Anti-idiotype (anti-ID) antibodies bind to idiotopes that are located in the variable region of another antibody and thus can be useful in highly sensitive and specific assays for antibody-based drugs. They may also have potential as therapeutic agents. Rapid access to custom anti-ID antibodies is essential, however, if novel assays and therapeutic candidates are to be developed within the accelerated timelines expected today. Sino Biological has the experience, high-throughput production capabilities, manufacturing capacity, and analytical expertise to rapidly provide custom anti-IDs.

Mirror-Image Antibodies

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

Many Types of Anti-idiotype Antibodies

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.

Important Agents for Preclinical and Clinical Antibody Development

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.

Potential for Therapeutic Use

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

Immunization and Screening Are Critical to Successful Anti-ID Antibody Development

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.

Anti-ID Antibody Services at Sino Biological

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 high-throughput recombinant technologies, ever-expanding numbers of manufacturing suites and process development laboratories, and standardized processes from gene synthesis through protein/antibody expression and purification to product shipping, Sino Biological is positioned to meet expectations for shortened delivery timelines. Indeed, the company has the capability to work on more than 1,000 projects at the same time. For example, 1,000 different recombinant antibodies can be delivered to a customer within three to four weeks. Sino Biological's manufacturing facility can also handle large-scale production at the gram level. Furthermore, the company provides one-stop custom antibody services covering the initial antigen design to final scale-up antibody production.

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.

At Sino Biological, both monoclonal and polyclonal anti-ID antibodies can be produced to support drug discovery, preclinical research, and clinical trials. Monoclonal anti-ID antibodies are widely used to detect the plasma concentrations of antibody drugs for PK/PD analysis. Polyclonal anti-ID antibodies purified directly from antisera can better simulate the real conditions in the blood samples of subjects, and thus are mainly used for immunogenicity assessments of antibody drugs. Based on comprehensive technology platforms, such as hybridoma, single B cell screening, phage display, and polyclonal antibody development platforms, Sino Biological provides mouse mAb, rabbit mAb, and rabbit pAb service packages for anti-ID antibody development. Sino Biological has completed more than 100 monoclonal and polyclonal anti-ID antibody projects with a success rate greater than 95% and has developed a large number of anti-ID antibodies with high affinity and specificity, as well as cross-reactivity with total IgG <2% or even no cross-reactivity, that fully meet the needs of customers for PK and ADA assays.

References

1. Stanova, Aliya K. et al. “Anti-Idiotypic Agonistic Antibodies: Candidates for the Role of Universal Remedy.” Antibodies (Basel). 9(2): 19 (2020).
2. Kohler, Heinz, Anastas Pashov, and Thomas Kieber-Emmons. “The Promise of Anti-idiotype Revisited.” Immunol. Sec. Vaccines and Molecular Therapeutics. 12 Apr. 2019.
3. Naveed, A., Rahman, S.U., Arshad, M.I. et al. “Recapitulation of the anti-Idiotype antibodies as vaccine candidate.” Med. Commun. 3: 1 (2018).
4. Harth, S., Frisch, C. “Recombinant Anti-idiotypic Antibodies in Ligand Binding Assays for Antibody Drug Development”. In: Posch, A. (eds.) Proteomic Profiling. Methods Mol. Bio vol 2261. (2021).