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idiotypic response, to other rat monoclonals against mouse cell surface antigens (Waldmann). The reason for the high immunogenicity of cell-bound antibody is not yet clear. While these animal experiments sometimes show dramatic effects, what of the human experience? This is less clearcut except in reversal of acute renal allograft rejection episodes because although more than 500 individuals worldwide have been treated with 17-1A antibody, there has not yet been a single controlled trial, so all the results must be considered anecdotal. There was general agreement, however, that if

The use of recombinant DNA techniques, cell fusion and novel bioprocessing in the pharmaceutical industry has assisted the development of many kinds of diagnostic and therapeutic products. Some directly affect the immune system (e.g. interleukir~s, intefferons, turnout necrosis factor, and colony stimulating factors). Others (e.g. peptides, cytokines, growth factors, H2-receptor antagonists, nonsteroidal anti-inflammatory drugs and neurohormonal agents) have immunological reactivity even though they are not designed to be immune modulators. The need to define the immunotoxico!ogical potential of these products during preclinical safety evaluation was among the topics discussed at a recent meeting*.

102

Immunology Today,vol. 8, No. 4, 1987

responses do occur, they often begin well after the monoclonal antibody treatment, suggesting that host responses may be important. Can any general conclusions be drawn ? Perhaps the most important is that we are indeed at the beginning of the use of monoclonal antibodies for immunomodulation. Clearly, they can have powerful effects, alone or with other agents. Equally clearly we need to know much more about many of the areas discussed in this workshop. We need to understand what makes a 'good' or 'bad' target antigen or epitope0 what host effector mechanisms can be recruited and how, what deLer-

mines the half-life of antibodies or conjugates, how to induce tolerance to antibodies or how to manipulate the anti-idiotype response in a favourable way. While it may be a long hard road, the results presented at the workshop encourage the belief that antibodies may one day be Ehrlich's magic bullet.

Peter Beverleyis in the ICRFHuman Tumour Immunology Group, University College London, UK; and G. Riethmulleris at the Institute for Immunology of the Universityof Munich, FRG.

hmunotoxicology and the new biotechnology from JayCavagnaro Active cells expand clonally and both differentiate and proliferate. Effector function may therefore be inhibited while affector function remains intact (L. Loose, Groton).

The immune system in toxicity testing The principal debate at the meetII i~j

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products of biotechnology can be adequately evaluated by traditional toxicity testing methods or whether no,eel approaches are required. Many toxicologists are wary of giving The major problems in this avalu- an impression of confusion and beation are the compounds' pancyto- lieve that 'there is nothing new'. toxicity and polypharmacological Nevertheless, original thinking is actions (G. Morgan, Philadelp>.ia). needed (H. Ozer, Chapel Hill). The Our knowledge of the biochemical immune system can be a sensitive pharmacology of these preducts is indicator of general toxicity as well limited and the site of action is often as an excellent source of information unknown. The challenge is to design about the mechanisms of action, inthe most specific and relevant ex- cluding toxicity, of the new biotech periment to answer questions of products. toxicity for the target organ. HowTraditional descriptive testing ever, the immune system as a target schemes in animals were developed organ is a complex network of cells for small molecular weight suband tissues and is therefore very stances in the context of regulatory different from the liver, kidney, heart guidelines. In the past, drug toxicity or brain. The immune system is anti- testing focused on physiological, gen driven and responds to a vast biochemical, pharmacological and number of antigens. The rate and formulation safety. Many of the cureffectiveness of the immune re- rent biotechnology products are norsponse can be shifted by xenobiotics. mal constituents of the body but they are administered at pharmaco* Themeetingwasheldat TvsonsCorner,Virginia, logical rather than phys;o~ogical USA,23-24 October,1986. levels, often by an unnatural route,

and may reach tissue sites not normally exposed. Many of the lymphokines may not be toxic but may activate numerous effector mechanisms, so that monitoring optimal activity becomes difficult. If the compounds are acting through indirect mechanisms 'more is certain!y not UI~LLt~I

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dose (MTD) may be an overdose because the response curve for immunological effects is often bellshaped (C. Pinsky, Frederick). Dose-effect relationships are also problematic when efficacy is inversely related to dose. If immunomodulation is the aim, the optimum dose in a toxicity study may be one that achieves maximum toxicity at the higher levels and by definition maximum inhibition of immunological function (Ozer).

