Chapter 2 FIGURE 1 Timeline of Clinical Development and Approval of CD20 Targeting Antibodies

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The handle http://hdl.handle.net/1887/68231 holds various files of this Leiden University dissertation.

Author: Engelberts, P.J. Title: CD20 as target for immunotherapy Issue Date: 2019-01-10

CD20 REVIEW

The evolution of immunotherapy: lessons learned from targeting CD20

"" Patrick J. Engelberts1, 2, Danita H. Schuurhuis1, Esther C.W. Breij1, Paul W.H.I Parren1, 2, 3

1 Genmab B.V., Uppsalalaan 15, 3584 CT Utrecht, The Netherlands

2 Department of Hematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands

3 Lava Therapeutics, Onderwijsboulevard 225, 5223 DE ‘s Hertogenbosch, The Netherlands

CD20 review 19

INTRODUCTION This review gives an extensive overview of pre-clinical and clinical investigations CD20 represents one of the most successful with CD20-targeting immunotherapeutic immunotherapy targets. The inclusion of rit- regimens, with the focus on lymphomas uximab (RTX; Rituxan®, MabThera®) into the and leukemias, and provides future per- treatment regimen of lymphoma patients spectives. significantly improved the overall survival of these patients. This revolutionized the im- CD20 EXPRESSION AND FUNCTION munotherapeutic landscape by showing the potential of monoclonal antibodies (mAbs). Expression on normal B cells and Since the initial approval of RTX in relapsed protein structure and/or refractory low-grade non-Hodgkin’s CD20 was initially described by Stashenko lymphoma (NHL), label expansions followed. et al. in 1980 as B1 [1], but has also been RTX continues to be the backbone for first- denoted as leukocyte surface antigen 16 line treatment of B-cell lymphoma and leu- (Leu-16), B1 polypeptide of 35-kDA (Bp35) kemia despite newly approved CD20 mAbs, [2] and membrane-spanning 4-domain and is one of only three antibodies on the family, subfamily A (MS4A) [3]. CD20 was WHO essential medicines list (www.who.int/ the first cell-surface antigen on human B medicine/publications/essentialmedicines; cells to be identified by a mAb and as such 6th list, Amended March 2017). Despite the became part of the cluster of differentiation success of CD20-targeted therapies, signifi- (CD). CD antigens are cell surface molecules cant numbers of patients still fail to respond expressed on leukocytes and other immune or become refractory upon treatment, even cells [4]. The CD nomenclature provides a though the target antigen generally contin- unified designation system for mAbs and ues to be expressed on their tumor cells. for the cell surface molecules they recog- This is stimulating research into new and nize. The 1st international workshop on improved CD20-directed immunotherapies human leukocyte differentiation antigens (see Figure 1, Table 1). CD20 expression is was in 1982 and since then, more than 350 restricted to B cells, but is not expressed on CDs have been assigned [5]. A subset of lymphoid progenitor cells, and expression is pre-defined CDs is used to phenotype the lost when B cells differentiate into plasma different human leukocyte subsets. CD20 is cells. CD20-targeting antibodies do not part of the CD-markers used to distinguish distinguish between malignant and non-ma- B-cells from other leukocytes [6]. lignant cells, but depletion of healthy CD20+ Although CD20 was discovered more than cells is well tolerated. Clinical research with three decades ago, its exact structure has CD20 antibodies was also expanded to other not been solved by crystallography due diseases in which a role for B cells was to the complex membrane topology of the hypothesized (e.g. rheumatoid arthritis (RA) protein. CD20 spans the membrane four and multiple sclerosis (MS)). times [7, 8], with a small extracellular loop of

CD20 review 21 ‘85 Naked antibody Radio-labeled antibody ‘86 (Glyco)-engineered antibody

1F5 ‘87 Bispecic antibody Bold FDA-approved Cancer 90Y-ibritumomab tiuxetan (2B8) ‘93 Auto-immune disease Rituximab (2B8) ‘94

‘95

‘96

Rituximab (2B8) ‘97 Rituxan

131I-tositumomab (B1) ‘98

‘00

‘01

90Y-ibritumomab tiuxetan (2B8) ‘02 Zevalin

131 ‘03 I-tositumomab (B1) Bexxar

‘04

Ocaratuzumab (2H7) ‘05 CD20bi (2B8) Rituximab (2B8) ‘06 Rituxan PRO131921 (2H7) ‘07 Ocrelizumab (2H7) Ofatumumab (2F2, OMB157) ‘08 TRU-015 (2H7) Obinutuzumab (GA101) Ofatumumab (2F2) ‘09 Veltuzumab (2B8) Arzerra FBTA05 (TPA10) ‘10 Ublituximab (CAT13 LFB-R603)

‘11

‘12

Obinutuzumab (BLY1) ‘13 Gazyva

REGN1979 ‘14

CD20 TDB (BTCT4465A, RG7828) ‘15

XmAb13676 ‘16

B001 Ocrelizumab (2H7) ‘17 CHO-H01 Ocrevus

GEN3013 ‘18

22 Chapter 2 FIGURE 1 Timeline of clinical development and approval of CD20 targeting antibodies. The figure provides an overview of clinical stage therapeutic antibodies. Timing of first entry into clinical development is shown below, and timing of FDA approval is shown in bold above timeline. Indications where anti-CD20 therapy is investigated /approved is indicated in symbols ( = cancer, = auto immune disease). The original clone from which the antibody was derived is shown in parenthesis, or (in case of bispecific molecule) the current development name is provided. Color coding refers to the antibody format used.

5 amino acids (aa)1 and a large extracellular served, but normal B cell development and loop of 44 aa The N- and C-terminus are antibody responses were retained [13]. In a located in the cytoplasm. patient that expressed non-functional CD20 Attempts to recombinantly produce soluble due to homozygous mutations, normal CD20 were only partially successful, since B-cell development occurred, although a binding of CD20-targeting mAbs required reduced capacity to elicit antibody re- specific conformations that were not responses to specific antigens was observed tained in the protein produced [9, 10]. RTX [14]. It has been well established that CD20 requires an intact disulfide bridge within is involved in mediating transmembrane the large extracellular loop to recognize calcium fluxes [15, 16] and is important for CD20. Reducing the disulfide bridge, by B-cell activation [17], differentiation [8, 17] treating the CD20 antigen with dithioth- and proliferation [17, 18]. reitol (DTT), resulted in loss of RTX binding, In normal physiology, CD20 is expressed on which was restored after re-oxidation [9]. more than 90% of the B cells in peripheral As no other disulfide bridge is present in blood and in lymphoid organs. Although it CD20, the small loop does not have such a has been suggested that CD20 was ex- stability-providing structure and thus can pressed at intermediate to low levels in a only partially be mimicked by constrained small subpopulation of normal CD3+ T cells peptides [11, 12] indicating the complexity [19-24], this was later found to be incor- and dynamics of the molecule. rect. CD20 expression in T cells was the result of trogocytosis, extraction of surface The exact function of CD20 remains elusive. molecules from other cells during contact Despite the close association of CD20 ex- [4, 25], a phenomenon that impacts diagnosis pression with B cell development, absence and mAb-based therapies of malignancies of CD20 protein only resulted in a mild [26, 27]. CD20 is first expressed at the pre-B phenotype. In CD20 knock out mice a small cell stage, before IgM is expressed on the reduction in number of B cells was ob- cell surface. The expression of CD20 con-

1 Inconsistencies exist in literature in the reported number of surface-exposed amino acids in the small loop. Based on the properties of the amino acids (hydrophobic Isoleucine and rigid proline), we define, in this review, IP-PI as the boundary of the transmembrane stretches, resulting in five surface-exposed amino acids in the small loop.

CD20 review 23 Bone Marrow CD20 expression Start BCR expression Light chain Start CD20 rearrangement expression

CLP B B B

pro-B cell pre-B cell naive B cell ALL

Blood 1st Antigen exposure CD20 expression B CLL

mature B cell non-mutated

Lymph node short-lived plasma cell CD20 expression P

Marginal Zone

B MCL Mantle Germinal center

B B B Somatic hypermutation Class switch

GC DLBCL FL

MZL

B 2nd Antigen exposure memory B cell

Blood Bone Marrow CD20 expression CD20 expression mutated CLL

End BCR expression ABC B P MM DLBCL End CD20 expression plasmablast plasma cell

24 Chapter 2 Bone Marrow FIGURE 2 Stages of B cell Development and associated malignancies. CD20 expression Start BCR expression B cells arise from common lymphoid progenitor (CLP) cells and undergo various maturation stages. Each step Light chain Start CD20 could give rise to a specific malignancy. ALL = Acute lymphatic Leukemia; CLL = Chronic lymphocytic leukemia; rearrangement expression FL = Follicular lymphoma; GC DLBCL = Germinal center Diffuse large B cell lymphoma; ABC DLBCL = Activated B cell Diffuse large B cell lymphoma; MM = Multiple myeloma.

CLP B B B pro-B cell pre-B cell naive B cell tinues throughout B cell maturation until EXPRESSION IN B CELL ALL the plasmacytoid immunoblast stage. CD20 MALIGNANCIES is not expressed on hematopoietic stem Blood 1st Antigen exposure cells, pro-B cells, plasma cells or on other A wide variety of studies have analyzed CD20 expression normal non-B cell lineage tissues [28, 29]. CD20 antigen expression in B cell malignan- B CLL The normal B cell development stages are cies and correlated this with expression on mature B cell non-mutated reflected in B cell malignancies, which often the normal B cell counterpart. More than Lymph node short-lived echo dominant clonal expansion of a specif- 90% of human B cell lymphomas and most plasma cell CD20 expression ic maturation stage (Figure 2) [6], although B cell leukemias express CD20. Expression P antigen exposure and stimulation may also of CD20 was found to be lower on B cell play a key role in the onset of B-cell malig- chronic lymphocytic leukemia (B-CLL) and nancies [30]. B cell acute lymphoblastic leukemia (B-ALL) Marginal Zone than on normal peripheral B cells or on In resting B cells, CD20 and the B-cell re- other malignant NHL [36-41]. In B-ALL, CD20 MCL B ceptor (BCR, i.e. surface-expressed immu- expression is lower and more heteroge- Mantle noglobulin (Ig)) are uniformly distributed in neous compared to other B-cell malignan- Germinal center the plasma membrane. CD20 is translocated cies. CD20 expression heavily depends on B B B to cholesterol- and sphingolipid-rich micro the differentiation stage of the originator Somatic hypermutation Class switch domains, referred to as lipid rafts or deter- cell and CD20 on B-ALL thus reflects the GC gent-insoluble glycolipid-enriched struc- low expression on pro-B-cells, early pre- DLBCL FL tures (DIGS), upon cross-linking by a subset B-cells and pre-B-cells from which the of CD20 antibodies, the so-called type I tumor derived [42]. B-CLL derives from MZL CD20 antibodies [31, 32], and/or engagement antigen-experienced B lymphocytes and of the BCR by antigen [33]. BCR and CD20 can be classified based on whether the cells co-localize initially, but rapidly translocate have undergone somatic hyper mutation B 2nd Antigen exposure to distinct lipid rafts, followed by endocyto- (mutated immunoglobulin heavy-chain memory B cell sis of the BCR [33]. A central feature of lipid variable region (IGHV)), or not (unmutated Blood Bone Marrow rafts is their ability to selectively include IGHV). Further stratification can be done CD20 expression CD20 expression mutated CLL or exclude membrane proteins. Transloca- based on other factors such as e.g. chromo- tion of CD20 and the BCR into lipid rafts is somal abnormalities and BCR signaling. This End BCR expression an extremely rapid process, and is directly heterogeneity of the disease is reflected ABC B P MM DLBCL End CD20 followed by phosphorylation of typical raft in CD20 expression, which for B-CLL cells expression [43-45] plasmablast plasma cell proteins like Lyn, which initiate the BCR sig- varies with the genetic subtype and naling cascades [34, 35]. the anatomical location of the tumor cells.

CD20 review 25 Indeed, CD20 expression levels were higher sion in pediatric B-ALL might offer at least on B-CLL cells obtained from peripheral a partial explanation for this difference blood than on cells isolated from bone mar- [59]. The addition of RTX to the hyperCVAD row and lymph nodes aspirates [46]. chemotherapeutic regimen (alternating In the WHO revision of the classification of cyclophosphamide, vincristine, doxorubicin lymphoid malignancies in 2016, a distinc- plus dexamethasone with methotrexate and tion is made in 35 different types of lym- cytarabine) for previously untreated B-ALL phoma and myeloma [47]. As it will go too patients, stratified for CD20 expression far to describe the role of CD20 expression (cut off > 20% CD20+ cells), impacted the in all of these subtypes, we will focus on overall survival (OS) for younger (age < 60 the more common types, such as follic- years; OS 75% vs 47%), but not older (age > ular lymphoma (FL), diffuse large B-cell 60 years; OS 64% vs 65%) patients [60]. This lymphoma (DLBCL), mantle cell lymphoma suggests that inclusion of CD20-directed (MCL), marginal zone lymphoma (MZL) and antibodies to the therapeutic regimen of multiple myeloma (MM). The level of CD20 B-ALL patients may improve therapeutic expression in these malignancies, from high outcome of at least a proportion of the to low, is DLBCL > FL > MCL > MZL > MM patients. However, it should be noted [38-40, 48]. Although generally plasma cells that the observed difference could also do not express CD20, it is well documented be based on patient selection, as it has that a small portion of MM are CD20-pos- been reported that CD20 expression was itive, yet expression is lower than on MZL absent in the high risk patient group with [49-53]. This is often associated with the translocation t(4;11) [56]. In standard-risk t(11;14) translocation and/or IgG isotype patients, CD20 expression was associat- [49, 50, 54, 55]. Waldenström Macroglobu- ed with poor survival [61]. As previously linemia (WM; where CD20 expression is described, CD20 expression in CLL (both in present, but lower than on other lymphoma percentage of cells expressing CD20 and in subtypes [46]), can be considered a pre- number of CD20 molecules expressed per stage of MM. cell) was lower compared to other B-cell lymphomas and normal peripheral B cells RELEVANCE OF CD20 [36, 38, 40, 41]. When taking into account that EXPRESSION IN MALIGNANCIES CD20 expression on B cells from lympho- FOR TREATMENT ma patients correlates both with response rate and overall survival when applying The prognostic role of CD20 expression in RTX-containing therapy [62], this would ALL is still cause for debate, as in child- not bode well for CD20-directed immu- hood-precursor B-ALL, CD20 expression is notherapies. Nevertheless, a clear benefit associated with increased survival, in con- of including CD20-targeting antibodies to trast to adult B-ALL where this is not the the treatment paradigm of patients with case [56-58]. The discovery that the addition CLL has been observed in various studies. of prednisone to the treatment regimen re- In previously untreated CLL patients, the sulted in the upregulation of CD20 expres- addition of CD20 antibodies to chlorambu-

