Preparation and Purification of Mice Monoclonal Antibody

The preparation and purification of mice antibody can be divided into cell fusion, screening and monoclonalization of positive hybridoma cells, and production and purification of monoclonal antibodies.

Cell fusion

The method of animal immunization is the same as that of antiserum. In order to ensure that the obtained B cells secrete strong antibody activity, intravenous enhanced immunization should be carried out three days before fusion. Mice myeloma cells (such as SP2/0-Ag14 cell line) should grow well in the logarithmic division stage when fused. B cells from spleen of mice obtained under sterile condition should be washed twice or three times with serum-free medium to remove mice production serum. The ratio of spleen cells to myeloma cells is generally 5:1 to 10:1. The commonly used fusion agent is 50% polyethylene glycol. After fusion, PEG is removed by centrifugation after slow dilution of culture medium. Then the cells are inoculated into 96-well plate by HAT medium (RPMI1640 contains 10%-20% fetal or calf serum and HAT suspension). Normally, clone growth can be seen under light microscope on the 3rd and 4th day after fusion, and screening can be carried out after the 10th day.

PEG is commonly used because of its low fusion efficiency, simple method and low cost, although the fusion rate of electrofusion is high, the number of cells in one fusion is small, and special equipment is needed, so it is seldom used. In the process of cell culture after fusion, the abdominal cavity cells of the same animal (i.e. feeder cells) can be added at the same time. The phagocytes can remove the debris of dead cells and contribute to the growth of hybridoma cells. Commercial hybridoma growth factor can also promote the growth of hybridoma cells.

Screening and Monoclonalization of Positive Hybridoma Cells

ELISA is the most commonly used screening method for hybridized cells after 10 to 14 days of culture (1 to 2 times of medium replacement). The positive cloned cells were subcloned by limited dilution method to ensure that the antibody secreting cells came from a single cell. Because the chromosomes of fused cells are easily lost, it is generally necessary to subclone them several times until all subclones from the same clone are positive, indicating that the cloned antibody-coding genes have been relatively stable and can be expanded for cultivation and construction.

Expanded production of monoclonal antibodies

The method of monoclonal cell line expansion culture and antibody preparation depends on the actual needs. At present, there are three commonly used methods to produce large quantities of monoclonal antibodies: mouse ascites preparation, bottle culture and hollow fiber reactor. The former is mostly used for laboratory preparation, and the latter two are suitable for industrial production. Ascites preparation method is low cost, but it is easy to inactivate antibodies due to the high amount of impurity proteins in ascites. Therefore, ascites should be purified as soon as possible after collection to prevent the degradation of antibodies. The supernatant obtained from flask culture was large in volume, but the concentration of antibody was low, and the cost of culture and purification of antibody was high. Compared with large bottle culture, the production of monoclonal antibodies using hollow fiber reactor is a more economical method, but the cost of equipment and equipment is larger.

For laboratory research or non-drug antibody production in mice, the antibodies obtained by ascites preparation and purification of protein A or protein G can basically meet the needs. The concentration of antibodies in the supernatant of hybridoma culture is generally in the order of microgram per milliliter, which can be directly used in immunoblotting and immunoprecipitation experiments. Using special serum-free medium for hybridoma culture can easily obtain the antibody requirements of general experiments.

Overview of Rabbit Monoclonal Antibody

Rabbit monoclonal antibody (RabMAbs): A new generation of monoclonal antibodies for research, diagnosis and treatment. Rabbit monoclonal antibody technology is currently difficult to replicate by replacing high or low affinity mouse monoclonal antibodies with high quality rabbit monoclonal antibodies.

Background

In 1995, Dr. Katherine Knight succeeded in obtaining myeloma-like tumors (plasmacytoma) in transgenic rabbits at Loyola University of Chicago. In the following years, Dr. Robert Pytela and Mr. Weimin Zhu improved the technology at UCSF to produce high-yield rabbit monoclonal antibodies. Epitomics has exclusive patents for rabbit monoclonal antibody technology and has developed a licensing platform for rabbit monoclonal antibodies for research, diagnostics and treatment.

