Major applications: hybridoma technology

Hybrid hybridoma refers to a phenomenon in which two or more cells combine to form a single cell. It can make the nucleus of two different sources express function in the same cell. Fusion of myeloma cells and immune lymphocytes (immunized B lymphocytes) forms hybridoma cells capable of secreting highly pure monoclonal antibodies.


Hybridoma technique

Lymphocyte hybridoma technology, also known as monoclonal antibody technology, is based on somatic cell fusion technology. Kohler and Milstein demonstrated that myeloma cells fuse with spleen cells of immunized animals to form a highly specific antibody that secretes high specificity against the antigen, a monoclonal antibody. The basis of this technology is cell fusion technology. Myeloma cells can be serially passaged in vitro, while spleen cells are terminal cells and cannot be propagated in vitro. For example, when a mouse myeloma cell is fused with a lymphocyte secreting an antibody or a factor, the fused cell has both the infinite reproductive characteristics of the tumor cell and the ability of the lymphocyte to secrete specific antibodies or factors, and overcomes the problem. The disadvantage is that immune lymphocytes cannot be propagated in vitro, and the fused cells are called lymphocyte hybridomas.


Principles and steps of hybridoma:

The basic principle of hybridoma technology is to fuse both cells while maintaining the main features of both. These two cells are antigen-immunized mouse spleen cells and mouse myeloma cells, respectively. The main feature of mouse spleen cells (B lymphocytes) immunized by specific antigens is its antibody secretion function, but it cannot be continuously cultured in vitro, and mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, the so-called immortality. Under the action of the selective medium, only the hybrid cells in which the B cells are fused with the myeloma cells can have the ability to continue to culture, and form a cell clone having both the secretory function of the antibody and the immortality of the cells.

The principle is clarified from the following three main steps:

(1) Cell selection and fusion

The purpose of establishing hybridoma technology is to prepare monoclonal antibodies specific for the antigen, so the fusion cell must select B cells that are immunized with the antigen, usually derived from the spleen cells of the immunized animal. The spleen is an important place for B cell aggregation. No matter what kind of immune stimulation, there will be obvious antibody responses in the spleen. The other side of the fusion cell is to maintain the proliferation of cells after cell fusion, and only tumor cells have this characteristic. Selecting cells from the same system increases the success rate of fusion. Multiple myeloma is a malignant tumor of the B cell line, so it is an ideal spleen cell fusion partner.

The use of cell fusion agents causes a certain degree of damage to the cell membrane, allowing cells to adhere to each other and fuse together. The best fusion effect should be minimal cell damage with the highest frequency of fusion. Polyethylene glycol (PEG 1 000~2 000) is currently the most commonly used cell fusion agent, and the general application concentration is 40% (W/V).

(2) Application of selective medium

Cell fusion is a random physics process. In a mixed cell suspension of mouse spleen cells and mouse myeloma cells, the cells will appear in various forms after fusion, such as fused splenocytes and tumor cells, fused splenocytes and spleen cells, fused tumor cells and tumor cells, unfused spleen cells, unfused tumor cells, and multimeric forms of cells, and the like. Normal spleen cells survive only 5-7 days in the culture medium, and no special screening is required; the multimeric form of the cells is also easy to die; and the unfused tumor cells require special screening and removal.

There are generally two ways to synthesize cellular DNA. The main route is to synthesize nucleotides from sugars and amino acids, and then to synthesize DNA. Folic acid is an important coenzyme involved in this synthesis process. The secondary route is the synthesis of DNA by the catalytic action of hypoxanthine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) in the presence of hypoxanthine and thymidine. There are three key components in the cell fusion selection medium, hypoxanthine (H), aminopterin (A) and thymidine (T), so the three heads are called HAT medium. Methotrexate is an antagonist of folic acid, which blocks tumor cells from synthesizing DNA by normal routes, and the tumor cells used for fusion are HGPRT-cell strains selected by toxic medium, so they cannot grow in this medium. Only the fused cells have the genetic properties of both parents, and can survive and multiply in HAT medium for a long time.

(3) Limited dilution and antigen-specific selection

In animal immunization, high purity antigens should be used. An antigen often has multiple determinants. The humoral immune response produced by an animal after being stimulated by an antigen is essentially the secretion of antibodies from a large number of B cell populations, while the B cells targeting the target antigenic epitope are only a small fraction. Since cell fusion is a random process, a considerable proportion of unrelated cell fusions in the already fused cells are screened for removal. The screening process is generally divided into two steps: one is antibody screening of fused cells, and the other is specific antibody screening based on this. The fused cells are fully diluted so that the number of cells allocated to each well of the culture plate is between 0 and several cells (30% of the wells are 0 to ensure a single cell in each well). The supernatant was subjected to ELISA to select highly secreting cells of the antibody; this process is often referred to as cloning. These positive cells were further cloned, and an antibody-positive cell line against the target antigen was identified by using an ELISA coated with a specific antigen, and then subjected to cryopreservation, in vitro culture, or intraperitoneal inoculation culture.

Hybridoma technology application:

Monoclonal antibodies are of great value not only in basic research in cell biology and cdc immunology, but also in combination with the application of gene therapy, and are widely used in practice. In medicine, monoclonal antibodies have been used in the diagnosis of diseases, and their advantages are accurate diagnosis and no cross-reactivity. For example, when monoclonal antibodies are used to diagnose hepatitis B and latent hepatitis B virus, false negatives are rarely missed. Monoclonal antibodies can also be used as a pharmaceutical carrier for the treatment of diseases. Monoclonal antibodies have specific affinity for target tissues, so they have specific localization and distribution characteristics in vivo. Combining anti-tumor drugs with monoclonal antibodies against certain tumors allows the drug to selectively concentrate on the tumor cells in the body, killing only the target cells without damaging the normal tissues, and greatly reducing the anticancer drugs. side effect. Therefore, drug-loaded monoclonal antibodies are known as “biological missiles.” Most of the monoclonal antibodies currently produced are mouse-mouse type, which is a heterologous protein for humans and therefore difficult to treat. In order to solve the human body’s rejection of heterologous monoclonal antibody proteins, scholars are working hard to develop human-human monoclonal antibodies, which are beneficial for the treatment of diseases.

The side effect of CART cell therapy

When the immune system is functioning normally, immune cells move around the body looking for things that don’t belong, like bacteria and viruses. These immune cells search for invaders using “receptors,” which can be thought of as antennae or feelers. When receptors find invaders in the body, special immune cells come in to destroy them. These special cells are called cytotoxic T cells.

