Antibody therapy alone and in combination with targeted drugs in chronic lymphocytic leukemia

Author's Accepted Manuscript

Antibody Therapy Alone and in Combination with Targeted Drugs in Chronic Lymphocytic Leukemia Tadeusz Robak, Jerzy Z. Blonski, Pawel Robak

www.elsevier.de/endend

PII: DOI: Reference:

S0093-7754(16)00016-6 http://dx.doi.org/10.1053/j.seminoncol.2016.02.010 YSONC51914

To appear in:

Semin Oncol

Cite this article as: Tadeusz Robak, Jerzy Z. Blonski, Pawel Robak, Antibody Therapy Alone and in Combination with Targeted Drugs in Chronic Lymphocytic Leukemia, Semin Oncol, http://dx.doi.org/10.1053/j.seminoncol.2016.02.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antibody Therapy Alone and in Combination with Targeted Drugs in Chronic Lymphocytic Leukemia

Tadeusz Robak1, Jerzy Z Blonski1, Pawel Robak2

Departments of Hematology 1 and Experimental Hematology2, Medical University of Lodz, Lodz, Poland

Short title: Monoclonal Antibodies for CLL

Corresponding author: Prof. Tadeusz Robak, Department of Hematology Medical University of Lodz 93-510 Lodz Ciołkowskiego 2 Poland Email: [email protected] ABSTRACT The development of nonchemotherapeutic agents, including monoclonal antibodies (mAbs) and other targeted drugs, makes chemotherapy–free treatment an attractive option for chronic lymphocytic leukemia (CLL). The classical mAb, rituximab, has been authorised for use in both first-line and second-line therapy for CLL. New mAbs directed against CD20, ofatumumab and obinutuzumab (GA-101), have also been approved for the treatment of this disease. Recently, several new mAbs with potential benefits over the approved anti-CD20 antibodies have been developed for use in CLL. Anti-CD37, anti-CD19 and anti-CD40 mAbs

are in early clinical trials and show promise in treating CLL. In addition, the combination of mAbs with B-cell receptor signaling pathway inhibitors and immunomodulatory drugs makes the chemotherapy-free option a reality today. Combination of antibodies with targeted drugs like ibrutinib, idelalisib or lenalidomide are expected to replace chemotherapy-based combinations for treating CLL in the near future. However, phase 3 trials should confirm the benefit of these new treatment strategies and establish their exact place in the therapeutic armamentarium for CLL. Key words: ABT-199, alemtuzumab, CLL, ibrutinib, idelalisib, lenalidomide, lumiliximab, obinutuzumab, ofatumumab, otlertuzumab, rituximab INTRODUCTION Despite significant progress in chronic lymphocytic leukemia (CLL) with fludarabine or chlorambucil-based immunochemotherapy, novel drugs are needed. Particularly for patients with del 17p and patients refractory to initial treatment, current therapeutic options are limited. In recent years, anti-CD20 monoclonal antibodies (mAbs), rituximab, ofatumumab and obinutuzumab have been approved for use in CLL therapy (Fig. 1, Table 1) [1]. In addition, several new mAbs have been developed to provide potential benefits over currentlyapproved anti-CD20 antibodies, and are now in the process of making the transition to the clinic (Fig 1,Table 2) [1,2]. Chimeric, humanized, and fully-human mAbs increase the potency and improve the tolerance of this class of therapeutic agents. Their antileukemic activity can further be enhanced by posttranscriptional modifications of the constant region, as well as by selection of glycosylated or defucosylated isoforms. The most promising newer mAbs are directed against CD37, CD19 and CD22. In addition, new chemotherapy-free treatment options have been introduced within clinical trials with the hope of improving

responses, impacting survival, and ultimately curing CLL. The most promising are B-cell receptor (BCR) signaling pathway inhibitors, such as ibrutinib and idelalisib [3]. Moreover, immunomodulatory drugs (IMiDs), particularly lenalidomide, and BCL-2 inhibitors (ABT199) are being explored in this disease. SINGLE ANTI-CD20 MONOCLONAL ANTIBODIES IN CLL Rituximab is the first mAb to be used both in monotherapy and in combination with chemotherapy for the treatment of CLL. New generations of anti-CD20 mAbs have augmented antitumor activity by increasing complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), and increasing Fc-binding affinity for the low-affinity variants of the FcγRIIIa receptor on immune effector cells (Table 1) [4]. Rituximab Rituximab (Mabthera®, Rituxan®, F. Hoffmann–La Roche) is a chimeric, humanmouse, anti-CD20 monoclonal antibody containing murine light- and heavy-chain variableregion sequences and human constant-region sequences [1]. The drug induces CDC and ADCC in addition to exerting a direct antiproliferative effect. In 1997, rituximab was the first mAb approved specifically for the treatment of patients with relapsed or refractory CD20positive low-grade non-Hodgkin’s lymphoma (NHL). Subsequently, the U.S. Food and Drug Administration (FDA) and European Medicinal Agency (EMA) granted approval to the use of rituximab in combination with chemotherapy for the treatment of both previously-untreated and previously-treated patients with CLL. In conventional doses of 375 mg/m2 weekly for 4 doses, rituximab demonstrates moderate clinical activity in patients with CLL (Table 3) [5,6]. However, higher doses or denser dosing regimens are more effective than standard doses, used routinely in other lymphoid malignancies [5-10]. O’Brian et al note a correlation between the dose of rituximab

and the clinical response [7]. The overall response (OR) rate ranged between 22% for lower doses and 75% for the highest doses, for pretreated CLL patients who received an initial dose of rituximab of 375 mg/m2, increasing to a fixed dose of between 500 and 2250 mg/m2 once weekly for 4 weeks, In another study, Byrd et al. administered rituximab at doses ranging from 250 mg/m2 to 375 mg/m2, 3 times weekly for 4 weeks, to give an OR rate of 45%, including 3% CR [6]. Rituximab has been also evaluated as a single drug in patients with previously untreated CLL. Hainsworth et al report an OR rate of 51% and complete response (CR) rate of 4% in a group of 44 symptomatic patients treated with four weekly doses of rituximab at 375 mg/m2 [5]. In a more recent study, rituximab was given at a dose 375 mg/m² weekly for 8 consecutive weeks in 34 early-stage patients, resulting in an OR rate of 82%, including 9% CR, and an 8-year overall survival (OS) rate of 74% [10]. Currently, monotherapy with rituximab is more commonly used in the US than in Europe both in the relapse and first-line settings. There has been also some exploration of rituximab as maintenance therapy in CLL [11-13]. In these studies, maintenance therapy with rituximab was successfully conducted for 6 months or longer. The results suggest that maintenance with rituximab prolongs progression-free survival (PFS) and improves the quality of response, particularly in patients with detectable disease after induction therapy. However, the value of maintenance with rituximab in CLL requires confirmation in randomized trials. Ofatumumab Ofatumumab (HuMax-CD20; Arzerra™, GlaxoSmithKline, plc/Genmab A/S) is a fully-human, IgG1 mAb that binds to both the small and large loops of the membrane CD20 antigen [14]. Ofatumumab stabilizes CD20 on lipid rafts, and hence induces CDC more effectively than rituximab. In preclinical studies, this antibody was found to exhibit more

potent in vitro activity against B-cell malignancies and prolonged survival in animal models compared to rituximab [14]. Ofatumumab monotherapy has shown promising efficacy in heavily pretreated patients in several studies (Table 3) [15-17]. In a pivotal, international study, ofatumumab at a total of 12 doses over a 28-week schedule was evaluated in 138 patients with fludarabine- and alemtuzumab-refractory CLL [16]. The study found the OR rates to be 58% and 47% in the fludarabine- and alemtuzumab-refractory groups and fludarabine-refractory CLL with bulky lymphadenopathy groups, respectively. Median PFS and OS times were 5.7 and 13.7 months in the fludarabine- and alemtuzumab-refractory groups, and 5.9 and 15.4 months, respectively, in the fludarabine-refractory patients with bulky lymphadenopathy. However, when used as monotherapy, ofatumumab was found to be inferior to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (Imbruvica™). In a multicenter, open-label, randomized, phase III trial, ofatumumab was compared with ibrutinib in patients with relapsed or refractory CLL (RESONATE) [17]. Overall, 43% of the patients in the ibrutinib arm demonstrated a PR, as compared with 4% in the ofatumumab group (P<0.001). In the ibrutinib group, an additional 20% of the patients had a PR with lymphocytosis. At a median follow-up of 9.4 months, ibrutinib significantly prolonged the duration of PFS (median not reached), when compared with the median duration of PFS with ofatumumab (8.1 months, p<0.001). Moreover, the OS rates at 12 months were 90% in the ibrutinib arm and 81% in the ofatumumab arm (P=0.001). In the ongoing trial, ofatumumab has been combined with high-dose methylprednisone followed by ofatumumab and alemtuzumab in patients with 17p del (NCT01465334). In 2009, the FDA granted accelerated approval to ofatumumab for the treatment of CLL patients refractory to fludarabine and alemtuzumab. Full approval for ofatumumab for use in CLL was granted in 2014, with the indication for use in combination with