Advantage

The emergence of rabbit monoclonal antibodies brings many benefits. First, rabbit antisera usually contain high affinity antibodies that recognize more types of epitopes than murine antisera. Second, rabbit monoclonal antibodies are capable of recognizing many antigens that do not produce immunity in mice. Third, because rabbit spleens are large, more fusion experiments can be performed, making it possible to screen high-throughput fusion cells.

Therapy

Rabbit monoclonal antibodies are used for treatment: By 2004, the market for therapeutic monoclonal antibodies is expected to be approximately $6.4 billion. It is expected that hundreds of therapeutic antibodies will be developed for the treatment of a variety of diseases in the next 10 years. Rabbit monoclonal antibody fusion cells and other proprietary Epitomics technologies provide an opportunity to release the energy of this new antibody and to create a new human monoclonal antibody for medical needs.

Diagnosis 

Rabbit monoclonal antibodies as diagnostic products: For several years, it has been known that the rabbit immune system provides the best antibody reagents for diagnostic purposes. Many small compounds and peptides produce an effective immune response in rabbits. Therefore, rabbit polyclonal antibodies are used in a variety of drug screening and clinical diagnostic kits.

Drug 

Rabbit monoclonal antibodies are powerful tools for drug development: complete sequencing of the human genome has greatly affected the progress of drug development. New drug development has shifted from traditional disease target selection to the systematic search for disease targets using genomic information. Protein is closer to the cause of disease than DNA or RNA. Monoclonal antibodies are the most powerful tool for detecting protein specificity and sensitivity. In addition, monoclonal antibodies are able to specifically counter or enhance the function of proteins, making them powerful tools for proteomics.

The advantages of rabbit monoclonal antibodies provide a large space for biomedical research and the development of antibody drugs. Due to the difficulty in finding specific antigens, the technically high barriers make the monotherapy therapeutic drugs difficult to be copied after the patent protection expires, and are not vulnerable to the price of generic drugs. Further development of rabbit monoclonal antibodies is expected to result in more sensitive and reliable diagnostic reagents and high quality antibody drugs.

After years of research, rabbit monoclonal antibodies have been used more and more in immunoblotting, immunohistochemistry, flow cytometry and other immunological studies.

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Application of Rabbit Monoclonal Antibody

Monoclonal antibody technology originated from the in vitro murine hybridoma technology created by Kohler et al. After years of research, murine monoclonal antibody has evolved from simple biological laboratory research and clinical diagnosis to therapeutic antibodies drugs. However, the main drawback of murine mAb is that the mouse immune system does not recognize certain immunogens, especially murine immunogens, and the affinity of murine monoclonal antibodies is not as high as that of rabbit-derived antibodies. However, due to the lack of technical difficulties in transfecting rabbit-derived plasmacytoma and virus in vitro in rabbit B cells, until 1995, Spieker-Polet et al. The rabbit plasmacytoma cell line was successfully obtained in c-myc/v-abl transgenic rabbits, and a stable rabbit-rabbit hybridoma was obtained, which made a breakthrough in rabbit monoclonal antibody technology. In recent years, rabbit monoclonal antibody technology has been improved day by day, and there are thousands of commercial rabbit monoclonal antibodies. This article reviews the current development of rabbit monoclonal antibody technology, technical advantages and the application prospects of rabbit monoclonal antibody technology in basic research, clinical diagnosis and treatment.

Development 

Since the establishment of murine monoclonal antibody technology, many rabbit monoclonal antibodies have been tried by hybridoma technology, but rabbit-mouse hybridoma cells obtained by heterologous myeloma fusion are unstable due to the lack of rabbit myeloma cell line. Can not secrete antibodies and other issues for a long time.

