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The Introduction of Fab Fragment Antibody

What is Fab fragment antibody?

With the rapid development of DNA recombination technology and the further understanding of antibodies, the third generation of antibodies, recombinant antibodies, following polyclonal antibodies and monoclonal antibodies, has emerged. Small molecule antibodies in recombinant antibodies have become the main members and research hotspots of the recombinant family because of their characteristics.The main small molecular antibodies are Fab fragment antibody, scFv fragment antibody, single domain antibody, bispecific antibody, double chain antibody, tribody and minibody.

Figure1.Fab fragment

Fab fragments, also known as antigen-binding fragments, are regions in the structure of antibodies that can bind to antigens. Fab fragment consists of a complete VH and CH1 domains of light and heavy chains. Both light chain and heavy chain have a constant region and a variable region, and there are disulfide bond links between light chain and heavy chain. Like monoclonal antibodies, the development of Fab antibodies has gone through four stages: murine, chimeric, humanized and fully human monoclonal antibody.

Structure of Fab

Natural polyclonal antibodies and monoclonal antibodies are immunoglobulin molecules. IgG molecule consists of two identical 5×104 Dolton heavy chains and two identical 2.5×104 Dolton light chains. Fab molecule is composed of light chain and heavy chain, about 5×104 Dolton. The interaction between the two chains and the disulfide bond formed play a stable role in the double-chain structure of Fab molecule.The antigen binding sites of Fab and IgG are composed of six complementarity determination regions (CDR), which are located on light chain and heavy chain respectively, and form the core of antigen binding sites. Among the three-dimensional structure of the variable region, two opposite parallel beta-folded peptide chains form the framework region. The outer side is connected by highly variable CDR rings, forming a sandwich structure. The inner side is connected by conservative disulfide bonds, which makes the CDR stand out.

Fab fragments are obtained mainly by chemical, enzymatic and genetic engineering methods. Chemical method is to use chemical reagents to act on the N-terminal of disulfide bond between heavy chains, open the disulfide bond, and obtain two identical antigen binding fragments. It is also a common method to digest full-length IgG antibodies with papain, pepsin and fig protease.Fab fragment antibodies of functional antibodies obtained by genetic engineering are expressed mainly through E. coli system, Pichia pastoris system and insect system, the most common of which is E. coli system. However, the low expression of Fab fragment antibody in E. coli system is the main factor restricting its large-scale production, so we will learn about the optimization measures of E. coli production today.

1. Optimization of host bacteria

Because the structural stability of Fab fragments is determined by the disulfide bond between the heavy chain Fd segment and the complete light chain, and the formation of disulfide bond requires oxidative environment, it is difficult to obtain functional antibody fragments in the cytoplasm of reductive environment. It is necessary to guide light chain and heavy chain Fd segment to the periplasmic cavity by signal peptide for correct folding. Recently, the cavity environment is particularly important.In addition, modification of strains, co-expression of catalysts and formation of oxidative environment in cytoplasm are also alternative measures. The difference of expression hosts has a significant effect on the total expression of Fab, mainly due to some proteases in host bacteria, especially periplasmase or outer membrane protein-related genes. The protease of host itself can degrade foreign proteins, and the deletion of outer membrane protein-related genes can release more Fab fragments into the culture medium, which is more conducive to purification and has practical application value in production.

2. Selection of expression vectors

When selecting expression vectors, we should start from promoter, replicator, selective label and fusion protein expression sequence, and select the appropriate combination of expression elements to express Fab fragment.

Selection of replicators

The choice of replicators depends on the number of copies. In general, it can be considered that plasmids with high copy number mean higher protein expression. However, a large number of plasmids exert more metabolic pressure on E. coli, which in turn reduces the growth and reproduction rate of E. coli, and consequently reduces the expression of the target protein. Therefore, high copy number does not represent high protein expression.

Promoter selection

In protein expression, a strong transcription promoter is needed to control the high level of expression, while avoiding the basic expression as far as possible.

