20; 30(18): 2190-2196.
18.
Polson AG, Yu SF, Elkins K, Zheng B, Clark S, Ingle GS, et al. Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma. Blood 2007 Jul 15; 110(2): 616-623.
19.
Dornan D, Bennett F, Chen Y, Dennis M, Eaton D, Elkins K, et al. Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 2009 Sep 24; 114(13): 27212729.
20.
Polson AG, Williams M, Gray AM, Fuji RN, Poon KA, McBride J, et al. AntiCD22-MCC-DM1: an antibody-drug conjugate with a stable linker for the treatment of non-Hodgkin's lymphoma. Leukemia 2010 Sep; 24(9): 15661573.
21.
Gazumyan A, Reichlin A, Nussenzweig MC. Ig beta tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization. J Exp Med 2006 Jul 10; 203(7): 1785-1794.
©
2015 Macmillan Publishers Limited. All rights reserved.
24
22.
Li D, Poon KA, Yu SF, Dere R, Go M, Lau J, et al. DCDT2980S, an anti-CD22monomethyl auristatin E antibody-drug conjugate, is a potential treatment for non-Hodgkin lymphoma. Mol Cancer Ther 2013 Jul; 12(7): 1255-1265.
23.
Advani R, Chen AI, Lebovic D, Brunvand MW, Goy A, Chang JE, et al. Final results of a phase I study of the anti-CD22 antibody-drug conjugate (ADC) DCDT2980S with or without rituximab in patients (Pts) with relapsed or refractory (R/R) B-cell Non-Hodgkin's Lymphoma (NHL). Blood 2013: Abstract 4399.
24.
Palanca-Wessels MC, Salles GA, Czuczman MS, Assouline SE, Flinn IW, Sehn LH, et al. Final results of a phase I study of the anti-CD79b antibody drug conjugate DCDS4501A in relapsed or refractory (R/R) B-cell NonHodgkin's Lymphoma (NHL). Blood 2013: Abstract 4400.
25.
Pfeifer M, Grau M, Lenze D, Wenzel SS, Wolf A, Wollert-Wulf B, et al. PTEN loss defines a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A 2013 Jul 9.
26.
Nogai H, Wenzel SS, Hailfinger S, Grau M, Kaergel E, Seitz V, et al. IkappaBzeta controls the constitutive NF-kappaB target gene network and survival of ABC DLBCL. Blood 2013 Sep 26; 122(13): 2242-2250.
27.
Zheng B, Fuji RN, Elkins K, Yu SF, Fuh FK, Chuh J, et al. In vivo effects of targeting CD79b with antibodies and antibody-drug conjugates. Mol Cancer Ther 2009 Oct; 8(10): 2937-2946.
28.
Johnston SR, Saccani-Jotti G, Smith IE, Salter J, Newby J, Coppen M, et al. Changes in estrogen receptor, progesterone receptor, and pS2 expression in tamoxifen-resistant human breast cancer. Cancer Res 1995 Aug 1; 55(15): 3331-3338.
©
2015 Macmillan Publishers Limited. All rights reserved.
25
29.
Vallejo-Gracia A, Bielanska J, Hernandez-Losa J, Castellvi J, Ruiz-Marcellan MC, Ramon y Cajal S, et al. Emerging role for the voltage-dependent K+ channel Kv1.5 in B-lymphocyte physiology: expression associated with human lymphoma malignancy. J Leukoc Biol 2013 Oct; 94(4): 779-789.
30.
Wenzel SS, Grau M, Mavis C, Hailfinger S, Wolf A, Madle H, et al. MCL1 is deregulated in subgroups of diffuse large B-cell lymphoma. Leukemia 2013 Jun; 27(6): 1381-1390.
31.
Weilemann
A,
Grau
M,
Erdmann
T,
Merkel
O,
Sobhiafshar
U,
Anagnostopoulos I, et al. Essential role of IRF4 and MYC signaling for survival of anaplastic large cell lymphoma. Blood 2015 Jan 1; 125(1): 124-132. 32.
Davis RE, Ngo VN, Lenz G, Tolar P, Young RM, Romesser PB, et al. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 2010 Jan 7; 463(7277): 88-92.
33.
Green TM, Young KH, Visco C, Xu-Monette ZY, Orazi A, Go RS, et al. Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. J Clin Oncol 2012 Oct 1; 30(28): 3460-3467.
34.
Johnson NA, Slack GW, Savage KJ, Connors JM, Ben-Neriah S, Rogic S, et al. Concurrent Expression of MYC and BCL2 in Diffuse Large B-Cell Lymphoma Treated With Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone. J Clin Oncol 2012 Oct 1; 30(28): 3452-3459.
35.
Scott DW, Wright GW, Williams PM, Lih CJ, Walsh W, Jaffe ES, et al. Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue. Blood 2014 Feb 20; 123(8): 1214-1217. ©
2015 Macmillan Publishers Limited. All rights reserved.
26
36.
Masque-Soler N, Szczepanowski M, Kohler CW, Spang R, Klapper W. Molecular classification of mature aggressive B-cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens. Blood 2013 Sep 12; 122(11): 1985-1986.
37.
Bartlett NL, Sharman JP, Oki Y, Advani RH, Bello CM, Winter JN, et al. A Phase 2 Study Of Brentuximab Vedotin In Patients With Relapsed Or Refractory CD30-Positive Non-Hodgkin Lymphomas:
Interim Results In
Patients With DLBCL and Other B-Cell Lymphomas Blood 2013; Abstract 848.
©
2015 Macmillan Publishers Limited. All rights reserved.
27
Figure legends
Figure 1: CD22 and CD79B expression in DLBCL. A-D. Immunohistochemical staining of (A) a CD79B-positive DLBCL case with strong CD79B expression (H-score 300; case: DLBCL_63), (B) a CD79B-positive DLBCL case with weak CD79B expression (H-score = 120; case: DLBCL_69), (C) a CD22positive DLBCL case with strong CD22 expression (H-score = 300, case: DLBCL_41) and (D) a CD22-positive DLBCL case with weak expression (H-score = 130; case: DLBCL_32). Magnification: 20x (A) or 40x (C-D). E. CD22 and CD79B expression in primary DLBCL patient samples. CD22 and CD79B expression was determined by immunohistochemistry and reported as Hscore. The black line indicates the mean expression of the respective surface marker.
