Analysis of immunogenicity of therapeutic protein drugs

  • The immunity of therapeutic protein drugs refers to the response of sexual protein and/or other disease treatments to self-treatment or the ability of related proteins to significantly alleviate or related events. The reaction has a wide range of effects, from the temporary emergence of drug resistance with no clinical significance, to the ability to change the efficacy of the drug or produce side-life drugs, and even serious to endangered. Therefore, immunogenicity research has always been an important part of drug research. -Non-clinical research-Clinical research-The whole life drug immunogenicity research monitored after marketing includes drug optimization clinical use program, drug clinical application program, drug clinical application, and even the success of drug research is reduced.


    Immunogenicity research has always been a difficult point in drug evaluation, involving a series of immunology, pharmacology, toxicology, clinical medicine, randomization, etc., with more content, increased intensity, less mature experimental methods, and inconsistent understanding in the industry. In this article, a simple drug immunization is carried out on the analysis of therapeutic protein immunity.


    The formation mechanism of immunogenicity and its influencing factors


    1. The formation mechanism of immunogenicity




    The body's immune response caused by therapeutic proteins includes both innate immunity and adaptive immunity (cellular immunity and humoral immunity). Innate immunity is mainly caused by microbial contamination, etc. Generally, there is less discussion, and more discussion and monitoring of the adaptive immune response of therapeutic proteins. Adaptive immunity includes not only the release of cytokines mediated by cellular immunity, but also the production of anti-drug antibodies mediated by humoral immunity.


    The general process of cellular immunity includes the differentiation of T cells and the release of cytokines, which can cause changes in the ratio of different T cell subtypes and changes in peripheral cytokine levels.


    Humoral immunity mainly works through anti-drug antibodies (ADAs) produced by B cells against therapeutic proteins. Anti-drug antibodies include not only binding antibodies (BAbs) that bind drugs, but also neutralizing ADAs (NAbs) that block their function. According to the production mechanism, it can be divided into two kinds of mechanisms: T cell dependent pathway and non-T cell dependent pathway. In fact, because immune complexes or aggregates can activate these two pathways to varying degrees, and both pathways will produce ADA, there is no obvious difference between the two mechanisms, and they will interact with each other. Increase the strength of the opponent's action.


    T cell dependent pathway


    The immune response generated by the T cell-dependent pathway is characterized by high intensity and long duration. After this response is initiated, the memory cells in the body will produce an immune response when the body encounters the same antigen again. When an antigen invades the body, antigen presenting cells (APCs) degrade the protein into linear antigen peptide fragments through phagocytosis or pinocytosis. The important antigen-presenting cells in the body include dendritic cells (inhibitory dendritic cells, iDCs), macrophages, and B cells. T cell activation requires CD4+ assistance and dual-signal co-activation: antigen peptides and major histocompatibility complex-2 (MHC-II) combine to form MHC II-antigen peptide complexes, which are expressed in APCs The surface is for TCR to recognize and bind; this process requires the costimulatory molecules (such as CD86 or CD80) on the surface of APCs to simultaneously bind with the costimulatory molecules (such as CD28) on the surface of T cells. The activated T cells proliferate, secrete specific cytokines to activate B cells, B cells proliferate and differentiate, and secrete ADA. The IgM type ADA with low affinity is initially formed. As B cells continue to differentiate, with the participation of cytokines, ADA can undergo class switching to produce IgG, IgA, or IgE type ADA. IgG type ADA has high affinity with drugs and is the most typical and common type of ADA. In addition, in the T cell-dependent pathway, a small number of B cells will differentiate into memory B cells with longer memory function, and secrete antibodies when the body encounters the same antigen stimulation again.


    T cell-independent pathway


    Without the participation of T cells, the body can also produce ADA for therapeutic protein aggregates, that is, a non-T cell dependent pathway. Drugs or ADAs immune complexes in a multimeric structure can directly bind to B cell receptors (BCR) to oligomerize and internalize BCR, activate B cells, and promote B cell differentiation and proliferation. Generate ADA. Without T cell assistance, the ADA produced by this pathway will not undergo class switching, so the ADA secreted by this pathway is mainly of IgM type, which has low affinity, short duration, poor specificity, and is not as strong as the T cell-dependent immune response. In addition, this pathway does not distinguish between self-antigens and non-self-antigens. Even with self-antigens, B cells may be activated to produce ADA if they form a compact multimeric form of 50 to 100 Å.


    1. Factors affecting immunogenicity


    Although all therapeutic protein drugs have the potential to induce immune responses, different therapeutic protein drugs have different possibilities of immune response. The production of immunogenicity is the result of a combination of multiple factors, such as: patient background (genetic, pre-existing state), drugs (structure, size, sequence, impurities, preparations, etc.), treatment methods (administration method, frequency, duration Etc.) Factors have an impact on the formation of immunogenicity. The United States Pharmacopoeia General Chapter 1106 sorts out these influencing factors, which can be divided into three aspects.


    2.1 Patient-related factors


    Everyone's immune status is different, and the differences in major histocompatibility and human leukocyte antigen (HLA) alleles are closely related to the individual's immune response. For example, patients with HLA-DRb-11, HLA-DQ-03, HLA-DQ-05 alleles are generally more likely to develop immunogenicity.


    The activation state of the patient's immune system is also related to the occurrence of immunogenicity. The immune system of patients with autoimmune diseases is in an activated state and is generally prone to produce immunogenicity, while the immune system of patients with tumors is generally in a suppressed state and is not prone to produce immunogenicity.


    Whether there is a history of exposure to related drugs also affects the occurrence of immunogenicity. For example, PEG is widely used in cosmetic products, and patients may often contact related products containing PEG and have pre-existing antibodies against PEG.


    2.2 Drug-related factors


    Drug structure


    Therapeutic protein is regarded as a foreign substance by the body's immune system. The efficacy of the drug is usually determined by the integrity of the epitope sequence on its structure. The oxidation, deamidation, deamination, degradation, conformation and other structural changes and sequence differences of the drug All are considered to be risk factors for immunogenicity because they can change the conformation of proteins, aggravate the aggregation of drugs, or change the kinetics of antigen uptake, processing and presentation of antigen peptides. The chemical changes of the above-mentioned drugs are similar to biotransformation and may occur in damaged physiological environments, such as inflamed tissues. Structural changes in recombinant endogenous protein drugs will also break immune tolerance, leading to the production of ADA.


    Glycosylation in the structure of therapeutic proteins can also affect the immunogenicity of the drug. The post-translational modification of the therapeutic protein is determined by the expression system or cell line of the selected product. Unlike other cell lines such as yeast and insect cell lines, the glycosyl structure of products obtained from mammalian cell lines is considered to be closer to the natural human glycosyl structure. Maintaining the same glycosylation modification as human is particularly important for recombinant human protein therapeutics. The type of glycosyl not only affects the function of the therapeutic protein, but also affects the stability of the product, the folding of the protein, and the optimization of biophysical characteristics. Therefore, the glycosylation structure needs to be detected during the production process. However, although some glycosylated structures are antigenic, their aggregation is reduced and immunogenic epitopes are blocked from time to time.


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