4
The Research Agenda: Implications for Therapeutic Countermeasures to Biological Threats

OVERVIEW

As with vaccines, not only are therapeutics an integral component of our biodefense arsenal, but making it publicly known that we are producing a constant stream of new, innovative antimicrobials would serve as a very strategic form of deterrence. Several issues related to antibiotic, antiviral, antitoxin, and antibody research and development were identified and discussed during this session of the workshop. In light of the plethora of bioterrorist agents that could be used against us, of utmost importance is deciding whether we should focus our efforts on the development of broad-spectrum or agent-specific antimicrobials. For example, one possible antiviral strategy is the development of family-specific antivirals. Increasing evidence suggests that common antiviral targets exist.

Our antibiotic arsenal is limited to only a handful of old antibiotics. Unfortunately, the general confidence in existing antibiotics and the complacency that was associated with infectious diseases in the 1960’s resulted in a lag in producing new classes of antibiotics. There are about twenty-five antibiotics currently in the early phases of clinical development. However, none of these are new classes of antibiotics, and none are broad-spectrum. In fact, there has been only one new class of antibiotic developed in the past two decades, and resistance to it emerged before it came to market. This is alarming given the increasing accessibility of the tools and knowledge needed to develop antibiotic-resistant strains of bioterrorist agents.

There is concern that the situation will become ever worse with the recent FDA changes in clinical trial design requirements. It is expected that the increased cost associated with larger clinical trials will discourage companies from



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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary 4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological Threats OVERVIEW As with vaccines, not only are therapeutics an integral component of our biodefense arsenal, but making it publicly known that we are producing a constant stream of new, innovative antimicrobials would serve as a very strategic form of deterrence. Several issues related to antibiotic, antiviral, antitoxin, and antibody research and development were identified and discussed during this session of the workshop. In light of the plethora of bioterrorist agents that could be used against us, of utmost importance is deciding whether we should focus our efforts on the development of broad-spectrum or agent-specific antimicrobials. For example, one possible antiviral strategy is the development of family-specific antivirals. Increasing evidence suggests that common antiviral targets exist. Our antibiotic arsenal is limited to only a handful of old antibiotics. Unfortunately, the general confidence in existing antibiotics and the complacency that was associated with infectious diseases in the 1960’s resulted in a lag in producing new classes of antibiotics. There are about twenty-five antibiotics currently in the early phases of clinical development. However, none of these are new classes of antibiotics, and none are broad-spectrum. In fact, there has been only one new class of antibiotic developed in the past two decades, and resistance to it emerged before it came to market. This is alarming given the increasing accessibility of the tools and knowledge needed to develop antibiotic-resistant strains of bioterrorist agents. There is concern that the situation will become ever worse with the recent FDA changes in clinical trial design requirements. It is expected that the increased cost associated with larger clinical trials will discourage companies from

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary pursuing new antibiotic development, especially when there are other therapeutic interests vying for the same resources. Although the FDA attempts to balance the demands of a public health emergency with their needs as a regulatory agency and offers several accelerated routes to licensure, including the proposed animal efficacy rule, there is still a sense that these regulatory processes need to be streamlined even more in order to accelerate drug discovery and development efforts and provide more incentive for the pharmaceutical industry. Our antiviral amamentarium is even more limited than our antibiotic arsenal. Cidofovir, for example, can only be administered intravenously and is highly nephrotoxic, making it unsuitable for mass casualty use. The clinical utility of ribavirin as an antiviral drug strategy for bioterrorist agents remains unclear. Antibiotics and antivirals are not the only potential therapeutic defense against bioterrorist agents. Basic research on the anthrax toxin system has led to some exciting prospects for antitoxin targeting. The most promising are the dominant negative inhibitors (DNIs), mutant forms of the protective antigen that block translocation of the virulence factors across the plasma membrane. Currently, DNIs are a very late stage product. If they can be proven efficacious in infected animal models, they could be produced and deployed very rapidly. There are several other approaches in much earlier stages of development. The use of recombinant monoclonal antibodies is another option which has been implicated for use against several biothreat agents, including anthrax, smallpox, and botulinum neurotoxins. For example, recent research has shown that a small mixture of recombinant monoclonal antibodies provides complete protection in mice against botulinum neurotoxin type A. Antibodies have several advantages as a bioweapons defense tool: they have been shown in multiple studies to be safe; ten have already been approved by the FDA and seventy more are in clinical trials, so their route to licensure is known; the technology and knowledge needed for production are readily available; their overall course through the discovery and approval process is much quicker than those of other types of therapeutics; and the technology platform used to produce and manufacture antibodies could be applicable to multiple agents. Finally, scaling up research and development of all of these various potential therapeutics will require an evaluation of the availability of and need for additional laboratory capacity. In particular, there are a very limited number of BSL-3 and 4 labs where nonhuman primate studies can be conducted. Hope was expressed that in the future the FDA will accept rodent data in lieu of nonhuman primate data, if it can be demonstrated that the efficacy is the same in rodents as in nonhuman primates. This would allow for more testing in a greater number of facilities, although it would still require at least BSL-3 capability. Aerosol testing requires BSL-4 capabilities, as well as trained, vaccinated personnel.