3. Vector design (target gene sequence)

The target gene sequence is the key factor affecting the expression level. The position order of light chain and heavy chain is different, and the expression level varies greatly. The classical vector design method is based on the same promoter and the ribosome binding sites of the light and heavy chains. The N-terminal of the heavy and light chains are added with their respective signal peptide sequences to guide the heavy and light chains into the periplasmic cavity for correct folding.

4. Choice of molecular chaperones

Because of the role of strong promoter, the cell resources of host bacteria are used to express the target protein. The level of target protein expression is often high. As the molecular chaperone of host bacteria cannot meet the correct folding of the peptide chain, inclusion bodies will be formed. Introducing exogenous molecular chaperones to assist with the correct folding of target proteins is a way to increase the production of active proteins and increase the production of active proteins.

5. Optimization of culture conditions

The secretion position of Fab antibody produced by Escherichia coli is closely related to the culture conditions. Specific culture conditions can make Fab molecules more secreted and expressed in the supernatant, possibly because the accumulation of Fab molecules in the periplasmic cavity causes the breakdown of the bacteria, or because the culture medium or culture temperature causes the permeability of the outer membrane of E. coli to increase.

6. Selection of culture medium

In the production of Fab fragment antibody, the composition of culture medium has an important influence on soluble expression. Buffer solution rich in metal ions and salt ions is more conducive to Fab expression, especially soluble expression. Metal ions can help fold enzymes form disulfide bonds and increase the expression of soluble proteins.

7. Purification of Fab antibody

In addition to constructing better vectors to increase the expression of Fab antibody, improving the purification process to enhance the purification efficiency can also improve the production of Fab.

Compared with full-length antibody, Fab antibody has attracted more and more attention. It has been widely used in the treatment of tumors, immune diseases and ophthalmic diseases. As a tracer diagnostic agent, it also has a good effect.
publié le samedi 17 août à 13:26, aucun commentaire.

Introduction to vaccine types and differences (part two)