Unfortunately, cancer cells are often able to hide from immune cells, which is why the cancer cells can grow out of control. Immunotherapy is a cancer treatment intended to make the body’s immune system able to detect and destroy cancer cells. Immune checkpoint inhibitors have been a successful immunotherapy approach because it pushes the immune system into high gear to fight cancer.

CAR T-cell therapy, however, is different. It is a type of immunotherapy called “adoptive cell immunotherapy.” As ASCO President Bruce E. Johnson, MD, FASCO, describes it, this technique “allows clinicians to genetically reprogram patients’ own immune cells to find and attack cancer cells throughout the body.”

In CAR T-cell therapy, a person’s T cells are removed and taken to a laboratory. The T cells are genetically changed so they will attack cancer cells. These CAR T cells are grown in large numbers and then injected into the patient. One of the remarkable things about this treatment is that it is a “living therapy.” CAR T cells typically have to be injected only once, because they go on to multiply in the body. CAR T cells continue fighting the cancer in the patient’s body, and their effectiveness may even grow over time.

Emily Emily, the first child to be cured with Chimeric antigen receptor T (CART) cell therapy, was received by President Obama in 2016. This interview made more people began to understand CART cell therapy and gave hope to more leukemia patients to survive.

Despite the success of CAR T cell therapy, this intervention carries the risk of serious side effects. These include neurotoxicity, which can lead to headaches, confusion and madness, as well as other neurological changes. But there is less awareness to them. A team at Brigham and Women’s HospiTal recently classified the neurological symptoms of patients treated with CAR-T cells to better understand their neurotoxic side effects. Although neurological symptoms are common (77% of patients have at least one symptom), they are also temporary. The findings were published in the journal Brain.

“The mechanism of CAR T cell-associated neurotoxicity is unclear and the symptoms are difficult to predict,” said Daniel Rubin, MD, Ph.D., the first author of the study. “We conducted this study to better define the specific neurological symptoms experienced by patients after CAR T cell therapy.”

To clarify the clinical signs of CAR-T-related neurotoxicity, the team was admitted to Dana-Farber/Brigham and Women’s Cancer between 2015 and 2018 at the Dana-Farber/Brigham and Women’s Cancer Center. An observational cohort study was performed in 100 lymphoma patients with CAR T-cell Therapy. The team evaluated symptoms from the start of CAR T cell therapy infusion to two months after infusion. In addition, all diagnostic assessments, including laboratory tests and imaging scans, were reviewed.

“We shared some clinical cases of early treatment. From a neurological point of view, these cases are very serious and unusual,” said neuroscientist Henrikas VaiTkevicius, MD. “This has spurred our interest in working with the oncology and T-cell treatment teams, allowing us to prospectively evaluate most patients rather than retrospectively.”

Their findings reveal the prevalence of neurological symptoms after CAR-T treatment. The most common symptom is encephalopathy, a brain disorder that causes confusion, headache, tremors, weakness, and language problems. Importantly, these effects are mostly reversible, and the symptoms almost always disappear over time.

In addition, researchers found a unique pattern of activity or inactivity in their research. Treatment-related neurological dysfunction often originates from areas where metabolic function appears to be inactive. This finding is important for the clinical evaluation of neurotoxicity and the application of imaging.

Rubin said: “Although neurological symptoms are common, imaging studies such as MRI, which is the basis of neurological diagnosis, are almost always normal. In contrast, diagnostic studies that directly assess neuronal function, such as electroencephalography (EEG), are more straightforward. And positron tomography (PET), which reliably detects and predicts neurological dysfunction.”

Next, the researchers will establish and validate a model to more accurately assess and diagnose CAR-T-related neurotoxicity.


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Antibody Immunogenicity Prediction

What is an antibody?

Antibody is a kind of immunoglobulin formed by the body under the stimulation of antigen, which can specifically bind to the antigen, participates in neutralizing toxins, sterilizing and lysing. Antibody production refers to a process in which an organism produces an immunoglobulin that is specifically reacted with a corresponding antigen by a plasma cell differentiated by B cells under the stimulation of an antigenic substance.


Since electrophoresis was first used to demonstrate that the antibody activity in serum is in the gamma globulin portion, the antibodies have been collectively referred to as two (gamma) globulins. It was later proved that the antibodies were not all in the γ region; and the globulins located in the γ region did not necessarily have antibody activity. In 1964, the World Health Organization held a special meeting to refer to antibody-related globulins as immunoglobulins (Ig). Such as myeloma protein, macroglobulinemia, cryoglobulinemia and other abnormal immunoglobulins present in the serum and “normal human” naturally occurring immunoglobulin subunits. Thus immunoglobulins are the concept of structure and chemistry, and antibodies are biological and functional concepts. It can be said that all antibodies are immunoglobulins, but not all immunoglobulins are antibodies.


Antigen has two basic properties, namely antigenicity and immunogenicity.

  1. Antigenicity refers to the ability of an antigen to specifically bind to an antibody or sensitized lymphocyte induced by it. The strength of antigenicity is closely related to the size of the antigen molecule, the chemical composition, the structure of the antigenic determinant, and the proximity of the antigen to the immune animal. It is generally believed that the larger the molecular weight of the antigen, the more complex the chemical composition, the more complete the three-dimensional structure and the farther the relationship with the immunized animal, the stronger the antigenicity will be.


  1. Antibody immunogenicitymeans the ability to stimulate the body to form specific antibodies or sensitized lymphocytes. That is to say, the antigen can stimulate specific immune cells, activate, proliferate, and differentiate immune cells, and finally produce antibodies to immune effector antibodies and sensitized lymphocytes. It also refers to the specific immune response of the body’s immune system to form antibodies or sensitized T lymphocytes after the antigen stimulates the body.


T cell epitope refers to an epitope recognized by a TCR, and the epitope component is a polypeptide after protein degradation. Such epitopes are generally not located on the surface of the antigen molecule, and the antigen-presenting cells must process the antigen into a small molecule polypeptide and bind it to the MHC molecule before being recognized by the TCR. Therefore, studying T cell epitopes not only helps to understand the immune response mechanisms of infectious diseases, autoimmune diseases, allergic reactions and tumors, but also promotes the design of computer-aided vaccines. The rapid development of bioinformatics provides an effective way to study T cell epitopes.