chlorambucil, for previously untreated patients who are not candidates for fludarabine-based therapy. Obinutuzumab Obinutuzumab (Gazyva™, GA-101, RO5072759, F. Hoffmann–La Roche) is a type II anti-CD20 antibody that results in lower CDC but significantly higher ADCC and direct cell death compared with rituximab and ofatumumab [18]. It is a novel, third-generation mAb characterized by an ability to stabilize CD20 on lipid rafts and hence induce high CDC. It is distinct from other anti - CD20 antibodies in current clinical use by its type II properties and glycoengineered Fc region. The antibody is based on proprietary GlycoMAb(®) technology, which incorporates glycoengineered antibodies that specifically increase ADCC and thereby increase immune-mediated target cell death. Obinutuzumab is currently the most effective CD20 antibody in CLL. In the phase I/II GAUGUIN study, obinutuzumab monotherapy was given in 33 patients with heavily pretreated relapsed/refractory CLL, 13 in phase I and 20 in phase II [19]. Overall response was 62% in phase I and 15% in phase II. All responses were partial responses. In phase II study median PFS was 10.7 months. The toxicities were similar to those of other anti-CD20 antibodies. Most common adverse events (AEs) were infusion-related reactions, neutropenia, and thrombocytopenia, with most AEs being of grade 1/2. Grade 3/4 neutropenia occurred in 7 patients in phase 1, and 4 patients in phase II. This study demonstrated that obinutuzumab can be safely administered to patients with relapsed/refractory CLL at doses up to 2000 mg. The results of a large randomized phase III trial testing three first-line chemo-immunotherapy regimes, i.e. combined obinutuzumab and chlorambucil, combined rituximab and chlorambucil and chlorambucil monotherapy, in patients with comorbidities have been recently reported (CLL11) [20]. Treatment with obinutuzumab–chlorambucil resulted in

higher rates of CR and significant improvements in PFS versus either rituximab + chlorambucil or chlorambucil therapy alone. Obinutuzumab has been approved by the FDA and EMA for use in combination with chlorambucil for previously untreated CLL [21]. Other anti-CD20 monoclonal antibodies New generations of anti-CD20 mAbs have been developed recently. The most promising second-generation mAbs, which are not approved yet for CLL, include ocaratuzumab and veltuzumab [4]. Ocaratuzumab Ocaratuzumab (AME-133v, LY2469298, Mentrik Biotech) is a type I, third-generation, humanized, Fab- and Fc-engineered IgG1 anti-CD20 mAb. Ocaratuzumab is the first Fcmutant mAb tested in clinical trial. It is characterized by 2 amino-acid changes introduced into its Fc portion. The antibody was optimized through protein engineering for increased affinity to CD20 and enhanced effector function in ADCC assays. Cell binding assays reveal that ocaratuzumab and rituximab compete for an overlapping epitope on the CD20 antigen [22]. In preclinical studies, in primary CLL cells in vitro, ocaratuzumab was found to induce direct cytotoxicity, CDC and antibody-dependent cellular phagocytosis (ADCP) similar to rituximab and ofatumumab [23]. Its ADCC is approximately 20 times more effective than rituximab at very low concentrations, which may facilitate sub-cutaneous dosing. Ocaratuzumab induces higher NK cell-mediated ADCC than rituximab and ofatumumab and similar to obinutuzumab. The safety, pharmacokinetics and efficacy of ocaratuzumab were assessed in phase I/II trials in patients with previously treated follicular lymphoma (FL) [22,24]. In these studies, ocaratuzumab was well tolerated and expressed therapeutic activity. Further studies are needed to establish the role of this antibody in the treatment of patients with CLL.

Veltuzumab Veltuzumab (IMMU-106, hA20, Immunomedics, Inc) is a 2nd generation humanized antiCD20 mAb with structural and functional differences to rituximab [25]. Veltuzumab differs from rituximab by one amino acid in the complementarity-determining region 3 of the variable heavy chain. It is very similar to rituximab in terms of antigen binding specificity, binding avidity and dissociation constant. However, veltuzumab has enhanced binding avidities and a stronger effect on CDC compared with rituximab in selected cell lines [26]. Veltuzumab is at least as effective as rituximab in NHL and CLL. Subcutaneous administration of veltuzumab was investigated in a multicenter, phase I/II study on previously untreated or relapsed CD20+ indolent NHL or CLL patients [27,28]. Recently, a multicenter, phase I/II study was undertaken to evaluate the safety and efficacy of veltuzumab given subcutaneously in patients with previously untreated or relapsed CLL with high levels of circulating leukemic cells [29]. In this study, 20 CLL patients received 4 doses of veltuzumab given every other week at 80, 160, or 320 mg or 16 doses of the drug administered twice weekly at doses of either 160 or 320 mg. After treatment, 15 of 18 evaluable patients achieved either PR or stable disease (SD) and the number of leukemic cells in peripheral blood decreased in all patients by 7.7 to 90.8%. These results indicate that veltuzumab administered subcutaneously is convenient, well tolerated and active in CLL patients. ALEMTUZUMAB Alemtuzumab (Campath®. Sanofi/Genzyme) is a recombinant humanized IgG1 mAb targeting the CD52 antigen. The drug is active in advanced, fludarabine-refractory CLL and has proven efficacy in patients with deletions of chromosome 17p (del 17p), and p53 mutations [30-33]. However, no benefit is observed in patients with bulky lymph node. In the pivotal CAM 211 trial, 93 patients with relapsed or refractory CLL, who had failed prior

therapy with fludarabine and an alkylating agent, were treated with stepped-up dosing followed by 30 mg three times weekly for a total of 12 weeks [32]. The response rate was 33%, including 2% CR and 31% PR. Median time to progression was 9.5 months for responders and 4.7 months overall. Alemtuzumab has proven to be even more active in treatment-naïve patients with CLL. Hillmen and colleagues performed a randomized, multicenter study in 297 patients that received either chlorambucil or alemtuzumab [34]. Patients receiving alemtuzumab demonstrated an increased OR rate compared to those receiving chlorambucil (83 % vs. 55 %; p<0.0001) and an increased CR rate (24 % vs. 2 %; p<0.0001). Moreover, elimination of minimal residual disease (MRD) occurred in 31% of complete responders to alemtuzumab and none to chlorambucil. Patients receiving alemtuzumab also experienced longer PFS compared to those receiving chlorambucil (median 14.6 and 11.7 months, respectively; p=0.0001). The currently approved schedule of alemtuzumab administration involves a dose of 3 mg delivered i.v. on day 1, escalating to 10 mg on day 2 and then to 30 mg three times weekly as tolerated, for a total of 8-12 weeks [35]. In addition, low-dose alemtuzumab is a valid therapeutic option for CLL patients [36]. Moreover, subcutaneous alemtuzumab demonstrates similar effectiveness and is safe for intravenous administration in fludarabinerefractory CLL patients [37]. Alemtuzumab is associated with a risk of toxicity, especially with bacterial and viral infections which are usually manageable with standard therapies. The most common alemtuzumab administration-related side effect is infusion toxicity, occurring mainly during the first several alemtuzumab infusion doses. However, subcutaneous administration significantly reduces the intensity of the first administration reaction with comparable therapeutic activity. Local reactions at site of injection are most common with this mode of administration. Cytopenias and infections are also common adverse events as a consequence

of profound cellular

immune suppression. Reactivated

Herpes

viruses

such as

cytomegalovirus (CMV) are the most frequent opportunistic infections. A recent analysis indicates that CMV reactivation occurred in 15-25% of refractory or relapsed CLL treated with alemtuzumab. At present, aggressive anti-infective prophylaxis is a mandatory procedure. In 2001, alemtuzumab was initially approved by the FDA to treat patients with CLL refractory to fludarabine and in 2007, regular approval for single-agent alemtuzumab for the treatment of CLL was granted. However, alemtuzumab was withdrawn by Sanofi from the markets and since September 4, 2012, alemtuzumab is no longer available commercially, but is provided free of charge through the Campath Distribution Program foundation for CLL patients. ANTI - CD19 ANTIBODIES CD19 is a 95-kDa transmembrane glycoprotein of the immunoglobulin superfamily containing two extracellular immunoglobulin-like domains and an extensive cytoplasmic tail. It is B-lymphocyte lineage specific surface receptor expressed from the earliest stages of preB cell development onwards until differentiation into plasma cells [38]. Since CLL cells retain CD19 expression and is not expressed on hematopoietic stem cells or other normal tissue, it represents an excellent target for antibody therapy of CLL patients. MOR00208 MOR00208 (XmAb5574, MorphoSys AG), is a humanized, Fc-engineered anti- CD19 mAb being explored for clinical applications in CLL and other B-cell lymphoid malignancies. In preclinical studies, MOR208 has shown antitumor activity including direct cytotoxicity, ADCC and ADCP against leukemic CLL cells [39]. In a phase I study, MOR208 exhibited preliminary efficacy in patients with high-risk, heavily pretreated CLL, prompting responses in 67% of patients and a tolerable toxicity profile [40]. On the basis of clinical criteria, 18 of