Knight et al. overexpressed v-abl and c-myc two oncogenes c on a transgenic service rabbit and placed them downstream of the heavy and light chain enhancers, and finally obtained myeloma-like tumors from the transgenic rabbits. A myeloma cell line was isolated and named 240E-1. The hybridoma obtained by fusing this strain of 240E-1 cells with rabbit B lymphocytes secretes a rabbit monoclonal antibody. This is the first time that a rabbit monoclonal antibody has been obtained by means of a rabbit-rabbit hybridoma. However, like the early murine myeloma cell line, 240E-1 cells also have two major problems: first, instability, after several passages or subclones, the hybridoma gradually loses the ability to secrete antibodies; secondly, the cell line itself Endogenous rabbit IgG was expressed. In order to solve the stability problem, Zhu et al. [22] improved the medium method and used repeated subcloning techniques to obtain a new cell line, named 240E-W, by 11 rounds of subcloning and screening. Chromosome typing experiments have shown that its chromosome number is more stable, with 79 to 90 pairs (mean 84 pairs), 240E-W is more stable than 240E-1 44 to 70 pairs (average 60 pairs). DNA fingerprinting and gene expression profiling also showed great differences between the two cells. The fusion cell strain was prepared with 240E-W, and the hybridoma cells obtained were more efficient and more stable. In order to solve the problem of endogenous IgG, Huang et al. obtained a cell line without the endogenous IgG heavy chain gene from the hybridoma of 240E-W cells and named it 240E-W2. Hybridoma cells obtained by fusion with 240E-W2 did not express detectable endogenous IgG heavy chains and improved fusion efficiency and hybridoma positive rate by various large-scale fusion experiments.

Applications

Application in the development of biomarkers

Rabbit monoclonal antibodies are widely used in various fields of life science research [28-31], and have unique advantages especially in immunohistochemistry and post-translational modification of proteins. To date, more than 100 rabbit monoclonal antibodies have been developed for the clinical diagnosis and research of IHC for cancer. The most representative ones are Her2 and c-Kit IHC diagnostic kits. In the field of protein modification research, such as protein phosphorylation, plays a crucial role in the disease-related protein signaling pathway. The rabbit monoclonal antibody using phosphorylated protein can clearly show the degree of phosphorylation of the protein. More than 200 phosphorylated specific rabbit monoclonal antibodies have been available.

Application in the development of therapeutic antibody drugs

Most therapeutic antibodies used in clinical practice require the step of in vitro affinity maturation to increase the affinity of the antibody. This process often takes 8 to 12 months and the final affinity is in the nanomolar range (10-9 mol/L). Rabbit monoclonal antibodies can reach 10-11 to 10-13 mol/L without the need for in vitro affinity maturation. This high affinity not only reduces the clinical use of antibodies, but also reduces the side effects caused by the use of large amounts of antibodies. But this high affinity advantage needs to be further confirmed in animal and clinical trials. In addition, humanization of rabbit monoclonal antibodies is easier than humanization of murine monoclonal antibodies. Because rabbit monoclonal antibody is mainly composed of a heavy chain and light chain genes, it is found that rabbits and human antibodies have higher homology, about 60% to 76%, by analyzing a large number of rabbit monoclonal antibody sequences, and mice. The homology with human antibodies is slightly lower, ranging from 57% to 72%, indicating that the development of drugs with rabbit monoclonal antibodies is more promising.

The Past and Present of High-throughput Sequencing

The term "high-throughput sequencing" I think most people won't be unfamiliar, because it is now a hot topic, but how much do you know about high-throughput?

The emergence of all things is inextricably linked, and it is impossible to appear out of thin air. The same is true for high-throughput sequencing. With the advancement of science and technology, people's exploration of microorganisms in the environment has never been terminated. Previously relying on traditional plate-pure culture methods, however, as many as 99% of the microorganisms could not be cultured and identified under the existing experimental conditions. Therefore, high throughput antibody production sequencing technology based on non-culture methods has emerged.

Differences between high throughput monoclonal antibody generation and general sequencing

"Ordinary gene sequencing" should refer to "conventional DNA sequencing", which is a method of sequencing by the Sanger method. It is very common to perform automatic sequencing directly with ABI 3730xl, which can basically achieve a read length of 600 bp to 800 bp. The concept of high-throughput sequencing is actually a relative concept. In 2000, sequencing on instruments such as 3700 and MegaBace was also high-throughput sequencing, which is relative to manual sequencing or running flat-plate glue. After 2005, high-throughput sequencing will refer to second generation sequencing, 454, Solexa (later changed to Illumina) and SOLiD and other second-generation sequencing, than the first generation of sequencing such as 3730. It has been improved by tens of thousands of times, even hundreds of millions of times, so it is called high-throughput sequencing.

