				Biopharmaceutical companies are developing clever technologies for plant made pharmaceuticals (PMPs). When will the promise for the future turn into reality?

Transgenic plants will eventually be adopted as manufacturing vehicles for biopharmaceutical products. The timing is dictated by the need to overcome several hurdles. The promise of PMPs, like most new technologies, precedes the practical reality. 

The two common arguments in favor of PMPs are reduction in manufactured costs and lack of human-infectious viral contaminants. Novel mammalian-cell expression systems and improved processes are pushing cell-culture productivity into the gram per liter range on a consistent basis. A ten-fold increase in expression level effectively turns a 1,000 L bioreactor into a 10,000 L bioreactor.  

Freedom from viral contaminants is a potentially powerful advantage. However, for PMPs to be accepted by the market, industry needs to focus on the hurdles and not the advantages.  

The first and foremost hurdle, at least among experienced biomanufacturers, is the regulatory unknown (also known as the fear of "being first"). There are draft FDA/USDA and EMEA documents focused on PMPs, and dozens of products in development.  The major achievement for transgenic systems, whether animal or plant, that paves the regulatory pathway for all will be approval of the first product. This could happen as early as March 2005, with EMEA approval of GTC Biotherapeutics' ATryn, a recombinant human thrombin expressed in the milk of goats. 

Secondly, it is not clear that total manufacturing costs will be reduced multi-fold, as often claimed. Capital requirements for the upstream part of a kilogram-level biomanufacturing facility will be reduced from the $100+ million range to perhaps the $10+ million range. A significant portion of the manufacturing costs, though, ranging from 30-60% depending upon the product, resides in downstream purification — a part of the biomanufacturing process not likely to be greatly influenced by the type of feed stream. 

Thirdly, and perhaps most importantly, there are issues regarding the public's (and industry's) perception of growing transgenic plants in open, unconfined spaces. Concern with the likelihood for gene flow among transgenic plants and their non-transgenic neighbors, along with the potential for transgenic "contaminants" appearing in the food chain, have led to strict recommendations from the USDA on buffer zones between transgenic and neighboring crops and, in some instances, moving them to locations far removed from traditional growing areas. Perhaps elimination of the gene flow issue will only be achieved by isolation in totally confined spaces such as greenhouses or underground mines. Mines, if comparable in capital and operational costs to greenhouses, might offer the best bet, since this involves a completely controlled environment — free of environmental pests and weather and seasonal issues — that can actually increase the annual amount of biomass generated per acre. 

Turning the issues into advantages will really drive adoption of PMPs into the mainstream of biomanufacturing. Someone will be "first" with a regulatory approval. Underground growth chambers will achieve economic parity.  

One remarkable potential advantage remains to be fully exploited; namely, the ability to genetically manipulate plants to perform post-translational modifications and, so, to control important structures such as glycans. Already, offending sugar residues (xylose and fucose) can be removed 

. Terminal galactose residues on glycans, typical of Mabs (not natively produced in plants) have been engineered into a variety of plant species. The ability to produce a protein with structures that more closely mimic the human molecule or, perhaps, improve upon the human molecule, will be what ultimately gives PMPs their place in the biomanufacturing sun.

 is vice president, Biotechnology Services at 

, P.O. Box 13341, Research Triangle Park, NC, 919.465.8149, Fax: 919.481.4908. 

Articles

Columns and Departments

Biopharmaceutical companies are developing clever technologies for plant made pharmaceuticals (PMPs). When will the promise for the future turn into reality?

PMPs — If Not Now, When?

The last few months of 2004 were marked by crises likely to affect pharmaceutical and biotech manufacturing for years to come. Safety data indicated that antidepressants may cause problems for young patients that warrant black box warnings, new labeling, and special packaging. Merck pulled its blockbuster COX-2 inhibitor Vioxx off the market because studies showed increased risk of cardiac events from long-term use of the drug. To cap it off, British regulators shut down Chiron's Liverpool vaccine plant due to product contamination, creating a serious shortage of flu vaccine in the US.

Members of Congress pointed fingers at lax FDA oversight, accusing the agency of trying to squelch staff warnings of these safety issues and ignoring signs of trouble at Chiron. Federal investigators sought evidence of unethical manufacturer behavior, as government officials and industry leaders weighed a range of messages emerging from these events:  					

The nation's adverse event reporting and post-marketing surveillance systems are limited and slow to identify looming safety problems.

Sponsors may need to provide much more safety data to bring to market treatments that patients may use for years.

The US vaccine supply system is antiquated, unreliable, and in need of a major overhaul to prevent shortages and inequitable access.

Perhaps regulators and policy makers should rethink efforts over the past decade to accelerate the drug approval process and streamline research requirements.

For some time now, the US health care system has been plagued by shortages of many vaccines for children and unpredictable supplies of influenza vaccines. Observers describe the nation's vaccine supply system as "very fragile" and "unreliable" — characteristics that have escalated with the departure of Wyeth Pharmaceuticals and other companies from flu vaccine production. Recent shortages prompted CDC to ramp up orders for this year, particularly to meet growing demand generated by the government's own flu vaccine campaign. 			

A key element in CDC's efforts to boost supply was enlisting San Francisco-based Chiron to provide over 40 million doses of flu vaccine this fall from its newly acquired Liverpool plant. The plan fell apart in October when British regulatory authorities suspended the facility's license due to contamination problems affecting the entire production run. While the Brits stood to lose less than 20% of their annual flu vaccine supply, an amount that other manufacturers could fill, the shutdown was devastating to the US, which was relying on Chiron for about one-half of its 100 million doses.  