3. Live-attenuated vaccine
This type of viral vaccine has more than 90% efficacy and its protection usually lasts for many years. Its outstanding advantage is that the pathogen produces an antigenic stimulus in the host replication, the amount, nature and location of the antigen are similar to the natural infection, so the immunogenicity is generally strong, and even does not require booster immunity. This outstanding advantage is also potentially dangerous: infection can be triggered in individuals with poor immunity; mutations may restore virulence. The latter can be more rationally attenuated with the understanding of the molecular basis of the pathogenic virulence, which may make it more effective to attenuate the virulence.
4. Inactivated vaccine
Compared with live attenuated vaccines, inactivated vaccines used non-replicating antigens (dead vaccines). Therefore, they are safe, but their immunogenicity is also weakened, and it is often necessary to boost immunity. It should be noted that not all pathogens can be highly effective vaccines after inactivation: some of these vaccines are highly effective, such as the polio vaccine (IPV) or hepatitis A vaccine for injection; others are inefficient, Short-lived vaccines, such as inactivated cholera vaccines, have almost been abandoned; there are some partially inactivated vaccines that are less effective and need to increase their protection rates and duration of immunization, such as traditional inactivated influenza and typhoid vaccine.
5. Vector vaccine
The vector vaccine introduces an antigenic gene into the body through a vector that is harmless to induce an immune response. It is characterized by a combination of strong immunogenicity of live attenuated vaccines and the accuracy of subunit vaccines. A significant benefit of this live vector vaccine is that it can effectively induce cellular immunity in vivo, which is currently not good enough to induce cellular immunity, and cellular immunity is promising in the context of some diseases that are particularly important. Important vectors used in the assay are variants of vaccinia virus, poliovirus, fowlpox virus, adenovirus, herpes virus, salmonella, Shigella, and the like. It is also possible to construct one or more cytokine genes at the same time, which can enhance the immune response or change the direction of the immune response.
The polysaccharide protein conjugate vaccine chemically binds the polysaccharide antigen to the carrier protein, and the antigen type changes from TIA to Thymus Dependent Antigen (TDA), which can stimulate children under 2 years old, the elderly and immunodeficiency produces an effective immune response and produces immune memory. The induced protective antibody is mainly immunoglobulin G, which is more effective than the polysaccharide vaccine-induced IgM (which can also stimulate IgG2 in human body) and can last for a long time with the incomparable advantages of polysaccharide vaccine
Hapten antigen, a small molecule, which alone does not induce an immune response, is not immunogenic, but is immunogenic when it is cross-linked or bound to a carrier such as a macromolecular protein or a non-antigenic polysine that induces an immune response. These small molecular substances can bind to the response effect product and are antigenic. They are only immunoreactive and not immunogenic, also called incomplete antigen. Most polysaccharides and all lipids are haptens. If the hapten is chemically combined with a molecule (carrier) of a pure protein, the pure protein will acquire new immunogenicity and stimulate the animal to produce the corresponding antibody. Once a hapten binds to a pure protein, it constitutes an antigenic cluster of the protein. Some chemically active group substances (such as penicillin, sulfonamides, etc.) which have a smaller molecular weight than the general hapten but have a specific structure are called simple haptens. When a simple hapten enters an allergic body, it binds to tissue proteins in the body. Become a complete antigen, this complete antigen can cause hypersensitivity reactions. In general, B lymphocytes recognize hapten determinants, and T lymphocytes recognize vector antigenic determinants.
6. Toxoid vaccine