27 patients (66.7%) showed a PR and the remaining 9 patients (33.3%) achieved stable disease (SD) with PFS for all patients being 199 days. Moreover, 30% of patients achieved a PR according to IWCLL 2008 criteria. Toxicity was acceptable, but infusion reactions were common. A phase II study of this agent in patients with CLL and other B-cell malignancies is ongoing (NCT02005289). Recently, the FDA has granted fast track designation to MOR208 to treat relapsed or refractory diffuse large B-cell lymphoma (DLBCL). MEDI-551 MEDI-551 (MedImmune/Astra Zeneca) is a humanized afucosylated humanized IgG kappa anti-CD19 mAb with antineoplastic activities [41]. The activity and toxicity of single-agent MEDI-551 was assessed in CLL patients previously treated with rituximab in a phase I/II, open-label, dose-escalation study (NCT00983619) [42]. Of 20 CLL patients evaluable for response, 4 achieved a PR and 13 reached SD. Infusion reactions were noted in 62% of the patients, and neutropenia in 23%. However, the only infusion reaction was considered treatment related. A phase II study of MEDI-551 in combination with bendamustine in relapsed CLL patients is ongoing (NCT01466153). SAR-3419 SAR-3419 (Sanofi-Adventis, NJ, USA) is an anti-CD19 composed of the humanized antibody huB4 conjugated to the maytansine derivative DM4 (N2'-deacetyl-N2'-(4-mercapto-4-methyl1-oxopentyl) maytansine) [43]. In refractory/relapsed patients with NHL, SAR3419 showed significant activity and low hematological toxicity [44]. SAR3419 holds promise as a novel and well-tolerated therapy in CLL and other B-cell lymphoid malignancies.

ANTI-CD23 ANTIBODY The CD23 antigen is a low-affinity Ig E receptor expressed on mature resting cells, some activated B cells and the majority of CLL cells [45]. Lumiliximab (IDEC-152; Biogen Idec) is a genetically-engineered chimeric macaque human anti-CD-23 IgA1 mAb. This antibody induces ADCC and CMC in CD23-bearing lymphoid cell lines [45]. In pre-clinical experiments, lumiliximab combined with fludarabine or rituximab demonstrated enhanced antitumor activity. In a phase I dose escalation study, lumiliximab was found to be well tolerated and demonstrate clinical activity in patients with relapsed or refractory CLL [46]. A phase I/II dose-escalation study with previously-treated CLL patients found a combination of lumiliximab with fludarabine, cyclophosphamide, and rituximab to give an overall response rate of 65%, with 52% of patients achieving a CR with the estimated median PFS 28.7 months [47]. Subsequently, a randomized trial comparing lumiliximab plus

fludarabine,

cyclophosphamide and rituximab (FCR) with FCR alone (LUCID trial) was initiated to define the value of this combination in relapsed CLL [48]. Unfortunately, the combination of lumiliximab with FCR was not significantly better than FCR alone in terms of OR rate (71% vs. 72%), CR rate (16% vs. 15%), and median PFS ( 24.6 vs. 23.9 months). In consequence, the study was stopped for lack of efficacy after interim analysis and development of this antibody terminated for this indication. ANTI-CD37 ANTIBODIES CD37 is one of the tetraspanin superfamily cell surface antigens, which are considered as targets for B cell malignancies [49]. It may be advantageous to target CD37 over CD20 in diseases in which the level of CD37 expression is higher than that of CD20. CD37 seems to be an ideal candidate as a therapeutic target for treatment of CLL and has aroused great interest in the investigation of anti-CD37 antibodies (Table 4).

Otlertuzumab Otlertuzumab (TRU-016, Emergent BioSolutions Inc.) is a novel humanized antiCD37 protein therapeutic produced using ADAPTIR™ Modular Protein Technology which has demonstrated antineoplastic activity in preclinical studies and clinical trials [50,51]. Otlertuzumab is an engineered protein including anti-CD37 variable regions linked to an immunoglobulin constant domain, which is produced by humanizing the precursor agent SMIP-016, a single chain monospecific protein that retains Fc mediated effector functions [52,53]. It is smaller than classic mAbs but has similar functionality and pharmacokinetic (PK) properties and can generate a unique signaling response. In addition, it mediates CDC and Fc-dependent cytotoxicity in a similar way to mAbs. Otlertuzumab has demonstrated a significantly greater ability to directly kill CLL cells than rituximab and greater Fc-mediated cellular cytotoxicity of CLL cells than either alemtuzumab or rituximab [54]. In a phase I study, otlertuzumab was found to encourage reductions in tumor lymphocyte blood counts, lymph node/spleen size and improvements in normal hematopoeitic function in patients with high risk CLL, even at low, non-saturating doses of CD37. Another phase I study investigated otlertuzumab in patients with relapsed/refractory CLL or small lymphocytic lymphoma (SLL) [55]. The drug was well tolerated with minimal infusion toxicity. Response occurred in 19 of 83 treated patients (23%) according to NCI-96 criteria. All responses were partial, and occurred more commonly in patients with symptomatic, untreated CLL. Recently, the preliminary results of a randomized trial of otlertuzumab + bendamustine vs. bendamustine alone were presented [56]. The OR rate in the otlertuzumab + bendamustine arm was 75%, with a CR rate of 48%, compared to an OR rate of 52% and a CR rate of 9% in the bendamustine arm. These results suggest that otlertuzumab

can be a valuable drug in CLL, but its success will depend on it demonstrating superiority over available mAbs and other emerging therapies in achieving and sustaining CRs and improving survival. Phase Ib study of otlertuzumab and rituximab or obinutuzumab in CLL is ongoing (NCT01644253). BI 836826 BI 836826 (MAb 37.1, Boehringer Ingelheim) is a chimeric IgG1-type of anti–CD37 molecule which has been Fc-engineered to improve ADCC activity and enhance affinity for Fc-gRIIIa. Both mAb 37.1 and its humanised version, MAb 37.2, deplete CLL cells in vitro more effectively than rituximab and alemtuzumab [57]. BI 836826 is under investigation in refractory/relapsed CLL in a phase I clinical trial (NCT01296932). IMGN529 IMGN529 (ImmunoGen, Inc. ) is a conjugate of the K7153A novel anti-CD37 antibody with the DM1 maytansinoid, a potent anti-microtubule agent, via the SMCC (succinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate) thioether linker [58]. IMGN529 combines the intrinsic proapoptotic and immune effector activities of its anti-CD37 antibody component with the cytotoxic potency of its DM1 maytansinoid payload through apoptosis induction, ADCC and CDC. The antibody component of IMGN529 was found to have stronger proapoptotic activity and comparable ADCC in vitro than rituximab. IMGN529 was evaluated in transgenic mice characterized by development of CLL-like disease with hCD37+ leukemic cells in the peripheral blood and lymphoid organs. In this model, IMGN529 rapidly eliminated peripheral blood leukemia and improved overall survival [59]. In human blood cells, IMGN529 resulted in potent and specific depletion of normal and CLL B cells and leukemic cells from CLL patients. IMGN529 may be a useful agent for the treatment of CD37-positive B-cell malignancies. A phase I study evaluating the safety and tolerability of

IMGN529 in patients with relapsed or refractory NHL has been recently initiated (NCT01534715). ANTI-CD40 ANTIBODIES CD40 is a transmembrane glycoprotein of the tumour necrosis factor (TNF) receptor superfamily. CD40 acts as a co-stimulatory molecule upon interaction with its ligand CD154 (CD40L) and is involved in B-cell activation, differentiation and the formation of germinal centres [60]. This molecule is highly expressed in B-cell malignancies, making it an attractive potential tumor target for antibody-based therapy. Lucatumumab Lucatumumab (HCD122, formerly CHIR-0.12.12; Novartis Pharmaceuticals) is a fully human, recombinant mAb of the IgG1 isotype targeting human CD40 and inhibiting the growth and survival of B-cell malignancies [61]. This antibody inhibits CD40L-CD40– dependent proliferation and survival, and exhibits stimulation of ADCC. In preclinical studies, lucatumumab was found to induce 18% greater average maximal lysis of B cells then rituximab [61]. Byrd et al have reported the results of phase I study with lucatumumab, in 24 relapsed and refractory CLL [62]. The drug had some clinical activity in this patient population with one PR and stable disease in 17 patients.

Currently no registered clinical

trials are underway with lucatumumab in CLL patients. Dacetuzumab Dacetuzumab (SGN-40, Seattle Genetics, Inc.) is the humanized IgG1 version of S2C6, a murine anti-human CD40 mAb. In contrast to lucatumumab, dacetuzumab does not prevent CD40/CD40L interactions. Dacetuzumab mediates ADCC and antibody- dependent cellular phagocytosis, and induces apoptosis in certain CD40-positive lymphoma lines [63].

Preclinical studies with dacetuzumab have shown synergy when combined with rituximab, lenalidomide or combination chemotherapy in animal models [64,65]. In a phase I study, five of twelve patients with refractory CLL demonstrated stable disease after 1 cycle of therapy [66].

