Novel Designs of Multivalent Anti-CD20 Humanized Antibodies As Improved Lymphoma Therapeutics

Research Article

Novel Designs of Multivalent Anti-CD20 Humanized Antibodies as Improved Lymphoma Therapeutics

Edmund A. Rossi,1 David M. Goldenberg,3 Thomas M. Cardillo,2 Rhona Stein,3 Yang Wang,2 and Chien-Hsing Chang1,2

1IBC Pharmaceuticals, Inc.; 2Immunomedics, Inc., Morris Plains, New Jersey; and 3Garden State Cancer Center, Center for Molecular Medicine and Immunology, Belleville, New Jersey

Abstract implicated (2). Although only about halfofpatients with relapsed NHL respond to rituximab, its importance lies in its representing a Multivalent antibodies, either monospecific or bispecific, may improve the efficacy of current therapeutic interventions category oftherapeutics, which has a strikingly different toxicity involving a single monoclonal antibody (mAb). We have profile than the typical cytotoxic drugs available. Its relative applied the Dock-and-Lock (DNL) method, a new platform paucity oflong-term toxicity and the short-term adverse event technology for the site-specific and covalent assembly of profile, being mostly associated with infusion reactions, show no modular components into stably tethered complexes of overlap with chemotherapy, thus providing a rational combination defined composition, to prepare a hexavalent, anti-CD20 ofimmunotherapy with chemotherapy without enhanced toxicity. antibody, designated Hex-hA20, which comprises six Fabs At present, next-generation anti-CD20 mAbs include human and humanized forms, with some claiming enhanced potency by with one Fc. We show that Hex-hA20 retains the binding antibody reengineering (1, 3–5). However, it is still unclear what activity of all six Fabs, associates with CD20 in lipid rafts, improvements are required to show better efficacy than affects antibody-dependent cell-mediated cytotoxicity, but not rituximab. Predicting on the basis oflaboratory results has been complement-dependent cytotoxicity, and inhibits prolifera- difficult, because despite 10 years of clinical use of rituximab and tion of Daudi, Raji, and Ramos cells in vitro at subnanomolar an expansive preclinical literature on the use ofanti-CD20 mAbs concentrations without the need for a cross-linking antibody. in lymphoma models in vitro and in vivo, how rituximab or other In addition, Hex-hA20 induces strong homotypical adhesion anti-CD20 mAbs kill lymphoma cells is still being debated (5) and is inefficient in stimulating calcium mobilization. Thus, among the three mechanisms proposed: complement-dependent Hex-hA20 exhibits biological properties attributable to both cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity type I and type II anti-CD20 mAbs, as exemplified by rituximab (ADCC), and apoptosis or growth inhibition induced by direct and tositumomab, respectively. Although Hex-hA20 has a signaling. short serum half-life, it shows antitumor efficacy in tumor- Dimeric forms of IgG made by genetic engineering (6–10) or bearing mice comparable with veltuzumab at equivalent chemical conjugation (11, 12) may become more effective doses. The versatile DNL method was also applied to generate antibodies than their monomeric counterparts as a result of two other multivalent anti-CD20 antibodies without the Fc increased valency, as well as enhanced effector functions. For region, Tri-hA20 and Tetra-hA20, comprising three and four example, a mAb for CD19 or CD20 that has little or no signaling Fabs of veltuzumab, respectively. Similar to Hex-hA20, these activity was shown to induce growth inhibition or apoptosis when were purified to near homogeneity and shown to have potent each is converted into an IgG-IgG homodimer (11, 12). Similar antiproliferative activity in vitro, thus indicating the need for observations ofattaining signaling activity or exhibiting higher clustering three or more CD20 molecules on the cell surface to cytotoxicity were reported for recombinantly produced fusion induce growth inhibition. [Cancer Res 2008;68(20):8384–92] proteins comprising three identical anti-HER2 (13) single-chain Fvs (scFv), four identical anti-CD22 (14, 15) scFvs, or four Introduction identical anti-CD20 (16) scFvs. In addition, a dextran-rituximab With the approval ofrituximab, a chimeric anti-CD20 monoclo- polymer was found to inhibit the growth of Raji xenografts in nal antibody (mAb), in 1997 for the treatment of relapsed or mice, whereas the homodimer or monomer ofrituximab was refractory low-grade or follicular non–Hodgkin lymphoma (NHL; ineffective (17). Therefore, we propose that making multivalent or ref. 1), a new era in mAb-based therapy of cancer was introduced multimeric mAbs ofa definedcomposition and proved homoge- whereas a profound management change in the treatment of neity should be promising for developing more potent anti-CD20 CD20-positive lymphomas occurred. It not only presented a novel agents, which may also help elucidate the most critical modality to treat B-cell malignancies but also provided an impetus mechanism ofaction in vivo. to treat autoimmune diseases where B-cell dysregulation is Veltuzumab or hA20 is a complementarity-determined region– grafted, anti-CD20 humanized mAb that exhibits CDC and ADCC in vitro, as well as inhibits cell proliferation and induces apoptosis upon cross-linking with a second antibody (18). Initial clinical Note: Supplementary data for this article are available at Cancer Research Online studies have shown good safety and efficacy results in NHL patients (http://cancerres.aacrjournals.org/). Requests for reprints: Chien-Hsing Chang, Immunomedics, Inc., 300 American and confirmed that low doses of veltuzumab are effective and that Road, Morris Plains, NJ 07950. Phone: 973-605-1330, ext. 108; Fax: 973-605-1103; all doses can be given in shorter infusion times than the approved E-mail: [email protected] or David M. Goldenberg, Garden State Cancer dose ofrituximab (19). Center, 520 Belleville Avenue, Belleville, NJ 07109. E-mail: [email protected]. I2008 American Association for Cancer Research. The Dock-and-Lock (DNL) method exploits the dimerization and doi:10.1158/0008-5472.CAN-08-2033 docking domain (DDD) and the anchoring domain (AD) that are