High-throughput sequencing technology (also known as next-generation sequencing technology) is a technology developed in recent years, which can sequence hundreds of thousands to millions of DNA molecules in parallel, and is highly efficient. With the advancement of technology, high-throughput sequencing technology is gradually being updated.

The past

Sanger method First generation sequencing technology (dideoxynucleotide end termination method): A DNA polymerase is used to extend primers bound to a template of a sequence to be determined until a chain termination nucleotide is incorporated. Due to the high cost, slow speed, low throughput, etc., it is gradually eliminated.

Present

Second-generation sequencing technologies (mainly including Roche 454 pyrosequencing, Illumina Solexa sequencing and ABI SOLID sequencing), each with the following advantages and disadvantages:

Roche 454 Pyrosequencing Technology - relies on bioluminescence for DNA sequence analysis.

Advantages: read sequence length (400 bp), de novo sequence can be performed;

Disadvantages: The number of duplicate bases cannot be determined.

Illumina Solexa sequencing technology - the principle is to reversibly terminate the chemical reaction.

Advantages: highly automated system, many read fragments, suitable for sequencing of a large number of small fragments (microRNA, lncRNA, etc.);

Disadvantages: The read sequence is short and not suitable for de novo sequence.

ABI SOLID sequencing technology - the core is the ligation reaction of four fluorescently labeled oligonucleotides.

Advantages: very high accuracy is read twice per base, especially for SNP detection; system flexibility allows for pooling of samples and segmentation of sequencing regions;

Disadvantages: The read length is limited by the connection reaction.

The above three sequencing methods have their own advantages and disadvantages. Currently, Illumina's sequencing technology is widely used in scientific research.

In order to meet the increasingly prominent research needs, the third generation sequencing technology (single molecule real-time DNA sequencing technology, sequencing process without PCR amplification) is also available: mainly for single-molecule fluorescence sequencing (SMRT) and nanopore sequencing (nanopore molecule).

Single-molecule fluorescence sequencing: a technique that relies on DNA polymerase-based side-synthesis sequencing, with a read length of up to 100,000 bp;

Nanopore Sequencing: A technical method that relies on exonuclease-based electrical signal sequencing with unlimited read length.

At present, due to the high cost, the single-reading long error rate is high, the bio-information analysis software is not rich enough, and the data accumulation is small, etc., and has not been widely used.

Applications

Transcriptome sequencing (RNA-Seq): study cell performance and function;

Methylation sequencing: epigenetic marker information;

Exome-Seq: study directed-enriched DNA;

Chromatin immunoprecipitation-depth sequencing (ChIP-seq);

Genomic sequencing

Digital gene expression profiling;

Sequence Capture technology: Combines chip and deep sequencing, captures the fragment to be tested with a chip probe, and analyzes the nucleic acid sequence using deep sequencing technology.

Clinical Application of Next Generation Sequencing Technology in the Detection of Infectious Diseases

With the completion of the Human Genome Project in 2003, gene sequencing technology continues to evolve, and next generation sequencing (NGS) technology has gradually grown into mainstream sequencing technology. Its continuous innovation in sequencing principles and continuous breakthroughs in technology have made it high. The application of flux gene sequence reading tends to individualized needs, and is expected to lead and replace the commonly used molecular diagnostic techniques.

First, NGS

NGS is also known as high throughput antibody production sequencing, including second-generation massively parallel sequencing and three-generation single-molecule sequencing. The second-generation sequencing includes Illumina's Solexa sequencing platform, Roche's 454 sequencing platform, and ABI's oligonucleotide-linked detection platform (SOLiD). Different platforms have different sequencing methods, but the principles have commonalities, all of which are genomes. The DNA is randomly spliced into small fragments of DNA, and the sequencing template is obtained for sequencing and data analysis by constructing a library, PCR amplification and other technical processes. Compared with Sanger sequencing, the flux is high, the speed is fast, and the cost is low, but the read length is generally Short shortcomings.