FDA officials rushed to England to see if there was some way to salvage any of the vaccine. Team Biologics investigators had inspected the Liverpool plant in June 2003 and found a number of manufacturing problems that appeared correctable. At that time, there was no evidence of contamination in finished doses, and Chiron said it would fix specific quality control shortcomings. But the plant had a history of manufacturing problems and had changed hands several times in the last five years. Chiron bought the facility from PowderJect Pharmaceuticals in July 2003 and spent $75 million to upgrade the facility — which may have been pouring money down a sinkhole.  

In August 2004, Chiron acknowledged contamination problems with some vaccine lots but assured FDA that it was an isolated situation, easily remedied. In fact, just a few days before the shut-down, Chiron CEO Howard Pien told a Senate committee that it had addressed its contamination issues and would deliver flu vaccine as promised. FDA officials were waiting for a Chiron report when Britain's Medicines and Healthcare Products Regulatory Agency closed the plant for three months because it determined that Chiron could not ensure product quality.  

The Chiron catastrophe spurred public interest in usually arcane production issues such as sterile vial filling and cell-based production. While US public health officials scrambled to find alternative supplies and encouraged prudent use of available doses, the situation spotlighted serious problems with the vaccine supply system. Health officials and policy makers wanted to know why FDA did not address Chiron's problems earlier. They examined why Adventis Pasteur and MedImmune are the only other licensed flu vaccine producers for the huge US market, a situation that limits possibilities for dealing with emergencies.  							

Wyeth exited the field in 2002 after years of losses due to low demand and low prices. Flu vaccine has to be reformulated each year and cannot be stored for future use. In its last production season (2002-3), Wyeth threw away 7 million of 21 million doses. European and other markets are more attractive to vaccine makers because some governments agree to purchase a certain amount of vaccine each year to reduce manufacturers' risk, and companies face fewer lawsuits and liability concerns abroad. Congress recently added vaccines for influenza and hepatitis A to the Vaccine Industry Compensation Program, but more may be needed to boost US vaccine production (see "Building Biodefenses"). While the federal government still is not likely to take over vaccine distribution and production, as some industry critics propose, this latest incident could prompt federal agencies to play a more prominent role in the vaccine market and to examine other ways to spur production:									

Expand government guarantees for vaccine purchase to cover unused doses that manufacturers often have to discard every year.

Provide grants and support for manufacturers adopting modern vaccine production techniques. NIH officials are offering up to $150 million in financial incentives to encourage pharma companies to develop modern flu vaccine production technologies, such as cell-based cultures and DNA engineering. Everyone agrees that it's vital to shift away from today's old-fashioned, egg-based systems, which are slow and difficult to change. 

Reform vaccine oversight and market. A report from the National Vaccine Advisory Committee, which appeared in October as the flu vaccine crisis was emerging, recommends dropping price caps on vaccines for children, improving vaccine coverage by payers and insurers, and seeking greater harmonization in US and foreign vaccine approval requirements to increase supplies in an emergency. 

Meanwhile, Congress and federal agencies will be focusing more on drug safety and quality issues in coming months. FDA and other regulatory authorities are reviewing extensive adverse-event databases to see whether the adverse effects of antidepressants on children show up in adults. Until now, researchers in the US and the UK have not found evidence of increased risk of suicidality in mature patients, but they don't know why there's such a big age-based difference. They want to see if different study designs can clarify these issues. 			

FDA also is scheduling an advisory committee meeting to address the larger question of how much long-term safety data is needed for therapies likely to be used by patients for years. Merck obtained conclusive data on Vioxx's safety problems from a three-year, post-approval study for preventing the recurrence of colorectal polyps. While the Vioxx experience may spur calls for more safety data on emerging therapies in this class, proposals to increase the length of studies and number of patients tested always create a dilemma. Overly extensive test demands can block a new drug from market if costs escalate too much, but new drug approvals based on limited data can subject FDA and marketers to attack if safety problems later emerge. 			 

Although biotech and pharma companies have long sought stronger liability protection for vaccines and drugs, legislation has been stuck on Capitol Hill for years. Congress approved Project BioShield in July only after dropping key liability provisions. Now Senate Judiciary Committee chairman Orrin Hatch (R-Utah) and Sen. Joseph Lieberman (D-Conn) are hoping to gain approval of a BioShield II bill next year that would create more incentives for industry to develop vaccines and other counterterrorism treatments.  			

Without added legal safeguards, manufacturers are likely to remain fairly lukewarm to the BioShield program, leaving the field to small biotech firms. HHS recently awarded $232 million in contracts to companies developing new vaccines against smallpox, plague, and tularemia, but the recipients are fairly small, and two are outside the US. The Senate Judiciary and Health committees held a hearing on BioShield II this fall to build support for tax incentives, intellectual property protections, and liability shields to spur more counterterrorism product development. Some health experts also want to extend BioShield protections to companies that develop antibiotics and general vaccines to protect the nation against a flu pandemic or other health crisis. 

's Washington editor, 7715 Rocton Avenue, Chevy Chase, MD 20815, 301.656.4634. 		

Regulatory Beat: Vaccine Manufacturing and Safety in the Spotlight

Because all protein therapeutics are potentially immunogenic, FDA has developed a risk assessment strategy for novel products in development and for major changes in manufacture and clinical use of licensed products. The first part of this series (

, November 2004) discussed FDA's assessment of critical factors regarding the consequences of immune responses to protein therapeutics. These factors were based on product origin (such as xenobiotic and mammalian cell) and the presence and biological function of endogenous protein counterparts. In this second part, host-specific and product-specific factors that positively or negatively impact immunogenicity are discussed. 