26 Chapter 2 cil treatment resulted in a greater efficacy from previously untreated patients) as for all tested CD20 antibodies compared a prognostic marker for outcome is well to chlorambucil alone [63, 64]. This was also documented, the role of CD20 expression found for relapsed/refractory CLL, where should not be over interpreted as many OFA + fludarabine (F) + chlorambucil (C) CD20-directed immune therapies require (O-FC) and RTX-FC (R-FC) both improved CD20 positivity, determined by immuno- outcome compared to patients treated with histochemistry (IHC) as one of the inclusion FC alone [65, 66]. criteria. In DLBCL, high CD20 expression was a prognostic for better overall survival inde- TWO TYPES OF CD20-SPECIFIC pendent of treatment (cyclophosphamide, MONOCLONAL ANTIBODIES doxorubicin, vincristine plus prednisone (CHOP) or R-CHOP) [67]. Inclusion of RTX Soon after the discovery of CD20, a dichot- to the CHOP treatment regimen further omy in the effector activity of CD20 mAbs increased the overall survival compared to was observed. Golay et al. described that CHOP alone, although this was dependent two CD20 mAbs, 1F5 and B1, both inhibited on CD20 expression and heterogeneity immunoglobulin (Ig) production of activated thereof [67-70]. B cells. However, only 1F5 was capable of activating resting B cells [17]. Deans et al. The prognostic significance of CD20 ex- showed that some CD20 mAbs can induce pression in Hodgkin’s lymphoma was found translocation of CD20 into lipid rafts [31], an only when present on the B cells [71], but activity which appeared to be independent not when expressed on Reed Sternberg cells of the epitope recognized [16]. Cragg and (the large multi-nuclear cells of B-cell origin Glennie proposed a functional classification that are a hallmark of Hodgkin’s lymphoma of CD20 antibodies into categories based and are used for diagnosis [72]), perhaps on their in vivo killing capacity. The type due to the fact that CD20 expression on the I antibodies RTX and 1F5 were shown to latter cells was much lower compared to use complement activation to eradicate that on morphologically normal B lympho- lymphoma xenografts, whereas the type cytes [73]. Besides the possible prognostic II antibody B1 did not use complement to role of CD20 expression on malignant cells, eradicate the lymphoma xenografts [77]. the presence of circulating CD20 (cCD20) The type I/II classification of CD20 antibod- in the sera of NHL patients was shown to ies provides insight into the mechanism negatively correlate with progression-free of action employed. Type I CD20 antibod- survival [74, 75]. Since the general consensus ies translocate CD20 in raft domains and is that CD20 cannot be shed, this cCD20 induce complement-dependent cytotoxicity likely reflects the remainder of killed CD20+ (CDC), whereas type II CD20 antibodies cells, a fact underscored by the association do not, but cause homotypic aggregation of cCD20 with tumor burden in CLL [76]. (HA), followed by programmed cell death Although the role of CD20 expression (often (PCD) to elicit cell death. The ability to determined by flow cytometry on samples recruit effector cells for antibody-depen-

CD20 review 27 dent cell-mediated cytotoxicity (ADCC) or RTX has a first-line approval for many of antibody-dependent cell-mediated phago- these indications. cytosis (ADCP) is shared by both classes of As many patients eventually relapsed CD20 antibodies. A molecular characteris- or became refractory to RTX treatment, tic, which may explain the difference was novel CD20-targeting antibodies have been identified by Niederfellner and colleagues, developed. New antibodies were often by comparing crystal structures of RTX and selected based on superior efficacyin vitro obinutuzumab (OBZ, GA101) in complex compared to RTX. In Table 1 the approved with CD20-derived peptides [11, 78]. They CD20 antibodies are listed, whereas in observed a ~70⁰ difference in orientation of Table 2 and Table 3 clinical studies with Fab fragments of RTX and OBZ binding to a CD20-directed molecules in the US and CD20 peptide due to their differential inter- Europe, respectively, are listed. action with asparagine residues at positions 171 and 176. It is thought that the angle at OFA, a next generation fully human CD20 which the antibody binds the CD20 strong- antibody targeting a different epitope than ly impacts its pharmacodynamic activity. RTX, was selected based on its superior CDC induction and efficacy at lower antigen CD20 ANTIBODIES FOR THE density compared to RTX [12, 84, 85]. OBZ, TREATMENT OF NHL a humanized antibody, was generated from one of the earliest CD20 mAbs (Bly- In 1997, RTX became the first CD20 anti- 1), and enhanced in its ability to induce body approved for clinical use. RTX rep- programmed cell death (PCD) and NK cell- resents a chimerized variant of the murine mediated lysis by protein engineering and anti-human CD20 mAb 2B8 (C2B8) [79]. glyco-engineering, respectively [86]. Both Since then, RTX has continued to transform mAbs are now FDA- and EMA-approved for the therapeutic landscape of lymphoma the treatment of CLL. Interestingly, despite and B-cell malignancies, with various large these completely different in vitro modes studies showing the undeniable benefit of action in destroying CD20+ cells, their of adding RTX to chemotherapeutic stan- therapeutic efficacy was quite compara- dards of care [80-83]. Originally approved ble. In previously untreated CLL patients, for relapsed, refractory indolent NHL, RTX treatment with OBZ plus chlorambucil (G-C) is currently approved for NHL (R-CHOP resulted in a median progression free sur- (aggressive NHL such as DLBCL) or R-CVP vival (PFS) of 26.7 months [63], whereas in (low grade NHL)), and CLL (R-FC). RTX, as a similar study OFA plus chlorambucil (O-C) a B cell-depleting agent, displays efficacy the median PFS was 22.4 months [64]. The beyond lymphoma with approval in the similarity in clinical outcome of CLL treat- autoimmune diseases rheumatoid arthritis ment with either OFA or OBZ also questions (RA) (in combination with methotrexate) the specific contribution of distinctin vitro and granulomatosis (in combination with mechanisms of action to in vivo efficacy. glucocorticoids) (summarized in Table 1). Various CD20-targeting antibodies, either novel or altered, have been preclinical-

28 Chapter 2 ly investigated to unravel the respective study where ublituximab plus ibrutinib was roles of PCD, ADCC, ADCP and CDC. Table 4 compared with ibrutinib alone in high-risk summarizes the antibody based mole- CLL patients. The combination resulted in a cules derived of selected CD20 antibodies 78% overall response rate (ORR), compared together with their reported mechanisms of to an ORR of 45% for ibrutinib alone. No action, a number of these antibodies have head-to-head comparison with other CD20 been evaluated in clinical settings (Figure 1, antibodies has been done, but it is expected Table 2, Table 3). that the FDA will approve this combination Interesting novel concepts, such as type I/ for high-risk patients soon, so clinical stud- type II intermediates which combine the ies are likely to expand [102]. Ocrelizumab mechanisms of action of both type I and (PRO-70769), another CD20 targeting anti- type II CD20 antibodies, such as CDC and body based on 2H7, was also tested in lym- PCD, were found to be very effective in phoma [91], where activity was observed in vitro and in vivo, even demonstrating supe- patients with relapsed/refractory FL after rior B cell depletion in the lymph nodes of prior RTX therapy, but this therapeutic area cynomolgus monkeys compared to RTX. So was abandoned to pursue multiple sclerosis far, these molecules have not entered clini- (MS) and other autoimmune indications. cal studies [87, 88]. Other studies were aimed Additional CD20-targeting antibodies, at enhancing specific attributes of antibody which display distinct activities in vitro and functions. Enhancing the affinity (reduced some of which bind to novel epitopes, have off-rate) [89], ADCC activity (de-fucosylation been described: 8E4 [103], 5S [104], OUBM [90, 91], or unknown method) [92], CDC [93, 94] [105], 1K1791 [106] and 7D8, 11B8 and 2C6 or all of them [95, 96] resulted in superior [12, 84, 85]. It will be interesting to see how in vitro efficacy compared to RTX. Of these these will compare in vivo to the currently compounds PRO131921 [97], ocaratuzu approved antibodies. Expiry of the patent mab [98] and veltuzumab [91] made it into protecting RTX has led to a widespread early clinical development, although none development of RTX biosimilars, some of of them have been investigated beyond a which have already obtained approval in phase 1/2 study. This is also the case for the EU (Table 1) or are under review by the CD20 small modular immune-pharmaceuti- FDA. cals (SMIP). SMIPs are IgG1-like molecules that contain a single-chain variable frag- CD20 ANTIBODIES USED FOR ment (scFv) as binding region instead of a TARGETED DELIVERY Fab fragment. 2LM20-4 [99] and TRU-015 [100], both showed superior efficacy inin Conjugation of CD20 antibodies with cyto- vitro ADCC and CDC compared to RTX. TRU- toxic payloads has been applied as a means 015 was studied in a phase 1/2 trial, but of generating more potent anti-CD20 mol- this trial was terminated before completion. ecules. Conjugated CD20 antibodies can be Ublituximab (LFB-R603, TG1101) [101] is divided into three groups. The first group a glyco-enhanced type I CD20 antibody consists of radio-immunoconjugates, aimed that showed promising results in a phase 3 at killing tumors by delivery of a radio-

CD20 review 29 active payload. Antibody-drug conjugates tory to RTX therapy, treatment with Zevalin represent a second group, aimed at killing resulted in a 67% ORR with an 11.8% of the tumor cells via intracellular deliv- median duration of response (DR). This led ery of cytotoxic, chemotherapy-like drugs. to the approval of Zevalin for patients with These molecules require internalization and relapsed or refractory low grade follicular processing in lysosomes for the toxic agent or transformed B cell non-Hodgkin’s lym- to be released. The third group comprises phoma. Studies in CLL found unacceptable antibody molecules labeled with an agent hematologic toxicity [110]. The therapeutic that is designed to enhance therapeutic efficacy of Zevalin can be mostly attributed efficacy without being cytotoxic, e.g. a to the radiolabel [111]. After all, ibritumom- cytokine. ab represents the mouse antibody parent (2B8) of RTX and the studies above there- RADIOLABELED CD20 ANTIBODIES fore highlight the increase in potency that may result from radiolabeling. 90Y-ibritumomab tiuxetan and 131I-tositumumab are radio-labeled CD20 antibodies The second approved radiolabeled an- and used as part of the FDA-approved radio ti-CD20 therapy was Bexxar. This thera- immunotherapy of respectively Zevalin® peutic regimen consists of a combination of and Bexxar® (Table 1) [107]. Zevalin, ap- tositumomab and 131I-tositumomab, which proved in 2002, was the first radiolabeled are applied in a dual administration with a anti-CD20 therapy. The therapeutic regi- dosimetric and a therapeutic part. Davies men of Zevalin contains two steps: step 1 wrote a comprehensive review on the includes a first infusion of RTX preceding clinical experience with Bexxar [112]. The 111In-ibritumomab tiuxetan to determine Bexxar regimen showed excellent clinical bio distribution; step 2 (7-9 days later) responses, especially in the first-line setting consists of a second infusion of RTX fol- [113], but was also effective in patients who lowed by 90Y–Zevalin. This was changed in had received prior RTX treatment [114]. As 2011 to two infusions of RTX followed by suggested above for Zevalin, the efficacy 90Y–ibritumomab tiuxetan, when the FDA of Bexxar was also mainly driven by the approved the removal of the 111In-ibritu- radiolabeled component [115]. Re-treatment momab tiuxetan imaging step, because the with Bexxar resulted in low efficacy, likely contribution to patient safety was found to due to the relative high level of human be negligible [108]. In a head-to-head com- anti-mouse antibodies (HAMA), because of parison between 90Y-ibritumomab tiuxetan immunogenicity of the mouse antibody in and RTX, the former showed a significantly humans [116]. Myelodysplastic syndrome higher ORR than RTX alone (80% vs 56%); (MDS) has also been reported as adverse the secondary endpoints, duration of re- event. However, in several studies no cor- sponse and time to progression, were not relation was observed between the radiola- significantly different between both arms bel and onset of MDS. The development of [109]. However, in a single-arm study of 30 MDS in NHL patients was similar for regi- patients who relapsed after, or were refrac- mens with or without a radiolabel [117]. The

30 Chapter 2 strong potential of Bexxar was highlighted ization has been described to be driven by in a follow-up study of a head-to-head com- BCR clustering and IgG Fc receptor (FcγR) parison of Bexxar and RTX in FL [118]. The expression, predominantly through the addition of Bexxar to the CHOP regimen interaction of type I CD20 antibodies with (CHOP-RIT) resulted in a significantly better FcγRIIb [127, 128]. Nevertheless, this has 10-year PFS compared to R-CHOP (56% vs been an area of controversy, as Vervoordel- 42%). OS was not significantly different be- donk et al. [129] demonstrated that the type tween the two treatment arms, neither was I CD20 antibody 1F4 did not internalize, the incidence of secondary malignancies irrespective of the isotype tested (IgG1, or MDS. Bexxar was withdrawn from the IgG2a, IgG2b and IgM), whereas the control market in February 2014, due to the low target CD19 did show antibody-induced number of patients being treated. internalization. Press et al. [130] demonstrated that the type II antibody B1 did not Some radio-immunotherapy (RIT)-relat- internalize. Despite contradictory studies ed studies in the pre-clinical setting have described in literature, it is now accepted been performed with the aim of validating that CD20 internalization occurs, but at a new radiolabels [119, 120] and/or novel relatively slow rate. Michel and Mattes [123] CD20-specific antibodies for use as carriers demonstrated that 111In-labeled anti-CD74 [121-123]. Radiolabeled antibodies have also or anti-MHC class II was internalized at been employed in mechanistic studies to in- a rate of 107 antibody molecules per 24 vestigate bio distribution and tumor pene- hours, but that 111In-labeled anti-CD20 tration of CD20 antibodies [120-122, 124, 125]. only reached ~4x106 intracellular mole- Radio-conjugates represent useful tools to cules per 48 hours. The fact that CD20 is study tumor penetration in vivo and might a relatively slow internalizing target was even be used to determine the tumor bur- further demonstrated in a study where den of patients in the more indolent setting, CD20 immunotoxin (IT) (CD20 antibody in the near future. Considering the wide conjugated to the plant toxin saporin S6) acceptance of unlabeled CD20 antibodies was compared to CD22 IT [131]. Even with combined with the negative perception of the higher drug–to-antibody ratio (DAR, RIT by physicians [126], it seems unlikely, i.e. the number of drugs attached to the however, that new therapeutic radioactive- molecule) for the CD20 IT, the CD22 IT was ly labeled anti-CD20 antibodies will enter much more potent as a result of the more the clinic, despite their strong therapeutic efficient internalization. potential. The use of CD20 as a target for anti- CD20-SPECIFIC ANTIBODY-DRUG body-drug conjugates may seem somewhat CONJUGATES (ADC) counterintuitive regarding its poor ability to internalize. However, because of the Although CD20 internalization occurs, this highly and selective expression pattern of is a relatively slow process, which is depen- CD20 and the notion that long-term incu- dent on several other antigens. Internal- bation may still lead to sufficient antibody

CD20 review 31 payload internalization to induce effec- BISPECIFIC ANTIBODIES tive killing, various type I CD20 targeting TARGETING CD20 antibodies, such as 1F5, RTX and OFA were conjugated using a variety of payloads and Bispecific antibodies (bsAbs) combine the tested for their ability to induce cell kill. In specificities of two antibodies in one mol- vitro and in vivo efficacy of OFA-vcMMAE ecule. A wide variety of bispecific formats (the tubulin disrupting agent monomethyl exists [136], from bivalent molecules, that auristatin E (MMAE) linked with a cleavable bind each target antigen monovalently, to valine-citruline (vc) linker to OFA) was in- tri- or tetravalent molecules, with an ability vestigated by Li et al. [132] and demonstrat- for multivalent binding of one or both of ed good tumor depletion in vivo. Similar re- the target antigens. BsAbs represent a sults were observed for RTX-vcMMAE [133], promising strategy to increase antibody although here the drug-linker facilitated function, as discussed in [137] and depicted internalization, since unlabeled RTX did not in Figure 3. accumulate in the cell to the same extent as RTX-vcMMAE or RTX-vcDOX (doxirubicin). BsAbs binding to two different epitopes This observation was in agreement with a on CD20 potentially have the advantage study that compared various B-cell targets of combining the MoA of both parental for suitability for an ADC approach [134]. antibodies in one molecule. For instance, Here the choice of linker influencedin vivo combining a type I and type II antibody efficacy, as only CD20-SPP-DM1 was able could lead to a type I/II antibody with su- to delay tumor outgrowth of Granta-519 perior efficacy by being able to apply more and CD20-MCC-DM1 was not. This was effector mechanisms (higher maximum found to be the case for multiple, but not response) compared to the combination of all, B cell targets. Another study, using a both parental antibodies or superior po- different drug, EPI (a N-(2-hydroxypropyl) tency (lower EC50/IC50) as both antibodies methacrylamide (HPMA) copolymer-epi- combined. This also applies to targeting two rubicin conjugate)), demonstrated in vivo antigens on one cell with a bsAb molecule. efficacy that was dependent on the amount Targeting two antigens potentially allows of RTX-bound drug [135]. increased specificity, by only targeting Taken together, there is evidence support- cells that express both antigens, or target a ing the in vivo suitability of CD20 as an ADC more heterogeneous population of cells, by target, albeit dependent on the drug/linker binding to cells expressing one antigen as combination. However, due to CD20’s slow well as binding to other cells expressing the internalization rate, the number of the pay- other antigen. If sufficient binding to one load molecules attached to the CD20 mAb target antigen is obtained via monovalent (DAR) needs to be high. binding, the other arm can be used for other purposes, such as delivery of a cytotoxic compound or recruitment of effector cells.