Preclinical testing In the biotech industry toxicology testing is often done early in a product's development (J. Kopplin, Emmeryville). The responsibility of preclinical safety testing thus becomes one of not only establishing the safety and potential toxicity of the test material but also evaluating the pharmacokinetics of the drug and monitoring the pharmacological responses. Such were the goals of the subchronic toxicity study in primates of granuiocyte-macrophage

(~) 1987, Elsevier Publications, Cambridge

0167 - 4919/87/$02.00

Immunology Today, vol. 8, No. 4, 1987

colony stimulating factor (R. Stoll, East Hanover). The areas of particular toxicological concern include: the intrinsic toxicity of the active drug; toxicity related to pharmacodynamic effects; biological toxicity; and toxicity of the contaminants (G. Zbinden, Schwerzenbach). Early on toxicologists were most concerned about matters such as contaminants, sensitization to host proteins, infection by pathogenic viruses and cell-transformation by viral oncogenes. They now realize that the purification processes which produce sufficient (multigram quantities) product to sustain clinical trials and to be marketed must produce material of pharmaceutical quality. Effective scale-up requires the definition of the product's chemistry at the lab bench (C. Prior, Clayton). The protein chemist must remove or minimize contaminants (e.g. nucleic acids, lipopolysaccharides, serum proteins) that might elicit toxicological or immunotoxicological responses. The toxicity problem with contaminants is best solved by rigorous quality control and sophisticated analytical procedures. A drug's side-effects may be idiosyncratic (intrinsic) and relate to the class of compounds and to the nature of the molecule (e.g. imI. I. I.~ I. I.l~l~,.I~,~ . . . ~ l I ll..l :": .l y. . . r~/l~ proteins). The evaluation of inuinsic toxicity requires animal models based on clinical experience with existing compounds (Zbinden). Studies on the mechanisms of toxic effects are essential. Standard toxicity tests in rodents, dogs or non-human primates give historical data and serve as potential methods for detecting unexpected effects. The most important effects relate to the pharmacodynamics of a compound at high doses, and when studying them the use of biologically and pharmacologically relevant models is more important than perhaps ever before (R. Cox, Vienna). The possibility of special risk human populations must be considered. Only when we learn what these products are doing in a well-defined immunological system can we speculate and extrapolate to man. Indirect responses not related to the pharmacodynamic effects present particularly difficult problems for the toxicologist. This 'biological toxicity' may include hypersensitivity, production of neutralizing anti-

bodies, cell transformation, hypercoagulability, platelet aggregation and activation of the clotting system (Zbinden). Relevant in-vitro tests for many of these effects have yet to be developed.

Immunogenicity of the new products Almost all of the compounds so far examined have been immunogenic in both preclinical and clinical studies. Most individuals given recombinant interleukin 2 develop binding antibodies, as demonstrated by treatment-related increases in antibody titer which decrease after treatment is discontinued. In the early trials, alpha interferon (IFN-~) elicited binding antibodies in more than 70% of recipients, while later trials with a more highly purified molecule sensitized fewer patients. Subcutaneous injection produced more binding antibody than did intramuscular (i.m.) or intravenous (i.v.) injection (J. Dean, Research Triangle Park). Following the administration of IFN-~ to rhesus monkeys, there was no evidence of toxicity which could be considered secondary to tile development of neutralizing antibodies nor was immunoprecipitation observed in tissues (J. Kamm, Nutley). Despite the development of antibody titers, there was no loss of clinical efficacy. in six years of experience performing subchronic studies with recombinant proteins, B. Marafino (Emmeryville) has never seen a case of toxicity in rodents or primates which could be associated with antibody. Even after growth hormone treatment of rodents, in which antibody titers were three logs higher than in monkeys or humans, no adverse effects were noted. Circulating antibody, even neutralizing antibody, does not appear to be toxic. Similarly, the presence of circulating IgE antibodies does not always correlate with the symptoms of allergic reactions (D. Flaherty, St. Louis). No local or systemic toxicity has been observed in patients sensitized to mouse antibody who have received a second i.v. injection (J. Weinstein, Bethesda). Of particular interest are the results of immuniza tion with mouse monoclonal antibodies directed against envelope ,~h,_=., coproteins and nuclear capsid proteins of Rift Valley fever virus: the antibody preparation with the greatest potential for proriucing neutralizing antibodies in humans