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary COUNTERMEASURES TO BIOLOGICAL THREATS: THE CHALLENGES OF DRUG DEVELOPMENT Gail H. Cassell,* Ph.D. Vice President, Infectious Diseases Drug Discovery Research and Clinical Investigation Eli Lilly and Company The Problem The diversity of existing biological weapons and the ever increasing possibilities preclude simple therapeutic countermeasures to bioterrorism. Furthermore, response possibilities are rather limited even for known threats. Although there are 13 viruses on the current list of potential biothreats, there is only one indicated antiviral—cidofovir—and it both requires intravenous administration and is highly nephrotoxic. More broadly, there are no truly broad spectrum antivirals, and only a limited number of antivirals for routine pathogens like influenza, herpes, hepatitis-B, and HIV. The situation is somewhat better but still worrisome with respect to antibiotics. There has been only one new class of antibiotics developed in the past two decades, and resistance emerged to this class before it entered the commercial market. This is a clear challenge to developing an armamentarium against biological pathogens. At first glance, the situation with respect to antibiotics currently in clinical development looks encouraging. About 25 antibiotics are in the first 3 stages of development, with several billion dollars devoted to their development. However, there are no new classes being pursued, nor are new broad spectrum antibiotics. Furthermore, most are quinolones, and 50 percent or more of the strains of E. coli in Beijng are resistant to quinolones as are many foodborne pathogens. In addition, quinolones are contraindicated for children, and neither quinolones nor tetracycline are acceptable for pregnant women. The Challenges So it would be mistaken to be sanguine about current antibiotic therapies to counter bioterrorism. Nor can one be optimistic about near-term prospects. Eli Lilly recently conducted a competitive analysis revealing that most of the large pharmaceutical companies, with the possible exception of Pfizer, are reducing their investment in antibiotic development. There are probably several reasons. A decade ago, we looked at new technologies like high throughput screening, combinatorial chemistry, and microarray assays, and anticipated a golden *   The information provided in this paper reflects the professional view of the author and not an official position of Eli Lilly and Company.

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary age of antibiotics. But today we have no new classes of antibiotics as a result of those efforts. It has become painfully apparent that discovering new antibiotics is not as easy as once believed. We have, for example, a plethora of targets. Numerous targets have been found with documented in vivo expression of antigens. But they are not necessarily what are called “drugable targets.” A target can be validated and essential for bacterial viability, but if there is not a chemical entity that will penetrate the bacterial cell wall and inhibit growth, you don’t have a real target. In addition, the chemical entity must be safe and not highly toxic. There is a 90 percent failure rate from the discovery of a target to the launch of a new antibiotic. This lack of success has likely damped further spending in this area. Moreover, there has been increased investment in antivirals. And, with the sequencing of the human genome, competition for resources within pharmaceutical companies has turned to other therapeutic areas where there are tremendous opportunities and great unmet medical needs with bigger market opportunities. In fact, infectious diseases, specifically those requiring antibiotic therapy, do not fare too well in financial analyses. Conclusion In short, our antibiotic armamentarium is limited, there is growing concern about an increasing number of potential new weapons, and there has been a marked increase in resistance to existing antibiotics. It seems clear that no public health response to bioterrorism is likely to prove effective without addressing the overall problem of antimicrobial resistance and the challenges of drug discovery and development. Finally, the best deterrent against the use of a biological weapon of mass destruction may be a constant stream of new, innovative antibiotics and antivirals. Knowledge of such commitment and successful developments would surely dissuade the efforts of our enemies in such an arena. THE FDA AND THE END OF ANTIBIOTICS David M. Shlaes, M.D., Ph.D. Vice President for Infectious Diseases Wyeth-Ayerst Research Robert C. Moellering Jr., M.D. Physician-in-Chief, Beth Israel Deaconess Medical Center Herrman Blumgart Professor of Medicine Harvard Medical School Antibacterial research has, for almost two decades now, been the “Cinderella” area in the pharmaceutical industry. The market for these products, used to