Toxoid vaccines have great significance when pathological changes in the disease are mainly caused by strong exotoxin or enterotoxin, such as tetanus and diphtheria vaccines. In general, the toxoid of enterotoxin is rarely successful. However, the heat-stable enterotoxin (LT) of enterotoxin-type Escherichia coli has a genetically engineered detoxified allosteric construct that promises to be an effective traveler diarrhea vaccine. Mutations corresponding to cholera toxin (CT) may become a more important vaccine. These two toxin variants can even induce good mucosal immunity and are promising mucosal immune adjuvants. Most of the currently used toxoid vaccines are manufactured using conventional techniques. These vaccines, such as diphtheria and tetanus vaccines, contain many impure components, and the formaldehyde treatment of toxins into toxoids also results in cross-linking with bovine-derived polypeptides from the culture medium, ultimately producing unnecessary antigens. Therefore, the study of a mutant, non-toxic pure molecule as a new vaccine can improve the quality and efficacy of these vaccines, such as the replacement of diphtheria toxin 52 at the glutamate to glycine, can lead to loss of toxicity, and can cross-react with diphtheria toxin.
Virus-like particles (VLPs) are assembled from viral structural proteins with self-assembly characteristics, and are similar in morphology to pathogens. VLPs do not contain viral nucleic acid substances, are not infectious, and cannot replicate autonomously; VLPs can repeated high-density expression of antigenic epitopes, resulting in a strong immune response, so VLPs are an ideal form of vaccine for a variety of diseases.  Enveloped virus-like particles (eVLPs) has the same size and structure as enveloped viruses and present antigens in their natural state for an improved immune response. Compared to non-enveloped VLPs, the former is relatively complex in structure, contain a host cell membrane, and the host cell membrane (envelope) is cross-linked to the target antigen displayed on the outer surface. Envelope VLPs can integrate multiple antigens of homologous or heterologous pathogens, producing envelope VLPs. Some structural proteins that require co-expression of the virus are assembled into granules that are released from the cell membrane in the form of budding. A variety of cell matrices can be used to produce enveloped VLPs, including: yeast cells, insect cells, mammalian cells, plant cells, and the like; in addition, chimeric envelope VLPs can also be prepared in vitro as virosomes.
The immunogenicity of VLPs can be enhanced by immunostimulating adhesion molecules and cytokines. CD40L is expressed on the surface of HIV VLPs and interacts with CD40-CD40L to localize and activate DCs, enhancing CD4+, CD8+ T cell responses. The virus-like particles can be used as an antigen carrier and an adjuvant delivery system, and the partial amino acid sequence of the hemagglutinin and neuraminidase of the influenza virus is inserted into the double-layer lipid globule membrane in a targeted manner, so that the two molecules can be combined with the host. The receptor-bound group on the surface of the immune cell is exposed on the surface, and the antigen or DNA encoding the antigen is encapsulated inside, which has the advantage of facilitating the directional transport of the antigen or drug molecule.
The discovery of vaccines is a landmark event in the history of human development. Because in a sense, the history of human reproduction is the history of human beings constantly fighting diseases and natural disasters. The most important means of controlling infectious diseases is prevention, and vaccination is considered to be the most effective measure. And it turns out that the variola virus that threatens humans for hundreds of years has been completely eliminated after the emergence of vaccinia vaccine, ushered in the first victory of human vaccines against the virus, and more firmly believes that vaccines control and eliminate infectious diseases. Since then, the vaccine family has expanded and developed over the past 200 years, and there are currently more than 20 vaccines for human disease prevention and treatment.
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publié le jeudi 15 août à 13:34, aucun commentaire.

