Optimization And Pharmacokinetics Of Therapeutic Antibodies
Since the mid-1990s, antibodies have emerged as an important new class of drug for therapeutic use across diverse clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases and cardiovascular medicine. The FDA approved antibody therapeutics include 14 unmodified IgG molecules, 2 radioimmunoconjugates, 1 antibody–drug conjugate and 1 Fab.
At least 150 additional antibodies are in various stages of clinical development. One of the strengths of antibody therapeutics is that they belong to a well established drug class that has a high success rate from the first use in humans to regulatory approval, the success rate amounting to 29% for chimeric antibodies, and 25% for humanized antibodies 1 . This compares favorably with the 11% success rate for small-molecule drugs 2 . Moreover, much of the development and clinical experience that is gained from the generation and optimization of one antibody is applicable to other antibodies, thereby streamlining certain activities and decreasing some of the many risks that are intrinsic to drug development. In general, antibodies are well tolerated by humans, although infusion reactions (particularly for the first dose) are common but usually manageable 3 . For example, most patients treated with rituximab (Rituxan; Genentech, Inc. and Biogen Idec Inc.; and MabThera; F.Hoffman- LaRoche Ltd) a CD20-specific monoclonal antibody, experience mild to moderate first infusion reactions that include fever and chills, and these reactions occur less frequently with subsequent doses. Infusion reactions with rituximab are commonly attenuated by premedication and by incremental increase in the rate of infusion of rituximab. A key strength of antibodies as therapeutics is that their clinical potential can readily be increased by improving their existing properties 4–6 .The limitations of antibody therapeutics, include the restriction of targets to those on the surface or exterior of host cells or invading pathogens. Advances in the expression of functional antibodies within cells known as intrabodies show that these antibodies are useful research tools, but the potential of intrabodies as therapeutics remains to be shown 7, 8 . Another limitation is that antibody drugs are expensive 9 , which limits their use to serious medical conditions. Many factors contribute to the high cost of antibody therapeutics, including the large expense of drug development in general, the high cost of manufacturing and the large total doses that are often required. In addition, the intellectual property that is often associated with the generation, optimization and production of antibodies commonly leads to a series of stacking royalty payments, which further increases the cost. Although antibody therapeutics is often safe and well tolerated, rare but serious adverse events have been reported for several antibodies 10, 11 . The development of potent antibody drugs has evolved into an iterative design process (Figure . 1).
Fig 1 - Iterative Design Of Antibody Therapeutics
The article attempts to review the design strategies involved 4–6 with an emphasis on IgG, because this is the format of antibody that is used by almost all approved antibody drugs 1 . In addition, antibody fragments are discussed, due to their growing clinical importance 1, 12 and the fragments are commonly used in the engineering of antibody properties 13 . This review article explores the complex relationship between the goals for antibody therapeutics, the available tools for antibody generation, optimization, and the tunable properties of antibodies.
The typical antibody or immunoglobulin (Ig) consists of two antigen-binding fragments (Fabs), which are linked via a flexible region (the hinge) to a constant (Fc) region (FIG. 2). This structure comprises two pairs of polypeptide chains, each pair containing a heavy and a light chain of different sizes. Both heavy and light chains are folded into immunoglobulin domains. The ‘variable domains’ in the amino-terminal part of the molecule are the domains that recognize and bind antigens; the rest of the molecule is composed of ‘constant domains’ that only vary between Ig classes. The Fc portion of the Ig serves to bind various effector molecules of the immune system, as well as molecules that determine the biodistribution of the antibody.