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary TABLE 4-1 Cure Rate Delta 90% 10% 80–89% 15% < 80% 20% treat acute, not chronic disease, is modest. There are many products available including many generics. For the most part, the market is growing only slowly and, except for the problem of resistance, is largely satisfied. Recently, when presenting proposals for Phase III trial designs for antibacterial compounds to the FDA and European regulatory bodies, a number of companies, both small and large, were told that the designs for equivalence studies had to target a 10% delta for the lower limit of the confidence interval. This requirement, seemingly innocent and technical, threw the pharmaceutical industry into a panic and probably contributed to the recent withdrawals by Lilly and Bristol-Myers-Squibb from the antibacterial discovery business. Why? Most clinical trials leading to approval of antibacterial drugs are based on equivalence studies. The delta statistically defines the boundary for equivalence. In the past, based on the 1992 points to consider document from the FDA (FDA, 1992), a step function for the delta has been used (Table 4-1). These boundaries have meant the study results must show there is a 95% (97.5 % one sided) probability that true cure rate for the new drug is not more than 10 to 20 % lower than the cure rate for the approved drug. In most reasonable sized studies the new drug has had to be as good as or better than the old one to be successful. The European regulatory authorities and the FDA are now suggesting that a 10% delta be used routinely in drug development (FDA, 2001b), and the FDA has now “disclaimed” the old step function on their web site (FDA, 2001a). It is not completely clear upon what data this suggestion is based, other than purely statistical considerations. In fact, a quick calculation will show that two independent trials successful at a 15% delta would result in approving a drug inferior at the 10% delta only 2% of the time. The concern that the FDA has expressed is over something called “biocreep.” In this concept, a slightly inferior experimental drug becomes the comparator for the next generation of compounds and so on until the experimental drugs of the future asymptotically approach the efficacy of placebo. However, one must wonder whether, for serious infections, this is any more than a theoretical concern, especially when most recent approvals (Synercid, Zyvox) have been based on comparison with standard therapy using older agents. One of the regulatory agency’s best weapons against biocreep is their control over the choice of comparators in clinical trials.

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary The deltas used in the step-function of the 1992 points to consider document were chosen, not based on scientific reasoning, but based on the size of trial that would be required given the cure rate (Pharmaceutical Research and Manufacturers of America [PhRMA], personal communication). The trial size required is very sensitive to efficacy rate, evaluability, b error (power) and the delta. For typical trials with an injectable antibiotic, the patient numbers under various assumptions are shown in Table 4-2. The increased numbers have implications for the ability to run a trial in a reasonable length of time, time of availability of the new drug to patients and physicians, time to market and overall costs of development. For example, Bristol-Myers-Squibb was told that they would be required to run a trial in acute bacterial meningitis at a 10% delta requiring enrolling over 700 patients (Roger Echols, BMS, personal communication). That would be the largest meningitis trial ever done, require about five years to accomplish this enrollment and would require enlisting over 90% of the patients from outside the United States. They declined based on the impracticality of the design and the fact that in the later years of the study, it was not clear that their comparator would still be considered the standard of care in the medical community. Similar concerns exist regarding our ability to run trials at a 10% delta for infections caused by resistant bacteria like vancomycin-resistant enterococci. TABLE 4-2 Number of patients for each indication with a one-sided 97.5% CI (assumes 75% evaluability) Indication Cure Rate 90% Power 10% delta 90% Power 15% delta A 85%     Number of Studies: 2   1532 688 B 80%     Number of Studies: 2   2248 1000 C 70%     Number of Studies: 2   2948 1316 D 65%     Number of Studies: 1   1598 710 Related to indication C       TOTAL   8326 3714     80% Power 10% delta 80% Power 15% delta TOTAL   6226 2770

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary Table 4-2 also shows the increase in numbers required if a 10% delta is required for all indications. Costs of a Phase III trial are directly related to the number of patients enrolled. Therefore, in the scenario above, the costs increase more than 100% going from a 15% to a 10% delta design and much more if one was starting at the old step function plan. This can be ameliorated to some extent by decreasing the b power to 80% from 90%. However, doing that results in a 32% risk of falsely concluding that the experimental compound is inferior to the comparator—a risk not acceptable for most companies. One might argue that large pharmaceutical companies can easily absorb these costs and, that if they want to sell a product, they should do so. However, just as in government agencies like NIH, proposed research in the pharmaceutical industry is subject to prioritization. In the case of the industry, business considerations play a large role in the process. Therefore, in most companies, programs with modest potential markets and large costs are automatically deprioritized unless there is some other, overriding strategic issue to be considered. Thus, one unintended result of promulgating these guidelines will be a decrease in the number of companies carrying out antibacterial research as was seen in the late 1980s and is occurring again now. PhRMA has suggested a number of alternate approaches to the FDA and the industry is more than willing to work with FDA, IDSA and other interested parties to address their concerns regarding clinical trial design in antibacterial development. However, the attempt by regulatory authorities to implement an across-the-board requirement for 10% delta trial designs has already wreaked irreparable damage to our ability to provide a reliable pipeline of new antibiotics for serious infections. We hope that the advisory committee of the FDA will understand these concerns and act appropriately. We would also ask that the European regulatory authorities reconsider their stance for the same reasons. THE ROLE OF ANTIVIRALS IN RESPONDING TO BIOLOGICAL THREATS C.J. Peters, M.D. Professor, Departments of Microbiology and Immunology and Pathology University of Texas Medical Branch at Galveston Many available viruses could be used to cause harm to others under many different scenarios. It is important to try to focus on some specific priorities to attempt to limit the problem to a tractable scope, yield maximum benefit in the short term and develop more comprehensive goals that we can hope to attain in the longer term. It is important to consider vaccines and drugs together as part of an overall antiviral strategy.