In view of the current research status of T cell epitopes, a literature has selected several hot issues to carry out antibody immunogenicity prediction research. Main tasks are as follows:


  • Combining sequence information and structural information to predict antigenic epitopes is the development direction of antigen epitope prediction. The HLA-A2 molecule is a kind of MHC class molecule ubiquitous in the human population. The HLA-A2 antigen peptide was selected as the research object, and the amino acid physicochemical properties of the antigen peptide and the energy term when combined with MHC were explored. In order to avoid the redundancy of the features used, affecting the efficiency and performance of the classification, the features were screened by the method of maximum correlation minimum redundancy (MRMR), and finally 50 features with high recognition ability were selected and constructed. A prediction method for HLA-A2 class molecular antigen peptides. The predicted results indicate that the selected features are capable of efficiently identifying antigen peptides bound by HLA-A2 molecules.


  • The I-Ag7 molecule and the HLA-DQ8 molecule are MHC class II molecules associated with type I diabetes in mice and humans. To predict the restriction epitopes of these two classes of MHC molecules, a GPS-MBA software package was developed. With improved Gibbs sampling algorithm, the core sequence of the epitope was obtained that were scored using GPS algorithm to construct a prediction model. After a comprehensive evaluation and comparison, GPS-MBA performed better than the same type of software. A large number of potential I-Ag7 and HLA-DQ8 epitopes can be predicted using this software. In addition, the Epitope Database for Type I Diabetes (TEDB) was designed, which contains all experimentally validated or predicted epitopes associated with type I diabetes.


  • In order to verify the negative selection hypothesis and study the immunogenicity of the antigenic epitope, the exogenous T cell epitope and the non-epitope peptide and the host protein sequence were analyzed for both human and mouse hosts, respectively. Using sequence alignment, it was found that only a very small number of epitopes were similar to the host protein sequence, which was lower than the number of non-epitope peptides similar to the host protein sequence. This result indicates that the exogenous T cell epitope is similar to the host protein sequence, suggesting that using an epitope with low similarity to the host protein sequence can help reduce the probability of cross-immunization and enhance the vaccine immunogen.


(4) In order to further develop the proteasome digestion site prediction software to improve the accuracy of predicting antigenic epitopes, an evaluation of existing prediction software has been carried out adopting a number of methods and software to predict proteasome cleavage sites. The more commonly used computer programs are PAProC, MAPPP and NetChop. Performance evaluation of the three using a unified data set shows that NetChop performs better than the other.

The key to successfully develop an ADC drug

Anti-drug conjugate (ADC) is an antibody that binds to cytotoxic drugs and targets cytotoxic drugs to tumors through the targeting of antibodies, thereby reducing the non-specific systemic toxicity of drugs commonly found in chemotherapy. The study of antibody-drug conjugate (ADC) can date back to 1980s.

A successful antibody drug conjugate drug includes four main parts: a suitable target (tumor antigen), a highly specific antibody, an ideal linker, and a highly cytotoxic drug.

Firgure. The model of ADC action

  1. The basis for target selection

Nowadays, ADC drugs are mainly used for anti-tumor. When selecting a target, the ideal target antigen should be overexpressed on the surface of tumor cells, but no expression or very low expression in normal tissues. Moreover, when the antibody and the target are aggregated in the ADC drug, it can be effectively internalized, and the drug is released into the cell to kill the target cell.

  1. Antibody specificity, affinity, and pharmacokinetics

The high affinity of the antibody and the target antigen is the core of the effective targeting of the ADC. It is generally believed that the affinity index KD 10 nM is a basic requirement for the antibody. On the basis of this, antibodies that are low in immunogenicity, long in half-life, and stable in blood are screened.

  1. Selection and intracellular drug release

The ideal linker can maintain stability in the blood and effectively release the drug in the target cells. The commonly used Linkers can be divided into two categories: cleavable linkers and non-cleavable linkers. The current study found that seven B cell receptors (CD19, CD20, CD21, CD22, CD79b, and CD180) have effective effects by cleavable linkers. In contrast, when non-cleavable linkers are used, only CD22 and CD79b antigens can bind to the antibody, effectively transport ADC to lysosomes, and release the drug to kill target cells. Therefore, when choosing which linker to be uses, the nature of the target should be considered.

  1. Selection of cytotoxic drugs

Since the antibody enters the body and can effectively enter the tumor site by about 0.003–0.08% of the total amount, it is necessary to have a highly effective and highly sensitive killing effect on the target cells (free drug IC50: 10-1 1–10-9M). ). There are two main types of commonly used drugs at present – microtubule inhibitors and DNA-damaging agents.

ADC development trend

  1. Site-specific conjugation

At present, the most advanced ADC drugs are using traditional no-specific conjugation. The biggest disadvantage is that the product obtained is a mixture of different drug molecules per antibody. Site-specific conjugated drugs, and more importantly, uniform data (eg, PK) for clinical evaluation is difficult to obtain. In oder to solve these shortcomings, site specific conjugation technology has become a hot spot for major companies. Using site specific conjugation techniques, the same number of drug molecules can be carried on each antibody to obtain a uniform ADC drug. It is conducive to pharmacodynamic research and evaluation. And in the clinical can get more stable and effective results. Among them, A mbrx’s Unatural Amino acid (pAcPhe) technology has more applications and promotion prospects.

  1. Multivalent ADC drugs

The development of antibody drugs and vaccines has progressed from monovalent drugs to multivalent drugs. ADC might also follow this development process, that is, to link several small molecules that are synergistic with each other in the same antibody to improve the drug’s efficacy. This requires a more sophisticated conjugation technology, to integrate two or more technologies. But now, the site-specific technology, excessively pursuits the coupling of a specific molecules at a specific site and neglects the diversity of coupling.

Practical and traditional techniques for multivalent coupling of drugs require simultaneous coupling of multiple drugs on one antibody. In this case, the singularity of the antibody itself to modify the linking group will result in a mixed product, and there is no guarantee that each antibody carries a different drug at the same time.

This problem can be solved by Site-specific techniques. When performing Site-specific modification, a variety of different coupling groups can be designed, which can use a group to carry out drug couples for the linker with the corresponding group.

Monoclonal antibodies and their conjugates are macromolecular substances. Large drug molecules are difficult to penetrate deep into the solid tumor through the capillary endothelium and through the extracellular space of the tumor. The use of antibody fragments, such as Fab, to prepare conjugates with lower molecular weight, may increase the permeability to the extracellular space and increase the amount of drug reaching deep tumor cells. “Small size or moderate miniaturization is an important way to develop ADC drugs.”