Figure 2: Anti-CD22-MMAE and anti-CD79B-MMAE are active agents for the treatment of DLBCL. A. Anti-CD22-MMAE displays potent in vitro activity across ABC and GCB DLBCL cell lines. The dashed red line indicates an IC50 value of 5 g/ml that was used as cut-off level to differentiate responding from non-responding cell lines. B. Anti-CD79B-MMAE displays potent in vitro activity across ABC and GCB DLBCL cell lines. The dashed red line indicates an IC50 value of 5 g/ml that was used as cut-off level to differentiate responding from non-responding cell lines. C. Determination of anti-CD79B antibody internalization rate in CD79B mutated (TMD8) and wild-type (WSU-DLCL2 and SUDHL-4) DLBCL cell lines. The vast majority of the anti-CD79B antibody is rapidly internalized in all three cell lines within the first hour of incubation. The internalization rate in TMD8 and WSU-DLCL2 cells are virtually identical and higher compared to the rate in SUDHL-4 cells. ©
2015 Macmillan Publishers Limited. All rights reserved.
28
D. Treatment with anti-CD22-MMAE induces a significantly higher rate of cell death compared with the anti-CD22-mAB in the DLBCL cell lines OCI-Ly7 and RL as measured by an increase of the sub-G1 fraction. Error bars depict the standard deviation. E. Treatment with anti-CD79B-MMAE induces a significantly higher rate of cell death compared with the anti-CD79B-mAB in the DLBCL cell lines OCI-Ly19 and RL as measured by an increase of the sub-G1 fraction. Error bars depict the standard deviation. *=
Cite this article as: M Pfeifer, B Zheng, T Erdmann, H Koeppen, R McCord, M Grau, A Staiger, A Chai, T Sandmann, H Madle, B Do¨rken, Y-W Chu, A I Chen, D Lebovic, G A Salles, M S Czuczman, M C Palanca-Wessels, O W Press, R Advani, F Morschhauser, B D Cheson, P Lenz, G Ott, A G Polson, K E Mundt, G Lenz, Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes, Leukemia accepted article preview 24 February 2015; doi: 10.1038/leu.2015.48. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 25 August 2014; revised 8 January 2015; accepted 13 February 2015; Accepted article preview online 24 February 2015
©
2015 Macmillan Publishers Limited. All rights reserved.
1
Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes
Matthias Pfeifer1, Bing Zheng2, Tabea Erdmann3,4, Hartmut Koeppen2, Ron McCord2, Michael Grau5, Annette Staiger6, Akiko Chai2, Thomas Sandmann2, Hannelore Madle3,4, Bernd Dörken1, Yu-Waye Chu2, Andy I. Chen7, Daniel Lebovic8, Gilles Andre Salles9, Myron S. Czuczman10, Maria Corinna Palanca-Wessels11,12, Oliver W. Press11, Ranjana Advani13, Franck Morschhauser14, Bruce D. Cheson15, Peter Lenz5, German Ott6, Andrew G. Polson2, Kirsten E. Mundt2,#, and Georg Lenz3,4,# 1
Department of Hematology, Oncology and Tumor Immunology, Charité Universitätsmedizin Berlin, Germany 2 Genentech, Inc., 1 DNA Way, South San Francisco, CA, USA 3 Translational Oncology, Department of Medicine A, Albert-Schweitzer-Campus 1, University Hospital Münster, Münster, Germany 4 Cluster of Excellence EXC 1003, Cells in Motion, 48149 Münster, Germany 5 Department of Physics, Philipps-University, Marburg, Germany 6 Department of Clinical Pathology, Robert-Bosch-Krankenhaus and Dr. Margarete FischerBosch-Institute of Clinical Pharmacology, Stuttgart, Germany 7 Oregon Health & Science University, Portland, OR, USA 8 University of Michigan, Ann Arbor, MI, USA 9 Hospices Civils de Lyon - Université de Lyon, Pierre-Bénite, France 10 Departments of Medicine and Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA 11 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 12 Seattle Genetics, Inc., Bothell, WA, USA 13 Stanford University Medical Center, Stanford University, Stanford, CA, USA 14 Hematology Department, EA 4481 GRIIOT, Centre Hospitalier Régional Universitaire de Lille, Lille, France 15 Lombardi Comprehensive Cancer Center, Georgetown University Hospital, Washington DC, USA #Corresponding authors
Corresponding authors: Georg Lenz, M.D. University Hospital Münster Translational Oncology Medical Department A Albert-Schweitzer-Campus 1 48149 Münster, Germany Phone: +49 251 83 56229 Fax: +49 251 83 52673 e-mail: [email protected]
©
Kirsten E. Mundt, PhD Genentech Inc. DNA Way 1 South San Francisco CA 94080, USA Phone: +1 650 225 5462 Fax: +1 650 467 7571 e-mail: [email protected]
2015 Macmillan Publishers Limited. All rights reserved.
2
Disclosure of Conflicts of Interest B.Z., H.K., R.M., A.C., T.S., Y.W.C., A.G.P., and K.E.M. are employees of Genentech. M.C.P.W. is an employee of Seattle Genetics. G.L. received research funding from Genentech.
©
2015 Macmillan Publishers Limited. All rights reserved.
3
Abstract Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubuledisrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment due to low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.