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary The Threat To ameliorate the adverse consequences of a bioterrorist (BT) attack, the scenario is everything and, equally, the scenario for every possible attack is unknowable. However, aerosol attacks have the greatest potential to cause mass casualties and also lend themselves to stealthy application (see chapter on aerosols). Only a limited number of viruses are known that grow to high titer and are stable and infectious in aerosols and thus lend themselves to this form of attack. Tables one and two list a number of human-pathogenic viruses that have often been considered as aerosol threats (Alibek, 2001; Ferguson, 1999; Alibek, 1998). The viral hemorrhagic fevers (VHF) (Table 4-3) are among the most dangerous (Peters, 2000). The VHF agents are lipid-enveloped RNA viruses with a genome size of around 1–2 million Daltons belonging to four different virus families (Peters and Zaki, 1999). They are zoonotic viruses and all are aerosol-infectious, either shown through formal studies in the laboratory and/or by the observation of frequent “unexplained” laboratory infections. As might be expected from their taxonomic diversity, they differ in individual strategies for maintenance in nature and in their pathogenesis of human disease. Several of these viruses were developed by the Soviets for use as strategic weapons for mass destruction, including Machupo, Lassa, Rift Valley fever, and Marburg viruses. At this time, technical difficulties may limit the prospects for weaponization of Crimean Congo HF and the hantaviruses, but like most problems, these are subject to solution. TABLE 4-3 Viral hemorrhagic fevers commonly mentioned in association with biological warfare or biological terrorism PRIMARY HEMORRHAGIC FEVERS (HF) ARENAVIRIDAE     Lassa Fever     South American HF (Argentine, Bolivian, etc) BUNYAVIRIDAE     Phlebovirus Rift Valley fever   Nairovirus Crimean Congo HF   Hantavirus HF with renal syndrome     Hantavirus pulmonary syndrome FILOVIRUS       Marburg HF     Ebola HF   FLAVIVIRUS     Yellow fever     Tick-borne HF (Kyasanur forest disease, Omsk, etc)

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary TABLE 4-4 Other viruses suggested to have potential in biological warfare or biological terrorism   Smallpox   Monkeypox   Nipah   “Viral encephalitides”   -Venezuelan equine encephalitis   -Other alphaviruses   -Tick-borne encephalitis virus   “Eradicated”: polio and measles   Influenza A: 1918 strain, Hong Kong H5N1, others The other candidate aerosol infectious viruses (Table 4-4) also are largely zoonotic with the exception of the very important BT agent, smallpox. The zoonotic agents can spread to close family contacts and to health care staff, but continuing chains of transmission are not a threat. Smallpox is quite different because its natural history is one of continuous inter-human transmission. The lack of a reservoir outside human-kind, the moderately higher transmissibility, and the existence of a highly effective vaccine that can be efficiently delivered combined to allow the eradication of the virus as a cause of natural disease. Monkeypox is another poxvirus which shares high aerosol stability and infectivity with smallpox but which has a much lower interhuman transmissibility and case fatality (Jezek and Fenner, 1988). Nipah virus is representative of a newly proposed genus of a very well-established family, Paramyxoviridae. Before the other human pathogen in this genus, Hendra virus, emerged in Australia in 1995 (Murray et al., 1998) the existence of the genus was unsuspected, but now its members are seen to be widely distributed among flying foxes (Macrochiroptera) and at least these two members have the potential to cross over into domestic animal species and also infect humans (Chua et al., 2000). Their future behavior is unpredictable, but analysis of the state of emerging infections in the world and the recent recognition of two serious episodes in the recent past suggest they are highly dangerous (Peters, 2001). The spread of Nipah virus among swine in Malaysia was progressive and could have resulted in an enormous economic, human, and political disruption if it had extended into Thailand and China. Stopping the march of the virus entailed destruction of more than one million pigs after it caused 265 human cases with a 40% case fatality and significant residual morbidity among survivors (Parashar, et al., 2000). The aerosol properties of this virus are unknown, but it appears to spread among pigs by small-particle aerosols or droplets. The great majority of human cases had close contact with living swine, but the occurrence of a small number of cases in persons living near pig farms but without actual contact with pigs also raises the question of aerosol infection of

