Five

IMMUNOLOGY

"Anaphylactic Shock Stress Without Benefit of Anesthesia"

The immune system is one of the most complex in the vertebrate organism, yet everyone knows what it is to be allergic to something, and we are familiar with what happens to sensitized people whose skin reacts with large swellings and wheals to insect bites and poison ivy or to the child who wheezes and may even strangle in the grip of asthma. A foreign protein, the "antigen," enters the body, binds to a receptor on the surface of a lymphocyte (one of the white blood cells), and together they cause a reaction which may be anything from completely innocuous to immediately fatal "anaphylactic shock." Medicines, such as the antihistamines, have been developed to deal with these acute reactions. Testing is necessary in their manufacture and this is fairly simple, although unfortunately animals like guinea pigs are generally used to investigate the possibility of anaphylactic shock. Thus in their Annual Report under the Animal Welfare Act for 1976, Bristol Laboratories of Syracuse, New York, reported that 500 guinea pigs were exposed to "anaphylactic shock stress without benefit of anesthesia" because in testing the drugs the "physiological state of the animal cannot be compromised by prior use of sedatives." (USDA/APHIS,1976, Bristol Labs.).

In Vitro Alternatives?

While it is true that the symptoms of anaphylaxis are all too plainly visible in the whole animal, might not the reaction be observed at a cellular or molecular level equally well, and directly on human cells for which the drug under investigation is ultimately intended? J. Humphrey indicates just this when he points out that principles worked out on experimental animals are often inapplicable to man but "fortunately the great improvements in tissue culture and micromethods have made it possible to do many of the tests used on mice, etc., with human peripheral blood lymphocytes or easily obtained lymphoid tissue such as tonsils." (Humphrey, J.,1978, p.109).

Demonstrating Anaphylactic Death in the Classroom - and the Alternative

In the Annual Report for 1976 under the Animal Welfare Act submitted by the Dept. of Pathology, School of Dental Medicine, University of Pennsylvania, the following justification is given for subjecting animals to pain or distress without using pain-relieving drugs:

"For classroom demonstrations guinea pigs are sensitized for systemic anaphylaxis and then challenged i.v.[intravenously] with antigen. Death in acute shock results within 5-10 minutes. This demonstration is used to impress students with need to avoid systemic allergic reactions in patients." (USDA/APHIS,1976, Pennsylvania, Univ.of).

Similar demonstrations were reported from Lehigh University Pennsylvania; Cleveland State University, Ohio; and Emporia Kansas State College. Chicago College of Osteopathy, using 12 guinea pigs and 12 rabbits, gave as an explanation: "to the best of our information, no comparable procedure available in an in vitro system."

However, instead of witnessing the agonizing "sacrifices" of live animals, students could have been impressed by a similar demonstration in a film entitled, "ANAPHYLAXIS IN GUINEA PIGS," a 1960 production of a university bacteriology department. A guinea pig sensitized to egg albumen receives a second injection of the protein and is shown undergoing paralysis, bronchial constriction and, finally, death by suffocation. While a question might be raised over the morality of subjecting even one guinea pig to such suffering for educational purposes, the fact that the film now exists offers a teaching alternative which could spare the lives of many other animals. (California, Univ.of, 1960).

Defending the Body: Cell Eaters, Antibody Manufacturers, Helper and Killer Cells

The functions of the cells which constitute the immune system of the body, and names which have been given them, are stimulating to the imagination. Foreign substances entering the body, the antigens, are seized on by the phagocytes, or "cell eaters." These inhabit the lymphoid tissue, especially the lymph nodes and the spleen, in whose cavities lurk the macrophages, or "big eaters." These cells prepare the antigens for presentation to the next line of defense, the lymphocytes, which loiter along the vessels of the lymphoid area or are swept through the blood vessels in a jostling throng of red blood cells.