The third-generation sequencing is based on the single-molecule real-time technology of American Pacific Biotechnology and the nanopore single-molecule sequencing technology of Oxford Nanopore, UK. It is characterized by no need for PCR amplification, distinguishing base signal differences by different means, and directly reading sequence information. Long reading and high throughput monoclonal antibody generation can directly sequence RNA and methylated DNA sequences. At present, there are shortcomings such as high error rate, high cost and insufficient bioinformatics analysis software.

Second, the current clinical application of NGS in infectious diseases

In recent years, there have been new changes in the spectrum of infectious diseases worldwide. On the one hand, the number of difficult infections is increasing. Traditional diagnostic methods cannot keep up with the rhythm of the continuous evolution and mutation of microorganisms. On the other hand, the spread of infectious diseases is obviously accelerated. These are all infections. The diagnosis and treatment of sexual diseases poses serious challenges. The rapid detection and diagnosis of clinical specimens of infectious pathogens has become an increasingly urgent need in the clinic. NGS technology plays an increasingly important role in infectious diseases because of its high speed, high accuracy and low cost.

1. Application in the detection of bacterial infections:

Clinically, the culture method is a standard for diagnosing bacterial infections. It takes a long time, has high technical requirements, and some bacteria grow harsh conditions, which increases the difficulty of successful culture. NGS technology is applied to the detection of bacterial infections. It can not only determine whether there is bacterial infection, but also directly classify the strains, obtain molecular serotypes, drug resistance gene profiles and other information, and analyze the evolution history, infection source and infection path of the strains. Helps with disease prevention, diagnosis and vaccine development.

In May 2011, Germany was the center of Europe, and a serious hemorrhagic intestinal infection broke out in Europe. Scientists carried out genome-wide sequencing of the pathogen strain and found that the causative culprit was the lethal Escherichia coli O104:H4, through the sequence Analyze the path and pathogenic mechanism of infection in human body, trace the source of infection path, and provide important information for the diagnosis and treatment of the disease. In June of the same year, similar infections occurred in France. It is generally believed that the German strain may be infected. The source, and the two methods of pathogens were completely consistent by traditional methods and molecular diagnostic methods, but the NGS sequence analysis found that the genetic diversity of Escherichia coli strains in Germany is much lower than that of French strains, thus confirming its evolution as a French pathogen. Support, clear source of infection. In 2013, similar cases were reproduced in Europe. After sequencing, the strains were found to be different from O104:H4. However, they have a variety of common pathogenic gene regions, and there is also the possibility of causing serious outbreaks. The sequencing results of related strains are helpful. To establish an effective disease warning.

In 2011, the National Institutes of Health in the United States had a carbapenem-resistant Klebsiella pneumoniae infection. Although early detection of infection prevention measures, 18 patients were infected, 11 infected patients died, comprehensive gene sequencing and epidemiology The analysis traces the source of infection and the route of infection, and promotes the control of nosocomial infection and transmission.

Due to the widespread use of genetic mutations and antibiotics, more and more bacteria have developed resistant strains. Among them, the resistant strains of Mycobacterium tuberculosis are difficult to culture and take a long time, and clinical testing is greatly limited. However, as a single-gene bacteria with low sequence diversity, gene sequencing technology has great advantages in detecting the virulence and drug resistance of its strains, and can quickly detect pathogen infection and its drug resistance, with the continuous maturity of technology. And the decline in cost, is expected to replace the existing culture and molecular diagnostic methods, can also analyze the molecular evolution rate and mode of the infection process through sequencing data, which has far-reaching significance for treatment and vaccine development.

2. Application in virus infection detection:

The diagnosis of viral infection is mainly based on antigen-antibody detection. Molecular diagnostic methods such as PCR detection and DNA probe are also widely used. NGS technology is mainly applied to genotype analysis, drug-resistant mutation detection and virus evolution in the host, as well as outbreaks of infected viruses. For rapid detection to assist with prevention, diagnostic treatment and vaccine research.