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary humans. Where will the next unexpected virus come from and what taxon will it belong to? Several alphaviruses causing viral encephalitis have been regarded as potential biological agents. The best established is VEE which was weaponized by the US and the Soviets. VEE also has the potential to establish itself as an endemic mosquito-borne disease in North America based on past epidemiological evidence (Weaver, 1997). Other less well-established threats will undoubtedly become more important in the future. These could even include common viruses such as polio and measles should they be eradicated (previous Forum). The problem is exemplified by smallpox virus. When vaccine was widely used and substantial immunity was maintained in the population, the threat of smallpox epidemics arising from isolated cases was less than it is today. Polio epidemics would be highly disruptive, but measles would probably be the worst threat. For example, measles epidemics during the U.S. Civil War were among the greatest impediments to expanding armies because of their heavy impact in both morbidity and mortality among recruits and staging centers for the two armies (Steiner, 1968). Influenza virus is infectious by aerosols and is capable of propagating efficiently among humans by aerosols even though other routes are also important (Kilbourne, 1975). In general, the impact of aerosol spread on control is huge. In the US we can deal effectively with fecal-oral, fomite, and large droplet transmission through our general level of sanitation or by using simple mask, eye protection, and hand washing measures. However, aerosols must be controlled with efficient filters on breathing air and the filters must be well-fitting and in use during the time of risk; any society would have difficulty dealing with an aerosol spread epidemic. The ability to produce recombinant influenza strains from natural strains or using synthesized genes is an accomplished feat (Neumann et al., 1999). The prediction of which viruses will be highly transmissible and lethal will come in the near future. Whether such viruses would be produced and could actually spread among human populations is another matter. The Solutions Biological warfare (BW) and terrorism present different challenges. BW scenarios could involve the use of biological weapons on the battlefield and would target a selected group which could therefore be immunized using their training schedules, different operational missions, and on-going military service to select and prioritize. A strategic biological warfare effort could also be directed against civilian populations, as was envisaged by the Soviets, and this scenario could possibly be countered through vaccine protection. However, a civilian population facing an ill-defined bioterrorist threat would be much harder to protect by immunization because of the problems of vaccine coverage and the inevitable adverse

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary events associated with all vaccines. The example of smallpox vaccine would be illustrative. Use of this vaccine in the defined and limited group of military persons in basic training was accomplished in the 1970’s with little morbidity among the vaccine recipients and little risk to non-military. If this program had been expanded to the entire civilian population with the consequent adverse effects (Koplan and Hicks, 1974), there would have been a huge backlash. The “swine flu “ vaccination program in 1976 provides an excellent example of the likely outcome (Neustadt and Fineberg, 1978). Widespread vaccination against an ill-defined threat would be associated with the adverse effects that will ensue from any vaccine and would bring the effort would be brought to a stop with serious negative medical and political consequences. Even a perfect vaccine would be tarred by the unfortunate events that occur by chance in a large, healthy population receiving no treatment at all. Thus, vaccines against viruses could be very useful but would likely only be used on a large scale in civilian populations in the face of a clear and present danger. Nevertheless, they are an important part of an over-all antiviral strategy because they provide protection for particular groups, including those studying the virus in the laboratory, antiviral drug developers, and those working with the virus in regions where it occurs naturally. Furthermore, availability of attenuated strains can be essential to expanding research activities, including antiviral drug development, to laboratories with lower levels of containment. Antiviral drugs could provide protection, subject to all the same problems of stockpiling and delivery as antibacterial agents now considered for use against such threats such as anthrax, plague, and tularemia. It is now clear that, with an adequate molecular and structural biological base, drug development capability, and financing, effective antivirals can be discovered and indeed even designed. Antivirals inhibiting enzymes active in nucleic acid synthesis or protein cleavage have been highly effective although with some price in toxicity. Other targets exist which might be less closely allied to host cell constituents and which might provide a greater therapeutic index, just as drugs such as penicillin inhibit gram positive bacterial cell wall synthesis, which has no counterpart target in mammalian cells. Monoclonal antibody strategies are also possible, but many of the protective targets are the neutralizing antibody epitopes, which are commonly virus specific. This can be overcome with multi-virus cocktails, but this in turn demands development and production efforts of multiple antibodies. Fortunately, the neutralizing epitopes are highly conserved and usually linked to virulence on the individual viruses of interest. The success of any antiviral strategy—drugs (including antibodies), vaccines, or combinations—will depend critically on the context in which it is used. Stockpiles of remedies will be essential, as well as expectant use of some vaccines. Equally important will be plans to deliver emergency vaccines or drugs in a timely fashion where they are needed. And most importantly, it will be incumbent on the physician to recognize the diseases and