Two thousand billion of these lymphocytes are present in the human body; every second about a million new lymphocytes arise in the bone marrow and the same number die. Some of these pass through the thymus gland and are known as T-cells, an equal number are not thymus related and are called B-cells. Stimulated by the presence of antigens, the latter produce antibodies, the defense protein molecules which at one end are so constructed that they can interlock with matching features on the surface of the antigens. This starts a militant operation to get rid of the "foreigners" chiefly bacteria and viruses.

The T-cells also defend against invaders, and are particularly effective against fungi, parasites, cancer cells and foreign tissue. There are different types of T-cells with specialized functions, and these also proliferate on the arrival of antigens. Among them are "killer" cells which attack and destroy the invaders directly, and "helper" cells which assist in B-cell differentiation and proliferation.

The next section introduces some immunoloqical research which has had fruitful results. The protagonists are an odd couple: a sheep and a mouse.

Sheep's Red Blood Cells: a Research Tool in Immunology

When sheep's red blood cells are injected into a mouse, they act as an antigen. B-and T-lymphocytes cooperate to produce antibodies which bind the sheep cells, against which the mouse has now become immunized. However, it would be difficult or impossible to repeat the experiment and come up with the identical antigen-antibody complex. Numerous different antibodies, providing they and the antigen have at least some parts (or "determinants") in common, may bind to the sheep's cells. Also, each individual mouse produces a different antigen-antibody combination, both in quality and quantity. Thus if one wanted to standardize the antigen-antibody complex, i.e., the antiserum, so that it could be given to any and all mice to immunize them against sheep's red blood cells, or if one wanted to isolate a specific antibody for analysis, it,would be necessary to use a technique which would eliminate the differences between animals and the variability due to the mixture of antibodies reacting with the antigen.

Of course, neither mouse nor man in nature is going to need an antiserum against sheep's red blood cells. The latter is just a convenient biological protein for research purposes. But, as will be seen presently, a procedure worked out with these cells can be applied to other antigens of more concern to humans - for instance, cancer or influenza virus, or "histocompatibility" antigens which are the ones which have to be neutralized if a tissue or organ graft is to "take."

In immunological work, the interests of humanitarians and researchers converge in their common desire to eliminate animals as much as possible from experiments. To the experimenters, it is the individual differences mentioned above which frustrate purification and reproduction of biological products, even though injection of antigens in any normal animal inevitably produces antibodies and some degree of immunity.

"Search and Destroy" - the Antibody and the Tumor Cell

An application of immunology in cancer research and therapy using chimpanzees as the animal model illustrates both the advantage of these new techniques and the humanitarian problem they can create. H. Seigler has developed an antiserum against the highly malignant and often fatal cancer melanoma by injecting human tumor cells into a chimpanzee. These cells contain a "tumor associated antigen" (TAA), and the ape injected with them develops an antibody against a human melanoma antigen. Seigler writes:

"There has long been interest in using tumor specific antibody to concentrate antitumor agents on tumor cells. In doing so, many of the harmful side effects of chemotherapeutic drugs might be avoided .... One recent study had rather dramatic success .... The antibody was conjugated with an akylating chemotherapeutic and administered to a melanoma patient with disseminated tumor. Following this treatment, the tumor systematically regressed .... We have utilized radio-labelled chimpanzee anti-human melanoma antibody in an effort to distinguish the specificity of the antibody and its ability to localize to the harbored tumor. Both immunofluorescent studies and autoradiography studies of the removed tumor tissue have demonstrated that the antibody did, indeed, fix specifically to the tumor cells."

Seigler has also found that the antibody is capable of clinching a diagnosis of melanoma by specifically combining with and thus identifying metastatic melanoma cells which the pathologist has not been able certainly to recognize. Once identified, the appropriate chemotherapy can be started. (Seigler, H., 1977).

Remarkable as this experiment and its results appear, it is unlikely that antibodies produced in another species could be pure and specific enough to be generally successful in treating humans. Also, chimpanzees should not be exposed to highly malignant tumors. A method was necessary which 1. would remove the procedure from the actual animal to cells in culture, 2. would permit the production of an antibody over a long enough period for the cells expressing it to be isolated ("cloned"), and 3. would result in its purification and its being made absolutely specific for the antigen against which it was to act.