32 Chapter 2 FIGURE 3 Diverse of applications of bsAb against CD20. E BsAb targeting CD20 currently under investigation aim to target two epitopes on 3 CD20 or two different antigens to target Tu two antigens either on the same cell (to 1 Tu increase specificity) or on different cells CAR-E (to address heterogeneity) (1), or to recruit effecter cells such as NK cells, T cells, macrophages and CAR-Effector (CAR-E) cells (3). Bispecific binding two antigens or two epitopes are also employed to increase specific payload delivery(2) . 2

Many bsAb formats have been described, trifunctional bsAb FBTA05 (Lymphomun, ranging from fragment-based bsAbs, (genet- bi20), many other formats, such as Fc/Fc*, ically fused antibody-binding domains (e.g. T cell dependent bispecific antibody (TBD), domain antibodies or VHH antibodies (also T cell specific antibodies (TCB), Xmab and termed nanobodies; Ablynx); bsAb lacking DuoBody® can be engineered to selectively an Fc domain)), to IgG molecules which render the Fc inert for Fc-driven effector can be either tetravalent (e.g. Fab4-IgG) or functions, while leaving FcRn binding intact. molecules with a regular IgG architecture (reviewed in [136, 138-140]). Applicability TARGETING OF TWO RECEPTORS of the specific bsAb format will mainly be driven by the desired effector functions of Li et al. [147], produced a tetravalent the compound and choice of half-life. Many CD20-targeting molecule, TetraM- of the current bispecific formats in clinical cAb-4-scFv, containing the binding regions evaluation for CD20 targeting are aimed at of both C2B8 (RTX) and 2F2 (OFA) with the recruiting T cells. Here, extension of half- intention to increase the binding avidi- life can be achieved by selection of an IgG- ty compared to the parental antibodies. based format that retains/has FcRn binding, Interestingly, it was observed that the such as knob-into-hole [141], DuoBody [142], ability of this molecule to induce PCD was XmAb [143] and Fc/Fc* (based on differential increased compared to that of the parental protein A affinity)[144] . Other platforms antibodies, both of which are type I CD20 circumvent short half-life by continuous antibodies that induce minimal PCD. When infusion, like BiTe [145] or fusion to albumin including a type II CD20 antibody, that [146]. IgG-based formats have the potential have higher capacity to induce PCD, in the to recruit Fc-mediated effector components construct (11B8/2F2 dual TetraMcAB) the such as complement and FcγR-express- activity could not be further increased, as ing cells of the innate immune system. shown in in vitro and in vivo assays [148]. Where Fc-mediated effector mechanisms The concern of modest therapeutic effi- are claimed as part of the potency of the cacy of mAbs, due to heterogeneity of

CD20 review 33 the tumor and possible antigen-negative only slightly better than the combination relapses, stimulated the research for so of IgG1 antibodies. To increase the poten- called T1xT2 bispecific molecules. HLA-DR cy of the Bs20x22, a hexavalent molecule and CD20 have been previously linked, as with 2 CD20 targeting arms and 4 CD22 they display similar cell aggregation and targeting arms or vice versa, also including antigenic clustering under specific condi- a functional Fc-region was generated. The tions [149] and when administered together addition of an Fc region led to an increase showed synergistic anti-lymphoma activity in PCD in vitro, but only a modest increase [150]. A bispecific dual-variable domain in in vivo efficacy, which was shown to immunoglobulin (DVD-Ig; a tetravalent depend on the presence of NK cells and/or IgG1 molecule containing a second variable neutrophils [153, 154]. The in vivo activity, as domain attached to each of the Fab re- measured by survival of xenograft mice, of gions) molecule targeting CD20 and HLA-DR a bispecific molecule targeting both CD20 induced increased HA, actin rearrangement and CD95 was superior to that of an Fc-en- and apoptotic cell death. Using confocal hanced CD20 antibody, which in turn was and light microscopy, it was demonstrated more effective than a chimeric CD20 mAb that HA induced by this bispecific molecule (all molecules were based on 2H7) [155]. resulted in increased cell-cell contact. The This was even more impressive considering DVD-Ig molecule also showed superior cell the fact that the bispecific molecule had a depletion capacity in a whole blood assay, shorter in vivo half-life than the chimeric where it was able to deplete more Raji cells and Fc-enhanced molecules (less than 2 than the parental antibodies alone or a hours for the bispecific and 4 hours for the combination thereof [151]. other antibodies, respectively). The shorter half-life was due to the size of the molecule Additional dual-antigen targeting bsAbs (both binding arms combined in one Fab- have been evaluated in the CD20 space, arm) and the absence of an FcRn binding such as targeting CD20 in combination with site.

CD22. The F(ab’)2 fragment of RTX chemically crosslinked to the F(ab’)2 fragment DELIVERY OF CYTOTOXIC of HB22.7 (Bs20x22) displayed similar PAYLOADS USING CD20 binding properties as the parental antibod- BISPECIFIC ANTIBODIES ies alone, but showed a greater potency in inducing apoptosis compared to the A second potential application of bsAb is parental antibodies alone or a combination the use as a vehicle to deliver a payload. thereof [152]. Bs20x22 was able to bind This was for example achieved by the de- simultaneously to both targets, even when sign of a bsAb molecule containing a CD20 expressed on different cells. Due to the targeting arm (2H7) in combination with an absence of an Fc region, Bs20x22 was not arm targeting a radiolabel (2H7-Fc-C825). capable of inducing CDC, ADCC and ADCP. This bispecific molecule showed superior In vivo, the bispecific compound inhibited in vivo bio distribution to the tumor (as tumor outgrowth in a Raji xenograft model detected by analysis of residual radioactiv-

34 Chapter 2 ity in tumor samples and normal organs) a tribody (a Fab fragment and two scFv compared to a CD20-targeting antibody molecules fused via a CH1 domain) [161]. that recruits a radioactive compound via This molecule showed enhanced ADCC a streptavidin/biotin linker (1F5-SA) [156]. in vitro compared to the parental CD20 Furthermore, it showed significantly better antibody 7D8 and RTX. This was shown to tumor-free survival in mice. Although it be mediated by an enhanced interaction would have been more accurate to compare with NK cells. Other recruitment strategies 2H7-Fc-C825 with 2H7-SA, this study does mainly focused on neutrophils and macro- show the feasibility of the concept. phages, either by targeting CD89 [162, 163] or CD47 [164, 165]. Fc receptor binding was Another method to combine an active diminished in these formats/strategies by compound with a bispecific molecule was introducing an N297A mutation in the Fc shown by using the dock-and-lock method, domain [162] or by using chemically cross- where IFN-alpha (IFNα) was linked to a linked F(ab’)2 molecules [163]. Stockmeyer CD20xHLA-DR bispecific antibody. Derived et al. showed that the concept of recruiting from the antibodies veltuzumab (CD20) and CD89-positive leucocytes worked in vitro L243 (HLA-DR), this molecule was termed and even outperformed Fc-receptor tar- 20-C2-2b. In vitro efficacy of this molecule geting bispecific molecules[163] . Li further was shown to be more potent than the demonstrated the potential of the bispecific parental antibodies or the combination molecule in an in vivo setting. Since mice do of both parental antibodies. Moreover not express CD89 either human PMNs were 20-C2-2b was able to deplete CD20-ex- added, or CD89 transgenic mice were used. pressing Daudi cells spiked in whole blood, In both models the bispecific molecule while sparing other cells such as T cells showed anti-tumor efficacy however did and endogenous B cells [157]. Whether it is not outperform RTX. clinically feasible to combine both the CD20 binding arm and an IFNα in one molecule T cell-recruiting bsAbs artificially link will have to be determined, but CD20 and T cells with CD20 molecules expressed on IFNα is a potent combination [158-160]. tumor cells and activate T cells upon this cross-linking, to kill the CD20-expressing RECRUITMENT OF EFFECTOR cells. The bsAb r2820 targets CD28 ex- CELLS pression on T cells to induce T cell activation and tumor cell killing [166]. This scFv The final and by far the most applied molecule induces kill of CD20-positive cells approach for CD20-directed bsAb is the by PBMCs. Interestingly, this compound generation of bispecific molecules that can was more effective when combined with recruit effector cells and direct the tumor a CD3xCD20 bispecific antibody[167] . Tar- antigens for tumor cell killing. The addi- geting CD3, by binding to CD3e (alone or in tion of a CD16 targeting Fab fragment to combination with CD3d), is the most widely two CD20 targeting scFv fragments (7D8) used method to recruit T cells to the tumor and an Fc fragment was investigated with antigen in a bsAb. This has been shown to

CD20 review 35 induce cytotoxic synapse formation and three with high-grade NHL), only modest target cell kill, independent of peptide responses of CD20bi in combination with presentation by MHC on the target cell and donor lymphocyte infusions (DLI) were ob- TCR specificity of the recruited T cells[168] . served, with stable disease for four months Redirecting T cell activity towards tumor as best response [177]. In another study antigens can induce dramatic regression of with FBTA05 in pediatric patients, 9 out advanced stage malignancy, as was shown of 10 patients achieved a clinical response for the CD3xCD19 bispecific blinatumomab with five CRs with a maximum duration in heavily pretreated NHL and ALL patients of 1424 days [178]. This was especially [169]. impressive regarding the heavy pre-treatment, including RTX-based regimens for 5 Various CD3xCD20 bsAb molecules such out of 10 patients. These first generation as an anti-CD20 x anti-CD3 diabody [170], CD3xCD20 bsAbs contained immunogenic anti-CD20-LHD-scFC anti-CD3 [171], 20-3S murine sequences, but HAMAs were only [172], BIS20x3 [173] and halfbody CD3xCD20 sparsely reported. In addition, both have [174] were mainly designed for concept, active Fc regions that are expected to platform or clone validation. While these induce non-specific T cell activation and studies were informative from a plat- are therefore undesired. Next generation form perspective, the majority of data on CD3xCD20 bsAbs therefore were designed validity of CD3xCD20 bsAbs comes from to contain an inert Fc tail (with respect to the clinical setting. CD3xCD20 bsAbs have effector function) or they completely lack been in clinical investigation since 2005. an Fc-region. REGN1979, a bsAb containing The first to be administered to patients was novel CD3 and CD20 antibodies generat- CD20bi (a heteroconjugate of the CD3 mAb ed in VelocImmune mice contains an IgG4 OKT3 and RTX). In a phase 1 study togeth- Fc-region to minimize Fc effector functions er with ex vivo anti-CD3 activated T cells [144]. Clinical investigation of REGN1979 (ATC) and autologous stem cell transplanta- started in a phase 1 dose escalation study tion (SCT), 9 out of 15 patients were in CR in NHL. In 2016, 25 patients have been after 90 days, with a median survival of enrolled of whom it was reported that 16 20.9 months [175]. The role of the bispe- patients discontinued treatment, most for cific molecule in this study is difficult to PD [179]. The low initial efficacy is likely assess, due to complexity of the treatment. due to the starting dose, as the dose ranged However, considering that these patients from 0.03-3.0 mg flat dose. In 2017, with were refractory NHL patients in the high dose levels at 5-7 mg flat REGN1979 dose, risk group, the results are impressive. The the ORR was reported to have increased next CD3xCD20 to be investigated in a to 45% [180]. Novel CD3xCD20 bsAbs are in clinical setting was FBTA05 (Lymphomun, development, such as XmAb-13676 [181], Bi20; a mouse-rat chimeric bsAb created RG7828 [141] (both are in phase 1/2 studies with hybridoma fusion technology) [176]. with no data reported), RG6026 [182] and In a small, dose-escalation study with six GEN3013 (expected to enter clinical studies patients (three with p53-mutated CLL and in 2018). It will be interesting to see wheth-

36 Chapter 2 er the differences in platforms on which to the activity of the CAR-T cells, partially these bispecific antibodies are based, will due to low CAR expression as a result of in- result in differences in clinical efficacy. efficient gene transfer[185] . Some of these issues have now been resolved with an op- CAR EFFECTOR CELLS timized vector and it will be interesting to see what the clinical results [186]. In another An alternative approach to redirect effec- clinical study, a phase 2 trial was conducted tor cells to the tumor is by gene therapy, evaluating CAR-T-20 in 11 patients with in which (patient’s) T cells are transfected lymphoma (NCT01735604). Here, 11 out of with a chimeric antigen receptor (CAR). 11 patients obtained a PFS with more than CARs represent scFvs fused to one or 50% achieving a six-month or longer PFS multiple co-stimulatory molecules that and one patient had a 27-month continuous are expressed on the surface of immune CR [187, 188]. Besides being very promising, effector cells such as T cells. Evolution these clinical studies also revealed draw- of CD20 CARs, like for CD19 CARs, was backs. It became clear that immune-privi- mainly driven by the signaling domains. leged sites such as the testis, are refractory In the first generation CARs, the activation to CAR-T treatment [187], but also other was mainly driven by only one signaling sites, such as lung and liver, showed out- domain (CD3ζ). In the second generation growth of CD20+ cells, despite the presence CARs, the signaling domain was extended of T cells [188]. Sufficient target expression to also include that of CD28 [183], and even is crucial, even though efficacy was ob- including the signaling domain of CD137 in served in vitro on cell lines expressing low the third generation CARs [184, 185]. This en- numbers of CD20, or on CLL cells (notorious hanced activation of the CAR-T cells by the for low CD20 expression) [189]. In patients inclusion of these co-stimulatory signaling treated with CAR-T cells, unexpectedly, domains in addition to that of CD3 signaling peripheral B cells were untouched by the domain enhanced the in vitro efficacy of the CAR-T cells, most likely due to their rela- CARs and led to a better clinical response as tively low CD20 expression level compared described below. to that on lymphoma cells [185].