Fig. 2 - Modular structure of immunoglobin
Mechanisms Of In Vivo Action
In antibody-based therapies, the goal is to eliminate or neutralize the pathogenic infection or the disease target, for example, bacterial, viral or tumor targets. Therapeutic antibodies can function by three principal modes of action: by blocking the action of specific molecules, by targeting specific cells or by functioning as signaling molecules. The blocking activity of therapeutic antibodies is achieved by preventing growth factors, cytokines or other soluble mediators reaching their target receptors, which can be accomplished either by the antibody binding to the factor itself or to its receptor. Targeting involves directing antibodies towards specific populations of cells and is a versatile approach; antibodies can be engineered to carry effector moieties, such as enzymes, toxins, radionuclides, cytokines or even DNA molecules, to the target cells, where the attached moiety can then exert its effect (for example, toxins or radionuclides can eliminate target cancer cells). The natural effector functions of antibodies are associated with binding to Fc receptors or binding to complement proteins and inducing complement dependent cytotoxicity (CDC) Targeting antibodies can retain such effector functions intact or they can be abolished during the design of the antibody, depending on the therapeutic strategy. The signaling effect of antibodies is predicated on either inducing crosslinking of receptors that are, in turn, connected to mediators of cell division or programmed cell death, or directing them towards specific receptors to act as agonists for the activation of specific cell populations. Another approach is to use antibodies as delivery vehicles for DNA and, more recently, to deliver antigens to certain immune cells that present processed antigenic peptides, or epitopes, to T cells, to activate a specific immune response against that antigen.
The terminal half-life of antibodies in plasma can be tuned over a wide range to fit clinical goals. It can range from several minutes for single domain antibodies and scFvs to several weeks for many IgG molecules (TABLE 1).
Table 1 - Pharmacokinetic Properties Of Antibody
Reduced plasma half
Use IgG with impaired affinity for FcRn
Less whole body exposure to antibody, improved target to non target ratio
Increased plasma half life
use IgG with increased affinity for FcRn (at pH 6.0), modify antibody fragment (PEGylation, binding to the molecules with a long half life such as IgG and serum albumin)
Increased plasma concentration might improve localization to target, increased efficacy, reduced dose or frequency of adminstration
Screen antibody panel, choose target antigen accordingly
Efficient effector functions
Select antibodies that can internalize from display libraries, screen antibodies panel with drug conjugated crosslink antibody
Improved efficacy for antibody drug conjugate or immunoliposomes
It might be desirable to increase the terminal half-life of an antibody to improve efficacy, to reduce the dose or frequency of administration, or to improve localization to the target. Alternatively, it might be advantageous to do the converse that is, to decrease the terminal half-life of an antibody to reduce whole body exposure or to improve the target-to-non-target binding ratios. Tailoring the interaction between IgG and its salvage receptor, FcRn, offers a way to increase or decrease the terminal half-life of IgG. Proteins in the circulation, including IgG, are taken up in the fluid phase through micropinocytosis by certain cells, such as those of the vascular endothelia. IgG can bind FcRn in endosomes under slightly acidic conditions (pH 6.0–6.5) and can recycle to the cell surface, where it is released under almost neutral conditions (pH 7.0–7.4) 14, 15 . Mapping of the Fc-region-binding site on FcRn80, 16, 17 showed that two histidine residues that are conserved across species, His310 and His435, are responsible for the pH dependence of this interaction. Using phage-display technology, a mouse Fc-region mutation that increases binding to FcRn and extends the half-life of mouse IgG was identified. Fc-region mutations that increase the binding affinity of human IgG for FcRn at pH 6.0, but not at pH 7.4, have also been identified. Moreover, in one case, a similar pH-dependent increase in binding (up to 27-fold) was also observed for rhesus FcRn, and this resulted in a twofold increase in serum half-life in rhesus monkeys compared with the parent IgG . These findings indicate that it is feasible to extend the plasma half-life of antibody therapeutics by tailoring the interaction of the Fc region with FcRn. Conversely, Fc-region mutations that attenuate interaction with FcRn can reduce antibody half-life, as has been elegantly shown by the pharmacokinetic optimization of an scFv–Fc to allow tumor imaging by positron emission tomography. Modulation of serum IgG concentrations has recently been achieved by tailoring the interaction between an IgG Fc region and FcRn. An IgG engineered to bind FcRn with higher affinity and reduced pH