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Mechanism of action of pancreatic cancer and related vaccines

With the improvement of people’s living standards and changes in life and work, more and more people are suffering from cancer, such as lung cancer, skin cancer, and liver cancer. In response to this situation, in addition to treating cancer, we should also prevent the occurrence of cancer from the source, such as the use of vaccine. Vaccine refers to a preventive biological product used for human vaccination to prevent and control the occurrence and prevalence of infectious diseases. The biological product refers to a preparation for prevention, diagnosis and treatment prepared by using a microorganism or a toxin thereof, an enzyme, a serum, or a cell of a human or an animal. With the development of science and technology, people’s understanding of cancer has deepened. Scientists believe that cancer vaccines can be developed to prevent and cure cancer. Cancer vaccines use the antigens of tumor cells to awaken the body’s immune system to fight cancer. There are many types of cancer vaccines, and there are different cancer vaccines for different cancers, such as lung cancer vaccines, throat cancer vaccines, pancreatic cancer vaccines, etc.

Pancreatic cancer:

Pancreatic cancer is a malignant tumor of the digestive tract, which is highly malignant and difficult to diagnose and treat. About 90% of ductal adenocarcinomas occur in the ductal epithelium. The morbidity and mortality of ductal adenocarcinomas has increased significantly in recent years and its 5-year survival rate is <1%, which is one of the most serious malignant tumors. The early diagnosis rate of pancreatic cancer is not high, its operative mortality rate is high, and the cure rate is very low. The incidence of this disease in men is higher than in women, the ratio of male to female incidence is 1.5:1 to 2:1. The incidence of man is greater than premenopausal women, and the incidence of postmenopausal women is similar to that of men.

Symptoms of pancreatic cancer:

Pancreatic cancer has no specific initial symptoms, but the most common symptoms are upper abdominal fullness, discomfort and pain. If a middle-aged person over the age of 40 complains of upper abdominal symptoms, in addition to considering hepatobiliary and gastrointestinal diseases, the possibility of pancreatic cancer should also be considered.

  1. Pain: Pain is the main symptom of pancreatic cancer. 60% to 80% of patients haveupper abdominal pain, and 85% of these patients with pain can not undergo surgeryor the disease has reached advanced stage. Pain is generally unrelated to diet and is persistent, ranging from fullness, dull pain to severe pain. There is also radiation pain, pancreatic head cancer mostly manifests as right side radioactive pain, while body tail cancer mostly manifests as left side radioactive pain.
  2. Jaundice:In the past, the diagnosis of pancreatic cancer often used painless jaundice as the first or necessary symptom of pancreatic cancer. The occurrence of jaundice has beenan important basis for the diagnosis of pancreatic cancer, thus making the patients lose the opportunity for early diagnosis and surgery. Today, painless jaundice is still the most common symptom of pancreatic cancer, but about 50% of patients with this symptom have the opportunity to undergo radical surgery. The appearance of jaundice in the morning and evening is closely related to the location of cancer, and jaundice often occurs in cancer of the head of the pancreas. Jaundice may have fluctuations, manifested as complete or incomplete obstructive jaundice. Carcinomas in the tail of the body or away from the biliary pancreatic duct may cause jaundice due to lymph node metastasis to the extrahepatic bile duct or adhesion or buckling near the bile duct.
  3. Gastrointestinal symptoms:The most common gastrointestinal symptoms areloss of appetite, nausea, vomiting, diarrhea, constipation or even nausea. Diarrhea is often steatorrhea.
  4. Weight loss, fatigue:Unlikeother cancers, pancreatic cancer often shows signs of weight loss and fatigue at the beginning of the cancer. These symptoms have nothing to do with the tumor site.
  5. Others:Pancreatic cancermay have high fever or even chills and other symptoms like cholangitis, so it is easy to confuse pancreatic cancer with cholelithiasis and cholangitis. Of course, when there is biliary obstruction and infection, there may be chills and high fever. Abdominal masses suggest that the cancer has reached an advanced stage, but sometimes the mass is a swollen liver and gallbladder, and pancreatic cancer is associated with a pancreatic cyst.

All of the above symptoms need to be differentiated from the symptoms of other digestive tract diseases, especially chronic pancreatitis. It is also necessary to pay attention to the identification of abdominal pain, because pancreatic cancer have abdominal pain, weight loss, fatigue and other symptoms. Chronic inflammation of the pancreas has been misdiagnosed and treated as cancer, and cancer has been misdiagnosed as inflammation, so other tests should be combined to identify these symptoms.

There are no very specific signs of pancreatic cancer, and although there is conscious pain, tenderness is not common in all patients. If there is tenderness, it is consistent with the site of conscious pain. The pancreas is located deep in the back of the abdomen, and if a lump is found there, the pancreatic cancer is mostly in the advanced stage. Chronic pancreatitis can also manifest as a mass, so it is difficult to distinguish it from pancreatic cancer. Pancreatic cancer can cause dilatation of the bile duct and gallbladder in the liver and cholestasis of the liver, so it can also manifest as swollen liver and gallbladder. Ascites usually occurs in the late stage of pancreatic cancer and is mostly caused by peritoneal invasion and spread of cancer. Ascites may be bloody or serous, and hypoproteinemia of advanced cachexia may also cause ascites. However, one thing to note is that pancreatic cancer complicated with pancreatic cyst rupture forms pancreatic ascites. It is characterized by an increase in the amylase and high protein content in ascites after water is released. At this time, ascites does not mean the late stage of pancreatic cancer, so don’t give up the chance of surgery.

The mechanism of action of pancreatic cancer vaccine:

Pancreatic cancer is a common malignant tumor of the digestive tract, which is highly malignant and has a very poor prognosis. Even with curative surgery, most patients have recurrence and metastasis early in the postoperative period. In order to change this situation, research on pancreatic cancer vaccines has begun in recent years. However, the regulatory T cell-mediated immune escape mechanism not only plays an important role in the development of tumors, but also inhibits the anti-tumor immune response of vaccines from the recognition, activation of differentiation to the entire stage of effect, thereby limiting the therapeutic effect of vaccine. .Clearing Treg and sensitizing the efficacy of pancreatic cancer vaccine has become an important direction in the treatment of pancreatic cancer in recent years.

