Immunogenicity of biological products

Overall, it is accepted that the administration of an exogenous protein to animals or humans has the potential to elicit an antibody response against the protein if the immune system recognizes the protein as foreign. Immunogenicity is a unique property of biological therapeutics that distinguishes biologicals from traditional small-molecule drug products. An immune response to a biological drug can occur in nonclinical animal species or in clinical trial subjects and patients, and the more the structure and amino acid sequence of the protein drug differs from the native protein, the greater the immunogenic potential of the drug.64 Immunogenic responses associated with protein drugs were first identified in diabetes patients administered insulins from animal (bovine or porcine) sources.65-67 In general, biological products that have a high degree of sequence homology to the native human protein are less likely to be immunogenic in humans; however, induction of antibody responses has occurred with biological therapeutics that are identical or nearly identical to the native human protein.68 The result of the immunogenic response can be any of the following: no effect; an alteration of the drug's pharmacokinetic profile; an abrogation of the pharmacological activity of the drug; or neutralization of the biological activity of the endogenous protein, potentially resulting in life-threatening consequences.6970 Additionally, antibody responses can potentially affect the interpretation of toxicology studies. For these reasons, immunogenicity of biological therapeutics is an important concern for clinicians, manufacturers, and regulatory agencies. The preclinical and clinical evaluation of the immunogenic potential of any biological drug is necessary during the drug development process.

Protein structure, manufacturing processes, impurities, host-cell proteins or contaminants, aggregate formation, and denatured proteins are all important factors that can influence the immunogenic potential of biologicals.64,71-73 In general, glycosylated proteins are less immunogenic than nonglycosylated proteins, which is presumably due to a higher exposure of antigenic sites on the protein backbone with nonglycosylated proteins.68,70,74 Factors related to the dosing regimen, such as dose schedule, frequency, and duration, can also influence the immune system's response to a protein drug. Typically, repeated administration is more immunogenic than a single dose, and immunogenicity increases with more frequent dosing and longer-term treatment.70,75

The route of administration is a particularly important factor that influences the immunogenic potential of biological therapeutics. As stated previously, most biological drug products are administered parentally, and the subcutaneous route is usually more immunogenic than intravenous or intramuscular administration.72,76-79 Underlying disease, concomitant medication, and the immune status of patients can also affect antibody responses to administered protein drugs. For example, cancer patients administered chemotherapeutic agents that cause myelosuppression may have a compromised immune system, and thus are less likely to mount an immune response to a biological therapeutic.64 Although these are some general considerations, immunogenicity can occur with any protein, even in conditions listed above where immunogenicity is less likely (i.e., single intravenous dose).

Most biological therapeutic products are human proteins or antibodies specific for a human protein. Therefore, it is not unexpected that the administration of a biological therapeutic to animals results in the production of antibodies against the drug. In general, the greater the dissimilarity between the human protein sequence and the animal protein sequence, the more likely the animal's immune system will elicit an antibody response to the drug.79,80 In some cases, antibody responses develop in nonhuman primates even though the sequence homology of biological therapeutics is generally more similar to nonhuman primates than to other species such as rodents and dogs. The production of antibodies in animals used in toxicology studies can affect the outcome of a toxicology study in various ways, such as altering drug elimination or its pharmacological activity. Since antibody responses can affect the outcome of toxicology studies and potentially generate misleading toxicity data and interpretations, measuring and characterizing antibody responses in repeated dose toxicity studies is critical.18,33,79 The development of antibodies in some animals in a toxicology study, however, does not necessarily invalidate the study, especially if the antibody responses are non-neutralizing and do not significantly alter the pharma-cokinetics of the drug. Therefore, it is important to determine whether the presence

1000 I,

1000 I,

FIGURE 6.2 Effect of clearing antibodies on serum drug levels.

of antibodies correlates with the pharmacology, pharmacokinetics, and toxicity of the product.

Three types of antibody responses can develop in nonclinical toxicology studies that can potentially alter the results or interpretation: (1) clearing/sustaining, (2) neutralizing, and (3) crossreactive antibodies that neutralize endogenous counterparts. Clearing antibodies bind to the protein therapeutic and increase plasma clearance of the drug.8182 Increased drug clearance leads to decreased distribution and exposure of target organs to the drug. Figure 6.2 illustrates the effect on serum levels of a biological drug in an animal that develops clearing antibodies compared to an animal that is antidrug antibody-negative.