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Figure 1. Schematic diagrams representing CH1-AD2-Fab module, CH1-DDD2-Fab module, CH3-AD2-IgG module, Tri-Fab, Tetra-Fab, and Hex-IgG (the DNL structure generated by reacting CH1-DDD2-Fab with CH3-AD2-IgG). Green, variable domains for the heavy (VH) and light (VL ) chains; gray, constant domains of the heavy (CH ) and light (CL ) chains; blue, DDD2 peptides; pink, AD2 peptides. involved in the natural binding between cyclic AMP–dependent Materials and Methods protein kinase and A-kinase anchoring proteins (20, 21) as a pair of Antibodies, reagents and culture media. Veltuzumab, epratuzumab linkers for specifically docking a DDD-containing module into an (hLL2, humanized anti-CD22), labetuzumab (hMN-14, humanized anti- AD-containing module, with the resulting binary complex further CEACAM5), and WR2, a rat anti-idiotype mAb to veltuzumab, were stabilized covalently by locking with disulfide bonds (22, 23). One provided by Immunomedics. Rituximab and tositumomab (murine anti-B1) distinct advantage ofthe DNL method, as noted previously (23), is were obtained from commercial supplies. The purity of rituximab or to provide a modular approach for generating multivalent tositumomab was determined by size-exclusion high-performance liquid antibodies ofmonospecificityor bispecificity via site-specific chromatography (SE-HPLC) to be >98%. Secondary antibodies were conjugation oftwo types ofrecombinantly produced fusion obtained from Jackson Immunoresearch. Mouse anti-human IgM was proteins that are derived from the same or different antibodies. obtained from SouthernBiotech. Antimouse Gr-1 ascites was kindly supplied by Dr. Myron Czuczman (Roswell Park Cancer Institute). The Here, we describe a veltuzumab-based hexavalent antibody (Hex- anti-mouse interleukin 2 (IL-2) receptor mAb, TMh-1, was purchased from hA20) made by DNL to consist ofa bivalent IgG linked to four BD Biosciences. All restriction endonucleases and other enzymes were monovalent Fabs, and the evaluation ofits antitumor activity purchased from New England Biolabs. Oligonucleotides were synthesized by against CD20-expressing human lymphoma cells in vitro and Sigma Genosys. PCR reactions were performed using Amplitaq polymerase in vivo. The versatile DNL method was also applied to generate two (Applied Biosystems) and a Perkin-Elmer GeneAmp PCR system 9600. Heat- other multivalent anti-CD20 antibodies without the Fc region: Tri- inactivated fetal bovine serum (FBS) was purchased from Hyclone. All other hA20, a trivalent construct composed ofthree hA20 Fabs, and cell culture media and supplements were from Invitrogen Life Technologies. Tetra-hA20, a tetravalent construct composed offourhA20 Fabs. Cell lines. Daudi, Raji, and Ramos were purchased from the American Type Culture Collection. Sp/ESF, a variant ofSp2/0-Ag14 engineered to All three multivalent anti-CD20 antibodies were purified to near 4 homogeneity and shown to retain the binding activity ofeach grow in serum-free medium, was used as the host cell for transfection. Generation of Hex-hA20. The pdHL2 vector has been used to mediate constituent Fab. In addition, testing ofTri-hA20 allowed us to the expression ofrecombinant IgGs in mammalian hosts (24). A plasmid establish that clustering three CD20 molecules on the cell surface via antibody binding is sufficient to induce growth inhibition. Schematic diagrams for Hex-IgG, Tri-Fab, Tetra-Fab, and the modules used for building them are shown in Fig. 1. 4 D. Nordstrom, E.A. Rossi, D.M. Goldenberg, C-H. Chang, unpublished data. www.aacrjournals.org 8385 Cancer Res 2008; 68: (20). October 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research shuttle vector was produced to facilitate the conversion of any IgG-pdHL2 Triton X-100 (100% lysis) and cells treated with complement alone vector into a CH3-AD2-IgG-pdHL2 vector. The expression vector encoding (background). CH1-DDD2-Fab-hA20 was generated from the CH3-AD2-IgG-hA20-pdHL2 by ADCC. Daudi cells were incubated with each test article in triplicate at A j excising the coding sequence for the CH1-Hinge-CH2-CH3 domains with SacII 5 g/mL for 30 min at 37 C and 5% CO2. Freshly isolated peripheral blood and EagI and replacing it with a 507-bp sequence encoding CH1-DDD2, mononuclear cells obtained from healthy volunteers were then added at a which was excised from the C-DDD2-hMN-14-pdHL2 expression vector (22) predetermined optimal effector to target ratio of 50:1. After a 4-h incubation, with the same enzymes. The expression vector CH3-AD2-IgG-hA20-pdHL2 or cell lysis was assessed by CytoTox-One (Promega). CH1-DDD2-Fab-hA20-pdHL2, each 30 Ag, was linearized by digestion with Calcium mobilization. Intracellular calcium was measured in Ramos SalI and transfected into Sp/ESF (2.8 106 cells) by electroporation (450 V, cells loaded with 20 Amol/L Fluo-3 AM (Invitrogen) using a Becton 25 AF). The pdHL2 vector contains the gene for dihydrofolate reductase, Dickinson FACScan and the FlowJo program (Tree Star, Inc.). For all thus allowing clonal selection, as well as gene amplification, with samples, a baseline was obtained for 60 s before adding each test article, methotrexate (MTX). After transfection, the cells were plated in 96-well which includes ionomycin and anti-human IgM as positive controls. To plates and selected in media containing 0.2 Amol/L MTX. Clones were evaluate the effect of cross-linking, cells were incubated with 1 Ag/mL of screened for CH3-AD2-IgG-hA20 or CH1-DDD2-Fab-hA20 productivity by a veltuzumab, rituximab, or tositumomab for further 15 min and stimulated sandwich ELISA using 96-well microtiter plates coated with WR2 to capture with an appropriate second antibody (50 Ag/mL final). the fusion protein, which was detected with horseradish peroxidase– Homotypical adhesion. Daudi cells (1.5 106/mL) were treated conjugated goat anti-human IgG F(ab’)2. Wells giving the highest signal were with veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at 1 nmol/L for expanded and ultimately used for production. 20 h and then examined with an inverted phase-contrast microscope.