 Consider Host-Specific Factors that Impact Immunogenicity Positively or Negatively.

Immunologic status and competence of the host.

 This is a critical factor in considering the risk of immune responses to therapeutic proteins. The incidence of hypersensitivity responses to specific biological drugs may be strongly affected by treatment of patients with steroids and concomitant treatment with chemotherapeutic agents.

 However, the incidence of hypersensitivity responses to drugs and environmental antigens does not appear to be diminished by intensive chemotherapy, despite the reduction of lymphocytes,

  although formal studies are lacking. However, patients who are undergoing chemotherapy or chemoradiotherapy are at much lower risk of mounting binding and neutralizing-antibody responses. This may be due to the severe depletion of CD4+ T cells which are replenished slowly and are required for development of T-dependent IgG antibody responses.

 For example, 95% of immune competent cancer patients generated neutralizing antibody to GM-CSF, while only 10% of immune-compromised cancer patients did so.

Similarly, 4% of healthy volunteers mounted neutralizing antibody responses against a pegylated, truncated MGDF, while only 0.5% of cancer patients did so.

In view of the relatively high incidence of food allergy directed to plant allergens, consideration should be given to the possibility of engendering hypersensitivity responses in patients treated with products derived from bioengineered plants bearing an allergen to which they are sensitive. Although this is currently a theoretical concern, the agency has anticipated this possibility due to the increasing exploration of plants as substrates for production of vaccines and biological therapeutics and has offered guidance.

 Subcutaneous (SC) administration is known to engender immune responses far more ably than either the intramuscular (IM) or intravenous (IV) routes. This may be due to the rapid egress of the product into tissues containing a high frequency of potent antigen presenting cells (APCs); the potential for product to form or stay in aggregates in the SC space compared to dispersal of aggregates in the high-flow, fluid-rich IV environment; and to the possible depot effect of SC injection. The increased incidence of pure red cell aplasia (PRCA) mediated by neutralizing antibodies to Eprex is a good case in point; it appears that the SC route is necessary, though not sufficient, to generate neutralizing antibody.  

 The effects of dose and frequency are not independent of other factors such as route of administration, product origin, and product-related factors that influence immunogenicity. For example, one would expect that for foreign proteins, frequent administration would enhance immunogenicity, especially when given by a more immunogenic route. In contrast, frequent administration of high doses of protein intravenously induces tolerance to Factor VIII in those patients who are least tolerant. Product-related factors, such as the presence of adjuvant and the type and level of product aggregates, may be crucial in determining whether a given dosing regimen proves immunogenic. 

Level of immune tolerance to endogenous protein.

 The completeness of immunologic tolerance to an endogenous protein determines how easily tolerance to it can be broken by  administration of a therapeutic counterpart. Thus, tolerance in both the T- and B-cell partners required for IgG-antibody production depends on numerous factors, especially the abundance of the protein.

 In this regard, T cells are tolerized at lower levels of soluble self-proteins than B cells,

 due to multiple mechanisms for negative selection of antigen-specific T cells in the thymus, including promiscuous expression of tissue-specific antigens.

B cells have been shown to be anergized (physically present, but functionally inactivated) when a model soluble protein is present at 10

 M but not when the protein is present at 10

 M in a transgenic model of B-cell tolerance.

 These serum levels correlate with the extent of B-cell receptor occupancy by the protein, with some degree of anergy observed at 5% and full anergy at ~45% occupancy of the available surface Ig receptors. Below the 5% threshold, the B-cell repertoire differs little from control mice. Additional factors that impact B-cell anergy and tolerance include the affinity of antibody for protein; the stage of B-cell development at which the B cell is exposed to antigen; antigen format, with cell-surface-presented antigen (arrayed antigen) being highly efficient at deletion of immature B cells; and concomitant signals (for example, costimulation and adjuvant activity).

How do these experimental data pertain to immune responses to therapeutic proteins? The finding that the immune system is less tolerant of low-abundance proteins, such as cytokines and growth factors, compared to high-abundance proteins is consistent with many lines of evidence: the presence of natural autoantibodies to cytokines and growth factors in healthy individuals;

 the development of antibody responses to cytokines during normal immune responses,

 and, finally, the ease with which immune responses can be generated to very-low-abundance endogenous factors by exogenous recombinant therapeutic products. For example, with thrombopoietin (TPO), present at picomolar levels, immune responses were relatively easily generated, in some cases requiring only two doses,

 whereas for albumin, the highest abundance protein, immune responses are difficult to detect.   

 Consider Product-Specific Factors that Impact Positively or Negatively on Immunogenicity.

 Immune responses to foreign proteins are expected and, as discussed above, may be anticipated for some self-proteins. However, the rapidity of development and the strength (titer) and persistence of the response depends on many factors, including previous and ongoing environmental exposure (streptokinase) and the mode of exposure (for example, an antigen introduced through the oral route may tolerize rather than immunize). Moreover, the presence of immunity-provoking factors in the product, such as product aggregates and materials with adjuvant activity, as well as the product's inherent immunomodulatory activity, are critical parameters in the generation of responses to both foreign and native protein. 

 Neodeterminants, such as those created from the fusion of a therapeutic protein with a partner antigen, may generate immune responses. Responses to the neodeterminant may spread to conserved segments of the molecule. As reported, this pathway may be responsible for the neutralization of the GM-CSF-IL-3 fusion molecule (PIXY 321) in which a specific neutralizing-antibody response to the fusion protein predominates over neutralizing responses to either the IL-3 or GM-CSF partners;

 mapping studies, however, were not performed to confirm that the specificity of the response was to neodeterminants resulting from the fusion. For fusion molecules in which both partners are self proteins, studies to define the antigenic site of antibody responses are highly recommended.  Fusion proteins involving a foreign protein and self protein are of particular concern because of the capacity of the foreign protein to act as a "helper" determinant, efficiently eliciting T-cell help to generate a response to the self-protein partner. 