OTHER MONOCLONAL ANTIBODIES WHICH MAY BE POTENTIALLY USEFUL IN CLL Other promising targets for mAbs which may be potentially useful in the treatment of CLL are CD22, CD74 and CD88. CD22 is a 135 kDa B-cell-specific transmembrane sialoglycoprotein expressed in about 70% of B cell lymphomas and leukemias but not expressed on plasma cells, memory B cells, stem cells, monocytes or T-cells [66]. CD22 is internalized into the cell when bound by the antibody and is currently used for immunotoxins as a target molecule. At least 6 different anti-CD22 immunotoxins are currently in clinical investigation, including inotuzumab ozogamicin, moxetumomab pasudotox, DCDT2980S and combotox [67]. Some of them have shown promising anti-CLL activity in preclinical studies. However, clinical data in CLL is not yet available [68]. CD74 is a type II transmembrane protein expressed on B cells which is associated with the α- and β-chains of HLA-DR. It is an attractive target for CLL due to increased expression on the surface of leukemic B-cells [69]. Milatuzumab (hLL1) is a humanized monoclonal antibody directed against CD74 that rapidly internalizes [70]. However, milatuzumab alone showed limited efficacy in relapsed and refractory CLL [71]. A milatuzumab-doxorubicin immunoconjugate is currently in the early phase of development for CLL (NCT00868478). CD84 expression regulates the survival of CLL cells in vitro and in vivo. This antigen may be a novel therapeutic target for therapeutic mAbs [72]. The in vitro experiments found

that inhibition of CD84 leads to reduced Bcl-2 expression and elevation of CLL cell death. This finding may suggest that CD84 could be another novel target for CLL therapy with mAbs. COMBINATIONS OF ANTIBODIES WITH OTHER TARGETED DRUGS During recent years, the combined use of mAbs with other targeted drugs for CLL therapy has been intensively investigated. In particular, B-cell antigen receptor (BCR) signal transduction inhibitors (ibrutinib and idelalisib) and immunomodulating drugs (lenalidomide), have been nominated as promising candidates for targeted CLL treatment. These drugs express high activity in CLL and are well tolerated. Their combinations with mAbs are under way to increase the rate and durability of responses, without increasing toxicity [3,73]. Clinical trials are currently underway using a combination of anti-CD20 mAbs with ibrutinib, idelalisib, lenalidomide or ABT-199, and early results have been reported (Table 5). Combinations with BTK and PI3K inhibitors Ibrutinib (PCI-32765; Imbruvica, Janssen – Cilag International NV / Pharmacyclics) is an irreversible covalent inhibitor of Bruton’s tyrosine kinase (BTK), a critical enzyme in the BCR signaling pathway. The drug used alone is associated with a high frequency of durable remissions in patients with previously untreated and relapsed or refractory CLL, including patients with high-risk genetic lesions [74]. A combination of ibrutinib with rituximab was investigated in single arm phase II trial in 40 patients with high-risk CLL, who displayed deletion 17p, TP53 mutation, deletion 11q or short PFS (<36 months) after previous first-line chemoimmunotherapy [75]. They received ibrutinib 420 mg p.o. daily continuously throughout the study, as well as rituximab 375 mg/m2 weekly for the first four weeks in cycle 1 and then monthly until cycle 6. The treatment was safe and well tolerated and induced high rates of durable responses. At a

median follow up of 14 months, the OR rate was found to be 95% for all patients and 90% in the 20 patients with del17p or TP53 mutation. The most frequently observed adverse events were grade 1/2 diarrhea, grade 1/2 bleeding events, nausea, vomiting and fatigue. These preliminary results indicate that the ibrutinib and rituximab combination should be further investigated in patients with high-risk CLL. Ibrutinib in combination with bendamustine and rituximab (BR) was also evaluated in 30 patients with relapsed or refractory CLL [76]. The treatment was well tolerated and highly active. OR was observed in 93% of the patients, and this included 5 CRs and 3 nodular PRs (nPRs). The estimated 12-month PFS was 90%. The randomized phase III HELIOS trial, investigating whether ibrutinib added to bendamustine and rituximab (BR) provides benefits over BR alone in patients with relapsed/refractory CLL/SLL, is ongoing [77]. Jaglowski et al reported interim data from a phase Ib/II trial investigating ibrutinib in combination with ofatumumab in patients with relapsed/refractory CLL/SLL who had received two or more prior therapies [78]. The treatment was well tolerated and highly active in patients with heavily pretreated relapsed/refractory CLL, who demonstrated a 100% OR rate. Recent studies have identified patients refractory to ibrutinib and characterized a BTK mutation (BTKC481S) responsible for ibrutinib resistance [79,80]. For these patients, new therapeutic targets and corresponding drugs with alternative mechanisms of action are urgently needed. Idelalisib (GS-1101, CAL-101, Zydelig, Gilead Sciences International Ltd. /Calistoga Pharmaceuticals), is a selective oral inhibitor of phosphatidylinositol 3-kinase P110d (PI3Kδ). Idelalisib used in monotherapy has shown substantial clinical activity and a favorable safety profile in heavily pretreated, refractory and high risk patients with CLL [81]. In addition, rituximab plus idelalisib achieved a response and survival benefit compared with rituximab alone in relapsed/refractory CLL patients with comorbidities [82]. The patients receiving

idelalisib and rituximab had improved rates of OR (81%) in comparison with rituximab monotherapy (13%, P<0.001). Median PFS was 5.5 months in the rituximab group, but was not reached in the idelalisib + rituximab group (P<0.001). Moreover, OS at 12 months was 92% for rituximab, compared to 80% for idelalisib + rituximab (P=0.02). A study of idelalisib in combination with rituximab in elderly patients with untreated CLL or SLL is ongoing (NCT01203930). Clinical trials evaluating the efficacy and safety of ofatumumab in combination with idelalisib for previously treated and untreated patients with CLL has also been initiated [83] (NCT02135133). Combinations with immunomodulatory drugs Lenalidomide (Revlimid; Celgene, Summit, NJ) is an immunomodulatory drug active in CLL in monotherapy and in combination with other agents. This drug induces activation of T and NK cells and in vitro enhances clearance of CLL cells by mAbs. Lenalidomide in monotherapy has demonstrated clinical efficacy in patients with relapsed disease and early data has been generated within clinical trials in a front-line setting [84-86]. However, lenalidomide is currently not approved for front-line therapy of CLL. In preclinical models, rituximab and lenalidomide have been observed to act synergistically against CLL cells. It has been documented that lenalidomide increases rituximab-mediated antibody-dependent NKand T-cell cytotoxicity and in vitro enhances clearance of CLL cells by mAbs [87]. In a phase II study, the lenalidomide and rituximab combination demonstrated high clinical activity when used as salvage therapy [88]. The OR rate was 66%, including 12% CR and 12% nPR. Median PFS was 17.4 months and estimated survival at 36 months was 71%. These results are comparable to currently employed chemotherapy and immunochemotherapy regimens in this patient population.

In addition, James et al report the results of a phase II study evaluating a combination of lenalidomide and rituximab as initial treatment in 69 CLL patients [89]. The patients were enrolled onto one of two age-specific groups, with arm A composed of patients younger than 65 years and arm B composed of patients 65 years or older. Lenalidomide was initially given at 2.5 mg/day and was escalated based on treatment tolerability to a maximum of 10 mg/day, for 21 days/cycle, for a maximum of seven cycles. Rituximab was administered at 50 mg/m 2 on day 29 of cycle 1, and an additional 325 mg/m2 on day 31, and another dose of 375 mg/m2 on day 33. Rituximab was then continued at 375 mg/m2 weekly throughout cycle 2 in a dosedense fashion and then was administered on day 1 of cycles 3 to 7. The combination was found to be effective, with the OR rate for younger patients being 95% with 20% CRs, and a 78% OR rate for the older patients, of which 11% were CRs. Median PFS values were 19 and 20 months respectively. These results are comparable to currently-employed chemotherapy and immunochemotherapy regimens. A phase I study of lenalidomide, rituximab and ibrutinib in relapsed/refractory CLL is also ongoing (NCT02200848). Ferrajoli et al explored the combination of lenalidomide and ofatumumab in patients with relapsed CLL who had received prior treatment with purine analogs [90]. In this phase II study, 36 patients received ofatumumab weekly for 4 weeks, followed by monthly administration during months 2–6 and then every second month for months 7–24. In addition lenalidomide was administered at a dose of 10 mg per day, starting on day 9 and continuing for 24 months. The overall response rate was 68%, including 24% CR and 44% PR. Median response duration was 22 months. Neutropenia was the most common toxicity but the severity of tumor flare reaction (TFR) was less than with single agent lenalidomide. Recently Costa et al performed a multi-center, phase II trial of sequential treatment with ofatumumab and lenalidomide in 34 patients with advanced, relapsed and refractory CLL [91]. Ofatumumab was given at a dose of 2000 mg on day 1 and lenalidomide 10 mg on days