©
2015 Macmillan Publishers Limited. All rights reserved.
4
Introduction Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category (1). This heterogeneity can partially be explained by the existence of different molecular subtypes identified by gene expression profiling (2-5). Applying the cell of origin classification, at least two major molecular subtypes can be distinguished. The germinal center B-cell-like (GCB) DLBCLs are derived from germinal center B-cells, whereas activated B-cell-like (ABC) DLBCLs originate from activated B-cells (2, 3). GCB and ABC DLBCLs depend on different oncogenic pathways that are frequently activated by somatic mutations (1). Importantly, the existence of molecular subtypes, as well as specific mutations appear predictive of response to pathway inhibitors such as ibrutinib, immunomodulatory agents such as lenalidomide or proteasome inhibitors (6-8). Thus, the assignment of DLBCL patients into molecular subtypes is becoming increasingly important to ensure that patients are selected for the most efficacious treatment regimens. Finally, ABC and GCB DLBCLs also show significant differences in outcome when treated with immunochemotherapy (9). The introduction of the anti-CD20 monoclonal antibody (mAB) rituximab into the therapy of DLBCL has significantly improved prognosis (1012). Nevertheless, a substantial number of patients relapse after first-line therapy. These relapsed or refractory DLBCL patients are characterized by dismal outcome (13). A novel therapeutic approach is the use of antibody drug conjugates (ADCs) in which cytotoxic drugs are attached to antibodies that are directed against antigens expressed on tumor cells (14). Recently this strategy has been shown to be very effective in the treatment of malignant lymphoma patients. The anti-CD30 ADC brentuximab vedotin achieved high response rates in patients with relapsed or
©
2015 Macmillan Publishers Limited. All rights reserved.
5
refractory CD30-positive Hodgkin’s lymphoma (HL) and anaplastic large-cell lymphoma (ALCL) (15-17). CD22 and CD79B are physiologically expressed in the vast majority of B-cells and therefore represent promising targets for ADCs. Preclinical data for an antiCD79B and an anti-CD22 antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) indicated efficacy in B-cell lymphomas including Burkitt lymphoma, mantle cell lymphoma, follicular lymphoma and DLBCL (18-20). However, these studies did not investigate whether ABC or GCB DLBCL or lymphomas characterized by specific mutations respond differentially to these agents. Of particular interest in this respect are mutations affecting CD79B that have been reported to impact B-cell receptor internalization in mouse B-cells (21). To this end, we analyzed the efficacy of anti-CD22-MMAE and anti-CD79B-MMAE in a large panel of cell lines derived from DLBCL patients of both molecular subtypes including cell lines characterized by CD79B mutations. To validate our in vitro findings, we investigated responses observed in DLBCL patients treated in two recent phase-I studies.
Material and Methods
Antibody drug conjugates The following ADCs were used in the present study. As previously described, pinatuzumab vedotin is an anti-CD22-MMAE ADC (DCDT2980S), whereas polatuzumab vedotin (DCDS4501A) represents an anti-CD79B-MMAE ADC (18, 22). Pinatuzumab vedotin and polatuzumab vedotin are the drug candidates used in two phase-I clinical trials in relapsed and refractory lymphoma patients (23, 24).
©
2015 Macmillan Publishers Limited. All rights reserved.
6
Cell lines Human DLBCL, ALCL and multiple myeloma (MM) cell lines were cultured as previously described in RPMI (Life Technologies, Carlsbad, CA, USA) with 10% FCS (Sigma-Aldrich, St. Louis, MO, USA), except for OCI-Ly1, OCI-Ly2, OCI-Ly3, OCILy4, OCI-Ly7, OCI-Ly10, OCI-Ly19, and TMD8 that were cultured in Isocove’s modified Dulbecco medium (Life Technologies) supplemented with 10% FCS (25, 26).
Viability assay, sub-G1 and cell cycle analysis Detailed protocols are available in the Supplemental Material and Methods.
Determination of CD22 and CD79B surface expression by flow cytometry Detailed protocols are available in the Supplemental Material and Methods.
CD79B antibody internalization assay Cells were pre-incubated with 10 g/ml of mouse anti-human CD79B antibody (clone SN8) (27) on ice for 30 minutes and washed once to remove excess unbound antibodies. Cells were subsequently incubated at 37°C allowing for internalization. To terminate internalization, cells were washed twice and stained with PE conjugated goat anti-mouse IgG antibody. Surface CD79B expression was measured by flow cytometry. Percent of internalization was calculated as % internalization (Tn) = (gMFI(T0)-gMFI(Tn))/gMFI(T0)*100 with gMFI = geometric mean fluorescence intensity.
Patient population
©
2015 Macmillan Publishers Limited. All rights reserved.
7
Both DCT4862g and DCS4968g were phase-I dose finding studies for anti-CD22MMAE or anti-CD79B-MMAE respectively, in patients diagnosed with relapsed or refractory B-cell lymphoma. These studies are registered at ClinicalTrials.gov under NCT01209130 and NCT01290549 respectively (23, 24). All patients gave informed written consent to participate in the clinical trials and to provide tissue for biologic studies. The respective institutional ethics review boards approved both trails. To correlate pre-clinical data with clinical results, we report in this manuscript CD22 and CD79B expression data as well as the correlation of expression and efficacy in DLBCL patients who were treated at doses of 1.8 mg/kg and above. The recommended phase-II dose was determined as 2.4 mg/kg for both CD22 ADC and CD79B ADC as single agents. The complete patient characteristics and overall results of these studies will be published in a separate manuscript.
CD22 and CD79B immunohistochemistry in primary DLBCL patient samples and DLBCL cell lines CD22 and CD79B immunohistochemical stainings were performed on the Ventana Discovery XT platform (Ventana, Tucson, AZ, USA) using the Ventana CC1 standard digestion pretreatment for antigen retrieval. The anti-CD22-mAB SP104 (Ventana) was used according to the manufacturer’s instructions. The anti-CD79B-mAB AT107-2 (0.25 g/ml; AbD Serotec, Oxford, UK) was incubated for 60 minutes at 37oC. Human tonsils were used as positive controls. The staining results are reported as Hscore as previously described that includes both numbers of positive tumor cells as well as the staining intensities (28, 29). The H-score was calculated for staining of tumor cells using the following formula: H-Score = (% at 0) * 0 + (% at 1+) * 1 + (% at 2+) * 2 + (% at 3+) * 3. Thus, this score produces a continuous variable that ranges from 0 to 300 (28, 29). A modified H©
2015 Macmillan Publishers Limited. All rights reserved.
8
score was used in the cell lines that displayed a rather uniform intensity of protein expression in a given case: % cells positive * predominant expression intensity (03).
Determination of molecular DLBCL subtypes Classification into molecular DLBCL subtypes was performed analogously to Wright et al. (3). The detailed algorithm is described in Supplemental Material and Methods and the data are summarized in Supplemental Figure 1.
Determination of IC50 concentrations and correlation analyses The IC50 concentrations were calculated as described in detail in the Supplemental Material and Methods.
Mutation analysis of CD79B in primary DLBCL patient samples Detailed protocols are available in the Supplemental Material and Methods. The primer sequences are summarized in Supplemental Table 1.