 Glycosylation may strongly modulate immunogenicity of the proteins to which they are attached, and the effects may be mediated via a number of possible pathways: immune responses may be generated directly to carbohydrate moieties consisting of unique mammalian xenogeneic sugars,

 Such foreign sugars may trigger innate immune responses through interaction with Toll-like receptors (such as TLR-2 and TLR-4 by yeast cell wall glycans or LPS,

 respectively) or through the abundant lectin-like molecules that pervade the mammalian proteome.

 Whether plant sugars from transgenic plant-produced proteins will prove immunogenic in humans is of great interest since oral administration of antigen is well known to induce tolerance, and consumption of plants is universal.  

Glycosylation may also indirectly alter the immunogenicity of the protein by minimizing protein aggregation, as appears to be the case with glycosylated IFN-β products.

 And, finally, glycosylation may "shield" immunogenic protein epitopes from the immune system, which — though beneficial for therapeutic proteins — poses a particular problem for successful vaccine development.

Antigenic organization (product aggregates). 

Protein aggregates have been appreciated as inducing immune responses to the monomeric form of the protein for over a half century.

 Speculation regarding how protein aggregates facilitate immune responses centers on their ability to cross-link sufficient numbers of B-cell receptors in a multivalent fashion, causing efficient B-cell activation

 and enhancing antigen processing and presentation, thereby efficiently recruiting the T-cell help critical for generating high-affinity IgG antibody.

 Since the biological activity of therapeutic proteins depends on protein conformation, the ability of protein aggregates to generate neutralizing antibody may depend on preservation of the native conformation of the molecule within product aggregates. Therefore, aggregates of denatured protein may be less potent in generating neutralizing antibody than in generating binding antibody. However, antibodies to linear determinants in the protein contact sites could theoretically neutralize the protein's bioactivity as well. They also could be generated 

Critical information is lacking regarding the types and quantities of aggregates sufficient to generate immune responses for specific protein therapeutics. This may differ dramatically, depending on product origin and the underlying level of tolerance. Product aggregates have been key to the development of immune responses to many therapeutic proteins including: type I IFNs,

 rHu IL-2 (which is formulated as an aggregate of an average size of 27 molecules

 It is obviously critical for manufacturers of therapeutic protein products to minimize aggregation and to use sensitive assays to detect the various types of aggregates that may arise.   

 It has recently been recognized that no single method of assessing aggregates provides a robust measure of protein aggregation.

 Indeed, exclusive use of size exclusion chromatography may preclude detection of higher molecular weight aggregates that fail to penetrate the gel. Therefore, consideration should be given to implementing several orthogonal methods, such as analytical ultracentrifugation,

 Fourier Transform Infrared spectroscopy (FTIR),

 Formulation components are chosen for their ability to preserve the native conformation of the proteins in order to maximize 

 activity (by preventing protein denaturation due to hydrophobic interactions among protein molecules and between protein and hydrophobic surfaces such as glass and air) as well as to prevent protein degradation due to, for example, oxidation and deamidation.

 Large proteins included as excipients in the formulation, such as human serum albumin (HSA), may impact immunogenicity positively or negatively. Although they are added for their ability to inhibit hydrophobic interactions, they may impact negatively by co-aggregating with product or by forming protein adducts. Indeed IFN-α-HSA aggregates have been demonstrated to engender immune responses to IFN-α.

 Formulation may engender adjuvant activity through micelle formation, which can form in relatively high concentrations of nonionic detergents such as Polysorbate 80.

 Non-ionic detergents are also known to enhance leaching of organic molecules and metal ions, which have potential adjuvant activity.

 Adjuvant activity can arise from multiple sources other than formulation. Indeed, adjuvants may arise from the presence of microbial host-cell proteins, oligonucleotides, or polysaccharides, which may exert direct adjuvant activity through Toll-like receptors or other pathogen-associated molecular pattern (PAMP) recognition molecules present on B cells and other APC populations.

 Adjuvant activity may also arise as a direct result of the biological activity of the product. Therefore, type I IFNs,

 are known to upregulate (through differing mechanisms) immune responses to themselves as well as to other biological therapeutics, endogenous proteins, and even small drug molecules,

while other products, such as anti-CD4 MAbs

 FDA has come to increasingly appreciate the fact that interactions between protein therapeutics and container closures may negatively impact product quality and immunogenicity. This is particularly apparent with the increasing use of prefilled syringes for patient self-administration. 

Syringes are delivery devices with multiple surfaces and materials that can interact with the product and alter product quality. Glass and air interfaces are hydrophobic surfaces that can mediate protein denaturation and aggregation. Moreover, glass syringes and movable surfaces (such as syringe plungers) are coated with silicone oil, which facilitates injection but also provides a surface on which proteins can denature and aggregate. Rubber surfaces contain organic leachables involved in vulcanization or plasticization that can leach into the product, particularly when the surfaces are not coated. Finally, metal wire is used to create the syringe hubs in glass syringes, and residual levels of such metals also may interact with the product.  

For example, one protein therapeutic was found to have increased levels of truncated product due to the leaching of metal ions from the syringe gasket, which activated a metalloprotease coeluting with the product. In another example, tungsten metal ions from the syringe barrel oxidized a therapeutic protein, causing product aggregation. Finally, organic leachables extracted from uncoated rubber stoppers by the polysorbate excipient may act as immune adjuvants, inducing neutralizing antibody to the product.  