In a clinical phase 1 study performed at the The occurrence of delayed adverse events, Fred Hutchinson Cancer Research Center such a cytokine release syndrome, which (NCT00621452), four patients were treat- may be observed 3-9 weeks after T-cell ed with cyclophosphamide (to achieve infusion [188] are also cause of concern. lymphocyte depletion) followed by three Although often manageable, new strategies infusions (2 to 5 days apart) of autologous to increase safety of CAR-T cell therapy CD20-specific CAR-T cells and SC IL-2. CAR-T are investigated. One strategy that is being cells were detectable in circulation, lymph explored is by inclusion of suicide switches, nodes and bone marrow, and a measurable such as caspase 9, which upon activation clinical response was obtained. However, result in the self-destruction of CAR-ex- both could not be definitively attributed pressing cells [190]. Another strategy em-

CD20 review 37 ployed is by generation of a generic CAR-T CD20 TARGETING AND construct (a scFv targeting PNE) that is only IMMUNOMODULATION active in the presence of a Fab fragment targeting CD20, containing a PNE motive The concept of immunomodulation for [191]. Here, the half-life of the Fab fragment CD20 antibodies encompasses the re- determines the efficacy and safety of the cruitment of immune effector cells as CAR-T cell therapy. CD20 itself may also mechanism of action, such as NK cells for be used as a suicide system for CAR-T cell ADCC and macrophages for ADCP. Also treatment. As T cells do not express CD20 complement plays a role, as C3a and C5a naturally, the forced expression of CD20 represent chemo-attractants for immune could provide a way to eliminate CAR-T cells. Pre-clinical and clinical studies further cells via RTX treatment [192]. investigated this route to improve the efficacy of CD20 antibodies. The first evidence Another point of concern is the possible for immunomodulation beyond a direct relapse of the lymphoma, due to outgrowth interaction of effector cells with RTX was of CD20-negative lymphoma cells. One pos- provided by Hilchey and colleagues [200]. sible solution is dual targeting with CAR-T They demonstrated that inclusion of RTX cells, such as with bispecific CAR-T cells resulted in a T cell-directed anti-lymphoma [193-195], which showed in vivo efficacy response. Later it was discovered that in to kill tumor cells expressing either target mice given anti-CD20 therapy, regulatory T alone or both targets together. Another cell (Treg) expansion was reduced and Th1 option is combined or sequential treat- expanse increased [201]. This was in con- ment with two or more different CAR-T trast to the previously reported effect on cell therapies for which several clinical T cell-dependent immunity [202]. However, studies are now recruiting (NCT03207178, currently there is no clear understanding of NCT03125577, and NCT02737085). the role that CD20 mAbs in play in the gen- Last but not least, manufacturing issues of eration of an adaptive memory response. CAR-T cell treatment are being addressed The administration of ex vivo expanded [196, 197]. During the manufacturing pro- immune cells, such as NK cells and T cells, cess steps such as e.g. T cell isolation, gene has also been studied. Although, especially transduction, and expansion need to be for NK cells, some increase in therapeutic performed under sterile conditions and activity was found, these were eventually as fast as possible to avoid the loss of the abandoned in favor of the pursuit of CD20 patient before start of treatment. CAR-T and CD20 CAR-NK cells. Although the concept of CAR-T cells targeting CD20 has In addition to CAR-T cells, also CAR-NK cells been around for over a decade, the general are under (early) pre-clinical investigation excitement and status of CD19 CAR-T cells [198, 199], but so far only in vitro analyses has not been achieved. While the initial are available. anti-tumor efficacy was promising, there are still many hurdles to take. Hurdles recently observed for CD19 CAR-T cells,

38 Chapter 2 where massive endogenous T cell prolifer- vitro studies do not readily predict thera- ation was observed after administration of peutic gain as both were different in MoA CAR-T cells [203], also need to be taken for employed but obtained comparable clinical CD20 CAR-T cells. In addition, the longevity outcome in a similar patient population and of persistence of CAR-T cells in the pa- treatment modality. Furthermore, head-to- tients’ needs to be addressed as continuous head clinical comparison of naked and ra- repression of CD20+ cells may raise new is- diolabeled mAbs showed results in favor of sues. As 10 new phase 1 studies with CD20 the radiolabeled mAbs. However, concerns CAR-T therapies were initiated in 2017, of MDS, secondary malignancies and com- some of these hurdles will be addressed. It plexities with respect to the supply-chain is expected that the inclusion of T cell ac- and administration ultimately led to the tivity into the treatment of lymphomas will demise of radiolabeled antibodies. be the direction CD20-based therapies are going, whether this will be with CAR-T cells Currently, many of the RTX bio-similars are or bispecifics remains to be seen. approved and it will be interesting to see whether this will make treatment cheaper OVERALL CONCLUSION and more accessible for RTX responders. Furthermore, the applicability of CD20 tar- The road traveled for CD20-targeted immu- geting antibodies beyond the treatment of notherapy has been long, but very fruitful. cancer will be an exciting development, as Strong clinical efficacy of CD20-specific it might teach us more on the role of B cells antibodies, together with the lack of serious in immunomodulation, something the vacci- safety issues associated with depletion of nation-like effects of RTX have hinted at. CD20-expressing cells makes CD20 an ideal It is expected that patients that have target, despite the relapses or resistance relapsed or are refractory to CD20 mAb that occur almost inevitably in any cancer therapy will have new CD20-targeting treatment. First, there is a benchmark in treatment paradigms to their exposure. place with RTX. Second, as depletion of CAR-T cells, CD3 bsAbs with or without CD20+ cells is safe, any flags raised during the combination of checkpoint inhibitors testing of novel compounds can be at- and/ or small molecules will be an exciting tributed to the compound/format and is not field to watch. CAR-T cells are expected to directly target-related. have potential in the future, but still many hurdles to take. It will be intriguing to see In the past decades, many lessons have whether the field is equipped with the tech- been learned with mAbs targeting CD20 in nical challenges associated with CAR-T cells, lymphoma and leukemias. Initially the focus seeing the lost confidence in radiolabeled was on mAbs and radiolabeled antibodies. mAbs was mostly associated with technical Pre-clinical data suggested that enhanc- challenges. The near future will be domi- ing singled-out effector functions of these nated by the CD3 as they have the promise mAbs would result in enhanced therapeutic of high therapeutic potential with manage- efficacy. OFA and OBZ showed us thatin able safety and, in contrast to CAR-T cell

CD20 review 39 technology, are much easier to produce and control. All in all, current development in CD20 immunotherapy sparks the hope for turning B cell malignancies in a controllable disease.

40 Chapter 2 Ref. [204] [12, 84, 205] [86, 206]

Non-Hodgkin’s Lymphoma (NHL). Lymphoma Non-Hodgkin’s (CLL). Leukemia Lymphocytic Chronic with Arthritis (RA) in combination Rheumatoid with moderately- in adult patients methotrexate inadequate RA who have severely-active to TNF antagonist one or more to response therapies. (GPA) with Polyangiitis Granulomatosis and Microscopic Granulomatosis) (Wegener’s in in adult patients (MPA) Polyangiitis with glucocorticoids. combination (CLL). Leukemia Lymphocytic Chronic the for with chlorambucil, In combination with patients untreated previously of treatment is based therapy whom fludarabine CLL for inappropriate. considered in who are patients of treatment extended For two at least after or partial response complete or progressive recurrent for therapy of lines CLL. with CLL patients of the treatment For and alemtuzumab. fludarabine to refractory (CLL). Leukemia Lymphocytic Chronic the for with chlorambucil, In combination untreated with previously patients of treatment lymphocytic leukemia. chronic (NHL). Lymphoma Non-Hodgkin’s followed with bendamustin In combination the treatment for monotherapy, GAZYVA by lymphoma (FL) who with follicular patients of a rituximab- to, refractory or are after, relapsed regimen. containing ------Indication of approval of Indication ------First Approval, Approval, First or EU US 1997 (US) 1998 (EU) 2009 (US) 2010 (EU) 2013 (US) 2014 (EU) Variant Chimeric IgG1 IgG1 optimized IgG1-Fc Clone C2B8 2F2 Bly-1 GA101 Brand name Brand (US) Rituxan (EU) Mabthera Arzerra Gazyva FDA/EMA approved CD20 antibodies. approved FDA/EMA

International non- International name (INN) proprietary (RTX) Rituximab (OFA) Ofatumumab (OBZ) Obinutuzumab TABLE 1 TABLE

CD20 review 41 Ref. [207] [208] [209] [210] [211] [212] [213]

Non-Hodgkin’s Lymphoma (NHL). Lymphoma Non-Hodgkin’s low- or refractory, relapsed CD20 positive, NHL who have or transformed follicular, grade, therapy, rituximab during or after progressed with rituximab-refractory including patients NHL. or primary progressive with relapsing patients (MS) multiple sclerosis of forms (NHL). Lymphoma Non-Hodgkin’s or follicular low-grade or refractory, Relapsed NHL follicular untreated NHL. Previously B-cell to response a partial or complete who achieve chemotherapy. first-line (NHL). Lymphoma Non-Hodgkin’s (CLL). Leukemia Lymphocytic Chronic and with Polyangiitis Granulomatosis Polyangiitis. Microscopic (NHL). Lymphoma Non-Hodgkin’s and with Polyangiitis Granulomatosis Polyangiitis. Microscopic (NHL). Lymphoma Non-Hodgkin’s B cell large lymphoma and diffuse Follicular NHL. (CLL). Leukemia Lymphocytic Chronic and with Polyangiitis Granulomatosis Polyangiitis. Microscopic (NHL). Lymphoma Non-Hodgkin’s (CLL). Leukemia Lymphocytic Chronic arthritis (RA). Rheumatoid and with Polyangiitis Granulomatosis Polyangiitis. Microscopic ------Indication of approval of Indication ------1 First Approval, Approval, First or EU US 2003 (US) 2017 (US) 2017 (US) 2002 (US) 2004 (EU) 2017 (EU) 2017 (EU) 2017 (EU) 2017 (EU) biosimilar biosimilar biosimilar biosimilar Variant Mouse IgG2a optimized IgG1-Fc mIgG1 RTX RTX RTX RTX Clone B1 2H7 2B8 C2B8 C2B8 C2B8 C2B8 Brand name Brand Bexxar Ocrevus Zevalin International non- International name (INN) proprietary tositumomab Ocrelizumab (OCRE) tiuxetan Ibritumomab Blitzima Ritemvia Rituzena Rixathon

42 Chapter 2 Ref. [214] [215] Non-Hodgkin’s Lymphoma (NHL). Lymphoma Non-Hodgkin’s arthritis (RA). Rheumatoid and with Polyangiitis Granulomatosis Polyangiitis. Microscopic (NHL). Lymphoma Non-Hodgkin’s (CLL). Leukemia Lymphocytic Chronic arthritis (RA). Rheumatoid and with Polyangiitis Granulomatosis Polyangiitis. Microscopic ------Indication of approval of Indication ------First Approval, Approval, First or EU US 2017 (EU) 2017 (EU) biosimilar biosimilar Variant RTX RTX Clone C2B8 C2B8 Brand name Brand International non- International name (INN) proprietary Riximyo Truxima 1) Oct-2013 FDA approval for Bexxar was withdrawn and Feb-2014 Bexxar was discontinued by the manufacturer by discontinued was Bexxar and Feb-2014 withdrawn was Bexxar for approval FDA 1) Oct-2013

CD20 review 43 First First date 2005 2007 2009 2010 2014 Recruitment Recruitment status Completed Withdrawn Completed Terminated Recruiting Highest level of of level Highest clinical investigation Phase I Phase I Phase I Phase I/II Phase I Clinical study design study Clinical T Cells With Activated Immune Consolidation (Anti-CD3 x Armed With OKT3 x Rituxan After Anti-CD20) Bispecific Antibody (CD20Bi) High for Transplant Cell Blood Stem Peripheral Lymphomas Risk CD20+ Non-Hodgkin’s With Allogeneic Immune Consolidation Armed With OKT3 x Rituxan T Cells Activated (Anti-CD3 x Anti-CD20) Bispecific Antibody Blood Peripheral Allogeneic (CD20Bi) After High Risk CD20+ Non- for Transplant Cell Stem (Phase I) Lymphoma Hodgkin’s and the side effects Phase I trial is studying by followed T cells treated give to way best with patients in treating transplant cell stem multiple myeloma. the of Study Phase I/II Dose-escalation Bispecific Trifunctional Investigational in Anti-CD20 x Anti-CD3 Antibody FBTA05 Infusion With Donor Lymphocyte Combination Chronic With CD20 Positive (DLI) in Patients and High (CLL), Low Leukemia Lymphocytic (NHL) After Non-Hodgkin´s Lymphoma Grade Transplantation Cell Stem Allogeneic Phase 1 Study Multi-Center An Open-Label, of and Tolerability the Safety Investigate to an Anti-CD20 x Anti-CD3 Bispecific REGN1979, With CD20+ in Patients Monoclonal Antibody, Treated Previously Malignancies B-Cell Antibody Therapy With CD20-Directed Clinical study # study Clinical NCT00244946 NCT00521261 NCT00938626 NCT01138579 NCT02290951 Sponsor Ann Barbara Cancer Karmanos Institute Ann Barbara Cancer Karmanos Institute Ann Barbara Cancer Karmanos Institute pharma Trion Regeneron - - - - - Format CD3x CD20 CD3x CD20 CD3x CD20 CD3x CD20 CD3x CD20 Clinical investigations in the US targeting Lymphoma and leukemia with CD20 mAbs. and leukemia Lymphoma targeting in the US investigations Clinical

CD20 compound Bispecific antibodies CD20Bi CD20Bi CD20Bi FBTA05 REGN1979 TABLE 2 TABLE

44 Chapter 2 First First date 2016 2015 2003 2008 2012 2016 ompleted C Recruitment Recruitment status Recruiting Recruiting Completed Recruiting Recruiting Highest level of of level Highest clinical investigation Phase I Phase I Phase I Phase I Phase I/II Phase I ), in ζ Clinical study design study Clinical the Evaluate to Study A Phase 1 Multidose in XmAb13676 of and Tolerability Safety Hematologic With CD20-Expressing Patients Malignancies Phase I/IB Trial Multicenter, An Open-Label, and Pharmacokinetics the Safety Evaluating as a Single BTCT4465A of Doses Escalating of in With Atezolizumab and Combined Agent B-Cell or Refractory With Relapsed Patients and Chronic Lymphoma Non-Hodgkin’s Leukemia Lymphocytic Of The Safety Evaluate To A Phase I Study Using Genetically Immunotherapy Cellular Cd8+ T CD20-Specific Autologous Modified CD20+ With Relapsed Patients For Clones Cell Indolent and the Safety Evaluate to Study A Pilot Using Immunotherapy Cellular of Feasibility Autologous CD20- Modified Genetically Relapsed With Patients For Cells Specific T and Indolent B Cell Mantle Cell or Refractory Lymphomas T Cells Autologous Redirected of Study Pilot A CD20-Specific Express to Transduced With in Patient Chimeric Immunoreceptor or Refractory CD20+ Resistant Chemotherapy and Lymphoma Leukemia T Autologous ACTR087, of Phase 1 Study Antibody Coupled Expressing Lymphocytes (CD16V-41BB-CD3 Receptors T-cell With in Subjects With Rituximab, Combination B-Cell or Refractory CD20-Positive Relapsed Lymphoma Clinical study # study Clinical NCT02924402 NCT02500407 NCT00012207 NCT00621452 NCT01735604 NCT02965157 - Sponsor Xencor Genentech Hutchinson Fred Research Cancer Center Hutchinson Fred Research Cancer Center PLA Chinese General Hospital Beijing Bio Bio healthcare Co.,Ltd technology - - Format CD3x CD20 CD3x CD20 CD20 CAR-T CD20 CAR-T CD20 CAR-T CD16 + CAR-T rituximab CD20 compound XmAb13676 BTCT4465A cells CAR-T

CD20 review 45 First First date 2016 2016 2016 2017 2017 2017 2017 2017 2017 - - - Recruitment Recruitment status Recruiting recrui Not ting recrui Not ting Recruiting recrui Not ting Recruiting Recruiting Recruiting Recruiting Highest level of of level Highest clinical investigation Phase I Phase I Phase II Phase I Phase I Phase II Phase I/II Phase I/II NA Clinical study design study Clinical in CAR-T of CD20-Targeted Research A Clinical Malignancies B Cell CD19-targeted of the Sequential Therapy for Therapy Cell CAR-T and CD20-targeted Lymphoma(DLBCL) B Cell Large Diffuse Sequential CAR-T of Research A Clinical Cell Stem Bridging Hematopoietic Relapse/ of in the Treatment Transplantation Malignancies B-cell Refractory Anti-CD19 and Anti- Sequential Infusion of T Receptor(CAR) CD20 Chimeric Antigen B-cell and Refractory Relapsed Against Cells Lymphoma an Anti- Expressing T Cells of Phase I Study CD19 and Anti-CD20 Bispecific Chimeric Malignancies With B Cell in Patients Receptor of Chimeric Antigen and Efficacy Safety Relapse/ Treating (CAR-T) T Cell Receptor CD19/CD20/CD22/CD30 Positive Refractory Non-Hodgkin Lymphoma 4SCAR19 of Therapy CAR-T Combination Plus 4SCAR20, 22, 38, and 123 Targeting Malignancies Hematological of the Safety Evaluate to A Phase I/II Study T Using Autologous Immunotherapy Cellular a CD20-Specific Express to Engineered Cells With Patients for Receptor Chimeric Antigen Non-Hodgkin B Cell or Refractory Relapsed Lymphomas T Cells CD19/CD20 tanCAR of Study Clinical Refractory and/or Chemotherapy in Relapsed and Lymphomas Leukemias B-cell Clinical study # study Clinical NCT02710149 NCT02737085 NCT02846584 NCT03207178 NCT03271515 NCT03196830 NCT03125577 NCT03277729 NCT03097770 Sponsor Southwest Hospital, China Southwest Hospital, China Southwest Hospital, China Shanghai Longyao Inc., Biotechnology Ltd. Beijing Doing Biomedical Co., Ltd. Shanghai Unicar-Therapy Bio-medicine Technology Co.,Ltd Shenzhen Geno-Immune Institute Medical Hutchinson Fred Research Cancer Center PLA Chinese General Hospital Format CD20 CAR-T CD20 CAR-T + CD19 CAR-T CD20 CAR-T CD20 CAR-T + CD19 CAR-T CD20/ CD19 CAR-T CD20 CAR-T CD20 CAR-T + CD19 CAR-T CD20 CAR-T CD20/ CD19 CAR-T CD20 compound

