Publisher Summary This chapter focuses on the important discovery that virus-specific cytotoxic T cells are dually specific for virus and for a self cell surface antigen encoded by the major histocompatibility complex (MHC). The initial work was carried out on the lymphocytic choriomeningitis virus system but it soon became evident that the same phenomenon applied to many other viruses. In addition, the same principle has been found to hold for other antigenic systems, such as trinitrophenyl coupled to cells, minor histocompatibility antigens, and the H-Y model. Graft rejection and the need for genetically homogeneous inbred mouse strains for cancer research led to the development of transplantation immunology and immunogenetics. The result is that the gene complex coding for major transplantation antigens is one of the better understood mammalian genetic regions. Cytotoxic T-cell specificity is comparable to serological specificity. Because quantification of specificity or cross-reactivity is difficult, and because of the technical limitations of these cytotoxic T-cell assays, results are interpreted with great reservation. MHC restriction reflects the fact that the effector function of T cells is determined by the kind of Self-H recognized together with the foreign antigen on cell surfaces: K and D are receptors for lytic signals, I determinants are receptors for cell differentiation signals that are delivered antigen-specifically by T cells.

MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness.

Hybridoma cells which secrete colorectal carcinoma-specific antibodies have been produced and used to study the antigenic structure of these tumor cells. Nineteen antibodies have been studied in detail, and 15 of these are colorectal carcinoma specific. Only two antibodies reactive with carcinoembryonic antigen (CEA) have been discovered and five other antibodies that react with distinct epitopes on the cell surface have been defined. Several antigens with distinct molecular characteristics have been shown to exist by use of hybridoma antibodies. Six hybridoma antibodies have been shown to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).

Colorectal carcinoma antigens detected by hybridoma antibodies

A legacy of molecular evolution is the formation of gene families encoding proteins that often serve related functions. One such family gaining recent attention is the tetraspanin superfamily, whose membership has grown to nearly 20 known genes since its discovery in 1990. All encode cell-surface proteins that span the membrane four times, forming two extracellular loops. Some of these genes are found in organisms as primitive as schistosomes and nematodes. Alternately known as the transmembrane 4 (TM4) superfamily or the TM4SF, 4TM, or tetraspan family, we propose here that the name tetraspanins be used for the purpose of standardization. What do the tetraspanins do? Awaiting definitive functional studies, we can only put together pieces of a puzzle that has been built by raising antibodies against these proteins and looking at their distribution, associations, and functions. A brief overview indicates that some tetraspanins are found in virtually all tissues (CD81, CD82, CD9, CD63), whereas others are h...

The tetraspanin superfamily: molecular facilitators.

Faraci, Frank M., and Donald D. Heistad. Regulation of the Cerebral Circulation: Role of Endothelium and Potassium Channels. Physiol. Rev. 78: 53–97, 1998. — Several new concepts have emerged in re...

Regulation of the Cerebral Circulation: Role of Endothelium and Potassium Channels

L INTRODUCTION Interest has burgeoned recently in the possibility of attacking tumor ceils with cytotoxic agents covalently linked to specific antibodies. This follows the innovation of cell hybridization techniques for producing monoclonal antibodies which has stimulated a world-wide search for antibodies with specificity for cancer cells. In anticipation that these endeavors will meet with success, a number of groups have attempted to devise ways of arming the antibody molecule to endow it with potent cytotoxic activity against the cells to which it can bind. Our decision to use toxins as the cytotoxic component ofthe conjugate was provoked by their supreme potency. One molecule of the bacterial exotoxin, diphtheria toxin, or of the plant toxins, abrin, from Abrus precatorius or ricin, from Ricinus communis, may be sufficient to kill a cell which it penetrates (Yamaizumiet al. 1978, Eiklid et al. 1980). This maximizes the chance of killing cancer cells which do not express specific antigens at high density or which reside in solid tumors not freely permeable to the conjugate. The modes of cytotoxic action of diphtheria toxin, abrin and ricin have been reviewed elsewhere (Collier 1976, Olsnes & Pihl 1976, Pappenheimer 1977, Gill 1978). Briefly, the toxins ali comprise two polypeptide chains, denoted A and B, which are linked by a disulfide bond and which, when separated, are virtually devoid of cytotoxic action. The toxins bind via a recognition site on the B-chain to receptors on the cell surface and the A-chain then penetrates (or is translocated across) the cell membrane into the cytosol where it terminates protein synthesis. Diphtheria toxin A-chain inactivates elongation factor 2 by

The preparation and cytotoxic properties of antibody-toxin conjugates.

