MUC1/X Protein Immunization Enhances Cdna Immunization in Generating Anti-MUC1 A/B Junction Antibodies That Target Malignant Cells Daniel B

Research Article

MUC1/X Protein Immunization Enhances cDNA Immunization in Generating Anti-MUC1 A/B Junction Antibodies that Target Malignant Cells Daniel B. Rubinstein,1 Maya Karmely,2 Ravit Ziv,2 Itai Benhar,3 Orit Leitner,5 Shoshana Baron,6 Ben-Zion Katz,6 and Daniel H. Wreschner2,4

1Food and Drug Administration, Silver Spring, Maryland; Departments of 2Cell Research and Immunology and 3Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel; 4Biomodifying, LLC, San Diego, California; 5Antibody Unit, Weizmann Institute of Science, Rechovot, Israel; and 6Department of Hematology, Sourasky Medical Center, Tel Aviv, Israel

Abstract MUC1 protein is a type I transmembrane protein (MUC1/TM) MUC1 has generated considerable interest as a tumor marker composed of a heavily glycosylated extracellular domain containing and potential target for tumor killing. To date, most anti- a tandem repeat array, a transmembrane domain, and a bodies against MUC1 recognize epitopes within the highly cytoplasmic domain (9, 14, 15). MUC1/TM is proteolytically cleaved h immunogenic A chain tandem repeat array. A major short- soon after its synthesis, generating two subunits, a and , which coming of such antibodies is that the MUC1 A chain is shed specifically recognize and bind each other in a strong noncovalent into the peripheral circulation, sequesters circulating anti- interaction (Fig. 1, MUC1/TM; refs. 9, 16–19). Cleavage of MUC1 tandem repeat array antibodies, and limits their ability to into the two subunits occurs in the SEA module (9, 18, 19), a highly even reach targeted MUC1-expressing cells. Antibodies recog- conserved domain found in several cell-tethered mucin-like nizing MUC1 epitopes tethered to the cell surface would likely proteins (20). Shedding of a subunit from the cell membrane be more effective. MUC1 A subunit binding the membrane- results in soluble tandem repeat–containing MUC1 in the tethered B subunit provides such an epitope. By use of a novel peripheral circulation, and it is this molecule that is used to protocol entailing immunization with cDNA encoding full- determine serum MUC1 levels in patients (21, 22). length MUC1 (MUC1/TM) followed by boosting with the The presence of the soluble a subunit MUC1 protein in the alternatively spliced MUC1/X isoform from which the tandem circulation presents a singular difficulty in delivering adequate repeat array has been deleted, we generated monoclonal amounts of anti-MUC1 antibodies to directly target MUC1- antibodies, designated DMC209, which specifically bind the expressing malignant cells. Because the most immunogenic part MUC1 A/B junction. DMC209 is exquisitely unique for this site; of MUC1 is the tandem repeat array, all anti-MUC1 antibodies amino acid mutations, which abrogate MUC1 cleavage, also generated to date almost exclusively recognize epitopes in that abrogate DMC209 binding. Additionally, DMC209 specifically immunogenic region. Sequestration of antitandem repeat anti- binds the MUC1 A/B junction on full-length MUC1/TM ex- bodies by the soluble, circulating MUC1 a subunit severely limits pressed by breast and ovarian cancer cell lines and on freshly the amount of antibody that can successfully bind MUC1 on the obtained, unmanipulated MUC1-positive malignant plasma cell surface. Furthermore, deposition of immune complexes of cells of multiple myeloma. DMC209 is likely to have clinical antitandem repeat antibodies and its soluble, circulating MUC1 application by targeting MUC1-expressing cells directly and target can lead to significant end-organ damage. as an immunotoxin conjugate. Moreover, the novel immu- In recent years, numerous efforts have been made to generate nization procedure used in generating DMC209 can be used effective anti-MUC1 antibodies using the full-length MUC1/TM to generate additional anti-MUC1 A/B junction antibodies, molecule as immunogen (23–25). The major obstacle hampering which may, analogously to Herceptin, have cytotoxic activity. those attempts is that immunization with the whole MUC1/TM Lastly, sequential immunization with MUC1/TM