January 13, 2023 PAO-01-23-CL-01
Antibodies that bind other antibodies were first discovered in the early 1900s.1 In the 1970s, Niels Jerne proposed that a network of idiotype and anti-idiotype (anti-IDs) antibodies existed.2 An idiotype is the collection of idiotopes contained in a single antibody, and anti-IDs are antibodies that bind to the idiotopes.3 The proposed network consists of antibodies, anti-IDs to those antibodies, anti-IDs to the anti-IDs, and so on. It is believed that, when new antibodies are generated in the body against foreign pathogens, anti-IDs are also spontaneously created.
Certain anti-IDs can block antigen binding and thus impede immunological responses, while others do not. The antigen-blocking anti-IDs may serve as a mirror image of the original antigen. In general, anti-IDs have the potential to alter the effects of many different immunogenic agents and thus may play a role in regulating many different cellular functions.1
Five different types of anti-ID antibodies have been identified to date.1 Ab2α anti-IDs bind to idiotopes of primary antibodies that do not participate in antigen recognition and therefore do not impact antigen binding. These anti-ID antibodies bind to free and bound antibody drugs and are non-inhibitory. Ab2β anti-IDs, however, bind to the idiotypic antibody involved in antigen recognition and thus can inhibit antigen binding and consequent immune responses, including stimulation of other anti-ID antibodies. They bind only to free antibody drugs and are considered to be inhibitory. Ab2γ anti-IDs bind close to the antigen-binding site of the primary antibody and thus may interfere with binding to the antigen. Ab2ε anti-IDs bind to both the primary antibody and its antigen when they are complexed together. Ab2δ anti-IDs recognize non-binding regions within the variable region of the heavy chains of primary antibodies.
Ab2β anti-IDs have attracted the most attention because they can serve as substitute antigens.1 It is important to note, however, that their behavior cannot be solely attributed to structural similarities: the similar binding interactions of Ab2β anti-IDs mediate their functional mimicry, but not because the three-dimensional structures of the paratopes of anti-ID antibodies are closely identical to the antigenic epitopes.
The safety and efficacy of all drug candidates must be established before application for marketing authorization. For antibody drugs, many different assays must be performed throughout preclinical development in animal models and clinical studies in humans. These assays range from determination of the pharmacokinetics (PK) and pharmacodynamics (PD) of the drug candidates to immunogenicity testing to toxicity analyses. Anti-ID antibodies can be useful in some of the assays required for antibody-based drug candidates.
Drug absorption, distribution, metabolism, and excretion (ADME) testing requires information on the concentration of the antibody drug candidate in the bloodstream and different bodily fluids and tissues over time. PK analyses leveraging anti-ID antibodies are used to determine the concentration of the antibody drug in animal and human bodies from the time of initial adsorption through distribution. They can also be used to determine the bioavailability of such drugs, as well as the time it takes for them to be cleared from the body.
Traditional PK analyses rely on the use of regular binding assays in which a plate is coated with the drug ligand and then exposed to the serum from the animal model or human patient. A secondary detection antibody (e.g., anti-IgG1) is then added that can detect the specific isotype. This approach, however, is not very sensitive or specific. There are two main reasons — the detection antibody also recognizes unrelated antibodies, which are at a high concentration in serum and bind nonspecifically to the plate, resulting in high background signals, and the serum can contain auto-antibodies to the drug ligand, resulting in false positive results.4
The concentrations of antibodies in human blood serum are approximately 10 mg/mL and antibody drugs are approximately 10 ng/mL, and thus analyses must be highly sensitive. Assays using anti-ID antibodies provide this high level of sensitivity and are specific to the target antibody drug. A bridging setup is commonly used — plates can be coated with anti-ID antibodies that capture one of the two separate arms of the antibody drug candidate, the other binding arm of which can be then recognized for detection by another labeled anti-ID antibody or the labeled drug ligand.
Antibody drugs can be free, partially bound, or fully bound to the drug targets in serum, so understanding what drug states are being detected is important. Based on different binding modes of anti-ID antibodies to antibody drugs, inhibitory, non-inhibitory, and complex-specific anti-ID antibodies can be used to measure free, total, and bound antibody drugs in serum, respectively.
