William M. Sutherland

PrLZ, a Novel Prostate-Specific and Androgen-Responsive Gene of the TPD52 Family, Amplified in Chromosome 8q21.1 and Overexpressed in Human Prostate Cancer

We report a previously unrecognized prostate-specific protein, PrLZ (prostate leucine zipper), a new member of the Tumor Protein D52 (TPD52) family. The gene for PrLZ was localized at chromosome 8q21.1, a locus most frequently amplified in human prostate cancer. Multiple tissue analyses demonstrated PrLZ predominantly in the prostate gland. Although its expression was enhanced by androgens in androgen receptor-expressing cells, PrLZ was detected in all of the human prostate cancer cell lines, regardless of androgen receptor status. Monoclonal anti-PrLZ antibodies were produced and intense immunohistochemical staining of PrLZ was observed in prostate epithelial cells in intraepithelial neoplasia and prostate cancer, whereas lower-level staining was detected in normal and benign epithelial components of the prostate gland. As the only prostate-specific gene identified in the most frequently amplified genomic region in prostate cancer, PrLZ may be the link between chromosome 8q amplification and malignant transformation of the prostate epithelia.

Targeting of Pseudomonas aeruginosa in the Bloodstream with Bispecific Monoclonal Antibodies

We examined the ability of a bispecific mAb reagent, consisting of a mAb specific for the primate erythrocyte complement receptor cross-linked with an anti-bacterial mAb, to target bacteria in the bloodstream in an acute infusion model in monkeys. In vitro studies demonstrated a variable level of complement-mediated binding (immune adherence) of Pseudomonas aeruginosa (strain PAO1) to primate E in serum. In vivo experiments in animals depleted of complement revealed that binding of bacteria to E was <1% before administration of the bispecific reagent, but within 5 min of its infusion, >99% of the bacteria bound to E. In complement-replete monkeys, a variable fraction of infused bacteria bound to E. This finding may have significant implications in the interpretation of animal models and in the understanding of bacteremias in humans. Treatment of these complement-replete monkeys with the bispecific reagent led to >99% binding of bacteria to E. Twenty-four-hour survival studies were conducted; several clinical parameters, including the degree of lung damage, cytokine levels, and liver enzymes in the circulation, indicate that the bispecific mAb reagent provides a degree of protection against the bacterial challenge.

Preparation of monoclonal antibodies to C3b by immunization with C3b(i)-Sepharose☆

We have prepared and characterized four monoclonal antibodies (MAbs) to human C3b of high specificity and affinity. Our procedure did not require a purified source of C3b for immunization. Instead, C3b and C3bi were deposited on Sepharose 4B via the alternative pathway of complement activation in normal human serum, and this C3b(i)-Sepharose served as the immunogen. C3b(i)-Sepharose was also prepared from a number of primate and non-primate sources, and this allowed us to demonstrate that the anti-human C3b MAbs cross-reacted with primate-derived C3b, but not with C3b from non-primates. The procedures we have developed may be useful in the further investigation of species-specific C3 fragment-binding proteins from both primate and non-primate sources.

Targeting of cancer cells with monoclonal antibodies specific for C3b(i).

Purpose: The goal of this research is to determine the feasibility of an immunotherapeutic approach based on the use of monoclonal antibodies (mAb) to target complement activation fragments on opsonized cancer cells. Methods: We investigated whether treatment of LNCaP and C4-2 human prostate cancer cell lines with normal human serum would allow for deposition of sufficient amounts of the complement-activation protein C3b and its fragments [collectively referred to as C3b(i)] such that these proteins could serve as cancer-cell-associated antigens for targeting by mAb. Radioimmunoassays, flow cytometry, and magnetic purging with specific immunomagnetic beads were used for the analyses. Results: In vitro opsonization of human prostate cancer cells with normal human serum resulted in deposition of C3b(i) in sufficient quantity (approx. 100,000 molecules/cell) for the cells to be targeted in a variety of protocols. We found that 51Cr-labeled and C3b(i)-opsonized cancer cells could be specifically purged at high efficiency (95%–99%) using anti-C3b(i) mAb covalently coupled to magnetic beads. Flow-cytometry experiments indicated that most normal white cells were not removed under similar conditions. Opsonization of cancer cells with sera from men with prostate cancer led to lower levels of cell-associated IgM and, subsequently, lower amounts of C3b(i) deposited than in normal subjects. Prototype experiments suggested that this deficiency could be corrected by addition of IgM from normal donor plasma. Conclusion: mAb directed against complement-activation products may provide new opportunities to deliver diagnostic and therapeutic agents selectively to cancer cells and tumor deposits. These opportunities may include ex vivo purging of C3b(i)-opsonized cancer cells prior to autologous bone marrow or stem cell transplantation.