cDNA acting molecule invariably results in an antibody response composed as a nonspecific adjuvant followed by protein of interest may almost in its entirety of antibodies recognizing epitopes on the prove to be a generalizable method to yield high-titer specific highly immunogenic tandem repeat array. For ultimate application antibodies. (Cancer Res 2006; 66(23): 11247-53) in in vivo targeting of MUC1-expressing tumor cells, such antibodies pose all the shortcomings inherent in antirepeat Introduction antibodies as detailed above. Antibodies recognizing MUC1 epitopes tethered to the cell MUC1 is a glycoprotein highly expressed in several human surface potentially obviate these difficulties. Although conceptually epithelial malignancies, including breast, prostate, ovarian, and simple, generation of monoclonal antibodies (mAb) to tethered pancreatic carcinomas, as well as on the malignant plasma cells of MUC1 first requires characterization of cell-bound, nonshedding multiple myeloma (1–6). Although alternative splicing can generate epitopes. The junction formed by the MUC1 a subunit binding the a variety of MUC1 isoforms (7–13), the most intensively studied membrane-tethered h subunit provides such an epitope. We recently investigated the mechanism whereby the cleaved junction composed of the MUC1 a and h subunits is formed (9). In Requests for reprints: Daniel H. Wreschner, Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-6407425; Fax: 972- the course of those studies, we analyzed the ‘cleavageability’ of the 3-6422046; E-mail: [email protected] or c/o A. Greenboim, Biomodifying LLC, MUC1/TM, MUC1/Y, and MUC1/X proteins (Fig. 1; ref. 9). The San Diego, CA 92122. Phone: 858-678-0731; Fax: 858-678-0791. I2006 American Association for Cancer Research. MUC1/Y and MUC1/X isoforms are generated from mRNAs spliced doi:10.1158/0008-5472.CAN-06-1486 at two distinct sites that use donor and acceptor sites located www.aacrjournals.org 11247 Cancer Res 2006; 66: (23). December 1, 2006

Figure 1. MUC1 protein isoforms and MUC1 fusion proteins. Left, cleaved transmembrane MUC1 protein containing the tandem repeat array (MUC1/TM). Alternative splicing uses a splice donor indicated by S.D.! resulting in one of two alternative splice acceptors (S.A. Y! and S.A. X!) resulting in the MUC1/X and MUC1/Y (MUC1/X D1-18) isoforms. The numbers (1, 18, and 63) refer to the location of amino acids in the extracellular domain of MUC1/X. The cleavage site in MUC1/X occurs between amino acids 62 and 63. Isoform MUC1/Y does not contain amino acids 1 to 18 (see also schematics in Fig. 3). As described in the text, antibody DMC209 recognizes the cleaved a/h junction present in both the MUC1/X and MUC1/TM proteins but does not react with the uncleaved MUC1/Y protein.

upstream and downstream to the tandem repeat array (7, 12), and tumor cells and HK293, human embryonic kidney cells, were grown in consequently, in both isoforms the tandem repeat array and DMEM. flanking sequences are spliced out (Fig. 1). The extracellular Generation of stable DA3 mouse mammary tumor cell transfectants domains both of MUC1/X and MUC1/Y are thus considerably less expressing MUC1/TM. DA3cells were cotransfected with the eukaryotic expression plasmids pCL-MUC1/TM or pCL-MUC/TM truncated at the complex than the large tandem repeat array–containing MUC1/TM cytoplasmic PvuII site and pSV2neo (coding for neomycin resistance). protein. In fact, the extracellular domain of the MUC1/X protein Expression constructs were transfected into cells using the calcium comprises only the 120-amino acid SEA module fused to the MUC1 phosphate procedure. Stable transfectants were selected with neomycin. 