CH3-AD2-IgG-hA20 and CH1-DDD2-Fab-hA20 were produced in roller The results were scored semiquantitatively according to Polyak and bottles, purified by Protein A (MabSelect, Amersham-Biosciences) and Deans (25). Protein L (CBind L, Fluka), respectively, and stored in PBS. To generate Hex- Animal studies. The pharmacokinetic analysis was performed in naive hA20, a mixture ofC H1-DDD2-Fab-hA20 (134 mg) and CH3-AD2-IgG-hA20 female Swiss-Webster mice (Taconic Farms) and compared with Hex-hA20 (100 mg) was treated with 1 mmol/L reduced glutathione at room and veltuzumab given either i.v. or s.c. using radio-iodinated samples temperature for 16 h, followed by 2 mmol/L oxidized glutathione for 24 h, (Supplementary Materials and Methods online). The in vivo efficacy was from which Hex-hA20 was purified by Protein A. DNL-20/14 was made evaluated in tumor-bearing SCID mice as described in the figure legends. similarly by reacting CH3-AD2-IgG-hA20 with CH1-DDD2-Fab-hMN-14 (22). Depletion ofnatural killer (NK) cells and neutrophils was performedas Generation of Tetra-hA20 and Tri-hA20. Tetra-hA20 was obtained by described (26), with the following modifications. Briefly, mice received i.p. A h A purifying the tetrameric form of CH1-DDD2-Fab-hA20 over a Superdex-200 injections ofantimouse Gr-1 ascites (100 L) and TM -1 mAb (100 g) 1 d column. Tri-hA20 was obtained by linking the dimeric form of CH1-DDD2- before inoculating Raji cells and three more weekly i.p. injections of Fab-hA20 covalently to CH1-AD2-Fab-hA20, which was produced as antimouse Gr-1 ascites on days 6, 13, and 20 to maintain neutrophil described for h679-Fab-AD2 (22). depletion, which was confirmed by fluorescence-activated cell sorting Molecular size. SE-HPLC was performed on a Beckman System Gold analysis ofblood samples taken fromone treated and one untreated mouse Model 116 with a Bio-Sil SEC 250 column (Bio-Rad) and 0.04 mol/L PBS on days 3, 13, and 20. Mice deemed to have succumbed to disease (pH 6.8) and 1 mmol/L EDTA as the mobile phase. progression, when hind-limb paralysis developed or ifthey otherwise SDS-PAGE. Reducing and nonreducing SDS-PAGE analyses were became moribund, were humanely sacrificed. Additionally, if mice lost >20% performed using 4% to 20% gradient Tris-glycine gels (Cambrex Bio ofinitial body weight, they were sacrificed.Survival curves were analyzed Science). using Kaplan-Meier plots (log-rank analysis) and Prism software. All animal Mass spectrometry. Matrix-assisted laser desorption/ionization time of studies were performed after approval of the local animal care and use flight (MALDI-TOF) mass spectrometry was performed in a sinapinic acid committee. matrix by Scripps Center for Mass Spectrometry. Competition ELISA. Microtiter plates were coated overnight with Results veltuzumab at 5 Ag/mL and blocked with PBS containing 2% bovine serum albumin (BSA) for 1 h. Hex-hA20 and veltuzumab, serially diluted in Hexavalent antibodies made by DNL. Hex-hA20 was readily triplicate, were each mixed with WR2 at 1 nmol/L and added to the coated obtained by mixing CH1-DDD2-Fab-hA20 and CH3-AD2-IgG-hA20 wells. The bound WR2 was quantified with peroxidase-conjugated goat anti- under redox conditions followed by purification with Protein A. rat IgG and O-phenylenediamine dihydrochloride. Both CH1-DDD2-Fab-hA20 and CH3-AD2-IgG-hA20 were produced Flow cytometry. Apoptosis, viable cell counting, cell binding, and off- with good yields as fusion proteins in myeloma cells, with rate measurements were performed by flow cytometry on a Guava PCA subsequent purification from culture supernatants by Protein L (Guava Technologies, Inc.) using the manufacturer’s reagents, protocols, and and Protein A, respectively. software. The purity ofHex-hA20 was shown by reducing SDS-PAGE, Scatchard analysis. The maximum number ofbinding sites per cell and the apparent affinity constants were determined by nonlinear regression which showed only three bands from the constitutive polypeptides analysis ofthe saturation binding data obtained with radio-iodinated (Fig. 2, lanes 1 and 2). In addition, nonreducing SDS-PAGE analysis samples and Raji cells using Prism software (GraphPad Software, Inc.). ofHex-hA20 confirmedits covalent structure, because no bands Samples were run in triplicate. Immunoreactivity ofeach radiolabeled corresponding to the monomeric form of CH3-AD2-IgG-hA20 were preparation was 90% or greater, as measured by binding to WR2. observed (Fig. 2, lanes 5 and 6). The molecular mass ofHex-hA20 Cell proliferation assay. The in vitro cytotoxicity was determined using was determined to be 368,475 Da by MALDI-TOF mass spectrom- 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- etry, which agrees well with the calculated molecular weight of 2H-tetrazolium (MTS), which is chemically reduced by metabolically active 362 kDa for Hex-hA20 from the deduced amino acid sequences of cells into formazan, and the intensity of the resulting color is proportional the constituent polypeptides. to the number ofliving cells. The presence ofa disulfide-linked dimer ofC -AD2-IgG-hA20 is CDC. Cells were seeded in black 96-well plates (Nunc) at 5 104 cells in H3 50 AL/well and incubated with serial dilutions (concentration range, 3.33 evident on nonreducing SDS-PAGE (Fig. 2, lane 7) and supported 10-8 to 2.6 10-10 mol/L) oftest and control mAbs in the presence of by its conversion to the monomer upon reduction with glutathione human complement (1:20 final dilution, Quidel Corp.) for 2 h at 37jC and (data not shown). The purity ofC H3-AD2-IgG-hA20 is indicated by

