Robert A. Henderson

MUC-1 epithelial tumor mucin-based immunity and cancer vaccines

Many obstacles still stand in the way to eliciting an effective immune response against cancer, even though several antigens and antigenic peptides have been identified as potential tumor targets. All of them, including the MUC-1 mucin, share the caveat of being normal cellular proteins. Unlike all the others, however, MUC-1 expressed on tumors can still be considered a truly tumor-specific antigen. Its expression on normal cells is hidden from the immune system, and its aberrant glycosylation on tumors creates new epitopes recognized by the immune system. Moreover, all other tumor targets identified so far are MHC-restricted peptides that can only be recognized by patients who carry a specific HLA type, or on tumors which continue to express particular HLA alleles. MUC-1 is powerfully different. Recognized as a native molecule independent of MHC, it is a universal immunogen and a universal target, and if made effectively immunogenic, it would be expected to elicit immune responses in all patients, and against numerous MUC-1 expressing human tumors. It may, in fact, be the extraordinary solution to an extraordinary problem of cancer immunity and immunotherapy.

Mechanisms contributing to the activity of integrins on leukocytes

Summary: Understanding how the integrins on leukocytes operate is important because these receptors control the activity of leukocytes in all phases of their lives. Thus integrins control leukocyte development and maturation in bone marrow, the circulation of naive cells in secondary lymphoid tissue, e.g. the lymph nodes, and leukocyte responses to inflammatory signals emanating from injured tissues. Using as an example LFA‐1, which is expressed by all leukocytes, we outline how the activity of this integrin is modified to meet the challenges posed by these leukocyte activities. Briefly, we discuss three means by which LFA‐1 is adapted to bind more efficiently to its chief ligand, ICAM‐1. LFA‐1 can undergo changes in conformation leading to increased affinity, can be clustered on the membrane and, finally, when activated can move into the lipid raft compartment of the membrane. The study of humans with the β2 deficiency syndrome termed leukocyte adhesion deficiency (LAD)‐1 and analysis of LFA‐1 null mice has given further insight into integrin activation mechanisms and the in vivo roles of LFA‐1 and other leukocyte integrins.

Myeloid cell function in MRP-14 (S100A9) null mice.

ABSTRACT Myeloid-related protein 14 (MRP-14) and its heterodimeric partner, MRP-8, are cytosolic calcium-binding proteins, highly expressed in neutrophils and monocytes. To understand the function of MRP-14, we performed targeted disruption of the MRP-14 gene in mice. MRP-14−/− mice showed no obvious phenotype and were fertile. MRP-8 mRNA but not protein is present in the myeloid cells of these mice, suggesting that the stability of MRP-8 protein is dependent on MRP-14 expression. A compensatory increase in other proteins was not detected in cells lacking MRP-8 and MRP-14. Although the morphology of MRP-14−/− myeloid cells was not altered, they were significantly less dense. When Ca2+ responses were investigated, there was no change in the maximal response to the chemokine MIP-2. At lower concentrations, however, there was reduced responsiveness in MRP-14−/− compared with MRP-14+/+ neutrophils. This alteration in the ability to flux Ca2+ did not impair the ability of the MRP-14−/− neutrophils to respond chemotactically to MIP-2. In addition, the myeloid cell functions of phagocytosis, superoxide burst, and apoptosis were unaffected in MRP-14−/− cells. In an in vivo model of peritonitis, MRP-14−/− mice showed no difference from wild-type mice in induced inflammatory response. The data indicate that MRP-14 and MRP-8 are dispensable for many myeloid cell functions.

Human Tumor Antigens Are Ready to Fly

Publisher Summary The chapter reviews the current knowledge of tumor antigens recognized by both humoral and cellular arms of the immune system and the promise that some of these antigens currently hold for cancer immunotherapy. The knowledge about tumor-associated antigens detected by antibodies can be and has been used in several ways: (1) the presence of the antigen has been used for detection and diagnosis of malignancy, (2) the antibody specific for the antigen has been conjugated to toxins, drugs, or radioisotopes and used for tumor therapy, (3) the antibodies have been made bispecific or multispecific and used to bring other effector mechanisms, such as T cell, natural killer (NK) cells, and macrophages, to the tumor site, and (4) tumor-associated antigens identified as growth factor receptors or other types of signaling molecules have been used to induce tumor cell death or differentiation when bound by the specific antibody. These uses are all grouped under the term “passive immunotherapy” and have the drawback that repeated treatments are necessary and very high concentrations of tumor-specific antibodies are required for each treatment to see a limited effect. “Active immunotherapy” is perceived to be more desirable based on the hypothesis that induction of an immune response to the tumor antigen would be long lived and would involve multiple components of the immune system. The chapter focuses primarily to antigens known to be immunogenic in patients; it is obvious that they are also candidate immunogens for eliciting immunity in patients through vaccination (“active immunity”). The ultimate success of a vaccine depends on the presence of fully immunocompetent T cells and B cells specific for the tumor antigen used as immunogens.