8-28, for up to 6 cycles. The OR rate was 47.6% and median OS was 21.5 months. However, neutropenia and thrombocytopenia were frequent adverse events, and TFR occurred in 43% of patients. The combination of lenalidomide and obinutuzumab in patients with recurrent CLL or SLL is also currently under investigation in one trial (NCT02225275), while the combination of MOR00208, an anti-CD19 mAb, with lenalidomide is being examined in another phase II trial for patients with relapsed or refractory CLL or older, untreated patients with CLL, SLL or prolymphocytic leukemia (NCT02005289). Combinations with BCL-2 inhibitors As BCL-2 is over expressed in CLL cells, it represents a good therapeutic target for effective drugs. ABT-199 (GDC-0199, RG7601, Genentech BioOncology/Roche) is a novel, second generation BCL-2 inhibitor with a high-affinity Bcl-2–selective BH3 mimetic, which has been recently developed by Abbott Laboratories [92]. ABT-199 can trigger apoptosis in vitro, even in del(17p) CLL cells. The results of a phase II, dose-escalation study of ABT-199 in highrisk relapsed/ refractory CLL were recently presented [93]. The response rate was 82% in patients with del(17p), and 78% in patients with fludarabine-refractory disease. A Phase Ib study evaluating the safety and tolerability of ABT-199 in combination with rituximab in subjects with relapsed phase II trial for patients with relapsed or refractory or older, untreated patients with CLL and SLL is ongoing (NCT01682616). Another trial compares ABT-199 plus rituximab with bendamustine plus rituximab in patients with relapsed or resistant CLL (NCT02005471).

CONCLUSIONS Despite advances in therapy, CLL remains an area with a significantly unmet medical need. In cases of patients with del 17p, and those who may be refractory to initial treatment with fludarabine-based immunochemotherapy, new therapeutic options are extremely limited. Consequently, there remains a need for new therapies that combine high levels of efficacy with improved tolerability. A major step forward in the treatment of CLL was the development and broad use of a mAb targeting CD20 and other antigens on the surface of B cells. Recent clinical data clearly demonstrates that new generations of mAbs have provided new opportunities for more effective therapies for patients with CLL. Immune-based approach with mAbs alone or their combinations with other targeted drugs represents a highly active approach for treatment of CLL patients, including those with high-risk cytogenetics. Treatment of relapsed CLL seems to be best supported by ibrutinib- or idelalisib-based therapies. Moreover, multi-drug non-chemotherapy regimens, based on combinations of mAbs, BCR signaling inhibitors, lenalidomide and/or BCL-2 inhibitors would be of interest as possible highly effective multi-drug regimens. The completion of trials with mAbs and other new agents in the near future will offer opportunity for chemotherapy-free treatment across all groups of CLL. These new strategies are aimed to preclude such chemotherapy toxicities as cytopenias and the increased risk of infectious disease and secondary neoplasia, while providing an equivalent or higher response rate and response duration. However, the efficacy and tolerability of chemotherapy-free treatment of CLL should be confirmed in large randomized trials. Given all these novel agents and targets, although their cost and potential toxicity should be taken into account, chemotherapy-free or at least chemotherapy-reduced concepts may become reality in the near future for patients suffering from CLL.

Acknowledgements This work was supported in part by the grant from the Medical University of Lodz (No 503/1-093-01/503-01) and by the Foundation for the Development of Diagnostics and Therapy, Warsaw, Poland. Conflicts of interest: TR has received consulting fees from F. Hoffmann–La Roche, Gilead, and Janssen and performed contracted research for F. Hoffmann–La Roche, GlaxoSmithKline, Janssen, Celgene, MorphoSys AG, Emergent BioSolutions, Pharmacyclics, Gilead and Seattle Genetics; JZB has performed contracted research for F. Hoffmann–La Roche, GlaxoSmithKline, Janssen, Celgene, Emergent BioSolutions, Pharmacyclics and Gilead; PR has performed contracted research for Janssen and Pharmacyclics.

REFERENCES 1. Robak T. Current and emerging monoclonal antibody treatments for chronic lymphocytic leukemia: state of the art. Expert Rev Hematol. 2014;7:841-57 2. Pallasch CP, Hallek M. Incorporating targeted agents into future therapy of chronic lymphocytic leukemia. Semin Hematol. 2014;51:235-48. 3. Byrd JC, Jones JJ, Woyach JA, Johnson AJ, Flynn JM. Entering the era of targeted therapy for chronic lymphocytic leukemia: impact on the practicing clinician. J Clin Oncol. 2014 Jul 21. pii: JCO.2014.55.8262. 4. Robak T, Robak E. New anti-CD20 monoclonal antibodies for the treatment of B-cell lymphoid malignancies. BioDrugs. 2011;25:13-25.

5. Hainsworth JD, Litchy S, Barton JH, et al. Single-agent rituximab as firstline and maintenance treatment for patients with chronic lymphocytic leukemia or small lymphocytic lymphoma: a phase II trial of the Minnie Pearl Cancer Research Network. J Clin Oncol. 2003; 21:1746–51. 6. Byrd JC, Murphy T, Howard RS, et al. Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity. J Clin Oncol; 2001;19: 2153-64. 7. O’Brien SM, Kantarjian H, Thomas DA, et al. Rituximab dose-escalation trial in chronic lymphocytic leukemia. J Clin Oncol 2001;19: 2165-70. 8. Huhn D, von Schilling C, Wilhelm M, et al. Rituximab therapy of patients with B-cell chronic lymphocytic leukemia. Blood. 2001;98:1326-31. 9. Itala M, Geisler CH, Kimby E, et al. Standard dose anti-CD20 antibody rituximab has efficacy in chronic lymphocytic leukaemia: results from a Nordic multicentre study. Eur J Haematol. 2002; 69:129-34. 10. Ferrajoli A, Keating MJ, O'Brien S, Cortes J, Thomas DA. Experience with rituximab immunotherapy as an early intervention in patients with Rai stage 0 to II chronic lymphocytic leukemia. Cancer. 2011;117:3182-6. 11. Del Poeta G, Del Principe MI, Buccisano F, et al. Consolidation and maintenance immunotherapy with rituximab improve clinical outcome in patients with B-cell chronic lymphocytic leukemia. Cancer. 2008;112:119-28. 12. Wiernik PH, Adiga GU. Single-agent rituximab in treatment-refractory or poor prognosis patients with chronic lymphocytic leukemia. Curr Med Res Opin. 2011; 27:1987-93.

13. Abrisqueta P, Villamor N, Terol MJ, et al. Rituximab maintenance after first-line therapy with rituximab, fludarabine, cyclophosphamide, and mitoxantrone (R-FCM) for chronic lymphocytic leukemia. Blood. 2013;122:3951-9. 14. Barth MJ, Czuczman MS. Ofatumumab: a novel, fully human anti-CD20 monoclonal antibody for the treatment of chronic lymphocytic leukemia. Future Oncol. 2013;9:1829-39. 15. Coiffier B, Lepretre S, Pedersen LM, et al. Safety and efficacy of ofatumumab, a fully human monoclonal anti-CD20 antibody, in patients with relapsed or refractory B-cell chronic lymphocytic leukemia: a phase 1-2 study. Blood. 2008;111:1094-100. 16. Wierda WG, Kipps TJ, Mayer J, et al. Ofatumumab as single-agent CD20 immunotherapy in fludarabine-refractory chronic lymphocytic leukemia. J Clin Oncol. 2010;28: 1749-55. 17. Byrd JC, Brown JR, O'Brien S, et al Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N Engl J Med. 2014;371:213-23. 18. Rioufol C, Salles G. Obinutuzumab for chronic lymphocytic leukemia. Expert Rev Hematol. 2014;7:533-43. 19. Cartron G, de Guibert S, Dilhuydy M-S, et al. Obinutuzumab (GA101) in relapsed/refractory chronic lymphocytic leukemia: final data from the phase 1/2 GAUGUIN study. Blood 2014;124:2196-202. 20. Goede V, Fischer K, Busch R, et al. Obinutuzumab plus Chlorambucil in Patients with CLL and Coexisting Conditions. N Engl J Med. 2014; 370:1101-10. 21. Cameron F, McCormack PL. Obinutuzumab: first global approval. Drugs. 2014;74:14754.

22. Forero-Torres A, de Vos S, Pohlman BL, et al. Results of a phase 1 study of AME-133v (LY2469298), an Fc-engineered humanized monoclonal anti-CD20 antibody, in FcγRIIIagenotyped patients with previously treated follicular lymphoma. Clin Cancer Res. 2012;18:1395-403. 23. Cheney CM, Stephens DM, Mo X, et al. Ocaratuzumab, an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia. MAbs. 2014;6:749-55. 24. Ganjoo KN, de Vos S, Pohlman BL, et al. Phase 1/2 Study of Ocaratuzumab, an Fcengineered humanized anti-CD20 monoclonal antibody, in low affinity FcγRIIIa patients with previously treated follicular lymphoma. Leuk Lymphoma. 2014 Jul 14. [Epub ahead of print]. 25. Goldenberg DM, Morschhauser F, Wegener WA. Veltuzumab (humanized anti-CD20 monoclonal antibody): characterization, current clinical results, and future prospects. Leuk Lymph. 2010;51:747-55. 26. Goldenberg DM, Rossi EA, Stein R, et al. Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody. Blood. 2009; 113:106270. 27. Morschhauser F, Leonard JP, Fayad L, et al. Humanized anti-CD20 antibody, veltuzumab, in refractory/recurrent non-Hodgkin's lymphoma:phase I/II results. J Clin Oncol. 2009; 27: 3346-53. 28. Negrea GO, Elstrom R, Allen SL, et al. Subcutaneous injections of low-dose veltuzumab (humanized anti-CD20 antibody) are safe and active in patients with indolent non-Hodgkin's lymphoma. Haematologica 2011;96:567–73.