Western blotting Western blotting was performed as previously described (30, 31).
Results
CD22 and CD79B expression in DLBCL To assess the expression pattern of the targets of anti-CD22-MMAE and anti-CD79BMMAE, we measured CD22 and CD79B cell surface expression of DLBCL derived
©
2015 Macmillan Publishers Limited. All rights reserved.
9
cell lines by flow cytometry. Cell lines derived from MM and ALCL that do not express either CD22 or CD79B were used as negative controls (Supplemental Figure 2A and 2B). As expected, virtually all DLBCL cell lines displayed high CD22 and CD79B surface expression compared to MM (p=2.2x10-7 for CD22 and p=8.6x10-6 for CD79B) and ALCL cell lines (p=2.2x10-7 for CD22 and p=1.3x10-5 for CD79B) albeit in a dynamic range (Supplemental Figure 2A and 2B and Supplemental Table 2). To analyze CD22 and CD79B cell surface expression in primary DLBCL, we performed immunohistochemical staining on 32 patient samples for CD22 and on 28 patient samples for CD79B obtained from patients treated in two recently completed phase-I trials investigating the efficacy of anti-CD22-MMAE and anti-CD79B-MMAE, respectively (NCT01209130 and NCT01290549). All specimens expressed CD79B, consistent with its role as a signal transducing subunit of the essential B-cell receptor (BCR; median H-score 250) (Figure 1A, 1B, and 1E and Supplemental Table 3). In contrast, the expression levels of CD22 determined by immunohistochemistry were more variable and unexpectedly low in several specimens (median H-score 90; Figure 1C, 1D, and 1E and Supplemental Table 4). These results are surprising, especially as previous assessment of CD22 surface expression in primary DLBCL samples using flow cytometry indicated CD22 expression on virtually all cases (20).
Activity of anti-CD22-MMAE and anti-CD79B-MMAE in different subtypes of DLBCL in vitro and in vivo Anti-CD22 and anti-CD79B ADCs have shown promising activity in preclinical models of various subtypes of malignant lymphoma (18). To specifically investigate the effects of anti-CD22-MMAE and anti-CD79B-MMAE in different molecular DLBCL subtypes, we assessed cell viability following treatment with these agents in a large panel of 27 DLBCL cell lines, previously assigned to either ABC or GCB DLBCL (7 ©
2015 Macmillan Publishers Limited. All rights reserved.
10
ABC DLBCL and 20 GCB DLBCL cell lines). We analyzed dose-dependent sensitivity of every cell line in the range of 0-50 µg/ml ADC and calculated the corresponding IC50. To differentiate responding from non-responding cell lines, we employed a cutoff value of IC50 ≤ 5 µg/ml for responders, based on the clinical exposure at trough drug levels being above 5 µg/ml for the recommended phase-II dose of 2.4 mg/kg for DLBCL (Figure 2A and 2B and Supplemental Table 5). Using this cut-off, we observed a similar pattern of responsiveness of DLBCL cell lines to both ADCs. 22 out of 27 (81%) cell lines were sensitive to anti-CD22-MMAE and 22 out of 27 (81%) cell lines showed toxicity following anti-CD79B-MMAE treatment. The vast majority of DLBCL cell lines responded to both anti-CD22-MMAE and anti-CD79B-MMAE respectively (Figure 2A and 2B and Supplemental Table 5). In contrast, a minority of cell lines was sensitive to either anti-CD22-MMAE or anti-CD79B-MMAE treatment. Only two cell lines (SUDHL-2 and WSU-NHL) were resistant to both drugs. For both agents, we could not detect a preferential response in either ABC or GCB DLBCL cell lines (p=0.10 for anti-CD22-MMAE and p=0.47 for anti-CD79B-MMAE; one-tailed two sample t-tests). These results demonstrate that anti-CD22-MMAE and anti-CD79BMMAE are active in DLBCL models irrespective of their molecular subtype. To validate if indeed both primary ABC and GCB DLBCL patients respond equally to anti-CD22-MMAE and anti-CD79-MMAE, we determined the molecular DLBCL subtype in 52 out of 87 (60%) patients treated in the recently completed phase-I trials using these agents. 26 patients (50%) were classified as GCB, 22 (42%) as ABC, and 4 (8%) were unclassifiable DLBCL (Supplemental Figure 1). Overall the response evaluation of 28 DLBCL patients treated with anti-CD22-MMAE and 29 patients treated with anti-CD79B-MMAE at doses of ≥ 1.8 mg/kg was available (Table 1). In these 57 patients, a molecular DLBCL subtypes classification was available in 29 patients (14 patients treated with anti-CD22-MMAE and 15 ©
2015 Macmillan Publishers Limited. All rights reserved.
11
patients treated with anti-CD79B-MMAE; Table 2). For the remaining patients we could not obtain sufficient material to perform a molecular subtype classification. The in vivo patient data confirmed our in vitro observations that both antiCD22-MMAE and anti-CD79B-MMAE are active agents in DLBCL. Treatment with anti-CD22-MMAE induced either a complete (CR) or a partial remission (PR) in 12 out of 28 (43%) patients at dose levels ≥ 1.8 mg/kg (Table 1). As suggested from our cell line data we observed activity in both ABC and GCB DLBCL patients. Three out of seven ABC (2 CRs) and two out of seven GCB DLBCL patients (2 CRs) responded to anti-CD22-MMAE (dose level ≥ 1.8 mg/kg; Table 2). Anti-CD79B-MMAE was similarly effective in DLBCL patients of both molecular subtypes. Responses were observed in 16 out of 29 (55%) patients. Four out of five ABC DLBCL patients responded (1 CR), whereas five out of nine GCB DLBCL obtained a PR (Table 2). Despite the small numbers of evaluable patients, these data suggest that anti-CD22MMAE and anti-CD79B-MMAE are active in both ABC and GCB DLBCL patients (Table 2).