30NH2-terminal amino acids (9). MUC1/Y is identical to MUC1/X, Transfectants expressing the MUC1 proteins were identified by immunoblot except for an 18-amino acid deletion at the SEA module N terminus analysis of cell lysates using affinity-purified polyclonal antibodies directed (9). Significantly, the MUC1/X isoform is cleaved at an identical site against the MUC1 cytoplasmic domain. as the full-length MUC1/TM protein and thereby results in the Generation of MUC1/X and MUC1/Y eukaryotic expression con- same noncovalent interaction of the a and h subunits (9). In structs and fusion proteins. The MUC1 fusion proteins used in this study, designated Flag-Yex-hFc and Flag-Xex-hFc, are depicted in Fig. 1. Standard contrast, the SEA module NH2-terminal truncation present in the MUC1/Y isoform results in a noncleaved protein (9). molecular biology methods were used to generate all constructs. Generation of HK293 transfectants expressing Flag-MUC1/Xex-hFc To generate antibodies directly targeting cancer cells, we and Flag-MUC1/Yex-hFc fusion proteins and purification of hFc-tagged a h surmised that antibodies specific for the MUC1 / junction fusion proteins. HK293(human kidney) cells were transiently transfected would target only membrane-bound MUC1. To circumvent using calcium phosphate with the eukaryotic pCMV3expression vectors generation of antitandem repeat array antibodies, we used the coding for the Flag-Xex-hFc, Flag-Yex-hFc, or mutant MUC1/X proteins (6 Ag cleaved MUC1/X protein (7, 9) both as immunogen and as DNA/25 cm2 flask). To obtain stable transfectants, neomycin-resistant screening reagent. We report here a novel procedure in which clones were isolated following addition of neomycin to the culture medium. mice were initially primed with MUC1/TM DNA to elicit an anti- Conditioned media containing the secreted MUC1 fusion proteins were MUC1/TM response. The resultant immune response contains spun at 15,000 rpm for 20 minutes, and the supernatant was filtered through A j antibodies reactive with the MUC1/X isoform. To further increase 0.45- m filter and stored at 75 C. Protein A-Sepharose 4 Fast Flow anti-MUC1/X titers, mice were boosted with MUC1/X protein. This (Amersham Pharmacia Biotech, Chalfont St. Giles, United Kingdom) was used to purify the COOH-terminally hFc-tagged Flag-Xex(Yex)-hFc proteins. resulted in exceptionally high anti-MUC1/X titers. Using this Immunization of mice and hybridoma production. Mice were protocol, we were successful in generating antibodies that immunized with consecutive i.d. DNA immunizations spaced at 21-day h recognize cleaved MUC1 a/ junction on the cell surface and intervals. The injected DNAs consisted of either the pCL-MUC1/TM or pCL- bind malignant cells expressing the full-length MUC1/TM. MUC1/X expression vector plasmids. Subsequently, mice were boosted with the extracellular domain of the MUC1/X protein injected with incomplete Freund’s adjuvant. Hybridomas were prepared by fusion of the myeloma cell Materials and Methods line with immune splenocytes and screened by ELISA assay (see above). Materials and antibodies. Reagents and chemicals were obtained from ELISA assay for determining binding of anti-MUC1 antibodies to the Sigma (St. Louis, MO), unless otherwise specified. The anti-MUC1 tandem MUC1/X and MUC1/Y proteins and to the MUC1 A/B junction. repeat antibodies (anti-epithelial membrane antigen, Mc5) were obtained ElisaImmunoAssay plates (Costar, Corning, NY) were coated with polyclonal from Chemicon International (Temecula, CA). goat anti-human Fc (Ga-hFc, 4 Ag/mL) followed by washing with PBS-

Cell culture. Cells were grown at 37jC and 5% CO2 in culture medium Tween 20 (0.05%) and blocking with PBS-Tween 20 plus 5% skimmed milk supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine, 100 (Blotto). Spent culture medium containing Flag-Xex(or Yex)-hFc proteins IU/mL penicillin, and 25 Ag/mL streptomycin. DA3mouse mammary (mutant and wild-type, see ref. 9 for detailed descriptions of these proteins

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Anti-MUC1 a/b Junction Monoclonal Antibodies and their mutants) was then applied to the wells to allow binding of the showed a 100-fold increase in anti-MUC1/X titers, from 1:300 (after Flag-Xex(Yex)-hFc proteins. Following incubation, samples were removed the MUC1/TM cDNA immunization alone) to a minimum of and the wells were washed with PBS-Tween 20. Wells were then incubated 1:30,000 following the MUC1/X protein boost (Fig. 2C). High titers with the mouse mAbs (or hybridoma supernatants) followed by horseradish were observed 18 days following the MUC1/X protein boost, which peroxidase (HRP)-conjugated anti-mouse antibody. increased even higher at day 32 (Fig. 2C). Mice