29. Kalaycio M, Negrea OG, Elstrom R, et al. Monotherapy with subcutaneous (SC) injections of low doses of humanized anti-CD20 veltuzumab is active in chronic lymphocytic leukemia (CLL). ASH Annual Meeting Abstracts. Blood 2012; 120: Abstract 192. 30. Stilgenbauer S, Dohner H. Campath-1H-induced complete remission of chronic lymphocytic leukemia despite p53 gene mutation and resistance to chemotherapy. N. Engl. J. Med.2002; 347: 452–3. 31. Lozanski G, Heerema NA, Flinn IW, et al. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood 2004; 103: 3278–81. 32. Keating MJ, Flinn I, Jain V, et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood, 2002; 99: 3554–61. 33. Zenz T, Häbe S, Denzel T, et al. Detailed analysis of p53 pathway defects in fludarabinerefractory chronic lymphocytic leukemia (CLL):dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood. 2009 ;114:2589-97. 34. Hillmen P, Skotnicki .B, Robak T, et al. Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia. J Clin Oncol. 2007; 25: 5616–23. 35. Osterborg A, Dyer MJ, Bunjes D, et al. Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia. European study group of CAMPATH-1H treatment in chronic lymphocytic leukemia. J Clin Oncol. 1997; 15:1567–74. 36. Cortelezzi A, Gritti G, Laurenti L, et al. An Italian retrospective study on the routine clinical use of low-dose alemtuzumab in relapsed/refractory chronic lymphocytic leukaemia patients. Br J Haematol. 2012;156: 481-9.

37. Stilgenbauer S, Zenz T, Winkler D, et al. Subcutaneous alemtuzumab in fludarabinerefractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol.2009;27:3994-4001. 38. Scheuermann RH, Racila E. CD19 antigen in leukemia and lymphoma diagnosis and immunotherapy. Leuk Lymphoma. 1995;18:385-97. 39. Awan FT, Lapalombella R, Trotta R, et al. CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain-engineered monoclonal antibody. Blood. 2010;115:120413. 40. Woyach JA, Avan F, Flinn IW, et al. A phase I trial of the Fc engineered CD19 antibody XmAb®5574 (MOR00208) demonstrates safety and preliminary efficacy in relapsed chronic lymphocytic leukemia. Blood 2014, Oct 9. pii: blood-2014-08-593269. 41. Ward E, Mittereder N, Kuta E, et al. A glycoengineered anti-CD19 antibody with potent antibody-dependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo. Br J Haematol. 2011;155:426-37. 42. Hamadani M, Forero A, Kipps TJ, et al. MEDI-551, an anti-CD19 antibody active in chronic lymphocytic leukemia (CLL) patients previously treated with rituximab. J Clin Oncol 2013;31 (suppl) : Abstract 7045. 43. Blanc V,

Bousseau1 A, Caron A et al. SAR3419: An Anti-CD19-maytansinoid

immunoconjugate for the treatment of B-cell malignancies. Clin Cancer Res 2011; 17:644858 44. Ribrag V, Dupuis J, Tilly H, et al. A dose-escalation study of SAR3419, an anti-CD19 antibody maytansinoid conjugate, administered by intravenous infusion once weekly in

patients with relapsed/refractory B-cell non-Hodgkin lymphoma. Clin Cancer Res. 2014 ;20:213-20. 45. Pathan NI, Chu P, Hariharan K, et al. Mediation of apoptosis by and anti-tumor activity of lumiliximab in chronic lymphocytic leukemia cells and CD23+ lymphoma cell lines. Blood 2008; 111: 1594-602. 46. Byrd JC, O’Brien S, Flinn IW, et al. Phase 1 study of lumiliximab with detailed pharmacokinetic and pharmacodynamic measurements in patients with relapsed or refractory chronic lymphocytic leukemia. Clin Cancer Res. 2007;13:4448-55. 47. Byrd JC, Kipps TJ, Flinn IW, et al. Phase 1/2 study of lumiliximab combined with fludarabine, cyclophosphamide, and rituximab in patients with relapsed or refractory chronic lymphocytic leukemia. 2010;115:489-95. 48. Awan FT, Hillmen P, Hellmann A, et al. A randomized, open-label, multicentre, phase 2/3 study to evaluate the safety and efficacy of lumiliximab in combination with fludarabine, cyclophosphamide and rituximab versus fludarabine, cyclophosphamide and rituximab alone in subjects with relapsed chronic lymphocytic leukaemia. Br J Haematol. 2014;167: 466–77. 49. Robak T, Robak P. Anti-CD37 antibodies for chronic lymphocytic leukemia. Expert Opin Biol Ther. 2014;14:651-61. 50. Pagel JM, Spurgeon SE, Byrd JC, et al. Otlertuzumab (TRU-016), an anti-CD37 monospecific ADAPTIR(™) therapeutic protein, for relapsed or refractory NHL patients. Br J Haematol. 2014 Aug 22. doi: 10.1111/bjh.13099. 51. Robak T, Robak P, Smolewski P. TRU-016, a humanized anti-CD37 IgG fusion protein for the potential treatment of B-cell malignancies. Curr Opin Investig Drugs. 2009;10:138390.

52. Zhao X, Lapalombella R, Joshi T, et al. Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical. Blood 2007;110: 2569-77. 53. Smolewski P, Robak P, Cebula-Obrzut B, et al. Pro-apoptotic effect of an anti-CD37 scFv-Fc fusion protein, in combination with the anti-CD20 antibody, ofatumumab, on tumour cells from B-cell malignancies. Eur J Cancer. 2014;50:2677-84. 54. Zhao XB, Trupti J, Lapalombella R, et al. NK Cells Contribute Significantly to the Innate Immune Effector Role of CD37-Specific SMIP in CLL and NHL. ASH Annual Meeting Abstracts. Blood 2006; 108: Abstract 135. 55. Byrd JC, Pagel JM, Awan FT, et al. A phase 1 study evaluating the safety and tolerability of otlertuzumab (TRU-016), an anti-CD37 mono-specific ADAPTIRTM therapeutic protein in chronic lymphocytic leukemia. Blood. 2014;123:1302-8. 56. Robak T, Hellman A, Kloczko J, et al. Phase 2 study of otlertuzumab (TRU-016), an antiCD37 ADAPTIRTM protein, in combination with bendamustine vs bendamustine alone in patients with relapsed chronic lymphocytic leukemia (CLL). ASH Annual Meeting Abstracts. Blood 2013; 122: Abstract 2860 57. Heider KH, Kiefer K, Zenz T, et al. A novel Fc-engineered monoclonal antibody to CD37 with enhanced ADCC and high proapoptotic activity for treatment of B-cell malignancies. Blood 2011;118: 4159-68. 58. Deckert J, Park PU, Chicklas S, et al. A novel anti-CD37 antibody-drug conjugate with multiple anti-tumor mechanisms for the treatment of B-cell malignancies. Blood 2013; 122:3500-10

59. Beckwith KA, Frissora FW, Stefanovski MR, et al. The CD37-targeted antibody-drug conjugate IMGN529 is highly active against human CLL and in a novel CD37 transgenic murine leukemia model. Leukemia. 2014;28:1501-10. 60. Law CL, Grewal IS. Therapeutic interventions targeting CD40L (CD154) and CD40: the opportunities and challenges. Adv Exp Med Biol. 2009;647:8–36. 61. Luqman M, Klabunde S, Lin K, et al. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells. Blood. 2008 ;112:711-20. 62. Byrd JC, Kipps TJ, Flinn IW, et al. Phase I study of the anti-CD40 humanized monoclonal antibody lucatumumab (HCD122) in relapsed chronic lymphocytic leukemia. Leuk Lymphoma. 2012;53:2136-42. 63. Law CL, Gordon KA, Collier J, et al. Preclinical antilymphoma activity of a humanized anti-CD40 monoclonal antibody, SGN-40. Cancer Res2005;65:8331–8. 64. Lewis TS, McCormick RS, McEarchern JA, et al. Preclinical analysis of the combined activity of SGN-40, anti-CD40 monoclonal antibody, with rituximab in Non-Hodgkin lymphoma. ASH Annual Meeting Abstracts. Blood 2008, 112:Abstract 1583. 65. Lapalombella R, Gowda A, Joshi T, et al. The humanized CD40 antibody SGN-40 demonstrates pre-clinical activity that is enhanced by. lenalidomide in chronic lymphocytic leukaemia. Br J Haematol. 2009;144:848-55. 66. Furman RR, Forero-Torres A, Shustov A, Drachman JG. A phase I study of dacetuzumab (SGN-40, a humanized anti-CD40 monoclonal antibody) in patients with chronic lymphocytic leukemia. Leuk Lymphoma. 2010;51:228-35.

