Background

Biologic drugs, comprising proteins, peptides, or other biologically produced molecules, may trigger the immune system's recoginition of them as foreign substances, potentially prompting an immune response. Immunogenicity assessment assesses the strength of this response by detecting the presence of natural antibodies.

The efficacy of a drug can be influenced by its immunogenicity. The immune response not only reduces drug's activity but also expedites its clearance from the body, thereby affecting its effectiveness. Immunogenicity may not only induce short-term side effects like inflammation but also impacting the patient's long-term health. Consequently, the immunogenicity of a biological drug profoundly influences its safety, ultimately determining its suitability for clinical use. A strong immune response can even be life-threatening, such as a severe allergic reaction.

Therefore, thorough study of the immunogenicity of biological drugs is paramount. Indeed, the immunogenicity of such drugs can dictate their clinical viability. Should a biological drug exhibit excessive immunogenicity, researchers may explore modifications to mitigate this issue, such as deleting the immunogenic epitope.

Immune Modeling

Modeling preclinical immunogenicity assessment is a crucial step. Researchers need to employ various models to examine factors such as tissue damage, immune response, drug toxicity, pharmacokinetics and pharmacodynamics, cross-reactivity, drug safety and tolerability, and long-term effects.

The two most commonly utilized models are the in vitro cell culture model and the animal model. The in vitro cell culture model generally employs human or animal-derived cell lines to evaluate immune responses at the cellular level. On the other hand, animal models are used to assess immune responses in vivo, often utilizing in laboratory animals such as mice, rats, and rabbits. Physiological, biochemical, pathological, and molecular biological techniques are used to evaluate side effects.

In Vitro Cell Culture Model

The in vitro cell culture model is a prevalent method for detecting immunogenicity. By cultivating cells under laboratory conditions, this model enables the observation and detection of various biological phenomena, including cell growth, differentiation, death, and changes related to inflammation, immune response, or disease. Several types of in vitro cell culture models, including:

• Primary cell culture model: Fresh tissue or organ slices are digested using various enzymes or mechanically dispersed, and the isolated cells are cultured.
• Cell line culture model: Cells isolated from tissues are continuously cultured in vitro until they exhibited "immortalized" characteristics such as unlimited growth and non-differentiation, forming a cell line.
• Three-dimensional tissue culture model: This complex model simulates tissue structure in the body, allowing cells to interact in three dimensions, form tissues, and observe how these tissues respond to drugs or other stimulants.
• Cell co-culture model: Different types of cells are co-cultured to interact and mimic the body's environment in the body.

These in vitro cell culture models facilitate the simulation and detection of immune responses in a controlled and actionable environment. Thy serve as a platform for testing different types of immune stimulation and their impact on the health and function of cells and tissues.

Fig. 1 In Vitro Cell Culture Model

3D Tissue Culture Model

The 3D tissue culture model, also known as the 3D-Culture model, is an experimental approach that mimics the in vivo environment. It more accurately reflects the cellular growth conditions within the body and has become increasingly prevalent in medical research in recent years. Various methods are commonly used to construct 3D tissue culture models:

• Polymeric culture method: This method based on cell aggregation behavior within a rotating ultra-low density culture environment, enabling spontaneous cell-cell interactions to form a three-dimensional spherical structure. It effectively simulates the cellular microenvironment in vivo.
• Suspension culture method: Cells are cultured in suspension by controlling the liquid flow state within the culture container. Common techniques include rotating wall culture, and plane oscillation culture.
• Culture ball method: Special cell culture plates, such as ultra-low adhesion culture plates, prevent cell attachment to the plate surface, promoting in the formation of tight cell masses within the culture matrix and , thus simulating tissue's three-dimensional structure.
• Bioprinting technology: This innovative approach precisely prints biological materials, such as cells and bioactive factors, according to preset three-dimensional models enabling the construction of complex tissues and organs.
• Colloidal culture method: Colloids, such as AGAR, collagen, and alginate, serve as the matrix for cell culture. By altering the physical properties of the colloidal matrix, such as hardness and pore size, researchers can regulate the cell growth environment, facilitating the formation of a three-dimensional structures.

When constructing a 3D cell culture model, researchers should choose the appropriate construction method based on their research purpose and experimental requirements. Moreover, given the complexity of three-dimensional culture models, adherence to standardized operating procedure during experimentation is essential to ensure the reliability of experimental results.

Animal Model

In immunogenicity evaluation, animal models serve as crucial tools for studying the body's immune response. In general, animal models can be divided into two categories: natural animal models and artificially induced animal models.

• Natural animal models: These models involve animals that naturally exhibit immune responses or disease models similar to humans under natural conditions. For example, pigs share similarities with humans in certain immune responses, making them valuable model animals in many immunogenicity studies.
• Artificially induced animal models: This type of model is mainly created through genetic engineering, surgery, and other methods to induce animals to produce immune responses or disease models similar to humans. For example, mice can be genetically modified or knocked out to develop human disease traits or similar immune responses.

Such models are designed to simulate human body responses, facilitating the assessment of the immunogenicity of natural or artificial immunogens. They play a pivotal role in immunogenicity assessment. However, due to inherent physiological and biochemical differences between animals and humans, results from animal models can only serve as reference points, necessitating further confirmation through clinical trials to assess their effects on humans.

Fig. 2 Mouse Animal Model

Detection Methods of Animal Models

The detection of animal models usually involves various techniques, including but not limited to:

• Histology and pathology: Examination of tissue sections to observe changes in cell and tissue structure, often involving morphological analysis under light or electron microscopy and immunohistochemical techniques to detect specific protein markers such as inflammatory or apoptotic markers.
• Molecular biology: Utilization of PCR, Western blotting, and other techniques to detect changes in the expression of specific genes or proteins.
• Biochemical analysis: Determination of levels of specific biochemical markers (such as enzymes, peptides, lipids, etc.) in blood or tissue samples to reflect the extent of tissue damage.
• Functional assays: For example, in a myocardial infarction model, assessment of tissue damage impact by measuring cardiac function parameters like heart rate, blood pressure, and left ventricular function.

Creative Biolabs

Creative Biolabs is backed by an advisory board comprising seasoned professionals in animal modeling, immunology, preclinical research, drug toxicology, analytical chemistry, and immunologic analysis. With substantial experience garnered from diverse projects, Creative Biolabs has earned considerable recognition and praise from clients. In Creative Biolabs' SIAT® immunogenicity tests, data on lymphocyte infiltration in various tissue sites are compiled, shedding light on interactions between drug-induced tissue damage and immune responses.

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