Introduction to Tumor Immunotherapy—Part One

Under normal circumstances, the immune system can identify and remove tumor cells in the tumor microenvironment, but in order to survive and grow, tumor cells can adopt different strategies to inhibit the body's immune system, unable to kill tumor cells normally, called immune escape. In order to better understand the multi-step complexity of tumor immunity, the concept of tumor-immune cycle is proposed. The tumor-immune cycle is divided into the following six steps: tumor antigen release; tumor antigen presentation; initiation and activation of effector T cells; T cell migration to tumor tissues; tumor tissue T cell infiltration; T cells recognition of tumor cells. Any abnormalities in these steps can lead to an anti-tumor-immune cycle failure and an immune escape. Different tumors can suppress the immune system's effective recognition and killing of tumor cells through abnormalities in different links to produce immune tolerance, and even promote the occurrence and development of tumors.
Tumors are the products of normal proliferation and migration in the body after the normal cells of the body have undergone mutation accumulation. Conventional tumor treatment is divided into two categories: surgery, which can remove solid tumors, but usually cannot eradicate tumors, and is not suitable for hematological tumors; radiotherapy and chemotherapy, although this treatment can effectively treat a variety of tumors, it can cause damage to the normal cells; at present, immunotherapy for tumors is increasingly becoming the hope of conquering cancer. At present, the main tumor immunotherapy programs include therapeutic tumor vaccines, immune checkpoint inhibitors, adoptive cellular immunotherapy, oncolytic viruses, etc.
1. Therapeutic Tumor Vaccines
For decades, cancer vaccines in development have become a form of immunotherapy that prevents cancer from developing or killing existing tumors by stimulating or restoring the body's own immune system.
The tumor vaccine is designed by applying various techniques to enhance the immune system's ability to recognize tumors by introducing tumor antigens into patients, to improve the immune microenvironment, and to stimulate powerful specific cellular immunity. In recent years, with the rapid development and cross-infiltration of related disciplines such as tumor pharmacology, molecular biology and immunology, as well as the development of tumor-associated antigens, carriers and adjuvants, various forms of tumor vaccines have been developed and clinical trials have been carried out. It mainly includes the following categories: tumor cell vaccine, which is divided into whole tumor cell vaccine and genetically modified tumor cell vaccine; tumor antigen vaccine, which is divided into tumor associated antigen (TAA) and tumor-associated antigen ( tumor specific antigen (TSA) vaccine, and tumor antigens including multiple layers, such as intact protein molecules, antigen peptides and purified DNA; DNA cancer vaccine, actually a kind of antigen vaccine, which is divided into naked DNA, plasmid DNA and viral vector DNA vaccine; autologous dendritic cell immunotherapy vaccine. TAAS, directly introduced into DC, which plays the role of antigen presentation, activates T lymphocytes and initiates the initial immune response. Bacterial vaccine is the earliest bacterial injection therapy.
In theory, tumor vaccine is one of the most effective and economical cancer treatment methods. A limited number of vaccine injections can bring long-term anti-tumor immunity effects. In fact, tumor vaccines have not been effectively confirmed in clinical studies. Until 2010, the US FDA approved the use of the Sipuleucel-T vaccine (or Provenge, manufactured by Dendreon, USA) for the treatment of asymptomatic or mild metastatic hormone-resistant prostate cancer, the first FDA-approved tumor treatment. The vaccine marks the official transition of the cancer vaccine from basic research to clinical application. Sipuleucel-T is an autologous cell therapy that mobilizes the patient's own immune system to fight disease. Its active component contains autologous peripheral blood mononuclear cells as well as recombinant fusion proteins of prostatic acid phosphatase and GM-CSF. The final products also include T cells, B cells, NK cells and other cells. However, existing studies have shown that the vaccine is only effective in a small number of patients with small tumors and low-grade malignancies. The current combined application of Sipuleucel-T and other immunologic or chemotherapeutic drugs continues.
2. Adoptive Cellular Immunotherapy
The immune response produces a complex interaction between the adaptive immune system and the natural immune system. B cells and T cells of the adaptive immune system use their receptors to recognize antigens, and T cells recognize antigen presenting cells (APC) to present antigenic peptides, a major histocompatibility complex (MHC). B cells directly recognize antigenic epitopes with highly specific adaptive immunity and immune memory. Natural immunity is non-specific immunity expressed by phagocytic cells, natural killer cells (NK), etc. The recognition of receptor-derived receptors (PRRs) identifies pathogen-associated molecular patterns shared by microorganisms and their products, such as lipopolysaccharide, peptidoglycan, and mannose, regulating the initiation, strength, and type of response of specific immune responses.
Dendritic cells (DCs) are a class of non-mononuclear phagocytic cells, which are important bonds between natural and adaptive immunity and are the most powerful APCs known in the human body. The production of anti-tumor immunity depends on the DC to capture the tumor antigen, process it intracellularly, and then present the antigen information to the immature T cells in the lymphoid tissue, thereby activating the specific immune response of the body. Activated T cells release cytokines and further stimulate T cell division, proliferation and maturation. A part of mature T cells differentiate into memory T cells to retain antigenic memory and rapidly respond to re-invasion of antigens, and most of them differentiate into effector cells with immune effects, such as regulatory T cells that produce cytokine-regulated immune responses, killer T cells that target and kill foreign cells, and effector T cells that stimulate B lymphocyte proliferation and antibody production. Dendritic cell-based vaccines can present tumor antigens to achieve a durable immune response.
To be continued in Part One…
publié le mercredi 14 août à 14:41, aucun commentaire.