66. Jasper GA, Arun I, Venzon D, et al. Variables affecting the quantitation of CD22 in neoplastic B cells. Cytometry B Clin Cytom. 2011;80:83-90. 67. Sullivan-Chang L, O'Donnell RT, Tuscano JM. Targeting CD22 in B-cell malignancies: current status and clinical outlook. BioDrugs. 2013;27:293-304. 68. Weldon JE, Xiang L, Chertov O, et al. A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity. Blood. 2009;113:3792-800 69. Stein R, Mattes MJ, Cardillo TM, et al. CD74: a new candidate target for the immunotherapy of B-cell neoplasms. Clin Cancer Res 2007;13(18 Pt 2):5556s-63s 70. Berkova Z, Tao RH, Samaniego F. Milatuzumab - a promising new immunotherapeutic agent. Expert Opin Investig Drugs. 2010;19:141-9. 71. Hertlein E, Triantafillou G, Sass EJ, et al. Milatuzumab immunoliposomes induce cell death in CLL by promoting accumulation of CD74 on the surface of B cells. Blood. 2010 ;116:2554-8. 72. Binsky-Ehrenreich I, Marom A, Sobotta MC, et al. CD84 is a survival receptor for CLL cells. Oncogene. 2014 ;33:1006-16. 73. Robak T, Robak P. BCR signaling in chronic lymphocytic leukemia and related inhibitors currently in clinical studies. Int Rev Immunol. 2013;32:358-76. 74. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med. 2013;369:32-42.

75. Burger JA, Keating MJ, Wierda WG, et al. Safety and activity of ibrutinib plus rituximab for patients with high-risk chronic lymphocytic leukaemia: A single-arm, phase 2 study. Lancet Oncol 2014;15:1090-9. 76. Brown JR, Barrientos JC, Barr PM, et al. Ibrutinib in combination with bendamustine and rituximab is active and tolerable in patients with relapsed/refractory CLL/SLL: final results of a phase 1b study. ASH Annual Meeting Abstracts. Blood 2013; 122:Abstract 525. 77. Hallek M, Kay NE, Osterborg A, et al. The HELIOS trial protocol: a Phase III study of ibrutinib in combination with bendamustine and rituximab in relapsed/refractory chronic lymphocytic leukemia. Future Oncol. 2014 Jun 5:1-9 78. Jaglowski SM, Jones JA, Flynn JM, et al. A phase Ib/II study evaluating activity and tolerability of BTK inhibitor PCI-32765 and ofatumumab in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and related diseases. ASCO Annual Meeting Abstracts. J Clin Oncol. 2012;30(Suppl 15):Abstract 6508. 79. Chang BY, Furman RR, Zapatka M, et al. Use of tumor genomic profiling to reveal mechanisms of resistance to the BTK inhibitor ibrutinib in chronic lymphocytic leukemia (CLL). 2013 ASCO Annual Meeting Abstracts. J Clin Oncol. 2013;31(suppl):Abstract 7014. 80. Cheng S, Guo A, Lu P, Ma J, et al. Functional characterization of BTK(C481S) mutation that confers ibrutinib resistance: exploration of alternative kinase inhibitors. Leukemia. 2014 Sep 5. doi: 10.1038/leu.2014.263. 81. Brown JR, Byrd JC, Coutre SE, et al. Idelalisib, an inhibitor of phosphatidylinositol 3kinase p110δ, for relapsed/refractory chronic lymphocytic leukemia. Blood. 2014;123:3390-7. 82. Furman RR, Sharman JP, Coutre SE, et al. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. N Engl J Med. 2014;370:997-1007.

83. Flinn I, Kimby E, Cotter FE, et al. A phase III, randomized, controlled study evaluating the efficacy and safety of idelalisib (GS-1101) in combination with ofatumumab for previously treated chronic lymphocytic leukemia (CLL). J Clin Oncol 31, 2013 (suppl; abstr TPS7131) 84. Badoux XC, Keating MJ, Wen S, et al, Lenalidomide as initial therapy of elderly patients with chronic lymphocytic leukemia. Blood 2011;118:3489-98, 85. Chanan-Khan A, Miller KC, Musial L, et al. Clinical efficacy of lenalidomide in patients with relapsed or refractory chronic lymphocytic leukemia: Results of a phase II study. J Clin Oncol 2006;24:5343-9 86. Chen CI, Bergsagel PL, Paul H, et al. Single-agent lenalidomide in the treatment of previously untreated chronic lymphocytic leukemia. J Clin Oncol 2011;29:1175–81. 87. Wu L, Adams M, Carter T, et al. lenalidomide enhances natural killer cell and monocytemediated antibody-dependent cellular cytotoxicity of rituximab-treated CD20+ tumor cells. Clin Cancer Res 2008;14:4650-7 88. Badoux XC, Keating MJ, Wen S, et al. Phase II study of lenalidomide and rituximab as salvage therapy for patients with relapsed or refractory chronic lymphocytic leukemia. J Clin Oncol. 2013;31:584-91. 89. James JF, Werner L, Brown JR, et al. Lenalidomide and rituximab for the initial treatment of patients with chronic lymphocytic leukemia: a multicenter clinical-translational study from the Chronic Lymphocytic Leukemia Research Consortium. J Clin Oncol. 2014; 32:2067-73. 90. Ferrajoli A, Falchi L, O'Brien S, et al. Combination of ofatumumab and lenalidomide in patients with relapsed chronic lymphocytic leukemia (CLL): Results of a phase ii trial. ASH Annual Meeting Abstracts. Blood 2012;120:Abstract 720

91. Costa LJ, Fanning SR, Stephenson J Jr, Et al. Sequential ofatumumab and lenalidomide for the treatment of relapsed and refractory chronic lymphocytic leukemia and small lymphocytic lymphoma. Leuk Lymphoma. 2014 Aug 17:1-15. [Epub ahead of print] 92. Vogler M, Dinsdale D, Dyer MJ, Cohen GM. ABT-199 selectively inhibits BCL2 but not BCL2L1 and efficiently induces apoptosis of chronic lymphocytic leukaemic cells but not platelets. Br J Haematol. 2013;163: 139-42. 93. Seymour JF, Davids MS, Pagel JM, et al. Bcl-2 Inhibitor ABT-199 (GDC-0199) monotherapy shows anti-tumor activity including complete remissions in high-risk relapsed/refractory (r/r) chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). ASH Annual Meeting Abstracts. Blood 2013; 122: Abstract 872.

Table 1. Characteristics of approved and novel anti CD-20 monoclonal antibodies for chronic lymphocytic leukemia MoAb

Drug characteristics

Rituximab (Rituxan,

Chimeric, human/mouse, IgG1 mAb Approved with by FDA and EMA for murine the treatment of

Mabthera, F.Hoffmann– La Roche)

Clinical status

light- and heavy-chain variable-region patients with previously treated and sequences and untreated CLL human constant-region sequences

Ofatumumab(HuMax- Type I, 2nd generation, human IgG1, Approved by FDA and EMA for CD20; binding to the Arzerra™,GlaxoSmithKline different CD20 epitope than rituximab, treatment of fludarabineplc/Genmab A/S) more and alemtuzumab - refractory CLL. effective at CDC and less at ADCC than rituximab Obinutuzumab,(GA-101, Type II, 3rd generation, glycoengineered, Approved by FDA and EMA for RO5072759,Hoffmann–La humanized IgG1, use with Roche) mAb with superior ADCC and superior chlorambucil in previously direct untreated CLL. cell-killing than rituximab Ocaratuzumab (AME- Type I, third-generation, humanized, FabPhase I study of subcutaneous 133v,LY2469298, and Fc- engineered IgG1 mAb ocaratuzumab in Mentrik Biotech) previously treated CD20+ B-cell malignancies (NCT01858181) Phase I/II study in previously Veltuzumab (IMMU-106,Type I, 2nd generationation, humanized IgG1 mAb, untreated or relapsed hA20, Immunomedics) binding to different CD20 epitope CD20+ than indolent NHL or CLL rituximab; enhanced (NCT00546793) ADCC and reduced CDC, enhanced affinity for FcγRIIIa Abbreviations: ADCC - antibody-dependent cell mediated cytotoxicity; CDC - complementdependent cytotoxicity; mAb - monoclonal antibody;

3 6

Table 2. Characteristics of emerging monoclonal antibodies and related agents currently in clinical trials in CLL or related diseases MoAb BI 836826 37.1;

Target Drug characteristics

Clinical status

(MAb CD37Chimeric IgG1-type mAb withOngoing phase I clinical trial in pretreated CLL Fc-engineered to improve ADCC ( NCT01296932).

