Case Study
- Anti-idiotype antibodies are powerful reagents for developing vaccine, pharmacokinetic (PK) and immunogenicity (anti-drug antibody) assays
 
An antibody that is directed against the antigen binding site of another antibody (i.e. where the antigen binding site is the epitope) is called an anti-idiotype antibody. There are three hyper-variable regions coincident with antigen binding sites: the Complementarity Determining Regions (CDRs). Since the anti-idiotype antibodies target the CDRs' (Fig. 1) antigen binding sites, the anti-idiotype antibodies may or may not compete with antigen. With these characteristics, anti-idiotype antibodies can be the powerful reagents for the following applications:

  • Vaccine development

  • Pharmacokinetic (PK) studies

  • Immunogenicity (anti-drug antibody) assays

 

( Fig. 1. Idiotype of antibody (Hypervariable regions) is the antigenic determinants )

  Anti-idiotype Antibody Application
 

1. Vaccine development

Anti-idiotype antibodies can be classified in two main groups, Ab2α & Ab2 β (Fig.2). Amongst them, the Ab2 β antibodies, which constitute about only 15-20 % of the total anti-idiotype antibody, carry an internal image of the nominal antigen.



(Fig. 2. General flow chart of anti-idiotype antibody network )

The Internal image of antigen-competing anti-idiotype antibodies can mimic antigens and thus may serve as immunogens for vaccine development, especially if the antigens are toxic or infectious.

 

2. Pharmacokinetic studies

The study of the tissue distribution and pharmacokinetics of antibody drugs requires the development of methodologies to differentiate the administered antibody drugs and endogenous antibodies. The most popular assay formats include Antigen Capture ELISA (Fig. 3), Antigen Capture Bridging Assay (Fig. 4) and Anti-idiotype Capture Sandwich ELISA (Fig. 5 & Fig. 6):



(Fig. 3 Antigen capture ELISA. ELISA wells are coated with antigen, incubated with serum samples, and detected with labeled anti-human IgG)



( Fig. 4 Antigen capture bridging assay. ELISA wells are coated with antigen, incubated with serum samples, and detected with labeled antigen)

The Antigen Capture ELISA and antigen capture bridging assay are the most conventional approaches for PK study of antibody drug. The disadvantage of these formats is the coated antigens may alter the conformation to some extent, and thus affect the binding affinities, and the resultant assay sensitivity. Additionally, for Antigen Capture Bridging Assays, the coated antigen is limited in order to provide optimal bridging and this will also affect the assay sensitivity.



(Fig. 5 Anti-idiotype capture sandwich ELISA. ELISA wells are coated with Strepavidin, incubated with biotin labeled anti-idiotype Ab (at C-terminus) and with serum sample, and detected with labeled anti-Hu Ig.)



(Fig. 6 Anti-idiotype capture sandwich ELISA.LISA wells are coated with anti-idiotype antibody as the capture, incubated with serum sample, and detected with labeled anti-Hu Ig)

The Anti-idiotype Capture Sandwich ELISA (Fig. 5 & Fig. 6), by using anti-idiotypes as the capture antibody, binds the antibody drugs specifically from the serum samples. If one has both antigen-competing and non-competing anti-idiotype antibodies one may quantitate the levels of antibody drugs in the circulation, both antigen-free and bound forms (1). Lofgren et. al. reported a study of "Comparing ELISA and Surface Plasmon Resonance for Assessing Clinical Immunogenicity of Panitumumab". They developed a panel of anti-idiotype mAbs, with affinities ranking from 1 ?M to 0.8 nM(2). Anti-idiotype mAbs are thus important tools for the clinical assay development, PK, and immunogenicity studies.