Activity of anti-CD22-MMAE and anti-CD79B-MMAE in CD79B mutated DLBCL Roughly 20% of primary ABC DLBCL patient samples harbor mutations affecting CD79B (32). Previous work suggested that these mutations might negatively affect BCR internalization in mouse models (21). Hence, we examined whether CD79B mutations would interfere with anti-CD79B-MMAE activity, as ADC internalization is crucial for its mechanism of action. To this end, we investigated the kinetics of internalization of the naked anti-CD79B antibody in the CD79B (Y196H) mutated cell line TMD8 compared to those in the CD79B wild-type cell lines SUDHL-4 and WSUDLCL2 (32). The vast majority of the anti-CD79B antibody was rapidly internalized in all three cell lines within the first hour of incubation. The internalization rate in TMD8 ©
2015 Macmillan Publishers Limited. All rights reserved.
12
and WSU-DLCL2 cells were virtually identical and higher compared to the rate in SUDHL-4 cells (Figure 2C) suggesting that the internalization rate in human CD79B mutated and wild-type lymphoma cells is comparable. This was further underscored as treatment of the two CD79B mutated cell lines HBL1 and TMD8 (32) with antiCD79B-MMAE induced toxicity in both cell lines. Both HBL1 and TMD8 were highly sensitive to anti-CD79B-MMAE treatment with IC50s of 0.02
g/ml and 0.16
g/ml,
respectively (Supplemental Table 5). This suggests that CD79B mutations do not seem to impair the efficacy of anti-CD79B-MMAE in vitro. Next, we analyzed if mutations in CD79B might impact response to antiCD79B-MMAE in primary patients. To evaluate this, we determined the CD79B mutation status in 35 primary DLBCL samples from patients treated in both trials. We identified two patient samples harboring either a CD79B Y196H or a CD79B Y196C mutation, respectively. In contrast, 33 patients had two wild-type alleles for CD79B. As the two patients with CD79B mutations were treated with anti-CD22-MMAE (both achieved a CR), we cannot evaluate the in vivo efficacy of anti-CD79B-MMAE treatment. However, our in vitro data suggest that anti-CD79B-MMAE should be active in both patients with wild-type or mutated CD79B.
Anti-CD22-MMAE and anti-CD79B-MMAE induce cell death in DLBCL To obtain a better understanding of the growth inhibitory effects of anti-CD22-MMAE and anti-CD79B-MMAE treatment observed in DLBCL cell lines, we investigated whether cell death or cell cycle arrest were induced. Treatment of the GCB DLBCL cell lines OCI-Ly7 and RL with anti-CD22-MMAE resulted in a significant increase in the sub-G1 fraction after 72 hours, indicating an increase in cell death (Figure 2D and Supplemental Figure 3A). Similarly, anti-CD79B-MMAE treatment induced cell death as measured by a sub-G1 peak increase in OCI-Ly19 and RL cells (Figure 2E and ©
2015 Macmillan Publishers Limited. All rights reserved.
13
Supplemental Figure 3B). We also observed an increase of the G2/M peak in RL and OCI-Ly7 cells treated with anti-CD22-MMAE and in RL cells treated with anti-CD79BMMAE (Supplemental Figure 3A and 3B), indicative of a mitotic arrest, consistent with the mechanism of action of a tubulin targeted agent. To assess the contribution of the respective antibody moieties to the ADC’s mechanism of action, we measured viability of DLBCL cell lines treated with the unconjugated anti-CD22 and anti-CD79B antibodies. The unconjugated antibodies did not induce any changes in cell viability or cell cycle suggesting that the toxic effect of these ADCs is mediated solely by MMAE (Figure 2D and 2E and Supplemental Figure 3A and 3B). To confirm the effect of MMAE on cell viability, we treated our panel of cell lines with MMAE in the absence of the antibody. Indeed, MMAE significantly reduced viability in all investigated cell lines (Supplemental Table 5), demonstrating the sensitivity of these cell lines to the payload drug MMAE. Co-expression of MYC and BCL2 is associated with adverse survival in DLBCL patients and expression of BCL2 family members such as MCL1 has been shown to affect response to conventional chemotherapy in DLBCL (30, 33, 34). Therefore, we analyzed if expression of either MYC and/or the BCL2 family members BCL2, BCL-XL or MCL1 is associated with either response or resistance to antiCD22 and anti-CD79B-MMAE. To this end, we determined the expression pattern of these proteins by Western blotting in cell lines that are characterized by either response to both agents or by response to only one agent or that are characterized by resistance to both drugs (Figure 3A). We detected BCL2 and MCL1 expression in only a fraction of the investigated cell lines, whereas BCL-XL and MYC were expressed in virtually all lines albeit at different levels (Figure 3A). We could not detect a preferential expression pattern in either one of the response groups (Figure 3A). ©
2015 Macmillan Publishers Limited. All rights reserved.
14
CD22 and CD79B surface expression and sensitivity to MMAE as predictors of response in DLBCL Next, we investigated whether the degree of cell surface expression of CD22 and CD79B correlates with response to anti-CD22-MMAE and anti-CD79B-MMAE in DLBCL cell lines. Indeed, we detected a significant correlation between antigen surface expression and response to both anti-CD22-MMAE (r=-0.75; p=6x10-6; Figure 3B) and anti-CD79B-MMAE (r=-0.68, p=1x10-4; Figure 3C). However, several cell lines responded to treatment with anti-CD22-MMAE and anti-CD79B-MMAE despite low surface target antigen expression (e.g. OCI-Ly1 and NUDUL-1 for antiCD22-MMAE treatment and OCI-Ly10, OCI-Ly3, OCI-Ly2, OCI-Ly4, and WSUDLCL2 for anti-CD79B-MMAE treatment; Supplemental Table 2 and 5). These data suggest that using a predefined cut-off level of surface target expression might not accurately predict which patients may respond to anti-CD22-MMAE or anti-CD79BMMAE treatment. In order to explore this possibility, we evaluated whether response to treatment in primary DLBCL patients correlates with CD22 and CD79B expression levels by immunohistochemistry. Response evaluation and immunohistochemistry was available for 18 anti-CD22-MMAE treated and 18 anti-CD79B-MMAE treated patients (dose level ≥1.8 mg/kg). The response to both ADCs was not correlated to the expression of the targets CD22 and CD79B respectively (p=0.2 for CD22 and p=0.8 for CD79B; chi-square test; Figure 3D and 3E). A low H-score did not predict resistance to these agents. Thus, the in vivo data supported our hypothesis that using a predefined CD22 or CD79B expression cut-off level for treatment selection might exclude patients who may still benefit from effective ADC therapies.