 The intent of packaging therapeutic products in prefilled syringes is to facilitate patient self-administration. The patient must store the product under appropriate conditions and administer it correctly. Given that most protein products are sensitive to heat, light, and mechanical agitation — which cause product denaturation and aggregation — it's worth considering whether, in the case of Eprex, self-administration with the attendant possibilities of storage or handling abuse, contributed to its immunogenicity. Did patients developing PRCA have excursions from normative storage or handling conditions? Clearly, the appropriate education of patients regarding product handling and administration is vital to ensure product quality. 

 References 1-29 can be found in Part 1 of this article. See the November 2004 issue of 

30.  Heywood GR, Rosenberg SA, Weber JS. Hypersensitivity reactions to chemo-immunotherapy with high dose IL-2. 

1995; 87:915-922; and personal communication of Alexandra S. Worobec, M.D. and Crystal Mackall, M.D. 

31.  Mackall CL. T-cell immunodeficiency following cytotoxic antineoplastic therapy: a review. 

32.  Ragnhammar P, Friesen H, Frodin J, Lefvert A, Hassan M, Osterborg A, et al. Induction of anti-recombinant human GM-CSF (E. Coli derived) antibodies and clinical effects in nonimmunocompromised patients. 

33.  FDA. Guidance for Industry: Drugs, Biologics, Medical Devices Derived from Bioengineered Plants for Use in Humans and Animals. Sept. 2002. Available at 

34.  Weigle WO. Analysis of autoimmunity through experimental models of thyroiditis and allergic encephalomyelitis. 

35.  Haribhai D, Engle D, Meyer, M, Donermeyer D, White JM, Williams C. A threshold for central T-cell tolerance to an inducible serum protein. 

36.  Goodnow CC. Transgenic mice and analysis of B cell tolerance. 

37.  Derbinski J, Kyewski B. Self representation in the thymus: an extended view. 

38.  Revoltella R. Natural and therapeutically induced antibodies to cytokines. 

39.  Soos J, Polsky R, Keegan S, Bugelski P, Herzyk D. Identification of natural antibodies to interleukin 18 in the sera of normal humans and three nonhuman primate species. 

40.  Turano, A., Balsari A, Viani E, Landolfo S, Zanoni L, Gargiulo F, et al. Natural human antibodies to g interferon interfere with the immunomodulating activity of the lymphokine. 

41.  Tiberio L, Caruso A, Pozzi A, Rivoltini L, Morelli D, Monti E, et al. The detection and biological activity of human antibodies to IL-2 in normal donors. 

42.  Miller L, et al. Abrogation of the hematological and biological activities of the interleukin 3/granulocyte macrophage colony stimulating factor fusion protein PIXY321 by neutralizing anti-PIXY antibodies in cancer patients receiving high dose carboplatin. 

43.  Cramer DV. Natural antibodies and the host immune responses to xenografts. 

44.  Bretthauer R, Castellino F. Glycosylation of pichia pastoris derived proteins.

45.  Gomord V, Faye L. Posttranslational modification of therapeutic proteins in plants. 

46.  Andersson K, Lidholm J. Characteristics and immunobiology of grass pollen allergens. 

47.  Roeder A, Kirschning C, Rupec R, Schaller M, Korting H-C. Toll-like receptors and innate antifungal responses. 

48.  Holmskov U, Thiel S, Jensenius J. Collectins and Ficolins: humoral lectins of the innate immune defense. 

49.  Runkel L, Meier W, Pepinsky RB, Karpusas M, Witty A, Kimball K, et al. Structural and functional differences between glycosylated and non-glycosylated forms of human Interferon-β. 

50.  Gribben J, Devereux S, Thomas N, Keim M, Jones H, Goldstone A, et al. Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF.

51.  Schauer R. Victor Ginsburg's influence on my research of the role of sialic acids in biological recognition. 

Immunogenicity

Immune responses to foreign proteins are expected and may be anticipated for some self-proteins. The rapidity of development and the strength and persistence of the response depends on many factors.

A Risk-Based Approach to Immunogenicity Concerns of Therapeutic Protein Products, Part 2: Considering Host-Specific and Product-Specific Factors Impacting Immunogenicity

As of late 2004, 26 modern antibody-based therapeutic agents have been approved in the European Union and the US. Some 500 such products are currently in development, ensuring that the number of approved antibody-based products will increase substantially over the coming years.  

Traditionally, polyclonal antibodies have been used to induce passive immunity. These antibody preparations are made by extracting human or animal serum exposed to the antigens of interest. Antibody preparations produced by such means are heterogeneous. Antibody levels vary from bleed to bleed and, when the producer animal dies, so does the product source. 

The mid 1970s development of hybridoma technology allowed production of large amounts of monospecific antibodies against virtually any antigen of interest.

 This technique fuses a mouse-derived (murine), antibody-producing lymphocyte with a transformed (cancerous) myeloma cell. A proportion of the daughter hybrid cells (hybridomas) continue to produce the murine antibody and also retain the immortal character of the parent myeloma cell. The hybridoma can be stored in a viable state for years, providing an almost inexhaustible seed supply of monoclonal antibody (MAb). The most striking attribute is that all the individual antibody molecules present in a monoclonal antibody preparation are identical (See "Antibody Structure" box on page 2). 

MAbs initially found wide application as 

 diagnostic reagents in immunoassays. Starting in the 1980s, several gained regulatory approval for 

use — either as therapeutic or diagnostic agents.