The pathogenicity of fixed rabies virus strains for adult mice depends on the presence of an antigenic determinant on the viral glycoprotein. Two virus-neutralizing monoclonal antibodies have been used to identify this determinant. All pathogenic strains of fixed rabies virus bind to these antibodies and are neutralized by them, whereas nonpathogenic strains fail to react with these monoclonal antibodies and are not neutralized by them. Antigenic variants of the rabies virus with altered glycoprotein were selected by growing virus in the presence of one monoclonal antibody, 194-2. All variants that lost their ability to react with this antibody and an additional antibody, 248-8, were found to be nonpathogenic for adult mice. Analysis of tryptic peptides of the glycoproteins of pathogenic parent virus and nonpathogenic variants and the amino acid sequence of a specific variant tryptic peptide revealed that the change in pathogenicity corresponded to an amino acid substitution at position 333 of the glycoprotein molecule. The nucleotide sequence of the nonpathogenic variant glycoprotein gene contained a base change that confirmed the single amino acid substitution in the tryptic peptide replacing arginine-333 in the parental glycoprotein. We conclude that arginine-333 is essential for the integrity of an antigenic determinant and for the ability of rabies viruses to produce lethal infection in adult mice.

https://www.pnas.org/content/pnas/80/1/70.full.pdf

Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus

We have constructed cell-specific cytotoxic agens by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-1A was modified by attachment of 3-(2-pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linked conjugates that retained the original binding specificity of the antibody moiety. the conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.

https://www.pnas.org/content/pnas/77/8/4539.full.pdf

Antibody-directed cytotoxic agents: use of monoclonal antibody to direct the action of toxin A chains to colorectal carcinoma cells

Rabbit anti-idiotypic antibodies (alpha Id Ab) were prepared against five murine monoclonal antibodies (mAb) specific for the rabies virus glycoprotein. Four of the mAb were directed against three known, type-specific, neutralizing sites on the glycoprotein, and the other mAb was directed against a topographically uncharacterized, nonneutralizing epitope. An absence of significant cross-reactivity among the alpha Id Ab for heterologous mAb suggested that the alpha Id Ab were highly specific for unique variable region determinants. The binding of three of the five alpha Id Ab to their homologous mAb could be inhibited by rabies virus-soluble glycoprotein, suggesting that the alpha Id Ab possessed subpopulations similar or adjacent to the antigen-binding site of the mAb. Two of the five alpha Id Ab injected into mice elicited a specific virus-neutralizing antibody response. Mechanisms to account for the induction of the virus-neutralizing antibody by alpha Id Ab are discussed.

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Pathogenic parental rabies virus and apathogenic variant virus were shown to differ in their ability to infect neurons in vivo and neuroblastoma cells in vitro. After intracerebral inoculation, the distribution of infected neurons in the brain was similar for both viruses, but the rate of spread throughout the brain, the number of infected neurons, and the degree of cellular necrosis were much lower in the case of apathogenic virus. After adsorption to mouse neuroblastoma cells, apathogenic virus was less rapidly internalized than pathogenic virus, and cell-to-cell spread of apathogenic variant virus was completely prevented by the addition of rabies virus-neutralizing antibody, whereas the spread of pathogenic virus was not affected.

Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro.

The specificities of monoclonal antibodies against melanoma cells were analyzed using radioimmunoassay, mixed-hemadsorption assay, and quantitative absorption on a variety of malignant and nonmalignant cells. Three of the six hybridomasecreted antibodies bound to the majority of melanoma cell lines, melanoma tumors, and astrocytoma cell lines as well as to all normal and Epstein-Barr virus-transformed lymphocytes tested. The binding pattern coincides with the presence or absence of the DR antigen on human cells. Conversely, two other antibodies, 19-19 and Nu4B, detected two different antigens common to melanoma and astrocytoma cells only. Cloning of melanoma cells resulted in establishment of DR-positive and Dr-negative clones, with the binding of Nu4B antibody retained in all.

Hilary Koprowski

Papers

Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus

Antibody-directed cytotoxic agents: use of monoclonal antibody to direct the action of toxin A chains to colorectal carcinoma cells

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro.

Specific Immunoreactivity of Hybridoma-secreted Monoclonal Anti-Melanoma Antibodies to Cultured Cells and Freshly Derived Human Cells