46 Chapter 2 First First date 2017 2017 2017 2006 2007 2008 2009 - ompleted ompleted ompleted ompleted C C C C Recruitment Recruitment status Recruiting Recruiting recrui Not ting Highest level of of level Highest clinical investigation Phase I Phase I/II Phase I Phase I/II Phase I/II Phase I/II Phase I/II and 4-1BB ζ Clinical study design study Clinical T Autologous Redirected of Phase 1 Study an Anti CD19 and Contain to Engineered Cells CD3 to Coupled Anti CD20 scFv With Relapsed Signaling Domains in Patients B CD19 or CD20 Positive and/or Refractory Malignancies Cell CAR T Cells CD19/CD20 Tan of Study Clinical Refractory and/or Chemotherapy in Relapsed and Lymphomas Leukemias B-cell the Safety, Evaluate to Phase Ia Study and Pharmacokinetics Tolerance, Recombinant of Pharmacodynamics Anti-CD20 Monoclonal Antibody Humanized Injection(B001) for With hA20 Immunotherapy of A Phase I Study 4 Consecutive for Weekly Once Administered With CD20+ Non-Hodgkin’s in Patients Weeks Lymphoma Subcutaneously of Phase I/II Study (hA20) in Patients Veltuzumab Administered or Lymphoma With CD20+ Non-Hodgkin’s Leukemia Lymphocytic Chronic With hA20 Immunotherapy of A Phase I Study 4 Consecutive for Weekly Once Administered With CD20+ Non- Hodgkin’s in Patients Weeks Lymphoma (IMMU-106, Veltuzumab of A Phase I/II Study Anti-CD20 Monoclonal hA20), a Humanized With Milatuzumab Combined Antibody, Anti-CD74 (IMMU-115, hLL1), a Humanized and in Relapsed Monoclonal Antibody, Lymphoma Non-Hodgkin’s B-cell Refractory Clinical study # study Clinical NCT03019055 NCT03185494 NCT03332121 NCT00285428 NCT00546793 NCT00596804 NCT00989586 - Sponsor Hospital Froedtert College & Medical Wisconsin of PLA Chinese General Hospital Shanghai Pharma Holding ceuticals Ltd Co., Immunomedics, Inc. Immunomedics, Inc. Immunomedics, Inc. Immunomedics, Inc. Format CD20 CAR-T CD20 CAR-T mAb mAb mAb mAb mAb CD20 compound Unlabeled antibodies B001 veltuzumab (hA20)

CD20 review 47 First First date 2010 2011 2014 2015 2006 2014 - - - Recruitment Recruitment status Terminated Terminated recrui Not ting recrui Not ting Completed recrui Not ting Highest level of of level Highest clinical investigation Phase I/II Phase II Phase I/II Phase I/II Phase I Phase I Clinical study design study Clinical Combination of Phase I/II Study (Anti-CD20) and Fractionated Veltuzumab (Anti-CD22) Radio Epratuzumab 90Y- With Follicular in Patients immunotherapy Lymphoma Double Blind, Placebo a Randomized, Multinational Phase Multicenter, Controlled, With in Subjects Finding Trial Range II Dose Arthritis Rheumatoid Severe to Moderate With Either Controlled Insufficiently Anti- Plus Methotrexate or Alone Methotrexate Treatment, Biological Factor Necrosis tumour Subcutaneous 3 Different Comparing Anti-CD20 Monoclonal Antibody of Dosages as an add-on Therapy Placebo to Veltuzumab Methotrexate to Anti- Humanized Combining Phase I/II Study Anti-CD22 (Epratuzumab) CD20 (Veltuzumab), With Monoclonal Antibodies and Both With in Adults Chemotherapy Intensive Acute B-precursor or Refractory Recurrent (ALL) Leukaemia Lymphoblastic Combined Veltuzumab of Phase I/II Study in Patients Tetraxetan With 90Y-Epratuzumab Non- Aggressive With Relapsed/Refractory, Lymphoma Hodgkin’s Phase 1/2 Dose- Multicenter, Open-Label, 2469298), (LY AME-133v of Study Escalation Weekly in Four Intravenously Administered With CD20+ Follicular in Subjects Doses, Non-Hodgkin’s or Refractory Relapsed Lymphoma Subcutaneous of A Phase I Study Anti- and Fc-engineered (Fab- Ocaratuzumab With CD20 Monoclonal Antibody) in Patients Malignancies B-Cell Treated CD20+ Previously Clinical study # study Clinical NCT01147393 NCT01390545 NCT01279707 NCT01101581 NCT00354926 NCT01858181 Sponsor Immunomedics, Inc. Takeda Immunomedics, Inc. Immunomedics, Inc. Applied Molecular Evolution Mentrik Biotech, LLC Format mAb mAb mAb mAb mAb mAb - CD20 compound Ocaratu zumab (AME-133v, Y2469298)

48 Chapter 2 First First date 2007 2016 2010 2012 2012 2013 - ot recrui ot ompleted ompleted ompleted C C C N Recruitment Recruitment status Terminated ting Recruiting Highest level of of level Highest clinical investigation Phase I/II Phase I Phase I Phase I/II Phase I/II Phase I Clinical study design study Clinical of Phase I/II Trial Multicenter, An Open-Label, PRO131921 of Doses Escalating of the Safety or Refractory With Relapsed in Patients Who Lymphoma Indolent Non-Hodgkin’s With a Prior Rituximab- Been Treated Have Regimen Containing With in Participants Ocrelizumab of A Study (NHL) Lymphoma Non-Hodgkin’s Follicular the safety, evaluate to is designed This study of efficacy and preliminary pharmacokinetics the anti-CD20 monoclonal antibody LFB-R603 B-cell or refractory with relapsed in patients who have lymphocytic leukemia chronic one prior fludarabine- at least received regimen. containing in Ublituximab of Phase I/II Study in (Revlimid®) With Lenalidomide Combination Malignancies Lymphoid With B-Cell Patients Refractory or Are Relapsed Who Have Antibody Therapy After CD20 Directed the of Phase I/II Study An Open Label in of Ublituximab and Safety Efficacy Non-Hodgkin Lymphoma With B-cell Patients Refractory or Are Relapsed Who Have Antibody Therapy After CD20 Directed the Efficacy Evaluating A Phase I/Ib Study a Third-Generation Ublituximab, of and Safety in Anti-CD20 Monoclonal Antibody, PI3k a Novel With TGR-1202, Combination or Bendamustin, and Ibrutinib Inhibitor; Delta Malignancies. With B-cell in Patients Clinical study # study Clinical NCT00452127 NCT02723071 NCT01098188 NCT01744912 NCT01647971 NCT02006485 Sponsor Genentech Roche Laboratoire de français Fractionnement et de Biotechnologies Therapeutics, TG Inc. Therapeutics, TG Inc. Therapeutics, TG Inc. Format mAb mAb mAb mAb mAb mAb CD20 compound PRO131921 Ocrelizumab Ublituximab (LFB-R603)

CD20 review 49 First First date 2015 2017 2017 2010 2011 2011 2012 - - ompleted ompleted ompleted C C C Recruitment Recruitment status Recruiting recrui Not ting recrui Not ting Terminated Highest level of of level Highest clinical investigation Phase I/II Phase I/II Phase I Phase I Phase III Phase I Phase III positive positive - Positive Positive - Clinical study design study Clinical in Pembrolizumab of Phase I/II Study (Ublituximab) With TG-1101 Combination With Relapsed- in Patients and TGR-1202 Leukemia Lymphocytic Chronic refractory (RT Transformation (CLL) or Richter’s With Ublituximab in Combination Venetoclax With in Patients (TGR-1202) and Umbralisib CLL/SLL or Refractory Relapsed of Study A Phase I Open-label, Multiple Dose as a Intravenously CHO-H01 Administered or With Refractory Subjects to Single Agent Lymphoma Follicular Relapsed and Evaluate to Double Blind RCT A Phase Ib, MabThera® of the PK, PD and Safety Compare in With CHOP, With TL011, in Combination With CD20+ DLBCL Subjects Randomized, An Open-labeled, Multi-center, Comparing Phase III Study Prospective CHOP With CHOP to CMAB304 in Combination in Patients Alone With CMAB304 Maintenance With DLBCL MK-8808 of Single Arm Study An Open-Label, With Advanced CD20 in Patients Lymphoma Follicular Open-label Randomized A Multicenter BIOCAD, CJSC BCD-020 (Rituximab, of Study Comparison in and Safety Efficacy Russia) Ltd., Roche Hoffmann-La (F. With MabThera of CD20 in Monotherapy Switzerland) Lymphoma Indolent Non-Hodgkin’s Clinical study # study Clinical NCT02535286 NCT03379051 NCT03221348 NCT01205737 NCT01459887 NCT01370694 NCT01701232 - Sponsor Therapeutics, TG Inc. Therapeutics, TG Inc. Cho Pharma Inc. Pharma Teva industries ceutical Ltd. Shanghai CP Guojian Pharma Co.,Ltd. ceutical Sharp & Merck Dohme Corp. Biocad - - - - Format mAb mAb mAb RTX-Bio similar RTX-bio similar RTX-bio similar RTX-bio similar CD20 compound CHO-H01 TL011 CMAB304 MK-8808 BCD-020

50 Chapter 2 First First date 2013 2011 2014 2014 2015 2016 - - ot recrui ot ompleted ompleted C C N Recruitment Recruitment status recrui Not ting Recruiting Unknown ting Highest level of of level Highest clinical investigation Phase I Phase III Phase III Phase I Phase II Phase III positive, positive, - positive DLBCL Patients positive - Clinical study design study Clinical and the Safety Assess to Phase I Trial GP2013 Monotherapy of Pharmacokinetics Patients in Japanese Weekly Administered Burden Tumor Low With CD20 Positive Lymphoma Non-Hodgkin’s Indolent B-cell Double-Blind Phase Controlled, A Randomized, and Safety the Efficacy, Compare to III Trial in MabThera® GP2013 vs. of Pharmacokinetics Advanced Untreated, With Previously Patients Lymphoma Follicular Stage Of Double-blind Study A Phase 3, Randomized, The First- For Rituximab Versus Pf-05280586 With Cd20 Patients Of line Treatment Lymphoma Follicular Burden, Tumor Low the of Study Escalation A Phase I Dose and Pharmacokinetics, Tolerability, Safety, SCT400, a Recombinant of Pharmacodynamics Chimeric Anti-CD20 Monoclonal Antibody in Non-Hodgkin’s With CD20+ B-cell Patients Lymphoma. and Open Randomized A Phase II, Multi-center, the PK, PD and and Compare Evaluate to Study in Patients SCT400 With Rituximab of Safety Lymphoma Non-Hodgkin’s With CD20+ B-cell Randomized, A Phase III, Multi-center, the Efficiency Compare to Study Controlled Chimeric SCT400(Recombinant of and Safety Experimental Anti-CD20 Monoclonal Antibody, Plus CHOP Rituximab Drug) Plus CHOP Versus in Untreated CD20 Clinical study # study Clinical NCT01933516 NCT01419665 NCT02213263 NCT02206308 NCT02456207 NCT02772822 - Sponsor Pharma Novartis ceuticals Pfizer Ltd. Sinocelltech - - - Format RTX-bio similar RTX-bio similar RTX-bio similar CD20 compound GP2013 PF- 05280586 SCT400

CD20 review 51 First First date 2015 2016 2017 2016 2016 2016 - ot recrui ot ompleted ompleted C N C Recruitment Recruitment status ting Recruiting Recruiting Recruiting Highest level of of level Highest clinical investigation Phase I/II Phase III Phase I Phase I Phase III Phase III - Clinical study design study Clinical group double-blind, parallel Randomized, between PK and PD profiles compare to study in patients (MabThera®) HLX01 and rituximab Lymphoma. with CD20+ B-cell Double-blind, Randomized, Multicenter, Compare to Study Phase III Clinical Parallel, of Rituximab and Safety the Efficacy in HLX01 and MabThera Biosimilar in Previously With CHOP, Combination With CD20+ DLBCL Subjects Untreated Open-label, A Phase Ia, Multi-centers, Evaluate to Study Clinical Dose-escalation and Pharmacokinetics Tolerability, Safety, HLX01 (a Potential of Pharmacodynamics With CD20 in Patients Biosimilar) Rituximab Lymphomas B-cell positive Double-blinded, Randomized, A Multicenter, the Assess to Study Controlled Parallel Recombinant of and Safety Pharmacokinetics Human Murine Chimeric Anti CD20 Monoclonal to (IBI301) Compared Antibody Injection B Cell in CD20 Positive Injection Rituximab Patients Lymphoma Double-blind, Randomized, A Multicenter, the Evaluate to Phase III Study Controlled, of IBI301 (Recombinant and Safety Efficacy Chimeric Anti-CD20 Monoclonal Antibody ) Versus With CHOP Regimen in Combination With CHOP Regimen in Combination Rituximab With Diffuse Patients in Treatment-naïve (DLBCL) Lymphoma B-cell Large Evaluating Double-Blind Study A Randomized, and Immunogenicity Safety the Efficacy, in With Rituximab ABP 798 Compared of Non- B-Cell With CD20 Positive Subjects (NHL) Hodgkin Lymphoma Clinical study # study Clinical NCT02584920 NCT02787239 NCT03218072 NCT02945215 NCT02867566 NCT02747043 Sponsor Shanghai Henlius Biotech Biologics Innovent Ltd. Co. (Suzhou) Amgen - - - Format RTX-bio similar RTX-bio similar RTX-bio similar CD20 compound HLX01 IBI301 ABT798

52 Chapter 2 First First date 2008 2013 2014 2015 2015 2007 - ompleted C Recruitment Recruitment status Completed Completed Completed recrui Not ting Withdrawn Highest level of of level Highest clinical investigation Phase I Phase I/II Phase I/II Phase I Phase I Phase I IL2 Immunocytokine Administered Administered IL2 Immunocytokine Clinical study design study Clinical and the side effects phase I trial is studying therapy cytokine fusion protein of dose best in treating rituximab after when given non-Hodgkin lymphoma. with B-cell patients DI- De-immunized of A Phase I/II Study Leu16- Non- With B-cell in Patients Subcutaneously (NHL) Hodgkin Lymphoma De- of Study Extension An Open-Label Immunocytokine DI-Leu16-IL2 immunized Non- With B-cell in Patients Administered (NHL) Hodgkin Lymphoma Evaluation of Pretargeted Anti-CD20 Radio Anti-CD20 Radio Pretargeted of Evaluation With BEAM Combined immunotherapy Stem and Autologous Chemotherapy High-Risk B-Cell for Transplantation Cell Malignancies investigators provide to is intended This study information and sponsor with the following drug MT- new the investigational regarding with relapsed/refractory 3724 in patients or Small Leukemia Lymphocytic B-cell Chronic Lymphoma: Lymphocytic and Safety Assess to Phase I Trial Xenogeneic CD20 DNA of Immunogenicity With B-Cell With Patients Vaccination Lymphoma Clinical study # study Clinical NCT00720135 NCT01874288 NCT02151903 NCT02483000 NCT02556346 NCT00561756 Sponsor Hope City of Medical Center Oncology, Alopexx LL Fred Hutchinson Hutchinson Fred Research Cancer Center Molecular Templates, Inc. Memorial Sloan Cancer Kettering Center - Format mAb (cytoki ne) RIT ADC Vaccine - CD20 compound Other DI-Leu16-IL2 B9E9-scFv- streptavidin ScFV-SLT-I A1 pINGmmi niCD20