 

3. Immunogenicity studies

An important part of the consideration of any biological therapeutic approach is evaluation of immunogenicity data. This requires a multidisciplinary approach; review experts evaluate the sensitivity and specificity of the assay itself and the characterization of the immune response, for example whether they neutralize the activity of the product. Clinical reviewers must consider whether the immune response alters the serum levels of the product, affect clinical safety and efficacy or both. (3)

The FDA recommends a multi-tiered approach to the testing of patient samples.
  • Tier 1: A rapid, sensitive screening assay
  • Tier 2: Putative positive samples should then be subjected to a confirmatory assay, such as ligand or antigen competition assay.
  • Tier 3: Further characterization: neutralization/class/isotype/titer (4, 5)
For immunogenicity screening assay development, the FDA recommends several formats:
  • (A) Direct binding ELISA (Fig. 7)
  • (B) Bridging ELISA (Fig. 8)
  • (C) Radioimmuno precipitation assay (RIPA)
  • (D) Surface Plasmon resonance (SPR)
  • (E) Electrochemiluminescence assay




( Fig. 7 Direct binding ELISA. Coating with antibody drug and detecting with labeled anti-hu Ig)



(Fig. 8 Bridging ELISA. Coating with drug and detecting with labeled antibody drug)

A positive control is necessary for immunogenicity screening assay development. If non-primate animals are immunized with a full length antibody drug in order to develop a positive control, the antibody response is likely to be directed against the FC region. Such a positive control may not be relevant for the anticipated immune response in human patients where the response to antibody drug is primarily to the variable regions. Ideally, the anti-idiotypic monoclonal or polyclonal antibody is the best positive control for immunogenicity assay development.

 

Problems on Anti-idiotype Antibody Generation

The percentage of anti-idiotype antibodies in hyper-immune anti-serum is very low. During the hybridoma screening process, an anti-idiotype clone, which co-exists with a non-anti-idiotype one, will be missed if conventional screening is applied; positive to the antibody drug, and negative to unrelated IgG. To generate successful anti-idiotype monoclonal antibodies, appropriate immunization and hybridoma screening strategies are critical. For the generation of anti-idiotype polyclonal antibodies, the key factors are immunization and affinity purification.

 
Successful Cases Studies at GenScript

The percentage of anti-idiotype antibodies in hyper-immune anti-serum is very low. During the hybridoma screening process, an anti-idiotype clone, which co-exists with a non-anti-idiotype one, will be missed if conventional screening is applied; positive to the antibody drug, and negative to unrelated IgG. To generate successful anti-idiotype monoclonal antibodies, appropriate immunization and hybridoma screening strategies are critical. For the generation of anti-idiotype polyclonal antibodies, the key factors are immunization and affinity purification.

 

( Fig. 9 Anti-idiotype mAb against a human IgM anti-Target A)

 

(Fig. 10 Anti-idiotype mAb against a human IgG1 anti-Target A)

 
References:

  • 1. Karl Erik Hellstrom, Dale E. Yelton, H. Perry Fell, Donna Beaton, Margit Gayle, Michael Maclean, Maria Kahn, and Ingegerd Hellstrom. (1990) "Epitope Mapping and use of Anti-Idiotypic Antibodies to the L6 Monoclonal Anticarcinoma". Cancer Research 50, 2449-2454.
  • 2. James A. Lofgren, Sripriya Dhandapani, Jason J. Pennucci, Christina M. Abbott, Daniel T. Mytych, Arunan Kaliyaperumal, Steven J. Swanson, and Michael C. Mullenix. (2007)."Comparing ELISA and Surface Plasmon Resonance for Assessing Clinical Immunogenicity of Panitumumab" The Journal of Immunology 178: 7467-7472.
  • 3. Karen Weiss, M.D., FDA. News along the Pike, December 3, 2003
  • 4. Anthony R. Mire-Sluis, Yu Chen Barrett, Viswanath Devanarayan, Eugen Koren, Hank Liu, Mauricio Maia, Thomas Parish, George Scott, Gopi Shankar, Elizabeth Shores, Steven J, Swanson, Gary Taniguchi, Daniel Wierda, Linda A. Zuckerman (2004). "Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology". Journal of Immunological Methods 289: 1-16.
  • 5. Guidance for Industry Assay Development for Immunogenicity Testing of Therapeutic Protein FDA, December 2009

 
   
 
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