©
2015 Macmillan Publishers Limited. All rights reserved.
15
To investigate if the discrepant results in cell lines and primary patient samples regarding the correlation of CD22 and CD79B expression and response to either antiCD22-MMAE or anti-CD79B-MMAE is related to the applied technique (flow cytometry vs. immunohistochemistry), we determined CD22 and CD79B expression in our panel of cell lines additionally by immunohistochemistry (Supplemental Table 6). Subsequently, we correlated response to treatment in the cell lines with the CD22 and CD79B H-score. For CD79B we could not detect a significant correlation between expression and response to anti-CD79B-MMAE treatment any more (r=0.21; p=0.3), whereas for CD22 only a weak correlation between CD22 expression and response to anti-CD22-MMAE was detectable (r=0.53; p=4x10-3; Supplemental Table 5 and 6). These data suggest that immunohistochemistry is less sensitive in accurately determining CD22 and CD79B expression levels compared to flow cytometry. Finally, we investigated whether sensitivity to MMAE correlated with sensitivity to either anti-CD22-MMAE or anti-CD79B-MMAE measured by the corresponding IC50 values in our panel of DLBCL cell lines. For anti-CD22-MMAE (r=0.25; p=0.2; Figure 3F) and for anti-CD79B-MMAE (r=0.30; p=0.1; Figure 3G) we did not detect a significant correlation, most likely due to the fact that virtually all cell lines were extremely sensitive to MMAE treatment (Supplemental Table 5).
Discussion ADCs represent a promising novel approach for the treatment of malignant lymphomas. Recently the anti-CD30 ADC brentuximab vedotin was approved for the treatment of relapsed CD30-positive HL and ALCL by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Previous work indicated that anti-CD22-MMAE and anti-CD79B-MMAE are toxic to preclinical ©
2015 Macmillan Publishers Limited. All rights reserved.
16
models derived from various malignant B-cell lymphomas (18-20). However, these studies did not investigate whether different molecular subtypes or lymphomas with specific, somatically acquired mutations respond preferentially to these agents. In the present study we demonstrated that the viability of the vast majority of ABC and GCB DLBCL cell lines was significantly impaired following anti-CD22-MMAE and antiCD79B-MMAE treatment. This was validated in two clinical studies, as both ABC and GCB DLBCL patients responded to these agents when treated for their relapsed or refractory disease: seven out of 12 (58%) ABC and seven out of 16 (44%) GCB DLBCL patients responded to treatment with an ADC. However, one has to be very cautious in drawing definitive conclusions from our phase-I studies given the small patient numbers. Nevertheless, the observation of responses to single agent antiCD22-MMAE and anti-CD79B-MMAE suggests that both DLBCL subtypes could be responsive to these agents. Our data are encouraging, especially as relapsed and primary refractory DLBCL patients typically experience dismal outcome (13). Similarly, CD79B mutations that predominantly occur in ABC DLBCL patients that are associated with inferior survival, did not impair response to anti-CD79B-MMAE in our in vitro models. This is interesting, as previous data in mouse B-cells suggested that mutations in the CD79B ITAM tyrosine residues elevate surface BCR expression by inhibiting receptor internalization (21). In our analyses, the internalization rate of the anti-CD79B antibody was similar in CD79B mutated TMD8 cells compared to two CD79B wild-type cell lines. These results were underscored as the CD79B ITAM mutations did not decrease the efficacy of anti-CD79B-MMAE in HBL1 and TMD8 cells. However, results of future studies in patients with mutated CD79B treated with anti-CD79B-MMAE need to be awaited to confirm these data in a clinical setting. The finding that anti-CD22 and anti-CD79B ADCs can successfully be utilized therapeutically in both ABC and GCB DLBCL patients is clinically relevant. In contrast ©
2015 Macmillan Publishers Limited. All rights reserved.
17
to anti-CD22 and anti-CD79B ADCs, various novel compounds such as the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib have been implicated to be preferentially active in ABC DLBCL patients (6). Similarly, the immunomodulatory drug lenalidomide had activity predominantly in relapsed or refractory ABC DLBCL patients in a recent phase-II trial (7). Thus, for the optimal use of these agents an accurate molecular diagnosis and assignment into cell of origin subgroups presumably will become a prerequisite. However, despite recent improvements using novel techniques such as the NanoString technology, the classification into ABC and GCB DLBCL is still not routinely done in clinical practice (35, 36). Thus, having agents such as anti-CD22-MMAE and anti-CD79B-MMAE that are equally effective in the treatment of both ABC and GCB DLBCL patients and for which molecular classification is not absolutely required might be advantageous. Interestingly, the anti-CD22 and anti-CD79B antibodies alone did not induce any toxicity in the investigated DLBCL cell lines. These data indicate that the toxic effects of the anti-CD22 and anti-CD79B ADCs are caused solely by MMAE. However, sensitivity to MMAE did not correlate with sensitivity to anti-CD22-MMAE or anti-CD79B-MMAE suggesting that additional features or the combination of different factors determine sensitivity to these agents. Potentially both target gene expression as well as sensitivity to MMAE are of major importance in dictating response to antiCD22 and anti-CD79B ADCs. We detected a significant correlation between sensitivity to anti-CD22-MMAE and anti-CD79B-MMAE and CD22 and CD79B surface expression determined by flow cytometry in DLBCL cell lines. However, anti-lymphoma responses of ADC treatment were observed in DLBCL cell lines with expression levels across the entire dynamic range of surface expression. Additionally, in our panel of cell lines CD79B expression determined by immunohistochemistry and response to anti-CD79B©
2015 Macmillan Publishers Limited. All rights reserved.
18
MMAE did not correlate, whereas for CD22 only a weak correlation between expression and response to anti-CD22-MMAE was detectable. Even more importantly CD22 and CD79B expression determined by immunohistochemistry did not correlate with response to anti-CD22-MMAE and anti-CD79B-MMAE in primary DLBCL patients indicating that the target expression levels detected by immunohistochemistry may not reliably predict patient response upfront. Even patients with no detectable levels of CD22 by immunohistochemistry responded. A similar phenomenon has previously been observed in patients with various CD30positive lymphoma subtypes treated with brentuximab vedotin (37). In these patients no correlation was observable between CD30 expression and response (37). Our data suggest that the expression levels required for activity of CD22 ADC may be below the level of detection of the immunohistochemistry assay used in our studies. Thus, our collective data indicate that patients with DLBCL should not be excluded from therapy with these agents based on low CD22 or CD79B expression by immunohistochemistry. In summary, anti-CD22-MMAE and anti-CD79B-MMAE represent active agents for the therapy of different DLBCL subtypes. Despite the small number of treated patients, we detected responses in both ABC and GCB DLBCL patients recapitulating the activity seen in vitro with representative cell lines.