Monoclonal Antibodies "Immortalized" by Cancer

A very ingenious and "elegant" experiment which has accomplished just this was developed in 1975 by C. Milstein and G. Köhler at the Medical Research Laboratory in Cambridge, England. It is known that a B-cell-derived malignant lymphoid cancer (myeloma) of mice will grow indefinitely in the laboratory. (Köhler, G.,1975). Cells from this if placed in a culture medium containing polyethelene glycol will fuse with normal B-lymphocytes from the spleen or lymph nodes of a mouse. This mouse has previously been injected with sheep's red blood cells and therefore the B-lymphocytes are producing antibodies against the sheep cells. The fused cells have immortality conferred on them by the continuously self-replicating cancerous part. The culture, however, contains not only the fused cells but also unfused myeloma and spleen cells. It is possible to get rid of the unfused cells, leaving only the desired fused or "hybridoma" cells, by juggling the chemicals in the broth medium in which the cells are grown so that it fails to nourish first the unfused myeloma. cells then the unfused spleen cells. These die out, leaving the hybrids energetically proliferating and secreting antibodies against the sheep's blood cells. (Staehelin, T.,1978, p.133).

At this point there is still a mixture of antibodies in the cell culture. Using special techniques such as autoradiography and isoelectric focusing it is possible to identify subpopulations or "clones" of B-cells, and to segregate the one among these which produces the specific antibody required. It is called "monoclonal" because all the cells of a clone are descended from one cell and make antibody molecules with identical connectors at one end which bind to a specific antigen. Even then, the culture has to be constantly monitored to prevent overgrowth by variant cells appearing through spontaneous mutations, but repeated subcloning will segregate the cells of desired specificity from these mutants. Antibodies from these are far purer than any that could be obtained from the serum of rabbits or other animals which had previously been injected with an antigen. Furthermore, the hybridomas can be frozen and preserved for future use.


Cell Culture as an Alternative

One method of assuring a stable and long-term source of the monoclonal antibody is by injecting these hybrid clones into mice to produce tumors from which the antiserum can be harvested. A humane alternative is not to use the whole animal but to continue the growth in cell culture and thence to collect the antibody. There is also the advantage of eliminating the contamination of whatever other antibodies there may be in normal mouse serum. There are many exciting applications of this research; in fact the possibility of making antibody molecules which will unerringly seek out and detect specific antigens has been called a revolution in immunology. It is not the simple antigen-antibody reaction such as occurs when an antigenic pollen hits the antibodies on the mucous membrane of the nose of a hay fever sufferer and produces the familiar symptoms. In that case there is likely to be a mass of different antibodies responding to a mixture of antigens. But for diagnostic purposes, for developing a medicine specifically designed for a particular disease germ or cancer (and delivering it to a certain location and nowhere else), for drawing fine distinctions between cellular and subcellular structures, this new technique is proving an exquisitely sensitive research and therapeutic tool.


The Next Step: of Mice and men

Dr. T. Staehelin, asked at the Roche Research Foundation Symposium in Basle (1977) to discuss the sensitivity of mouse antibodies to human antigens, replied that the hope of the future lay in using human lymphocytes in an in vitro (test tube) immune response.

"This approach may eventually turn out to be a necessity and may have a great advantage .... If it is possible to fuse human lymphocytes with an established human lymphoblastoid or plasmacytoma cell line, then we may be able to produce monoclonal human antibodies. Of course the potential here would not only be diagnostic, but eventually even therapeutic." (Ibid., p. 139) .

Köhler and Milstein in the experiment described above fused mouse lymphocytes with a mouse myeloma cell line. Staehelin's 1977 prediction referring to the possibility of using human cells to produce antibodies from hybrids has already come to pass in this rapidly expanding field of research. H. Koprowski and others (Koprowski, H.,1978, p.17) took lymphocytes from the cerebrospinal fluid of a patient with a herpes zoster encephalitis.

These were hybridized with mouse myeloma cells and were found to segregate into clones that produced human antibodies and clones that did not.