sustained high anti- MUC1/X protein titers up to 6 months following the initial single Results MUC1/X protein boost (data not shown). Immunization with MUC1/TM cDNA but not with MUC1/X Generation of DMC209 mAbs that recognize the cleaved A/B cDNA induces anti-MUC1 A/B junction antibodies. Mice were junction. To best generate anti-a/h junction-specific mAbs, we immunized either with cDNA expression vectors coding for the proceeded to hybridoma formation using spleen cells from mice MUC1/TM protein (containing the a chain tandem repeat array) or showing the highest anti-MUC1/X antibody titers. Spent hybrid- with cDNA coding for the MUC1/X isoform from which the tandem oma media from 10 96-well plates containing fused spleen- repeat is deleted (Fig. 1). Following four consecutive DNA myeloma cells were assayed for antibodies recognizing the cleaved immunizations, sera were assayed for antibody directed against MUC1 a/h junction. To identify such antibodies, hybridoma MUC1/X. Because MUC1/X is cleaved at an identical site as the full- supernatants were screened in parallel against MUC1/X protein length MUC1/TM molecule, it served as the screening protein to and the MUC1/Y isoform (Fig. 1). Whereas MUC1/X is cleaved at an identify anti-MUC1 a/h junction antibodies. This provided several identical site as in the full-length MUC1/TM protein resulting in advantages. Because the MUC1/X extracellular domain lacks the interacting a and h subunits, MUC1/Y, which does not cleave, does central tandem repeat array, antibodies directed against the highly not yield the MUC1 a/h junction epitope. We selected for immunogenic tandem repeat array epitopes are not detected. More hybridomas that were MUC1/X positive and MUC1/Y negative. importantly, as MUC1/X is composed solely of two small Screening 950 hybridoma supernatants yielded two hybridomas interacting MUC1/X a and h subunits that form the native a/h highly reactive with MUC1/X protein but nonreactive with MUC1/ junction, only antibodies directed against epitopes common to Y. Limiting cell dilution resulted in pure clones, which were both MUC1/X and MUC1/TM are detected. The MUC1 a/h designated DMC209, an IgM antibody, and DMC111, an Igg2a junction is one such major epitope, making this screening antibody. procedure a simple detection system for antibodies binding this Detailed epitope analysis of DMC209 and DMC111 reactivity. critical region of interest. Analysis of the specificity of the mAbs generated by clones DMC209 Unexpectedly, all mice ( five of five) immunized with the and DMC111 confirmed that both were MUC1/X positive and MUC1/X cDNA failed to raise significant anti-MUC1/X antibody MUC1/Y negative (Fig. 3A and B). Furthermore, DMC209 and titers (Fig. 2A and B). In contrast, five of five mice immunized with DMC111 were also nonreactive with the noncleaved MUC1/Xex D1- MUC1/TM cDNA generated highly reproducible, albeit modest, 11 protein (Fig. 3F) but reacted with cleaved D1-7 MUC1/Xex anti-MUC1/X antibody titers (Fig. 2A and B). These results indi- protein (Fig. 3E). MUC1/Xex refers to the extracellular domain of cated that, whereas immunization with MUC1/TM cDNA induces the MUC1 protein, whereas mutants of the MUC1/Xex proteins antibodies recognizing epitopes common to both the MUC1/X and containing deletions of amino acids 1 to 7 or 1 to 11 are designated MUC1/TM proteins, immunization with MUC1/X cDNA failed to by D1-7 and D1-11 (amino acid 1 is indicated in Fig. 1 next to the elicit anti-MUC1/X antibodies. MUC1/X protein; also see Fig. 3). To further assess the importance As a major objective of this study was to generate mAbs of MUC1 cleavage, the immunoreactivity of DMC111 and DMC209 recognizing the a/h junction, mice with much higher anti-MUC1/X was tested against MUC1/Xex mutants that harbor a mutation at titers were required. To generate high-titer anti-MUC1/X, we the critical Ser63 residue (S63; see Fig. 3). MUC1 cleavage occurs boosted the MUC1/TM cDNA-primed mice with MUC1/X protein. directly upstream to this serine residue appearing within the Following a single MUC1/X protein boost, all immunized mice sequence GSVVV, thereby generating an NH2-terminal a subunit

