Creative Biolabs offers comprehensive immunogenicity assessment services for biotherapeutic drug candidates. Relying on the outstanding academic group and profound expertise, Creative Biolabs has successfully established the proprietary Sensitive Immunogenicity Assessment Technology® (SIAT®) system that can be utilized to evaluate the immunogenicity potential for a broad range of biotherapeutic drug candidates.

Background

Biotherapeutic drugs, including protein, enzyme, antibody, antibody-drug conjugate (ADC), are of significant value in the treatment of various diseases. Compared to small molecule drugs, the advantages of biotherapeutic drugs are mainly manifested by prolonged half-life, high target specificity, and low intrinsic toxicity. However, the use of biotherapeutic drugs can lead to unwanted immune responses such as the generation of anti-drug antibody (ADA) in patients, depending on multiple patient-related and product-related factors. The immune responses to biotherapeutic drugs decrease the efficacy of the drugs due to faster clearance of the drugs as antigens. In addition, unwanted immune system activation raises concerns of patient safety for issues such as rapid and enormous cytokine release. Therefore, immunogenicity risk management of biotherapeutic drugs and biosimilar biologics is critical for successful development. On the other hand, due to the enormous investment and cost in the whole process of developing a biotherapeutic drug candidate, it is worthwhile and necessary to assess the immunogenicity at the early stage of the drug discovery process. Above all, immunogenicity assessment is one of the major requirements for IND application.

Fig.1 Example roadmap for immunogenicity prediction. (Jawa et al., 2013)

Featured Services of SIAT® System

• In Silico Immunogenicity Assessment

SIAT® in silico immunogenicity assessment is able to analyze and predict the potential immunogenicity of biotherapeutic drug candidates. The protein sequences are analyzed by modern bioinformatics combined with experimental human HLA binding data to estimate the binding affinity of the peptide to HLA, which can predict the potential of the peptide for T cell activation. Our SIAT® in silico immunogenicity assessment service is the best fit for the early discovery and exploring stage.

• In Vitro Class II HLA Binding Assay

Although the in silico models can predict the binding affinity of peptides in biotherapeutic drug candidates to class II HLA alleles, the accuracy of these prediction algorithms is not always in accordance with physical binding data. Therefore, after having defined the epitopes of interest in silico, further in vitro assays are needed to validate these predictions, among which class II HLA/MHC binding assay is the most widely used.

• Ex Vivo Immunogenicity Assessment

Creative Biolabs offers SIAT® ex vivo immunogenicity assessment service that utilizes human peripheral blood mononuclear cells (PBMCs) to provide an immune response model. This model closely resembles the immune response in vivo. The responses of T cells or B cells to candidate biotherapeutic drugs are evaluated. Multiple techniques such as ELISPOT, ELISA, and Flow Cytometry are used according to different needs.

• In Vivo Immunogenicity Assessment

SIAT® in vivo immunogenicity assessment employs the standardized immunogenicity assessment protocols with a personalized approaching strategy. HLA transgenic mice or humanized mouse models are available at our facility and collaboration partners. These mouse models provide functional and reliable elements of human immune systems without putting patients at risk.

• Anti-drug antibody (ADA) Assay

Anti-drug antibody (ADA) has been observed in many preclinical and clinical studies, resulting in significant impacts in efficacy, toxicology, and pharmacokinetics of biotherapeutic drugs. The detection and confirmation of ADAs, as well as neutralizing antibodies (NAbs), comprise a fundamental part of immunogenicity assessment. Following current FDA and EMEA guidelines and AAPS white papers, SIAT® system can detect ADAs by using a variety of leading instrumentation platforms, such as ELISA/RIA, surface plasmon resonance (SPR) and electrochemiluminescence (ECL).

• Immunogenicity Assay for Preclinical Study

The immunogenicity data in preclinical studies is essential to the safety and efficacy evaluation of a protein therapeutic. Currently, we have established a variety of immune modeling assays to determine the immunogenicity in animal models, and test their roles in cell activation. In general, the T cell immune responses to biopharmaceuticals have been quantified for real-time kinetic measurements. Meanwhile, antigen processing and MHC affinity of T cells have been also analyzed in humanized mouse models.

• In Vitro Human Systems

The in vitro human system has been considered as the most suitable model for detecting the immunogenicity of potential biotherapeutic molecules in preclinical studies. Creative Biolabs has generated an in vitro human cell-based assay based on two-dimensional (2D) cell culture and three-dimensional (3D) cell culture technologies. Human cell-based assays play an important role in stimulating the cellular microenvironment to reveal in vivo cell mechanisms, including cell differentiation, cell growth, and cell activation. As a consequence, these models are attractive systems for evaluating immunogenicity in vitro.