Chimpanzee dendritic cells with potent immunostimulatory function can be propagated from peripheral blood

We have established dendritic cell (DC) cultures from chimpanzee peripheral blood mononuclear cells (PBMC) by using recombinant human (rh) granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and rh interleukin‐4 (IL‐4) and demonstrate that these cells have all the characteristics of DC as described for other species. We consistently can obtain 1×107 DC per 100 ml of blood, a yield of 5% DC as compared to 0.1 to 0.5% DC reported in fresh human PBMC. The cultured DC have a varied morphology with typical cytoplasmic extensions. Phenotypically, the blood‐derived DC lack expression of most lineage antigens, but express CD83, an antigen specifically expressed on human blood DC. Chimpanzee DC express very high levels of major histocompatability complex class II antigens, adhesion and costimulatory molecules. Consistent with this phenotype of a powerful antigen‐presenting cell, chimpanzee DC generate allogeneic mixed leukocyte responses 15 to 20 times more potent than that elicited by macrophages, Epstein–Barr virus‐transformed lymphoblasts and fresh PBMC. In addition, chimpanzee DC very efficiently present tetanus toxoid to PBMC‐derived CD4+ T cells as compared to macrophages and PBMC. The DC generated by culturing chimpanzee PBMC with rhGM‐CSF and rhIL‐4 thus closely resemble human blood‐derived DC propagated in the same manner. This technology provides a powerful animal model with which to apply DC to clinical studies with relevance to human disease. In particular, chimpanzee DC can be tested as immunotherapeutic agents for cancer, and be studied in relation to the pathogenesis of human immunodeficiency virus (HIV) infection.

Retroviral expression of MUC-1 human tumor antigen with intact repeat structure and capacity to elicit immunity in vivo.

MUC-1 mucin is an epithelial cell antigen whose aberrant expression plays a role in autoimmunity and tumor immunity and is thus an attractive candidate for immunotherapy of gene therapy. Because the MUC-1 cDNA is composed almost entirely of 60-bp tandem repeats and is susceptible to homologous recombination, it presents a special challenge to cloning and expression in viral vectors. Nevertheless, we have been successful in constructing a retroviral vector (MFG-MUC-1) with a 22-tandem repeat MUC-1 cDNA. Both stable and transient packaging cell lines are capable of producing high-titer retroviruses that can transfer the expression of MUC-1 to murine 3T3 cells. Transduced cells express uniformly high levels of MUC-1 on their surface, and western blot analysis reveals that the molecule expressed is of full length and extensively glycosylated. We have used the MFG-MUC-1 vector to stably transduce an immortalized murine dendritic cell line and show that immunization of mice with transduced cells elicits specific immune responses to mucin. The ability of this vector to transfer expression of the MUC-1 tumor antigen to potent antigen-presenting cells is expected to be of use in the immunotherapy of epithelial cancers.

Sequence analysis of peptides presented to the immune system by class I and class II MHC molecules

Cytotoxic T lymphocytes (CTL) are an arm of the immune system concerned with recognition of host cells that express new antigens as a result of viral infection. CTL do not recognize new antigens directly, but only as short peptides bound to cell surface class I molecules of the major histocompatability complex (MHC) (Germain, 1986; Monaco, 1992; Morrison et al., 1983). It is thought that a small fraction of newly synthesized self and viral proteins are degraded into small peptides in the cytoplasm, and these are then transported into a subcellular compartment, the endoplasmic reticulum, where they become bound to a groove on the top of class I molecules, and are subsequently transported to the cell surface for presentation to cytotoxic lymphocytes. Class I molecules are glycoproteins consisting of light (12 kDa) and heavy (47 kDa) chains. Each cell presents up to 105 copies of six different class I molecules. Since the cell is also synthesizing several thousand different proteins, each type of class I molecule is expected to present several thousand peptide fragments to the immune system at any particular moment. CTL bind to the class I molecules and check the nature of the peptides being presented. If they are of foreign origin, the CTL become activated, multiply, and lyse the infected cell. If only self peptides are presented, the CTL remain inactive and continue their surveilance of other cells in the neighborhood.