Figure 2. A and B, anti-MUC1/X antibodies generated in mice immunized with DNA coding for either MUC1/TM or MUC1/X. Mice were immunized either with expression vectors containing cDNA coding for the MUC1/TM protein (MUC1/TM, group II) or with cDNA encoding for MUC1/X from which the tandem repeat sequence is deleted (MUC1/X, group I). Sera were assayed for antibody titers directed against the cleaved MUC1/X protein as described in Materials and Methods.Allmice immunized with MUC1/TM cDNA showed significant anti-MUC1/X antibody titers, whereas no anti-MUC1/X antibodies were detected in mice immunized with MUC1/X cDNA. A, antibody titers from mice immunized with MUC1/TM cDNA (black lines) and from mice immunized with MUC1/X cDNA (red lines; k = 1,000). B, anti-MUC1/X immunoreactivity of sera from groups I and II mice at a 1:100 dilution. C, anti-MUC1/X titers in mice primed with MUC1/TM DNA followed by a single MUC1/X protein boost. Mice previously primed with MUC1/TM cDNA (A and B) were boosted with a single MUC1/X protein immunization. Sera were assayed for anti-MUC1/X reactivity as described in Materials and Methods 18 or 32 days following the protein boost (violet and green lines, respectively). Bars, SDfor titers from individual mice. www.aacrjournals.org 11249 Cancer Res 2006; 66: (23). December 1, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research terminating at the G (glycine) residue and a h subunit initiating antibody. Henceforth, our reported analyses are restricted to the with the S63residue. We previously found that only S63 !C and cleavage-dependent antibody DMC209. S63!T mutant proteins, in addition to MUC1/X, generated cleaved Binding of DMC209 to MUC1/TM on intact cells. DMC209 MUC1 proteins, whereas all other mutations at this residue recognizes the cleaved a/h junction located in the MUC1/X resulted in noncleaved proteins (9). These analyses showed that, protein. As this cleavage site is identical to that found in the full- whereas DMC209 and DMC111 bound equally well to the wild-type length MUC1/TM protein (9), it was of critical importance to MUC1/X protein and to the cleaved S63!C and S63!T mutant determine whether DMC209 binds to the MUC1/TM protein as proteins (Fig. 3C), all other S63mutant uncleaved MUC1/X displayed on intact cells. We approached this issue by first proteins (Fig. 3D) were completely nonreactive with DMC209. In examining whether mouse cells not expressing human MUC1/TM contrast, DMC111 reacted equally well with both the cleaved become reactive with DMC209 following transfection with human MUC1/X proteins (wild-type and cleaved S63!C and S63!T MUC1/TM. Nontransfected parental mouse mammary tumor cells mutant proteins; Fig. 3C) and the uncleaved S63mutant proteins were found to be completely nonreactive with DMC209 (Fig. 4I, A), (Fig. 3D). These results confirm that the epitopes recognized by the although DMC209 clearly bound to the same cells expressing either two mAbs are different. More importantly, only DMC209 is the full-length MUC1/TM protein (Fig. 4B) or an isoform lacking completely dependent on MUC1 cleavage for its reactivity. the cytoplasmic domain of MUC1/TM (Fig. 4I, C). To confirm that Significantly, DMC209 was nonreactive with both the MUC1 a MUC1/TM is expressed on these cells, antibody to the a chain and h subunits when each was tested in isolation (Fig. 3G and H); tandem repeat array showed, as expected, MUC1 expression on the DMC209 reacts with the two subunits only when presented as transfected cells (Fig. 4II, B). interacting entities. These analyses provided strong evidence that Having established that the DMC209 mAbs bound MUC1/TM in DMC209 is cleavage dependent and that it recognizes the junction transfected cells, we proceeded to confirm that the antibodies also of the interacting a and h subunits. Further analyses (described bind tumor cell lines known to express MUC1/TM. HEY (an ovarian below and data not shown) revealed robust binding of DMC209 cancer cell line) and the breast cancer cell lines T47D and MCF-7 to cells expressing MUC1/TM, the major MUC1 isoform, and all showed significant DMC209 binding (Fig. 4I, D-F). significantly less binding with the noncleavage-dependent DMC111 DMC209 binds MUC1-expressing multiple myeloma cells