Acknowledgements
This work was supported by research grants to G.L. from Genentech and the Deutsche Krebshilfe. In addition, this work was supported by the Deutsche Forschungsgemeinschaft, DFG EXC 1003 Cells in Motion - Cluster of Excellence,
©
2015 Macmillan Publishers Limited. All rights reserved.
19
Münster, Germany, as well as by a Doctoral Scholarship to M.G. from the PhilippsUniversity Marburg.
©
2015 Macmillan Publishers Limited. All rights reserved.
20
Disclosure of Conflicts of Interest B.Z., H.K., R.M., A.C., T.S., Y.W.C., A.G.P., and K.E.M. are employees of Genentech. M.C.P.W. is an employee of Seattle Genetics. G.L. received research funding from Genentech.
Supplementary information is available at Leukemia´s website.
©
2015 Macmillan Publishers Limited. All rights reserved.
21
References 1.
Nogai H, Dorken B, Lenz G. Pathogenesis of Non-Hodgkin's Lymphoma. J Clin Oncol 2011 May 10; 29(14): 1803-1811.
2.
Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403: 503-511.
3.
Wright G, Tan B, Rosenwald A, Hurt EH, Wiestner A, Staudt LM. A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A 2003 Aug 19; 100(17): 99919996.
4.
Shipp MA, Ross KN, Tamayo P, Weng AP, Kutok JL, Aguiar RCT, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nature Medicine 2002; 8: 68-74.
5.
Monti S, Savage KJ, Kutok JL, Feuerhake F, Kurtin P, Mihm M, et al. Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood 2005 Mar 1; 105(5): 1851-1861.
6.
Wilson W, Gerecitano J, Goy A, de Vos S, Kenkre V, Barr P, et al. The Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), Has Preferential Activity in the ABC Subtype of Relapsed/Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): Interim Results of a Multicenter, Open-Label, Phase 2 Study Blood 2012; Abstract 623.
7.
Hernandez-Ilizaliturri FJ, Deeb G, Zinzani PL, Pileri SA, Malik F, Macon WR, et al. Higher response to lenalidomide in relapsed/refractory diffuse large B-
©
2015 Macmillan Publishers Limited. All rights reserved.
22
cell lymphoma in nongerminal center B-cell-like than in germinal center B-celllike phenotype. Cancer 2011 Nov 15; 117(22): 5058-5066. 8.
Dunleavy K, Pittaluga S, Czuczman MS, Dave SS, Wright G, Grant N, et al. Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma. Blood 2009 Jun 11; 113(24): 60696076.
9.
Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 2008 Nov 27; 359(22): 2313-2323.
10.
Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002; 346(4): 235242.
11.
Pfreundschuh M, Schubert J, Ziepert M, Schmits R, Mohren M, Lengfelder E, et al. Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60). Lancet Oncol 2008 Feb; 9(2): 105-116.
12.
Habermann TM, Weller EA, Morrison VA, Gascoyne RD, Cassileth PA, Cohn JB, et al. Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J Clin Oncol 2006 Jul 1; 24(19): 3121-3127.
13.
Gisselbrecht C, Glass B, Mounier N, Singh Gill D, Linch DC, Trneny M, et al. Salvage regimens with autologous transplantation for relapsed large B-cell lymphoma in the rituximab era. J Clin Oncol 2010 Sep 20; 28(27): 4184-4190.
14.
Leslie LA, Younes A. Antibody-drug conjugates in hematologic malignancies. Am Soc Clin Oncol Educ Book 2013: 108-113. ©
2015 Macmillan Publishers Limited. All rights reserved.
23
15.
Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 2004 Oct 15; 10(20): 7063-7070.
16.
Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med 2010 Nov 4; 363(19): 1812-1821.
17.
Pro B, Advani R, Brice P, Bartlett NL, Rosenblatt JD, Illidge T, et al. Brentuximab vedotin (SGN-35) in patients with relapsed or refractory systemic anaplastic large-cell lymphoma: results of a phase II study. J Clin Oncol 2012 Jun 20; 30(18): 2190-2196.
18.
Polson AG, Yu SF, Elkins K, Zheng B, Clark S, Ingle GS, et al. Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma. Blood 2007 Jul 15; 110(2): 616-623.
19.
Dornan D, Bennett F, Chen Y, Dennis M, Eaton D, Elkins K, et al. Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 2009 Sep 24; 114(13): 27212729.
20.
Polson AG, Williams M, Gray AM, Fuji RN, Poon KA, McBride J, et al. AntiCD22-MCC-DM1: an antibody-drug conjugate with a stable linker for the treatment of non-Hodgkin's lymphoma. Leukemia 2010 Sep; 24(9): 15661573.
21.
Gazumyan A, Reichlin A, Nussenzweig MC. Ig beta tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization. J Exp Med 2006 Jul 10; 203(7): 1785-1794.
©
2015 Macmillan Publishers Limited. All rights reserved.
24
22.
Li D, Poon KA, Yu SF, Dere R, Go M, Lau J, et al. DCDT2980S, an anti-CD22monomethyl auristatin E antibody-drug conjugate, is a potential treatment for non-Hodgkin lymphoma. Mol Cancer Ther 2013 Jul; 12(7): 1255-1265.
23.
Advani R, Chen AI, Lebovic D, Brunvand MW, Goy A, Chang JE, et al. Final results of a phase I study of the anti-CD22 antibody-drug conjugate (ADC) DCDT2980S with or without rituximab in patients (Pts) with relapsed or refractory (R/R) B-cell Non-Hodgkin's Lymphoma (NHL). Blood 2013: Abstract 4399.
24.