5% CO2. Viable cells were then quantified using the Vybrant Cell Metabolic the observation ofonly two bands (Fig. 2, lane 3), corresponding to Assay Resazurin kit (Invitrogen). Controls included cells treated with 0.25% the constituent polypeptides (heavy chain-AD2 and n chain) on

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Figure 2. SDS-PAGE analysis of protein A–purified samples under reducing (lanes 1–4) and nonreducing (lanes 5–8) conditions. Lanes 1 and 5, Hex-hA20 (2 Ag); lanes 2 and 6, Hex-hA20 (0.5 Ag); lanes 3 and 7, CH3-AD2-IgG-hA20; lanes 4 and 8, veltuzumab. Left, positions of prestained Mr standards; right, arrows, positions of Hex-hA20, dimeric CH3-AD2-IgG-hA20, monomeric CH3-AD2-IgG-hA20, veltuzumab, heavy chain-AD2, heavy chain, Fd-DDD2, and n light chain are indicated as Hex, (IgG)2, IgG, H-AD2, H, d-DDD, and K, respectively.

reducing SDS-PAGE. These results are corroborated by the HPLC cell lines, Raji, Ramos, and Daudi, with an EC50 of0.064, 0.15, and data shown in Supplementary Fig. S1A-D. 0.15 nmol/L, respectively. In contrast, veltuzumab in the absence of Binding analysis. As shown by competition ELISA in Fig. 3A, a cross-linking antibody showed detectable potency in all three cell Hex-hA20 has a 3-fold higher avidity than veltuzumab, suggesting lines only at >10 nmol/L. As expected, cross-linking ofveltuzumab that at least three or more ofthe six Fab components are capable of with goat anti-human Fc resulted in significant inhibition of binding simultaneously to the WR2 antiidiotype antibody. The proliferation. For Hex-hA20, cross-linking did not increase its binding ofHex-hA20 to CD20 on live cells was compared with that potency further in Daudi. Additional experiments compared the ofveltuzumab by flowcytometry. Figure 3 B shows that Hex-hA20 effects of Hex-hA20, Tri-hA20, and Tetra-hA20 on cell proliferation resulted in 40% to 50% greater fluorescence intensity than over 5 days by viable cell counting. Representative results are shown veltuzumab when probed by PE–anti-Fab. In contrast, the signal in Fig. 4B for Raji and Ramos, which show the ability of Tri-hA20, observed for Hex-hA20 with PE–anti-Fc was lower than that of Tetra-hA20, and Hex-hA20 to prevent 50% or greater cell veltuzumab. These results are consistent with Hex-hA20 having four proliferation without cross-linking at concentrations as low as more Fabs than veltuzumab, but only one Fc like veltuzumab. 0.5 nmol/L. Similar results were obtained for Daudi (data not Additional evidence for the higher valency and avidity of Hex-hA20 shown). The cell counting assay also showed that Hex-hA20 (EC50 = is provided by Scatchard analysis ofbinding to Raji cells (Fig. 3 C), 0.17 nmol/L) was considerably more potent than anti-B1 (EC50 = which shows that the apparent association constant ofHex-hA20 4.65 nmol/L) when evaluated with Ramos for 3 days. [f3.9 108 (mol/L) 1]isf4-fold higher (P < 0.0001) than that of Apoptosis and the roles of caspases and calcium. Raji cells veltuzumab [f1 108 (mol/L) 1], whereas an F test determined were treated with the three multivalent anti-CD20 constructs at that the saturation binding curves for rituximab and veltuzumab 0.5 or 5 nmol/L and analyzed with the Guava Nexin assay after were similar (P = 0.1859). More importantly, the number of 24 hours (Fig. 5A, left). Treatment with Tri-hA20, Tetra-hA20, or receptors per cell calculated from the maximum number of binding Hex-hA20 resulted in more cells in early apoptosis (12–16%) f sites (Bmax) obtained for Hex-hA20 was found to be 1/4 ofthat compared with the bivalent veltuzumab (6–9%) and the untreated obtained with veltuzumab (f100,000 versus f437,5000), suggest- control (3%). Comparable results were obtained with Daudi and ing that all six Fabs ofHex-hA20 are capable ofbinding to CD20 on Ramos cells (data not shown). the cell surface, because the same number of CD20 molecules would The extent ofapoptosis was also assessed forRaji cells treated require thrice as