in fresh bone marrow aspirates. For ultimate clinical application, antibodies to the MUC1 a/h junction must be able to target MUC1- expressing malignant cells. To investigate whether DMC209 antibodies can selectively bind tumor cells in an in vivo–like setting, malignant plasma cells in freshly obtained bone marrow aspirates from patients with multiple myeloma were used. The heterogeneous cell population in the aspirates allows direct assessment of DMC209-binding specificity: DMC209 should bind multiple myeloma cells, which express MUC1, whereas not bind, or minimally bind, non-MUC1-expressing cells present in the same aspirate sample. To do these analyses, antibodies DMC209, CD38, CD45, and CD138 were labeled with different fluorophores, and the cell aspirate was analyzed by flow cytometry. DMC209 cell binding in the aspirate material can thereby be readily compared with simultaneous staining with anti-syndecan (CD138) and CD38 both well-recognized myeloma markers (26). As expected (27), DMC209- positive myeloma cells were CD45 negative (data not shown; ref. 27). Plotted side scatter (reflecting cell granularity) versus red fluorescently labeled DMC209-positive cells assessed the composi- tion of the mixed cell populations present in the bone marrow aspirate (Fig. 5, left). The discrete population of cells with significant DMC209 reactivity was gated and designated R1 (red dots within the R1 gate, Fig. 5, left). The location of the DMC209- positive cells was identified in the CD38/CD138 scatter plot (Fig. 5, right, red dots). Results with freshly obtained bone marrow Figure 3. Detailed analysis of the epitopes recognized by DMC209 and aspirates from three multiple myeloma patients revealed DMC209 DMC111 mAbs. Wild-type MUC1/Xex and MUC1/Yex proteins (A and B; ‘‘ex’’ + designates the extracellular domains of the MUC1 proteins, see Fig. 1) as well immunoreactivity in 26% of CD138 cells in one aspirate and in as point-mutated MUC1/Xex (C and D) and internally deleted MUC1/Xex (E and 37% in another, whereas expression was low (5%) in the third F) were assayed for their reactivity with the DMC209 and DMC111 antibodies. aspirate (patients I, II, and III, respectively; Fig. 5). DMC209 seems The extracellular domains of the MUC1/X and MUC1/Y proteins (MUC1/Xex and MUC1/Yex) were purified as secreted proteins comprising Flag and hFc epitopes to identify a subpopulation of malignant plasma cell population in at their NH2 terminus and COOH terminus, respectively. ElisaImmunoAssay patients I and III. In these samples, a mixture of DMC209-positive plates were coated with polyclonal goat anti-human Fc (Ga-hFc). Flag-Xex(or Yex)-hFc proteins (mutant and wild-type; see ref. 9 for detailed descriptions of (Fig. 5, right, red dots) and DMC209-negative (Fig. 5, right, black these proteins and their mutants) were then applied to the wells to allow binding. dots) cells is seen in the CD38/CD138-positive population. In The location of the numbered amino acids is indicated in Fig. 1, MUC1/X protein. addition, two samples that were negative for anti-MUC1 tandem Wells were incubated with the mouse mAbs DMC111 or DMC209 followed by HRP-conjugated anti-mouse antibody to detect bound antibody. +, binding of repeat array mAb H23were also DMC209 negative (data not antibodies DMC111 and DMC209; , lack of binding; nr, not relevant. shown). DMC209 binding to MUC1-expressing malignant plasma

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Figure 4. DMC209 flow cytometric analyses of MUC1/TM-expressing cells. Parental nontransfected mouse mammary tumor cells and cells transfected with cDNA coding for full-length MUC1/TM or MUC1/TM with its cytoplasmic tail truncated (DA3/PAR, DA3/TM, and DA3/ TM minus CYT, respectively) were reacted with DMC209 and analyzed by flow cytometry as described in Materials and Methods. Similarly, the ovarian cancer cell line HEY and breast cancer cell lines T47D and MCF-7 were analyzed following incubation with DMC209. Red and purple tracings, reaction of cells with the fluorescently labeled secondary antibody alone or with primary DMC209 antibody followed by secondary antibody, respectively. II, green line, reactivity with an anti-MUC1 tandem repeat mouse mAb.