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary play (Marks and Marks, 1996 ). For phage display, repertoires of antibody genes are cloned into a bacterial phage vector where the antibodies are displayed on the surface of the bacteriophage fused to one of the phage coat proteins (Marks et al., 1991). While it is not technically possible to display full length IgG antibodies on phage, it is possible to display smaller single chain Fv (scFv) or Fab antibody fragments. Such antibody fragments contain the antigen recognition of the IgG (the variable domains), but lack the constant regions. To perform phage display, repertoires of antibody heavy and light chain variable domain genes are assembled and cloned into a phage vector to create libraries of scFv or Fabs displayed on the phage surface. The source of the variable region genes can be any species, including immunized humans. Once phage libraries are constructed, binding phage antibodies can be isolated from non-binding phage by a variety of types of affinity chromatography. Binding phage antibodies can be detected by ELISA, and the antibodies characterized with respect to affinity, epitope recognized, sequence, and biologic activity. Given the high transformation efficiency of bacteria, it is possible to make libraries of millions to billions of different antibodies, allowing immortalization of the entire immune response to an antigen. As a result, hundreds to thousands of antibodies are generated, allowing isolation of high affinity antibodies to rare epitopes. In contrast, generation of hybridomas captures only a fraction of the immune response, due to the inefficiency of the fusion process. Other advantages of antibody phage display include the ability to make antibodies from immunized humans and to engineer antibody affinities to values not achievable by hybridoma technology (Schier et al., 1996). Neutralization of Botulinum Neurotoxin by Monoclonal and Oligoclonal Antibody Recombinant mAb could provide an unlimited supply of antitoxin free of infectious disease risk and not requiring a source of human donors for plasmaphoresis. Such mAb must be of high potency in order to provide an adequate number of doses at reasonable cost. In some instances, the potency of polyclonal antibody can be recapitulated in a single mAb (Lang et al., 1993). In the case of BoNT, potent neutralizing mAb have yet to be produced: single mAb neutralizing at most 10 to 100 times the 50% lethal dose (LD50) of toxin in mice (Pless et al, 2001; Hallis et al., 1993). To generate mAb capable of neutralizing BoNT serotype A (BoNT/A), we generated scFv phage antibody libraries from immunized humans, mice, and mice transgenic for the human immunoglobulin locus (Amersdorfer et al., 1997; Amersdorfer et al., in press). After screening more than 100 unique mAb from these libraries, three groups of scFv were identified which bound non-overlapping epitopes on the BoNT/A binding domain (HC) and which neutralized toxin in vitro (prolonged the time to neuroparalysis in a murine hemidia-

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary phragm model) (Amersdorfer et al., 1997; Amersdorfer et al., in press). In vitro toxin neutralization increased significantly when two scFv binding non-overlapping epitopes were combined. The small size of the scFv, however, precluded study of in vivo toxin neutralization, due to the rapid clearance of the 25 kDa scFv from serum (Colcher et al., 1990). To evaluate in vivo BoNT neutralization, IgG were constructed from the variable domain genes of three BoNT/A scFv that neutralized toxin in vitro. No single IgG significantly neutralized toxin in vivo, but combining mAb led to potent toxin neutralization. The most potent mAb pair protected mice challenged with 1500 LD50s of toxin, while combining all three mAb protected mice challenged with 20,000 LD50s of toxin (per 50 mg of antibody administered) (Nowakowski et al., 2002). The potency of the three antibody combination (oligoclonal Ab) was formally titered using the standard mouse neutralization bioassay and was determined to be 45 International Units/mg of Ab. One International Unit (IU) neutralizes 10,000 LD50s of BoNT/A toxin, yielding a potency of 450,000 LD50s /mg of Ab. This is 90 times more potent than the hyperimmune human globulin used to treat infant botulism (Arnon, 1993) and approaches the potency of hyperimmune mono-serotype horse type A anti-toxin (Sheridan et al., 2001). The increase in potency appears to result primarily from a large increase in the affinity of the oligoclonal Ab for toxin compared to the individual mAb (Nowakowski et al., 2002), and also to greater blockade of the toxin surface which interacts with cellular receptors (Mullaney et al., 2001). Such mechanisms may be generally applicable to many antigens in solution, suggesting that oligoclonal Ab may offer a general route to more potent antigen neutralization than mAb. The precise contribution of these two mechanisms to the increase in potency is unknown. It is also unknown as to whether engineering the affinity of one of the mAbs to a value approaching that of the oligoclonal Ab would yield a similar increase in potency as combining mAbs. Conclusions, Obstacles, and Future Research Needs Recombinant oligoclonal Ab offers a safe and unlimited supply of drug for prevention and treatment of BoNT/A intoxication. With an elimination half-life of up to 4 weeks, Ab would provide months of protection against toxin. Since the current oligoclonal Ab consists of either chimeric or human IgG, production could be immediately scaled to produce a stockpile of safe anti-toxin. Alternatively, we have already replaced the chimeric S25 IgG with a fully human IgG and increased potency of the oligoclonal Ab more than 2 fold. Work is ongoing to replace chimeric C25 with a fully human homologue. Chimeric, humanized, and human mAb represent an increasingly important class of therapeutic agents whose means of production are known. The high potency of the oligoclonal Ab makes it possible to manufacture millions of doses of antitoxin from a single manufacturing facility which could be stockpiled for future use. Ten mAb have