dependence potently inhibits the interaction of endogenous IgG with FcRn, thereby resulting in a rapid reduction in the IgG concentration in mice. These FcRn-blocking antibodies, which are known as Abdegs (antibodies that increase IgG degradation), have potential therapeutic application for the reduction of IgG concentrations, for example, in patients with antibody-mediated diseases 18 . Using antibody fragments instead of antibodies reduces the terminal half-life of the therapeutic from many weeks to a few hours or less, which is too short for many therapeutic applications 19 . The terminal half-life of antibody fragments can be extended many folds by endowing them with the ability to bind long-lived molecules, such as IgG 20 and serum albumin 21 . Antibody fragments with a wider range of terminal half-lives (from a few hours to weeks) can be generated through PEGylation (that is, conjugation to polyethylene glycol, PEG) 22 . At least two PEGylated antibody fragments have progressed to clinical trials 23 , including certolizumab PEGol (Cimzia; UCB; a humanized TNF-specific Fab′conjugated to PEG). Certolizumab PEGol has a terminal half-life of 14 days in patients, which is comparable to the parent IgG, and it is well tolerated. Regulatory submission for certolizumab PEGol is now complete, and this followed preliminary reports of favorable safety and efficacy data in two pivotal Phase III clinical trials for the treatment of Crohn’s disease. The potential advantages of PEGylated fragments over IgG include a lack of any undesirable Fc-mediated effects, a reduction in the risk of immunogenicity and a moderate reduction in the cost 24 .
A prerequisite for effective antibody targeting is that the antibodies should be able to penetrate tissues, and small antibody fragments are better than complete antibodies in this respect. Recent research has shown that high affinity fragments are retained in the periphery of the tumor, whereas the medium-affinity antibodies penetrate throughout the tumour 25 . Furthermore, bivalent low-affinity fragments penetrate better and more uniformly than high-affinity fragments 26 . Fortunately, antibody fragments can be selected for optimal affinity and specificity by all of the in vitro selection processes 27, 28 . The therapeutic efficacy of an antibody, however, also depends on its stability as well as its immunogenicity. Strategies for the engineering of antibody stability with regards to antibody fragments have been described 29 .
Optimization Of Antibody
Fig. 3 - Optimization Of Antibody
Antibodies are commonly optimized by selection from genetically diverse display libraries of antibody-fragment variants or by structure based design followed by expression, purification and functional characterization of individual variants (figure 3). These selection and design strategies are complementary and are particularly powerful when used in combination, as exemplified by the tailoring of the interaction between the Fc region of IgG and FcRn 33 . Phage-display libraries are the most commonly used display technology for antibody generation and optimization 13, 30, 31 . However, other display technologies such as yeast, mRNA and ribosome-display libraries are gaining in popularity for the selection and optimization of antibodies 13 . Display libraries display single-chain variable-domain antibody fragments (scFvs) or Fabs, and contain the encoding DNA or RNA. They have high genetic diversity or repertoire size (commonly 109–1013). These technologies allow the selective recovery of clones that bind a target antigen from a library, and they provide the means to amplify the selected clones for further rounds of selection or analysis. The genetic diversity in these libraries is commonly created by cloning the epertoire of the immunoglobulin heavy-chain and light-chain variable gene segments from naive or immunized individuals. Alternatively, this diversity can be achieved by using synthetic DNA to randomize the complementarity determining regions (the antigen-binding loops) or by a combination of these two approaches. The binding step can be undertaken with the target in solution, immobilized on a surface or on cells. After extensive washing, specifically bound clones are recovered and amplified for the next round of selection. In an optional step, additional genetic diversity can be introduced during clone amplification: for example, by error-prone PCR. Structure-based design uses three-dimensional structural information, sometimes in combination with sophisticated computational methods, to predict the site and type of useful mutation. The ideal starting point for design is a high-resolution structure of an antibody in complex with the relevant binding partner: that is, antigen, complement, IgG receptor (FcγR) or FcRn. However, even a molecular model of an antibody or antigen–antibody complex can sometimes provide a useful starting point. Extensive mutational analysis has increased our understanding of, and provided a means to modulate, the interaction of the Fc region of IgG with various FcγRs80, 142, FcRn80 and complement component 1q (C1q).