CD20 review 53 First First date 2003 2017 2017 2007 - - ompleted C Recruitment Recruitment status recrui Not ting recrui Not ting Completed Highest level of of level Highest clinical investigation Phase II Phase I Phase I/II Phase I/II positive NHL positive - Clinical study design study Clinical and Safety Evaluate to A Phase II Study of Specific Immunotherapy, Efficacy KLH and to Conjugated Idiotype Recombinant the Anti-CD20 Antibody, GM-CSF Following With Patients Treated in Previously Rituximab, Lymphoma Non-Hodgkin’s Follicular Neoantigen a Personalized of Study A Pilot Rituximab Front-Line Following Vaccine Cancer Lymphoma in Follicular (NHL) Vaccine: NANT Non-Hodgkin Lymphoma With in Subjects Immunotherapy Combination Relapsed CD20 CpG 7909, Rituximab of A Phase I/II Trial Radio Zevalin and Y-90 Immunotherapy, With Previously Patients for immunotherapy CD20+ Non-Hodgkin Lymphoma Treated Clinical study # study Clinical NCT00071955 NCT03361852 NCT03169790 NCT00438880 Sponsor Genitope Corporation Dana-Farber Cancer Institute Inc. NantKwest, Clinic Mayo Format Vaccine CD20 compound combination

54 Chapter 2 First First date 2008 2012 2016 2017 2013 2014 2017 2016 Recruitment Recruitment status Terminated Recruiting Recruiting Recruiting Unknown Recruiting Recruiting Recruiting Highest level of of level Highest clinical investigation Phase I/II Phase I Phase II Phase I Phase I/II Phase I/II Phase I Phase I Positive B-Cell B-Cell Positive - Clinical study design study Clinical Killer Cells Natural MT2007-12 Allogeneic With CD20 Positive in Patients With Rituximab or Chronic Non-Hodgkin Lymphoma Relapsed Increase to Strategies Leukemia. Lymphocytic Killer Natural to Cells CLL Tumor Sensitivity of Cytolysis Cell-Immune-Mediated Blood Cord In Allogeneic Killer Cells Natural Transplantation Therapy Killer (NK) Cell Natural Personalized Blood Transplantation in Cord NAM Expanded Testing A Phase I Trial Donor Related or Mismatched Haploidentical a Short by Followed Killer (NK) Cells Natural Relapsed/ of the Treatment for IL-2 of Course and Relapsed/ Multiple Myeloma Refractory Non-Hodgkin Lymphoma Refractory CD20+ of CD20 antibody and efficacy The safety in CIK transfusion by followed usage lymphomas. and/or chemo resistant refractory T Lymphocytes Autologous of Study Pilot Cytotoxicity Cell With Antibody-Dependent With CD20 in Patients Malignancies Phase 1 Study of ACTR707, an Autologous T an Autologous ACTR707, of Phase 1 Study With Rituximab, in Combination Product, Cell or Refractory CD20+ With Relapsed in Subjects Lymphoma B Cell AR160 (Abraxane/Rituximab of A Phase I Trial 160nm Nanoparticle) in Relapsed/Refractory Including Transformed Lymphomas B Cell Lymphoma Follicular Clinical study # study Clinical NCT00625729 NCT01619761 NCT02727803 NCT03019666 NCT01828008 NCT02315118 NCT03189836 NCT03003546 Sponsor Masonic Cancer University Center, Minnesota of Anderson M.D. Center Cancer Anderson M.D. Center Cancer Masonic Cancer University Center, Minnesota of PLA General Hospital National Univer sity Hospital, Singapore Unum Therapeutics Inc. Clinic Mayo Format Ab + effector cells - CD20 compound + NK RTX cells + T cells RTX RTX coated coated RTX nano parti cles

CD20 review 55 First First date 2010 2009 2015 2008 Recruitment Recruitment status Prematurely Ended Prematurely Ended ongoing ongoing Phase Phase I Phase I/II Phase I Phase II - Clinical study design study Clinical the of study Phase I/II dose-escalation bispecific trifunctional investigational anti-CD20 x anti-CD3 antibody FBTA05 with donor lymphocyte in combination with CD20 positive infusion (DLI) in patients and (CLL), low lymphocytic leukemia chronic non-Hodgkin´s lymphoma (NHL) high grade transplantation. cell stem allogeneic after the of study Phase I/II dose-escalation bispecific trifunctional investigational in anti-CD20 x anti-CD3 antibody FBTA05 lymphocytic chronic or refractory relapsed leukemia and Tole Safety Assess to A Phase 1 Study an anti-CD20 x anti- REGN1979, of rability and CD3 bispecific monoclonal antibody, death-1 an anti-programmed REGN2810, in Patients (PD-1) monoclonal antibody, Malignancies with B-cell first- multicenter, Open, non-controlled, of doses using escalating in-man study stage with advanced LFB-R603 in patients lymphocytic leukemia B-Chronic EudraCT Number: EudraCT 2009-014641-88 2006-006694-24 2015-001697-17 2008-002601-40 - - - - Sponsor Medizinische Fa der Techni kultaet schen Universitaet Muenchen Biotech Fresenius GmbH Phar Regeneron Inc maceuticals, LFB BIOTECHNO LOGIES - - - Format CD3x CD20 CD3x CD20 CD3x CD20 mAb - Clinical investigations in Europe directed at Lymphoma and leukemia with CD20 mAb. and leukemia at Lymphoma directed in Europe investigations Clinical

CD20 com pound Bispecific antibodies FBTA05 FBTA05 REGN1979 Ublituximab TABLE 3 TABLE

56 Chapter 2 First First date 2008 2009 2005 2010 Recruitment Recruitment status ongoing completed completed completed Phase Phase I/II Phase I/II Phase I/II Phase III - - - - Clinical study design study Clinical with Immunotherapy of A Phase I/II Study Veltuzumab Administered Subcutaneous with CD20+ Non-Hod (hA20) in Patients Lymphocytic or Chronic Lymphoma gkin’s Leukemia humanized combining Phase I/II Study anti-CD22 (epra anti-CD20 (veltuzumab), monoclonal antibodies and both tuzumab) with recurrent in adults with chemotherapy leukaemia lymphoblastic acute B precursor (ALL)- MARALL dose-escalating An open-label, multicenter, rhuMAb 2H7 3-weekly phase I/II trial of non-Hodgkin’s with follicular in patients lymphoma stu double-blind, controlled A randomized, pharma pharmacokinetics, evaluate dy to of GP2013 and efficacy safety codynamics, with rheumatoid in patients and rituximab standard to or intolerant arthritis refractory anti-TNF three DMARDs and one or up to therapies EudraCT Number: EudraCT 2008-000434-47 2008-002286-32 2004-004110-17 2010-021184-32 - Sponsor Immunomedics, Inc. Queen Mary Me of University dical London Products Roche Limited AG Hexal - Format mAb mAb mAb RTX-Bio similar - CD20 com pound antibodies Naked Veltuzumab 2H7 GP2013

CD20 review 57 First First date 2011 2014 2015 2013 2014 Recruitment Recruitment status Prematurely ended ongoing ongoing Terminated ongoing Phase Phase III Phase I/III Phase III Phase III Phase III ------Clinical study design study Clinical Parallel-Group, A Phase 3, Randomized, to Double-Blind Study Active-Controlled, of CT-P10 and Safety the Efficacy Compare in Com Administered Each With MabThera, Vincris bination With Cyclophosphamide, With (CVP) in Patients and Prednisone tine, Lymphoma. Follicular Advanced Parallel-Group, A Phase 1/3, Randomized, to Double-Blind Study Active-Controlled, Pharmacoki of Equivalence Demonstrate for Efficacy of and Noninferiority netics Each With Rituxan, in Comparison CT-P10 With Cyclop in Combination Administered and Prednisone Vincristine, hosphamide, Follicular With Advanced (CVP) in Patients Lymphoma Parallel-Group, A Phase 3, Randomized, Double-Blind Study Active-Controlled, between and Safety Efficacy Compare to with Low in Patients and Rituxan CT-P10 Lymphoma Follicular Burden Tumour double-blind, multi-center, A randomized, compare multi-national Phase III trial to of BI 695500 plus che and safety efficacy plus chemo rituximab versus motherapy follicular with untreated in patients therapy lymphoma. non-Hodgkin’s Double-blind, Parallel-group, Randomized, Mabio of Trial Bioequivalence Comparative MabThera to Compared nCD20 (Mabion SA) in Pa Roche) Hoffman-La by (rituximab Lymphoma B-cell Large with Diffuse tients EudraCT Number: EudraCT 2011-002813-12 2013-004493-96 2014-005324-10 2011-002908-33 2013-005506-56 - Sponsor Inc CELLTRION, Inc. CELLTRION, Inc. CELLTRION, Ingel Boehringer heim International mbH Mabion - - - - - Format RTX-Bio similar RTX-Bio similar RTX-Bio similar RTX-Bio similar RTX-Bio similar - CD20 com pound CT-P10 BI 695500 MabionCD20

58 Chapter 2 First First date 2014 2014 2015 2016 2005 2015 2012 Recruitment Recruitment status ongoing ongoing completed ongoing completed ongoing Ongoing Phase Phase III Phase III Phase III Phase III unknown phase I/II unknown ------Clinical study design study Clinical DOUBLE-BLIND A PHASE 3, RANDOMIZED, RITUXI VERSUS OF PF-05280586 STUDY TREATMENT THE FIRST-LINE MAB FOR LOW WITH CD20-POSITIVE, OF PATIENTS LYMPHOMA FOLLICULAR TUMOR BURDEN, Evalu Double-Blind Study A Randomized, and Immunogeni Safety ating the Efficacy, with Rituximab ABP 798 Compared city of B-Cell with CD20 Positive in Subjects (NHL) Non-Hodgkin Lymphoma double-blind, A Phase III, randomized, evaluate multi-national trial to multi-center, versus of BI 695500 and safety efficacy tre immunotherapy as a first-line rituximab burden tumor with low atment in patients lymphoma follicular Double-blind, Multi-center, A Randomized, the Efficacy, Evaluate to Multi-national Trial SAIT101 of and Immunogenicity Safety, Immunothe as a First-line Rituximab Versus Tumor with Low in Patients Treatment rapy Lymphoma Follicular Burden the Effect Evaluate to Study A Multicenter on Primary (IDEC-102) Rituximab of and Response, Recall Response, Humoral Spe Immunity to Acquired of Maintenance cific Antigens the tumor-targeting of A Phase I/II study human L19IL2 monoclonal antibody-cyto with in combination kine fusion protein Diffuse or refractory in relapsed Rituximab (DLBCL) Lymphoma B-cell Large and Dosi PET/CT-Imaging 89Zr-Rituximab immuno Radio and 90Y-Rituximab metry lymphoma in CD20+ B-Cell therapy EudraCT Number: EudraCT 2014-000132-41 2013-005542-11 2014-004544-36 2016-001966-27 2004-004774-85 2014-001949-25 2011-005474-38 - Sponsor Inc Pfizer Inc. Amgen Ingel Boehringer heim International mbH Biotech Archigen Limited Idec Inc Biogen S.p.A. Philogen Jules Institut Bordet - - - - Format RTX-Bio similar RTX-Bio similar RTX-Bio similar RTX-Bio similar vaccine RIT - CD20 com pound PF- 05280586 ABP 798 BI 695500 SAIT101 Other RTX RTX+L19Il2 RTX

CD20 review 59 Reference [204] [84] [86] [31] [87] [88] [89] [90] [116] [92] [93] [94] [95] Summary I Type I Type enhanced II, ADCC Type engineering, glycol through for angle modified elbow PCD increased Bexxar II, backbone of Type epitope I/II, overlapping Type with rituximab I/II Type RTX of I, CDR grafting Type in CDR3 with 1aa difference glycan through enhanced ADCC engineering, through enhanced ADCC engineering protein ADCC Increased CDC enhanced ADCP and CDC enhanced ADCC, engineering protein through through CDC, ADCC Enhanced engineering protein Mechanism of Action Action Mechanism of reported ADCP CDC, ADCC, ADCP CDC, ADCC, PCD ADCP, ADCC, PCD ADCP, ADCC, PCD ADCP, CDC, ADCC, PCD ADCP, CDC, ADCC, ADCP CDC, ADCC, ADCP CDC, ADCC, ADCP CDC, ADCC, ADCP CDC, ADCC, ADCP CDC, ADCC, ADCP CDC, ADCC, PCD ADCP, CDC, ADCC, Highest level of of level Highest investigation approved approved approved Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Phase 2 Pre-clinical investigations Phase 1 Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Format Chimeric IgG1 Human IgG1 IgG1-Fc Humanized optimized Murine IgG2 IgG1 IgG1 IgG1 IgG1 optimized IgG1-Fc (P247I/E332I/A339Q) IgG1 IgG1/IgG3 chimer IgG1-S267E/H268F/ S324T + G326A/ I322E IgG1-H57DE/ H102YK/L93NR Clone(s) C2B8 2F2 Bly1 B1 1.5.3 BM-ca hA202 C2B8 2B8 TGLA C2B8 2H7 C2B8

CD20 targeting antibodies in pre-clinical research. in pre-clinical antibodies CD20 targeting

CD20 compound Monoclonal antibodies rituximab ofatumumab obinutuzumab tositumomab 1.5.3 BM-ca Veltuzumab BLX-300 Ocaratuzumab LY2469298) (AME-133v, TGLA 113F 2H7-EFT +AE (PRO131921) Triple TABLE 4 TABLE

60 Chapter 2 Reference [96] [99] [100] [216] [118] [103] [104] [105] [106] [84] [84] [84] [217] [122] Summary glycan through enhanced ADCC engineering, CDC enhanced engineering protein through molecule IgG like molecule IgG like I Type I Type CDC Increased IgG IgM to cloned from with epitope overlapping ofatumumab unique germline I Type II Type I Type Bexxar® Zevalin® rituximab MoA rituximab Mechanism of Action Action Mechanism of reported CDC both for enhanced and ADCC ADCP CDC, ADCC, ADCP CDC, ADCC, PCD ADCP, CDC, ADCC, ADCP CDC, ADCC, PCD ADCP, CDC, ADCC, CDC PCD ADCP, CDC, ADCC, PCD ADCP, CDC, ADCC, ADCP CDC, ADCC, PCD ADCP, ADCC, ADCP CDC, ADCC, cell induced Radiolabel APCD death + ADCC, and PCD cell induced Radiolabel death + Highest level of of level Highest investigation Pre-clinical investigations Pre-clinical investigations Phase 1 studies Phase 3 Phase 3 Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations approved* approved* Format IgG1 (SMIP) IgG1-variant (SMIP) IgG1 variant optimized IgG1-Fc IgG1 IgG1 scFv-IgG IgG1 IgG1 IgG1 IgG1 IgG1 mIgG2a-I131 mIgG1-Y90 Clone(s) C2B8 2H7 2H7 CAT- 13.6E12 2H7 8E4 1-28 OUMB6 OUMB3 1K1791 7D8 11B8 2C6 B1 2B8 CD20 compound RTX-EFTAE 2LM20-4 TRU-015 (TG-1101, Ublituximab LFB-R603, EMAB-6) Ocrelizumab (PRO70769) Hu8E4 5S hOUMB6 hOUMB3 1K1791 HuMab-7D8 HuMab-11B8 HuMab-2C6 Antibody conjugates Tositumumab-I131 Ibritumomab-Y90