cells and lack of binding to non-MUC1-expressing cell lineages in from which the tandem repeat array is deleted (7, 9) represents an the same freshly obtained, unmanipulated aspirate sample isoform far less complex than the complete tandem repeat array– underscore the specificity of MUC1 a/h junction binding in containing MUC1/TM molecule. Because both MUC1/TM and distinguishing MUC1-expressing tumor cells. MUC1/X are cleaved at an identical site and result in the same noncovalent interaction of the a and h subunits (9), we reasoned Discussion that immunization with cDNA coding for MUC1/X would elicit anti-a/h junction antibodies, avoiding formation of antibodies to Tumor-associated MUC1 has been extensively studied as a the tandem repeat array. tumor marker and as a target in cancer therapeutics (28–33). In an Contrary to expectations, immunization with cDNA encoding attempt to target MUC1, a variety of anti-MUC1 antibodies have the MUC1/X protein failed to elicit strong anti-MUC1/X antibody been generated, almost all of which react with epitopes contained within the highly immunogenic tandem 20-amino acid repeat sequence of the MUC1 a chain. Antibodies binding the tandem repeat array do have potential advantages for therapeutic use because the presence of multiple repeat MUC1 epitopic immu- nogens may result in a large number of anti-MUC1 antibodies binding MUC1 at distinct sites. This approach, however, has serious drawbacks, limiting the clinical use of antitandem repeat antibodies. The level of soluble circulating a subunit MUC1 containing the tandem repeat array is elevated in patients with MUC1-expressing malignancies such that circulating a chain can bind and sequester much or all of the antibodies directed against the repeat sequence. Consequently, the amount of anti-MUC1 even reaching the targeted MUC1-expressing cancer cells may be very limited. Furthermore, formation and deposition of MUC1-anti-MUC1 circulating immune complexes can result in severe end-organ damage. These shortcomings likely contributed to the limited success of antitandem repeat MUC1 antibodies in clinical trials to date (28, 33–36). To circumvent the inherent shortcomings of antitandem repeat antibodies, we elected to generate anti-MUC1 antibodies uniquely targeting a cell-bound MUC1 epitope. Because the MUC1 h subunit is stably tethered to the cell membrane and strongly binds the a subunit (Fig. 1; refs. 9, 16–19), the region comprising the junction of the two cleaved subunits represents a potentially valuable cell- Figure 5. Flow cytometry analysis of DMC209 reactivity with freshly obtained a h bone marrow cells from patients with multiple myeloma. Bone marrow aspirates bound MUC1 epitope. Antibodies specific to the / junction should from three separate patients (patients I, II, and III) were simultaneously analyzed directly target MUC1-expressing cells and escape sequestration by with DMC209 and with antibodies to CD138 and CD38, each labeled with circulating tandem repeat array–containing MUC1 a subunit. fluorochromes fluorescing at different emission wavelengths. Left, side scatter h analyses were done against DMC209. DMC209-positive cells were gated and To generate anti-a/ junction antibodies, we immunized mice designated R1 (left, red dots), and their location in the CD38/CD138 scatter plot with cDNA encoding the MUC1 a/h junction. The MUC1/X protein was identified (right, red dots). www.aacrjournals.org 11251 Cancer Res 2006; 66: (23). December 1, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research immune responses, whereas mice immunized with MUC1/TM a/h junction structure resulting from cleavage. This is the precise cDNA generated significant and highly reproducible anti-MUC1/X specificity required of an antibody for use in targeting MUC1- antibody titers. Furthermore, immunizing mice primed with expressing cells: the antibody should react only with membrane- MUC1/TM cDNA with a single boost of MUC1/X protein led to tethered MUC1 composed of cleaved and interacting a and h exceptionally high anti-MUC1/X antibody titers. We concluded that subunits. That DMC209 answers to these specifications is immunization with MUC1/TM cDNA induces antibodies, which illustrated by its binding to cell transfectants expressing