• Immunogenicity Analysis of Immune-Related Diseases

Up to now, various types of biopharmaceuticals, especially for antibodies and therapeutic protein, have proven the efficacy in specific disease treatments. However, the immunogenicity of biopharmaceuticals still needs to be evaluated in different disease models, such as tumors, autoimmune diseases, infectious diseases. Creative Biolabs offers a series of immunogenicity analyses targeting specific disease types. We can help client to test the immunogenicity in disease models, and to further provide no immunogenicity version of biopharmaceuticals for clinical use.

• Immunogenicity Risk Assessment

At Creative Biolabs, we bring novel services to evaluate immunogenicity risk and provide a panel of assays to identify the risk factors and actual causes for immunogenicity in different kinds of candidate therapeutic drugs. Our platform consists of in silico and in vitro strategies to analyze the interactions among immune cells, immunogenicity risk, and lead targets. Pilot studies conducted by our labs have shown that physicochemical structural properties, immunological tolerance, administration route are key factors for causing unnecessary immunogenicity in pre-clinical and clinical studies.

Fig.2 SIAT® Immunogenicity System.

Creative Biolabs is one of the leading service providers for biotherapeutic drug development. Our scientists have gained extensive experience through years of cooperation with customers across the world. Our comprehensive SIAT® system offers in silico, in vitro, ex vivo, in vivo immunogenicity assessment and ADA assay services. We also provide de-immunization service if high immunogenicity potential is identified. All these services will assist customers in candidate selection and optimization in different phases of new biotherapeutic drug development. Contact us to discuss your project and experience the great value of our services.

Published Data

• Immunogenicity Assessment of Engineered Therapeutic Antibodies by Assessing IL-2 Secretion from CD4+ T Cells

Fig. 3 Induction of IL-2-secreting CD4+ T cells by therapeutic antibodies on Day 3. (Yoshiyuki Arata, 2023)

The study presented in the article evaluates the immunogenicity of engineered therapeutic antibodies by assessing interleukin-2 (IL-2) secretion from CD4+ T cells. This rapid in vitro method can predict the potential for anti-drug antibody (ADA) formation, a critical factor in the efficacy and safety of biotherapeutics. The results show that the assay effectively mirrors clinical outcomes regarding ADA incidence, as demonstrated by the response rates of antibodies known for their immunogenic profiles. Immunogenicity assessment in this study was crucial for determining how engineered antibodies might stimulate immune responses, particularly through the activation of CD4+ T cells that secrete IL-2, an early indicator of immunogenic potential. This assay allows for a quick and predictive measure of how a therapeutic antibody might interact with the immune system before extensive clinical trials.

FAQ

1. What is immunogenicity and why is it important to assess in biotherapeutic drug candidates?

   Immunogenicity refers to the ability of a substance, such as a biotherapeutic drug, to provoke an immune response in the body. This assessment is crucial for biotherapeutic candidates because an immune response can affect the drug's efficacy, safety, and pharmacokinetics. Detecting potential immunogenic reactions early in the drug development process helps mitigate risks associated with adverse reactions in patients.

2. What role does protein aggregation play in the immunogenicity of biotherapeutic drugs?

   Protein aggregation can significantly increase the immunogenicity of biotherapeutic drugs. Aggregated proteins can be more readily recognized by the immune system as foreign, leading to an enhanced immune response. Therefore, assessing and controlling aggregation during the formulation and stability testing phases is critical to minimize immunogenic potential.

3. How do post-translational modifications affect the immunogenicity of biotherapeutics?

   Post-translational modifications (PTMs) such as glycosylation, oxidation, deamidation, and phosphorylation can alter the physical and chemical properties of a protein, potentially making it more immunogenic. Differences in PTMs between a biotherapeutic and endogenous proteins, or batch-to-batch variations in these modifications, can trigger immune responses. Immunogenicity testing must therefore evaluate the impact of PTMs on the drug's immunogenic profile.

4. Can immunogenicity be predicted during the early stages of biotherapeutic development?

   While predicting immunogenicity with complete accuracy in the early stages is challenging, certain in silico and in vitro techniques can provide early indications. These include computational tools for predicting T-cell epitopes and in vitro assays using human immune cells to assess the immunogenic potential. These predictions, however, must be validated with clinical data as the development progresses.

5. What is the difference between binding antibodies and neutralizing antibodies in the context of immunogenicity?

   Binding antibodies are those that bind to a biotherapeutic drug but do not necessarily interfere with its biological activity. In contrast, neutralizing antibodies bind to the drug and inhibit its therapeutic function, directly impacting its efficacy. Immunogenicity assessments typically screen for both types of antibodies, but special attention is given to the detection and characterization of neutralizing antibodies due to their clinical implications.

6. What are the challenges associated with immunogenicity testing in clinical trials?

   Immunogenicity testing during clinical trials faces several challenges, including the variability in patient responses, the sensitivity and specificity of assays, and the interpretation of how immunogenicity impacts clinical outcomes. Additionally, the presence of drug in patient samples can interfere with immunogenicity assays, making it difficult to detect anti-drug antibodies. Overcoming these challenges requires robust assay development and validation, as well as careful analysis of clinical data.

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