CD20 review 61 Reference [123] [119] [122] [120] [121] [124] [125] [131] [132] [133] [133] [134] [135] [147] Summary induced cell cell induced cell induced cell induced rituximab MoA rituximab MoA rituximab antibody MoA Mechanism of Action Action Mechanism of reported cell induced Radiolabel death cell induced Radiolabel death Radiolabel death + Radiolabel death + Radiolabel death + Imaging only Imaging only cell induced conjugate death cell induced conjugate death cell induced conjugate death cell induced conjugate death cell induced conjugate death cell induced conjugate death PCD ADCP, CDC, ADCC, Highest level of of level Highest investigation Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical - Format mIgG2a-111In mIgG2a-At211 IgG1 IgG1 IgG1,IgG1,IgG1, mIgG2 IgG1-99mTc IgG1 IT IgG1-saporin-S6 IgG1-MMAE IgG1-MMAE IgG1-DOX IgG1-DM1 IgG1-HPMA-epiru bicin TetraMcAb Clone(s) 1F5 1F5 C2B8 C2B8 2F2, Bly-1, C2B8, B1 C2B8 Bly-1 C2B8 2F2 C2B8 C2B8 C2B8 C2B8 C2B8, 2F2 - CD20 compound 1F5-111In [211At]1F5-B10 90Y-DOTA-RTX 177Lu-CHX-A-DTPA-RTX 89ZR-obinutuzumab 89ZR-ofatumumab 89ZR-rituximab mab 89ZR-tositumumo rituxi Technetium-99m mab CY7-obinutuzumab CD20 IT OFA-MMAE RTX-MMAE RTX-DOX CD20-SPP-DM1 CD20-MCC-DM1 RTX-P-EPI Bispecific antibodies C2B8(ScFvHL)4 2F2(ScTvHL)4

62 Chapter 2 Reference [148] [151] [152] [153] [153] [155] [156] [160] [161] [163] [159] [166] [218] [176] Chemical crosslinking Chemical Summary DVD-Ig Hexavalent Hexavalent arm Fab Bivalent crosslinking Chemical DVD-Ig Fv Bispecific single chain heteroconjugation Quadroma effector effector effector PCD Mechanism of Action Action Mechanism of reported PCD ADCP, CDC, ADCC, PCD ADCP, CDC, ADCC, PCD ADCP, CDC, ADCC, PCD ADCP, CDC, ADCC, ADCC kill directed T-cell ADCP kill directed T-cell kill + directed T-cell mediated Fc functions kill + directed T-cell mediated Fc functions Highest level of of level Highest investigation Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations phase I phase I/II Pre-clinical Pre-clinical investigations ) β - 2 2 2 α xF(ab’) 2 Format 11B8/2F2(ScF vHL)4-Fc Ig DVD F(ab') HexAb HexAb BS9520 2H7-Fc-C825 IgG1-4x(IFN Tribody F(ab’)2xF(ab’) DVD-Ig scFv IgG1+mIgG2 mIgG2a x rIgG2b - Clone(s) 11B8, 2F2 C2B8, HLA- DR Rituximab/ HB22.7 hA20 epra tuzumab hA20 2H7, CD95 2H7, C825 hA20 7D8 1F5, CD89 2B8, B6H12 2H7, 9.3 x OKT3 RTX x TPA10 26II6 R1xCD20 α CD20 compound 11B8/2F2(ScFvHL)4-FC Ig DVD CD20-HLA-DR Bs20x22 20-22 20-20 CD20xCD95 CD20xDOTA 20-2b [(CD20)2xCD16] Fc CD20-CD47 r2820 CD20Bi Lymphomun, FBTA05, Bi20

CD20 review 63 Reference [144] [198] [141] [158] [170] [171] [172] [150] [174] Summary Quadroma half DVD-Ig Mechanism of Action Action Mechanism of reported kill directed T-cell kill directed T-cell kill directed T-cell kill directed T-cell kill directed T-cell kill directed T-cell kill directed T-cell kill directed T-cell kill directed T-cell kill directed T-cell Highest level of of level Highest investigation phase I/II phase I/II Phase I Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Pre-clinical investigations Format IgG1/4 chimeric Triple-F BsAb IgG1 generated using knob-into-holes technology CD20 TCB DuoBody Diabody fusion IgG1-LHD-scFv x ScFv F(ab’)2 IgG1 half DVD-Ig Clone(s) x REG30 REG32 x unknown sp34 2H7 x UCHT1 x Bly-1 SP34 7D8 x SP34 HIT3a x HI47 x unknown OKT3 ha20 x OKT3 x Bly-1 CLB-T3/4 x unknown OKT3 Anti-CD3 CD20 compound REGN1979 XmAb-13676 RG7828 RG6026 GEN3013 anti-CD20 x diabody CD3xCD20 bsAb 20-3S BIS20x3 halfbody

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66 Robak, T., et al., Rituximab plus fludarabine 76 Manshouri, T., et al., Circulating CD20 is and cyclophosphamide prolongs progression- detectable in the plasma of patients with free survival compared with fludarabine and chronic lymphocytic leukemia and is of cyclophosphamide alone in previously treated prognostic significance. Blood, 2003. 101(7): p. chronic lymphocytic leukemia. J Clin Oncol, 2507-13. 2010. 28(10): p. 1756-65. 77 Cragg, M.S. and M.J. Glennie, Antibody 67 Johnson, N.A., et al., Diffuse large B-cell specificity controls in vivo effector mechanisms lymphoma: reduced CD20 expression is of anti-CD20 reagents. Blood, 2004. 103(7): p. associated with an inferior survival. Blood, 2738-43. 2009. 113(16): p. 3773-80. 78 Klein, C., et al., Epitope interactions of 68 Suzuki, Y., et al., Association of CD20 levels monoclonal antibodies targeting CD20 and their with clinicopathological parameters and its relationship to functional properties. MAbs, prognostic significance for patients with DLBCL. 2013. 5(1): p. 22-33. Ann Hematol, 2012. 91(7): p. 997-1005. 79 Grillo-Lopez, A.J., et al., Rituximab: the 69 Lee, L., et al., Impact of rituximab on treatment first monoclonal antibody approved for the outcomes of patients with diffuse large b-cell treatment of lymphoma. Curr Pharm Biotechnol, lymphoma: a population-based analysis. Br J 2000. 1(1): p. 1-9. Haematol, 2012. 158(4): p. 481-8. 80 Marcus, R., et al., CVP chemotherapy plus 70 Choi, C.H., et al., Prognostic Implication of Semi- rituximab compared with CVP as first-line quantitative Immunohistochemical Assessment treatment for advanced follicular lymphoma. of CD20 Expression in Diffuse Large B-Cell Blood, 2005. 105(4): p. 1417-23. Lymphoma. J Pathol Transl Med, 2016. 50(2): p. 96-103. 81 Hiddemann, W., et al., Frontline therapy with rituximab added to the combination of 71 Tzankov, A., et al., Prognostic significance of cyclophosphamide, doxorubicin, vincristine, CD20 expression in classical Hodgkin lymphoma: and prednisone (CHOP) significantly improves a clinicopathological study of 119 cases. Clin the outcome for patients with advanced-stage Cancer Res, 2003. 9(4): p. 1381-6. follicular lymphoma compared with therapy with CHOP alone: results of a prospective 72 Kuppers, R. and M.L. Hansmann, The Hodgkin randomized study of the German Low-Grade and Reed/Sternberg cell. Int J Biochem Cell Biol, Lymphoma Study Group. Blood, 2005. 106(12): 2005. 37(3): p. 511-7. p. 3725-32.

68 Chapter 2 82 Herold, M., et al., Rituximab added to first-line 91 Cheney, C.M., et al., Ocaratuzumab, an Fc- mitoxantrone, chlorambucil, and prednisolone engineered antibody demonstrates enhanced chemotherapy followed by interferon antibody-dependent cell-mediated cytotoxicity in maintenance prolongs survival in patients with chronic lymphocytic leukemia. MAbs, 2014. 6(3): advanced follicular lymphoma: an East German p. 749-55. Study Group Hematology and Oncology Study. J Clin Oncol, 2007. 25(15): p. 1986-92. 92 Lv, M., et al., Novel anti-CD20 antibody TGLA with enhanced antibody-dependent cell- 83 Salles, G., et al., Rituximab combined with mediated cytotoxicity mediates potent anti- chemotherapy and interferon in follicular lymphoma activity. Cancer Lett, 2010. 294(1): lymphoma patients: results of the GELA- p. 66-73. GOELAMS FL2000 study. Blood, 2008. 112(13): p. 4824-31. 93 Sato, F., et al., A complement-dependent cytotoxicity-enhancing anti-CD20 antibody 84 Teeling, J.L., et al., Characterization of new mediating potent antitumor activity in the human CD20 monoclonal antibodies with humanized NOD/Shi-scid, IL-2Rgamma(null) potent cytolytic activity against non-Hodgkin mouse lymphoma model. Cancer Immunol lymphomas. Blood, 2004. 104(6): p. 1793-800. Immunother, 2010. 59(12): p. 1791-800.

85 van Meerten, T., et al., HuMab-7D8, a 94 Moore, G.L., et al., Engineered Fc variant monoclonal antibody directed against the antibodies with enhanced ability to recruit membrane-proximal small loop epitope of CD20 complement and mediate effector functions. can effectively eliminate CD20 low expressing MAbs, 2010. 2(2): p. 181-9. tumor cells that resist rituximab-mediated lysis. Haematologica, 2010. 95(12): p. 2063-71. 95 Li, B., et al., Characterization of a rituximab variant with potent antitumor activity against 86 Mossner, E., et al., Increasing the efficacy of rituximab-resistant B-cell lymphoma. Blood, CD20 antibody therapy through the engineering 2009. 114(24): p. 5007-15. of a new type II anti-CD20 antibody with enhanced direct and immune effector cell- 96 Wirt, T., et al., An Fc Double-Engineered CD20 mediated B-cell cytotoxicity. Blood, 2010. Antibody with Enhanced Ability to Trigger 115(22): p. 4393-402. Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity. 87 Bornstein, G.G., et al., Development of a new Transfus Med Hemother, 2017. 44(5): p. 292- fully human anti-CD20 monoclonal antibody for 300. the treatment of B-cell malignancies. Invest New Drugs, 2010. 28(5): p. 561-74. 97 Morschhauser, F., et al., Results of a phase I/ II study of ocrelizumab, a fully humanized anti- 88 Nishida, M., et al., BM-ca is a newly defined type CD20 mAb, in patients with relapsed/refractory I/II anti-CD20 monoclonal antibody with unique follicular lymphoma. Ann Oncol, 2010. 21(9): p. biological properties. Int J Oncol, 2011. 38(2): 1870-6. p. 335-44. 98 Chan, J.K., C.S. Ng, and P.K. Hui, A simple guide 89 Goldenberg, D.M., et al., Properties and to the terminology and application of leucocyte structure-function relationships of veltuzumab monoclonal antibodies. Histopathology, 1988. (hA20), a humanized anti-CD20 monoclonal 12(5): p. 461-80. antibody. Blood, 2009. 113(5): p. 1062-70. 99 Nickerson-Nutter, C., et al., Distinct in vitro 90 Gasdaska, J.R., et al., An afucosylated anti-CD20 binding properties of the anti-CD20 small monoclonal antibody with greater antibody- modular immunopharmaceutical 2LM20-4 result dependent cellular cytotoxicity and B-cell in profound and sustained in vivo potency in depletion and lower complement-dependent cynomolgus monkeys. Rheumatology (Oxford), cytotoxicity than rituximab. Mol Immunol, 2011. 50(6): p. 1033-44. 2012. 50(3): p. 134-41.

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106 Nishida, M., et al., Novel humanized anti-CD20 114 Horning, S.J., et al., Efficacy and safety of monoclonal antibodies with unique germline VH tositumomab and iodine-131 tositumomab and VL gene recruitment and potent effector (Bexxar) in B-cell lymphoma, progressive after functions. Int J Oncol, 2008. 32(6): p. 1263-74. rituximab. J Clin Oncol, 2005. 23(4): p. 712-9.

107 Jacene, H.A., et al., Comparison 115 Davis, T.A., et al., The radioisotope of 90Y-ibritumomab tiuxetan and contributes significantly to the activity of 131I-tositumomab in clinical practice. J Nucl radioimmunotherapy. Clin Cancer Res, 2004. Med, 2007. 48(11): p. 1767-76. 10(23): p. 7792-8.

108 Conti, P.S., et al., The role of imaging with (111) 116 Gregory, S.A., The iodine I-131 tositumomab In-ibritumomab tiuxetan in the ibritumomab therapeutic regimen: Summary of safety in 995 tiuxetan (zevalin) regimen: results from a patients with relapsed/refractory low grad (LG) Zevalin Imaging Registry. J Nucl Med, 2005. and transformed LG non-Hodgkin’s lymphoma 46(11): p. 1812-8. (NHL). Journal of Clinical Oncology, 2004. 22.14: p. 6732-6732.

117 Armitage, J.O., et al., Treatment-related myelodysplasia and acute leukemia in non- Hodgkin’s lymphoma patients. J Clin Oncol, 2003. 21(5): p. 897-906.

70 Chapter 2 118 Shadman, M., et al., Continued Excellent 128 Vaughan, A.T., et al., Activatory and inhibitory Outcomes in Previously Untreated Patients Fcgamma receptors augment rituximab- With Follicular Lymphoma After Treatment mediated internalization of CD20 independent With CHOP Plus Rituximab or CHOP Plus (131) of signaling via the cytoplasmic domain. J Biol I-Tositumomab: Long-Term Follow-Up of Phase Chem, 2015. 290(9): p. 5424-37. III Randomized Study SWOG-S0016. J Clin Oncol, 2018. 36(7): p. 697-703. 129 Vervoordeldonk, S.F., et al., Preclinical studies with radiolabeled monoclonal antibodies for 119 Green, D.J., et al., Astatine-211 conjugated to treatment of patients with B-cell malignancies. an anti-CD20 monoclonal antibody eradicates Cancer, 1994. 73(3 Suppl): p. 1006-11. disseminated B-cell lymphoma in a mouse model. Blood, 2015. 125(13): p. 2111-9. 130 Press, O.W., et al., Retention of B-cell-specific monoclonal antibodies by human lymphoma 120 Kameswaran, M., et al., Synthesis and Preclinical cells. Blood, 1994. 83(5): p. 1390-7. Evaluation of (177)Lu-CHX-A”-DTPA-Rituximab as a Radioimmunotherapeutic Agent for 131 Polito, L., et al., Two Saporin-Containing Non-Hodgkin’s Lymphoma. Cancer Biother Immunotoxins Specific for CD20 and CD22 Show Radiopharm, 2015. 30(6): p. 240-6. Different Behavior in Killing Lymphoma Cells. Toxins (Basel), 2017. 9(6). 121 Yoon, J.T., et al., Evaluation of Next-Generation Anti-CD20 Antibodies Labeled with Zirconium 132 Li, Z.H., et al., Preclinical studies of targeted 89 in Human Lymphoma Xenografts. J Nucl therapies for CD20-positive B lymphoid Med, 2018. malignancies by Ofatumumab conjugated with auristatin. Invest New Drugs, 2014. 32(1): p. 122 Johari Doha, F., et al., Development of DOTA- 75-86. Rituximab to be Labeled with (90)Y for Radioimmunotherapy of B-cell Non-Hodgkin 133 Law, C.L., et al., Efficient elimination of B-lineage Lymphoma. Iran J Pharm Res, 2017. 16(2): p. lymphomas by anti-CD20-auristatin conjugates. 619-629. Clin Cancer Res, 2004. 10(23): p. 7842-51.