many veltuzumab to occupy them. The data with 5 nmol/L Hex-hA20 or veltuzumab over a 24-hour period using obtained from the off-rate measurements (Fig. 3D) also indicate the Guava MultiCaspase assay (Fig. 5A, right). The results at 24 hours that Hex-hA20 dissociates f3-fold slower than veltuzumab, which were 17% and 6% for Hex-hA20 and veltuzumab, respectively, which remarkably dissociates f2-fold to 3-fold slower than rituximab.5 agree with those determined by the Guava Nexin assay. Antiproliferative activity. Based on the MTS assay (Fig. 4A), The effect of Z-VAD-FMK (a broad-spectrum caspase inhibitor) Hex-hA20 strongly inhibited proliferation in three Burkitt lymphoma on apoptosis induced by Hex-hA20 was examined in Ramos, and the results (Supplementary Fig. S2) indicate that Z-VAD-FMK at 100 Amol/L completely prevented the apoptosis induced by antihuman IgM, but not that induced by Hex-hA20, suggesting 5 D.M. Goldenberg, E.A. Rossi, R. Stein, T.M. Cardillo, M.S. Czuczman, F.J. that both caspase-dependent and caspase-independent pathways Hernandez-Ilizaliturri, H.J. Hansen, C-H. Chang. Properties and structure-function relationships ofveltuzumab (hA20), a humanized anti-CD20 monoclonal antibody occur for Hex-hA20, as reported for rituximab with cross-linking (mAb), submitted for publication. (27). Although CD20 clustering is presumably achievable either www.aacrjournals.org 8387 Cancer Res 2008; 68: (20). October 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research indirectly by cross-linking the antigen-bound veltuzumab with a Membrane localization of CD20/Hex-hA20. Veltuzumab second antibody or directly via multivalent engagement ofHex- translocates CD20 into lipid rafts, as shown previously (28) by hA20, the former, but not the latter, leads to a rapid rise in Western blot analysis of the cell lysates in fractions isolated from intracellular calcium levels (Supplementary Fig. S3). sucrose density gradient centrifugation. The distribution of CD20 Effector functions. CDC activity was evaluated in vitro using at the cell surface upon binding to veltuzumab or Hex-hA20 was human complement and Daudi cells (Fig. 5B, left). Veltuzumab examined further with immunofluorescence microscopy using exhibits potent CDC activity. Surprisingly, Hex-hA20 fails to induce cholera toxin subunit B-Alexa Fluor 488 as the reporter for CDC in Daudi cells. Because CH3-AD2-IgG-hA20 induces CDC with ganglioside GM-1, a common lipid raft marker (29). As shown in similar potency as veltuzumab, modification of the carboxyl Supplementary Fig. S5A-D, incubation ofDaudi cells with termini ofveltuzumab by the addition ofthe small AD2 peptide veltuzumab or Hex-hA20 led to the formation of membrane does not affect CDC, suggesting that the addition of the four Fab- patches or caps with punctuate spots, which were superbly DDD2 groups apparently prevents complement fixation, despite matched by the images obtained with cholera toxin subunit B, the ability ofHex-hA20 to bind C1q (data not shown). Hex-hA20 indicating the localization ofCD20 in lipid rafts.Although the and veltuzumab have comparable ADCC (Fig. 5B, right). Thus, the fluorescent patterns observed were similar, veltuzumab seemed to Fc ofHex-hA20 induces ADCC. form larger and fewer patches than Hex-hA20. Homotypical adhesion. Hex-hA20, Tetra-hA20, and Tri-hA20 Serum stability. Hex-hA20 was found to have the same stability induced homotypical adhesion ofDaudi cells, resulting in >50% of in serum as veltuzumab, maintaining 86% binding activity after cells having medium-size to large-size aggregates, whereas under 11 days (Supplementary Fig. S6). These results are similar to those the same conditions, the extent ofcell aggregation observed for ofthe bispecificTri-Fab complexes reported previously (22). veltuzumab was similar to that ofthe untreated control Pharmacokinetic analysis. Using radio-iodinated preparations, (Supplementary Fig. S4). it was found that Hex-hA20 cleared f4.5 times faster than