Quantitative studies of heteropolymer-mediated binding of inactivated Marburg virus to the complement receptor on primate erythrocytes.

Previous in vitro and in vivo experiments in our laboratory have demonstrated that cross-linked bispecific monoclonal antibody (mAb) complexes (Heteropolymers, HP) facilitate binding of prototype pathogens to primate erythrocytes (E) via the E complement receptor, CR1. These E-bound immune complexes are safely and rapidly cleared from the bloodstream. In order to generate a robust bispecific system for HP-mediated clearance of real pathogens such as Filoviruses, we have developed the necessary methodologies and reagents using both inactivated Marburg virus (iMV) and a recombinant form of its surface envelope glycoprotein (rGP). We identified mAbs which bind rGP in solution phase immunoprecipitation experiments. HP were prepared by chemically cross-linking an anti-CR1 mAb with several of these anti-Marburg virus mAbs and used to facilitate binding of iMV and rGP to monkey and human E. These HP mediate specific and quantitative binding (> or = 90%) of both antigens to monkey and human E. Binding was also demonstrable in an indirect RIA. E with bound Marburg virus were probed with 125I labeled mAbs to the Marburg surface glycoprotein and more than 100 mAbs are bound per E. It should be possible to adapt this general approach to other pathogens, and experiments underway should lead to an in vivo test of HP-mediated clearance of Marburg virus.

CPK accumulation in fusion-blocked quail myocytes

The accumulation of creatine phosphokinase (CPK) activity during the development of quail (C. coturnix japonica) myoblasts in culture was determined under both fusion permissive and impermissive (low Ca2+) conditions. When fusion is prevented by decreasing the [Ca2+] of growth restrictive medium, accumulation is initiated at the same time, follows the same kinetics and attains the same steady-state level as that in muscle fibers which form in the same medium with the normal level of Ca2+. Individual clones, in which complete homogeneity of cell type can be assured, were employed to establish levels of CPK activity per nucleus at the prefusion stage and in clones in which every nucleus is included within a syncytium. Cross-comparisons were made between these values and the activity measurements derived from mass cultures. These comparisons indicate no significant effect of fibroblast contamination or of myogenic cells of more advanced or retarded stages on the sign or magnitude of increase in CPK activity. Based on the parameters measured and the qualitative evidence that the same isozyme shift occurs in myocytes as in muscle fibers there is no reason to assume that differentiation is less complete or regulated differently in fusion-blocked myocytes. These studies indicate the utility and justification of employing this cell system and the biochemical criterion employed to examine myogenic differentiation on a cell by cell basis.

Clearance of blood-borne pathogens mediated through bispecific monoclonal antibodies bound to the primate erythrocyte complement receptor

Abstract The primate erythrocyte complement receptor facilitates both the immune adherence reaction and the immune complex clearance properties of primate erythrocytes. These phenomena have been studied for more than 40 years. However, it has only recently become apparent that these characteristics of primate erythrocytes may be useful in the generation of a therapy based on bispecific monoclonal antibodies. Our approach uses bispecific monoclonal antibody constructs (heteropolymers) that promote binding of specific target pathogens to primate erythrocytes via the complement receptor. Once bound to the erythrocytes, the pathogen-heteropolymer complex should be cleared from the circulation, phagocytosed and destroyed in the liver. Results with several prototype target pathogens in monkey models indicate it may be possible to use this technology to develop a robust and general therapy for the treatment of diseases associated with blood-borne pathogens.

Dynamics of a human seminal vesicle specific protein: Charakteristik eines Bläschendrüsen-spezifischen Proteins des Menschen

Summary The present paper is concerned with the temporal alterations and tissue localization of a seminal antigen scrected by the human seminal vesicle. This antigen is recognized by antibody MHS‐5, which is one of a set produced in mice by immunization with human sperm. The respective clone produced an antibody of the IgG1 subtype, which reacted with seminal fluid from over 400 normal donors and 21 semen samples from vasectomized men. Incubation of seminal vesicle secretion with either prostatic fluid or prostate specific antigen (PSA) resulted in degradation on the antigen. The experiments showed that MHS‐5 antigen is a substrate for the serine protease PSA: Immunohistochemical studies suggested that MHS‐5 is a “sperm‐coating” antigen and is exclusively synthesized and secreted by the seminal vesicle.