Boehringer Ingelheim)

activity and enhance affinity for Fc-gRIIIa

Otlertuzumab (TRUCD37Antibody-based 016; homodimeric Emergent Product

single-chain Phase I study in relapsed/refractory CLL or SLL

therapeutic protein producedcompleted; on phase II the platform otlertuzumab/bendamustine vs.

Development, Seattle of ADAPTIR™ LLC) antibody-

study

of

consistingbendamustine of alone ongoing (NCT01188681).

derived, single-chain fragments

variable

linked to Ig constant domain IMGN529 (ImmunoGen )

Phase I study in relapsed/refractory NHL CD37Coniugate of anti-CD37 antibody K7153A ongoing (NCT01534715). with the maytansinoid, DM1.

MOR208 (XmAb5574, MorphoSys AG)

Phase a I study in relapsed/refractory CLL CD19Humanized IgG1 mAb with engineered completed; phase constant fragment designed to enhance FcgammaRIIIa

(Fc)–domain II study in patients with CLL and NHL ongoing (NCT02005289). binding

of

MEDICD19Humanized 551(MedImmune/ humanized IgG Astra Zeneca)

afucosylated Phase I/ II, dose-escalation study in relapsed or refractory advanced B-cell malignancies in previously treated with kappa anti-CD19 mAb rituximab ongoing (NCT00983619); phase II study of MEDI-551 combined with bendamustine in (NCT01466153) .

relapsed

CLL

ongoing

3 7

Lucatumumab ((HCD122, ;Novartis Pharmaceuticals)

CD40Fully human IgG1 isotype mAb Phase I study in relapsed CLL completed. targeting human CD40, blocking CD40/CD40L interactions

Dacetuzumab (SGNPhase I study in pre-treated CLL completed CD40Humanized anti-CD40 IgG1 mAb 40, (NCT00283101) Seattle Genetics) Milatuzumab (hLL1, Immunomedix)

Phase I study in pre-treated CD74Humanized monoclonal antibody completed(NCT00603668);

Phase I/II study of hLL1-DOX in relapsed NHL and CLL ongoing ( NCT00868478).

Abbreviations: ADCC - antibody-dependent cell mediated cytotoxicity; CDC - complementdependent cytotoxicity; CLL – chronic lymphocytic leukemia; Ig – immunoglobulin; mAb monoclonal antibody; NHL – non-Hodgkin lymphoma.

Table 3. Clinical trials of anti-CD20 monoclonal antibodies used alone in chronic lymphocytic leukemia N

Patiet characterisics

OR

CR

PFS

Hainsworth R 375 mg/m2 weekly, x 4 et al 2003 [5]

66

Untreated

51%

4%

18.6 months

Itala et al R 375 mg/m2 weekly, x 4 [9]

24

Refractory/relapsed 35%

0

Huhn et al R 375 mg/m2 weekly, x 4 2001 [8]

30

Refractory/relapsed 25%

0

12.5 weeks (3 months) 20 weeks

O’Brien et R escalated doses 375- 40 al [7] 2250 mg/m2 weekly x 4

Refractory/relapsed 36%

0

8ms

Study

Treatment

CLL

(2275%)

Wiernik & R 375 mg/m2 weekly, x 8 23* Refractory/relapsed 90.9% 63.6% 28.5 + escalating doses up to

3 8

Adiga[12]

3g/m2 per dose progressing pts

in

months**

Byrd et al R 375 mg/m2 3 times 33 [6] weekly for 4 weeks

Refractory/relapsed 45%

Furman et R 375 mg/m2 followed by 110 Relapsed al 2014 500 mg mg/m2 every 2 weeks for 4 doses and then [82] every 4 weeks for 3 doses, for a total of 8 infusions

13%

3%

10 months

0

5.5 months

Coiffier et OF - 100 - 2000 mg 33 50% 0 al. 2008 weekly, x4 Relapsed/refractory for higher [15] doses Wierda et OF dose 1 - 300 mg; doses FA-ref and BF-ref 58% al. 2010 2 to 12 - 2,000 mg 138 CLL. [16]

Byrd et al OF dose 1 - 300 mg; Relapsed 2014 [17] doses 2 to 12 - 2,000 mg 196 refractory Cartron et OB 400 - 1200 mg x8 al 2014 [19]

33

Relapsed refractory

and 4.1%

NR

5.7months in FA-ref group; 5.9 in BF-ref group 0 8.1months

or 62% 0 phase I;30% phase II

10.7 months

Abbreviations: R - rituximab; OF- ofatumumab; OB – obinutuzumab; FA-ref - fludarabineand alemtuzumab-refractory; BF-ref- fludarabine-refractory CLL with bulky (> 5 cm) lymphadenopathy; NR-not reported; OR – overall response; CR – complete response; PFS – progression-free survival;* 10 patients were previously untreated; ** 11 patients received maintenance with rituximab

3 9

Table 4. Clinical trials of novel monoclonal antibodies in chronic lymphocytic leukemia Study

Treatment

Patient

N OR

characteristics

Median response duration

Byrd et al. Otlertuzumab 0.03 - 20 Relapsed/refractory 23%- PR 2014 [55] mg/kg in dose-escalation 83 phase and 10 - 30 mg/kg in dose-expansion phase x 8, weekly + x4 monthly

289 days

Hamadani MEDI-551 - 3 cycles et al 2013 [ 42]

NR

Relapsed/refractory, 20% previously treated 26 with rituximab

Woyach et MOR00208 0.3 mg/kg - Relapsed/refractory 66.7% al. 2014 12 mg/kg x 9 27 [40]

199 days

Byrd et al Lumiliximab 125 -500 Relapsed/refractory Reduction of NR 2 2007 [46] mg/m weekly × 4 or 500 46 lymphocytosismg/m2 3 × first week, 91%, no PR or weekly × 3 or 500 mg/m2 3 CR × per week × 4wks Byrd et al Lucatumumab 0.3 mg/kg, Relapsed 20012[62] 1.0 mg/kg, 3.0 mg/kg, 4.5 mg/kg or 6.0 mg/kg.

17 pts- stable 76 days 26 disease 1 pt nodular PR 230 days.

Furman et Dacetuzumab 3, 4, 6, or al. 2010 8 mg/kg x 6 doses over 5 [66] weeks

5 pts- stable NR 12 disease

Relapsed

Abbreviations: NR-not reported; OR – overall response; R-rituximab; CR – complete response ; NR – not reported; PFS – progression free survival;

4 0

Table 5. Clinical trials of anti-CD20 monoclonal antibodies combined with other targeted drugs in chronic lymphocytic leukemia Study

Treatment

Patient characteristics

N

OR

CR Median PFS

Burger et al. Ibr (420mg/d) + R 375 mg/m2 weekly in cycle 1, then once per cycle until 2014 [75] cycle 6, followed by single-agent Ibr.

Previously treated 40 high-risk (n=36), and untreated with 17p del or TP53 mutation (n=4)

95%

8%

78% PFS

at

18 months

et B(70 mg/m2) + R(375 30 mg/m2 or 500 mg/m2 on Relapsed/refractory D1) x 6 cycles + fixed dose Ibr (420 mg/day)

93%

5 pts

90% PFS at 12 months

Ibr 420 mg daily, in 2824 day cycles, until disease et al. 2014[ progression + 300 mg on Relapsed/refractory d 1 cycle 2, followed by 2000 mg on D8, 15, and 78] 22 of cycle 2, D1, 8, 15, and 22 of cycle 3, and on D1 of cycles 5-8.

100%

NR

NR

et R 375 mg/m2 followed Relapsed by 500 mg mg/m2 every 2 al 2014 [82] weeks for 4 doses and then every 4 weeks for 3 doses, for a total of 8 infusions + Id (150 mg/d) if progression 300 mg x2/d

81%

0

Median not reached; 93% at 24 weeks

66%

12% 17.4

Brown al. 2013 [76] Jaglowski

Furman

110

Badoux et R 375 mg/m2 weekly 59 al 2013 [88] during cycle one and on day 1 of cycles 3- 12 + L Relapsed/refractory 10 mg/d until clinical benefit James et al L 2.5 – 5 mg/day for 21 Treatment-naive days/cycle + R 50 + 325 2014 [89] + 375mg/m2 in cycle 1 and

40<65 95% yrs 78% 29>65

months

19 20% months 20

4 1

375 mg/m2 cycles 2 - 7

yrs

Ferrajoli et O- weekly x 4 weeks Relapsed (300 mg week 1; 1,000 al 2013 [90] mg weeks 2-4; monthly during ms2–6 and every other m during months 7– 24 + L - 10 mg PO/day from day 9 for 24 months

34

Costa et al O 2000 mg d 1 + L 10 21 2014 [91] mg d s 8-28 x 6 Relapsed/refractory

11% months 22

68%

24% months

47.6% 0

NR

Abbreviations: L- lenalidomide; Ibr- ibrutinib;Id – idelalisib; NR-not reported; O ofatumumab; B- bendamustin; OR – overall response; R-rituximab; CR – complete response ; PFS – progression free survival;

4 2

Caption to the figure 1. Fig 1. Schematic presentation of targeted drugs

approved or potentially useful in CLL

and their targets.

4 3