Daniel B. Rubinstein

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

Combinatorial Ig libraries with phage display allow in vitro generation of human Ig fragments without the need to maintain hybridomas in ongoing cell culture or to select circulating Ig from human serum. Identifying tumor‐associated antigens on the surface of intact tumor cells, as opposed to purified proteins, presents a challenge due to the difficulty of preserving complex 3‐D epitopic sites on the cell surface, the variable expression of antigens on different malignant cell types and the stereotactic interference of closely associated proteins on the intact membrane surface limiting accessibility to antigenic sites. A combinatorial Ig library of 1010 clones was generated from the cDNA of PBMCs derived from patients with breast adenocarcinoma. Following subtractive panning, the library was enriched for Ig (Fab fragment) binding to intact adenocarcinoma cells and the resultant Fabs were screened against a cDNA expression library, itself generated from breast cancer cells. Using this approach, we isolated clones from the cDNA library expressing gC1q‐R, a glycoprotein comprising the major structure of C1, the first component of the complement system. gC1q‐R is a 33 kDa glycoprotein expressed not only on the cell surface but also intracellularly, with motifs that target it to mitochondria and complete homology with HABP and human HeLa cell protein p32, which is copurified with pre‐mRNA SF2. Sequencing of the gene encoding tumor‐associated gC1q‐R did not reveal any consistent tumor‐specific mutations. However, histochemical staining with anti‐gC1q‐R MAb demonstrated marked differential expression of gC1q‐R in thyroid, colon, pancreatic, gastric, esophageal and lung adenocarcinomas compared to their nonmalignant histologic counterparts. In contrast, differential expression was not seen in endometrial, renal and prostate carcinomas. Despite high expression in breast carcinoma, gC1q‐R was also expressed in nonmalignant breast tissue. Although the precise relation of gC1q‐R to carcinogenesis remains unclear, our finding of tumor overexpression and the known multivalent binding of gC1q‐R to not only C1q itself but also a variety of circulating plasma proteins as well as its involvement in cell‐to‐cell interactions suggest that gC1q‐R may have a role in tumor metastases and potentially serve in molecule‐specific targeting of malignant cells.

Targeting Retroviral Vectors to CD34-Expressing Cells: Binding to CD34 Does Not Catalyze Virus-Cell Fusion

We have attempted to engineer murine leukemia virus (MuLV)-based retroviral vectors to specifically transduce cells expressing human CD34, an antigen present on the surface of undifferentiated hematopoietic stem cells. A number of chimeric ecotropic MuLV envelope (Env) proteins were constructed that contained anti-CD34 single-chain antibody variable fragments (scFvs). The scFv-Env proteins were generated either by replacing the receptor-binding domain of Env with the scFv or by inserting the scFv into the N terminus of the Env protein. Only chimeric Env proteins with scFv insertions between amino acids 6 and 7 were incorporated into viral particles, and coexpression of native MuLV Env did not rescue incorporation-defective proteins. In addition, the efficiency of incorporation varied with the specific anti-CD34 scFv that was used. Retroviral vectors containing the scFv-Env proteins bound to CD34+ cells and transduced NIH 3T3 cells expressing human CD34 (3T3-CD34 cells) at approximately twice the efficiency of the parental NIH 3T3 cells. However, the introduction of the mutation D84K, which prevents binding to the ecotropic MuLV receptor mcat-1, prevented transduction of both NIH 3T3 and 3T3-CD34 cells. Complementation cell-cell fusion assays [Zhao et al. (1997). J. Virol. 71, 6967-6972] in 3T3-CD34 cells revealed that although the scFv-Env proteins could contribute postbinding entry functions when bound to mcat-1, they were unable to do so when bound to CD34. Taken together, these data suggest that although the interaction with CD34 effectively increased the concentration of virus on 3T3-CD34 cells, entry could occur only through an interaction with mcat-1; CD34 alone was not capable of triggering the appropriate postbinding changes that lead to viral entry.