Biotechnology overview: therapeutic glycoprotein production in various expression systems


In the past few years, the biopharmaceutical industry has turned to mammalian cell expression systems for the production of biological agents. Of all the mammalian cell expression systems to date, CHO cells are the most widely used, accounting for 70% of recombinant protein production, and most of the proteins are monoclonal antibodies. This article introduces the production of therapeutic glycoproteins by the cell line of mammalian cells, and outlines the screening system and gene expression system of CHO cell lines.

Figure. Host cells of glycoprotein production

  1. Chinese hamster ovary cells (CHO)

CHO cells are widely used in the production of glycoproteins, which are determined by their numerous advantages, such as high protein production rate, suitable for large-scale industrial suspension culture, and can adapt to various serum-free and chemically defined media. In addition, the sugar chains of recombinant glycoproteins produced by CHO cells are highly similar to humans themselves, and have better compatibility and biological activity in humans. In addition, since many viral genes that enter CHO cells are not expressed, they are highly resistant to human virus-infected CHO cells, thereby minimizing the rate of protein infection by viruses and reducing biosafety risks. In addition, different gene amplification systems have been developed in CHO cells to achieve higher protein yields. More than half of the FDA and EU-approved monoclonal antibodies have been produced by CHO cells in recent years.

Although CHO cells have many advantages in glycoprotein production, they are not catalyzed for certain types of human proteins, such as alpha-2,6-sialylation and alpha-1,3/4-fucosylation. Moreover, for some glycosylated CHO cells that are not expressed by humans, such as hydroxyacetoxy-neuraminic acid (Neu5Gc) and galactose-α1,3-galactose (α-gal), even if the content of both is not more than 2%, but may also be immunogenic to humans. Even after improvement by metabolic engineering, CHO cells have some limitations on the production of γ-carboxylic acid recombinant proteins, such as coagulation factors.

  1. Human cell lines

One way to produce humanized glycoproteins is to use human cell lines for protein production. It can be guaranteed that even if it is not the ideal glycoprotein type, it at least won’t cause an immune response. The most widely used strains are HEK293 and HT-1080 cells from human embryonic kidney and fibrosarcoma. Xigris is produced by HEK293 and is the first FDA and EMA approved protein synthesized by human cell lines. The production of four proteins (Agalsidase alfa, Epoetin delta, Idursulfase and Velaglucerase alfa) in HT-1080 cells was approved by the FDA or EMA, although these products were withdrawn from the market due to commercial factors. Compared with Velaglucerase alfa produced by HT-1080 cells, CHO cells have similar glycosylation performance, and their in vitro activity, stability and potency are comparable. Many of the therapeutic proteins produced using human cell lines in 2014 were approved by the FDA or EMA, such as rFVIIIFc and rFIXFc for the prevention of hemophilia A and B bleeding episodes. These proteins expressed in the HEK293 cell line have higher levels of tyrosine sulfation and glutamate gamma carboxylation compared to CHO cell lines, and there is no Neu5Gc and a-gal glycosylation.

At present, some human cell lines are also used in preclinical studies and/or recombinant glycoprotein production, such as PER.C6 cells, which can obtain high yield proteins even without amplification of related genes. Both MOR103 and CL184 are therapeutic proteins produced by PER.C6, and clinical 1/2 studies have been conducted. HKB-11 cells obtained by fusion of HEK293S cells and human B cells exhibited high concentrations of protein production and α2,3 and α2,6-sialic acid linkages. The other two cell lines, proteins produced by CAP and HuH-7 cells, are currently in clinical trials and exhibit similar levels of glycosylation as human proteins.

  1. Other mammalian cell lines

Young hamster kidney cells (BHK) are commonly used in vaccine production. Currently only two recombinant glycoproteins are produced by BHK cells, namely factors VIIa and VIII. These macromolecular proteins are challenging for BHK cells due to their large levels of glycosylation and sulfation.

Murine myeloma cells (NS0 and Sp2/0), derived from tumor cells that no longer synthesize native immunoglobulins, can also be used to produce commercial monoclonal antibodies such as cetuximab and palivizumab. In 2015, the FDA approved three monoclonal antibodies, Dinutuximab, Necitumumab and Elotuzumab, produced by murine cells, for the treatment of different cancers. In addition, the level of Neu5Gc and α-gal glycosylation expressed by murine cells is higher than that of mouse cells, thus increasing the risk of immunogenicity.

  1. Non-mammalian cell lines and other expression systems

Although the use of mammalian systems to produce recombinant proteins has been a trend for nearly a decade, other expression systems are also available for recombinant protein production. Since these biological cells lack the required related enzymatic mechanism and are not suitable for the production of proteins containing sugar chains, they are often used for protein production without glycosylation.

Bacterial expression systems have the advantage of rapidly proliferating and highly expressed proteins. However, proteins tend to form aggregates, and extraction procedures are necessary because of the loss of chaperone protein. Some commercial proteins, such as asparaginase and collagenase without sugar chains are commonly produced by bacterial expression systems.

Yeast expression system has the same advantages as bacteria, rapid proliferation and high protein expression, but these proteins often have high mannose sugar type, which may be immunogenic and not effective for human body, such as gram plasmin.

For both plant and insect cells, both can produce proteins with complex glycosylation, but their type structure is quite different from that of humans. In fact, plants express sugar chains with α1,3-fructo and β1,2-xylose as cores, which are completely different from humans and may be immunogenic to humans. The N-glycotype produced by insect cells is either a high mannose type or an oligosaccharide type, and sialylation is absent in the glycosylation levels of both plant and insect cells. Glycosylation genetic engineering is also used in plant and insect cells to produce proteins. In 2012, the FDA approved the first therapeutic recombinant protein, taliglucerase alfa, produced by plant cells. The therapeutic proteins approved for adoption in insect systems are the human papillomavirus vaccine, prostate cancer vaccine and influenza vaccine. Recently, some therapeutic proteins have also been obtained in transgenic animals. Like other mammalian expression systems, the glycosylation structure of transgenic animals expressing proteins is somewhat different from that of humans. The therapeutic protein produced by the first transgenic animal in the market is the antithrombotic drug recombinant human antithrombin III, obtained from transgenic goat milk. Two more proteins were subsequently approved, namely recombinant C1-Esterase inhibitors obtained from transgenic rabbit milk and recombinant humanized lysosomal acid lipase obtained from transgenic eggs.