Conversely, sustaining antibodies can slow the rate of plasma clearance of the drug, resulting in prolonged drug exposure, which can also confound interpretation of the toxicology study.75 Neutralizing antibodies bind to or near the target-binding domain of the biological drug, which can interfere with its ability to bind its target receptor and, ultimately, reduce the pharmacological activity and efficacy.80 83 The primary concern for the development of clearing or neutralizing antibodies in animals used in toxicology studies is the potential for lower exposure of target organs to the biological drug product, resulting in fewer treatment-related toxicities. Such studies are likely not predictive of the potential for human toxicity. Cross-reactive antibodies can also bind and neutralize the biological, but of more concern, this type of antibody can also bind and neutralize the biological function of the endogenous protein, resulting in toxicity. For example, the subcutaneous administration of recombinant human thrombopoietin (rhuTPO) to rhesus monkeys led to the development of cross-linking antibodies that neutralized the function of the monkeys' endogenous thrombopoietin, resulting in thrombocytopenia.68 84 As illustrated in Figure 6.3, a transient increase in platelet counts, which would be the expected pharmacological response, occurs between Days 14 and 21. Subsequently, platelet counts

Study Day

FIGURE 6.3 Effect of cross-linking antibodies on platelet profiles in rhesus monkeys injected with various doses of rhuTPO for 14 days. A transient dose-dependent increase followed by a rapid decrease in platelet counts is observed. All animals were positive for anti-TPO antibodies from Day 21 onwards. (Reprinted from Koren, E., et al., Current Pharm. Biotechnol, 3, 349, 2002. With permission from the International Association for Biologi-cals, Switzerland.)

Study Day

FIGURE 6.3 Effect of cross-linking antibodies on platelet profiles in rhesus monkeys injected with various doses of rhuTPO for 14 days. A transient dose-dependent increase followed by a rapid decrease in platelet counts is observed. All animals were positive for anti-TPO antibodies from Day 21 onwards. (Reprinted from Koren, E., et al., Current Pharm. Biotechnol, 3, 349, 2002. With permission from the International Association for Biologi-cals, Switzerland.)

are considerably reduced and even fall below baseline levels. Similar to the effect seen after administration of rhuTPO, rhesus monkeys administered recombinant rhesus TPO also had a reduction in platelet counts with coinciding antibodies.84

Another potential consequence of the production of antidrug antibodies that can affect the outcome of a toxicology study is antibody-antigen complex formation and deposition in various tissues, which could lead to immune complex-mediated toxicity.78 For example, glomerulonephritis was observed in cynomolgus monkeys administered recombinant human interferon-y (rHuIFN-y) intramuscularly. These monkeys had detectable anti-rHuIFN-y antibodies, and thus this lesion, which morphologically resembled an immune complex glomerulitis, may have been secondary to the deposition of anti-rHuIFN-y antibody complexes in renal glomeruli.85

Antibody responses can occur in humans administered biological therapeutics and, in some cases, have consequences similar to those observed in nonclinical toxicology studies. The clinical sequelae of antibody production in humans can vary from no effect to life-threatening syndromes, with the latter being a relatively rare occur-rence.68 Clinical consequences that can occur in humans administered biologicals are reduced drug exposure or loss of efficacy of the drug through the development of clearing or neutralizing antibodies.86-88 Clinical outcomes of greater concern include infusion-related reactions or the induction of an anaphylactic response, which have been reported for various biologicals.15,89

The most concerning clinical effect of antibody responses in humans is the production of crossreactive antibodies that neutralize the biological activity of the patient's own endogenous protein that mediates a unique biological function. For example, administration of a particular formulation of recombinant erythropoietin to humans resulted in pure red blood cell aplasia in some patients. This toxicity correlated with the development of anti-erythropoietin antibodies, which presumably crossreacted and neutralized endogenous erythropoietin.90,91 Since the development of antidrug antibody responses in humans can have serious clinical consequences, the detection and characterization of antibody responses using highly sensitive and reproducible assays is essential in the development of any therapeutic protein product.92

Overall, animal models, including nonhuman primate models, are not reliably predictive of the immunogenic potential of biological products in humans.33,80 The limited predictive power of animal models for human immunogenicity is because most biological therapeutic products are human proteins, and thus will likely induce an antibody response when administered to animals. In many cases, animal models even overpredict the antibody response that is observed in humans.80 Animal models, however, can be useful in predicting the relative immunogenicity of various biological drugs in humans. A rhesus monkey model, for example, was useful in predicting the relative immunogenicity of different forms of human growth hormone in humans.93 Additionally, some animal studies have been predictive of the clinical consequences of antibody responses in humans. In the case of TPO, administration of human and homologous TPO to various animal species was predictive of the development of antibody-mediated thrombocytopenia observed in humans administered recombinant TPO.84,94 Numerous efforts are ongoing to develop more sophisticated approaches to reliably evaluate the immunogenic potential of biological therapeutics. For instance, the use of transgenic mice that are immunologically tolerant to the human protein they have been genetically engineered to express are a promising model that may be a better predictor of the immunogenic potential of biological therapeutics in humans.64,79,80

Immunogenicity remains a challenge in the development of biological therapeutics intended for use in humans. Continued development of more sensitive assays for the detection and characterization of antibody responses, the generation of more predictive models of immunogenicity, as well as nonclinical and clinical monitoring of potential clinical consequences of antibody responses are all necessary measures to ensure the safety and efficacy of biologicals.

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