Figure 3. Analysis of Hex-hA20 binding. A, competition ELISA showing Hex-hA20 has a higher avidity than veltuzumab for binding to WR2. Hex-hA20 (o)or veltuzumab (n) were incubated at varying concentrations in the presence of WR2 for competition of binding with immobilized veltuzumab. The percentage of inhibition was plotted versus mAb concentration, and EC50 values were generated with Prizm software. B, binding to Daudi cells as determined by flow cytometry using PE-conjugated anti-human Fab (PE–anti-Fab) or PE-conjugated anti-human Fc (PE–anti-Fc). All incubations and washes were performed at 4jC. Daudi cells were suspended at 1 106 cells/mL in 1% BSA-PBS and incubated with Hex-hA20, veltuzumab, or labetuzumab for 1 h. The cells were washed with 1% BSA-PBS, incubated with a 1:200 dilution of PE–anti-Fab or PE–anti-Fc for 30 min, washed once more, and analyzed on a Guava PCA using the Guava Express software application. C, Scatchard analysis using radio-iodinated Hex-hA20 (n), veltuzumab (E), or rituximab (o) and Raji cells. D, dissociation from Daudi or Raji cells. Hex-hA20 (.), veltuzumab (n), and rituximab (E) were labeled with PE using a Zenon R-phycoerythrin Human IgG Labeling kit (Invitrogen Corp.). Cells were suspended in CM (phenol red–free RPMI 1640 supplemented with 10% FBS at 1 106 cells/mL), and 5 105 cells were incubated with each PE-labeled antibody at 65 nmol/L for 30 min at room temperature. The cells were washed twice with CM to remove unbound antibody, resuspended in 1.5 mL of CM in the presence of 1 Amol/L CH1-DDD2-Fab-hA20 at 37jC, and analyzed for cell-bound PE-labeled antibody at several time points on a Guava PCA using the Guava Express software application. The dissociation half-life was determined by nonlinear regression using Prism software.

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Figure 4. Inhibition of cell proliferation. A, in vitro antiproliferation determined by the 4-d MTS assay for Raji, Ramos, or Daudi. Cells were treated with Hex-hA20 (o), veltuzumab (E), or veltuzumab plus goat anti-human Fc (n). Daudi cells were also treated with Hex-hA20 plus goat anti-human Fc (5). Briefly, cells were placed in 96-well plates at 5,000 cells per well in complete RPMI 1640. Five-fold serial dilutions of Hex-hA20, veltuzumab, or veltuzumab cross-linked with goat-anti human Fc were added to triplicate wells at final concentrations ranging from 2 108 to 6.4 1012 mol/L. The plates were incubated for 4 d, after which 20 ALof CellTiter 96 Aqueous One Solution Reagent (Promega Corp.) was added, and the incubation was continued for an additional 4 h before reading the plates at 490 nm. B, in vitro antiproliferation determined by the viable cell counting assay for Ramos (left) or Raji (right). Cells were seeded in T-flasks at 1 105 cells/mL and treated with veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at the indicated concentrations. Viable cell densities (VCD) were determined daily over 5 d by flow cytometry using Guava Viacount. On day 3, cultures were split 1:2 to maintain logarithmic growth. Cells were plotted as viable cells per milliliter measured on days 3, 4, and 5 for veltuzumab (gray lines and symbols) at 15 nmol/L ( ), 2.5 nmol/L ( ), or 0.5 nmol/L ( ), Tri-hA20 ( ) at 0.5 nmol/L, Tetra-hA20 (o) at 0.5 nmol/L, and Hex-hA20 (4) at 0.5 nmol/L.