The current situation of gene therapy industry

Many factors help gene therapy succeed

The outbreak of gene therapy began in the early 1990s, and if we look back at the success stories of gene therapy at that time, luck might be the main force. After nearly 30 years of scientific and technological development, gene therapy has become more and more mature, and the success rate has been continuously improved, mainly due to several factors:

  1. Improvement of viral vectorsenhances the effectiveness and safety of the treatment, such as the most widely used lentiviral vectorsand adeno-associated virus vectors;
  2. The development of vector preparation and identification technology has greatly improved the purity and efficacy of the vector, which not only improves the success rate of cell transfection, but also reduces the incidence of adverse reactions;
  3. The accumulation of basic biological knowledge is increasing, which makes scientists understand the target cells, tissues and organs more deeply, can more accurately predict the effects and side effects of gene therapy, and prepare solutions in advance;
  4. More detailed clinical observations and more effective molecular monitoring also help scientists use more accurate evidence to grasp the efficacy and safety of gene therapy;
  5. Affected by the death of American boys in 1999, scientists have been more cautious about the clinical trials of gene therapy since 2000, and have also improved the design of clinical trials, such as recruiting patients who show only early symptoms to participate in the trial, rather than progressing to patients with advanced disease, this also increases the success rate of clinical trials to some extent.

Technology and talent barriers in the industry

Although gene therapy has made some progress in some areas, there are still many problems to be solved. The technical difficulties in gene therapy are mainly how to improve the effectiveness and reduce the safety risks. Like many emerging technologies, the key technologies and talents that can address industry pain points are currently the biggest barriers in the gene therapy industry.

For gene therapy based on transgenic technology, the technical bottlenecks encountered at this stage are mainly:

(1). Most of viral vectors lack target, and cannot specifically infect diseased cells, even though different subtypes of adeno-associated viruses (AAV) are partially selective for tissues, they are far from the level of precise-specific recognition. Therefore, when performing “in vivo” treatment, the clinical application range can only be local fixed-point injection;

(2). Retroviruses and lentiviruses, which are widely used in clinical practice, insert their own genome into the genome of the host cell after infecting the host cell. The insertion position is random, and there is a potential for insertional mutation and malignant transformation of cells. Although adeno-associated viruses are non-integrating viruses, there is still the possibility of insertion into the host genome;

(3). The ideal gene therapy should be able to regulate the therapeutic gene at an appropriate level or manner depending on the nature and severity of the disease. However, the existing gene delivery system has a limited capacity and cannot accommodate the whole gene or complete regulatory sequence. Therefore, only the gene expression regulatory elements carried by the virus can be borrowed, resulting in the expression level of the target gene being unable to be regulated, and the expression level under normal physiological conditions cannot be achieved;

(4). The viral vector has certain toxicity and immunogenicity, and is easily removed by the human immune system after being injected into the patient, and also causes side effects;

(5). There is still room for further improvement in the efficiency of gene delivery of viral vectors. For gene therapy based on gene editing technology, the key technical difficulties are mainly a. gene editing technology, especially the CRISPR technology is available in a short time and there are too many uncertain factors; scientists have limited cognition of function and regulatory networks on human genes. Changing genes may cause unforeseen security problems. b. The efficiency of gene editing system of introduction into cells and the efficiency of gene editing are not high enough and cannot be applied clinically in a large scale; c. Gene editing systems, like viral vectors, are also not cell-targeting.

Regardless of GM technology or gene editing technology, optimizing and upgrading the carrier systems are the most direct, effective and unavoidable ways to solve the technical problems faced by current clinical applications. In addition, gene therapy is mostly an individualized treatment plan, and real commercial optimization requires great effort.

An overview of new progress in stem cell culture manufacturing technology (part one)

Stem cells are primitive cells that can survive for long periods of time and have the ability to self-reproduce and multi-directionalize. They almost present in all tissues. In recent years, with the deepening of research by scientists, stem cells have seen hope in the treatment of various diseases, such as blood system diseases, nervous system diseases, cardiovascular diseases, autoimmune diseases, and endocrine diseases.

Stem cell technology is one of the most advanced and hottest directions in medical research. It has developed rapidly in recent years and has achieved exciting results. At the same time, scientists have made great achievements in the cultivation and manufacture of stem cells. Scientists from the Massachusetts General Hospital developed a new program that could revolutionize the process of adult stem cell culture.

In this article, we made an overview of new technologies or progress in stem cell manufacturing.

1. SCTM: developed method for producing adult stem cells initially.


Scientists at the University of Queensland in Australia have developed the first method of producing adult stem cells in the world. This research will profoundly affect patients with a series of serious diseases.

The study was conducted in collaboration with a number of research institutions, including the University of Queensland’s Australian Institute of Bioengineering and Nanotechnology, led by Nicholas Fisk, a professor at the University of Queensland Clinical Research Center.

Mesenchymal stem cells (MSCs) can be used to repair bones and potentially repair other organs. This study reveals a new approach to producing mesenchymal stem cells.

Professor Fisk said, “We use a small molecule, SB431542, a transforming growth factor-β (TGF-β) pathway inhibitor, to induce embryonic stem cells for 10 days (to produce mesenchyme). Stem cells are produced at a much faster rate than other studies reported in the literature. This technique can also be applied to less controversial induced pluripotent stem cells (iPSCs).

2. Biofabrication: A major breakthrough in 3D printing of human embryonic stem cells.

DOI: 10.1088/1758-5082/5/1/015013

In a new study, a team from Scotland first used a new three-dimensional printing technique to arrange human embryonic stem cells (hESCs). The results of the study were published on February 5, 2013 In the Biofabrication journal, the paper titled “Development of a valve-based cell printer for the formation of human embryonic stem cell spheroid aggregates”. This breakthrough is expected to allow people to use hESCs to construct three-dimensional tissues and structures, thereby accelerating and improving the drug testing process.

In the field of biomanufacturing, the fabrication of three-dimensional tissues and organs by combining artificial solid structures and cells has made great progress in recent years. However, in most previous studies, animal cells were used to test different printing methods in order to fabricate these structures.

Dr. Will Wenmiao Shu, co-author of the paper and researcher at Heriot-Watt University in Scotland, said, “As far as we know, this is the first time to print hESCs. Using hESCs to create three-dimensional structures will allow us to build more accurate human tissue models, and these human tissue models are required for drug development and toxicity testing in vitro. Because most drug development is directed at human disease, the use of human tissue makes sense.”

In the long term, this new method of printing may lay the foundation for the integration of hESCs into artificially constructed organs and tissues that are ready to be transplanted into patients with different diseases.