Palanca-Wessels MC, Salles GA, Czuczman MS, Assouline SE, Flinn IW, Sehn LH, et al. Final results of a phase I study of the anti-CD79b antibody drug conjugate DCDS4501A in relapsed or refractory (R/R) B-cell NonHodgkin's Lymphoma (NHL). Blood 2013: Abstract 4400.
25.
Pfeifer M, Grau M, Lenze D, Wenzel SS, Wolf A, Wollert-Wulf B, et al. PTEN loss defines a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A 2013 Jul 9.
26.
Nogai H, Wenzel SS, Hailfinger S, Grau M, Kaergel E, Seitz V, et al. IkappaBzeta controls the constitutive NF-kappaB target gene network and survival of ABC DLBCL. Blood 2013 Sep 26; 122(13): 2242-2250.
27.
Zheng B, Fuji RN, Elkins K, Yu SF, Fuh FK, Chuh J, et al. In vivo effects of targeting CD79b with antibodies and antibody-drug conjugates. Mol Cancer Ther 2009 Oct; 8(10): 2937-2946.
28.
Johnston SR, Saccani-Jotti G, Smith IE, Salter J, Newby J, Coppen M, et al. Changes in estrogen receptor, progesterone receptor, and pS2 expression in tamoxifen-resistant human breast cancer. Cancer Res 1995 Aug 1; 55(15): 3331-3338.
©
2015 Macmillan Publishers Limited. All rights reserved.
25
29.
Vallejo-Gracia A, Bielanska J, Hernandez-Losa J, Castellvi J, Ruiz-Marcellan MC, Ramon y Cajal S, et al. Emerging role for the voltage-dependent K+ channel Kv1.5 in B-lymphocyte physiology: expression associated with human lymphoma malignancy. J Leukoc Biol 2013 Oct; 94(4): 779-789.
30.
Wenzel SS, Grau M, Mavis C, Hailfinger S, Wolf A, Madle H, et al. MCL1 is deregulated in subgroups of diffuse large B-cell lymphoma. Leukemia 2013 Jun; 27(6): 1381-1390.
31.
Weilemann
A,
Grau
M,
Erdmann
T,
Merkel
O,
Sobhiafshar
U,
Anagnostopoulos I, et al. Essential role of IRF4 and MYC signaling for survival of anaplastic large cell lymphoma. Blood 2015 Jan 1; 125(1): 124-132. 32.
Davis RE, Ngo VN, Lenz G, Tolar P, Young RM, Romesser PB, et al. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 2010 Jan 7; 463(7277): 88-92.
33.
Green TM, Young KH, Visco C, Xu-Monette ZY, Orazi A, Go RS, et al. Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. J Clin Oncol 2012 Oct 1; 30(28): 3460-3467.
34.
Johnson NA, Slack GW, Savage KJ, Connors JM, Ben-Neriah S, Rogic S, et al. Concurrent Expression of MYC and BCL2 in Diffuse Large B-Cell Lymphoma Treated With Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone. J Clin Oncol 2012 Oct 1; 30(28): 3452-3459.
35.
Scott DW, Wright GW, Williams PM, Lih CJ, Walsh W, Jaffe ES, et al. Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue. Blood 2014 Feb 20; 123(8): 1214-1217. ©
2015 Macmillan Publishers Limited. All rights reserved.
26
36.
Masque-Soler N, Szczepanowski M, Kohler CW, Spang R, Klapper W. Molecular classification of mature aggressive B-cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens. Blood 2013 Sep 12; 122(11): 1985-1986.
37.
Bartlett NL, Sharman JP, Oki Y, Advani RH, Bello CM, Winter JN, et al. A Phase 2 Study Of Brentuximab Vedotin In Patients With Relapsed Or Refractory CD30-Positive Non-Hodgkin Lymphomas:
Interim Results In
Patients With DLBCL and Other B-Cell Lymphomas Blood 2013; Abstract 848.
©
2015 Macmillan Publishers Limited. All rights reserved.
27
Figure legends
Figure 1: CD22 and CD79B expression in DLBCL. A-D. Immunohistochemical staining of (A) a CD79B-positive DLBCL case with strong CD79B expression (H-score 300; case: DLBCL_63), (B) a CD79B-positive DLBCL case with weak CD79B expression (H-score = 120; case: DLBCL_69), (C) a CD22positive DLBCL case with strong CD22 expression (H-score = 300, case: DLBCL_41) and (D) a CD22-positive DLBCL case with weak expression (H-score = 130; case: DLBCL_32). Magnification: 20x (A) or 40x (C-D). E. CD22 and CD79B expression in primary DLBCL patient samples. CD22 and CD79B expression was determined by immunohistochemistry and reported as Hscore. The black line indicates the mean expression of the respective surface marker.
Figure 2: Anti-CD22-MMAE and anti-CD79B-MMAE are active agents for the treatment of DLBCL. A. Anti-CD22-MMAE displays potent in vitro activity across ABC and GCB DLBCL cell lines. The dashed red line indicates an IC50 value of 5 g/ml that was used as cut-off level to differentiate responding from non-responding cell lines. B. Anti-CD79B-MMAE displays potent in vitro activity across ABC and GCB DLBCL cell lines. The dashed red line indicates an IC50 value of 5 g/ml that was used as cut-off level to differentiate responding from non-responding cell lines. C. Determination of anti-CD79B antibody internalization rate in CD79B mutated (TMD8) and wild-type (WSU-DLCL2 and SUDHL-4) DLBCL cell lines. The vast majority of the anti-CD79B antibody is rapidly internalized in all three cell lines within the first hour of incubation. The internalization rate in TMD8 and WSU-DLCL2 cells are virtually identical and higher compared to the rate in SUDHL-4 cells. ©
2015 Macmillan Publishers Limited. All rights reserved.
28
D. Treatment with anti-CD22-MMAE induces a significantly higher rate of cell death compared with the anti-CD22-mAB in the DLBCL cell lines OCI-Ly7 and RL as measured by an increase of the sub-G1 fraction. Error bars depict the standard deviation. E. Treatment with anti-CD79B-MMAE induces a significantly higher rate of cell death compared with the anti-CD79B-mAB in the DLBCL cell lines OCI-Ly19 and RL as measured by an increase of the sub-G1 fraction. Error bars depict the standard deviation. *=