123 Michel, R.B. and M.J. Mattes, Intracellular 134 Polson, A.G., et al., Antibody-drug conjugates accumulation of the anti-CD20 antibody 1F5 in for the treatment of non-Hodgkin’s lymphoma: B-lymphoma cells. Clin Cancer Res, 2002. 8(8): target and linker-drug selection. Cancer Res, p. 2701-13. 2009. 69(6): p. 2358-64.

124 Camacho, X., et al., Technetium-99m- or Cy7- 135 Zhang, L., et al., A new construct of antibody- Labeled Rituximab as an Imaging Agent for drug conjugates for treatment of B-cell non- Non-Hodgkin Lymphoma. Oncology, 2017. Hodgkin’s lymphomas. Eur J Pharm Sci, 2017. 92(4): p. 229-242. 103: p. 36-46.

125 Lin, X., et al., Near-infrared fluorescence imaging 136 Spiess, C., Q. Zhai, and P.J. Carter, Alternative of non-Hodgkin’s lymphoma CD20 expression molecular formats and therapeutic applications using Cy7-conjugated obinutuzumab. Mol for bispecific antibodies. Mol Immunol, 2015. Imaging Biol, 2014. 16(6): p. 877-87. 67(2 Pt A): p. 95-106.

126 Schaefer, N.G., et al., Radioimmunotherapy in 137 Kontermann, R.E., Dual targeting strategies non-Hodgkin lymphoma: opinions of nuclear with bispecific antibodies. MAbs, 2012. 4(2): p. medicine physicians and radiation oncologists. J 182-97. Nucl Med, 2011. 52(5): p. 830-8.

138 Brinkmann, U. and R.E. Kontermann, The 127 Lim, S.H., et al., Fc gamma receptor IIb on target making of bispecific antibodies. MAbs, 2017. B cells promotes rituximab internalization and 9(2): p. 182-212. reduces clinical efficacy. Blood, 2011. 118(9): p. 2530-40.

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143 Chu, S., S. Lee, and R. Rashid, Immunotherapy 152 Tuscano, J.M., et al., The Bs20x22 anti- with Long-Lived Anti-CD20 × Anti-CD3 CD20-CD22 bispecific antibody has more Bispecific Antibodies Stimulates Potent T Cell- lymphomacidal activity than do the parent Mediated Killing of Human B Cell Lines and of antibodies alone. Cancer Immunol Immunother, Circulating and Lymphoid B Cells in Monkeys: 2011. 60(6): p. 771-80. A Potential Therapy for B Cell Lymphomas and Leukemias. Blood, 2014. 124(21): p. 3111. 153 Gupta, P., et al., Multiple signaling pathways induced by hexavalent, monospecific, anti-CD20 144 Smith, E.J., et al., A novel, native-format and hexavalent, bispecific, anti-CD20/CD22 bispecific antibody triggering T-cell killing of humanized antibodies correlate with enhanced B-cells is robustly active in mouse tumor models toxicity to B-cell lymphomas and leukemias. and cynomolgus monkeys. Sci Rep, 2015. 5: p. Blood, 2010. 116(17): p. 3258-67. 17943. 154 Rossi, E.A., et al., Hexavalent bispecific 145 Pang, X., et al., Treatment of Human B-Cell antibodies represent a new class of anticancer Lymphomas Using Minicircle DNA Vector therapeutics: 1. Properties of anti-CD20/CD22 Expressing Anti-CD3/CD20 in a Mouse Model. antibodies in lymphoma. Blood, 2009. 113(24): Hum Gene Ther, 2017. 28(2): p. 216-225. p. 6161-71.

146 Muller, D., et al., Improved pharmacokinetics of 155 Nalivaiko, K., et al., A Recombinant Bispecific recombinant bispecific antibody molecules by CD20xCD95 Antibody With Superior Activity fusion to human serum albumin. J Biol Chem, Against Normal and Malignant B-cells. Mol Ther, 2007. 282(17): p. 12650-60. 2016. 24(2): p. 298-305.

147 Li, B., et al., Development of novel tetravalent 156 Green, D.J., et al., Comparative Analysis of anti-CD20 antibodies with potent antitumor Bispecific Antibody and Streptavidin-Targeted activity. Cancer Res, 2008. 68(7): p. 2400-8. Radioimmunotherapy for B-cell Cancers. Cancer Res, 2016. 76(22): p. 6669-6679.

148 Li, B., et al., Construction and characterization of a bispecific anti-CD20 antibody with potent 157 Rossi, E.A., et al., A bispecific antibody- antitumor activity against B-cell lymphoma. IFNalpha2b immunocytokine targeting CD20 Cancer Res, 2010. 70(15): p. 6293-302. and HLA-DR is highly toxic to human lymphoma and multiple myeloma cells. Cancer Res, 2010. 70(19): p. 7600-9.

72 Chapter 2 158 Sivaraman, S., et al., Effect of interferon-alpha 168 Offner, S., et al., Induction of regular cytolytic T on CD20 antigen expression of B-cell chronic cell synapses by bispecific single-chain antibody lymphocytic leukemia. Cytokines Cell Mol Ther, constructs on MHC class I-negative tumor cells. 2000. 6(2): p. 81-7. Mol Immunol, 2006. 43(6): p. 763-71.

159 Trinh, K.R., et al., Anti-CD20-interferon- 169 Goebeler, M.E. and R. Bargou, Blinatumomab: a beta fusion protein therapy of murine B-cell CD19/CD3 bispecific T cell engager (BiTE) with lymphomas. J Immunother, 2013. 36(5): p. unique anti-tumor efficacy. Leuk Lymphoma, 305-18. 2016. 57(5): p. 1021-32.

160 Rossi, E.A., et al., CD20-targeted tetrameric 170 Xiong, D., et al., Efficient inhibition of human interferon-alpha, a novel and potent B-cell lymphoma xenografts with an anti-CD20 immunocytokine for the therapy of B-cell x anti-CD3 bispecific diabody. Cancer Lett, 2002. lymphomas. Blood, 2009. 114(18): p. 3864-71. 177(1): p. 29-39.

161 Glorius, P., et al., The novel tribody [(CD20)(2) 171 Lu, C.Y., et al., Tetravalent anti-CD20/CD3 xCD16] efficiently triggers effector cell-mediated bispecific antibody for the treatment of B cell lysis of malignant B cells. Leukemia, 2013. lymphoma. Biochem Biophys Res Commun, 27(1): p. 190-201. 2016. 473(4): p. 808-813.

162 Li, B., et al., CD89-mediated recruitment of 172 Rossi, D.L., et al., A new class of bispecific macrophages via a bispecific antibody enhances antibodies to redirect T cells for cancer anti-tumor efficacy. Oncoimmunology, 2017. immunotherapy. MAbs, 2014. 6(2): p. 381-91. 7(1): p. e1380142. 173 Withoff, S., et al., Characterization of BIS20x3, 163 Vugmeyster, Y., et al., Depletion of B cells by a a bi-specific antibody activating and retargeting humanized anti-CD20 antibody PRO70769 in T-cells to CD20-positive B-cells. Br J Cancer, Macaca fascicularis. J Immunother, 2005. 28(3): 2001. 84(8): p. 1115-21. p. 212-9. 174 Bardwell, P.D., et al., Potent and conditional 164 van Bommel, P.E., et al., CD20-selective redirected T cell killing of tumor cells using Half inhibition of CD47-SIRPalpha “don’t eat DVD-Ig. Protein Cell, 2018. 9(1): p. 121-129. me” signaling with a bispecific antibody- derivative enhances the anticancer activity of 175 Lum, L.G., Phase I Dose Esclation of Activated T daratumumab, alemtuzumab and obinutuzumab. Cells (ATC) Armed with Anti-CD3 × Anti-CD20 Oncoimmunology, 2018. 7(2): p. e1386361. Bispecific Antibody (CD20Bi) after Stem Cell Transplant (SCT) In Non-Hodgkin’s Lymphoma 165 Piccione, E.C., et al., A bispecific antibody (NHL). Blood, 2010. 116(21): p. 488. targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma 176 Stanglmaier, M., et al., Bi20 (fBTA05), a novel cells. MAbs, 2015. 7(5): p. 946-56. trifunctional bispecific antibody (anti-CD20 x anti-CD3), mediates efficient killing of B-cell 166 Otz, T., et al., A bispecific single-chain antibody lymphoma cells even with very low CD20 that mediates target cell-restricted, supra- expression levels. Int J Cancer, 2008. 123(5): p. agonistic CD28 stimulation and killing of 1181-9. lymphoma cells. Leukemia, 2009. 23(1): p. 71-7. 177 Buhmann, R., et al., Immunotherapy of recurrent 167 Brandl, M., et al., Bispecific antibody fragments B-cell malignancies after allo-SCT with Bi20 with CD20 X CD28 specificity allow effective (FBTA05), a trifunctional anti-CD3 x anti-CD20 autologous and allogeneic T-cell activation antibody and donor lymphocyte infusion. Bone against malignant cells in peripheral blood and Marrow Transplant, 2009. 43(5): p. 383-97. bone marrow cultures from patients with B-cell lineage leukemia and lymphoma. Exp Hematol, 1999. 27(8): p. 1264-70.

CD20 review 73 178 Schuster, F.R., et al., Immunotherapy with the 187 Zhang, W.Y., et al., Treatment of CD20-directed trifunctional anti-CD20 x anti-CD3 antibody Chimeric Antigen Receptor-modified T cells FBTA05 (Lymphomun) in paediatric high-risk in patients with relapsed or refractory B-cell patients with recurrent CD20-positive B cell non-Hodgkin lymphoma: an early phase IIa trial malignancies. Br J Haematol, 2015. 169(1): p. report. Signal Transduct Target Ther, 2016. 1: 90-102. p. 16002.

179 Bannerji, R., Phase 1 Study of REGN1979, an 188 Wang, Y., et al., Effective response and delayed Anti-CD20 x Anti-CD3 Bispecific Monoclonal toxicities of refractory advanced diffuse large Antibody, in Patients with CD20+ B-Cell B-cell lymphoma treated by CD20-directed Malignancies Previously Treated with CD20- chimeric antigen receptor-modified T cells. Clin Directed Antibody Therapy. Blood, 2016. Immunol, 2014. 155(2): p. 160-75. 128(22): p. 621. 189 Watanabe, K., et al., Target antigen density 180 Bannerji, R., et al., Safety and Preliminary governs the efficacy of anti-CD20-CD28-CD3 Clinical Activity of REGN1979, an Anti-CD20 zeta chimeric antigen receptor-modified effector x Anti-CD3 Bispecific Antibody, in Patients CD8+ T cells. J Immunol, 2015. 194(3): p. 911- with B-NHL Previously Treated with CD20- 20. Directed Antibody Therapy. American Society of Hematology, 2017. 130(Suppl 1): p. 1550. 190 Budde, L.E., et al., Combining a CD20 chimeric antigen receptor and an inducible caspase 9 181 S.Y., C., S.H. Lee, and R. Rashid, Immunotherapy suicide switch to improve the efficacy and safety with long-lived Anti-CD20 x Anti-CD3 Bispecific of T cell adoptive immunotherapy for lymphoma. Antibodies Stimulates Potent T Cell-mediated PLoS One, 2013. 8(12): p. e82742. Killing of Human B cell lines and of Circulating and lymphoid B cells in Moneys: A potential 191 Rodgers, D.T., et al., Switch-mediated activation therapy for B cell lymphomas and Leukemias. and retargeting of CAR-T cells for B-cell Blood, 2014. 124: p. 311. malignancies. Proc Natl Acad Sci U S A, 2016. 113(4): p. E459-68. 182 Bacac, M., CD20 Tcb (RG6026), a Novel “2:1” T Cell Bispecific Antibody for the Treatment of 192 van Meerten, T., et al., The CD20/alphaCD20 B Cell Malignancies. Blood, 2016. 128(22): p. ‘suicide’ system: novel vectors with improved 1836. safety and expression profiles and efficient elimination of CD20-transgenic T cells. Gene 183 Zheng, Y., et al., Potential therapeutic Ther, 2006. 13(9): p. 789-97. strategy for non-Hodgkin lymphoma by anti- CD20scFvFc/CD28/CD3zeta gene tranfected T 193 Martyniszyn, A., et al., CD20-CD19 Bispecific cells. J Exp Clin Cancer Res, 2010. 29: p. 121. CAR T Cells for the Treatment of B-Cell Malignancies. Hum Gene Ther, 2017. 28(12): p. 184 Chen, F., et al., Construction of Anti-CD20 1147-1157. Single-Chain Antibody-CD28-CD137-TCRzeta Recombinant Genetic Modified T Cells and its 194 Zah, E., et al., ADDENDUM: T Cells Expressing Treatment Effect on B Cell Lymphoma. Med Sci CD19/CD20 Bispecific Chimeric Antigen Monit, 2015. 21: p. 2110-5. Receptors Prevent Antigen Escape by Malignant B Cells. Cancer Immunol Res, 2016. 4(7): p. 185 Till, B.G., et al., CD20-specific adoptive 639-41. immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB 195 Zah, E., et al., T Cells Expressing CD19/CD20 domains: pilot clinical trial results. Blood, 2012. Bispecific Chimeric Antigen Receptors Prevent 119(17): p. 3940-50. Antigen Escape by Malignant B Cells. Cancer Immunol Res, 2016. 4(6): p. 498-508. 186 Lee, S.Y., et al., Preclinical Optimization of a CD20-specific Chimeric Antigen Receptor Vector and Culture Conditions. J Immunother, 2018. 41(1): p. 19-31.

74 Chapter 2 196 Zhu, F., et al., Closed-system manufacturing 206 Robak, T., GA-101, a third-generation, of CD19 and dual-targeted CD20/19 chimeric humanized and glyco-engineered anti-CD20 antigen receptor T cells using the CliniMACS mAb for the treatment of B-cell lymphoid Prodigy device at an academic medical center. malignancies. Curr Opin Investig Drugs, 2009. Cytotherapy, 2017. 10(6): p. 588-96.

197 Lock, D., et al., Automated Manufacturing of 207 Zelenetz, A.D., A clinical and scientific overview Potent CD20-Directed Chimeric Antigen Receptor of tositumomab and iodine I 131 tositumomab. T Cells for Clinical Use. Hum Gene Ther, 2017. Semin Oncol, 2003. 30(2 Suppl 4): p. 22-30. 28(10): p. 914-925. 208 Frampton, J.E., Ocrelizumab: First Global 198 Chu, Y., et al., Targeting CD20+ Aggressive B-cell Approval. Drugs, 2017. 77(9): p. 1035-1041. Non-Hodgkin Lymphoma by Anti-CD20 CAR mRNA-Modified Expanded Natural Killer Cells 209 EMA, Zevalin In Vitro and in NSG Mice. Cancer Immunol Res, 2015. 3(4): p. 333-44. 210 EMA, Blitzima.

199 Oberschmidt, O., S. Kloess, and U. Koehl, 211 EMA, Ritemvia. Redirected Primary Human Chimeric Antigen Receptor Natural Killer Cells As an “Off-the-Shelf Immunotherapy” for Improvement in Cancer 212 EMA, Rituzena. Treatment. Front Immunol, 2017. 8: p. 654. 213 EMA, Rixathon. 200 Hilchey, S.P., et al., Rituximab immunotherapy results in the induction of a lymphoma idiotype- 214 EMA, Riximyo. specific T-cell response in patients with follicular lymphoma: support for a “vaccinal effect” of 215 EMA, Truxima. rituximab. Blood, 2009. 113(16): p. 3809-12.

216 de Romeuf, C., et al., Chronic lymphocytic 201 Deligne, C., et al., Anti-CD20 therapy induces a leukaemia cells are efficiently killed by an memory Th1 response through the IFN-gamma/ anti-CD20 monoclonal antibody selected for IL-12 axis and prevents protumor regulatory improved engagement of FcgammaRIIIA/CD16. T-cell expansion in mice. Leukemia, 2015. 29(4): Br J Haematol, 2008. 140(6): p. 635-43. p. 947-57.

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