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Blood, Vol. 113, Issue 24, 6161-6171, June 11, 2009 This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma Blood Rossi et al. 113: 6161 Supplemental materials for: Rossi et al Supplemental Methods Expression vectors The pdHL2 mammalian expression vector has been used to mediate the expression of many recombinant IgGs1 and DNL modules.2,3 CH3-AD2-IgG-v-mab-pdHL2 and CH3-AD2-IgG-v-mab-pdHL2 were generated from v-mab-IgG-pdHL2 and e-mab-IgG-pdHL2, respectively, as described previously.2 CH3-AD2-IgG-h734-pdHL2 was generated similarly. CH1-DDD2-Fab-v-mab-pdHL2 and CH1-DDD2-Fab-e-mab-pdHL2 were constructed from the cognate IgG-pdHL2 vectors as follows. The coding sequences for the CH1-CH3 domains were excised by digestion with SacII and EagI restriction endonucleases and replaced with a 507 base-pair sequence encoding CH1-DDD2, which was excised from an existing vector, CH1-DDD2-Fab-hMN-14-pdHL2, described previously.3 Transfection and selection of stable IgG-AD2– and Fab-DDD2–secreting cell lines CH3-AD2-IgG-pdHL2 or CH1-DDD2-Fab-pdHL2 vectors, each 30 µg, were linearized by digestion with Sal I and transfected into Sp/ESF (2.8 × 106 cells) by electroporation (450 volts, 25 µF). The pdHL2 vector contains the gene for dihydrofolate reductase, thus allowing clonal selection as well as gene amplification with methotrexate (MTX). Following transfection, the cells were plated in 96-well plates and selected in media containing 0.2 µM MTX. Clones were screened for CH3-AD2-IgG-v-mab, CH3-AD2-IgG-e-mab, CH1-DDD2-Fab-v-mab or CH1-DDD2-Fab-e-mab productivity by a sandwich ELISA using 96-well microtiter plates coated with WR2 or WN to capture the fusion protein, which was detected with horseradish peroxidase-conjugated goat anti-human IgG F(ab′)2. Wells giving the highest signal were expanded and ultimately used for production. Fusion proteins were produced as batch cultures in roller bottles, which were seeded at 2 × 105 cells/ml and incubated at 37°C under 5% CO2 until the cell viability dropped below 25% (~10 days). Culture broth was clarified by centrifugation, filtered, and concentrated up to 50-fold before further purification. CH3-AD2-IgGs and CH1-DDD2-Fabs were purified by Protein A (MabSelect, Amersham-Biosciences) and Protein L (CBind L, Fluka) chromatography, respectively, and stored in 1 mM EDTA/PBS. Generation of hexavalent IgG-based DNL structures Hexavalent IgGs were created with DNL by mixing a CH3-AD2-IgG module with a 205% mole-equivalent of a CH1-DDD2-Fab module to ensure that the former was fully conjugated in the reaction. The reaction mixtures were incubated with 1 mM reduced glutathione (GSH) at room temperature for 16–24 h and followed by 2 mM oxidized glutathione for 24 h, from which hexavalent IgGs were purified by Protein A. Table 1 (printed article) lists the modular components and the eight hexavalent antibodies prepared from these components for the present study. Calcium mobilization Intracellular calcium was measured in Ramos cells loaded with 20 µM Fluo-3 AM (Invitrogen) using a Becton Dickinson FACScan and the FlowJo program (Tree Star Inc., San Carlo, CA). For all samples, a baseline was obtained for 60 sec before adding each test article, which included 1 µg/ml of 22–20, 20–22, v-mab, rituximab, or e-mab. Ionomycin and anti-human IgM were used as positive controls. To evaluate the effect of crosslinking, cells were incubated with a MAb for an additional 15 min and then stimulated with goat anti-human IgG Fc-specific second antibody (50 µg/ml final). Homotypic adhesion Daudi cells (1.5 × 106/ml) were treated with v-mab, e-mab, 22–20, or 20–22 at 50 nM for 16 h, and then examined with an inverted phase-contrast microscope (Olympus BX60). The results were scored semi-quantitatively according to Polyak and Deans.4 Serum stability Blood specimens were collected under a protocol approved by the New England Institutional Review Board (Wellesley, MA). 