 The major target indication of modern antibody preparations is cancer, although several products aimed at additional indications also have been approved (Table 1).  

RCSB Protein Data Bank, 15c8, http://www.rcsb.org/pdb

The binding of an antibody to the antigen against which it is produced is extremely specific. All human (and indeed other) cells display a range of surface antigens. Some are found on a range of cell types, while others — termed unique surface antigens (USAs) — are specific to a given cell type. Antibodies produced against unique surface antigens bind selectively to the surface of these cells. In effect these antibodies are "magic bullets" capable of selectively targeting specific cell types such as cancer cells, virally infected cells, or microbial cells at an infection site.  			

Antibodies generally display favorable safety profiles. Humanized antibodies (described below), in particular, are well tolerated at high doses. Furthermore, engineered antibodies can be quite versatile. Slight changes in the six to ten amino acids within the complementarity-determining region (Figure 1) can retarget the antibody, creating a new potential therapeutic. Common drug development and manufacturing approaches can be employed to bring the product to market.   			

Binding of an antibody to a cell surface can potentially trigger cellular destruction via immunological effector functions associated with the antibody's F

 region (for example, phagocytic destruction and complement activation). Antibody-mediated cellular destruction generally involves conjugating an antibody to a cytotoxic agent, usually a radionuclide, a toxin, or potentially an enzyme capable of converting an inactive prodrug into an active cytocidal agent (Figure 2). The antibody delivers the cytocidal agent directly to the surface of the target cell — and defines antibody-mediated cellular targeting.   

Figure 2. Antibody-Based Therapeutics (a) Antibodies bind selectively to specific cell types if they are raised against a unique surface antigen (USA). (b) Effector molecules can be conjugated to the antibody. Radioactive tags are employed to either detect or destroy the target cell. (c) Toxins can also be used as cytocidal agents. (d) Enzymes (E) can selectively convert a harmless prodrug into a cytocidal agent at the cell surface.

ANTIBODY-MEDIATED CANCER DIAGNOSIS AND THERAPY

When a cell is transformed (becomes cancerous), it usually begins high-level expression of several genes that were previously either unexpressed or expressed at extremely low levels. Some of the resultant proteins are found on the cell surface and are termed tumor surface antigens (TSAs). TSAs potentially represent unique surface antigens, and antibodies raised against specific TSAs are likely to be approved for oncological applications.   			

Several anti-TSA antibodies conjugated to radioactive tags have been approved for the detection and treatment of various cancers (Table 1). Conjugation of radioactive tags to anti-TSA MAbs facilitates targeted tumor destruction via the ionizing effects of radioactivity. 

Methods of conjugating radioactive tags to MAbs are well established. The most common radioisotopes used are β emitters. The medium-energy radioactivity they emit penetrates through several cell layers, which is suitable for the treatment of large tumors. This can be particularly advantageous in the treatment of tumors containing a proportion of TSA-negative cells. 

The most popular radionuclides studied have been isotopes of iodine (

Y is particularly popular as it emits high-energy radiation, namely β

 particles, which have a mean path length of up to 5 mm and hence deep tissue penetration. 

Y also has a relatively short half-life (64 hr) and does not emit γ radiation, which permits outpatient therapy.  

Radiolabelled antibodies can also be used for 

 diagnostic imaging (immunoscintigraphy) of cancer and other medical conditions. This requires radioisotopes emitting γ rays of suitable mean energy. Metastable technetium (

In) are used most often, and a number of products have been approved. 

Several trials are investigating the use of toxins conjugated to anti-TSA antibodies as targeted therapeutic agents. A variety of toxins or toxin subunits derived from plants, bacteria, and fungi have been used, including the ricin A chain and 

 exotoxin. Some antibody-TSA complexes are quickly internalized upon binding of the antibody to a cell surface. In the case of antibody-toxin conjugates this process is essential to cellular destruction. Upon their internalization, most of the antibody-toxin conjugate molecules are destroyed in the lysosomes; however, a small number survive and can subsequently induce cell death. 

MAbs also find application in the detection and treatment of medical conditions other than cancer. Simulect is a chimeric antibody that specifically binds to the α-chain of the interleukin-2 (IL-2) receptor. Binding inhibits activated T-lymphocytes, which are involved in the acute rejection of transplanted tissue. Other products aimed at preventing transplant rejection include Zenapax and Orthoclone OKT3. Additional product indications include psoriasis, asthma, rheumatoid arthritis, and Crohn's disease (Table 1). 

Table 1. Modern Antibody Preparations Approved in the EU and the US			

Many, if not most, first-generation MAb-based therapeutic products proved to be clinical disappointments. Poor efficacy was due to a number of factors. The single most important limitation was their immunogenicity. First-generation MAbs were invariably intact murine monoclonals and, as such, were highly immunogenic when administered to humans. Human anti-mouse antibodies were detected in up to 80% of patients within 14 days of MAb administration — the HAMA response. Repeat administration induced a response in most other patients, limiting the therapeutic efficacy of murine monoclonals to one or two doses. 			

In addition to the universal obstacle of immunogenicity, first-generation MAbs displayed complications relating to tumor penetration and target specificity. Intact antibodies are large and bulky macromolecules that often cannot effectively penetrate a solid tumor mass. This limits their therapeutic efficacy to the outermost layers of transformed cells. The specificity of antibody-mediated tumor detection or destruction obviously depends upon identifying and targeting a TSA uniquely expressed on the target cells. Absolute antigen specificity is desirable but rarely attained in practice. For example, antibodies can also target healthy tissue if it expresses a surface antigen closely related to the TSA. All too often, this can lead to significant and unacceptable side effects. 