By the usual purification techniques it should be possible to select those clones whose antibodies are specific to the encephalitis antigens, with a potential of diagnostic, therapeutic or research use.


And Finally, the All-Human Hybridoma

Most of the hybridoma research reported up to the present has used mouse myeloma cell culture as the "immortal" part of the antibody producing system. But human lymphocytes can also be transformed into cancer and, hence, into immortal cell lines, by exposure in the laboratory to a virus called Epstein-Barr. This creates a "lymphoblastoma," and there seems no reason why this could not be linked with normal human lymphocytes producing specific antibodies. If the donor of the latter were suffering from a cancer, such as melanoma, one would expect the antibody to be specific for a melanoma tumor virus; if the individual had been immunized with tetanus toxoid, one would look for a tetanus antibody. Even without the intervention of animals, therefore, a hybridoma capable of producing antibodies of exquisite specificity to any known antigen can be made.

F. Melchers and his co-editors of "Lymphocyte Hybridomas" conclude the Preface with this paragraph:

"Monospecific antibodies have been the dream of many immunologists for long .... Lymphocyte hybridomas have made the dream become a reality. It is, therefore, predictable that lymphocyte hybridoma cultures are likely to replace many rabbits in many research laboratories." (Melchers, F.,1978, p.XVII).

A Laboratory Without Animals: the Patient's Body

But the last word on this subject has not by any means been said, so varied are the possibilities of immunological research. The investigations reported above were chiefly concerned with developing an antibody, or antiserum, which might ultimately be injected into a patient to combat a cancer-bearing tumor associated antigen (TAA) known to be the specific target for that antibody. S. Leong and associates have taken cells from a melanoma sufferer, combined them with an immunological strengthener or adjuvant known as BCG, and injected them, not into a chimpanzee, as Seigler did in the experiment described above, but back into the patient himself. This produces an antibody against his particular TAA, although one too weak to arrest the disease. Nevertheless, this antibody can be a useful tool. Leong and associates have also been working with cultures of human melanoma cells and have isolated various tumor associated antigens from the membrane of these cells. If the patient's antibody is extracted and combined with these antigens, it can pick out the one which is identical with the patient's, and this can be purified and developed into a vaccine. This in turn can be injected into the patient and may be able to mobilize enough antiserum in his blood to destroy the cancer. (Leong, S., 1978).

Epstein-Barr Virus to the Rescue?

Of course it is not possible to risk injecting live or even inactivated cancer cells as a vaccine to produce antibody against cancer into anyone other than a patient who already has the disease. It is possible, as we have seen, to grow specific antibodies in culture outside the body, but this is a roundabout way to produce them. It's much more effective if the body acts as its own laboratory and makes antibodies just as it does in response to a shot of tetanus vaccine. In the case of cancer, the solution may come through one of those tumor associated antigens on the membrane of the cancer cell.

The Epstein-Barr virus, which causes relatively innocuous mononucleosis - the "kissing disease" of the young - is also responsible for a lymphoid cancer, or lymphoma. (Its ability to transform laboratory cultures of human lymphocytes into cancer was mentioned above). Furthermore, a tumor associated antigen is a normal inhabitant both on the surface coat of the virus and on the lymphoma cell membrane, although this antigen itself is neither infectious nor cancer-producing. However, if injected into an animal, it does produce antibodies and renders the animal immune to mononucleosis. If animals, why not humans? and this may lead to protection against the lymphoma too - which especially affects African children - and against Epstein-Barr nasopharyngeal cancer, a distressing affliction of Chinese men. (Anon.,1980d)

Neither this vaccine, nor the anti-melanoma antiserum developed through hybridization is yet available (in early 1980) for therapy. But should cancer actually turn out to be an immunological aberration, we are surely "getting warm" in this scientific game of hide-and-seek. And if, as seems possible, anticancer viruses of the future can be grown on human cell cultures, rather than in animal models, then the rabbits, and monkeys and ourselves, for different reasons, have much to look forward to.


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