22–20 and 20–22 were diluted to 10 µg/ml in freshly pooled human sera and aliquots were either immediately frozen at −80°C for day 0 samples or incubated for 5 days at 37°C. Day 0 and day 5 serum samples were diluted 400-fold and assayed for bispecific binding using a sandwich ELISA with plates coated with WR1 (Rat IgG1, 5 µg/well). Captured bsAb was detected with WN (Rat IgG2a) and quantified with peroxidase-conjugated goat anti-rat IgG2a (Zymed) and o-phenylenediamine dihydrochloride. This assay specifically measures only intact bsAbs. If a molecule is cleaved, it either will not bind to the immobilized WR1 or not be detected by the WN probe. PK in mice Two groups of 28 naοve female BALB/c mice (Taconic Farms; Germantown, NY) were weighed prior to injection and grouped four to a cage such that the mean and standard deviations between the two groups of mice were not significantly different. One group was co-administered 125I-22-20 (10 ∝Ci, 50 ∝g, 136 pmoles) and 131I-e-mab (35 ∝Ci, 20 ∝g, 136 pmoles) as a single i.v. injection. The second group of mice was likewise co-administered 125I-20–22 (10 ∝Ci, 50 ∝g, 136 pmoles) and 131I-v-mab (35 ∝Ci, 20 ∝g, 136 pmoles). Animals were sacrificed at various time-points post-injection (5-min, 2-, 6-, 24-, 48-, 72-, and 168-h). Blood samples were counted in a calibrated -counter set for both 125I (channels 15–80) and 131I (channels 260–470). Additionally, a cross-over curve was made from 131I to correct for any 131I counts crossing over into the 125I channels. For PK analysis, blood concentrations (pmole/ml) of each reagent as determined from the percent injected dose values were plotted and analyzed using the WinNonLin PK software package (v5.1; Pharsight Corp.; Mountain View, CA). A non-compartmental analysis provided the best-fit model for the data. Therapeutic efficacy in Burkitt lymphoma xenograft models Studies were performed in female C.B.17 homozygous severe combined immune deficient (SCID) mice of approximately 20 g (7-week-old when received from Taconic, Germantown, NY). For the Burkitt lymphoma model, each mouse was inoculated i.v. with 1.5 × 107 Daudi cells on day 0. All mice were weighed and randomly assigned to treatment groups such that the average weights between all the groups were not significantly different. Animals monitored daily were humanely sacrificed when hind-limb paralysis developed or if they became otherwise moribund. Additionally, mice were sacrificed if they lost more than 20% of initial body weight. Survival curves were analyzed using Kaplan-Meier plots (log-rank analysis), using the Prism (v4.03) software package (GraphPad Software, Inc.). In one study, groups of 6 mice were treated with either a single i.p. injection of 4 ∝g of 22–20 or 20–22 (10 pmol). Control groups of mice were administered either a 4 ∝g i.p. injection of 20–14, 22–14, 22–22 or 1.6 µg (10 pmol) of v-mab. Untreated control mice received a 200 ∝l i.p. injection of saline. All mice received therapy 1 day post-Daudi inoculation. In the other study, groups of 6 mice were administered 10 ∝g i.v. doses of 22–20, 734–20, 22–14, or a combination of 734–20 and 22–14 (10 µg each) on days 1, 4, and 7. Natural killer