Some of these difficulties have been circumvented thanks to advances in antibody engineering. Chimeric, humanized, and human monoclonals greatly reduce or essentially eliminate immunogenic responses while, in the case of cancer, much smaller, antigen-binding antibody fragments enhance tumor penetration. 			

Cloning and expression of antibody or antibody-fragment genes and cDNA is now routine. Initial attempts to reduce the immunogenicity of murine monoclonals entailed producing chimeric antibodies by recombinant DNA technology. Several such products gained approval, mainly in the 1990s. They are made by splicing the gene sequences coding for the mouse-variable regions (these contain the antigen-binding complementarity determining regions) to the constant regions of a human antibody. The resultant hybrid structure is designed to retain the desired antigen-binding specificity of the parent murine monoclonal and be less immunogenic, since only a minor part of the chimeric structure is murine in origin. In practice, reduced immunogenicity is observed, and chimeric products display extended serum half-lives (200-250 hr, as compared to 30-40 hr in the case of intact murine monoclonals).  

Antibody engineering also allows the production of "humanized" antibodies,

 in which only the gene sequences coding for the CDRs of the murine antibody replace the CDR sequences in human antibody genes. The resultant antibody is entirely human except for these short sequences and has essentially the same biochemical characteristics as native human antibodies, as confirmed in practice. These antibodies display serum half-lives virtually indistinguishable from native human antibodies (14-21 days).   

Although not antibodies themselves, recombinant DNA technology has also facilitated the development of two fusion proteins that contain antibody domains fused to other proteins (marketed under the trade names Enbrel and Amevive respectively; Table 1).

There are two ways of generating antigen-binding Fab or F(ab)

 fragments. The older way is by direct proteolytic cleavage of intact antibodies. Genetic engineering also allows the generation of F

-like fragments by linking gene sequences coding for the V

 domains with a "linker" sequence coding for a short peptide, anchoring the two domains together.

 Such fragments, due to their smaller size, are capable of more readily penetrating solid tumors and should therefore show increased efficacy for treating cancers. Direct studies have confirmed their enhanced ability to penetrate tumors. Intact IgG molecules take 54 hr to travel 1 mm through a solid tumor, whereas Fab fragments can travel the same distance in 16 hr.

Despite this potential advantage, the use of antibody fragments for solid tumor therapy has been quite limited. These fragments have very short half-lives. While rapid clearance would be an advantage for 

 diagnostic use (facilitating rapid removal of the conjugated radioactive tag), it is usually a disadvantage in a therapeutic application. 

The development of chimeric and, in particular, humanized antibodies have mostly overcome issues of antibody antigenicity in humans. It is now possible to produce fully human antibodies via recombinant DNA technology — either via transgenic animals or via phage display technology. Research undertaken in the 1990s has led to the development of mice strains capable of producing a fully human antibody repertoire. This is achieved by inactivating endogenous murine immunoglobulin genes and effectively replacing them with human immunoglobulin loci. This approach has been commercialized by companies such as Abgenix (the XenoMouse), Medarex (HuMab mouse), and Kirin (TC mouse).

An alternative means of producing fully human antibodies entails the expression of human antibody libraries in phage, viral particles that replicate in bacteria. The phage display libraries can easily be screened for an antibody that selectively binds the antigen of interest.

 Several such antibodies are in clinical trials and are likely to be approved over the coming years.    

All modern antibody preparations are produced using mammalian cell lines, either hybridoma cells in the case of monoclonals or Chinese hamster ovary (CHO) or related cell lines in the case of most engineered products. Systems based on mammalian cell lines are relatively expensive. Estimates of the cost of product synthesis are in the vicinity of $300/g antibody. High production costs are slowing the adoption of antibody-based products, as therapeutic regimes often require ongoing administration of tens of milligrams of product per dose. (Comparative dosage levels of interferons, for example, would typically be in the microgram range.)  			

Looking to lower costs, a wide variety of engineered antibodies have been produced in various transgenic systems. These include the milk of transgenic goats, the eggs of transgenic chickens, cow blood, and a range of transgenic plants including wheat, rice, soy, and tobacco.

 Although some published projections suggest that the cost of synthesis could be below $50/g, there is no approved product to support the data. Upfront developmental costs and additional technical and regulatory issues are delaying production of biopharmaceuticals in transgenic animals. Plant-based production also suffers from a number of complications, including radically altered product glycosylation patterns.  

Genetic engineering facilitates the design and production of bispecific antibodies and antibody fragments (BsAbs). As the name implies, bispecific antibodies are engineered to bind to two different, specific antigens. Such antibodies can be designed so that one antigen-binding site targets a unique surface antigen on a cancer cell while the other either binds a therapeutic agent or attracts immune effector cells to the target cell surface.

 Bispecific antibodies have some disadvantages, including short half-lives and the potential to trigger serious adverse effects due to the induction of cytokine release. 			

The production of a desired therapeutic antibody 

 represents an elegant means of overcoming technical complications such as short half-life of injected product. A number of research groups are pursuing this strategy. One obvious practical approach is the introduction of encapsulated or otherwise protected antibody-producing cells into the body.

Another is gene therapy, although the same technical difficulties that beset gene therapy generally also apply in this instance.

 Gene therapy would also allow the expression of antibodies within individual cells, allowing antibodies to target and hence inactivate (or perhaps activate) specific intracellular molecules and processes relevant to disease progression. By attachment of appropriate cellular signal sequences, such intracellular antibodies (intrabodies) can target not only the cell cytoplasm but also specific intracellular organelles.