Goals For Antibody Therapeutics
Currently available antibody therapeutics establishes this class of drugs and point to design goals (table 2) for future antibodies.
Table 2 - Strategies For Designer Antibodies
Minimize non human sequence (chimerazation or humanization of mouse antibody
Increased efficacy , improved safety, more efficient effector function, long terminal half life
ANTIGEN BINDING SPECIFICITY
Improved selectivity for antigen
Select from display libraries, screen antibody panel, use structure based design
Prerequisite for target therapy
Increased species cross reactivity
Select from display libraries, screen antibody panel, use structure based design
Facilitates preclinical development (efficacy testing, toxicology)
ANTIGEN BINDING AFFINITY
Select from display libraries, use structure based design
Increased efficacy , reduced dose or frequency of administration, increase potency of ADCC
Select from display libraries, use structure based design
Increase localization to tumours, more homogenous distribution in tumours
One need is to improve the efficacy of antibody therapeutics, as is indicated by anticancer therapy, for which antibodies are, as yet, seldom (if ever) curative. For example, a pivotal Phase III clinical trial for the treatment of metastatic colorectal cancer showed that — when included in a chemotherapy regimen (consisting of irinotecan (Camptosar; Pfizer Inc.), fluorouracil and leucovorin) —bevacizumab (Avastin; Genentech, Inc. and F.Hoffman-LaRoche Ltd; a vascular endothelial growth factor (VEGF)-specific monoclonal antibody) extended the median survival of patients from 15.6 to 20.3 months 32 . Therefore, in oncology, one of the goals for increasing the efficacy of antibodies is to improve their antitumor activity and to improve patient survival, by increasing the frequency of partial responses or, more preferably, complete responses, and by extending the duration of responses 32,33 . To improve safety, it might be useful to focus on limiting first-infusion reactions or more serious target-related adverse events. For example, the acute and severe influenza-like syndrome that is associated with administration of muromonab-CD3 (Orthoclone OKT3; Ortho Biotech Products a CD3-specific monoclonal antibody) is Fc-mediated and is largely overcome by attenuating the interaction between the Fc region of the antibody and the receptors for the antibody (IgG receptors; FcγRs) expressed by the patient. In general, increasing the potency of the antibody or extending its half-life in plasma might allow the dose or frequency of administration to be reduced, which might have the associated benefits of improved quality of life and/or convenience for the patient, and/or reduced cost of the drug. The high cost of antibody therapeutics that are produced in mammalian cells — commonly Chinese hamster ovary cells, NS0 mouse myeloma cells or hybridoma cells — might be reduced by using alternative hosts for production. For example, Escherichia coli is an established host for the generation of protein therapeutics and is gaining acceptance for the production of antibody fragments. Numerous other hosts are being explored for the production of antibodies, and these include other microorganisms, insect cells, and transgenic plants and animals 34. .
Antibodies are highly specific, naturally evolved molecules that recognize and eliminate pathogenic and disease antigens. The past 30 years of antibody research have hinted at the promise of new versatile therapeutic agents to fight cancer, autoimmune diseases and infection. Technology development and the testing of new generations of antibody reagents have altered our view of how they might be used for prophylactic and therapeutic purposes. The therapeutic antibodies of today are genetically engineered molecules that are designed to ensure high specificity and functionality. Some antibodies are loaded with toxic modules. Whereas others are designed to function naturally, depending on the therapeutic application.
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M.Pharm (Pharmaceutics), Lecturer, Department of Pharmaceutics, Rajiv Academy for Pharmacy, Mathura, Delhi Mathura bypass, Chhattikara 281006, India
Corresponding author – email@example.com
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Prof. Kamla Pathak
M.Pharm (Pharmaceutics),Ph.D., Department of Pharmaceutics , Rajiv Academy for Pharmacy, Mathura Delhi Mathura bypass, Chhattikara 281006, India