Hepatitis B virus is the main cause of chronic liver disease such as hepatitis, cirrhosis and liver cancer. The nucleoside antiviral drugs commonly used in the treatment of viral infections have the advantages of less toxic and side effects and good effects, but the possibility of virus resistance mutations in long-term treatment is resistant. The drug can cause serious cases such as viral rebound and hepatitis attack, even liver decompensation and acute liver failure. The drug resistance gene locus can be detected by NGS, and the treatment plan can be adjusted according to drug resistance in time to achieve better curative effect.

AIDS is a disease that seriously threatens the public health of our country. Its pathogen, HIV, is a highly mutated virus. The current guidelines recommend that the viral load declines or the treatment failure needs to be changed before and during antiviral therapy. The patients in the program were tested for drug resistance, and the results provided an important reference for the development and adjustment of the treatment plan. NGS can detect local HIV genotype resistance mutations, clarify the distribution of drug resistance genotypes and their subtypes, provide guidance for the selection and replacement of clinical therapeutic drugs, and accurately understand the evolution and mutation of HIV in the host. Possibly, it provides a new idea for the design of antiviral drugs and the development of vaccines.

In 2014, West Africa broke out the largest Ebola virus infection in the record, with a long outbreak, high infections and deaths, and caused great panic worldwide. Through the combination of NGS technology and data analysis, the researchers identified their genetic diversity, vulnerability and epidemiological characteristics, tracked the source of infection and the path of infection, and predicted the process of viral infection of the human body and the body's immune response. The evolution of the adaptive mutation in the host reduces the chance of being recognized by immune cells, and has greatly promoted the prevention, control, diagnosis, treatment and vaccine development of the epidemic.

3. Role in the detection of fungal and other pathogen infections:

With the increase in the use of antibiotics, hormones and immunosuppressive drugs, the number of patients with malignant tumors and organ transplants has increased, and the incidence of invasive fungal infections has gradually increased. The mortality rate is over 50%. Rapid and accurate diagnosis is the key to treatment. . Traditional culture methods have become increasingly unable to meet clinical needs. In recent years, with the rapid development of NGS technology as a technical support, the genome-wide data of a series of important pathogenic fungi have been published one after another, making humans aware of these pathogenic fungi. To a whole new level. Through gene sequencing detection, the establishment of local fungal genomics data and comparative analysis can not only carry out rapid and accurate diagnosis of fungal infections, but also discover functional genes unique to different fungi, study its pathogenic mechanism and drug resistance site mutations, and discover new ones. The drug acts on the target site, thereby developing an antifungal drug with a broad spectrum, low adverse reactions, and no cross-resistance.

In the clinical detection of other pathogen infections other than bacteria, viruses and fungi, NGS currently plays a complementary role. When clinical and laboratory are unable to identify pathogens, high-throughput sequencing technology and bioinformatics analysis can be used to confirm the diagnosis. Infect pathogens and establish a local database for rapid diagnosis and further clinical research. In 2009, China completed the whole genome sequencing and functional analysis of the first multi-cellular human parasite, analyzed the composition of its functional genes, and explored the process of its co-evolution with the host, which effectively promoted the diagnosis, treatment and prevention of schistosomiasis; In addition, domestic and foreign scholars also through the genome-wide sequencing of Plasmodium, mapping its genetic map, studying the genetic differences and mutations of different Plasmodium, providing ideas for the development of new drugs and vaccines.

4. The clinical significance of metagenomic detection:

The human body has a large number of microorganisms that play an indispensable role in metabolic activities, researching microorganisms in the human body, exploring its interaction with the human body, and the relationship between microorganisms and diseases, exploring new ways of disease prevention and control, and promoting Human health is of great significance. Most microorganisms cannot be studied by traditional culture and identification. Metagenomics is a discipline that studies the genome of microbial populations. The technology does not rely on the cultivation of microbial communities in the sample, but directly performs nucleic acid extraction and determines all the samples in the sample. The nucleic acid sequence of the microorganism is analyzed and functionally identified. It not only can understand the microbial community's own quantity and composition, but also the microbial community itself and its evolutionary relationship with the host, and study the microbial community structure and function under different states. The effects of analyzing the structural and functional characteristics of normal human flora and disease states.