cell and neutrophil depletion Two groups of 5 mice had their NK cells and neutrophils depleted as described previously.5 Briefly, NK-cell/neutrophil-depleted mice were pretreated with 100 ∝l i.p. of anti-mouse Gr-1 ascites (gift from Dr. Myron Czuczman, Roswell Park Cancer Institute, Buffalo, NY) and 100 ∝g anti-mouse IL-2 receptor antibodies (TM®-1; BD Biosciences; San Jose, CA) beginning on day 3. In order to maintain neutrophil depletion, mice received additional i.p. injections of anti-mouse Gr-1 ascites on days 4 and 11. Depletion was confirmed on days 1 and 11 by FACS analysis of blood samples taken from 1 pretreated mouse and 1 non-depleted “normal” SCID mouse. Three additional groups (n=5) were not NK/neutrophils-depleted. On days 1, 3, 5, and 9, groups of depleted and non-depleted mice received i.v. injections (230 ∝g) of either 22–20 or 20–22. Saline or 100 µg of e-mab were administered to non-depleted mice. Supplemental Results Hexavalent bsAbs made by DNL CH3-AD2-IgG modules were produced in cells that were stably transfected with a vector encoding the secreted polypeptides represented in Fig. 1B of the printed article, which self-assemble to form CH3-AD2-IgG (Fig. 1D, printed article). Protein A-purified CH3-AD2-IgG-v-mab was shown previously by SE-HPLC analysis to contain two peaks attributed to monomeric and a disulfide-linked dimer, which is converted to the monomeric form upon reduction with glutathione during the DNL reaction.2 Consistent with the previous results for CH3-AD2-IgG-v-mab, SE-HPLC analysis of Protein A-purified CH3-AD2-IgG-e-mab resolved monomer (8.86 min) and disulfide-linked dimer (7.93 min) peaks (Fig. S1A, left). The CH3-AD2-IgG-734 module produced a similar SH-HPLC profile (not shown). The presence of a disulfide-linked dimer of CH3-AD2-IgG-e-mab and CH3-AD2-IgG-v-mab was also evident on non-reducing SDS-PAGE (Fig. S1B, lanes 9 and 10). The purity of CH3-AD2-IgG-e-mab and CH3-AD2-IgG-v-mab was indicated by the observation of only two bands (Fig. S1B, lanes 1 and 2), corresponding to the constituent polypeptides (heavy chain-AD2 and kappa chain), on reducing SDS-PAGE. Similar SDS-PAGE results were obtained for CH3-AD2-IgG-734. CH1-DDD2-Fab modules were produced in myeloma cells that were stably transfected with vectors encoding the secreted polypeptides depicted in Fig. 1A of the printed article, which self-assemble to form stable CH1-DDD2-Fab dimers (Fig. 1C, printed article). Protein-L–purified CH1-DDD2-Fab-v-mab, CH1-DDD2-Fab-e-mab, and CH1-DDD2-Fab-hMN-14 were shown by SE-HPLC analysis to consist of three peaks (Fig. S1A center shows CH1-DDD2-Fab-v-mab), of which the two peaks at 9.45 min and 8.55 min were attributed to the dimeric and tetrameric forms of CH1-DDD2-Fab, respectively, and the third peak at 11.28 min was from the free kappa chain that binds Protein L and co-purifies with the product. The tetrameric form is reduced to the dimeric form during the DNL reaction.3 The purity of CH1-DDD2-Fab-v-mab and CH1-DDD2-Fab-e-mab is demonstrated by the observation of only two bands (Fig. S1B, lanes 3 and 4), corresponding to the constituent polypeptides (Fd-DDD2 and kappa chain), on reducing SDS-PAGE. Similar SDS-PAGE results were obtained for CH1-DDD2-Fab-hMN-14. The calculated molecular size of 22–20 and 20–22, as well as the control structures (20–20, 22–22, 20–14, and 734–20) from the deduced amino acid sequences of the constituent polypeptides is ~362 kDa. Size exclusion HPLC analysis of protein A-purified 22–20 showed a predominant peak at 7.83 min (Fig. S1A right), consistent with a