were less effective in passive protection studies in animals (J. Dalrymple, Frederick). Pharmacologically administered foreign proteins (e.g. monoclonal antibodies) may induce hypersensitivity (types I, II, I!1) or antibodies, which in turn may produce antiidiotypic antibodies. Interaction with idiotopes not on the antibody molecule but on the receptors of lympffoid cells may perturb the immune response. Neutralizing antibodies to exogeneous IFN may also neutralize endogenously produced IFN and impede the immune mechanisms relying on it. Exogeneous IFN may also modulate MHC antigen expression (J. Cruse, Jackson). Theoretical adverse consequences of aberrant antigen expression may include autoimmune disease.

Toxicity of (ytokines In trials of treatment with interferons and interleukin 2 clinical benefits have been noted but adverse reactions are common and have included fever, malaise, chills, dyspnea, chest pain and tightness, wheezing, nausea, vomiting, tremor, and skin rast'es. Other toxic manifestations have included hypotension, tachycardia, acute renal failure, diffuse capillary leakage, profound weight gain and puirronary edema. "We can ask whether the potential benefits outweigh the potential for harm onJy when we have a good understaqding of the benefits and risks" (Kamm). Many of the recombinant molecules are p!eiotropic and act as messengers and modifiers of a number o r immune responses (Ozer). Some may be useful only in combination with other agents and so may present a complicated toxicological picture. The toxicity endpoints for these compounds are not well defined. They may lack a clear dose-response curve, and often the response that should be measured is unknown. The colony stimulating factors may be unique in giving no evidence of toxicity at doses that are pharmacologically important. However, this too presents a problem in phase I clinical trials where doses are judged in terms of an MTD. A further complication, and probably of greatest importance from a regulatory point of view, is that some new therapeutic products are unique to each patient (e.g. anti-idiotypic anti-

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bodies, lymphokine-activated killer cells). Guidelinesfor toxicity testing The recent rapid advances in biotechnology have required close communication between regulatory agencies and industry. The United States Food and Drug Administration (FDA) is strongly advocating a 'case by case approach' to safety testing of biotechnology products and the Administration, under the auspices of The White House Office of Science and Technology Policy, has developed guidelines to promote a coherent regulatory framework for the biotechnology industry. Both the Environmental Protection Agency (EPA) and the FDA have issued a number of documents and guidelines to help the manufacturers develop reasonable testing protocols. Japan is also reviewing its regulatory approaches to safety testing of biotechnology products (Y. Harada, Osaka). Risk assessment at the EPA will

I m m u n o l o g y Today, vol. 8, No. 4, 1987

constitute a series of approximations which, with expert advice, will evolve as studies are reviewed (R. Sjoblad, Washington). Applications to the FDA are often reviewed by re~eo~ch scientists who have been working with similar projects for a number of years (B. Burlington, Bethesda). We are only now beginning to learn the hazards that could be associated with the new biotechnology products. The lessons are coming from empirical evaluation, rational extrapolation of experimental pharmacodynamic models, utilization of pharmacokinetic data, biomechanistic considerations and scientific speculation (Zbinden). The challenge of these products is that for many the biochemical pharmacology has still to be defined (Dean). The new products have dictated a departure from the normal procedures of toxicology. These procedures may be insufficient because they will be expected to evaluate not only the toxicity but the pharmacokinetics and the pharmacological re-

sponses of the test material as well. Preclinical approaches to screening may permit modifications of potentially toxic substances (e.g. treatment of tumour necrosis factor with cyclooxygenase inhibitors). However, we must question to what extent the presence or absence of effects are unique to the animal models used. It may also be more relevant in some circumstances to test in an animal whose immune system is disturbed (e.g. zinc deficiency diets, various cancers, old age). Efforts are being made to develop both in-vivo and in-vitro tests in which to compare clinical experiences. While animal models may not predict all such experiences, they may provide information that will give us further insight into the complexities of the immune system as a target organ of toxicity. Joy Cavagnarois a SeniorStaff Scientistat Hazleton LaboratoriesAmerica, Vienna, VA 22075, USA

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