veltuzumab when given i.v. (Supplementary Fig. S7A), resulting in a survival than the saline control (P = 0.0034), with the MST of59, 41, 3.7-fold lower mean residence time for Hex-hA20 compared with and 19 days for Hex-hA20, veltuzumab, and untreated, respectively. veltuzumab (127 hours versus 472 hours). When given s.c. Importantly, a better treatment outcome was observed for Hex- (Supplementary Fig. S7B), Hex-hA20 cleared at a much faster rate hA20 than for veltuzumab (P = 0.05) at this higher mole-equivalent (T1/2 f3 days) than veltuzumab (T1/2 >9 days), with both having dose (465 Ag Hex-hA20 versus 200 Ag veltuzumab). the same Tmax of24 hours. However, the Cmax was only halfas high for Hex-hA20 compared with veltuzumab (12.9 nmol/L versus 25.7 nmol/L). Pharmacokinetic variables determined for Hex-hA20 Discussion and veltuzumab are provided in Supplementary Table S1. The DNL method uses both recombinant engineering and site- In vivo efficacy. In the multiple-dose study with the Daudi specific conjugation chemistry to provide a facile, efficient, and model, mice were treated with Hex-hA20 at 30 Ag (q7dx2) and 6 Ag modular platform technology for the production of multivalent (q7dx2). As shown in Fig. 6A, both treatments resulted in antibodies that can be either monospecific or bispecific, resulting significantly improved median survival time (MST) when compared in novel constructs which are different from other designs reported with the saline control (66.5 and 42 versus 21 days; P < 0.0001). in the literature (13–16, 30–34). Among the various approaches for Although no significant difference in survival was observed generating multivalent antibodies, chemical cross-linking is between mice given Hex-hA20 and veltuzumab at the higher doses, generally considered impractical for developing commercial it seemed that mice receiving Hex-hA20 at the lower doses had products, whereas recombinant engineering often leaves uncer- significantly lower MST than those receiving veltuzumab at an tainty that every scFv or Fab in the resulting structures is capable equivalent dose (P = 0.0044). of binding to its antigen simultaneously with full affinity (16, 30). We also examined the role ofeffectorcells in inhibiting tumor The DNL method described herein seems to adequately resolve growth using the Raji model (Fig. 6B). In those animals depleted of these concerns. First, as shown by SE-HPLC, SDS-PAGE, and mass NK cells and neutrophils, there was no difference between the spectrometry, each veltuzumab-based, multivalent construct gen- saline control and mice treated with veltuzumab or Hex-hA20 erated by DNL has a defined composition of the expected (MST = 17 days for all three groups). In contrast, nondepleted mice molecular size and can be readily purified to near homogeneity. that received Hex-hA20 or veltuzumab had significantly improved Second, a unique feature shared by the various types of multivalent www.aacrjournals.org 8389 Cancer Res 2008; 68: (20). October 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research antibodies made by DNL is the ability ofeach Fab constituent in Based on their efficacy in certain in vitro assays, anti-CD20 mAbs the respective structure to engage its cognate antigen either in the have been classified by Cragg and colleagues (35) as either type I, form of immobilized antiidiotype antibody or on the target cell represented by rituximab, or type II, represented by tositumomab surface, as shown collectively for Hex-hA20 by competition ELISA, (36). Type I anti-CD20 mAbs are characterized by potent CDC, flow cytometry, and Scatchard analysis. Moreover, the fact that rapid calcium mobilization upon the addition ofa second antibody Tri-hA20, but not veltuzumab, can potently inhibit the proliferation (37, 38), weak induction ofhomotypical adhesion (39), enhanced ofCD20-positive cells in vitro is consistent with the notion that all association ofCD20 with lipid rafts(40), and a general requirement three Fabs in Tri-hA20 are capable ofsimultaneously binding to of cross-linking for efficient antiproliferation or apoptosis (39). In CD20, resulting in clustering ofCD20 and the onset ofsignal contrast, type II anti-CD20 mAbs lack CDC, fail to bring CD20 into transduction, which leads us to conclude that a minimum valency lipid rafts (3, 4), induce a high level of homotypical adhesion (39), of 3 is required for an anti-CD20 antibody to effectively induce and do not stimulate calcium mobilization upon further cross- growth inhibition without cross-linking. A similar observation was linking (37). A further differentiation is that apoptotic cell death reported by Miller and colleagues (30) that, at 2 to 3 nmol/L, mediated by type I mAbs involves both caspase-dependent and trivalent and tetravalent anti-CD20 constructs made by linearly caspase-independent pathways, but only caspase-independent stitching two or more Fab groups ofrituximab were able to induce pathways seem to be associated with type II mAbs (4). However, maximal apoptosis (up to 25%) ofthe B-lymphoma cell line, WIL2- both type I and type II anti-CD20 mAbs are effective in ADCC and S, without requiring further cross-linking. More recently, tetrava- apparently bind to the same region ofthe extracelluar loop ofCD20 lent anti-CD20 antibodies made by genetically fusing an identical on the plasma membrane (41–43). Interestingly, at saturation, the pair ofscFvs in tandem to the hinge C H2-CH3 were also shown by Li number by which a type II mAb binds to each CD20-positive B cell and colleagues (16) to have significant antiproliferative and only approaches halfofthat observed with a type I mAb (3, 4), apoptotic activities in Daudi and Raji lymphoma cells in the suggesting that a type II mAb may act virtually as a tetravalent absence ofa second antibody, despite the lack ofdirect evidence antibody by engaging two adjacent extracellular loops ofCD20 with that all ofthe fourscFvs are engaged in antigen-binding. each antigen-binding site.