protein of 350–380 kDa. MALDI-TOF mass spectrometry gave a molecular mass of 368.5 kDa for 22–20 (data not shown). The purity of 22–20 and 20–22 was demonstrated by reducing SDS-PAGE, which showed only three bands from the constitutive polypeptides (Fig. S1B lanes 5 and 6); namely, the heavy chain-AD2 (~55 kDa), Fd-DDD2 (~26 kDa), and the kappa chain (~25 kDa). In addition, non-reducing SDS-PAGE analysis confirmed their covalent structure, as bands corresponding to the monomeric form of CH3-AD2-IgG or CH1-DDD2-Fab were not observed (Fig. S1B, lanes 13 & 14). Similar SDS-PAGE results were obtained for 20–20, 22–22, 20–14 and 734–20 (not shown). REFERENCES 1. Qu Z., Griffiths GL, Wegener WA., et al. Development of humanized antibodies as cancer therapeutics. Methods. 2005;36:84–95 2. Rossi EA, Goldenberg DM, Cardillo TM, Stein R, Wang Y, Chang C-H. Novel designs of multivalent anti-CD20 humanized antibodies as improved lymphoma therapeutics. Cancer Res. 2008;68:8384–92. 3. Rossi EA, Goldenberg DM, Cardillo TM, McBride WJ, Sharkey RM, Chang C-H. Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting. Proc Natl Acad Sci USA. 2006;103:6841–6846. 4. Polyak MJ, Deans JP. Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure. Blood. 2002;99:3256–3262 5. Hernandez-Ilizaliturri, FJ, Jupudy V, Ostberg J, et al. Neutrophils contribute to the biological antitumor activity of rituximab in a non-Hodgkin’s lymphoma severe combined immunodeficiency mouse model. Clin Cancer Res. 2003;9(16 Py 1):5866–5873. Files in this Data Supplement: Table S1.Comparison of normal tissue biodistribution of 22–20 and e-mab in naοve BALB/c mice (PDF, 34.2 KB) Table S2. Comparison of normal tissue biodistribution of 20–22 and v-mab in naοve BALB/c mice (PDF, 34 KB) Figure S1. Biochemical characterization of 22–20, 20–22, and constituent AD2 and DDD2 modules (JPG, 219 KB) - (A) SE-HPLC analysis of CH3-AD2-IgG-e-mab, CH1-DDD2-Fab-e-mab and 22–20, showing the retention times and the presence of the dimeric and monomeric forms in CH3-AD2-IgG-e-mab and CH1-DDD2-Fab-e-mab. (B) SDS-PAGE analysis of purified samples under reducing (lanes 1–8) and non-reducing (lanes 9–16) conditions: lanes 1 & 9, CH3-AD2-IgG-e-mab; lanes 2 & 10, CH3-AD2-IgG-v-mab; lanes 3 & 11, CH1-DDD2-Fab-e-mab, lanes 4 & 12, CH1-DDD2-Fab-v-mab; lanes 5 & 13, 22-20; lanes 6 & 14, 20–22; lanes 7 & 15 e-mab; lanes 8 and 16, v-mab. The positions of pre-stained Mr standards are indicated on the left. The positions of 22–20/20–22, dimeric CH3-AD2-IgG, monomeric CH3-AD2-IgG, e-mab/v-mab, dimeric CH1-DDD2-Fab, heavy chain-AD2, heavy chain, Fd-DDD2 and kappa light chain are indicated as DNL, (IgG)2, IgG-AD2, IgG, (Fab)2, H-AD2, H, d-DDD2, and K, respectively, on the right with arrows. Figure S2. In vitro growth inhibition (JPG, 73.6 KB) - Ramos cells were plated at 5,000/well in 96-well plates and incubated with various concentrations of v-mab, 20–22, 22–20, 20–14, or 734–20. After four days of culture, the relative viable cell density was measured using an MTS assay. Dose-response curves and EC50 values were generated with Prism software using a sigmoidal dose-response (variable slope) non-linear regression. Figure S3. Homotypic aggregation of Daudi cells induced by 22–20 and 20–22 (JPG, 500 KB) - Cells were treated for 16 hours with 50 nM of 22–20, 20–22, e-mab, or v-mab and visualized by phase contrast microscopy. Figure S4. Stability of 22–20 and 20–22 in fresh pooled human sera (JPG, 93.7 KB) - This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef

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