3. Biofabrication: Handheld 3D “printing pen” can efficiently print human stem cells


Recently, in a research report published in the international magazine Biofabrication, researchers from Australia successfully used a hand-held 3D printing pen to successfully map human stem cells with high survival rate in free mode. The new device developed by the researchers can help surgeons perform personalized cartilage transplants during surgery.

The researchers pointed out that the use of hydrogel-type “bio-ink” to carry and support human stem cell growth, and the use of a lower light source to coagulate “bio-ink”, the survival rate of stem cells transported by the pen will exceed 97%. This new type of 3D printing pen also greatly helps tissue engineering research, for example, it can print out cells layer by layer to build artificial tissue for transplantation.

However, in some cases, such as during cartilage repair, the precise geometric properties of the implant may not be accurately applied to the surgical procedure, which makes the preparation of the artificial cartilage tissue graft become complex and difficult; the new print pen acts like a surgeon’s hand and can accurately fill a custom-made stent or graft into a missing part of the patient’s body. Researcher Professor Choong said that the development of this new type of device is the result of a joint effort between scientists and clinicians, and will bring unprecedented changes to improved research and patient care.

To be continued in Part Two…

An Evaluation of Current in Vitro Assays

Abstract: The so-called in vitro assay refers to in vitro studies using microorganisms, cells or biomolecules outside of their normal biological background. In contrast, in vivo assays were performed in animals (including humans) and all plants. Examples of in vitro assays include: the isolation, growth and identification of cells from multicellular organisms in cell or tissue culture; subcellular components (e.g. mitochondria or ribosomes); cells or subcellular extracts (e.g. wheat germ or mesh); erythrocyte extracts; purified molecules such as proteins, DNA or RNA; and commercial production of antibiotics and other drugs. Viruses that replicate only in living cells are studied in cell or tissue culture in the laboratory, and many animal virologists refer to this work as separating them from the entire body of the animal in vitro. In vitro preclinical testing allows for species-specific, simpler, more convenient, and more detailed analysis than in vivo testing using whole organisms. In vitro assay meaning is of great significance. Just as the entire animal research has gradually replaced human trials, and in vitro trials are gradually replacing research on whole animals. This article will use several specific examples to explore the true face of in vitro assays and the research status in recent years.

Keywords: in vitro test, research progress, evaluation

In vitro assay of cytotoxicity of dental restorative materials

A number of in vitro assays have been applied to the biocompatibility evaluation of dental restorative materials with the aim of minimizing costly and influential animal testing and in vivo testing.

The cytotoxicity assay using in vitro cell culture is one of the most widely used in vitro assay methods and also an important assay index in the biocompatibility evaluation system of oral materials.

The assay allows the cultured cells to be contacted with the material to be tested for a certain period of time, and then the amount of growth or growth and the activity state of the cells are measured to evaluate the cytotoxicity of the material to be tested.

The cytotoxicity assay is fast and cost-effective and much less expensive than animal testing and in vivo testing. Being highly standardized and experimentally controlled and with reproducible results, the cytotoxicity assay can be tightly controlled to target specific scientific issues.

Embryo toxicity in vitro test

  • What is embryo toxicity?

Embryo toxicity usually refers to the selective toxic effect of exogenous factors on the embryo, which is manifested by the loss of embryos in the early stage of implantation or after implantation. Any toxicity observed during the prenatal period may be related or unrelated to the toxic effects of the mother. Although embryo toxicity is triggered in the uterus, it is manifested in prenatal or postnatal development, including gestational egg wilting, fetal death, fetal growth restriction, functional defects and deformities.

  • Embryo toxicity assay methods

The in vivo assay is a toxicity and toxicological assay performed on the whole animal. The three-stage assay recommended by the International Coordinating Committee (ICH) requires the use of rug registration technology:

  • Fertility and early embryo development toxicity test.
  • Embryonic one-child developmental toxicity assays (denatogenic sensitiveperiod reproductive toxicity test).
  • Perinatal toxicity assays. The assay requires the use of a large number of assay animals, high cost, long assay period, and has difficulty in quickly assessing the toxicity of a large number of substances, and cannot meet the three principles of reduction, substitution and optimization.

The in vitro assay has the advantages of simplicity, economy, short assay period, controllable assay conditions, and easy measurement of dose-response relationship and exclusion of maternal interference. It has been widely used in the screening of embryo toxicity and the study of teratogenic mechanisms. In recent years, with the rapid development of molecular biology, embryology, developmental toxicology and reproductive toxicology, a variety of in vitro assay systems for embryonic reproductive development toxicity have been established. In the late 1990s, the European Institute of Alternative Methods recommended three in vitro embryo developmental toxicity screening assays with high efficacy as the preferred method for in vitro screening, namely in vitro whole embryo culture (WEC) test, embryonic cell micromass culture (MM) assay and embryonic stem cell test (EST).


In vitro assay of bioavailability of heavy metals on human body

To assess the bioavailability of heavy metals in the soil to humans, there are usually two methods, animal testing and in vitro preclinical testing. In the animal test, the dose-response relationship can be studied by artificially feeding the feed mixed with heavy metal contaminated soil, thereby obtaining the toxicity threshold value for the animal, and then introducing the uncertainty factor to consider the possible intra- and inter-species differences, so as to obtain the human body’s toxicity threshold, and ultimately determine the maximum allowable intake of the human body. The results of animal testing are generally considered to be fairly reliable, but the application of this method is limited by its relatively long assay period and high trial costs. The emergence and development of in vitro methods provides an economical, rapid and effective means of assessment.

On the one hand, after in vitro screening and screening for soils with serious pollution levels that may endanger human health, toxicological studies can be carried out in combination with animal experiments. On the other hand, in vitro methods can be used to quickly verify the effectiveness of soil remediation measures.

Skin drug allergy reaction in vitro test

In vitro preclinical testing is a strong evidence for the verification of skin drug allergies. Because of its high safety, sensitivity and specificity, it is a powerful complement to skin assays and challenge assays. Skin drug allergies are classified into immediate (mainly IgE-mediated) drug allergy and non-synaptic (non-IgE-mediated) drug allergy.