The new chapter in tumor immunotherapy—immunological checkpoint inhibitors

Because of its significant survival benefit, tumor immunotherapy has become a new effective treatment option for cancer followed by surgery, chemotherapy, radiotherapy and targeted therapy. As one of the methods for tumors, immunological checkpoint inhibitors have been approved for the treatment of a variety of advanced tumors. Although some cancer patients can benefit from it, the immune-related adverse reactions, primary drug resistance and secondary drug resistance during the clinical use of the inhibitor limit its wide clinical application. Biomarker studies related to the efficacy of immunological checkpoint inhibitors are helpful for the screening and individualized treatment of patients, and are of great significance for the regulation of immunosuppressive monotherapy or combination therapy.
With the deepening of basic research on tumor immunity, researchers found that the immune system can not only identify and eliminate tumor cells, but also help tumors escape immune attacks. Immune escape is one of the basic features of tumors. Tumors gradually acquire the ability of immune escape by their own genes, epigenetic changes and domestication of the surrounding environment. The ligand receptor binding at the immune checkpoint inhibits activated T lymphocytes, which is the key cause of immune escape. Therefore, researchers try to block the binding of ligand receptors at the immune checkpoint to activate immune cells and achieve anti-tumor effects, thus opening a new chapter in tumor immunotherapy.
Immunological checkpoint inhibitors have been approved for their use against advanced or metastatic melanoma, non-small cell lung cancer, kidney cancer, classic Hodgkin's lymphoma, urothelial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma and the treatment of solid tumors with high microsatellite instability or mismatch repair defects. Some of the representative inhibitors include cytolytic T lymphocyte-associated antigen 4 (CTLA-4), programmed death receptor 1 (PD-1) and their ligand (PD-L1) antibodies.. However, there are two problems in clinical application:
a.25% to 30% of patients will develop immune-related adverse reactions, including skin reactions, colitis, endocrine diseases and liver damage, and the incidence of grade 3 to 4 adverse reactions account for 6% to 10%, more serious patients will lead to death;
b.60% to 80% of cancer patients appear indications of primary drug resistance, and clinical study follow-up results show that 30% of patients appear secondary drug resistance.
Biomarker research related to immunological checkpoint inhibitors can help us achieve patient screening and individualized treatment, avoid over-medication, reduce the occurrence of adverse reactions, and ultimately improve patients' quality of life and prolong patient survival. Therefore, it is particularly urgent and important to study and identify biomarkers related to the efficacy of immunological checkpoint inhibitors.
1.Mechanism of action of immunological checkpoint inhibitors
The immune checkpoint is a class of molecules that negatively regulate the immune response of the body in a physiological condition. It maintains the body's immune tolerance and reduces the damage of the tissue during the immune response. From the uptake, processing and presentation of tumor antigens to the activation, migration, infiltration and killing of T lymphocytes, each step of the tumor's immune cycle is regulated by immune checkpoints. One of the mechanisms by which tumors evade immune attacks is to down-regulate immune responses using immune checkpoints.
Taking two types of immunological checkpoint inhibitor approved by the US Food and Drug Administration (FDA) as example: a. CTLA-4 is expressed in T lymphocytes of the central lymphoid organ, which regulates the activation of T lymphocytes in the early stages of the tumor immune cycle. The signal of T lymphocyte activation is that the receptor on the surface of T lymphocytes specifically binds to the antigen; the co-stimulatory molecule can bind to the leukocyte differentiation antigen 28 (CD2(a) and present on the cell surface of the ligand leukocyte differentiation antigen 80/ 86 (CD80/CD86). CTLA-4 competitively binds to the ligand of its homologous analog, CD28, thereby negatively regulating the activation of T lymphocytes. B. The 2PD-1 molecule is mainly expressed on the surface of immune cells in peripheral tissues, especially in the tumor microenvironment. The PD-1 molecule on the surface of T lymphocytes binds to the PD-L1 molecule on the surface of tumor cells, which weakens the immune response signal and ultimately inhibits the killing of tumor cells by T lymphocytes. Therefore, anti-CTLA-4 antibody and anti-PD-1/PD-L1 antibody can effectively relieve the braking effect of the immune checkpoint on the anti-tumor immune response of T lymphocytes, thereby achieving the effect of treating tumor.
New immune checkpoint molecules such as indoleamine-2,3 plus dioxygenase (IDO), lymphocyte activating gene 3 (LAG3) and T cell membrane protein 3 (TIM-3) have also been found to be involved in tumor immune escape, The actual clinical efficacy of the inhibitors needs to be tested in the results of large-scale prospective clinical studies.
To be continued in Part Two…
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publié le mardi 13 août à 14:50, aucun commentaire.