Figure 5. A, apoptosis measured by Guava Nexin (left) showing percentage of early apoptotic cells (Annexin V–PE positive/7-AAD negative) induced in Raji after 24-h incubation with veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at 0.5 nmol/L (black columns) or 5 nmol/L (gray columns); apoptosis measured by Guava MultiCaspase (right) for which Raji cells were cultured in the presence of Hex-hA20 (5 nmol/L), veltuzumab (5 nmol/L), or anti-IgM (5 Ag/mL) and analyzed at 3, 7, 16, and 24 h by flow cytometry after staining with SR-VAD-FMK. Cells were plated at 2 105 cells/mL in fresh media and incubated at 37jC with each test article at the indicated concentrations for up to 24 h, and duplicate wells were processed for Guava analysis. B, CDC (left) was measured in Daudi cells for Hex-hA20 (o), epratuzumab ( ), veltuzumab (n), or CH3-AD2-IgG-hA20 ( ) in the presence of human complement. The percentage complement control (number of viable cells in the test sample compared with cells treated with complement only) was plotted versus the log of the nanomolar concentration. ADCC (right) was measured for Hex-hA20, veltuzumab, epratuzumab, or labetuzumab at 5 Ag/mL using Daudi as the target cells and freshly isolated peripheral blood mononuclear cells from two donors as the effector cells. A 100% lysis reference was generated by the addition of detergent to wells containing target cells only. The bar graphs show percentage of lysis obtained for each of the two donors.

Cancer Res 2008; 68: (20). October 15, 2008 8390 www.aacrjournals.org

Figure 6. Efficacy of Hex-hA20 in human lymphoma xenograft models. A, Daudi cells (1.5 107) were injected i.v. into SCID mice on day 0. On days 1 and 8, groups of mice (n = 9–10) were given either Hex-hA20 at two different doses (30 or 6 Ag) or equimolar amounts of veltuzumab (12.4 or 2.4 Ag). B, SCID mice were depleted of NK cells and neutrophils before the administration of Raji cells with antimouse Gr-1 ascites and TMh-1 mAb specific for mouse IL-2 receptor, as described in Materials and Methods. On day 0, Raji cells (1 106) were injected i.v. into both depleted and nondepleted mice. Hex-hA20 (465 Ag) or veltuzumab (200 Ag) was given i.v. on days 3, 5, 7, and 11, whereas the control group received saline.

We note that Hex-hA20 exhibits biological properties attribut- than the parental veltuzumab, except when a higher mole- able to both type II (for example, negative for CDC and calcium equivalent dose (such as 465 Ag) was given. Because mouse mobilization; positive for antiproliferation, apoptosis, and homo- complement was reported to be inactive in mediating CDC ofthe typical adhesion) and type I (for example, positive for trafficking to DHL-4 cell line in vitro by rituximab (26), the shorter half-life of lipid rafts). Thus, one effective approach to converting a type I anti- Hex-hA20 rather than the lack ofCDC may explain why the in vivo CD20 mAb to a type II can be achieved by making the type I mAb antitumor efficacyofHex-hA20 was not superior to that ofthe multivalent. Preliminary investigation ofthe signaling pathway bivalent veltuzumab, suggesting that more frequent or higher doses indicates that Hex-hA20 induces caspase-dependent, as well as ofHex-hA20 may be indicated. It is also noted that cross-linking of caspase-independent, apoptosis. Additional studies are in progress veltuzumab may occur in vivo via binding to effector cells (44), thus to identify the subcellular events associated with the binding of mimicking the advantage ofHex-hA20 over veltuzumab in vitro. CD20 by Hex-hA20 or Tri-hA20, which may reveal unequivocally The observation that neither veltuzumab nor Hex-hA20 is effective the molecular factors that account for the antiproliferative potency in mouse models depleted with NK cells and neutrophils ofa multivalent anti-CD20 antibody with definedcomposition. emphasizes the importance ofADCC for in vivo killing oftumor When tested against human lymphoma xenografts in mice, the cells. Whether a multivalent anti-CD20 construct can be made by therapeutic activity ofHex-hA20 was comparable, but not better, DNL to retain CDC and show more potent antitumor effects in vivo www.aacrjournals.org 8391 Cancer Res 2008; 68: (20). October 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research is worthy to determine. On the other hand, because CDC has been Acknowledgments suggested to be a major factor in infusion-related toxicity for Received 5/29/2008; revised 8/13/2008; accepted 8/14/2008. rituximab, it is tempting to speculate that Hex-hA20 may be a Grant support: National Cancer Institute grant P01-CA103985 from NIH (D.M. viable alternative where reduced side effects are desired. Goldenberg). The costs ofpublication ofthis article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement in accordance Disclosure of Potential Conflicts of Interest with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Meiyu Loo, Diana Delaney, Diane Nordstrom, and John Kopinski for E.A. Rossi, D.M. Goldenberg, T.M. Cardillo, Y. Wang, and C-H. Chang: Employment excellent technical assistance, Dr. Xiaochuan Chen for advice on measuring and ownership interest, Immunomedics, Inc. and IBC Pharmaceuticals, Inc. R. Stein cytoplasmic calcium by flow cytometry, and Dr. Francisco Hernandez-Ilizaliturri for disclosed no potential conflicts of interest. the protocol ofdepleting NK cells and neutrophils in SCID mice.

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Edmund A. Rossi, David M. Goldenberg, Thomas M. Cardillo, et al.

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