Data from the Pharmaceutical Research and Manufacturers of America suggests its member companies had about 75 antibody-based products undergoing clinical evaluation in 2003.

 When characterized by product type, antibody-based products therefore were second only to vaccines, of which there were 98 in development. Globally about 500 antibody products are in development.

 Datamonitor suggests that the total market value is set to grow very substantially, reaching more than $17 billion before the end of the decade. The global market value in 2002 was estimated at $5.4 billion.

Special Section

As of late 2004, 26 modern antibody-based therapeutic agents have been approved in the European Union and the US. Some 500 such products are currently in development, ensuring that the number of approved antibody-based products will increase substantially over the coming years.

Modern Antibody-Based Therapeutics

				In the biopharmaceutical industry, outsourcing many facets of development, approval, and manufacture is as commonplace as it is practical. In this young industry, few companies possess all the internal capabilities or experience in every specialty required to discover, create, and commercialize a new product. The use of contract services is often driven by the desire to manage costs and enhance or maintain productivity. Project management is less commonly outsourced because the potential application and impact of doing so is misunderstood. However, firms that have retained professional project managers realize significant value, such as reduced time to market, less costly manufacturing processes, and better information flow among worldwide R&D teams. 

Start-ups and big pharmaceutical companies alike recognize the potential to generate beneficial new drugs. However, like the large molecules with which biotech firms work, biotech ventures and partnerships are complex — and their complexity increases exponentially with the number of players. The addition of third-party manufacturers, CROs, contract analytical labs, co-development partners, and other parties makes it much more difficult to control budgets, maintain timelines, facilitate decision making, and ensure effective communications. 			

While technically proficient in their respective areas of expertise, biotech firms often lack an ingredient necessary for success — strong project management experiences and leadership skills. Biotech projects require leaders who can bring together both internal and external stakeholders to reach a common objective. This is why the project management function is absolutely critical: Someone needs to keep an eye on the big picture while tracking the details. It takes experience and discipline to create and manage the project plan, track follow-up, communicate status and issues, resolve conflicts, mitigate risks, verify compliance to requirements, and coordinate different experts with potentially conflicting priorities and agendas. 

Coordination between senior management, contract manufacturers, external consultants, bench-level scientists, and other stakeholders increases productivity, job satisfaction, and ultimately the probability of project success. This role cannot be the part-time job of a technical team lead (or even the full-time job, depending on the person's qualifications). Project management is a specific set of skills and processes administered to meet the specific complexities of a project. In today's increasingly competitive environment, excellence in execution becomes even more critical. 

The first step is evaluating your company's current ability to accomplish its priorities. Is your staff already over-committed? How often are strategic initiatives or tactical projects behind schedule or over budget? 

It is no surprise that for many companies the answers are "yes" and "too often." These stumbling blocks can be at least partially overcome through sophisticated project management practices. Some further questions that can help you assess the project management expertise within your organization include:   					

Does your organization have a clearly defined process for managing tasks?

Do you recruit and dedicate staff specifically to manage projects?

Are your project managers formally trained and involved in continuing skill improvement?

Have you collected and analyzed empirical data that categorize and describe projects and identify project management costs?

It is unlikely that your company has taken its internal project management to this level. Your focus is on your core competency, biotechnology, which is no guarantee of excellence in managing teams or coordinating and executing strategies — the forte of a trained, professional project manager. 			

The reality at many corporations is that project management is relegated to engineers, scientists, or researchers; it is assigned on the basis of availability; and it is often added on to a technical staffer's other daily tasks. The problems can multiply: if internal staff members (even internal project managers) are spread too thin, execution suffers as commitments are missed and timelines get pushed back. Companies of all sizes can suffer from this misalignment between their staff's skills and the requirements of the roles they are asked to fulfill. 

However, if you hire a professional, full attention will be paid to your project without the encumbrance of other responsibilities or internal politics. Professionals also bring business management expertise to the table, along with tested protocols and facilitation experience. Facilitation depends on leadership — character, behavior, and attitude — which is as important as methodology. Leadership is a different skill than organizational ability — one more reason that project management should not be left in the hands of the untrained.  

Project management is a means to an end. Fully developed, the knowledge, tools, skills, and protocols necessary for successful coordination and completion of any project make project management a profession in its own right — especially when practiced as a core competency. 

You can find general information about project management service providers on the Internet, or you can consult professional publications and organizations such as the Project Management Institute (PMI; 

). Your network of colleagues can be a good resource as well. 			

Because the vast majority of firms provide project management as one of many services, there is currently no freestanding listing of project management companies under the North American Industry Classification System (NAICS). Search larger categories if you are interested in hiring a firm for a particular type of project. For example, for a construction project, research data on architectural and engineering firms. For help with a product launch, seek consultants that offer project management services as part of product development. But be advised — this method will help you find an engineer or a consultant, not a project manager in the purest sense.  

Next, narrow your search to your geographic location and other basic criteria, such as experience and type of clients. Then dig deeper. Many companies stop their evaluation too early, asking only about costs and experience specific to their industry. Additional criteria are discussed below. 

Project management is a specific set of skills and processes administered to meet the specific complexities of a project.

BioPharm International-12-01-2004

PMPs — If Not Now, When?

Regulatory Beat: Vaccine Manufacturing and Safety in the Spotlight

A Risk-Based Approach to Immunogenicity Concerns of Therapeutic Protein Products, Part 2: Considering Host-Specific and Product-Specific Factors Impacting Immunogenicity

Modern Antibody-Based Therapeutics

Project Management Services Matter

StreetTalk: 2004 Round-Up