Some Key Genes You Should Know About Cancer

Genes are all the nucleotide sequences needed to produce a polypeptide chain or functional RNA. Genes support the basic structure and performance of life, and store all the information of life's race, blood type, gestation, growth, apoptosis and other processes. The interdependence of environment and heredity deduces the important physiological processes such as the reproduction of life, cell division and protein synthesis. All life phenomena, such as organism's birth, growth, decline, disease, aging and death, are related to it. It is also an intrinsic factor that determines life and health. Therefore, genes have dual attributes: materiality (mode of being) and informedness (fundamental attribute).
Genes have two characteristics. One is that they can reproduce themselves faithfully to maintain the basic characteristics of living things. The other, in the reproduction of offspring, genes can be "mutation" and mutation, when the zygote or mother is affected by the environment or genetic, the genome of the offspring will occur harmful defects or mutations. Some diseases, in a particular environment will occur hereditary, also known as genetic diseases. Under normal conditions, life changes on the basis of heredity, and these changes are normal changes.
Oncogenes are a class of genes that are inherent in human or other animal cells (as well as oncoviruses). They are also called transformed genes, which, when activated, can promote the carcinogenesis, invasion and metastasis of normal cells. The ways of oncogene activation include point mutation, gene amplification, chromosome rearrangement, virus infection and so on. The result of its activation is an increase in its number or function, which leads to excessive cell proliferation and other malignant characteristics, thus forming malignant tumors.
Recent studies have found that STC2 expression is significantly increased in gastric cancer, ovarian cancer, liver cancer and other malignant tumors. Although the mechanism of action in the tumor is still unclear, it can play a role in promoting growth, proliferation, invasion and metastasis in a variety of tumors. Dr Paul Huang, head of the proteomics team at cancer research institute London,pointed out that the active status of F12 and STC2 genes was closely related to the mortality of breast cancer patients. Female cancer patients with high activity of F12 gene and low activity of STC2 gene had a 32% probability of dying within 10 years, while those with low activity of F12 gene and high activity of STC2 gene had a 10% probability of dying within 10 years. The biological characteristics and mechanism of STC2 gene in breast cancer have not been clearly studied.
SRC is a proto-oncogene and a member of the non-receptor protein tyrosine kinase family. Abnormal SRC expression and activity often cause the occurrence and development of some cancers such as breast cancer, colon cancer, pancreatic cancer, prostate cancer and lung cancer. The relationship between its expression and breast cancer was first found in mice, and studies in cell lines further showed that SRC gene plays an important role in the growth of breast cancer cells. The role of human proto-oncogene SRC in cancer development remains unclear, mainly because human cancer SRC mutations are rare and wild-type SRC is less pathogenic.
SKP2 was first cloned from human fibroblasts by Zhang et al in 1995. Fluorescence in situ hybridization confirmed that Skp2 gene was located in the 5pl3 region of human chromosome, with a total length of 31962bp. SKP2, a protein product encoded by the SKP2 gene, is composed of 436 amino acid residues and has a molecular weight of 45kD, hence the name p45. The SKP2 gene could be a new target for treating cancer, forcing cancer cells to age, stop dividing and die by blocking its activity, researchers at the university of Texas M.D. Anderson cancer center and memorial Sloan Kettering cancer center reported in the journal nature. Improved understanding of the SKP2 gene and its association with cell aging could lead to the development of novel drug agents that inhibit tumor development in a variety of common cancers.
RAD51 gene, as a DNA repair gene, plays a key role in maintaining gene stability. RAD51 gene mutation plays an important role in the occurrence and development of various tumors. Tumor resistance to radiotherapy and chemotherapy drugs was also associated with its mutation. In addition, Rad51 gene may interact with other genes to jointly influence the occurrence and development of tumors.
PLAC1(placenta-specific 1 gene) has the characteristics of cancer-testis antigen (CT), which can be expressed in various tumor tissues, but hardly in normal tissues (except placenta and testis). PLAC1 encodes a membrane protein that has certain immunogenicity and can cause humoral and cellular immunity in tumor patients. The current results show that PLAC1 represents a new class of tumor-related antigen, which may be a target of tumor immunotherapy.
publié le lundi 12 août à 09:04, aucun commentaire.