Antibody Targeting of Cell-Bound MUC1 SEA Domain Kills Tumor Cells

The cell-surface glycoprotein MUC1 is a particularly appealing target for antibody targeting, being selectively overexpressed in many types of cancers and a high proportion of cancer stem-like cells. However the occurrence of MUC1 cleavage, which leads to the release of the extracellular α subunit into the circulation where it can sequester many anti-MUC1 antibodies, renders the target problematic to some degree. To address this issue, we generated a set of unique MUC1 monoclonal antibodies that target a region termed the SEA domain that remains tethered to the cell surface after MUC1 cleavage. In breast cancer cell populations, these antibodies bound the cancer cells with high picomolar affinity. Starting with a partially humanized antibody, DMB5F3, we created a recombinant chimeric antibody that bound a panel of MUC1+ cancer cells with higher affinities relative to cetuximab (anti-EGFR1) or tratuzumab (anti-erbB2) control antibodies. DMB5F3 internalization from the cell surface occurred in an efficient temperature-dependent manner. Linkage to toxin rendered these DMB5F3 antibodies to be cytotoxic against MUC1+ cancer cells at low picomolar concentrations. Our findings show that high-affinity antibodies to cell-bound MUC1 SEA domain exert specific cytotoxicity against cancer cells, and they point to the SEA domain as a potential immunogen to generate MUC1 vaccines.

MUC1/X Protein Immunization Enhances cDNA Immunization in Generating Anti-MUC1 α/β Junction Antibodies that Target Malignant Cells

MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most antibodies against MUC1 recognize epitopes within the highly immunogenic α chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 α chain is shed into the peripheral circulation, sequesters circulating antitandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recognizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 α subunit binding the membrane-tethered β subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding full-length MUC1 (MUC1/TM) followed by boosting with the alternatively spliced MUC1/X isoform from which the tandem repeat array has been deleted, we generated monoclonal antibodies, designated DMC209, which specifically bind the MUC1 α/β junction. DMC209 is exquisitely unique for this site; amino acid mutations, which abrogate MUC1 cleavage, also abrogate DMC209 binding. Additionally, DMC209 specifically binds the MUC1 α/β junction on full-length MUC1/TM expressed by breast and ovarian cancer cell lines and on freshly obtained, unmanipulated MUC1-positive malignant plasma cells of multiple myeloma. DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly and as an immunotoxin conjugate. Moreover, the novel immunization procedure used in generating DMC209 can be used to generate additional anti-MUC1 α/β junction antibodies, which may, analogously to Herceptin, have cytotoxic activity. Lastly, sequential immunization with MUC1/TM cDNA acting as a nonspecific adjuvant followed by protein of interest may prove to be a generalizable method to yield high-titer specific antibodies. (Cancer Res 2006; 66(23): 11247-53)

Anti-CD34(+) fabs generated against hematopoietic stem cells in HIV-derived combinatorial immunoglobulin library suggest antigen-selected autoantibodies

Bone marrow suppression associated with HIV infection does not appear to be solely due to direct viral cytopathic effects. Autoantibodies may play a role in myelosuppression, however it is unclear whether autoantibodies produced in HIV infection represent a primary pathogenic process or merely reflect polyclonal B cell activation. To address these questions, we generated combinatorial immunoglobulin libraries using the pComb3 phagemid from an HIV+ individual with evidence of circulating autoantibodies. From one library, three anti-CD34 Fabs were identified using fresh CD34+ cells as antigenic targets by a method of phage subtraction. The anti-CD34 Fabs are specific by immunoblotting and Elisa and are of high affinity, with calculated Kds in the range of 10(-7) -10(-8) M. Nucleic acid sequencing revealed all three to be of the VH3 family and to have lambda light chains with some gene segments expressing little somatic mutation, while other segments were somatically mutated in patterns suggestive of antigen selection. These findings indicate that (1) A subset of HIV-associated anti-CD34 autoantibodies are monospecific and antigen-selected and are not merely a consequence of polyclonal B cell activation and elevated Ig levels in HIV. Autoreactivity in HIV therefore includes both polyspecific, low affinity antibodies as well as monospecific antigen-selected high affinity antibodies. (2) Although bone marrow suppression in HIV is likely to be multifactorial, autoantibodies to hematopoietic stem cells may contribute to its pathogenesis. (3) Library sampling of VH gene family rearrangements shows no evidence for under-representation of the VH3 family in the immune dysregulation of HIV infection. Phage subtraction is corroborated to be an effective means of identifying, cloning, and characterizing antibodies to hematopoietic differentiation antigens.