Similarly, determination of the immunogenicity of antibody drugs requires detection of low levels of anti-drug antibodies (ADA) generated as part of natural immune responses to these drug candidates. In ADA assays, a bridging format is also widely used — a plate is coated with the antibody drug, which captures any anti-drug antibodies present in the animal or human patient serum. Then, anti-drug antibodies are detected by exposing them to the antibody drug candidate that is appropriately labeled. Due to the similarity between anti-ID antibodies and anti-drug antibodies, anti-ID antibodies can be used as a positive control or reference standard in ADA assays.
Given the higher sensitivity and specificity possible with assays based on anti-ID antibodies, anti-ID antibodies are now considered to be reagents critical for both the preclinical and clinical assessment of antibody drug candidates.
In addition to their value as analytical reagents, anti-ID antibodies are also believed to have potential as therapeutic agents, as they mimic the antigenic structure and potentiate antibody and cell-mediated immunity.3 Several studies have explored their use as therapeutic vaccines against infectious diseases and cancer and as treatments for autoimmune diseases and other disorders.1,2 In particular, anti-ID antibodies have been identified that mimic the effects of cytokines, neurotransmitters, hormones, various toxins, and illegal drugs (notably opiates — they bind opiate receptors, thereby preventing the binding of illegal drugs and the consequent biological effects).1
It is true that, to successfully develop anti-ID antibody production processes, both thorough immunization and screening processes must be employed.
Immunogens can either be full-length antibodies or digested F(abʹ)2 fragments. Full-length antibodies are more likely to cross-react with human total IgG and isotype IgG, thereby complicating the screening of anti-ID antibodies. Conversely, full-length antibody preparation is cost-effective, because enzyme digestion is not required, and the produced anti-ID antibodies have a higher probability of being recognized and bound by the original antibody. It is necessary to choose more advantageous immunogens according to particular needs in practice.
In addition to routine immunization, Sino Biological has established rapid immunization technology platforms for urgent projects. For fast mouse mAb production, the immunization period is shortened from 8–12 weeks to 4–5 weeks. For fast rabbit polyclonal antibody (pAb) production, the products can be delivered in only 45 days from rabbit immunization to antibody purification, which requires 75 days using standard processes.
Various types of screening tools can be employed to screen for inhibitory or non-inhibitory anti-ID antibodies, as well as anti-ID antibodies that bind to antibody drug–antigen complexes. ELISA is the primary method of screening, and it typically takes 2–3 rounds of screening to identify suitable clones for their downstream application. Anti-ID antibodies are first validated for its binding to the antibody drug but not an isotype control. However, this alone would be insufficient to identify the inhibitory idiotype, and competitive ELISA in the presence of the antigen is necessary to obtain Ab2β and Ab2γ. To identify anti-ID antibody pairs, additional bridging assays should also be performed.
Sino Biological offers the world’s largest selection of bioactive recombinant proteins and antibodies (monoclonal, polyclonal, and newer formats). The company also provides custom manufacturing and research services. Leveraging
The experience that Sino Biological has gained developing manufacturing processes for thousands of antibodies from well-known, conventional IgGs to novel, highly complex multifunctional molecules enables the company to overcome the challenges that arise as projects progress and still develop cost-effective processes within short timelines that afford high yields of high-purity products. The experience characterizing all these different types of antibodies also makes it possible for Sino Biological to develop unique analytical methods for challenging antibodies.
Anti-ID antibody production is a custom service offered to clients. In order to speed up clients’ drug development, Sino Biological provides a full range of custom anti-ID antibody production services from antigen preparation and anti-ID antibody development to detection method establishment and kit development. The company has strong expertise and experience in targeting diverse modalities, including full-length mAb, F(ab')2, Fab, scFv, VHH, ADC, bispecific antibody, and Fc-fusion protein. Customized anti-ID antibodies can be used to set up PK or ADA assays to determine specific antibody drug or ADA levels in samples.
Na Li (Lina), Ph.D., QIHC CM, is a technical account manager at Sino Biological. She completed her Ph.D. research at the Singapore-MIT Alliance for Research and Technology. She has multiple years of experience as an antibody-based assay development scientist and has led pre-clinical projects involving multiple research areas including cancer therapy, toxicology, and infectious diseases. Lina joined Sino Biological in 2022 as a technical account manager, helping clients from various biotechs, academic research groups, and pharmaceutical companies with protein and antibody development.