New Treatment for Skin Cancer - Skin Cancer Vaccine (Part Two)

2. Melanoma
Melanoma is the most aggressive malignant tumor in skin cancer. In the past 20 years, the incidence rate worldwide has risen sharply, increasing at a rate of 3.1% per year. The incidence rate in China is relatively low, but in recent years, the growth has accelerated, with about 20,000 new cases each year. In situ melanoma with a thickness < 1 mm has a 5-year survival rate of over 95%. Despite this, 20% progressed to metastatic melanoma. Once metastasis occurs, the prognosis is extremely poor, with a 5-year survival rate of less than 10%. Metastatic melanoma is not sensitive to radiotherapy and chemotherapy and is one of the most difficult tumors to be effectively treated. Melanoma is highly immunogenic and immunotherapy is the most promising treatment. In recent years, breakthroughs have been made in immunotherapy, especially immunomodulatory antibodies. Develop specificity for cancer development vaccines and cancer, providing great potential for the treatment of cancer patients. CTLA-4 monoclonal antibody Ipilimumab was used to treat metastatic melanoma, and overall survival (OS) was significantly benefited. It was approved by the US FDA on March 25, 2011.
2.1. Cytokines
Cytokines, a class of immunomodulatory small molecule proteins secreted by immune cells, enhance antitumor immune responses by inducing activation and proliferation of immune cells. Cytokines for the treatment of melanoma mainly include interleukins and interferon alpha.
2. 1.1. Interleukin
High-dose IL-2 is the first immunization drug approved by the US FDA for inoperable or metastatic melanoma. The objective response rate (ORR) of IL-2 treatment is low, but recent studies suggest that IL-2 can be used as an immunoadjuvant to enhance the efficacy of specific immunotherapy. Phase III clinical trials showed a significant increase in ORR (16% vs 6%) and a benefit from progression-free survival (PFS) in combination with high-dose IL-2/gp100 compared with high-dose IL-2 (2. February vs 1. June, P=0.00(a); The median OS has an extended trend (17 August vs 11. January, P=0.06). Another characteristic of high-dose IL-2 is severe adverse reactions and high mortality. It is limited to patients with good organ function in experienced specialists in experienced treatment facilities. The main causes of adverse reactions were "cytokine storm" and "systemic autophagy syndrome." Animal model studies have found that the combination of IL-2 and autophagy inhibitors can limit adverse reactions and prolong antitumor effects. Clinical studies have found that melanoma intra-injection of IL-2 has a high ORR (> 60%) and a moderate adverse effect. More interestingly, after intratumoral injection of IL-2 recurrence, chemotherapy was still effective (effective 36.7%). Thus, IL-2 combination therapy has the advantage of improving the remission rate and reducing adverse reactions, and becomes the main treatment for melanoma.
2.1. 2. Interferon alpha (IFNα;)
IFNα, the only effective drug for melanoma adjuvant therapy. The two commonly used subtypes, FNα-2α and IFNα-2b, differ only in two amino acids. There is almost no difference between the two, in terms of binding receptors, mode of action, and adverse reactions. IFNα is mainly used for adjuvant treatment of high-risk patients after melanoma surgery, and is rarely used for metastatic melanoma. A large number of clinical studies have shown that IFNα can prolong disease-free survival (DFS), but has no advantage in improving survival rate, and serious adverse reactions. Polyethylene glycol IFNα-2b (IFNα-2b, Sylatron), modified IFNα, long half-life (40-60 hours). Phase III clinical trial (EORTC 18991) found that Sylatron significantly improved postoperative relapse-free survival (RFS) for stage III melanoma, with an average of 34.8%. Therefore, on March 29, 2011, the FDA approved Sylatron for the adjuvant treatment of melanoma with microscopic or gross lymph node metastasis within 84 days of postoperative (including lymph node dissection). Subgroup analysis showed that Sylatron reduced the risk of recurrence, distant metastasis, and death in patients with microscopic lymph node involvement and primary ulcer.
2.2. Vaccine
Sipuleucel-T (Provenge), an autologous dendritic cell vaccine, was approved by the US FDA in April 2011 for the treatment of hormone-resistant asymptomatic or metastatic prostate cancer patients. Provenge's success marks a new era in vaccine treatment. Melanoma is highly immunogenic and its associated antigen-targeted vaccine is a valuable study. At present, melanoma therapeutic vaccines mainly include protein/polypeptide vaccines, recombinant vector vaccines, whole-cell vaccines, and dendritic cell vaccines.
To be continued in Part Three…
publié le dimanche 11 août à 04:14, aucun commentaire.

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