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary been approved by the FDA for human therapy and more then 70 other Mab therapeutics are in clinical trials (Reichert, 2001). As a result, the process of scaling production and manufacturing, as well as the necessary toxicology and clinical safety testing requirements are known. This should result in a rapid development timeline, especially compared to vaccines or small molecule drugs. The major challenges and obstacles to development are FDA regulatory issues related to combining mAbs and a predicted worldwide shortage of IgG manufacturing capacity (Reichert, 2001). Oligoclonal Ab would be applicable to the other BoNT toxin serotypes and these antibodies should be generated as rapidly as possible. Oligoclonal antibody may also be able to potently neutralize other class A agents as well. Anthrax toxicity is toxin mediated, and polyclonal Ab has been shown to be protective for this agent (Little et al., 1997; Beedham et al., 2001). Vaccinia immunoglobulin can be used to prevent or treat smallpox or complications arising from vaccination of immunocompromised hosts (Feery, 1976). Ab may also be useful for plague and the hemorrhagic fevers (Hill et al., 1997; Wilson et al., 2000). Given the threats posed by these agents, rapid generation and evaluation of oligoclonal Ab for their neutralization is warranted. MEETING THE REGULATORY AND PRODUCT DEVELOPMENT CHALLENGES TO ADDRESS TERRORISM Andrea Meyerhoff,* M.D. Director, Anti-terrorism Programs Office of the Commissioner, Food and Drug Administration FDA’s mandate in anti-terrorism warrants a balance between its requirements as a regulatory agency and the demands of a public health emergency. We attempt to achieve this balance by facilitating the availability of safe, effective drugs, vaccines, and medical devices in a manner that is consistent with our legal responsibilities as a regulatory agency. Organization of FDA Anti-Terrorism Programs The FDA is divided into five centers which are organized based on the type of products that are regulated. Three of these centers regulate products that deal with medical care: CDER (Center for Drugs); CBER (Center for Biologics), which regulates vaccines; and CDRH (Center for Devices and Radiation Health), which regulates a range of medical devices from diagnostic assays to mechanical ventilators. *   This statement reflects the professional view of the author and should not be construed as an official position of the Food and Drug Administration.

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary The Director of Anti-terrorism Programs is housed in the Office of the Commissioner, which is not housed in any particular center but rather coordinates across these centers. There is a designated anti-terrorism point of contact (POC) within each center and with whom the director liaisons on antiterrorism issues. For many products under development, there is already a relationship established with the appropriate regulating center. New products that are seeking regulatory guidance and old products that may have an anti-terrorism application are often routed through the Anti-terrorism Programs first and then passed on to the appropriate POC. Anti-terrorism Programs works with the POC to coordinate all efforts that are relevant to each particular stage of product development. Existing Regulatory Mechanisms for Enhanced Product Availability There are several existing regulatory mechanisms that can be invoked to address issues of anti-terrorism product availability. They apply to a number of different phases of product development, from the early pre-IND (i.e., before the drug is introduced into human trials) to the review of the NDA (i.e., new drug application for marketing approval): Pre-IND meeting is an attempt to begin early dialogue between the sponsor and the review division and provide regulatory guidance in preparing IND (investigational new drug) applications. IND applications include a set of data that are shown to the agency before the product is used for the first time in humans. The entire body of data are reviewed by all of the various disciplines that are brought to bear at that stage, and missing pieces of data are identified. Pre-IND meetings are regarded as resources for developers. There is no set period in the pre-IND phase when this meeting must occur. Some sponsors approach the FDA quite early; others meet immediately before they submit the IND just to make sure that everything is okay. IND regulations refer to the set of regulations that determine how a product will be used when it is initially introduced into a human population. IND regulations may also be viewed as a mechanism for making an investigational product available. IND regulations have three basic components: an informed consent form; review of the protocols for planned use by an institutional review board; and a plan for the collection of safety and efficacy data from the human population in which the product is going to be used. “Streamlined” IND is not an official regulatory term, but it serves the purpose of addressing the requirements of the IND regulations while simultaneously making a product available to a large population in an emergency setting. Streamlined INDs are in place for both biologic and drug products, and the template can be used for other products as well if the need should arise. The animal efficacy rule was proposed and published in the Federal Register in October 1999. It is intended to apply when a disease cannot be studied in

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary humans, that is when the disease is either very rare or it would be unethical to introduce the disease into a human population. Clearly, diseases due to biological agents of intentional use would fit into this category. This rule provides the framework for which efficacy data could be derived from an animal model of disease and is intended to address efficacy only. The safety of the drug still needs to be studied in the human population. The rule is based on the use of a scientifically valid animal model and generally requires the use of two species. In cases where a well-established species is already recognized as a scientifically valid model for disease, it would be decided on a case-by-case basis whether efficacy data is needed from a second species as well. Currently, this rule is still only proposed and has not been used (the approval of ciprofloxacin for anthrax invoked accelerated approval, not the animal rule); finalization is anticipated within the next few months. Accelerated approval (sometimes referred to as subpart H regulation) refers to a set of regulations that permit the use of a surrogate marker for the purposes of demonstrating efficacy of a product if the product is considered reasonably likely to provide an improvement in mortality or serious morbidity. Still, postmarketing data would need to be collected to verify the surrogate. This is the regulatory approach that was taken for ciprofloxacin for anthrax which was initially approved for human use in the mid-1980s, so there was already a fairly well-developed set of human pharmacokinetic data and a very large safety database. The surrogates in this case were human serum levels of ciprofloxacin which have been shown to be associated with improved survival in monkeys that have been exposed to aerosolized B. anthracis spores. Serum level in humans have been shown to reach or exceed weight-adjusted levels in monkeys. The labeled regimen for post-exposure inhalational anthrax is a sixty-day dosing period. Safety databases of patients who received the drug for more than sixty days, patients who received the drug for sixty days, patients who received it for less fewer than sixty days, and patients who received other antibiotics all show similar adverse event rates. GI events are the most common, with a slightly incidence of higher abdominal pain and rash in the sixty-day group. However, patients who received the drug for sixty days showed no previously unidentified adverse events associated with the shorter, more usual seven to fourteen day dosing periods. There is also a substantial pediatric safety database which supports the approval of ciprofloxacin for post-exposure inhalational anthrax indication in pediatric patients. Priority review is a request that is made by the applicant at the time of NDA filing. It is generally used for products that are considered to have special public health significance and results in a review process that is shortened to six months rather than the usual ten or twelve. In addition to accelerated approval, ciprofloxacin for anthrax also received priority review.