MUC1, recognize epitopes common to both the MUC1/X and MUC1/TM MUC1-expressing cancer cell lines, and MUC1-expressing multiple proteins that can be significantly enhanced by subsequent MUC1/X myeloma cells. DMC209 activity in bone marrow aspirates is protein immunization. This cDNA/protein two-step ‘heteroimmu- perhaps the most directly informative. It shows that, in an nization’ protocol successfully yielded junction-specific DMC209 unmanipulated heterogeneous cell population, DMC209 specifically mAbs, which solely target cell-tethered MUC1, and represents a binds the MUC1-expressing malignant plasma cells of myeloma novel paradigm to generate antibodies against MUC1-expressing while remaining nonreactive with the other cell lineages present in malignant cells. the sample. In addition, DMC209 reactivity in the aspirates is Beyond specifically generating anti-MUC1 a/h junction anti- concordant with immunoreactivity of anti-CD138 (anti-syndecan), bodies, our findings may have far-reaching implications for a well-recognized plasma cell marker (37). Our finding of CD138- producing high-titer antibodies against intractable protein immu- positive cells differentially expressing the DMC209 epitope is nogens, which heretofore have elicited poor immune responses. We concordant with previous findings showing variability of MUC1 surmise that the cis-localization of the highly immunogenic expression on the plasma cells of multiple myeloma (3, 38, 39). upstream tandem repeat array in the MUC1 sequence potentiates Significantly, DMC209 recognizes a distinct subpopulation of the development of antibodies against the poorly immunogenic multiple myeloma cells, suggesting heterogeneity within the downstream a/h junction region. Once antibody synthesis against malignant plasma cell population. Cellular heterogeneity may have the a/h junction region is initiated by primary immunization with implications in the variable clinical course of the disease. MUC1/TM cDNA, markedly elevated antibody titers were obtained These results suggest that the novel cDNA/protein immunization by a subsequent protein boost. The MUC1 tandem repeat array protocol described can yield high titers of antibodies to the MUC1 cDNA may serve as a general molecular adjuvant potentiating a/h junction to directly target MUC1-expressing malignant cells. It production of antibodies directed to protein domain(s) encoded by further opens the possibility of generating therapeutically useful downstream sequences. In our specific case, the downstream antibody-immunotoxin or antibody-radioisotope conjugates. sequences encoded the MUC1 a/h junction region, but one could Finally, MUC1 acts as a signaling receptor protein that undergoes envisage replacement of this region with any cDNA encoding a extensive cytoplasmic tyrosine phosphorylation analogous to the protein of interest followed by boost immunization with that same epidermal growth factor receptor family of proteins (9, 13, 27, protein. High titers of antibody may thereby be obtained against 40–43). The intriguing possibility exists therefore that, just as protein domains that by themselves elicit poor immune responses. Herceptin mAb targets erbB2-expressing adenocarcinomas (44, 45), Characterization of monoclonal anti-MUC1 a/h junction anti-MUC1 a/h junction-specific antibodies may have direct or antibody DMC209 showed that its immunoreactivity was clearly indirect cytotoxic activity. Such investigations are presently being dependent on the cleavage status of the MUC1 protein: all amino undertaken. acid point mutations at S63of the GSVVV cleavage site that abrogated cleavage (9, 19) also abolished DMC209 reactivity. As Acknowledgments DMC209 immunoreactivity is preserved with cleaved G[S63C]VVV and G[S63T]VVV point mutants, we conclude that DMC209 is likely Received 5/2/2006; revised 8/17/2006; accepted 9/19/2006. The costs of publication of this article were defrayed in part by the payment of page a conformational antibody that does not recognize a specific amino charges. This article must therefore be hereby marked advertisement in accordance acid sequence at the cleavage site but rather recognizes the MUC1 with 18 U.S.C. Section 1734 solely to indicate this fact.

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