Ablating MUC1+ tumor and cancer stem cells by targeting the MUC1 oncoprotein with configurational-specific monoclonal antibodies and peptides.

e13088 Background: MUC1, a glycoprotein overexpressed by breast, intestinal, pancreatic, and gastric adenocarcinomas is a heterodimer with an extracellular α chain that is noncovalently bound to a ...

Abstract 2444: Targeting the cell-bound MUC1-SEA module with monoclonal antibodies and peptides to ablate MUC1+ malignancies

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The MUC1 oncoprotein is overexpressed in a variety of high-incidence malignancies including breast, colon, and pancreatic carcinoma and by the malignant plasma cells of myeloma. It is a heterodimer composed of a large extracellular α chain that is noncovalently bound to a membrane-bound β subunit. The α-chains ‘on-again-off-again’ interaction with the β-chain results in intracellular signaling. A variety of anti-MUC1 antibodies have been described to date, almost all of which recognize the immunogenic tandem-repeat-array of the MUC1 α-subunit which is shed into the peripheral circulation. Because it circulates freely, the α subunit actively sequesters anti-α chain Abs, precluding their even reaching and adequately targeting MUC1+ malignant cells. To circumvent this problem we identified the MUC1 SEA module that comprises the junction of the extracellular α-subunit with the cell-bound β-subunit as a valuable cancer cell target since it invariably remains attached to the cell surface. We generated a series of anti-MUC1 α/β junction IgGγ1 monoclonal Abs, denominated DMB mAbs, which bind with exceptionally high affinity to MUC1-expressing human breast cancer cells. The antibodies were non-reactive when used as probes in Western blot analyses of the MUC1 protein resolved on denaturing SDS gels, suggesting that the epitopes recognized by the DMB mAbs are configurational. The novel mAbs bound well to MUC1-expressing cancer cells such as T47D and ZR-75 and to the extracellular domain of the MUC1/X protein which comprises only the SEA module that is fused C-terminally to the thirty N-terminal amino acids of the MUC1 protein. The DMB mAbs were much less reactive with recombinant SEA module alone, which corresponds to the MUC1/X extracellular domain that lacks the 30 N-terminal amino acids. Unexpectedly, all DMB mAbs showed high reactivity with mutant ‘SEA module 4G’ protein that comprises an additional 4 glycine residues upstream to the GSVVV cleavage site resulting in a non-cleaved protein. From these results we conclude that the native structure of the cleaved SEA module associated with the MUC1/X protein is different to that of the SEA module that lacks the N-terminal 30 amino acids, and more similar to mutant ‘SEA module 4G’ protein. To reconcile these seemingly contradictory findings, we hypothesized that the N-terminal portion of the MUC1 protein binds to the SEA module, thereby forming epitopes recognized by the DMB mAbs. Based on these structural findings, small peptides were derived from the MUC1 N-terminus and appear to selectively bind the MUC1 SEA module. We conclude that (1)The anti-MUC1 α/β junction Abs and peptides described here bind to specific configurationally-determined MUC1 α/β junction epitopes, and (2)Abs and peptides targeting those epitopes are likely to prove biologically-significant and clinically useful in selectively targeting and killing MUC1+ malignant cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2444.