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary Recent FDA Anti-Terrorism Initiatives: Drug Development Recent FDA antiterrorism-specific initiatives, most of which involve anthrax, include: In early November, 2001, the FDA published a Federal Register notice recognizing that doxycycline and penicillin are also approved for anthrax. The Federal Register notice was published because product labels do not contain specific dosing information for post-exposure inhalational anthrax, even though scientific data support this labeling. The Federal Register notice states this, provides the dosing recommendations, and invites applications from manufacturers of these drugs to request labeling supplements. This was done as a way to expand the options of products available to manage what was clearly a growing population of people who had been exposed to aerosolized spores of B. anthracis. Because of potential side effects, drug intolerance, other medications, and any of a number of other reasons why people cannot take a particular class of drugs, having more available options expands our ability to manage large populations of exposed individuals. There are a number of ongoing efforts among several government agencies to provide regulatory guidance for the development of animal models to be used in the evaluation of drugs specifically for diseases related to bioterrorism. There has been much ongoing collaboration with DOD laboratories and the NIH to establish guidelines and goals for studying these products in animal models. The FDA has been considering other products besides antimicrobials that could be made available for the treatment of clinically apparent inhalational anthrax. REGULATION AND PRODUCTION OF RECOMBINANT HUMAN ANTIBODIES Kathryn E. Stein,* Ph.D. Director, Division of Monoclonal Antibodies Center for Biologics Evaluation and Research Food and Drug Administration The FDA is fully prepared to deal with the issue of monclonal antibody cocktails and, in fact, has had relevant policies in place since 1994. At that time, it was anticipated that manufacturers might develop antibody cocktails directed at either different epitopes on a particular antigen or different antigens on a particular organism. From a safety and efficacy perspective, these policies consider *   This statement reflects the professional view of the author and should not be construed as an official position of the Food and Drug Administration.

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Biological Threats and Terrorism: Assessing the Science and Response Capabilities - Workshop Summary cocktails as single products. However, there must be a rationale for the use of each component in the cocktail as well as a means for determining the dose of each component. These data could come from preclinical animal models, for example, or in vitro neutralization or other tests. The real question is, what kinds of antibodies should be included in the cocktails? For example, because the murine Fc region is the most immunogenic part of a monoclonal, both chimeric and humanized antibodies, with human Fc regions, have been engineered and shown to exhibit much less immunogenicity in humans than whole murine antibodies. With regards to how antibodies are produced, there is some concern that phage display may create combinations of heavy and light chain genes that would raise unusual issues regarding immunogenicity. One could envision a mixed antibacterial and antiviral cocktail comprised of antibodies to a diverse assortment of potential bioterrorist agents. However, more research is needed to identify protective factors and determine which virulence factors the antibodies should target. (See Marks for further discussion on antibody options.) There are many antibodies currently being researched in academe that could be developed into cocktails. There needs to be greater partnerships among academe, government, and industry in order to bring the intellectual property to the antibody engineers so that these products can be developed. There are ways to lyophilize monoclonal antibodies such that the cocktails could be stable at room temperature and on the shelf of every emergency room, although formulation needs to be further studied. The FDA is willing to consider any proposed products. Limitations on monoclonal cocktails pertain mostly to production. The worldwide capacity for mammalian cell culture has reached its maximum. In order to increase production to build a stockpile for prophylaxis or treatment in the event of a large-scale bioterrorist attack, we must either build more manufacturing facilities or buy or renovate already existing facilities. Such large-scale production will likely require government assistance and funding. REFERENCES D. Shlaes: Food and Drug Administration (FDA). Division of Anti-Infective Drug Products. Points to Consider, Clinical Development and Labeling of Anti-Infective Drug Products. 1992. Online. www.fda.gov/cder/present/anti-infective798/biostats/tsld005.htm Food and Drug Administration (FDA). Division of Anti-Infective Drug Products. Points to Consider, Clinical Development and Labeling of Anti-Infective Drug Products, Disclaimer of 1992 Points to Consider Document. March 08, 2001a. Online. www.fda.gov/cder/guidance/ptc.htm. Food and Drug Administration (FDA). International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, Guideline, Choice of Control Group and Related Issues in Clinical Trials. May 2001b. Online. www.fda.gov/cder/guidance/4155fnl.htm

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