For IgE-mediated rapid-acting drug allergy, there are two main detection methods. For IgE-mediated allergic reactions to immediate-onset drugs, serum-specific IgE antibody (sIgE) assays are the most common in vitro assays. Serum sIgE antibody assays were performed using the Pharmacia fully automated CAP system and were graded based on serum sIgE antibody levels. The higher the drug-specific slgE level, the stronger the clinical relevance is. The other is the basophil activation test. In the immediate hypersensitivity reaction of drugs, Fc receptors with high affinity IgE on the plasma membrane of basophils can specifically bind IgE, and degranulate release heparin, histamine and other active transmitters. BAT is to observe whether it is activated by suspicious drugs after stimulating basophils.

For non-IgE-mediated delayed-type drug allergies, there are four main detection methods:

  • Lymphocyte transformation test(LTT)
  • Lymphocyte activation test (LAT)
  • Cytotoxicity test
  • Cytokine test


[1] Genschow E, Spielma nn H, Seholz G, et al. The ECVAM international validation study on in vitro embryo toxicity tests: results of the definitive phase and evaluation of prediction models [J]. ATLA, 2002, 30(2):151-178.

[2] Romano A, Torres MJ, Castells M, et al. Diagnosis and management of drug hyper sensitivity reactions[J]. J Allergy Clin Immunol, 2011, 127(3 Suppl): S67-72.

Three reasons to answer why is cancer so difficult to treat

Why is cancer so difficult to treat?

In many people’s minds, cancer and AIDS are the two most horrific diseases. If you ask me, which one will be conquered first between the two? My answer is definitely AIDS.

Why so? Why is cancer so difficult to treat? There are three main reasons in my opinion.

The first reason is that cancer is an “endogenous disease” and cancer cells come from the patient itself, are part of the patient’s body.

For “exogenous diseases”, such as bacterial infections, we have antibiotics and the effect is very good. Why is antibiotics work so well? Because it is only toxic to bacteria and has no effect on human cells, and can be used at very high concentrations to kill all bacteria, while patients are “unscathed”.

But it’s not that simple to conquer cancer. Although cancer cells are broken human cells, they are still human cells. Therefore, to fix them, we are almost destined to be “to win at a great cost”. This is the “side effect” that everyone often hears. For example, traditional chemotherapy drugs can kill fast-growing cells, which is useful for cancer cells, but unfortunately, many normal cells in our body are also growing rapidly, such as hair follicle cells under the scalp. Hair follicle cells are essential for hair growth. Chemotherapy drugs kill cancer cells and kill hair follicle cells. This is why patients with chemotherapy lose their hair. Hematopoietic stem cells responsible for hematopoiesis and maintaining the immune system are also killed, so the immune system of chemotherapy patients is very weak and extremely susceptible to infection. The epithelial cells of the digestive tract are also killed, so the patient has severe diarrhea, no appetite, and so on. Because of these serious side effects, chemotherapy drugs cannot be used in large doses, the concentration must be strictly controlled, and they cannot be used continuously. It is necessary to take a course of treatment. In fact, doctors constantly weigh and even compromise between curing cancer and maintaining the basic life of patients. If chemotherapy drugs can continue to be used in large doses like antibiotics, the cancer has long been cured. This is the main reason why I think AIDS will be overcome first than cancer. After all, AIDS is an “exogenous disease” caused by HIV. In theory, we may find drugs that only kill HIV without affecting human cells.

The second reason is that cancer is not a single disease, but a combination of tens of thousands of diseases.

There are no two identical leaves in the world, and there are no two identical cancers in the world. For example, lung cancer is the number one cancer killer in China and the United States. China now has nearly 600,000 lung cancer patients per year, and the United States has 160,000. I was often asked: is there any new medicine for lung cancer in the United States? I said: there are, but it is only useful for a small number of patients. For example, Novartis’s latest lung cancer drug, Ceritinib, has recently been approved by the FDA, which has a good effect on 3% to 5% of lung cancer patients. But why did the new drug that was spent 10 years of research only work for a small number of patients? According to the pathological classification, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer.

Is that the complete classification of lung cancer? No. We know that cancer is caused by genetic mutations. A recent systematic gene sequencing study showed that the average number of mutations per patient in lung cancer is close to 5,000! The combination of mutations varies from person to person, and each patient’s genome is specific. The 600,000 lung cancer patients are actually more like 600,000 different diseases. Of course, this is not to say that we need 600,000 different drugs to treat lung cancer. Most of these thousands of mutations do not work for cancer cell growth. Only a few mutations are critical. As long as we capture these key genes, we are likely to develop more effective drugs. But in any case, new anti-cancer drugs developed by pharmaceutical companies, even if they are panacea, cannot cure all lung cancer patients. Going back to the question just now, why is Novartis’ new drug, Ceritinib, effective only for 3% to 5% of lung cancer patients? Because Ceritinib targets the mutated ALK gene, only 3% to 5% of lung cancer patients have ALK mutations. For lung cancer patients without ALK mutations, the drug is completely ineffective. Ceritinib is currently preparing for clinical trials in China and expects that in the near future, Chinese patients with ALK mutations will be able to use this drug.

Because of the diversity of cancers, pharmaceutical companies are almost destined to develop drugs for only a few patients at a time. What is the development cost of each new drug? According to data, it needs at least 10 years and 2 billion dollars! Such a large amount of time and money investment has led us to make slow progress. To overcome all cancers, even if it is not in the foreseeable future, there is still a long way to go.

The third reason is that cancer can quickly develop drug resistance.

This is a common cause for cancer and AIDS, and it is the root cause of AIDS. Many people may have heard of super bacteria. Staphylococcus aureus infection is fatal before the onset of antibiotics, which can cause sepsis. But after humans discovered penicillin, Staphylococcus aureus was not so terrible. However, the evolution of biology is incomparably magical. Because we abuse penicillin, while it kills 99.999999% of bacteria, a certain bacterium suddenly develops new genetic mutations, which evolved resistance. They are no longer afraid of penicillin and become very dangerous. So, humans tried to find stronger antibiotics, such as vancomycin. But now there are both Staphylococcus aureus resistant to penicillin and vancomycin, which is super bacteria.

Biological evolution is a double-edged sword. Nature gives us this ability to adapt to different environments, but cancer cells not only retain the basic evolutionary ability, but also stronger. For the drugs we give it, the cancer cells are constantly changing, and try to avoid the drugs and survive. In Ceritinib clinical trials, people found that many cancer cells discarded the mutated ALK gene after a few months of treatment, and produced new mutations to help the cancer grow. The cancer cells evolve so fast. I cannot help thinking that how humans are insignificant in front of nature.