Intestinal microbial metagenomics is closely related to human metabolism and immunity. It has become a hot research topic in recent years. The relationship between intestinal flora and metabolic diseases such as diabetes and obesity has been continuously studied. The interaction between drugs and intestinal microflora is also being explored.

Third, NGS technology is currently used in clinical testing problems

There are still many practical problems in the routine application of NGS technology in clinical tests. The complexity of clinical specimens may result in too little pathogen information and data loss. Pathogen data may be mixed in the normal flora and it is difficult to distinguish. There are no different specimens yet. Uniform provisions for pre-sequencing processing and parameter setting; high-throughput sequencing will generate large amounts of data, requiring huge data analysis. If there is a lack of local databases, the cost and time of computing work is difficult to control; for disease, sequencing of genomic data The relationship with disease is still unclear. Access to genomic information alone is not sufficient to clarify the cause of the disease and the process of disease development. Accurate analysis and data mining are needed to decipher the causes of infectious diseases and achieve precise treatment and intervention. Further exploration is needed to ensure the accuracy of the application in clinical practice.

With the continuous updating of sequencing technology, the cost of antibody sequence database will continue to decrease. Once a complete genetic database is established, NGS technology will make greater achievements in the identification of pathogenic microorganisms, reduce the occurrence and spread of infectious diseases, and become a routine clinical test. Technology to promote medical development.

Next-generation Sequencing Assisted Immunotherapy and Revealed PD-1 Antibody Resistance

The research team of Prof. Chuanhe Zhiji and Prof. Taniguchi, from the Institute of Industrial Science, Osaka University, Japan, successfully used an electrical test method to successfully identify a molecule of a nucleic acid base that constitutes DNA (deoxyribonucleic acid).

The results of this study show that scientists can identify the identity of a unit of nucleic acid base based on the current value between the nanoelectrodes, and also verify the basic principles of next-generation DNA sequencing. The next generation of DNA sequencing technology can be a leap forward.

Not long ago, New England Journal of Medicine published an article entitled "Unmasking PD-1 Resistance by Next-Generation Sequencing", which describes how to expose the resistance of PD-1 antibodies with next-generation antibody sequence technology.

In recent years, the FDA has approved PD-1 antibodies for the treatment of various types of cancer, including melanoma, lung cancer, kidney cancer, classic Hodgkin's lymphoma, and head and neck cancer. However, even the most sensitive tumors (such as melanoma) are naturally resistant.

In a new study published in NEJM, scientists describe how to use genome-wide sequencing to identify the mechanisms behind tumor-producing immune resistance in PD-1 antibody treatment. The melanoma patients who participated in the study responded to pembrolizumab treatment for more than 6 months after relapse.

Tumors can resolve after blocking the PD-1/PD-L1 pathway and may be involved in the activation of tumor-infiltrating T cells. This small trial population consisted of four patients. Their initial response to PD-1 antibodies was a significant increase in tumor-infiltrating CD8-T cells. However, CD8-T cells were usually confined to the tumor margin when the disease relapsed.

In this study, the results of whole-genome antibody protein sequencing of tumor cell DNA integrated in vitro functional studies, gene expression analysis, and immunophenotypic data. The genetic variation detected in the study is related to two signaling pathways.

Changes in the first pathway result in a lack of response to interferon-gamma in tumor cells, involving functional deletion mutations in the genes encoding Janus kinase 1 (JAK1) and Janus kinase 2 (JAK2). The study found that the tumor did not detect JAK mutations at baseline, but this mutation occurred during recurrence. In vitro studies have shown that JAK mutations cause tumor cells to completely lose sensitivity to interferon-gamma.

The change of the second pathway is related to the escape mechanism of cytotoxic T cells (missing of MHC class I expression on the surface of tumor cells), specifically related to the gene encoding β-2-microglobulin (B2M). Truncating mutation.

Although the number of patients in this study is small, the conclusion is compelling. This is the first study to reveal that the drug resistance of PD-1 antibody immunotherapy is related to tumor cell gene mutation. Previously, scientists have uncovered the non-genetic or immunological mechanisms behind resistance to PD-1 therapy. The genomic mechanism revealed by this study is also complementary to previous studies.

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